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balb / c female mice ( 68 week old ) were obtained from jackson laboratory . animal use conformed to institutional guidelines of weill cornell medical college and the feinstein institute for medical research . mice were immunized with multi - antigenic peptide coupled to dweysvwlsn ( mp , 100 g ) , control polylysine core ( mc , 100 g ) , scrambled wsdyevwlsn ( ms , 100 g ) , or aaaaavwlsn ( ma , 100 g , anaspec ) as previously described15 . we used mouse monoclonal aab r4a or control igg2b ( 200 g , southern biotech ) , human monoclonal aab g11 , human control aab b1 or human anti - dna ab d9 ( 150 g ) , obtained by isolating peptide - reactive or non - reactive b cells and expressing the ig heavy and light chain genes in 293 cells39,40 . the d form of dweysvwlsn peptide ( 200 g ) or irrelevant peptide nemqssrlre was given i.p . to mice at the time of intravenous r4a administration , and again 24 h and 48 h later . eu - labeled monoclonal abs ( perkin elmer ) were intravenously administered ( 100 g ) to gestating dams at e14 . fetal brains exposed to eu - r4a were stained with ab to cd-31 to identify blood vessels . for tunel assays , coronal sections ( 20 m thick ) of e15 brains were processed according to the manufacturer ( r&d systems ) and counterstained with methyl green . for ph3 assays of mitotic cells , frozen brain sections ( 20 m ) were blocked with 10% normal goat serum and incubated overnight at 4c with ph3 ab ( 1:1000 ; chemicon , millipore , billerica , ma ) followed by red x goat anti - rabbit igg ( jackson immuno research laboratories ) . measurements of the cp and the cerebral width were performed on nestin ( 1:200 ; chemicon ) and dapi ( 1:1000 ; invitrogen , carlsbad , ca ) stained e15 coronal sections , with openlab software ( improvision , perkinelmer ) . quantification of ph3 + and tunel+ cells was performed using automated image analysis programs on an axio - image microscope ( zeiss , thornwood , ny ) . anatomic regions were identified using within section coordinates and positive cells were counted in comparable areas of interest . negative geotaxis was measured23 in offspring born from mph ( n = 19 ) , mpl ( n = 14 ) and mc ( n = 15 ) dams at ages p11 , p15 or p16 and p20 . mice were placed with their heads facing downward on an incline ( 45 ) and the time to turn 180 and climb the incline was measured ( maximum 30 s , if the animal did not reach the top of the incline in the allotted time , a score of 30 s was assigned ) . each mouse was given three trials and the mean latency was calculated . adult male offspring from mph ( n = 10 ) , mpl ( n = 8) and mc ( n = 10 ) groups underwent a behavioral battery15,16 ( described in supplementary fig . we used a blinded format and randomized the order in which the cohorts of animals were tested in different tasks . the novel object recognition task27 consisted of three phases . in the first ( sample ) , mice explored two identical objects for 5 min . in the second ( delay ) , mice stayed in their home cage for 10 min . test objects replaced sample objects , so that one was identical to those in the sample phase ( familiar object ) and the other was different ( novel object ) . for the final phase ( choice ) , mice inspected the test objects for 5 min . object exploration was scored automatically with the pc - based anymaze software ( stoelting ) . we defined a3 as the exploration time for the familiar object , and b1 as the exploration time for the novel object , and measured the discrimination ratio , ( b1a3 ) / ( b1+a3 ) . the topological task28 consisted of five sessions ( 5 min ) with varying intersession intervals ( 3 min , 10 min , 3 min and 10 min ; sequentially ) . four different objects were arranged in a square . in sessions 1 and 2 , mice were familiarized to this arrangement . before session 3 , two objects were transposed , creating a novel configuration ii . animals were expected to explore displaced objects more avidly during session 3 and 4 . before session 5 , the two objects that had remained unchanged were switched , thus creating configuration iii . we defined d1 as the sum of the times exploring the displaced objects in configurations ii and iii ( sessions 3 and 5 ) and d2 as the sum of the times exploring the non - displaced objects ( sessions 3 and 5 ) . topological processing was measured with the ratio , ( d1d2 ) / ( d1+d2 ) . the fear extinction task26 initially required animals to associate the conditioned stimulus ( cs , 20-s tone , 5 khz , 80 db ) with the unconditioned stimulus ( us , foot - shock , 1 s , 0.6 ma ) , as previously described16 . extinction learning was tested 24 h later by exposing mice to 20 trials of cs alone in a novel chamber , with an intertrial interval of 120 s. to determine fear extinction , we measured the amount of freezing during each tone , using freezeframe software ( actimetrics ) . eight months after birth , mice that had been subjected to behavioral studies were anesthetized and perfused . the anterior striatum , and the anterior and posterior commissure were the internal landmarks to block and section the tissue in order to determine the volume of neocortical areas , hippocampus and amygdala . tissue was stained with cresyl violet and mouse anti - neun ( 1:1000 ; chemicon ) . internal cortical coordinates were the interhemispheric fissure and the anterior corpus callosum ; for hippocampus and amygdala ( basolateral and lateral nuclei ) , the coronal perimeters were captured on video screen by ibas software ( zeiss ) from the dorsal third ventricle to the posterior commissure . for neuron size determinations , video capture of neurons was performed using an optical dissector randomly sampling each region of interest . statistical comparisons were performed with kruskal - wallis and fisher 's analysis of variance , student 's t test ( unpaired , two - sided ) , mann - whitney u test , and kolmogorov - smirnov non - parametric test . we used the software packages spss ( spss inc . ) , statistica ( statsoft ) and origin ( originlab ) for our analyses . balb / c female mice ( 68 week old ) were obtained from jackson laboratory . animal use conformed to institutional guidelines of weill cornell medical college and the feinstein institute for medical research . mice were immunized with multi - antigenic peptide coupled to dweysvwlsn ( mp , 100 g ) , control polylysine core ( mc , 100 g ) , scrambled wsdyevwlsn ( ms , 100 g ) , or aaaaavwlsn ( ma , 100 g , anaspec ) as previously described15 . we used mouse monoclonal aab r4a or control igg2b ( 200 g , southern biotech ) , human monoclonal aab g11 , human control aab b1 or human anti - dna ab d9 ( 150 g ) , obtained by isolating peptide - reactive or non - reactive b cells and expressing the ig heavy and light chain genes in 293 cells39,40 . the d form of dweysvwlsn peptide ( 200 g ) or irrelevant peptide nemqssrlre was given i.p . to mice at the time of intravenous r4a administration , and again 24 h and 48 h later . eu - labeled monoclonal abs ( perkin elmer ) were intravenously administered ( 100 g ) to gestating dams at e14 . fetal brains exposed to eu - r4a were stained with ab to cd-31 to identify blood vessels . for tunel assays , coronal sections ( 20 m thick ) of e15 brains were processed according to the manufacturer ( r&d systems ) and counterstained with methyl green . for ph3 assays of mitotic cells , frozen brain sections ( 20 m ) were blocked with 10% normal goat serum and incubated overnight at 4c with ph3 ab ( 1:1000 ; chemicon , millipore , billerica , ma ) followed by measurements of the cp and the cerebral width were performed on nestin ( 1:200 ; chemicon ) and dapi ( 1:1000 ; invitrogen , carlsbad , ca ) stained e15 coronal sections , with openlab software ( improvision , perkinelmer ) . quantification of ph3 + and tunel+ cells was performed using automated image analysis programs on an axio - image microscope ( zeiss , thornwood , ny ) . anatomic regions were identified using within section coordinates and positive cells were counted in comparable areas of interest . negative geotaxis was measured23 in offspring born from mph ( n = 19 ) , mpl ( n = 14 ) and mc ( n = 15 ) dams at ages p11 , p15 or p16 and p20 . mice were placed with their heads facing downward on an incline ( 45 ) and the time to turn 180 and climb the incline was measured ( maximum 30 s , if the animal did not reach the top of the incline in the allotted time , a score of 30 s was assigned ) . each mouse was given three trials and the mean latency was calculated . adult male offspring from mph ( n = 10 ) , mpl ( n = 8) and mc ( n = 10 ) groups underwent a behavioral battery15,16 ( described in supplementary fig . we used a blinded format and randomized the order in which the cohorts of animals were tested in different tasks . the novel object recognition task27 consisted of three phases . in the first ( sample ) , mice explored two identical objects for 5 min . in the second ( delay ) , mice stayed in their home cage for 10 min . test objects replaced sample objects , so that one was identical to those in the sample phase ( familiar object ) and the other was different ( novel object ) . for the final phase ( choice ) , mice inspected the test objects for 5 min . object exploration was scored automatically with the pc - based anymaze software ( stoelting ) . we defined a3 as the exploration time for the familiar object , and b1 as the exploration time for the novel object , and measured the discrimination ratio , ( b1a3 ) / ( b1+a3 ) . the topological task28 consisted of five sessions ( 5 min ) with varying intersession intervals ( 3 min , 10 min , 3 min and 10 min ; sequentially ) . four different objects were arranged in a square . in sessions 1 and 2 , mice were familiarized to this arrangement . before session 3 , two objects were transposed , creating a novel configuration ii . animals were expected to explore displaced objects more avidly during session 3 and 4 . before session 5 , the two objects that had remained unchanged were switched , thus creating configuration iii . we defined d1 as the sum of the times exploring the displaced objects in configurations ii and iii ( sessions 3 and 5 ) and d2 as the sum of the times exploring the non - displaced objects ( sessions 3 and 5 ) . topological processing was measured with the ratio , ( d1d2 ) / ( d1+d2 ) . the fear extinction task26 initially required animals to associate the conditioned stimulus ( cs , 20-s tone , 5 khz , 80 db ) with the unconditioned stimulus ( us , foot - shock , 1 s , 0.6 ma ) , as previously described16 . extinction learning was tested 24 h later by exposing mice to 20 trials of cs alone in a novel chamber , with an intertrial interval of 120 s. to determine fear extinction , we measured the amount of freezing during each tone , using freezeframe software ( actimetrics ) . eight months after birth , mice that had been subjected to behavioral studies were anesthetized and perfused . the anterior striatum , and the anterior and posterior commissure were the internal landmarks to block and section the tissue in order to determine the volume of neocortical areas , hippocampus and amygdala . tissue was stained with cresyl violet and mouse anti - neun ( 1:1000 ; chemicon ) . internal cortical coordinates were the interhemispheric fissure and the anterior corpus callosum ; for hippocampus and amygdala ( basolateral and lateral nuclei ) , the coronal perimeters were captured on video screen by ibas software ( zeiss ) from the dorsal third ventricle to the posterior commissure . for neuron size determinations , video capture of neurons statistical comparisons were performed with kruskal - wallis and fisher 's analysis of variance , student 's t test ( unpaired , two - sided ) , mann - whitney u test , and kolmogorov - smirnov non - parametric test . we used the software packages spss ( spss inc . ) , statistica ( statsoft ) and origin ( originlab ) for our analyses .

systemic lupus erythematosus ( sle ) is an autoimmune disease mediated by autoantibodies ( aabs ) and preferentially affecting women of childbearing age . since the offspring of mothers with sle exhibit a high frequency of learning disorders1 - 5 , we hypothesized that maternally transferred aabs that bind dna and the n - methyl - d - aspartate receptor ( nmdar)6 - 12 could play a pathogenic role during fetal brain development . here we describe a maternal sle murine model wherein pregnant dams harbored dna - specific , nmdar - specific aabs throughout gestation . high titers of these aabs in maternal circulation led to histological abnormalities in fetal brain and subsequent cognitive impairments in adult offspring . these data support a paradigm in which in utero exposure to neurotoxic aabs causes abnormal brain development with long - term consequences . this paradigm may apply to multiple congenital neuropsychiatric disorders .

METHODS Animals and immunization Immuno-histology of fetal brains Behavioral assessment Adult Histology Statistical analysis Supplementary Material

locally advanced breast cancer ( labc ) is defined by presence of a large primary tumor ( > 5 cm or t3 ) , associated with or without skin or chest - wall involvement ( t4 ) or with fixed ( matted ) axillary lymph nodes or with disease spread to ipsilateral internal mammary or supraclavicular nodes in the absence of any evidence of distant metastases . labc accounts for 10 - 20% in the west , while in india , it accounts for 30 - 35% of all cases . labc encompasses a wide spectrum of malignant breast tumors with varying presentation and poses a significant therapeutic challenge . the introduction of neoadjuvant chemotherapy ( nact ) in labc offered us advantages like initiation of early systemic therapy , delivery of drugs through intact vasculature , down - staging of tumors , which makes inoperable tumors operable and renders tumors suitable for breast conserving surgery ( bcs ) . it also helps in vivo assessment of response . national surgical adjuvant breast and bowel project ( nsabp)-18 and milan trials have shown that there were no difference in disease free survival ( dfs ) and overall survival between the patients who had received nact when compared to the patients who had received postoperative adjuvant chemotherapy . there are very few indian studies of nact in labc published until date . keeping this in mind , we have conducted a retrospective analysis to see the outcome of nact in labc patients at a tertiary care center . we retrospectively reviewed the case files of 148 patients of stage iii labc patients treated with nact followed by surgery and radiotherapy from january 2006 to december 2010 . the pathologic diagnosis was confirmed by fine - needle aspiration cytology or core needle biopsy performed before treatment . complete metastatic workup with chest x - ray , ultrasound abdomen , and bone scan of each patient was done . fac 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . ac t adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 cmf cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1 . adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 adriamycin - 60 mg / m iv day 1 . cyclophosphamide - 600 mg / m iv day 1 paclitaxel - 175 mg / m iv day 1 cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . clinical response ( cr ) to nact was assessed according to world health organization criteria . patients with positive or close margins were given boost with ir 192 implant or electron beam or conformal radiotherapy . postmastectomy radiation dose was 35 gy/15 # to chest - wall and 40 gy/15 # to the supraclavicular fossa . tamoxifen or letrozole was given to hormone receptor positive patients for 5 years according to the menopausal status . fac 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . ac t adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 cmf cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1 . adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 adriamycin - 60 mg / m iv day 1 . cyclophosphamide - 600 mg / m iv day 1 paclitaxel - 175 mg / m iv day 1 cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . cyclophosphamide - 600 mg / m iv day 1 . methotrexate - 40 mg / m iv day 1 . 5 fu - 600 mg / m iv day 1 . clinical response ( cr ) to nact was assessed according to world health organization criteria . patients with positive or close margins were given boost with ir 192 implant or electron beam or conformal radiotherapy . postmastectomy radiation dose was 35 gy/15 # to chest - wall and 40 gy/15 # to the supraclavicular fossa . tamoxifen or letrozole was given to hormone receptor positive patients for 5 years according to the menopausal status . median age at presentation was 46 years ( range : 22 - 72 years ) . 84 ( 56.7% ) patients presented with no axillary or single mobile ipsilateral axillary lymphnode , whereas 64 ( 43.30% ) patients had n2 or n3 disease . patient characteristics majority of the patients ( 60.8% ) patients received only anthracycline - based chemotherapy and 52 ( 35.1% ) patients received combination of anthracycline and taxane - based chemotherapy with median number of cycles being six . six patients received cmf based chemotherapy due to preexisting cardiac morbidity ( i.e. , coronary artery disease in three patients , dilated cardiomyopathy in one patient , systolic dysfunction with ejection fraction < 50% in two patients ) and of older age . these patients were of > 70 years of age and their mean karnofsky performance status ( ps ) was 70 . most of the patients ( 80.4% ) responded to nact either in the form of complete or partial response ( pr ) . complete cr was seen in18% patients and pr was seen in 62% patients , 13% patients had stable disease ( sd ) and 7% patients had progressive disease ( pd ) after nact . pathological complete response ( pcr ) was seen in 24 ( 16.20% ) patients [ table 2 ] . there was no significant difference in response when anthracycline and taxane - based chemotherapy was compared . most of the patients ( 78% ) underwent axillary dissection up to level ii . previously performing a mastectomy after nact was our institutional protocol . there was no statistically significant difference in dfs between the patients undergoing bcs and mastectomy . in 10 patients surgery median number of axillary lymphnodes dissected was 11 ( range : 3 - 19 ) . lymphovascular space invasion was positive in 43 ( 29.1% ) patients and extracapsular extension ( ece ) was present in 27 ( 18.2% ) patients . at a median follow - up period of 44 months , 36 patients ( 24.3% ) developed relapse of which six patients developed locoregional recurrence ( lrr ) while 28 patients developed distant metastases and two patients had recurrence in the contralateral breast . among six patients with lrr , three patients developed local recurrence , one patient developed axillary lymphnode recurrence , another two developed supraclavicular nodal relapse . patients with n0 or n1 disease had better 5 year dfs when compared to patients with n2 or n3 disease ( 84% vs. 48% ; p = 0.04 ) [ figure 1 ] . patients who had responded to chemotherapy ( cr + pr ) had significantly better 5 year dfs than nonresponders ( sd + pd ) ( 80% vs. 15% p = 0.02 ) . the patients who had achieved a pathological tumor size of <3 cm had better dfs when compared to patients with pathological tumor size of > 3 cm ( 93% vs. 22% p = 0.03 ) [ figure 2 ] . presence of ece was also associated with higher distant relapse . 5 year dfs was 24% in patients who had ece as compared to 89% in patient who had no ece . age , menopausal status , hormone receptor profile , lymphovascular space invasion , margin positivity had no significant impact on dfs as shown in table 3 . meier curve showing impact of nodal status on disease free survival kaplan meier curve showing impact of pathological tumour size on disease free survival kaplan meier curve showing impact of extracapsular extension on disease free survival impact of different prognostic factors median age at presentation was 46 years ( range : 22 - 72 years ) . 84 ( 56.7% ) patients presented with no axillary or single mobile ipsilateral axillary lymphnode , whereas 64 ( 43.30% ) patients had n2 or n3 disease . majority of the patients ( 60.8% ) patients received only anthracycline - based chemotherapy and 52 ( 35.1% ) patients received combination of anthracycline and taxane - based chemotherapy with median number of cycles being six . six patients received cmf based chemotherapy due to preexisting cardiac morbidity ( i.e. , coronary artery disease in three patients , dilated cardiomyopathy in one patient , systolic dysfunction with ejection fraction < 50% in two patients ) and of older age . these patients were of > 70 years of age and their mean karnofsky performance status ( ps ) was 70 . most of the patients ( 80.4% ) responded to nact either in the form of complete or partial response ( pr ) . complete cr was seen in18% patients and pr was seen in 62% patients , 13% patients had stable disease ( sd ) and 7% patients had progressive disease ( pd ) after nact . pathological complete response ( pcr ) was seen in 24 ( 16.20% ) patients [ table 2 ] . there was no significant difference in response when anthracycline and taxane - based chemotherapy was compared . breast conserving surgery was possible in 42 ( 28.4% ) cases . 96 ( 64.9% ) most of the patients ( 78% ) underwent axillary dissection up to level ii . previously performing a mastectomy after nact was our institutional protocol . there was no statistically significant difference in dfs between the patients undergoing bcs and mastectomy . in 10 patients surgery most ( 82.4% ) patients had invasive ductal carcinoma . only 12 ( 8.1% ) patients had positive margins . median number of axillary lymphnodes dissected was 11 ( range : 3 - 19 ) . lymphovascular space invasion was positive in 43 ( 29.1% ) patients and extracapsular extension ( ece ) was present in 27 ( 18.2% ) patients . at a median follow - up period of 44 months , 36 patients ( 24.3% ) developed relapse of which six patients developed locoregional recurrence ( lrr ) while 28 patients developed distant metastases and two patients had recurrence in the contralateral breast . among six patients with lrr , three patients developed local recurrence , patients with n0 or n1 disease had better 5 year dfs when compared to patients with n2 or n3 disease ( 84% vs. 48% ; p = 0.04 ) [ figure 1 ] . patients who had responded to chemotherapy ( cr + pr ) had significantly better 5 year dfs than nonresponders ( sd + pd ) ( 80% vs. 15% p = 0.02 ) . the patients who had achieved a pathological tumor size of <3 cm had better dfs when compared to patients with pathological tumor size of > 3 cm ( 93% vs. 22% p = 0.03 ) [ figure 2 ] . 5 year dfs was 24% in patients who had ece as compared to 89% in patient who had no ece . age , menopausal status , hormone receptor profile , lymphovascular space invasion , margin positivity had no significant impact on dfs as shown in table 3 . meier curve showing impact of nodal status on disease free survival kaplan meier curve showing impact of pathological tumour size on disease free survival kaplan meier curve showing impact of extracapsular extension on disease free survival impact of different prognostic factors there is paucity of literature pertaining to outcome of nact in labc in india where majority of breast cancer patients present with advanced disease . the aim of the study was to assess the use of nact in patients with labc at an indian tertiary care center . the median age of presentation in our study is 46 years , which is quite comparable with other studies . raina et al . in an early breast cancer study reported median age of 47 years whereas min et al . 42.6% of our patients were premenopausal , which is slightly lower than other studies by yadav et al . and a study by raina et al . showed estrogen receptor ( er ) positivity of 64% . there is a lot of variation in the number of cycles of chemotherapy that are given in neoadjuvant setting in the literature . investigators have administered either 3 - 4 cycles of chemotherapy or chemotherapy was continued up to maximal response . the advantage of giving chemotherapy up to maximal response is that if the patient has achieved good cr in less than planned cycles , continuation of further chemotherapy consolidates the complete response by maintaining the dose intensity . another advantage of continuing nact up to maximal response is that it may be possible that a fixed number of cycles may not be enough to achieve the amount of response necessary to do bcs and if chemotherapy is continued , further regression may continue . majority of our patients achieved maximal response after six cycles of nact . most of the patients ( 60.8% ) received anthracycline based chemotherapy as per institutional protocol . however , with increasing popularity of taxanes considerable amount of patients ( 35.1% ) received combined anthracycline and taxane - based chemotherapy . when docetaxel has been compared head on with anthracycline based chemotherapy it seems to show a better response rate in selected patients as reported in a small series . the nsabp trial has shown that use of taxanes with doxorubicin sequentially did show a better response rates in terms of superior partial and complete response both in er positive and negative patients . 80.4% of our patients responded to nact ( clinical cr + pr ) with pathological cr being 16.2% . our study showed a slightly lower pcr possibly because majority of patients received anthracycline based chemotherapy and we did not include patients with early breast cancer . the rates of bcs after nact in the present study is comparable to those reported in the literature . hence , bcs can be a good option after nact in labc patients instead of radical surgery . when we analyzed different prognostic factors , we found that response to chemotherapy was an important determinant of dfs . patients who responded to chemotherapy had significantly better dfs when compared with patients who had stable or pd after nact ( 80% vs. 15% ; p = 0.02 ) . deo et al . showed similar results in their study . in the present study , prognosis for patients with n0 or n1 disease was favorable compared with patients with advanced nodal disease ( 5 year dfs 84% vs. 48% ; p = 0.04 ) which was in concordance with the finding described by giordano . . showed pathological tumor stage did not have significant impact on lrr free survival . in a study by chen et al . it has been seen that 5 year lrr free survival was less in patients who had residual tumor size of > 2 cm after nact as compared to patients who had residual tumor size of < 2 cm . the current study showed patients who achieved pathological tumor size of <3 cm had better 5 year dfs than patients who had pathological tumor size of more than 3 cm ( 5 year dfs 93% vs. 22% ; p = 0.03 ) . brenner et al . showed borderline significance of ece in terms of survival , whereas yadav et al . we found presence of extensive ece was associated with decreased 5 year dfs ( 25% vs. 89% ; p = 0.025 ) . in the present study lrr was seen in 4% patients and ipsilateral breast tumor recurrence ( ibtr ) was seen only in three patients . the institut curie reported ibtr rates of 16% at 5 years and 22% at 10 years for patients who underwent bcs after nact . bonadonna et al . reported a 5 year ibtr rate of 7% after bcs and nact . in addition , cance et al . recently reported an ibtr rate of 10% among patients with advanced primary tumors treated with bcs after nact . most of our patients underwent modified radical mastectomy after nact which might explain very less ibtr rates in the present study . patient selection criteria , different therapeutic approaches , type of surgery , chemotherapeutic regimens used are probably the major factors responsible for the variation in published rate of lrr . the limitation of this study is its retrospective nature and its short follow - up . however bcs rates can be further increased with sequential use of taxanes in the future , which may also help us to get the real picture of locoregional relapse pattern after bcs in labc . the present study demonstrates clinicopathological variables such as nodal status , response to chemotherapy , pathological tumor size and presence of ece had significant impact on dfs .

background : introduction of neoadjuvant chemotherapy ( nact ) has dramatically changed the management of locally advanced breast cancer ( labc ) . however , very few randomized trials of nact have been carried out specifically in labc patients in our country . in this retrospective analysis , we presented our experience with nact in labc patients.materials and methods : medical records of 148 patients of stage iii labc patients treated with nact , followed by surgery and radiotherapy from january 2006 to december 2010 were reviewed . clinical and pathological responses to different chemotherapy regimens were assessed according to world health organization criteria . various factors influencing response to nact and clinical outcome were identified and analyzed.results:a total of 90 ( 60.8% ) patients received anthracycline - based chemotherapy and 52 ( 35.1% ) patients received mixed anthracycline and taxane - based chemotherapy.119 patients ( 80.4% ) responded to nact either in the form of complete or partial response ( pr ) . complete response was seen in 27 ( 18.2% ) patients and 92 ( 62.2% ) patients showed pr after nact . pathological complete response was seen in 24 ( 16.2% ) patients- . at a median follow - up period of 44 months 36 patients ( 24.3% ) developed relapse of which six patients developed locoregional recurrence , while 28 ( 18.9% ) patients developed distant metastasis . nodal status , response to chemotherapy , pathological tumor size <3 cm and extracapsular extension ( ece ) came out to be important prognostic factors in this study.conclusion:neoadjuvant chemotherapy is a reasonable alternative to upfront surgery in the management of labc . clinicopathological variables such as nodal status , response to chemotherapy , pathological tumor size and presence of ece had significant impact on disease free survival .

INTRODUCTION MATERIALS AND METHODS Chemotherapy regimens RESULTS Patient characteristics Chemotherapy regimens Response to neoadjuvant chemotherapy Surgery Postoperative histopathology findings Pattern of failure Prognostic factors and survival DISCUSSION CONCLUSION

hearing loss is among the three most prevalent conditions worldwide , with around 636.5 million people suffering from a certain degree of hearing loss1 . it is estimated that in brazil , 6.8% of the population suffers from disabling hearing loss , i.e. , a hearing threshold level for the better ear of 41 db hl or greater , averaged at frequencies of 0.5 , 1 , 2 , and 4 khz . of these individuals , 5.3% are children between 4 and 9 years old , 36.2% are elderly , and 19.5% are adults between 20 and 59 years old2 . in the state of so paulo , a population - based study showed that the prevalence of hearing loss was 44% , being higher for elderly male individuals3 . a population - based cross - sectional study performed in rio de janeiro showed that , for the elderly , the prevalence of hearing loss in the better ear was 42.9%4 . hearing loss can hinder or even impede acquisition and development of oral language , as well as academic and social development in children . in addition , it can decrease the quality of life and work and educational opportunities of adult individuals . this condition also has an impact on society in terms of loss of productivity of affected individuals as well as costs of treatment , education , and rehabilitation of people with special needs . thus , healthcare actions focused on prevention , early identification , and treatment of hearing problems also have social and economic implications . the brazilian national health system ( sistema nico de sade , sus ) has incorporated the treatment of the hearing impaired in previous decades . such action was supplemented by the establishment of the national hearing healthcare policy in 20045 and , more recently , by the care network for people with disabilities within the sus6 . this network is organized into the components of primary care , specialized rehabilitation care , and emergency hospital care . primary care services are provided through healthcare basic units ( ubs ) , which prioritize certain strategic actions intended to enhance access for and attention to people with disabilities , including the promotion of early identification services . in brazil it is estimated that 910 out of 1000 school aged children have permanent hearing loss in at least one ear8 . the health in school program ( pse ) , initiated in 2007 as a joint action between the ministries of health and education , aims to incorporate the school community into projects and programs that make use of healthcare and education in order to comprehensively deal with the issues that compromise the development of brazilian children and teenagers . the primary task of the pse is the assessment of the health condition of students , including hearing status . in order to do so , audiometric procedures , among others , are recommended9 . for adults and the elderly , hearing loss onset is often insidious , complicating self - perception of the problem and , consequently , the request for treatment . performing hearing screening in this population would enable timely identification and corresponding referral to rehabilitation services , in order to minimize secondary consequences such as functional decline , depression , and social isolation . in brazil , there are still no large scale hearing screening programs targeting school aged children , adults , and the elderly . when screenings are conducted in this population , they are isolated events . the related costs can be attributed to the cost of the professional performing the procedure , the necessary time to perform it , and the cost of the necessary equipment . in order to decrease screening costs , a simple , fast , and effective method using low cost equipment that can be easily operated by a primary care professional it is also necessary to emphasize the difficulty of access to hearing health for populations living far from urban centers . thus , a relevant aspect of this scenario is the development of computer - based systems that can be used in telehealth models . different low cost systems for conducting audiometric screening11 12 13 or audiological assessment at distance ( 14 - 15 ) have been published in the literature . such systems use tdh-39 supra - aural headphones or er3a insert phones , which can increase device cost . one study was found on the use of a software - based screening audiometer that utilized a conventional circumaural phone with a 3.5 mm plug . however , this system was automatic and hearing responses were queried with 3 db increments , making the test relatively lengthy with a duration of about 15 minutes ( 10 ) . the telessade ( ts ) audiometer was developed to perform audiometric screening using off - the - shelf usb interface headphones . this characteristic has a relevant impact on acquisition and replacement costs ( the prototype costs less than 50 usd ) and also facilitates access of the user to the ts audiometer . a wide variety of usb headphones are available and the user can chose a model that is considered more convenient , once that set of headphones has been calibrated for use with the ts audiometer . the fact that only off - the - shelf electronic devices are used also facilitates the availability of the ts audiometer , as the eventual manufacturer will not require a specific infrastructure for manufacturing and distribution . the aim of this study was to evaluate the ts audiometer for use during audiometric screening . this randomized prospective study was conducted in the speech language pathology and audiology clinic of the x school , university x , and was approved by the relevant research ethics committee ( process number 021/2010 ) . once an informed consent form was signed by 60 individuals , aged 18 years or older , they were voluntarily enrolled in the study and segregated into 2 groups ( table 1 ) : group a : for this group , employees and graduate students of the bauru school of dentistry were invited to participate in the study . this group was composed of 30 adults with age varying from 18 to 41 years old . group b : for this group , individuals who had ent or audiological complaints and were referred to the clinic were invited to participate in the study . this group was comprised of 30 individuals with age ranging from 23 to 85 years old . there were 12 adults and 18 elderly subjects ( aged 60 years or older ) . the ts audiometer is integrated by software developed in visual basic.net 2005 ( net framework 2.0 ) for windows xp and a set of usb headphones . the system can store the registration information of the location where screening is performed , the information of the healthcare professionals ( users ) and screened populations , as well as the corresponding evaluation results . the audiometer uses a microsoft lifechat lx-3000 headset with the following specifications : ( a ) bilateral earphones ( 20 hz20 khz ) ; ( b ) embedded microphone ( 100 hz10 khz ) ; ( c ) embedded usb sound board with 16 bit precision ; ( d ) plug and play , i.e. , no other software installation is needed for normal use ; and ( e ) incorporated volume control . as for every usb device , the headset is identified by the host computer by its vendor i d and product i d , therefore allowing the software to apply the corresponding calibration parameter or impede the performance of the audiometric procedure if the corresponding parameters are not available . following fda recommendations for signature of electronic records 16 , the identity and electronic signature of the healthcare professional are protected by a password only known and modifiable by the healthcare professional . additionally , a change control , or audit trail , is not deemed necessary , as once they are electronically signed , the assessment results can no longer be modified . in this study , the ts audiometer was installed and tested in an asus eeepc900 laptop , given its relatively low cost and portability . this laptop has a 8.9 " screen , a celeron m353 processor , a 4 gb hard drive , 1 gb of ddr ii ram , an intel uma video board , a 1.3 megapixel webcam , a windows xp operating system , 802.11b / g wlan , usb / vga / earphone / mic / network inputs / outputs , embedded loudspeakers , a battery autonomy of approximately 2.5 hours , dimensions of 22.5 17 2 cm , and a weight of 0.99 kg . the ts audiometer includes a calibration user interface . these values are stored in the system parameters database and they are used every time an audiometric screening is performed . the device ( computer and headphones ) was calibrated by an engineer with experience in conventional audiometer calibration according to the applicable requirements of the standard project abnt / cb 03/ce-03:029.01 - 022/1 . the frequencies 2508000 hz were used to calibrate the left and right earphones of the microsoft lifechat lx-3000 headset . during calibration , the ts audiometer software was used to provide acoustic stimulation . with regard to the calibration , it must be noted that the usb headset included its own sound board , i.e. , its own analog and digital input / output stages . therefore , the calibration parameters were characteristic of the headset model and independent of the host computer model or type . the frequencies 250 , 500 , 1000 , 2000 , 3000 , 4000 , 6000 , and 8000 hz were calibrated ranging from 10 db hl ( minimum stimulation level ) to 70 db hl ( maximum stimulation level ) in 5 db steps for both the right and left channels . another feature contemplated during development of the ts audiometer was the real - time estimation of ambient noise levels during performance of the screening procedure . as audiometric screening is not necessarily conducted in an audiometric booth or an acoustically prepared room , the ts audiometer uses the microphone embedded in the headset , placed in the upright position , to determine the ambient noise level . the software application converts the sampled noise level into a db scale in order to assess if the real - time ambient noise level allows screening to be performed . the system will automatically display a message to the evaluator when the ambient noise level is greater than 60 db spl to make him / her aware that minimization of noise level is desirable . the system user interface will flag the responses obtained under an ambient noise level greater than 60 db spl and also stores the average ambient noise level measured during an evaluation on its database . this is intended to minimize the occurrence of false positives induced by the lack of an appropriate acoustic environment . as the screenings were conducted in a sound booth , audiometric screening was conducted in groups a and b with 2 different pieces of equipment : the sd 50 audiometer ( siemens ) coupled with supra - aural thd-39 headphones and the ts audiometer . the screenings were conducted on the same day by 2 different audiologists , each one operating one audiometer . the audiologists did not share the results obtained with each other and they were not aware of the patients ' hearing complaints . pure tones at frequencies of 500 , 1000 , 2000 , and 4000 hz were presented at a 25 db hl level . the participant was instructed to raise his / her hand each time the acoustic stimuli were heard . in the audiometric screening , fail when the subject did not respond to one or more stimuli presented in one or both ears . following audiometric screening , and regardless of the result ( pass or fail ) , all participants underwent otologic inspection , pure - tone threshold audiometry , and speech audiometry procedures . for such purposes , sd 50 ( siemens ) or midimate 622 ( madsen ) audiometers were used . air conduction hearing thresholds were obtained with tdh-39 earphones for the inter - octaves of the frequencies between 250 and 8000 hz , for both ears . when the air conduction hearing thresholds were greater than 20 db hl , bone conduction audiometry was also performed for the frequencies 500 , 1000 , 2000 , 3000 , and 4000 hz . hearing thresholds were determined by applying the ascendant - descendant strategy . at each pure - tone detection response , the presentation level was reduced by 10 db until the individual no longer responded to the stimuli . then , the presentation level was increased in 5 db steps until a response was detected . the hearing threshold , at each frequency , was the lowest level at which the individual could detect 50% of the stimuli presented . all procedures were performed in a sound booth , where the noise levels were within the specified ranges of the ansi 1999 standard 18 . concordance analysis and cohen 's kappa coefficient were used to analyze the screening results between the conventional and ts audiometers for groups a and b. the precision of the ts audiometer screening instrument was evaluated through specificity , sensitivity , positive predictive value , and negative predictive value analysis . the sensitivity was defined as the percentage of ears that failed screening with the ts audiometer among those in which hearing loss was observed with pure - tone threshold audiometry . the specificity was defined as the percentage of ears that passed screening among those with normal hearing results . in this study , air conduction audiometric thresholds less than or equal to 20 db hl were considered as normal hearing . the positive and negative predictive values were defined as the probability of a patient having hearing loss if they failed screening and the probability of a patient having normal hearing if they passing screening , respectively . with regard to group b , the number of individuals that failed the screening was 27 with the ts audiometer and 26 with the conventional audiometer . within group b , the other 26 participants presented different degrees of sensorineural hearing loss ( figure 2 ) , as per the pure - tone and speech audiometry results . mean and standard deviation of audiometric thresholds for participants who presented hearing loss ( n = 26 ) . in group a ( table 2 ) , all tested ears passed the screening . although the sample size was limited , this observation is supported by the fact that the participants were predominantly young females , a population for which the prevalence of hearing loss is smaller 2 3 . the concordance between the screening results obtained with the conventional and ts audiometers was excellent . in group it is worth noting that this group of participants had some kind of otologic complaint , with 18 of them reporting hearing difficulties . this group was also mostly composed of elderly subjects , for whom a greater prevalence of hearing loss is observed 2 3 4 . the number of failures at the frequency of 500 hz was less than that for the other frequencies , which contradicts results reported in the literature 10 . this can be explained by the fact that the screening was conducted in a sound booth , this being a limitation for the generalization of the data from the current study . in fact , hearing screening is generally conducted in a non - acoustically isolated room . consequently , ambient noise can exert a masking effect over emitted signals , which is more significant at low frequencies , thus affecting screening results at these frequencies . for this reason , several screening protocols exclude the 500 hz test , although this frequency is relevant for the assessment of the impact of middle ear condition on hearing sensitivity . in table 3 , the kappa coefficients show an excellent concordance between the evaluators for the screening results obtained with the conventional and ts audiometers at the frequencies of 500 hz to 3000 hz , and substantial concordance for the frequency of 4000 hz 19 . the inter - evaluator reliability allows verification of the degree of correspondence between the independent evaluations of 2 or more evaluators classifying the same phenomena . therefore , it is a relevant indicator of the quality of the screening procedure with the ts audiometer . previously , kappa coefficients of 0.790.93 were observed when evaluating the concordance between audiometric screenings conducted with a portable audiometer and a conventional audiometer20 . another study10 indicated a kappa coefficient of 0.20 between screening performed with a software - based audiometer and a conventional one . the fact that this result is lower than the one obtained in the current study could be related to the population characteristics ( school aged children ) and the impact of ambient noise at the frequency of 500 hz , since when this was excluded from the analysis , a kappa value of 0.62 was obtained . another important difference is that an automatic screening procedure was employed in the previous study , in contrast with the conventional procedure utilized in the current study . in table 4 , it can be observed that audiometric thresholds for group a were within normal values . audiometric thresholds greater than 25 db hl were found for at least one frequency in 65.4% of the ears tested in group b. for this group , 26 participants ( 86.6% ) presented sensorineural hearing loss , with a greater involvement of the high frequencies ( figure 2 ) . this high incidence of hearing loss is due to the fact that the participants of this group were recruited from a specialized hearing healthcare clinic . it is relevant to note that all of the subjects who presented hearing loss underwent a complete audiological assessment and received corresponding treatment , including fitting of hearing aids . therefore , the sensitivity and specificity of the screening procedures with the conventional and ts audiometers ( table 5 ) were calculated using the results obtained with pure - tone threshold audiometry as a reference . it was not possible to calculate the sensitivity of the procedures for group a due to the fact that none of the participants failed the screening or presented hearing loss with pure - tone audiometry . for group b , the sensitivity and specificity values of the ts audiometer were similar to those of the conventional audiometer ( table 5 ) . elevated sensitivity and specificity values avoid the occurrence of false negatives and false positives , respectively . however , it must be remembered that no procedure is entirely accurate , i.e. , no procedure has a sensitivity and specificity equal to 100% . the sensitivity and specificity values of the ts audiometer were similar to those found in the literature . the sensitivity and specificity of a portable screening audiometer were reported to be 91.898.5% and 88.096.3% , respectively20 . studies involving the audioscope device with pure - tone sweep ( 500 to 4000 hz ) for hearing screening demonstrated a sensitivity of 9497% and a specificity of 6980%11 . with regards to affordable audiometers , the ts audiometer presented higher sensitivity and specificity values than those found in the literature . sensitivity and specificity values of 86.7% and 75.9% were verified with a remote automatic audiometric screening method ( 20 db hl pure - tone sweep ) , based on the use of a computer and tdh-39 earphones 13 . automatic hearing screening conducted with a low cost audiometer and a circumaural phone showed a sensitivity of 100% and a specificity of 49% 10 . the differences observed between the literature and the present study can be related to the stimuli loudness level applied in the current study ( 25 db hl ) , attenuation differences in the earphones utilized , and , mainly , the screening method ( automatic vs. manual ) and screening room acoustics ( non - acoustically isolated room vs. audiometric booth ) used . under operational conditions ( field application ) , the performance indicators of a test procedure are modified by the frequency of occurrence of a medical condition within the population ( prevalence ) . therefore , the predictive value of the procedure has a significant relevance , i.e. , the probability of occurrence of a medical condition given a positive or negative result . in the current study , the positive and negative predictive values of the ts audiometer were equal to 94.9% and 91.5% , respectively , being similar to those obtained with a conventional audiometer ( table 5 ) . this means that if a subject fails screening with the ts audiometer , there is a 94.9% chance of them actually suffering from hearing loss and a 5.1% ( 100 - 94.9 ) chance of them having normal hearing . if the subject passes screening , there is 91.5% chance of them having normal hearing and an 8.5% ( 100 - 91.5 ) chance of them having hearing loss . for group b , the observed predictive values of the ts audiometer were greater than those found in the literature for other affordable audiometers , which presented positive and negative predictive values of 48.1% and 95.7%13 and 56% and 43%10 , respectively . these findings are justified by the differences observed in the sensitivity and specificity of the ts audiometer when compared to other audiometers , and the fact that the screened population was from a specialized hearing healthcare clinic , where a higher number of subjects with hearing loss were registered . screening programs are justified if there is evidence that sustains 3 standard criteria : ( a ) negative consequences of the medical condition must be sufficiently significant in order to justify the screening effort ; ( b ) an effective treatment for the detected medical condition is available ; and ( c ) an accurate , practical , and convenient screening test is available11 . although this study is limited by the number of participants , the results obtained suggest that the ts audiometer meets the necessary criteria for a screening procedure the specificity , sensitivity , and positive and negative predictive values of audiometric screening conducted with the ts audiometer were high and similar to those obtained with a conventional audiometer .

summary introduction : the implementation of hearing screening programs can be facilitated by reducing operating costs , including the cost of equipment . the telessade ( ts ) audiometer is a low - cost , software - based , and easy - to - use piece of equipment for conducting audiometric screening . aim : to evaluate the ts audiometer for conducting audiometric screening . methods : a prospective randomized study was performed . sixty subjects , divided into those who did not have ( group a , n = 30 ) and those who had otologic complaints ( group b , n = 30 ) , underwent audiometric screening with conventional and ts audiometers in a randomized order . pure tones at 25 db hl were presented at frequencies of 500 , 1000 , 2000 , and 4000 hz . a fail result was considered when the individual failed to respond to at least one of the stimuli . pure - tone audiometry was also performed on all participants . the concordance of the results of screening with both audiometers was evaluated . the sensitivity , specificity , and positive and negative predictive values of screening with the ts audiometer were calculated . results : for group a , 100% of the ears tested passed the screening . for group b , pass results were obtained in 34.2% ( ts ) and 38.3% ( conventional ) of the ears tested . the agreement between procedures ( ts vs. conventional ) ranged from 93% to 98% . for group b , screening with the ts audiometer showed 95.5% sensitivity , 90.4% sensitivity , and positive and negative predictive values equal to 94.9% and 91.5% , respectively . conclusions : the results of the ts audiometer were similar to those obtained with the conventional audiometer , indicating that the ts audiometer can be used for audiometric screening .

Introduction Method Results Discusion Conclusion

this retrospective , hospital - based , observational study was carried out from august 2014 to july 2015 after obtaining necessary permission from the institutional ethics committee . diagnosed all patients admitted to the department of clinical haematology of scb medical college during the study period being treated with l - asp - based chemotherapy ( mcp 841 protocol ) all newly diagnosed indoor patients on induction therapy with the mcp 841 protocol , of either gender and up to 40 years of age , were included in the analysis . patients on any other drug other than those in the mcp 841 protocol that could have altered the blood glucose levels were excluded from the study . patients having abnormal plasma glucose level before starting therapy with l - asp were also excluded from the study . patients bed head tickets were collected from the record section of the hospital . under the mcp 841 protocol , a patient is on l - asp intramuscular every other day on days 220 of the first induction therapy . time of onset of hypo / hyperglycemia , persistence of plasma glucose alteration , and patient outcome were noted . fasting plasma glucose ( fpg ) levels were noted before the start of treatment with l - asp , on day 6 and day 12 of l - asp therapy and after the stoppage of l - asp therapy . differences in adr characteristics of native and pegylated l - asp were also noted and compared . the serum amylase levels of all these patients were estimated to exclude the possible pancreatitis . paired sample t - test was applied using medcalc version 12 - 1993 - 2013 medcalc software bvba ( acacialaan 22,8400 ostend , belgium ) to compare the fpg of patients before treatment with l - asp and on day 6 of l - asp treatment . paired sample t - test was applied using medcalc version 12 - 1993 - 2013 medcalc software bvba ( acacialaan 22,8400 ostend , belgium ) to compare the fpg of patients before treatment with l - asp and on day 6 of l - asp treatment . out of the 146 cases included for analysis , 129 were on native l - asp and only 17 were on pegylated l - asp . twenty - one percent of patients showed altered plasma glucose level out of 146 cases ( thirty in number ) . a total of 17 patients developed hypoglycemia ( fpg < 65 mg / dl ) whereas only 13 patients developed hyperglycemia ( fpg > 100 mg / dl ) . the serum amylase levels of all these patients were normal which showed the absence of pancreatitis . the median age in the hypoglycemia group was 5.5 years which is much less than that in the hyperglycemia group ( 19.5 years ) . the male : female ratio was 13:4 in hypoglycemia group and 10:3 in hyperglycemia group [ table 1 ] . demographic characteristics of patients on l - asparaginase of patients who were on native l - asp , 20.9% developed either hypo- or hyper - glycemia . on the contrary , in patients who were on pegylated l - asp all of them had hyperglycemia ( fpg > 100 mg / dl ) [ figure 1 ] . percentage of patients with plasma glucose alteration out of the total thirty patients who developed abnormality in fpg , 56.67% developed hypoglycemia whereas 43.33% had hyperglycemia . in majority of the cases , the fpg abnormality developed after taking three doses of l - asp in a particular cycle of chemotherapy . the number of patients who developed hypo- and hyper - glycemia after administration of native l - asp was 17 and 10 , respectively . on the contrary , all patients in hypoglycemic group had normal plasma glucose level before starting the treatment ( mean = 77.5 mg / dl ) . most of them developed hypoglycemia ( mean = 60.6 mg / dl ) on day 6 which returned to normal ( mean = 67.3 ) after stoppage of l - asp treatment . similarly , patients in hyperglycemia group had a mean plasma glucose of 86.5 mg / dl . on day 6 , majority of the patients hyperglycemia persisted even after the stoppage of l - asp treatment ( mean = 121.6 mg / dl ) . when the mean plasma glucose levels before the start of treatment with l - asp were compared with the values on day 6 by paired t - test , the p values for the hypo- and hyper - glycemia groups were found to be 0.0012 and 0.0309 , respectively . l - asp , an enzyme , has a wide application in many therapeutic protocols , including all . during all treatment , glucose levels are routinely monitored because many patients develop hyperglycemia , presumably because of glucocorticoids and l - asp . unexpectedly , many of our patients experienced repeated fasting hypoglycemia during induction and maintenance therapy . since altered fpg level can change the clinical outcome of patients , it was thought imperative to probe the exact course of hypo / hyperglycemia so that appropriate measures can be taken . most patients were given native l - asp and only a few were given pegylated l - asp , may be due to cost factor . approximately , one - fifth of the patients on l - asp therapy developed abnormal plasma glucose levels . most of the patients who developed hyperglycemia belonged to higher age group ( median age = 19.5 years ) compared to patients who have developed hypoglycemia ( median age = 5.5 years ) . this may be due to the fact that , in patients with higher age group , pancreatic reserve is less compared to younger age group . however , there is no significant difference between male to female ratio so far l - asp - induced abnormal plasma glucose levels ( hypo- or hyper - glycemia ) are concerned . in this study , as per the american diabetes association 2016 guidelines , fpg 100125 mg / dl is taken as impaired fasting glucose and > 126 is diabetic . hypoglycemia is taken as < 65 mg / dl because research in healthy adults shows that mental efficiency declines slightly but measurably as blood glucose falls below 65 mg / dl . fpg abnormality was observed in 20.9% of the cases in native l - asp group whereas only in 17.6% in pegylated l - asp group . we could find only cases of hyperglycemia in pegylated l - asp group whereas incidences of both hypoglycemia and hyperglycemia were observed in native l - asp - treated group . almost all the patients developed statistically significant abnormal glucose level after 3 dose of l - asp which may be due to the fact that the destruction of the endocrine pancreas is dose - dependent . however , though hypoglycemia returned to normal level after the end of the chemotherapy cycle , hyperglycemia persisted for longer duration almost till the end of observation in our study in most patients . given the time relation between l - asp administration and abnormal plasma glucose levels , we presume that our patients experienced l - asp - induced hypoglycemia . tanaka et al . in 2012 have reported that inappropriate insulin secretion , normal free fatty acids , and low ketone bodies during severe hypoglycemia indicate hyperinsulinism , suggesting that l - asp - induced hypoglycemia is a result of insulin hypersecretion . there were no other drugs that could have potentially induced hypoglycemia during the study period . glucocorticoids are known to induce hyperglycemia through insulin resistance and gluconeogenesis during induction therapy for childhood all . we suspect that parallel use of glucocorticoids may mask the hypoglycemic effect of l - asp . misgar et al . in 2014 have reported a case of 15-year - old boy who developed l - asp - induced hypoglycemia . use of l - asp has recently become even more common in the adult all regimens . we believe that in addition to the effect of l - asp on leukemic cells , it is important to pay more attention to its pharmacological features and physiological mechanisms . greater knowledge of these effects will enable clinicians to understand and develop appropriate strategies for l - asp use in chemotherapy regimens . hsu et al . in 2002 reported one case of diabetic ketoacidosis and persistent hyperglycemia as long - term complications of l - asp - induced pancreatitis . mondal et al . in 2010 reported two cases of diabetic ketoacidosis with l - asp therapy . quintanilla - flores et al . in 2014 reported one case of acute pancreatitis and diabetic ketoacidosis following l - asp / prednisone therapy in all . hence , to the best of our knowledge , there are only two reported cases of hypoglycemia induced by l - asp therapy by tanaka et al . of tokyo , japan , and misgar et al . of kashmir , however , in this retrospective study of ours , we have got 17 hypoglycemia as well as 13 hyperglycemia cases indicating the adverse effects of l - asp . our study reveals that a significant percentage of cases ( 21% ) have developed abnormal plasma glucose level among the study population . most of them developed hypoglycemia and they were in lower age group , whereas the higher age group patients developed hyperglycemia . in comparison to peg l - asp , native one was associated with more incidences of abnormal fpg . in majority of the cases , the fpg abnormality developed after taking three doses of l - asp in induction phase of mcp 841 protocol . identification of hypoglycemia as an adverse effect will enable clinicians to understand and develop appropriate strategies for l - asp use in chemotherapy regimens . in addition to the effect of l - asp on leukemic cells , it is important to pay more attention to its pharmacological features and physiological mechanisms .

objectives : the objective of this study was to evaluate any abnormal change in plasma glucose levels in patients treated with l - asparaginase ( l - asp)-based chemotherapy regimen in patients of acute lymphoblastic leukemia ( all).materials and methods : this retrospective , hospital - based study was conducted in patients of all , admitted to the clinical haematology department of a tertiary care hospital of odisha from august 2014 to july 2015 . indoor records of 146 patients on multi - centered protocol-841 were evaluated for any alteration in plasma glucose level , time of onset of hypo / hyperglycemia , and persistence of plasma glucose alteration.results:twenty-one percent of patients showed abnormal plasma glucose level . most of these patients developed hypoglycemia and were of lower age group . most of these patients developed hypoglycemia and were of lower age group , whereas a majority of higher age group patients developed hyperglycemia . in majority of the cases , abnormal glucose developed after three doses of l - asp . hypoglycemia subsided whereas hyperglycemia persisted till the end of our observation period.conclusions:l-asp produces more incidences of hypoglycemia than hyperglycemia in a good number of all patients towards which clinicians should be more vigilant . however , hyperglycemia persists for a longer duration than hypoglycemia .

Materials and Methods Statistical Analysis Results Discussion Conclusions Financial Support and Sponsorship Conflicts of Interest

the latest several decades have witnessed the progress in the treatment of severe sepsis and septic shock , acute organ dysfunction and consequential multiple organ dysfunction , however , cardiovascular dysfunction due to severe infection is still a major contributor to sepsis related morbidity and mortality . cardiovascular dysfunction induced by severe sepsis and septic shock is characterized by signs of distributive shock and septic cardiomyopathy consisting of bi - ventricular myocardial contractility impairment and diastolic dysfunction . the characteristics of septic cardiomyopathy include left ventricular dilatation , depressed ejection fraction and recovery during 710 days . in severe sepsis and septic shock , myocardial depression is the manifestation of septic cardiomyopathy and may attribute to the overwhelming production of inflammatory cytokines , mitochondrial dysfunction , and decreased myofibrillar sensitivity to calcium . rivers et al . suggested in 2001 that early goal - directed therapy ( egdt ) was effective for severe sepsis management , however , several recent studies , including the process and arise trials in 2014 and the promise trial in 2015 indicated that egdt did not improve outcomes compared to usual care . international sepsis guidelines have been adopted worldwide , and it is widely accepted that the standard treatment for sepsis should concentrate on infection control and optimization of hemodynamic parameters by fluid resuscitation and vasopressor therapy including noradrenaline and vasopressin . in addition , using dobutamine to increase the cardiac index is recommended by international sepsis guidelines . however , several studies have demonstrated that the use of dobutamine to increase cardiac output did not improve microcirculation , peripheral perfusion , or the outcome of septic shock patients , and even increased the 90-day mortality rate . another inotropic agent is levosimendan , a ca sensitizer and inodilator , which has been used successfully in the management of acute heart failure . levosimendan not only has inotropic and vasodilator effects , but also has anti - inflammatory and anti - apoptotic effects . in addition , meta - analysis has shown that levosimendan reduced mortality in critically ill patients and chronic advanced heart failure patients . therefore , the aim of the present study was to compare the effects of levosimendan and dobutamine on biomarkers of myocardial injury and systemic hemodynamics in patients with septic shock in the icu . this pilot study was conducted in the medical - surgical icu at tongde hospital of zhejiang province in hangzhou , china between march 2014 and january 2016 . it was conducted in strict accordance with the protocol approved by the ethics committee of tongde hospital of zhejiang province(hangzhou , china ) . all participants were recruited by tongde hospital of zhejiang province and they all ( or their guardians ) signed informed consents prior to enrollment . patients with low cardiac output ( left ventricular ejective fraction ( lvef 45% ) were enrolled within the first 24 hours from the onset of septic shock after having established normovolemia ( cvp=12 to 15 mmhg ) and mean arterial pressure ( map ) of at least 65 mmhg using norepinephrine , if needed . red blood cells were transfused when hemoglobin concentrations decreased to below 7 g / dl to elevate systemic oxygen supply . inclusion criteria were : ( 1 ) all participants were diagnosed with septic shock and established normovolemia , and used norepinephrine to maintain map of at least 65 mmhg ; ( 2 ) all participants had lvef 45% after fluid resuscitation and vasopressor therapy . exclusion criteria included : ( 1 ) onset of septic shock > 24 hours ; ( 2 ) patients younger than 18 years of age ; ( 3 ) lvef 45% before fluid resuscitation ; ( 4 ) patients with pre - existing cardiomyopathy , valvular heart disease , or heart failure ; ( 5 ) present or suspected acute coronary syndrome within recent two weeks ; ( 6 ) pregnancy ; and ( 7 ) ventricular outflow tract obstruction . all patients received mechanical ventilation using a volume - controlled mode with a tidal volume of 6 to 8 ml / kg of predicted body weight , and were sedated with midazolam and fentanil . according to the international sepsis guidelines , for septic shock treatment we used norepinephrine to maintain a mean arterial pressure ( map ) of at least 65 mmhg despite quantitative fluid resuscitation ( the variation of cvp from 12 to 15 mmhg ) within the first 24 hours from the onset of septic shock . after these goals were achieved , cardiac ultrasound scans were used to measure lvef immediately . finally , 38 patients with low cardiac output ( lvef 45% ) were enrolled and randomized ( by the use of a table of random numbers ) into two groups : a group receiving 0.2 g / kg / minute ( without a loading bolus dose ) of levosimendan ( levosimendan group , n=19 ) , and a group receiving 5 g / kg / minute of dobutamine ( dobutamine group , n=19 ) . during the 24-hour drug intervention period , all patients also received fluid therapy and norepinephrine to maintain normovolemia and map of more than 65 mmhg . after this period , levosimendan and dobutamine were discontinued and the attending icu physicians decided whether dobutamine should be started based on the hemodynamic status of the patients . in addition , in order to avoid the interference of depurative extracorporeal circulation on biomarkers of myocardial injury , all patients did not receive continuous blood purification ( cbp ) before or within 48 hour after enrollment . the treating physicians and echocardiographers were not blinded to the experimental procedure or the echocardiographic and laboratory results during the study . transthoracic echocardiography was performed before and again after fluid resuscitation and vasopressor therapy , and 24 hours after inclusion . all echocardiograms were performed by an expert echocardiographer not involved in patient care , using a vivid e9 ultrasound scanner and acquiring lvef ( modified simpson s rule ) . all patients were monitored using a pulse - indicated continuous cardiac output ( picco ) system ( pulsion medical system , munich , germany ) . they were required to have both a left femoral artery catheter and a right central venous catheter . the correct placement of the catheter insertion was further confirmed by chest radiography . a 5-french thermistor - tipped catheter ( pv2013l16 , pulsion medical system , german ) was inserted into the femoral artery and a central venous catheter ( cs-277202-e , arrow , usa ) was placed into a central vein ( jugular or subclavian vein ) ; both were connected to the picco system . thermodilution parameters and pulse contour parameter were obtained with the picco monitor , based on triplicate injections of 15 ml of cold isotonic saline 0.9% ( < 8c ) via the central venous catheter , and were recorded as the average of the three measurements . the corresponding ventilator function and perfusion parameters were observed and kept constant during the 6-hour period preceding the measurements . blood gas samples , including lactate samples , were obtained from the arterial catheters via 3 ml heparinized syringes ( pl67bp ; bd diagnostics , plymouth , uk ) anaerobically and analyzed on blood gas bedside machines ( abl800 : radiometer , copenhagen , denmark ) . the changes in heart rate ( hr ) , cvp , map , stroke volume index ( svi ) , cardiac index ( ci ) , left ventricular stroke work index ( lvswi ) , systemic vascular resistance index ( svri ) , intrathoracic blood volume index ( itbvi ) , global end diastolic volume index ( gedi ) , extravascular lung water index ( evlwi ) , oxygen delivery index ( do2i ) , oxygen consumption index ( vo2i ) , lvef , lactate , positive end - expiratory pressure(peep ) , and norepinephrine dose were recorded at baseline and 24 hours after randomization . we collected 3 ml venous blood samples from the two groups of patients at baseline and 24 hours after randomization and sent samples immediately to department of biochemistry at tongde hospital of zhejiang province to measure plasma levels of heart - type fatty acid binding protein ( hfabp ) , troponin i ( tni ) , and brain natriuretic peptide ( bnp ) . pass software ( version 11 ; ncss , llc ) was used to calculate sample size . sample size was determined by two - sample t - test power analysis using preliminary data obtained in our laboratory with the following assumptions : of 0.05 ( two - tailed ) , power of 80% , differences in the mean of hfabp between patients in the levosimendan and the dobutamine groups of 3.4 ng / ml , and a standard deviation of 1.4 ng / ml . therefore , we calculated that a sample size of 17 would provide 80% power of detecting a difference at a 0.05 level of significance . the multivariate liner regression analysis was used to assess the effect of levosimendan on biomarkers of myocardial injury ( hfabp , tni , and bnp ) and heart function ( levf and ci ) after controlling for age , gender , apacheii scores , sofa scores , and baseline values in the septic shock patients . data were expressed as mean standard deviation of the mean ( sdm ) for quantitative variables , and as count and percentages for qualitative values . distributions of the discrete variables were compared between the two treatment groups with either the chi - square test or fisher exact tests . two sample t - test was used to compared between the two groups ; and paired t - test was used for continuous variables before and after treatment . chicago , il , usa ) was used for statistical analysis ; all tests were 2-tailed and p<0.05 was considered to be statistically significant . this pilot study was conducted in the medical - surgical icu at tongde hospital of zhejiang province in hangzhou , china between march 2014 and january 2016 . it was conducted in strict accordance with the protocol approved by the ethics committee of tongde hospital of zhejiang province(hangzhou , china ) . all participants were recruited by tongde hospital of zhejiang province and they all ( or their guardians ) signed informed consents prior to enrollment . patients with low cardiac output ( left ventricular ejective fraction ( lvef 45% ) were enrolled within the first 24 hours from the onset of septic shock after having established normovolemia ( cvp=12 to 15 mmhg ) and mean arterial pressure ( map ) of at least 65 mmhg using norepinephrine , if needed . red blood cells were transfused when hemoglobin concentrations decreased to below 7 g / dl to elevate systemic oxygen supply . inclusion criteria were : ( 1 ) all participants were diagnosed with septic shock and established normovolemia , and used norepinephrine to maintain map of at least 65 mmhg ; ( 2 ) all participants had lvef 45% after fluid resuscitation and vasopressor therapy . exclusion criteria included : ( 1 ) onset of septic shock > 24 hours ; ( 2 ) patients younger than 18 years of age ; ( 3 ) lvef 45% before fluid resuscitation ; ( 4 ) patients with pre - existing cardiomyopathy , valvular heart disease , or heart failure ; ( 5 ) present or suspected acute coronary syndrome within recent two weeks ; ( 6 ) pregnancy ; and ( 7 ) ventricular outflow tract obstruction . all patients received mechanical ventilation using a volume - controlled mode with a tidal volume of 6 to 8 ml / kg of predicted body weight , and were sedated with midazolam and fentanil . according to the international sepsis guidelines , for septic shock treatment we used norepinephrine to maintain a mean arterial pressure ( map ) of at least 65 mmhg despite quantitative fluid resuscitation ( the variation of cvp from 12 to 15 mmhg ) within the first 24 hours from the onset of septic shock . after these goals were achieved , cardiac ultrasound scans were used to measure lvef immediately . finally , 38 patients with low cardiac output ( lvef 45% ) were enrolled and randomized ( by the use of a table of random numbers ) into two groups : a group receiving 0.2 g / kg / minute ( without a loading bolus dose ) of levosimendan ( levosimendan group , n=19 ) , and a group receiving 5 g / kg / minute of dobutamine ( dobutamine group , n=19 ) . during the 24-hour drug intervention period , all patients also received fluid therapy and norepinephrine to maintain normovolemia and map of more than 65 mmhg . after this period , levosimendan and dobutamine were discontinued and the attending icu physicians decided whether dobutamine should be started based on the hemodynamic status of the patients . in addition , in order to avoid the interference of depurative extracorporeal circulation on biomarkers of myocardial injury , all patients did not receive continuous blood purification ( cbp ) before or within 48 hour after enrollment . the treating physicians and echocardiographers were not blinded to the experimental procedure or the echocardiographic and laboratory results during the study . transthoracic echocardiography was performed before and again after fluid resuscitation and vasopressor therapy , and 24 hours after inclusion . all echocardiograms were performed by an expert echocardiographer not involved in patient care , using a vivid e9 ultrasound scanner and acquiring lvef ( modified simpson s rule ) . all patients were monitored using a pulse - indicated continuous cardiac output ( picco ) system ( pulsion medical system , munich , germany ) . they were required to have both a left femoral artery catheter and a right central venous catheter . a 5-french thermistor - tipped catheter ( pv2013l16 , pulsion medical system , german ) was inserted into the femoral artery and a central venous catheter ( cs-277202-e , arrow , usa ) was placed into a central vein ( jugular or subclavian vein ) ; both were connected to the picco system . thermodilution parameters and pulse contour parameter were obtained with the picco monitor , based on triplicate injections of 15 ml of cold isotonic saline 0.9% ( < 8c ) via the central venous catheter , and were recorded as the average of the three measurements . the corresponding ventilator function and perfusion parameters were observed and kept constant during the 6-hour period preceding the measurements . blood gas samples , including lactate samples , were obtained from the arterial catheters via 3 ml heparinized syringes ( pl67bp ; bd diagnostics , plymouth , uk ) anaerobically and analyzed on blood gas bedside machines ( abl800 : radiometer , copenhagen , denmark ) . the changes in heart rate ( hr ) , cvp , map , stroke volume index ( svi ) , cardiac index ( ci ) , left ventricular stroke work index ( lvswi ) , systemic vascular resistance index ( svri ) , intrathoracic blood volume index ( itbvi ) , global end diastolic volume index ( gedi ) , extravascular lung water index ( evlwi ) , oxygen delivery index ( do2i ) , oxygen consumption index ( vo2i ) , lvef , lactate , positive end - expiratory pressure(peep ) , and norepinephrine dose were recorded at baseline and 24 hours after randomization . we collected 3 ml venous blood samples from the two groups of patients at baseline and 24 hours after randomization and sent samples immediately to department of biochemistry at tongde hospital of zhejiang province to measure plasma levels of heart - type fatty acid binding protein ( hfabp ) , troponin i ( tni ) , and brain natriuretic peptide ( bnp ) . pass software ( version 11 ; ncss , llc ) was used to calculate sample size . sample size was determined by two - sample t - test power analysis using preliminary data obtained in our laboratory with the following assumptions : of 0.05 ( two - tailed ) , power of 80% , differences in the mean of hfabp between patients in the levosimendan and the dobutamine groups of 3.4 ng / ml , and a standard deviation of 1.4 ng / ml . therefore , we calculated that a sample size of 17 would provide 80% power of detecting a difference at a 0.05 level of significance . the multivariate liner regression analysis was used to assess the effect of levosimendan on biomarkers of myocardial injury ( hfabp , tni , and bnp ) and heart function ( levf and ci ) after controlling for age , gender , apacheii scores , sofa scores , and baseline values in the septic shock patients . data were expressed as mean standard deviation of the mean ( sdm ) for quantitative variables , and as count and percentages for qualitative values . distributions of the discrete variables were compared between the two treatment groups with either the chi - square test or fisher exact tests . two sample t - test was used to compared between the two groups ; and paired t - test was used for continuous variables before and after treatment . chicago , il , usa ) was used for statistical analysis ; all tests were 2-tailed and p<0.05 was considered to be statistically significant . figure 1 shows a flow diagram that quantifies progress through the trial . of the 72 patients that did not meet the inclusion criteria , five patients declined to participate , 11 patients were excluded from the study for other reasons ( six because of an onset of septic shock > 24 hours , three because of lvef 45% before fluid resuscitation , and two due to acute coronary syndrome within the recent two weeks ) . age , gender , apache ii scores , sofa scores , and type of infection were balanced between the two groups at admission . there were no difference in the duration of mechanical ventilation , length of stay in icu and hospital , or 28-day mortality between the two groups ( all p>0.05 ) . as shown in table 2 , no significant differences were observed in hr , cvp , map , svri , itbvi , gedi , peep , and norepinephrine dose ( all p>0.05 ) . compared to dobutamine , levosimendan increased ci ( p=0.001 ) , svi ( p=0.030 ) , lvswi ( p=0.002 ) , and lvef ( p=0.018 ) , and decreased evlwi and lactate ( p=0.012 and p=0.022 , respectively ) after 24 hours of intervention . in addition , levosimendan improved do2 and vo2 of systemic tissues ( p=0.040 and p=0.031 , respectively ) . compared with the dobutamine group , concentrations of hfabp , tni , and bnp were significantly decreased at 24 hours after intervention in the levosimendan group ( p<0.001 , p=0.025 , and p<0.001 , respectively , see figures 24 . ) multivariate regression analysis showed that compared to the dobutamine group , after adjusting for the patients age , gender , apache ii , sofa , and baseline of hfabp , tni , bnp , lvef , and ci , the levosimendan group had significantly decreased levels of hfabp , tni , and bnp ( p<0.001 , p=0.03 and p<0.001 , respectively , tables 35 ) , and increased lvef and ci ( p=0.03 and p<0.001 , respectively , see tables 6 , 7 ) figure 1 shows a flow diagram that quantifies progress through the trial . of the 72 patients that did not meet the inclusion criteria , five patients declined to participate , 11 patients were excluded from the study for other reasons ( six because of an onset of septic shock > 24 hours , three because of lvef 45% before fluid resuscitation , and two due to acute coronary syndrome within the recent two weeks ) . age , gender , apache ii scores , sofa scores , and type of infection were balanced between the two groups at admission . there were no difference in the duration of mechanical ventilation , length of stay in icu and hospital , or 28-day mortality between the two groups ( all p>0.05 ) . as shown in table 2 , no significant differences were observed in hr , cvp , map , svri , itbvi , gedi , peep , and norepinephrine dose ( all p>0.05 ) . compared to dobutamine , levosimendan increased ci ( p=0.001 ) , svi ( p=0.030 ) , lvswi ( p=0.002 ) , and lvef ( p=0.018 ) , and decreased evlwi and lactate ( p=0.012 and p=0.022 , respectively ) after 24 hours of intervention . in addition , levosimendan improved do2 and vo2 of systemic tissues ( p=0.040 and p=0.031 , respectively ) . compared with the dobutamine group , concentrations of hfabp , tni , and bnp were significantly decreased at 24 hours after intervention in the levosimendan group ( p<0.001 , p=0.025 , and p<0.001 , respectively , see figures 24 . ) multivariate regression analysis showed that compared to the dobutamine group , after adjusting for the patients age , gender , apache ii , sofa , and baseline of hfabp , tni , bnp , lvef , and ci , the levosimendan group had significantly decreased levels of hfabp , tni , and bnp ( p<0.001 , p=0.03 and p<0.001 , respectively , tables 35 ) , and increased lvef and ci ( p=0.03 and p<0.001 , respectively , see tables 6 , 7 ) septic cardiomyopathy , a kind of cardiovascular dysfunction induced by severe sepsis and septic shock , is manifested by low cardiac output and is closely related to higher mortality in septic shock patients [ 1618 ] . inotropic agents including dobutamine and levosimendan may show benefits in the treatment of cardiovascular dysfunction induced by sepsis . the major findings of the present study were that levosimendan increased lvswi and lvef , decreased evlwi and lactate , and improved tissue perfusion , hemodynamics , and cardiac function . however , levosimendan did not reduce the days on mechanical ventilation , lengths of stay in icu and hospital , or 28-day mortality . elevated concentrations of tni and bnp are frequently observed in patients with severe sepsis and septic shock even in the absence of an acute coronary syndrome ( acs ) . demonstrated the relationship between elevated bnp level and severity of sepsis regardless of congestive heart failure ; their findings also supported the utility of bnp level as a marker for mortality in septic shock . studies have shown that tni was sensitive and superior to creatinine kinase - mb ( ck - mb ) for the detection of myocardial injury in septic shock and after coronary angioplasty with or without stenting . hence , bnp and tni have been identified as cardiac biomarkers and predictors of cardiac dysfunction and death in septic patients and successfully used in icus . however , it is difficult to distinguish septic patients with or without cardiac dysfunction related to septic shock via utilization of bnp alone . furthermore , tiruvoipati et al . suggested tni was related to lower ejection fraction and higher need for inotropic / vasopressor support , but did not independently predict mortality in critically ill patients with severe sepsis . hfabp is a more sensitive and specific biomarker of myocardial injury than tni and bnp , which make it superior to tni and bnp for the assessment of recurrent or persistent myocardial injury . zhang et al . found that serum hfabp was frequently elevated among patients with severe sepsis and was associated with sepsis - related myocardial dysfunction , and elevated hfabp independently predicted 28-day mortality in severe sepsis .therefore , hfabp , tni , and bnp were utilized as biomarkers of myocardial injury for septic shock patients in the present study . this study demonstrated that both dobutamine and levosimendan reduced the concentrations of biomarkers of myocardial injury in critically ill patients with septic shock . but hfabp , tni , and bnp were lower in the levosimendan group than in the dobutamine group , which suggested that levosimendan could play a more important role in attenuating septic myocardial injury . wu et al . conducted a study in patients with acute myocardial infarction ( ami ) who received emergency percutaneous coronary intervention ( pci ) and found that the tni concentration decreased much more in the levosimendan group than in the placebo group , suggesting that the myocardium was suffering less damage . in myocardium injury and stunning , the activation of the ca - dependent protease results in degradation of tni partially and selectively during reperfusion . because levosimendan has the ability to increase responsiveness to calcium while keeping the cytosolic ca concentration unchanged , levosimendan prevented the degradation of tni , which could prevent the contractile dysfunction of myocardium stunning and damage . studies found that bnp levels were elevated in severe sepsis and septic shock patients and elevated bnp were related to myocardial dysfunction , global tissue hypoxia , and mortality . several mechanisms probably account for the elevated bnp levels in sepsis , including neuro - hormonal activation , volume resuscitation , and sepsis - induced biventricular dilation , as well as stimulation of lipopolysaccharide or pro - inflammatory cytokines , acute lung injury , and acute respiratory distress syndrome induced sepsis . levosimendan was found to have significantly decreased bnp levels at day 1 and day 3 in patients with decompensated heart failure ( dhf ) and renal dysfunction . feola et al . demonstrated that bnp concentrations decreased more significantly in a levosimendan group than a furosemide group . kyrzopoulos et al . suggested that the decreasing effect of levosimendan treatment on bnp was associated with its decreasing effect on end - diastolic cardiac wall tension . immunomodulatory and anti - inflammatory properties of levosimendan might also contribute to its effect on decreased bnp . these probably mechanisms of decreased tni and bnp concentrations could explain the changes of tni and bnp in our study . hfabp , a stable low molecular weight protein found in the cytoplasm of myocardial cells , is a sensitive and specific biomarker of myocardial injury . it is frequently elevated in patients with severe sepsis and septic shock and is associated with sepsis - related myocardial dysfunction . the probably mechanisms leading to elevated hfabp concentrations in sepsis and septic shock cases may be related to sepsis - induced myocardial dysfunction and elevated levels of free fatty acids due to an increased catabolism of glycogen and lipids during multiple organ dysfunction syndrome ( mods ) associated inflammatory responses . in addition , decreased renal function also contributes to the elevation of hfabp . in this study , compared to dobutamine , levosimendan significantly decreased hfabp levels in patients with cardiomyopathy associated with severe sepsis and septic shock . to date first , levosimendan improves inotropic function due to binding to the ca saturated troponin c of myocardial thin filament and diastolic function because it does not promote calcium flux into the cell . the improvement in systolic and diastolic function and myocardial oxygen supply atttributed to levosimendan may decrease hfabp concentration via preventing myocardial dysfunction associated sepsis . second , the anti - inflammatory and anti - apoptotic effects enable levosimendan to reduce catabolism of glycogen and lipids via mediating inflammatory responses and decreasing the level of hfabp during sepsis and septic shock . finally , levosimendan decreases hfabp through reducing renal injury and improving renal function and because levosimendan leads to augmentation of renal perfusion , increases in renal blood flow and increases glomerular capillary surface area and glomerular filtration rate ( gfr ) . the present study demonstrated that levosimendan had no adverse effects on hemodynamics , on the contrary , it elevated lvef , svi , and ci and did not increase the dose of norepinephrine needed . in addition , decreased evlwi and lactate , and increased do2i and vo2i in patients with myocardial dysfunction suggests that levosimendan improved pulmonary vascular permeability and tissue perfusion via elevated cardiac output , and the driving pressure of blood flow entering into microcirculation . in our study , however , although there were no significant difference in mechanical ventilation time , icu length of stay , hospital length of stay , or 28-day mortality between the two groups , all of these were still shorter in the levosimendan group than the dobutamine group . the number of septic shock patients investigated in our study was small and the study period was relative brief , therefore , the risk of positive results in a study with numerous secondary variables has to be taken into account . second , our study did not show a statistically significant difference in 28-day mortality between the two groups and the difference was 5.2% , suggesting that the effect size was much lower than that assumed in the sample size calculation . post hoc sample size calculations showed that 1,305 patients per group would be required to show a statistically significant difference in 28-day mortality between the two groups . it is impossible to achieve a study like this in a single center ; a multi - centers investigation would be required in the future . finally , it is also indispensable to make clear the molecular biologic mechanisms of decreased hfabp in septic shock patients treated by levosimendan . the number of septic shock patients investigated in our study was small and the study period was relative brief , therefore , the risk of positive results in a study with numerous secondary variables has to be taken into account . second , our study did not show a statistically significant difference in 28-day mortality between the two groups and the difference was 5.2% , suggesting that the effect size was much lower than that assumed in the sample size calculation . post hoc sample size calculations showed that 1,305 patients per group would be required to show a statistically significant difference in 28-day mortality between the two groups . it is impossible to achieve a study like this in a single center ; a multi - centers investigation would be required in the future . finally , it is also indispensable to make clear the molecular biologic mechanisms of decreased hfabp in septic shock patients treated by levosimendan . compared with dobutamine , this study demonstrated levosimendan reduced the level of biomarkers of myocardial injury including hfabp , tni , and bnp , increased lvef , strengthened systolic function , and improved systemic hemodynamics significantly in patients with septic shock ; but there were no significant difference in duration of mechanical ventilation , length of stay in icu , or 28-day mortality between the two treatment groups . in conclusion , although levosimendan improved the biomarkers of myocardial injury and hemodynamic parameters , our study failed to show any improvement in clinically relevant outcomes like length of icu stay , duration of mechanical ventilation , and 28-day mortality .

backgroundwe aimed to investigate the effect of levosimendan on biomarkers of myocardial injury and systemic hemodynamics in patients with septic shock.material/methodsafter achieving normovolemia and a mean arterial pressure of at least 65 mmhg , 38 septic shock patients with low cardiac output ( left ventricular ejective fraction ) , levf 45% ) were randomly divided into two groups : levosimendan dobutamine . patients in the levosimendan and dobutamine groups were maintained with intravenous infusion of levosimendan ( 0.2 g / kg / minute ) and dobutamine ( 5 g / kg / minute ) for 24 hours respectively . during treatment we monitored hemodynamics and lvef , and measured levels of heart - type fatty acid binding protein ( hfabp ) , troponin i ( tni ) , and brain natriuretic peptide(bnp ) . in addition , the length of mechanical ventilation , intensive care unit ( icu ) stay , hospital stay , and 28-day mortality were compared between the two groups.resultsthe levosimendan group and the dobutamine group were well matched with respect to age ( years , 55.41 7.5 versus 50.213.6 ) and gender ( males , 68.4% versus 57.9% ) . levosimendan - treated patients had higher stroke volume index ( svi ) , cardiac index ( ci ) , lvef , and left ventricular stroke work index ( lvswi ) , and lower extravascular lung water index ( evlwi ) compared to dobutamine - treated patients ( p<0.05 ) . hfabp , tni , and bnp in the levosimendan group were less than in the dobutamine group ( p<0.05 ) . there was no difference in the mechanical ventilation time , length of stay in icu and hospital , and 28-day mortality between the two groups.conclusionscompared with dobutamine , levosimendan reduces biomarkers of myocardial injury and improves systemic hemodynamics in patients with septic shock . however , it does not reduce the days on mechanical ventilation , length of stay in icu and hospital , or 28-day mortality .

Background Material and Methods Ethics statement Study population Experimental procedure Standard echocardiographic examination Hemodynamics monitoring and lactate Outcome measures Statistical analysis Results Demographic and clinical characteristics of the study population The changes of hemodynamics and lactate in the different groups Levosimendan decreased the plasm levels of biomarkers of cardiac injury induced by septic shock Discussion Limitations of the study Conclusions

protein protein interactions are essential to the function of the ubiquitous xenobiotic - metabolizing cytochrome p450 monooxygenases , or cyps . classic studies have demonstrated the importance of cyp interactions with the accessory proteins cytochrome p450 reductase ( cpr ) and cytochrome b5 ( b5 ) for both electron transfer and allosteric modulation . however , it has recently become clear that cyp enzymes may participate in additional interactions with other protein partners , such as progesterone receptor membrane component 1 ( pgrmc1 ) , human serum albumin ( hsa ) , and even other cyp isoforms . these interactions have been shown to alter cyp function and , in some cases , lead to the production of toxic metabolites . in this review , we attempt to summarize the available evidence from the primary literature and offer some future perspectives on the study of noncanonical protein protein interactions with cytochrome p450 enzymes . cytochrome p450 reductase ( cpr ) was identified as the primary cyp electron transfer partner by the late 1960s . the term cytochrome p450 reductase is a bit of a misnomer , as this enzyme participates as a reducing partner in a number of different reactions , including those catalyzed by heme oxygenases i and ii , squalene monooxygenase , and certain fatty acid desaturases . initially characterized as a cytochrome c reductase , cpr was eventually found to be localized in the endoplasmic reticulum , where it functions as a redox partner with cyps that are also located there . cpr is a diflavin ( fmn , fad ) containing reductase that receives its electron - reducing equivalents from nadph . the cpr electron transfer protein shuttles two electrons to the cyp during the reaction cycle , with the second electron transfer thought to be the rate - limiting step . cpr is known to be post - translationally modified and has an apparent molecular weight between 76 and 80 kda , depending on post - translational modifications , the species - specific isoform , and detergent solubilization procedures . in addition to the fmn , fad , and nadph binding domains , cpr also contains a 60 amino acid hydrophobic n - terminal membrane anchoring region , which is essential for full activity with native redox partners , such as cytochrome p450 enzymes . a number of important crystal structures of cpr and several of its mutants have been produced , including those demonstrating important conformational changes that take place upon reduction and cyp binding . the first crystal structure of cpr was obtained using an n - terminally truncated sequence of the rat isoform . this structure demonstrated that the nadph fad binding domain also contained an important linker sequence that could help position the fmn and fad domains in close proximity for efficient electron transfer . the linker sequence distinguishes cpr from the closely related , single - domain ferrodoxin reductases . it is worthwhile to note that this particular n - terminally truncated construct was not able to support cyp reduction , even though it successfully reduced cytochrome c. more recent x - ray crystal structures have provided some insight into cyp the crystal structure of a cpr mutant with a four amino acid deletion in the hinge region between the fmn binding domain and the rest of the protein demonstrated remarkable flexibility in this region by crystallizing in three different conformations . although this particular mutant was not able to catalyze intramolecular electron transfer between the fad and fmn prosthetic groups , it was able to successfully reduce cyps , given enough reducing equivalents . the three different conformations observed suggest that cpr inherently possesses a high degree of conformational plasticity , especially in the c terminus of the hinge region , allowing it to efficiently interact with a variety of cyp isoforms and other enzymes . other structures obtained from protein x - ray crystallography and nmr have confirmed this conformational flexibility and the ability of cpr to transition between closed and open forms , which had also been predicted by computational molecular dynamics studies . another intriguing possibility is that subtle structural differences between cyp isoforms may regulate interaction with cpr and cytochrome b5 . seminal work examining structural and functional variation in the cpr gene has been conducted by both the masters and kranendonk groups , underscoring the importance of cpr in drug disposition and disease . several mutations in the gene encoding cpr are known to be associated with specific disease states , such as a rare form of congenital adrenal hyperplasia known as antley amino acid changes that are implicated in these conditions include y181d , a457h , y459h , v492e , and r616x . surprisingly , despite the large number of cyps present in the human genome ( 57 , excluding possible pseudogenes ; http://drnelson.uthsc.edu/hum.html ) , there is only one gene encoding cpr . additionally , the total cyp content of the human liver is estimated to be between 5- and 25-fold higher than that of cpr , suggesting differential expression . the fact that cyps and cpr interact in a 1:1 stoichiometry raises an unusual conundrum : exactly how do cyps and cpr interact to promote efficient oxidation of substrates ? from a structural perspective , it stands to reason that all of the different cyp isoforms must share a common functional cpr binding surface . indeed , several studies have proposed that the binding interaction between cyp and cpr takes place between the positively charged proximal surface near the cyp heme and the negatively charged fmn and fad / nadph domains of cpr , suggesting that their interaction is primarily driven by electrostatics . however , there is some controversy in this regard , as other studies have suggested that hydrophobic interactions are more predominant . these discrepancies may be the result of the different experimental approaches employed ( e.g. , x - ray crystallography vs site - directed mutagenesis and stopped - flow fluorescence spectroscopy ) . cpr complex is remarkably low , between 5 and 110 nm , depending on the particular isoform used and whether substrate is present , implying a very high - affinity interaction . upon initial examination , a tight - binding interaction may seem counterintuitive ; however , a rapid kon or koff rate would allow the system to work more efficiently due to swift equilibration . clearly , the interaction can be modulated by the presence of substrates and/or inhibitors , as well as other cyp isoforms , as noted below . another compounding factor is the variety of methods , often indirect , that have been used to determine cyp separate from its function as an electron transfer partner , cpr may also act as an allosteric effector to induce conformational changes in cyps that modulate the rate of electron transfer . a number of useful structural studies conducted with the bacterial model cyp isoform , cyp101 ( p450cam ) , have been useful to inform our understanding of how cpr might interact with mammalian cyps in order to modulate electron transfer . trans isomerization redox switch in cyp101 ( p450cam ) that is triggered by binding to the reduced form of cyp101 s cognate electron transfer partner , putidaredoxin ( pdx ) . these conformational changes account for most of the significant structural perturbations observed by nmr upon complex formation . conformational plasticity may be one mechanism by which the binding affinity of a cyp and its redox partner are modulated : if the subset of conformational states occupied by the reduced electron transfer protein is the same as that selected upon binding to a cognate cyp , then the entropic cost ( i.e. , free energy ) of binding would be lowered , resulting in a higher affinity . in a unique approach to examine cyp electron transfer partner interactions , hiruma and colleagues utilized paramagnetic nmr in conjunction with a site - specific lanthanide label to solve the solution structure of the cyp101pdx complex . this cyp101pdx complex structure was , in turn , validated by simultaneous determination of the x - ray crystal structure of the same complex . in contrast to reports of the interaction between mammalian cyps and cpr , here hydrophobic networks dominated the binding interface between the two proteins . the data suggested a minor conformational substate ( or substates ) that could represent an encounter complex between the two proteins , as postulated by an earlier study . additionally , the retrieved structural parameters were consistent with an electron transfer rate theoretically faster than that observed experimentally , suggesting the involvement of a gating process in electron transfer . similar allosterically induced conformational changes have also been observed with mammalian cyps and cytochrome b5 . taken together , these studies underscore the dynamic nature between cyps and their electron transfer partners , both from a functional standpoint as well as a structural one . seemingly small structural changes , such as a single amino acid substitution , could have dramatic functional consequences on the ability of cpr to efficiently deliver its electrons to a particular cyp isoform . recent studies have examined how genetic variation in the human cpr gene can manifest itself in the form of alterations in the isoform dependent cyp - mediated metabolism of certain substrates ( table 1 ) . interestingly , the effects of specific cpr mutations seem to be cyp isoform dependent , suggesting a unique interaction surface between individual cyp isoforms and cpr . in a study examining the effect of the 35 most common cpr mutations on the activity of cyp1a2 and cyp2c19 , miller and colleagues discovered that while most mutations either decreased the activity of both cyp1a2 and cyp2c19 , or eliminated activity all together ( most notably the a287p and r457h mutations ) , certain mutations enhanced cyp activity . in most cases , the enhancements were specific to the cyp isoform examined and the particular cpr mutation . for cyp1a2 , activity enhancement was observed with cpr mutants g213e , r406h , and a462 t . in the case of cyp2c19 , increased activity was reported for cpr mutants m263v and g504r . most surprisingly , the q153r cpr mutation , a rare disease - causing mutation that inhibits steroidogenic activity , increased the activity of both cyp1a2 and cyp2c19 to 144 and 284% , respectively . the authors rationalized these results by suggesting that many of the cpr mutants with reduced activity had decreased fad binding or lay in a region known to be important for cpr cyp contacts . the cpr gain - of - function mutants were more difficult to explain , but miller and colleagues suggest that , at least in the case of the q153r mutant , electron transfer rates to the cyp may be affected by the close proximity of glutamine residue 153 to the fmn moiety . the change in electrostatic charge in the q153r mutant may accelerate electron transfer from cpr to the cyp . whatever the mechanism involved , it is clear that there are important functional consequences for subtle alterations in the interactions between cpr and cyp isoforms . a follow - up study examined the effects of cpr mutations on the ability of cyp2d6 to metabolize different substrates . interestingly , for any particular cpr mutation , different effects were seen depending on the substrate used . the q153r cpr mutation mentioned above had a slight stimulatory effect on the cyp2d6-mediated metabolism of 2h-1-benzopyran-3-carbonitrile,7-(ethoxy - methoxy)-2-oxo-(9cl ) ( eomcc ) , yet it almost doubled the rate of metabolism of dextromethorphan . this observation may speak to the promiscuous nature of drug - metabolizing cyps and the multiple substrate binding sites within the active site . additional work with the cyp3a4 isoform also indicated that it was susceptible to functional variation due to cpr mutations . consistent with the previous findings for cyp2d6 and cyp1a2 , cyp3a4 activity is decreased in combination with most of the cpr mutants examined , but it is increased with the q153r mutant . this effect was again substrate - specific for the substrates testosterone and quinidine , and nominal activity was observed for midazolam and erythromycin oxidation . a study utilizing the full - length cyp and cpr mutant isoforms simultaneously expressed in insect sf9 cells via a baculovirus vector recapitulated the results obtained with the bacterially expressed isoforms ( referenced above ) . a result unique to this study was the observation that some of the reduced - function cpr mutants regained activity when coexpressed with cyp isoforms in the sf9 insect cells . this suggests that mutant cpr association with a cyp may help to stabilize the cpr protein into an active conformation , thereby rescuing activity . this is an intriguing possibility , given the postulate by pochapsky regarding the ability of a cyp to select the active conformation of a particular electron transfer partner . additional studies have pointed to the importance of the n - terminal hydrophobic anchoring sequence in supporting cyp - mediated oxidation . hayashi and colleagues found that an n - terminally truncated form of rat cpr failed to support cyp1a1-mediated 7-ethoxycoumarin o - deethylation , although it efficiently reduced cytochrome c and rat heme oxygenase i. furthermore , structural studies have demonstrated that crystallized , yet catalytically active , yeast cpr has a substantially different structure than the conventional n - terminally truncated catalytically inactive rat enzyme , reinforcing the importance of this sequence in supporting efficient electron transfer from cpr to cyp . cpr genetic variants may also have effects on different cyp isoform alleles as well . in a study examining the cyp2c9 alleles cyp2c9.1 , cyp2c9.2 , and cyp2c9.3 , tracy and colleagues found increased metabolic oxidation of the substrates diclofenac , flurbiprofen , and tolbutamide with four different mutant cpr enzymes tested ( q153r , a287p , r457h , and a503v ) . this was in contrast to the predominate effect seen previously with cyp1a2 , cyp2d6 , cyp2b6 , and cyp3a4 , where a decrease in activity was reported with these cpr mutants . the authors noted that since the effects they observed led to an increase in the vmax , but not a change in the km , it was most likely that the increase in activity observed was due to enhanced electron transfer and not an increase in cyp substrate affinity . variation in the cpr gene can lead directly to toxicity for certain drugs that are metabolized through reduction . the herbicide paraquat produces toxicity through the generation of superoxide anion during redox cycling . examined the effect of six common cpr mutations in patients with impaired steroidogenesis on the redox cycling of paraquat in a cho cell model system . in five out of the six cpr mutants examined , paraquat toxicity was diminished . although not explicitly confirmed in this study , the authors inferred that the mutants either affected the catalytic activity or expression levels of cpr , thereby reducing its ability to activate paraquat . in the c569y mutant , the observed paraquat toxicity was equivalent to that with the wild - type enzyme , implying a rescue of function with this particular mutation . the authors suggest that variation in the cpr gene may be a primary susceptibility determinant in paraquat toxicity . similarly , in a study investigating the toxicity of mitomycin c , wang et al . found that cpr mutations may be useful as biomarkers to predict the therapeutic response to this anticancer drug . mitomycin c is a common chemotherapeutic agent that is metabolically activated by either a one- or two - electron reduction step . cpr is the primary enzyme responsible for the one electron reduction , which produces the semiquinone anion radical that is thought cause damage to cellular dna . of six cpr mutants that were examined ( y181d , a287p , r457h , v492e , c569y , and v608f ) , cellular toxicity of mitomycin c was decreased in all with the exception of the c569y mutant , demonstrating that a high degree of cpr functional activity is needed to activate mitomycin c. in particular , the v608f and y181d mutants lacked any ability to activate mitomycin c , presumably due to altered association with the flavin moiety . in addition to mitomycin c , the impact of cpr variants has also been examined with tacrolimus , warfarin , and other drugs , illustrating the importance of this particular enzyme in drug metabolism and elimination . cytochrome b5 ( b5 ) is a small ( 17 kda ) heme - containing integral membrane protein located on the cytosolic side of the endoplasmic reticulum and was originally identified in 1950 in silkworm larva . although the majority of b5 is membrane - bound , a certain fraction is present as a soluble , cytosolic form in erythrocytes , where it catalyzes the reduction of methemoglobin . although mutations in the b5 gene itself are quite rare , a few have been traced to the congenital abnormality of 17,20-lyase deficiency . despite the fact that it can act as an electron donor to cyp enzymes , b5 is primarily involved in lipid biosynthesis , delivering electrons to microsomal desaturases that synthesize steroids , fatty acids , and plasmalogens . in the early 1970s , estabrook and mannering first demonstrated that b5 stimulated the metabolism of several cyp substrates . however , at this time , the functional role b5 played in cyp - catalyzed oxidation was still somewhat unclear . early reports suggested that it could be involved in delivery of the second electron in the cyp catalytic cycle , thereby accelerating the rate - limiting step in cyp - mediated catalysis . remarkably , a more contemporary report from the wolf laboratory in a hepatic cpr deletion mouse model established that b5 and b5 reductase can act as efficient cyp electron donors , without the need of cpr . despite its role in electron transfer , the effects seen from b5 are highly variable and dependent on the particular reaction conditions used , as addition of b5 can either inhibit or have no effect on some reactions rather than stimulate them . furthermore , several studies have demonstrated that addition of apo ( heme free ) b5 can stimulate the activities of many cyps , including cyp3a4 , cyp2c9 , and cyp17 . it has been postulated that , in certain cases at least , b5 exerts the majority of its effects in the absence of electron transfer . it is still not completely clear if b5 functions solely as an electron donor , allosteric modulator , electron sink , or an uncoupling inhibitor . it is entirely plausible that its function may be contextual , depending on the particular isoform / substrate combination with which it is interacting . a number of elegant studies over the years have established that electrostatic interactions are the basis for formation of the cyp waskell and colleagues mapped out the binding interface between cyp2b4 and b5 through extensive site - directed mutagenesis of both proteins and identified r122 , r126 , r133 , k139 , and k433 of cyp2b4 as residues critical to their interaction . recently , a number of structural and dynamic 2d nmr studies have provided additional insight into the cyp embedded in lipid bicells , ramamoorthy , waskell , and colleagues were able to take advantage of rotating frame separated local field ( slf ) nmr . the authors employed two - dimensional proton evolved local - field ( 2d pelf ) pulse sequences to accurately measure a broad range of heteronuclear dipolar couplings , allowing for a complete mapping of b5 protein dynamics in a native lipid bilayer environment . notably , the authors discovered that the hydrophobic n - terminal helix of b5 precessed in a cone with an approximate 8 fluctuation in the tilt of the helix , which is estimated to be 13 , suggesting that the lipid - embedded hydrophobic n - terminus is more mobile than originally thought . this added flexibility may allow this region of the protein to interact with a diverse variety of cyp isoforms . there is a growing appreciation of how inherent protein flexibility can allow proteins to adopt non - native states , which may allow them to gain new functions or interact with different protein partners . , ramamoorthy and colleagues utilized a modified inept technique , dipolar enhanced polarization transfer ( drept ) , to detect an increased range of residual dipolar couplings from isotopically labeled b5 that allowed them to characterize the amino acid side chain motions of b5 while embedded in lipid bicells . two - dimensional nmr methodologies have also been useful in providing explanations for the specific rate enhancements seen with certain cyp substrate pairs . the bifunctional steroidogenic enzyme cyp17a1 performs both steroid hydroxylation , which is unaffected by b5 , and an androgen - forming lyase reaction that is enhanced 10-fold in its presence . work by the scott group examining the interactions between an isotopically labeled b5 construct and cyp17a1 led to the observation that the cyp17a1b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the cyp17a1 active site than when the lyase substrate 17-hydroxypregnenolone is bound . this suggests that , at least in this case , the b5 reaction rate enhancement may be primarily driven by substrate affinity . in a follow - up study , in which cyp17a1 was isotopically labeled and the b5 construct was unlabeled , titration of b5 into cyp17a1pregnenolone complex induced a set of conformational substates closely resembling those of the cyp17a117-hydroxypregnenolone complex without b5 . this result implied that b5 may also be able to allosterically induce catalytically productive conformations in cyp17a1 , even in the absence of substrate . these studies as well as others have culminated in a complete structural and catalytic model for the cyp ahuja et al . determined the structure of full - length , membrane bound b5 in the presence of cyp2b4 . this structure , determined for the first time in the presence of a phospholipid bilayer , confirmed the electrostatic nature of the cyp b5 interaction , revealing a large number of charge pairings between surface residues on cyp2b4 and b5 that facilitate the interaction between the two proteins . however , it also hinted at the importance of hydrophobic interactions for the complex that are mediated by the phospholipid bilayer . interestingly , the authors described an increase in the affinity between cyp2b4 and b5 in the presence of a small molecule substrate or inhibitor , similar to that observed by estrada et al . for the cyp17a1b5 interaction . additionally , their results suggest a pathway for electron transfer between cyp2b4 and b5 mediated through a salt bridge with arg125 of cyp2b4 and the heme propionates of b5 . this study illustrates the power of 2d nmr techniques to obtain detailed structural data for the interactions between two protein partners . finn and colleagues created a liver - specific b5 conditional knockout mouse in order to better define the functional importance of the cyp b5 interaction.in vitro metabolism studies demonstrated the complete lack of nadh - mediated metabolism and a 50 to 90% decrease in nadph - mediated metabolism for most of the drug substrates examined . additionally , a dramatic increase in the half - life for these particular drug substrates was noted , thus establishing that b5 is a major contributor to the in vivo metabolism of many drugs and xenobiotics , albeit in a substrate- and isoform - selective manner . a follow - up study demonstrated altered clearance of several anticancer agents in the same knockout mouse model . therefore , mutant b5 alleles that are deficient in their ability to interact with , and transfer electrons to , hepatic cyps could lead to altered clearance rates for certain drugs , which may contribute to drug - induced toxicities . furthermore , varying levels of b5 function important for drug oxidation can also produce altered metabolite ratios , potentially increasing the production of toxic products in certain instances . in a study conducted by the hollenberg group , b5 was determined to be essential for the formation of protein adducts to cyp3a5 from metabolic activation of the contraceptive agent 17-ethynylestradiol . in the absence of b5 , the reactive 17-oxirene - related species was not formed , leading to cyp enzyme protection . an additional concern from a toxicological point of view is the potential for the generation of reactive oxygen species ( ros ) due to increased uncoupling . indeed , using a reconstituted system containing both lipid and cpr , b5 has been shown to generate superoxide and hydroxyl radicals in the presence of cr(vi ) , leading to double - stranded dna breaks in vitro.(109,110 ) the fact that cpr was needed to observe the effect suggests the importance of the interaction between b5 and cpr to generate these ros . additionally , ros generated by b5 have been demonstrated to increase lipid peroxidation in reconstituted biological membranes , which may in turn lead to cellular damage and death . therefore , there are multiple mechanisms through which altered b5 levels and/or function can exert a toxicological effect . surprisingly , cyps also exhibit protein protein interactions with other cyps . in the early 1990s , research by coon , davydov , and others demonstrated that cyps can form homomeric complexes . work in the ensuing 2 decades has established that multiple cyp isoforms can interact with one another in both homomeric and heteromeric complexes and that these complexes can often have significant effects on cyp - mediated oxidation of substrates ( for recent reviews , please see references ( 117 ) and ( 118 ) ) . for simplicity , we have grouped the cyp cyp interactions into those described by homomeric ( two or more molecules of the same isoform ) and heteromeric ( two or more molecules of different isoforms ) . pioneering studies of homomeric cyp cyp interactions were conducted by davydov in the early to mid-1990s , examining the response of cyp2b4 to pressure induced conformational changes . davydov and his colleagues discovered that approximately 35% of the ferrous cyp2b4 co - bound complex was refractory to conversion into the inactive p420 form upon application of pressures in excess of 2 kbar . however , this refractory population was reduced to less than 5% upon addition of 0.2% triton n-101 , a detergent that created conditions known to produce monomers . these results implied that a significant portion of active cyp ( i.e. , cyp able to be reduced and bind co ) was present in the oligomeric state . later work by this same group would demonstrate that sodium dithionite or cpr induced reduction kinetics in cyp3a4 are multiphasic under conditions favoring cyp3a4 oligomers but monophasic under conditions favoring monomers . these studies are suggestive of active cyp arranged in homomeric oligomers in the phospholipid bilayer . confirmation of a direct physical interaction between individual cyps in a live cell membrane was achieved in seminal studies by kemper and colleagues using fluorescently labeled cyp2e1 and cyp2c2 in cells transfected with murine cyp cdna . they found that while fluorescence resonance energy transfer ( fret ) occurred between individual cyp2c2 molecules in a membrane , it could not be detected between cyp2e1 monomers , representing a homomeric self - association with cyp2c2 but , interestingly , not cyp2e1 . the cyp2c2 dimers were later confirmed to exist in murine hepatocyte endoplasmic reticulum membranes , indicating the in vivo relevance of such associations . taken together , these studies laid the groundwork for analysis of the functional consequences of cyp cyp homomeric interactions . in an elegant kinetic study conducted by jamakhandi et al . , the authors found that metabolism data for cyp2e1 fit best to either a quaternary ( cyp cpr cyp cpr ) , trimeric ( cyp cyp cpr ) , or dimeric ( cyp cyp ) complex model where only the binary ( cyp cpr ) complex was active . the authors found that simply varying the individual concentrations of cyp or cpr in a reconstitution system produced contradictory results , which did not correspond to the simplest functional ( 1:1 ) complex . simultaneously , job titration analysis revealed an asymmetric plot , indicative of higher - order molecular complexes . in an attempt to identify the catalytically relevant species , both cyp and cpr concentrations were titrated simultaneously , resulting in a two - dimensional data surface . when the data were globally fit to 32 different candidate models ( including binary , ternary , and quaternary cyp cpr complexes ) , it revealed that the most likely model was one composed of the binary complex ( cyp cpr ) , the quaternary complex ( cyp cpr cyp cpr ) , and the homodimer ( cyp cyp ) . a second , although less likely , possibility was a model that involved a weakly bound ternary complex ( cyp - cyp interestingly , the results implied that the binary complex was the only catalytically active state . therefore , in this case , even though cyp2e1 was shown to be capable of forming cyp cyp complexes that could have an effect on catalytic activity , the binary cyp more recently , a comprehensive study by the backes group examining the effect of homomeric cyp complex formation on the activity of cyp1a2 , cyp2b4 , and cyp2e1 found that , at least in the case of cyp1a2 , cyp cyp homomeric complexes contribute to altered catalytic activity seen with this enzyme under conditions that would promote dimer formation . to understand how cyp homomeric complexes might affect function , the catalytic activities of several individual cyps , including cyp2b4 , cyp2e1 , and cyp1a2 , were assessed in reconstituted systems as a function of cyp concentration . interestingly , although oxidation mediated by cyp2b4 as a function of cyp cpr best fit to a simple hyperbolic michaelis menten equation , cyp2e1 and cyp1a2 demonstrated atypical ( non - michaelis menten ) kinetics that fit best to a sigmoidal curve , indicating a high degree of cooperativity with these particular isoforms . this non - michaelis menten kinetic behavior could be converted back to a hyperbola simply by increasing the ionic strength of the buffer , suggesting that the cyp cyp complex in the membrane using a variety of biophysical techniques , including chemical cross - linking and bioluminescence resonance energy transfer ( bret ) . in yet another study , cyp interaction was found to affect enzymatic function by a direct modulation of allosteric ( non - michaelis menten ) enzyme kinetics . both homotropic and heterotropic cooperativity have long been known to occur with hepatic drug - metabolizing cyp isoforms , particularly cyp3a4 . recently , davydov and colleagues reported that stimulation of cyp3a4-mediated oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin ( 7-bfc ) by -naphthoflavone in human liver microsomes ( hlm ) was highly dependent on the degree of cyp3a4 oligomerization . the amount of enzyme activation seen was directly correlated with the surface density of the enzyme . while no activation was detectable at high lipid / cyp ratios ( 750 ) , activation reached greater than 225% at a lipid / cyp ratio of 140:1 . this suggests that allosteric activation requires at least some degree of homomeric cyp cyp interaction . cyp complexes , at least in the dimeric form , it is still not entirely clear what role these complexes may play in vivo in the detoxification and elimination of xenobiotic agents , especially under conditions of cyp induction or endoplasmic reticulum ( er ) stress . one suggested physiological function basis for the formation of cyp cyp complexes is their ability to prompt a hypothetical functional allosteric mechanism future studies with relevant in vivo model systems may shed some light in this area . the identification of the existence of homomeric cyp complexes has led to interest in the possibility of heteromeric cyp complexes as well . using the same methodology that revealed homomeric interactions between individual cyp2c2 molecules , kemper and colleagues identified additional heteromeric interactions between cyp2c2 and cyp2e1 . this implied that differences in homo- and heteromeric oligomerization state between isoforms may help explain some of the relative differences seen in activity . indeed , this was found to be the case in complexes formed between cyp1a2 and cyp2b4 in reconstituted systems . in a reconstituted system containing cpr , lipid , and both cyp1a2 and cyp2b4 enzymes , a small increase in catalytic activity for the cyp2b4 substrate benzphetamine was detected . however , activity against another cyp2b4 substrate , 7-pentoxyresorufin - o - dealkylation ( prod ) , was dramatically inhibited . inhibition of prod was also dependent on cpr levels , with the inhibitory effect being more pronounced at subsaturating cpr concentrations , suggesting competition for reductase binding between the two isoforms . in a follow - up study , backes and colleagues determined that it was likely that a ternary complex containing cpr , cyp1a2 , and cyp2b4 was formed , for cyp2b4 activity was dramatically reduced at subsaturating reductase concentrations . this result suggested an alternative model where , under substoichiometric conditions , cpr is specifically bound to cyp1a2 and at higher cpr concentrations , reductase binding to cyp1a2 becomes saturated , resulting in the formation of a quaternary complex in which cpr binds to both cyp1a2 and cyp2b4 enzymes . in a more recent study examining the interaction between cyp2c9 and cyp2c19 in reconstituted systems , kupfer and colleagues found evidence of a metabolic interaction between even these highly related cyp isoforms . previously , using in vitro systems reconstituted with purified enzyme , cyp2c19 was the most active isoform for methoxychlor - o - demethylation . however , in hlm , cyp2c9 performed the bulk of methoxychlor - o - demethylation . interestingly , while an equimolar ratio of cpr to cyp2c9 supported maximal rates of methoxychlor - o - demethylation , the rate of methoxychlor - o - demethylation by cyp2c19 was not fully saturated in the same system ; even when reconstituted with a 9-fold molar excess of cpr , cyp2c19 methoxychlor - o - demethylation and s - mephenytoin hydroxylation kinetics were not fully saturated ( as compared with that in hlm ) . however , when a binary reconstitution system was prepared by mixing cyp2c9 and cyp2c19 enzymes , methoxychlor - o - demethylation and s - mephenytoin hydroxylation by cyp2c19 were inhibited dramatically , with the amount of inhibition dependent on the amount of cpr and substrate used , yet increasing concentrations of cpr were generally able to overcome the inhibitory effects . by contrast , in the incubation containing only cyp2c9 , diclofenac hydroxylation was activated by the presence of cyp2c19 , thus demonstrating that cyp cyp interactions can modulate the catalytic rates of a variety of oxidation reactions but that this modulation is highly dependent on the substrate undergoing metabolism , with some substrates activating metabolism and others inhibiting it . surprisingly , subramanian and colleagues found that while addition of cyp2d6 to a reconstituted incubation of cyp2c9 and ( s)-flurbiprofen increased the ks by 20-fold , it had no effect on the km , but it decreased the vmax by 50% . one possible explanation for this discrepancy is that affinity constants determined by heme spin state ( i.e. , optical spectrum ) perturbation may mask total ligand binding because they measure only substrate interactions that perturb the heme iron . it is possible that , in the presence of an additional cyp isoform , the spin state shift may be less complete , thereby not reflecting the effects of additional ligand binding . the authors additionally noted that the effects on metabolism were dependent on the order of addition of the enzymatic constituents , with the greatest rate enhancements observed when cpr was added before the second cyp , again suggesting a competition for reductase binding . in yet another study , yamazaki et al . discovered similar types of enzymatic stimulation between cyp1a2 and other isoforms , including cyp3a4 , cyp2c9 , and cyp2d6 , which indicate a more generalizable phenomenon among the hepatic cyps . as more substantial biophysical and functional evidence has accrued demonstrating heteromeric interaction between individual cyp isoforms , attention has shifted to structurally defining the mechanism of this interaction . two different studies conducted in 2010 by the backes and tracy laboratories were instructive in this regard . the backes group set out to answer the question of whether two different cyp isoforms need to be located in the same membrane to functionally interact , thereby potentially excluding long - range allosteric effects . their experimental system consisted of incorporating cyp1a2 and cyp2b4 into vesicles with cpr individually and together as a cyp cyp complex . if the two cyps functionally interacted in the same membrane , then metabolic stimulation ( or inhibition ) would be observed only when both cyps were present in the same vesicle , and a mixture of both the individual cyp1a2cpr and cyp2b4cpr vesicles would simply produce an additive effect . indeed , this is exactly what the authors observed prior to confirming this functional interaction between these two cyps with cross - linking and coimmunoprecipitation experiments . work from the tracy lab examined the effect of the hydrophobic n - terminal anchor sequence on mediating the interaction between cyp2c9 and cyp3a4 . using a reconstituted enzyme system containing both cyp2c9 and cyp3a4 , the authors demonstrated that cyp2c9 mediated ( s)-naproxen metabolism was inhibited as much as 80% by the presence of cyp3a4 , while the km remained unchanged . the degree of inhibition was directly proportional to the cyp3a4 concentration and indirectly proportional to the cpr concentration , suggesting a competition for reductase . oddly , cyp2c9 did not seem to alter cyp3a4-mediated testosterone metabolism . in order to study the role of the hydrophobic n - terminal anchor sequence , the experiments were performed with both n - terminally truncated and full - length cyp isoforms . there was no evidence for metabolic inhibition when the full - length cyp3a4 and cyp2c9 isoforms were incubated in the presence of the truncated form of the other cyp isoform , but evidence for inhibition was found only when both full length forms were present . this indicated that interaction between the hydrophobic n - termini of the two cyp isoforms was required for functional inhibition . it is possible that , in this context , the hydrophobic n - termini may orient the cyps in the membrane . finally , the authors conducted coimmunoprecipitation studies to demonstrate that the two cyps were directly interacting in the membrane . it has now been firmly established that cyp s form both homomeric and heteromeric complexes with functional consequences . the difficult work of defining how these complexes interact structurally and what their in vivo roles may be now lies ahead . of all the protein protein interactions in which cyps have been documented to participate , perhaps the most enigmatic is that with nonenzymatic proteins , including the progesterone receptor membrane component 1 ( pgrmc1 ) and bovine and human serum albumin ( bsa / hsa ) . although these proteins do not provide reducing equivalents to the cyp catalytic cycle , they are still capable of modulating cyp - mediated metabolism . presently , these mechanisms remain to be fully elucidated ; intriguingly , they may involve direct delivery of a drug substrate to the particular cyp . below , we provide a brief summary of what is currently known regarding these cyp protein protein interactions . despite its rather precise identification , the progesterone receptor membrane component 1 is a somewhat misleading name , as it neither binds progesterone directly nor does it share any homology with progesterone receptors . in contrast , pgrmc1 shares a high degree of structural homology with cytochrome b5 . pgrmc1 has been demonstrated to either increase or decrease the rate of cyp - mediated metabolism , depending on the cyp pgrmc1 also seems to play a role in the development of certain cancers , as recent reports suggest that it may be the inscrutable receptor , a known biomarker of tumor cell proliferation . in mammals , there exist two additional pgrmc1 family isoforms , prgmc2/dg6 and neudesin , although it is not known if they also interact with cyp enzymes . prgmc1 is highly expressed in the human liver and typically colocalized in the smooth endoplasmic reticulum ( ser ) with cyp proteins . it has been shown to interact directly with cyp enzymes , including cyp3a4 , cyp2c9 , cyp7a1 , cyp21a2 , and cyp51 . pgrmc1 activates progesterone oxidation mediated by cyp21a2 and is essential for cyp51 activity in the cholesterol synthesis pathway . min and colleagues reported that the coexpression of pgrmc1 with cyp21a1 enhanced progesterone 21-hydroxylation in cos-7 cells , while a heme - deficient pgrmc1 mutant had no effect . in an experiment with the yeast homologue , dap1 , hugues et al . showed that both cyp51a1 and cyp61a1 can be positively regulated by the presence of dap1 . remarkably , prgmc1 has been revealed to exhibit isoform - selective effects in its interaction with cyps . oda found that pgrmc1 generally reduced cyp activities , either through decreased vmax and km values ( in the case of cyp3a4 ) or simply through a decreased vmax ( in the case of cyp2c9 ) ; however , in the case of cyp2e1 , it had no effect . coimmunoprecipitation studies suggested that the mechanism behind the differential effects may be differences in binding affinity between pgrmc1 and individual cyp isoforms . szczesna - skorupa and kemper found similar results in their studies of the interactions of pgrmc1 with cyp2c2 , cyp2c8 , and cyp3a4 in transfected hepg2 cells . in each case , pgrmc1 downregulated cyp activities , which could be rescued by increased expression of cpr . pgrmc1 was found to have a high affinity for cpr , but only in the absence of cyp2c2 , indicating competition between pgrmc1 and cyp2c2 for binding to cpr . in contrast to what was observed with the drug - metabolizing cyp isoforms , cyp51 activity was reduced with decreasing levels of pgrmc1 , indicating that a separate mechanism may be in place for the interaction of pgrmc1 with cyps involved in steroid synthesis . the mechanism of cyp activation or inhibition by pgrmc1 is highly debatable ; however , it is conceivable that it may involve drug delivery or sequestration , as a hydrophobic binding pocket has been identified on pgrmc1 . in any case , much work remains to elucidate the structural and functional details of the cyp human serum albumin ( hsa ) is a 65 kda monomeric , slightly basic globular protein that circulates as a major component of blood plasma and is the most abundant protein in the human body . hsa functions primarily as a carrier for steroids , fatty acids , and thyroid hormone . additionally , it plays a role in stabilizing osmotic pressure by regulating extracellular fluid volume . hsa binds a large variety of drugs and xenobiotic agents and has a major impact on their pharmacokinetics and disposition . under normal physiological circumstances , hsa however , it is synthesized in the liver and is present in the ser of hepatocytes , so it is conceivable that hsa could come into direct contact with cyp isoforms . moreover , the extent of drug or toxin protein binding has long been known to effect in vitro - to - in vivo extrapolations of disposition . therefore , in an effort to improve in vitro - to - in vivo predictions , researchers began to add either hsa or bovine serum albumin ( bsa , a cheaper and more well - characterized substitute ) to both microsomal incubations and recombinant baculovirus - expressed enzymes . this accomplished the desired effect of improving pharmacokinetic predictions by increasing the in vitro intrinsic clearance rate , clint ( vmax / km ) to more closely match what is seen in vivo . this effect is often mediated through a decrease in the substrate km , and it has been observed for a number of different cyp isoform / drug combinations , including cyp2c8 , cyp2c9 , and cyp1a2 . however , for some cyp isoforms , including cyp2c19 and cyp2d6 , bsa was found to be inhibitory . established that the degree of activation or inhibition observed was dependent on the concentration of bsa used , with lower concentrations activating and higher concentrations being inhibitory . in addition , bsa s cyp activation / inhibition profile was demonstrated to deviate from that observed with hsa , indicating the importance of the albumin source for comparison studies and in vitro - to - in vivo extrapolations . a number of theories have been proposed to account for the effect seen with albumin , including direct drug delivery , allosterically induced conformational change , and inhibitory long - chain unsaturated fatty acid sequestration . although no single theory fits the data precisely , recent evidence seems to lend credence to long - chain unsaturated fatty acid sequestration . of course , because none of these theories are mutually exclusive , it may be that multiple mechanisms are involved rather than a single predominant mechanism with any particular drug / cyp isoform . lastly , other proteins related to albumin , such as human intestinal fatty acid binding protein ( ifabp ) , have also been demonstrated to improve in vitro - to - in vivo predictions based on microsomal incubations , suggesting a more generalizable phenomena with this class of proteins . in addition to clearly defining the particular mechanisms of albumin cyp interaction , future work in this area will also need to delineate exactly which members of this protein family interact with individual cyp isoforms . protein interaction will no doubt further enhance our ability to correlate in vitro metabolism to that observed in vivo . the cyp pathway of oxidative transformation of drugs and other xenobiotics is critical for the elimination of toxicity in the host organism . proper functioning of this pathway relies on effective formation of protein protein complexes among the cyps , their electron transfer partners , and other proteins that can influence their function . mutations in either the cyp or an interacting protein that effect the structure or stability of the cyp or its protein partner can lead to deficiencies or alternative pathways of biotransformation , which in turn may produce toxic metabolites . therefore , a comprehensive understanding of the many different types and functions of protein protein interactions that cyps undergo is necessary to predict and treat such toxicities . new techniques protein interactions that are beginning to be developed will be a boon for research in this area . additionally , from a pharmacological point of view , modulating protein protein interactions may provide an avenue for therapeutic intervention . conceivably , cyps might also interact with phase ii , or drug conjugating , enzymes such as glucuronidases , sulfotransferases , or gluthione - s - transferases to modulate their activity as well . protein interactions increases , we will not only improve our ability to predict and prevent toxic cyp drug interactions but also gain a new appreciation for the many unique attributes of this versatile enzyme family .

through their unique oxidative chemistry , cytochrome p450 monooxygenases ( cyps ) catalyze the elimination of most drugs and toxins from the human body . protein protein interactions play a critical role in this process . historically , the study of cyp protein interactions has focused on their electron transfer partners and allosteric mediators , cytochrome p450 reductase and cytochrome b5 . however , cyps can bind other proteins that also affect cyp function . some examples include the progesterone receptor membrane component 1 , damage resistance protein 1 , human and bovine serum albumin , and intestinal fatty acid binding protein , in addition to other cyp isoforms . furthermore , disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites . in this review , we summarize the available evidence for cyp protein protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein protein interactions with cyp enzymes .

Introduction Cytochrome P450 Interactions with Cytochrome P450 Reductase and Cytochrome b5 Cytochrome P450 Interactions with Other CYP Isoforms Cytochrome P450 Interactions with Other Proteins Conclusions and Future Perspectives

methods and any associated references are available in the online version of the paper at http://www.nature.com / naturemethods/. difference in optimal collision energies for identification and quantification supplementary note real - time filtering supplementary table 1 summary of quantification and identification results supplementary protocol implementing quantmode on commercial mass spectrometers

we describe a mass spectrometry method , quantmode , which improves the accuracy of isobaric tag based quantification by alleviating the pervasive problem of precursor interference co - isolation of impurities through gas - phase purification . quantmode analysis of a yeast sample contaminated with interfering human peptides showed substantially improved quantitative accuracy compared to a standard scan , with a small loss of spectral identifications . this technique will allow large - scale , multiplexed quantitative proteomics analyses using isobaric tagging .

METHODS Supplementary Material

with the improvement of diagnostic imaging , the incidence of uia diagnosis is increasing , along with the national cost containment pressures associated with treatment and management . economic considerations are important in the cost - benefit evaluation of treatments , and influence healthcare coverage , reimbursement , and policy . while many studies have already compared the treatment modalities for intracranial aneurysms , surgical clipping and endovascular coiling , in terms of the procedural invasiveness , clinical and functional outcomes , and durability , few studies have investigated the economic costs for such treatments23713 ) . plus , treatment costs show distinct disparities between countries313).during the process of informed consent for clipping or coiling , uia patients are also concerned about the cost implications . therefore , the authors performed a cost comparison , and investigated the principal cost determinants of uia treatment in south korea . this study conducted a retrospective review of data from a series of consecutive patients who underwent surgical clipping or endovascular coiling of a uia at the authors ' institution between january 2011 and may 2014 . the exclusion criteria included a recent ( < 1 month ) subarachnoid hemorrhage caused by a concomitant aneurysm , giant aneurysm with a diameter > 2.5 cm , poor preoperative neurological state affecting postoperative management , and another disease or medical condition significantly increasing the hospital costs . the medical records were reviewed to obtain relevant clinical information , and all the radiological data were obtained using an electronic picture archiving and communication system . the treatment decision , surgical coiling versus endovascular coiling , was made primarily based on the findings of the digital subtraction angiography . surgical treatment was favored over endovascular treatment for patients with the following findings : 1 ) difficult navigation of the microcatheter into the aneurysm19 ) , 2 ) very small ( <3 mm ) aneurysm149 ) , 3 ) complex and wide - neck aneurysm requiring y or x stent - assisted coiling610 ) , 4 ) aneurysm with an arterial branch incorporated into the sac58 ) , 5 ) fusiform or complex aneurysmal configuration , or 6 ) aneurysmal compression of an adjacent cranial nerve11 ) , as long as the patients had no problems related to surgical accessibility or comorbidity . all the patients completed an angiographic examination , chest x - ray , and laboratory tests as a preoperative evaluation before admission , and were admitted to hospital one day before the surgical or endovascular procedure . the operative and perioperative management procedures were uniformly applied to each uia patient . for the patients who underwent surgical clipping , a postoperative computed tomography ( ct ) and ct angiography another ct scan was performed on the day of discharge to check for the occurrence of any subdural hygromas . for most surgical patients , the length of the hospital stay was 3 - 6 days . in the case of elderly patients , a longer hospital stay was usually required due to fatigue and general weakness . meanwhile , for the patients who underwent endovascular coiling , diffusion - weighted magnetic resonance imaging was commonly performed on day 1 following the procedure to evaluate any procedure - related thromboembolic phenomena . the length of the hospital stay after coiling was 3 - 4 days for most patients , irrespective of the patient 's age . the treatment costs for each patient were calculated based on the expenses incurred during the time spent as an inpatient for aneurysm treatment , including the operative and postoperative costs . the operative costs covered the neurosurgical or endovascular procedure , anesthesia , and all the materials ( e.g. , aneurysm clip , endovascular coil , and other surgical supplies ) , while the perioperative costs included the costs for the bed occupancy in the intensive care unit and regular hospital ward , radiological imaging studies , laboratory tests , and all the medications . the costs affected by patient selection ( e.g. , bed occupancy in the regular hospital ward ) were made uniform to the basic lowest cost . all the costs are all presented in korean won and us dollars based on an international conversion rate of 1100=$1 . the statistical analyses were performed using an spss software package ( version 18.0 for windows ; spss inc . , chicago , il , usa ) . plus , scientific data analysis and graphing software ( sigmaplot , version 10.0 ; systat software inc . , chicago , il , usa ) was applied for the statistical linear regression analysis . an independent t - test was used for the mean comparison between the surgical and endovascular groups . the correlation between the aneurysm diameter and the total hospital costs was assessed using a pearson correlation analysis . this study conducted a retrospective review of data from a series of consecutive patients who underwent surgical clipping or endovascular coiling of a uia at the authors ' institution between january 2011 and may 2014 . the exclusion criteria included a recent ( < 1 month ) subarachnoid hemorrhage caused by a concomitant aneurysm , giant aneurysm with a diameter > 2.5 cm , poor preoperative neurological state affecting postoperative management , and another disease or medical condition significantly increasing the hospital costs . the medical records were reviewed to obtain relevant clinical information , and all the radiological data were obtained using an electronic picture archiving and communication system . the treatment decision , surgical coiling versus endovascular coiling , was made primarily based on the findings of the digital subtraction angiography . surgical treatment was favored over endovascular treatment for patients with the following findings : 1 ) difficult navigation of the microcatheter into the aneurysm19 ) , 2 ) very small ( <3 mm ) aneurysm149 ) , 3 ) complex and wide - neck aneurysm requiring y or x stent - assisted coiling610 ) , 4 ) aneurysm with an arterial branch incorporated into the sac58 ) , 5 ) fusiform or complex aneurysmal configuration , or 6 ) aneurysmal compression of an adjacent cranial nerve11 ) , as long as the patients had no problems related to surgical accessibility or comorbidity . all the patients completed an angiographic examination , chest x - ray , and laboratory tests as a preoperative evaluation before admission , and were admitted to hospital one day before the surgical or endovascular procedure . the operative and perioperative management procedures were uniformly applied to each uia patient . for the patients who underwent surgical clipping , a postoperative computed tomography ( ct ) and ct angiography another ct scan was performed on the day of discharge to check for the occurrence of any subdural hygromas . for most surgical patients , the length of the hospital stay was 3 - 6 days . in the case of elderly patients , a longer hospital stay was usually required due to fatigue and general weakness . meanwhile , for the patients who underwent endovascular coiling , diffusion - weighted magnetic resonance imaging was commonly performed on day 1 following the procedure to evaluate any procedure - related thromboembolic phenomena . the length of the hospital stay after coiling was 3 - 4 days for most patients , irrespective of the patient 's age . the treatment costs for each patient were calculated based on the expenses incurred during the time spent as an inpatient for aneurysm treatment , including the operative and postoperative costs . the operative costs covered the neurosurgical or endovascular procedure , anesthesia , and all the materials ( e.g. , aneurysm clip , endovascular coil , and other surgical supplies ) , while the perioperative costs included the costs for the bed occupancy in the intensive care unit and regular hospital ward , radiological imaging studies , laboratory tests , and all the medications . the costs affected by patient selection ( e.g. , bed occupancy in the regular hospital ward ) were made uniform to the basic lowest cost . all the costs are all presented in korean won and us dollars based on an international conversion rate of 1100=$1 . the statistical analyses were performed using an spss software package ( version 18.0 for windows ; spss inc . , chicago , il , usa ) . plus , scientific data analysis and graphing software ( sigmaplot , version 10.0 ; systat software inc . , chicago , il , usa ) was applied for the statistical linear regression analysis . an independent t - test was used for the mean comparison between the surgical and endovascular groups . the correlation between the aneurysm diameter and the total hospital costs was assessed using a pearson correlation analysis . surgical clipping was applied to 222 patients , where a single aneurysm was clipped in 188 patients and 2 - 3 multiple aneurysms were clipped in 34 patients . while most patients underwent a superciliary keyhole approach ( n=180 , 81.1% ) , the others experienced a pterional approach ( n=31 ) , interhemispheric approach ( n=8 ) , or combined pterional and interhemispheric approach ( n=3 ) . meanwhile , the demographic and aneurysmal characteristics of the 376 patients who underwent surgical clipping ( n=188 ) or endovascular coiling ( n=188 ) for a single uia are summarized in table 1 . the ages , genders , and aneurysm locations were comparable between the surgical and endovascular groups . however , the diameters of the endovascularly - treated aneurysms were larger than those of the surgically - treated aneurysms , although the difference was small ( meansd , range : 5.92.8 mm , 2.2 - 16.0 mm vs. 5.12.2 mm , 2.0 - 14.0 mm ) when comparing the total hospital costs for surgical clipping of a single uia ( n=188 ) and endovascular coiling of a single uia ( n=188 ) , surgical treatment [ meansd : 8,280,0001,490,000 ( $ 75271355 ) ] showed significantly lower total hospital costs than endovascular treatment [ meansd : 11,700,0003,050,000 ( $ 106362773 ) , p<0.001 ] ( table 2 ) . the median and interquartile range ( iqr ) of the total hospital costs for surgical versus endovascular treatment were 7,770,000 ( $ 7064 ) and 1,440,000 ( $ 1309 ) vs. 11,440,000 ( $ 10400 ) and 3,460,000 ( $ 3145 ) , respectively . thus , the median of the total hospital costs for coiling was 47% higher than that for clipping . the higher total hospital costs for endovascular treatment were due to higher operative costs , including the medical equipment and endovascular coils . the operative costs for surgical clipping [ meansd : 5,800,000680,000 ( $ 5273618 ) ] were significantly lower than the operative costs for endovascular coiling [ meansd : 9,310,0002,800,000 ( $ 84642545 ) , p<0.001 ] . the median and iqr of the operative costs for surgical versus endovascular treatment were 5,600,000 ( $ 5091 ) and 730,000 ( $ 664 ) vs. 9,140,000 ( $ 8309 ) and 3,430,000 ( $ 3118 ) , respectively . the price of one endovascular coil was approximately 550,000 ( $ 500 ) , while the price of one surgical clip was approximately 140,000 ( $ 127 ) . in addition , endovascular treatment of a single aneurysm required a considerable increase in the number of coils according to the diameter of the aneurysm , whereas most surgical cases only required one to three aneurysm clips . notwithstanding , there was no statistical difference in the perioperative costs between the surgical and endovascular groups [ meansd : 2,480,0001,070,000 ( $ 2255973 ) vs. 2,400,0001,310,000 ( $ 21821191 ) ] . in the multi regression analysis , the factors significantly associated with the total hospital costs for endovascular treatment of a single uia were the aneurysm diameter ( p<0.001 ) and patient age ( p=0.014 ) , while the patient gender and aneurysm location were not associated . in the case of surgical treatment , the only factor significantly associated with the total hospital costs was the aneurysm diameter ( p<0.001 ) , while the patient age , gender , and aneurysm location were not associated . the pearson correlation analysis of the endovascular group showed a strong positive correlation ( r=0.77 ) between the aneurysm diameter and the total hospital costs ( fig . 1a ) , whereas only a weak positive correlation ( r=0.16 ) was revealed between the patient age and the total hospital costs . meanwhile , the pearson correlation analysis of the surgical group showed a weak positive correlation ( r=0.26 ) between the aneurysm diameter and the total hospital costs ( fig . a simple linear regression analysis was used to develop an equation for predicting the dependent variable ( y , total hospital costs ) given a value ( x , aneurysm diameter ) for the independent variable . for the endovascular group , this equation was y ( )= 6,658,630 + 855,250x ( mm ) or y ( $ ) = 6053.3 + 777.5x ( mm ) . the value of r was 0.6004 . thirty four patients were surgically treated for two ( n=27 ) or three ( n=7 ) uias during a single operation , however , no patient received endovascular treatment for multiple uias during a single session . the mean value of the total hospital costs for the surgical treatment of two uias was 10,810,0002,960,000 ( $ 98272691 ) , while the mean value for the surgical treatment of three uias was 11,540,0001,430,000 ( $ 104911300 ) . for both treatment groups , no direct mortality or permanent morbidity adversely affecting the glasgow outcome scale ensued from the treatments . two patients in the surgical group developed venous infarction and transient somnolence after clipping a pericallosal artery aneurysm , yet both patients recovered without neurological deficits . meanwhile , a patient in the endovascular group developed a thromboembolism and transient leg monoparesis after coiling an anterior communicating artery aneurysm . surgical clipping was applied to 222 patients , where a single aneurysm was clipped in 188 patients and 2 - 3 multiple aneurysms were clipped in 34 patients . while most patients underwent a superciliary keyhole approach ( n=180 , 81.1% ) , the others experienced a pterional approach ( n=31 ) , interhemispheric approach ( n=8 ) , or combined pterional and interhemispheric approach ( n=3 ) . meanwhile , the demographic and aneurysmal characteristics of the 376 patients who underwent surgical clipping ( n=188 ) or endovascular coiling ( n=188 ) for a single uia are summarized in table 1 . the ages , genders , and aneurysm locations were comparable between the surgical and endovascular groups . however , the diameters of the endovascularly - treated aneurysms were larger than those of the surgically - treated aneurysms , although the difference was small ( meansd , range : 5.92.8 mm , 2.2 - 16.0 mm vs. 5.12.2 mm , 2.0 - 14.0 mm ) when comparing the total hospital costs for surgical clipping of a single uia ( n=188 ) and endovascular coiling of a single uia ( n=188 ) , surgical treatment [ meansd : 8,280,0001,490,000 ( $ 75271355 ) ] showed significantly lower total hospital costs than endovascular treatment [ meansd : 11,700,0003,050,000 ( $ 106362773 ) , p<0.001 ] ( table 2 ) . the median and interquartile range ( iqr ) of the total hospital costs for surgical versus endovascular treatment were 7,770,000 ( $ 7064 ) and 1,440,000 ( $ 1309 ) vs. 11,440,000 ( $ 10400 ) and 3,460,000 ( $ 3145 ) , respectively . thus , the median of the total hospital costs for coiling was 47% higher than that for clipping . the higher total hospital costs for endovascular treatment were due to higher operative costs , including the medical equipment and endovascular coils . the operative costs for surgical clipping [ meansd : 5,800,000680,000 ( $ 5273618 ) ] were significantly lower than the operative costs for endovascular coiling [ meansd : 9,310,0002,800,000 ( $ 84642545 ) , p<0.001 ] . the median and iqr of the operative costs for surgical versus endovascular treatment were 5,600,000 ( $ 5091 ) and 730,000 ( $ 664 ) vs. 9,140,000 ( $ 8309 ) and 3,430,000 ( $ 3118 ) , respectively . the price of one endovascular coil was approximately 550,000 ( $ 500 ) , while the price of one surgical clip was approximately 140,000 ( $ 127 ) . in addition , endovascular treatment of a single aneurysm required a considerable increase in the number of coils according to the diameter of the aneurysm , whereas most surgical cases only required one to three aneurysm clips . notwithstanding , there was no statistical difference in the perioperative costs between the surgical and endovascular groups [ meansd : 2,480,0001,070,000 ( $ 2255973 ) vs. 2,400,0001,310,000 ( $ 21821191 ) ] . in the multi regression analysis , the factors significantly associated with the total hospital costs for endovascular treatment of a single uia were the aneurysm diameter ( p<0.001 ) and patient age ( p=0.014 ) , while the patient gender and aneurysm location were not associated . in the case of surgical treatment , the only factor significantly associated with the total hospital costs was the aneurysm diameter ( p<0.001 ) , while the patient age , gender , and aneurysm location were not associated . the pearson correlation analysis of the endovascular group showed a strong positive correlation ( r=0.77 ) between the aneurysm diameter and the total hospital costs ( fig . 1a ) , whereas only a weak positive correlation ( r=0.16 ) was revealed between the patient age and the total hospital costs . meanwhile , the pearson correlation analysis of the surgical group showed a weak positive correlation ( r=0.26 ) between the aneurysm diameter and the total hospital costs ( fig . a simple linear regression analysis was used to develop an equation for predicting the dependent variable ( y , total hospital costs ) given a value ( x , aneurysm diameter ) for the independent variable . for the endovascular group , this equation was y ( )= 6,658,630 + 855,250x ( mm ) or y ( $ ) = 6053.3 + 777.5x ( mm ) . thirty four patients were surgically treated for two ( n=27 ) or three ( n=7 ) uias during a single operation , however , no patient received endovascular treatment for multiple uias during a single session . the mean value of the total hospital costs for the surgical treatment of two uias was 10,810,0002,960,000 ( $ 98272691 ) , while the mean value for the surgical treatment of three uias was 11,540,0001,430,000 ( $ 104911300 ) . for both treatment groups , no direct mortality or permanent morbidity adversely affecting the glasgow outcome scale ensued from the treatments . two patients in the surgical group developed venous infarction and transient somnolence after clipping a pericallosal artery aneurysm , yet both patients recovered without neurological deficits . meanwhile , a patient in the endovascular group developed a thromboembolism and transient leg monoparesis after coiling an anterior communicating artery aneurysm . previous cost comparisons of surgical clipping and endovascular coiling of intracranial aneurysms have revealed distinct disparities between different countries . the international subarachnoid aneurysm trial ( isat ) , primarily conducted in europe , showed no significant cost difference between surgical and endovascular groups at a 1-year or 2-year follow - up13 ) . while the endovascular patients had higher costs related to the initial intervention and subsequent procedures due to recurrences , this was offset by lower costs related to the shorter hospital stay and fewer complications . however , a national analysis using a nationwide inpatient sample in the united states showed significantly higher total hospital costs for surgical clipping of both ruptured and unruptured aneurysms when compared with endovascular coiling3 ) . in contrast , the present study showed significantly lower costs for surgical clipping of uias , attributable to the lower device cost of clips over coils and good surgical results with minimal complications . in the present surgical case series , the advantage of lower economic costs was also enhanced by the reduced surgical invasiveness of a keyhole approach used on 81% of the patients . radovanovic et al.12 ) previously reported that minimally invasive surgery reduced hospital costs by decreasing the operative time and length of the hospital stay when compared with conventional surgery . however , in the present study , the impact of minimally invasive surgery on the treatment costs was not critical , as postoperative costs including hospital bed charges are not high in south korea . since the total hospital costs associated with endovascular coiling of a uia varied predictably as a function of the aneurysm diameter , the costs for endovascular coiling of a uia can be estimated before the procedure using the suggested equation provided by a simple linear regression analysis , where r is a statistical measure of the closeness of the data to the fitted regression line . thus , when the value of r is 0.6 , 60% of the data is on the straight line . first , it is based on a retrospective review of a case series from a single institution . as the surgical techniques and results affecting treatment costs can vary significantly between medical institutions , it is difficult to make generalizations . second , it does not include any long - term economic impact related to the surgical and endovascular treatment of uias . for example , the number of postoperative angiograms , outpatient services , and retreatments can be higher in the case of endovascular treatment . in south korea , the surgical clipping of uias was found to incur lower total hospital costs than endovascular coiling when the surgical results were favorable without significant complications . plus , a strong positive correlation was noted between an increase in the aneurysm diameter and a dramatic increase in the costs of endovascular coiling .

objectivea cost comparison of the surgical clipping and endovascular coiling of unruptured intracranial aneurysms ( uias ) , and the identification of the principal cost determinants of these treatments.methodsthis study conducted a retrospective review of data from a series of patients who underwent surgical clipping or endovascular coiling of uias between january 2011 and may 2014 . the medical records , radiological data , and hospital cost data were all examined.resultswhen comparing the total hospital costs for surgical clipping of a single uia ( n=188 ) and endovascular coiling of a single uia ( n=188 ) , surgical treatment [ meanstandard deviation ( sd ) : 8,280,0001,490,000 ] resulted in significantly lower total hospital costs than endovascular treatment ( meansd : 11,700,0003,050,000 , p<0.001 ) . in a multi regression analysis , the factors significantly associated with the total hospital costs for endovascular treatment were the aneurysm diameter ( p<0.001 ) and patient age ( p=0.014 ) . for the endovascular group , a pearson correlation analysis revealed a strong positive correlation ( r=0.77 ) between the aneurysm diameter and the total hospital costs , while a simple linear regression provided the equation , y ( )=6,658,630 + 855,250x ( mm ) , where y represents the total hospital costs and x is the aneurysm diameter.conclusionin south korea , the total hospital costs for the surgical clipping of uias were found to be lower than those for endovascular coiling when the surgical results were favorable without significant complications . plus , a strong positive correlation was noted between an increase in the aneurysm diameter and a dramatic increase in the costs of endovascular coiling .

INTRODUCTION MATERIALS AND METHODS Patients Decision of treatment modality Perioperative management Treatment costs Statistical analysis RESULTS Patients Comparative cost analysis for single UIA Multi regression analysis for single UIA Pearson correlation analysis and simple linear regression analysis Cost of surgical clipping for multiple UIAs Clinical outcomes DISCUSSION CONCLUSION

intensive care requires not only the use of sophisticated equipment but also highly skilled and dedicated healthcare staff . as such , the intensive care unit ( icu ) takes a significant proportion of the total healthcare cost , and therefore patients with prolonged icu length of stay ( los ) can have serious cost implications . moreover , patients with prolonged icu - los can also lead to a shortage of icu beds and result in operations being cancelled . regarding the population of cardiac surgery patients , the prediction of their icu - los is a fact of great significance , based on the need for containing the burgeoning costs involved in cardiac operations , saving resources and icu costs . in addition , the cardiac surgery icu - los is a significant healthcare index that has been correlated with poor patient outcome , such as increased expenditure and higher morbidity and mortality rates . furthermore , the icu - los is a valuable indicator of the quality and the effectiveness of the provided care . the above data could interpret the scientific interest for the early prediction of the length of hospitalization in cardiac surgery icus . various predictive models for prolonged icu stay after cardiac surgery have been developed , during the last two decades [ 5 , 6 ] , and several papers have attended preoperative risk factors associated with prolonged icustay . these efforts are now more than ever in season and necessary because of the global financial crisis , that has affected negatively the healthcare systems ' operation , worldwide , and demands a more efficient use of the icu resources . the aim of this study was to identify the preoperative and intraoperative predictors for prolonged icu - los , among cardiac surgery patients . this identification could lead to the early recognition of the high - risk patients for longer icu hospitalization and , also , could provide an important aid to nursing administrators and leaders for the appropriate resource planning and effective choice of the operation list , with the low - risk patients being scheduled for surgery before the high - risk candidates . we conducted an observational cohort study from september 2010 to january 2011 among 194 patients who were admitted to the 8-bed cardiac surgery icu of a general , tertiary hospital of athens , greece . the inclusion criteria into the study were a priori defined as follows : ( a ) patient age 18 years old and ( b ) a minimum icu - los of 24 hours . based on the predefined inclusion criteria 44 patients were excluded from the study , and 150 met the inclusion criteria and constituted our study sample . a short structured questionnaire on basic sociodemographic and clinical patient characteristics was used for data collection purposes . the collected sociodemographic data included patient age , gender , height , weight , and body mass index ( bmi ) . in addition , the collected clinical characteristics were the history of chronic pulmonary disease and diabetes , the type of surgery , the preoperative serum creatinine ( cr ) levels , the blood glucose levels during the intraoperative period , the use of intraaortic balloon pump ( iabp ) preoperatively , the application of cardiopulmonary bypass ( cpb ) , the preoperative ejection fraction of the left ventricle ( eflv ) , the duration of surgery and cpb , the presence of atrial fibrillation ( af ) preoperatively , the emergency procedure , the ischemic time , the transfusion of red blood cells ( rbcs ) , the euroscore values and the icu - los . we defined the icu - los as the duration of the hospitalization of patients from their admission to the icu until their discharge . euroscore is the most valid and reliable stratification model for predicting the perioperative risk of cardiac surgery patients in north america , europe , and japan . it was developed between 1995 and 1999 and was first introduced into clinical practice in 1999 , while the logistic algorithm of euroscore is available since 2003 . it includes three wide categories of risk factors : the patient- ( age , female sex , chronic pulmonary disease , extracardiac arteriopathy , neurological dysfunction disease , previous cardiac surgery , serum creatinine preoperatively , active endocarditis , critical preoperative state ) , the cardiac- ( unstable angina , left ventricular dysfunction moderate : left ventricular ejection fraction ( lvef ) 3050% or poor : lvef < 30% , recent myocardial infarct ( < 90 days ) , pulmonary hypertension , and the operation- ( emergency , other than isolated coronary artery bypass grafting , surgery on thoracic aorta , postinfarct septal rupture ) associated factors . the sum of the various risk factors results in a total score which represents the predicted probability of mortality and ranges between 0 and 100% ( logistic euroscore ) [ 8 , 9 ] . one of the researchers , the same each time , obtained the data based on the review of both medical and nursing patient records . euroscore was calculated for each patient based on data collected by the hospital records . in total finally , the measurement of the serum glucose levels was carried out for each patient , intraoperatively , immediately after the induction of anesthesia and was repeated every 30 minutes . continuous variables are presented as median ( interquartile range , ir ) , while categorical variables are presented as absolute and relative frequencies . the kolmogorov - smirnov test and graphs ( histograms and normal q - q plots ) were used to test the normality of the distribution of the continuous variables . distributions of continuous variables were extremely skewed and were converted into binary variables using median values as the criterion for separation . for bmi the criterion for separation was the international limit of 24.9 kg / m . for serum cr levels the cut - off point was the 1.4 mg / dl , a value that constitutes the limit between the normal and the increased cr levels of the biochemistry laboratory of the hospital in which our study was carried out . in addition , the cut - off for prolonged icu - los ( 2 days ) was chosen based on the median value and the literature data [ 3 , 10 ] . chi - square test and fisher 's exact test were used to identify differences between groups . kendall 's tau rank correlation coefficient ( r ) was used to estimate correlation between continuous variables . variables that were statistically significant ( p < 0.05 ) in bivariate analysis were entered into the backward stepwise multivariate logistic regression analysis with icu - los as dependent variable . multivariate analysis was used to control potential confounding variables . only variables with 95% confidence interval for adjusted odds ratio that did not cross 1 were considered to have an independent and significant association with icu - los . criteria for entry and removal of variables were based on the likelihood ratio test , with entrance and removal limits set at p < 0.05 and p > 0.05 . we estimated crude and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for the predictive factors related to the icu - los . a correlation matrix was assessed prior to conducting the multivariate logistic regression analysis to check for collinearity among the independent variables . kendall 's tau rank correlation coefficient ( r ) was used to estimate correlations between variables . all tests of statistical significance were two - tailed , and p - values of less than 0.05 were considered significant . statistical analysis was performed using the statistical package for social sciences software ( ibm spss statistics 19.0 for windows , spss inc . , researchers received patients ' consent after being informed about the type and purpose of the study . each potential subject adequately informed of the aims , method , the anticipated benefits , and potential risk of the study . the potential subject informed of the right to refuse to participate in the study or to withdraw consent to participate any time without reprisal . the investigation was carried out in accordance with the ethical standards of the responsible institutional committee for human experimentation and with the helsinki declaration of 1975 , as revised in 2008 . precautions took place to protect the privacy of research subjects and the confidentiality of their personal information . the methods of the study were restricted to observation and recording patient data and no part of the standard care was omitted . the majority of patients were males ( 68% ) , and the median age was 66 ( ir 15 ) years old . most patients had either received a coronary artery bypass grafting ( 44.7% ) or had undergone a valve disease surgery repair or replacement ( 27.3% ) . 17.3% and 48% of patients had serum cr levels higher than 1.4 mg / dl and blood glucose levels higher than 130 mg / dl , respectively . preoperative af was presented in 11.7% of patients , while the median logistic euroscore value was 5.4 ( 7.3 ) % . the median icu - los was 2 ( ir 3 ) days , and the overall 30-day mortality was 13.3% . table 2 provides the main predictive factors ' ( including demographic characteristics and perioperative - related factors ) distribution and their relation with icu - los ( below or above median ) . the preoperative serum cr > 1.4 mg / dl , the mean intraoperative blood glucose levels > 130 mg / dl , the duration of surgery and the duration of cpb longer than 250 and 125 min , respectively , the presence of af preoperatively , the emergency procedure , the transfusion of rbc and the logistic euroscore values > 5.4% were associated with icu - los more than 2 days at the level of 5% ( p a correlation matrix was assessed prior to conducting the multivariate logistic regression analysis to check for collinearity among the independent variables . because duration of surgery and duration of cardiopulmonary bypass were strongly correlated ( r = 0.66 , p < 0.001 ) , we chose to include only the duration of surgery in the multivariate model . we found a positive association between perioperative risk ( high euroscore ) and icu - los ( or = 4.3 , 95% ci = 2.28.4 , p < 0.001 ) while this association remained statistically significant after adjusting for gender , age , bmi , chronic pulmonary disease , diabetes , type of surgery , preoperative iabp , cbp , eflv , duration of surgery , cardiopulmonary bypass time , emergency , ischemic period , and rbc ( or = 2.6 , 95% ci = 1.25.6 , p = 0.017 ) . in addition to the above , patients with preoperative serum cr > 1.4 mg / dl had an almost 3 times greater probability to have icu - los > 2 days ( or = 3 , 95% ci = 18.7 , p = 0.049 ) , and also patients with mean intraoperative blood glucose > 130 mg / dl had an almost 3 times greater probability for longer icu - los ( or = 3 , 95% ci = 1.46.4 , p = 0.004 ) . finally , patients with af preoperatively were associated with an almost 6.3 times greater probability of having an icu - los of more than 2 days . the main findings of our study were the strong association of three preoperative ( serum cr levels , preoperative af , and logistic euroscore ) and one intraoperative factor ( glucose levels ) with the prolonged icu - los of cardiac surgery patients . in particular , patients with preoperative renal dysfunction , high perioperative risk , history of af or high mean glucose levels intraoperatively have greater possibility to stay in the cardiac surgery icu more than 2 days . as aforementioned , the increased serum cr levels , preoperatively , were statistically significantly correlated with prolonged icu hospitalization . in line with our findings , herman et al . , in a recent study of 3489 cabg patients in a hospital of canada , concluded that the history of renal failure is among the independent predictors for prolonged hospitalization into the intensive care setting . in addition , ranucci et al . , in a retrospective study of 9120 cardiac surgery patients revealed the increased preoperative cr levels among the main determinants of late discharge from the icu . the history of renal failure was among the independent risk factors for extended cardiac surgery icu hospitalization . similar conclusions were reached by the studies of bucerius et al . and ghotkar et al . . however , in a recent retrospective study of 298 patients who underwent aortic arch surgery , the authors found that the increased serum cr levels were associated with prolonged icu stay , through the univariate analysis . nevertheless , contrary to the finding of this study , the multivariate analysis did not indicate significant association between these two variables . in addition , wong et al . correlated the high cr levels postoperatively and not preoperatively with increased cardiac surgery icu hospitalization . we could find easily a logical interpretation regarding the previous correlation , based on the fact that patients with preexisting renal dysfunction or / and failure are characterised as high - risk patients with higher clinical severity and morbidity rates . in addition , these patients have higher incidence of postoperative complications , such as hemorrhage , reoperation , prolonged mechanical ventilation , strokes , and necessity for renal replacement therapy [ 1517 ] . moreover , the literature review reveals that the renal dysfunction during the preoperative period , among cardiac surgery patients , is a strong and independent indicator of increased in - hospital and 30-day mortality . consequently , their stay for a longer period in the icu in order to achieve clinical stability and to discharge from the critical care environment safely is rather reasonable . another important finding of this study was the significant correlation between the preoperative af and the prolonged icu - los . , in a prospective study of 10759 patients undergoing cabg , observed that 20 variables ( 15 preoperative and 5 intraoperative ) , including the presence of af preoperatively , have a strong association with prolonged icu - los . moreover , the authors of other studies [ 19 , 20 ] found that the no - sinus rhythm or the rhythm disturbances , preoperatively , including af , are significant predictors of prolonged stay in the cardiac surgery icu . the recent developments in invasive cardiology , the improvement of surgical techniques , and the improved pharmacological treatments have contributed to the application of surgical treatment among elder patients with advanced cardiovascular disease and significant comorbidity [ 13 , 21 ] . therefore , af appears very frequently preoperatively in patients who are candidates for cardiac surgery , increasing the perioperative risk and the occurrence of postoperative complications . preoperative af significantly worsens the heart functional status , increasing the incidence of postoperative complications , such as delirium , stroke , and low cardiac output syndrome , which lead to negative healthcare patient outcomes , including higher mortality and prolonged icu and in - hospital stay [ 2123 ] . in addition , a point of great interest is that while almost 30% of patients undergoing cardiac surgery will develop postoperative af , if patients have history of af ( preoperative af ) , this probability becomes almost 2 times greater ( 60% ) [ 24 , 25 ] . the above data in conjunction with the correlation between the postoperative af and the prolonged cardiac surgery icu - los allow us to interpret the association of the preoperative af with the prolonged icu hospitalization , a finding which was revealed by our study . in addition , via the multivariate analysis , we found that patients with high euroscore have significantly greater probability to stay longer in the icu than patients with lower perioperative risk . , in a recent study of 313 consecutive cardiac surgery patients , concluded that there is a strong association between high perioperative risk and the prolonged icu - los . in particular , patients with high logistic euroscore values had an almost 1.9 times greater probability to stay in the icu longer than 2 days . several studies have reached to similar findings , revealing a positive association between increased perioperative risk ( high euroscore values ) and the increased cardiac surgery icu - los [ 10 , 26 , 27 ] . in addition , several investigators using risk stratification models other than euroscore , such as parsonnet score , noted , in line with our findings , strong evidence between the high perioperative risk and the increased icu - los [ 6 , 12 , 28 ] . the above correlation could be justified by the fact that variables , which are included in euroscore , constitute significant risk factors and predictors of the increased hospitalization in the critical care environment . older age , female gender , chronic pulmonary disease , extracardiac arteriopathy , neurological dysfunction , high preoperative cr levels , previous cardiac surgery , unstable angina , poor left ventricular ejection fraction , recent myocardial infarction , emergency operation and procedures other than isolated coronary artery bypass grafting are parameters which are included in euroscore model and simultaneously have been independently and separately correlated with increased icu - los . indeed , a recent systematic review and validation study of the performance of 14 icu - los prediction models among cardiac surgery patients revealed that the euroscore model has significantly high performance in terms of discrimination , accuracy , and calibration . among the 14 prediction models , these data indicate that euroscore is a valid and reliable tool for predicting the cardiac surgery icu - los . moreover , we identified the significant correlation of one intraoperative variable with the prolonged icu - los . in particular , patients with mean serum glucose levels > 130 mg / dl , intraoperatively , had greater probability to stay longer in the icu . in accordance with our results , ouattara et al . in a prospective cohort study of 200 diabetic patients undergoing coronary artery bypass grafting concluded that the poor glycemic control intraoperatively is correlated with significant increase in the icu - los . hyperglycemia during the perioperative period has been associated with negative patient outcome and constitutes an independent risk factor for higher morbidity and mortality . moreover , the poor glycemic control has been associated with postoperative complications , such as sternal wound infection , impaired wound healing , adverse renal and pulmonary events , increased incidence of af and myocardial infarction [ 29 , 3135 ] . however , in contrary to the findings of the present study , the literature review shows that many investigators have not achieved to correlate the intraoperative hyperglycemia with the prolonged icu - los among cardiac surgery patients [ 3538 ] . in addition , lazar et al . in a controlled randomized trial of 141 diabetics patients who underwent coronary artery bypass graft surgery correlated the inadequate intra- and postoperative glycemic control with increased total postoperative in - hospital stay and not icu stay separately . the significant shortage of randomized controlled trials and the different definitions of the perioperative hyperglycemia in conjunction with the different intensity and duration of the applied serum glucose therapy do not allow safe conclusions and could justify the inconsistence of the literature findings [ 39 , 40 ] . finally , the median icu - los of our study was 2 days , a finding that is in line with the international literature , based on the fact that most researchers have demonstrated the 2 days as the cutoff point for the increased cardiac surgery icu - los [ 3 , 10 ] . our study allows us to reach significant conclusions regarding the preoperative and intraoperative factors that could influence the icu - los among cardiac surgery patients . in addition , to the best of our knowledge , this study is among the few greek studies aiming to investigate parameters that could predict the length of icu stay and consequently to contribute to administrative decisions regarding the plan of the operative list , the provided care , and the icu resources ' allocation . however , our study has some limitations . firstly , the small sample size in conjunction with the fact that this study was carried out in one cardiac surgery centre limits the generalization of the finding to the general cardiac surgery population and affects the external validity of the study . secondly , the icu - los is often a subjective outcome that affected , except from clinical ( e.g. , patient severity , complications ) and by nonclinical factors , such as the icu and the ward capacity of the institution , the opinion of the intensive care specialists or a busy operation schedule . moreover some hospitals are more comfortable transferring patients out to a lower acuity nursing environment than other hospitals . finally , the different cut - off points of the prolonged icu - los impede the ability to compare the findings of the corresponding international literature . further research is needed to identify more predictive variables for the identification of patients at risk for prolonged cardiac surgery icu hospitalization . demographic and clinical preoperative and intraoperative patient characteristics are significant predictors of the icu - los among cardiac surgery patients . patients with renal dysfunction , history of af , high euroscore values , and intraoperative hyperglycemia seem to have significantly greater probability of staying for more than 2 days in the intensive care environment . the early recognition of the nonmodified parameters ( history of renal dysfunction and af , high euroscore ) could lead to the early identification of patients at risk . in addition , the management of the modified predictor ( intraoperative hyperglycemia ) , through the maintenance of normoglycemia during the operation , could erase a significant risk factor for prolonged icu - los in the cardiac surgery setting . nursing administrators may use this knowledge for the appropriate use of the limited icu , human and financial , resources . in addition , they have the opportunity for a more efficient and effective planning of the operative list , selecting low - risk patients in periods with significant restriction of the icu beds availability . this chance has great importance for healthcare systems , such as the greek , that are characterized by limited resources , nursing staff and icu - beds shortage , mainly within the public sector .

the prediction of intensive care unit length of stay ( icu - los ) could contribute to more efficient icu resources ' allocation and better planning of care among cardiac surgery patients . the aim of this study was to identify the preoperative and intraoperative predictors for prolonged cardiac surgery icu - los . an observational cohort study was conducted among 150 consecutive patients , who were admitted to the cardiac surgery icu of a tertiary hospital of athens , greece from september 2010 to january 2011 . multivariate regression analysis revealed that patients with increased creatinine levels preoperatively ( odds ratio ( or ) 3.0 , p = 0.049 ) , history of atrial fibrillation ( af ) ( or 6.3 , p = 0.012 ) and high euroscore values ( or 2.6 , p = 0.017 ) had a significant greater probability to stay in the icu for more than 2 days . in addition , intraoperative hyperglycemia ( or 3.0 , p = 0.004 ) was strongly associated with longer icu - los . in conclusion , the high perioperative risk , the history of af and renal dysfunction , and the intraoperative hyperglycemia are significant predictors of prolonged icu stay . the early identification of patients at risk could allow the efficient icu resources ' allocation and the reduction of healthcare costs . this would contribute to nursing care planning depending on the availability of healthcare personnel and icu bed capacity .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Limitations 6. Conclusion

colorectal cancer ( crc ) is one of the most prevalent cancer , along with lung and breast cancers . in 2012 , approximately 1.4 million new cases and 700,000 crc - related deaths were estimated worldwide ( 1 ) . over the last decade , crc incidence and mortality in the united states have been on a steady decline as a result of advances in its prevention , screening , and treatment . nevertheless , it is still the second leading cause of cancer deaths ( 2 ) . moreover , there has been a significant rise in crc incidence in newly developed or economically transitioning countries , such as south america , eastern european countries , and most parts of asia ( 3 ) . although crc is highly curable at an early stage , approximately 50% of patients with crcs eventually develop metastatic disease ( 4 ) . five - year survival rate is approximately 10% in patients with a stage iv disease ( 5 ) . there are many known risk factors for crc including age , sex , smoking , excessive alcohol consumption , high intake of red and processed meat , obesity , diabetes , inflammatory bowel disease , inherited genetic disorder , and family history of crc ( 6789101112 ) . among them , ulcerative colitis and crohn 's disease increase the overall risk of colitis - associated cancer ( 14 ) . approximately 20% of patients with inflammatory bowel disease develop colitis - associated cancer within 30 years of disease onset ( 15 ) . inherited crc , including lynch syndrome and familial adenomatous polyposis , represent less than 5% of crc cases ( 16 ) . in this review , we briefly review currently available drugs for the treatment of colorectal cancer and summarize clinical studies that use cell - based cancer immunotherapy as promising therapeutic strategy . we also suggest the use of cytokine - induced killer ( cik ) cells as an additional therapeutic approach for metastatic colorectal cancer . most crcs occur without a known genetic risk factor and significant family history . only a small proportion ( 5% ) of inherited crcs , which is also known as familial crc , have been thoroughly characterized ( 17 ) , and additional 20~25% are yet to be completely understood ( 17 ) . crc develops over the course of more than 10 years , and entails multistep genetic events for tumor progression . a multistep genetic model of colorectal carcinogenesis colorectal cancers occur as a result of oncogenes activation , along with the inactivation of tumor suppressor genes by mutations or loss of heterozygosity ( loh ) ( 18 ) . mutations in at least four to five genes are required for the formation of a malignant tumor ( 18 ) . the molecular pathogenesis of colorectal cancer is commonly classified into two main pathways of genomic instability : chromosomal instability ( cin ) and microsatellite instability ( msi ) pathways . cin leads to an imbalance in chromosome number ( aneuploidy ) such as chromosome gains or losses ( including loh events at specific tumor suppressor gene loci ) , and is observed in 80~85% of sporadic crcs ( 18192021 ) . a mutation coupled with loh of the adenomatous polyposis coli ( apc ) tumor suppressor gene , followed by activating mutations of v - ki - ras2 kirsten rat sarcoma viral oncogene homolog ( kras ) , lead to the initial appearance of adenoma from normal colonic epithelium ( 18 ) . apc mutations are typically observed in earliest stages of tumors , including in adenomas as small as 0.5 cm ( 22 ) . this indicates that a functional loss of apc , which is part of the wnt signaling pathway , plays a role in early tumorigenesis . in contrast , msi , which is observed in 15~20% of sporadic crcs ( 21 ) , is a hypermutable phenotype caused by impaired dna mismatch repair ( mmr ) system to correct errors such as single - base mismatches and insertion - deletion loops that spontaneously occur during dna replication ( 2324 ) . msi arises from impaired dna mmr genes , such as mutl homolog 1 ( mlh ) 1 , muts homolog ( msh ) 2 , and to a lesser degree msh6 ( 2526 ) . faulty dna fidelity caused by defects in dna mmr causes frameshift and point mutations , mainly in repetitive sequences ( microsatellites ) ( 26 ) . lynch syndrome is a well - established hereditary predisposition to colorectal cancer caused by a germline mutation in a dna mmr gene . however , 12% of msi exhibit sporadic crc with cin ( 27 ) . because pathways are not mutually exclusive , crc can display features of multiple pathways . i colon cancer , surgery is the definitive treatment without the use of adjuvant chemotherapy . benefit of adjuvant chemotherapy for patients with a stage ii disease is highly debatable due to minimal gains in overall response . however , adjuvant chemotherapy is generally acceptable for the use patients with node - positive cancer following surgery ( high - risk stage ii disease ) ( 29 ) . adjuvant chemotherapy is required for all patients with a stage iii colon cancer after surgical resection , which have a high risk of recurrence of 15~50% ( 30 ) . conventional chemotherapeutic agents include 5-fluorouracil ( 5-fu ) with leucovorin ( lv ) , capecitabine ( an orally administered prodrug of 5-fu ) , oxaliplatin , and irinotecan . adjuvant chemotherapy using the 5-fu / lv regimens following surgery provides significant reduction in mortality by 22% and improves event - free survival by 35% ( 31 ) . to improve disease - free survival ( dfs ) and overall survival ( os ) , currently , folfox ( 5-fu / lv and oxaliplatin ) or folfiri ( 5-fu / lv and irinotecan ) is widely used for standard first- or second - line treatment in patients with node - positive colon cancer following surgical resection ( 3233 ) . however , folfox and folfiri demonstrated greater toxicities , including grade 3 or higher acute toxicity , compared to 5-fu / lv alone ( 34 ) . in 2004 , targeted therapeutic agents for metastatic colorectal cancer , such as anti - epidermal growth factor receptor ( egfr ) antibodies ( cetuximab and panitumumab ) , vascular endothelial growth factor ( vegf ) inhibitors ( bevacizumab , ziv - aflibercept , and ramucirumab ) , and a multikinase inhibitor ( regorafenib ) , were approved by the us food and drug administration ( fda ) for combinational use with standard chemotherapy treatment ( 35 ) . currently , there are various regimens of combinational therapies using targeted therapeutic agents and conventional chemotherapy drugs ( 36 ) . treatment strategy is often dependent on tumor - specific factors , such as kras and/or v - raf murine sarcoma viral oncogene homolog b ( braf ) mutation , and condition of patients and their disease progression following first - line treatment ( 3336 ) . since ras mutations lead to continuous activation of signaling pathways downstream of egfr , the use of anti - egfr antibodies is effective in patients with wild - type kras exon 2 , but not in patients with kras and/or braf mutations . thus , approximately 30~40% of crcs are associated with kras gene mutation and do not respond to anti - egfr antibodies ( 37 ) . over the past decade , multidisciplinary advances in the treatment of metastatic colorectal cancer with targeted therapy have statistically improved progression - free and overall survivals . despite these advances , only a relatively small effect on survival outcomes was observed , especially in patients with distant metastatic disease . dendritic cell ( dc ) therapy is one of several strategies in therapeutic cancer vaccines . sipuleucel - t , a dc - based cancer vaccine , has been approved by the us fda for advanced prostate cancer in 2011 . the goal of dc vaccination is to elicit anti - tumor response by inducing tumor - specific effector t cells . dcs are generated ex vivo by culturing hematopoietic progenitor cells or monocytes with a combination of cytokines and then pulsing them with antigens ex vivo ( 38 ) . there are various type of cancer antigens for dcs loading , including mutated and non - mutated antigens ( 39 ) . now , we can find 9 clinical trials for the treatment of patients with crc from 2004 to 2015 ( table 1 ) . antigens that are most commonly used for colorectal cancer include carcinoembryonic antigen ( cea ) peptides ( 4041 ) , melanoma - associated antigen ( mage ) from allogeneic melanoma cell lysates ( 4243 ) , and autologous cell lysates from biopsy material ( 44 ) . while cea expression in normal colon epithelial cells is relatively low , it is over expressed in most colorectal carcinomas as well as in many cancers ( 45 ) . therefore , cea has been the major immunological target of dc - based cancer vaccines for colorectal cancer . clinical trials of cea - pulsed dcs demonstrated its immune - stimulatory capacity and it was well tolerated in patients without any observable toxicities . however , the overall clinical response was rather unimpressive ( 404146 ) , which may reflect severely impaired immune functions in patients with excessive tumor burdens and tumor immunoediting mechanisms . nevertheless , several clinical studies of mage - pulsed dcs showed 24~40% clinical benefit rate with durable responses and tumor regression ( 4243 ) . moreover , regulatory t cell levels declined upon dc vaccination ( 42 ) . recently , hunyadi j. and colleagues demonstrated that autologous tumor cell lysates - loaded dcs led to an increase in 6-years survival rate in colorectal cancer patients and more efficient induction of t - lymphocytes proliferation in vitro when compared to cea - pulsed dcs ( 44 ) . however , because of limited number of patients , additional evaluations in large - scale clinical trials are needed . although significant advances have been made over the past decade , further studies are required to fully determine the potential antitumor effects of dc vaccination for colorectal cancer . adoptive cell therapy ( act ) for metastatic melanoma was first described in 1988 ( 47 ) . in act , tumor - infiltrating lymphocytes ( tils ) are collected from solid tumor specimen , and are activated and expanded ex vivo . subsequently , tils are administered intravenously to the autologous patient . in 2002 , dudley me et al . demonstrated that host immunodepletion using cyclophosphamide and fludarabine prior to til infusion resulted in dramatic improvement in clinical outcomes for patients with metastatic melanoma ( 48 ) . although act has been evaluated in various cancers , only a small number of act trials for crc have been performed . in a phase i clinical trial , 14 patients with colorectal liver metastases received act with tils in combination with high - dose il-2 ( 49 ) . unfortunately , no significant difference in dfs rates was observed between tils therapy and conventional chemotherapy . a novel act approach for crc was conducted in two separate studies ; sentinel lymph node ( sln ) t lymphocytes were used in a pilot study ( seven with stage ii - iii and nine with stage iv crc ) ( 50 ) and in a phase i / ii study ( 46 with stage i - iii and nine with stage iv crc ) ( 51 ) , respectively . all patients with stage iv disease responded to the treatment with the following outcomes : four stable disease , one partial response , and four complete response with no detectable cancer cells remaining . the overall survival of act - treated patients was also significantly improved to 2.6 years compared to 0.8 years in conventionally - treated control patients . the latest phase i / ii study also showed favorable clinical outcomes ( 51 ) . the median overall survival of sln - t lymphocytes - treated and control groups in stage iv patients were 28 and 14 months , respectively . in the two studies , no significant toxicity was observed following sln - t lymphocytes treatment . these studies demonstrated that sln - t lymphocyte immunotherapy is indeed feasible and safe for patients with metastatic crc . however , surgeon 's proficiency may be required for the intraoperative sentinel node detection . in a phase i study of autologous genetically engineered t cells , all patients developed severe colitis despite showing a decrease in serum cea levels by cea - specific t cells ( 52 ) . cytokine - induced killer ( cik ) cells are ex vivo - expanded lymphocytes used for cancer immunotherapy . cik cells are generated by culturing peripheral blood mononuclear cells ( pbmcs ) with a combination of il-2 and anti - cd3 monoclonal antibodies for 14 days ( 53 ) . cik cells consist of heterogeneous cell population , mainly cd3cd56 , cd3cd56 , and a minor population of cd3cd56 cells . importantly , cd3cd56 cells are the distinguishing cell population among the cik cells and have the most potent cytotoxic function ( 53 ) . cd3cd56 cells originated from cd3cd8cd56 cells but not from cd3cd56 cells during the ex vivo expansion ( 54 ) . cik cell cytotoxicity is mediated by perforin release and dependent on several activating receptors such as nkg2d , nkp30 , and dnam-1 ( 5556 ) . cik cells also exhibit non - specific and non - mhc - restricted cytotoxicity ( 56 ) . over the past decade , cik cell therapy has been evaluated in numerous clinical studies in patients with various types of cancer , such as hepatocellular carcinoma , non - small cell lung cancer , renal cell carcinoma , and gastric cancer ( 57 ) . cik cell therapy can be used as a postoperative adjuvant treatment as well as a palliative treatment following standard therapies . cik cell therapy was evaluated in a limited number of clinical studies in patients with colorectal cancer . in a retrospective study , 21 of 96 colorectal cancer patients who underwent surgery as well as adjuvant chemotherapy received one to three cycles of cik cell transfusion for immunotherapy ( 58 ) . patients in the cik - treated group had significant improvement in their 2-year dfs rates than those in the control group ( 59.6524.80% vs 29.356.39% ) . other studies used cik in a combination therapy with dcs , specifically tumor lysate - pulsed dcs . in 2014 , two clinical studies of dc vaccine and cik cell combinational therapy for colorectal cancer patients were published . one study demonstrated that overall survival rates were significantly improved in the dc - cik therapy compared to the control group ( p=0.04 ) ( 59 ) . moreover , most patients that received dc - cik therapy displayed improvement in quality of life , including physical strength , appetite , sleep , and body weight . gao , d. and colleagues evaluated the clinical benefits of dc - cik therapy in 54 gastric and colorectal patients following surgery with or without chemo - radiotherapy ( 60 ) . the study demonstrated that dfs and 5-year survival of colorectal cancer patients were significantly prolonged in dc - cik treatment groups ( dfs rate : 66% and 5-year survival rates : 75% ) when compared to patients in control groups ( dfs rate : 8% and 5-year survival rates : 15% ; p<0.01 ) . il-12 levels were significantly increased in patients that received the dc - cik treatment . besides fever , since they did not compare the efficacy of combination therapy with each alone , we could not clarify whether combination therapy is better than each therapy . however , when we considered the efficacy of the single therapy of dcs and cik cells described above , we could presume that the combination of cik and dc might exert better antitumor activity than each alone . here , we provide additional evidence that cik cell therapy can effectively prevent growth of colorectal cancers in a xenograft mouse model . hypaque density gradient centrifugation , washed three times with pbs , and cultured in the presence of immobilized anti - cd3 antibody ( 5 g / ml ) and recombinant human il-2 ( 700 u / ml ) for five days . the cell suspension was further incubated in complete medium containing il-2 only ( 170 u / ml ) for nine days . thereafter , medium containing il-2 was replenished every 2 or 3 days . on day 14 cik cells comprised of 94% cd3 , 3% cd3cd56 , 16% cd3cd56 , 14% cd4 , and 81% cd8 cells ( fig . 1a ) . to examine the cytotoxicity of cik cells , two cell lines , sw620 and k-562 were used . sw620 cells are metastatic human colon cancer cell lines derived from lymph node ( 61 ) . k-562 cell lines are human leukemic cells commonly used as reference target cells of nk cells due to the very low level of mhc class i antigens on their cell membrane ( 62 ) . a 4-h cr release assay revealed that cik cells were able to kill 14% , 22% , and 36% of sw620 cells at effector : target ratios of 10:1 , 30:1 , and 100:1 , respectively ( fig . moreover , cik cells showed strong cytotoxicity against k562 cells , a known nk - sensitive target . a nude mouse xenograft assay was used to examine the in vivo antitumor activity of cik cells . sw620 cells ( 610 ) in 300 l of pbs were injected subcutaneously on day 0 , followed by intravenous once - a - week injection of cik cells and adriamycin ( adr ) . on day 25 , mice were sacrificed and the tumor mass and body weight ( to determine toxicity ) were measured . in control mice , sw620 cells grew to a tumor volume of 473190 mm ( n=7 ) ( fig . cik cells injected at doses of 110 , 310 , and 1010 cells per mouse were able to inhibit tumor growth by 7% , 53% , and 73% , respectively . cik cells injected at doses of 110 , 310 and 1010 cells per mouse reduced tumor weight by 10% , 54% and 73% , respectively ( fig . thus , our preclinical efficacy data showed that cik cells were able to kill sw620 cells in vitro and in vivo , suggesting that cik cells might be a good immunotherapy candidate for colorectal cancer . patients with metastatic colorectal cancer have significant risk of recurrence following surgery and conventional adjuvant therapy . although advances in therapeutic agents for crc have reduced the risk of recurrence and increased overall survival , patients with metastatic disease have a poor prognosis and a particularly low 5-year survival rates of less than 10% . additionally , current combination therapy is occasionally discontinued due to a grade 3~4 toxicity with negative effects on quality of life ( 63 ) . overall quality of life should be considered for patients with stage iv disease for palliative therapy . also , elderly patients with stage iii disease often avoid adjuvant chemotherapy due to anxiety of old age , comorbidities , side effects , and perceived minimal benefit ( 64 ) . clinical and preclinical studies of crc treatment with dcs , t cells , and cik cells showed promising outcomes , although only limited information have been available till recently . among the immune effector cells , cik cells have certain advantages in a clinical application : they are relatively easy to generate and expanded in large - scale from pbmcs , their non - mhc - restricted cytotoxic activity could eliminate mhc class i - negative tumor cells , and importantly , cik cell therapy causes mild , transient , and easy - to - manage side effects . over the last decades , cik cell therapy has been evaluated in numerous clinical studies in patients with various cancers . in most patients , combination therapy using conventional agents and cik cells showed superior clinical outcomes than standard therapy alone . clinical studies previously reported by others and our preclinical data suggest that immunotherapy of crc with cik cells can be a promising strategy to limit the growth and metastasis of crc .

colorectal cancer is the third leading cancer worldwide . although incidence and mortality of colorectal cancer are gradually decreasing in the us , patients with metastatic colorectal cancer have poor prognosis with an estimated 5-year survival rate of less than 10% . over the past decade , advances in combination chemotherapy regimens for colorectal cancer have led to significant improvement in progression - free and overall survival . however , patients with metastatic disease gain little clinical benefit from conventional therapy , which is associated with grade 3~4 toxicity with negative effects on quality of life . in previous clinical studies , cell - based immunotherapy using dendritic cell vaccines and sentinel lymph node t cell therapy showed promising therapeutic results for metastatic colorectal cancer . in our preclinical and previous clinical studies , cytokine - induced killer ( cik ) cells treatment for colorectal cancer showed favorable responses without toxicities . here , we review current treatment options for colorectal cancer and summarize available clinical studies utilizing cell - based immunotherapy . based on these studies , we recommend the use cik cell therapy as a promising therapeutic strategy for patients with metastatic colorectal cancer .

INTRODUCTION THE MOLECULAR PATHOGENESIS OF COLORECTAL CANCER CURRENT TREATMENT OF COLORECTAL CANCER DENDRITIC CELL THERAPY FOR COLORECTAL CANCER T CELL THERAPY OF COLORECTAL CANCER CYTOKINE-INDUCED KILL CELL THERAPY FOR COLORECTAL CANCER CONCLUSION

the majority of swallowed indigestible foreign bodies pass through the gastrointestinal tract without complications [ 1 , 2 , 3 , 4 ] . however , there are three physiological narrowings involving the pylorus , duodenal c - loop and ileocecal valve . foreign bodies longer than 10 cm , such as a toothbrush , can not negotiate the duodenal c - loop due to its fixed retroperitoneal position . these objects should be endoscopically removed as soon as possible to avoid pressure necrosis and gastrointestinal perforation [ 5 , 6 , 7 ] . if endoscopic removal fails or there is evidence of obstruction or perforation , laparoscopic gastrotomy should be performed . an 18-year - old caucasian woman with no previous history of related medical problems was admitted to the department of internal medicine , division of gastroenterology , clinical hospital split because she had accidentally swallowed a toothbrush . the patient admitted she had been using the toothbrush to induce emesis . on presentation , 2 h after ingestion , she was asymptomatic and her vital signs were within normal limits . a plain abdominal x - ray study confirmed the presence of the foreign body in the left upper abdominal quadrant ( fig . informed written consent for upper gastrointestinal endoscopy was obtained from the patient and her parents . esophagogastroduodenoscopy revealed the toothbrush in the stomach with its head positioned against the gastric fundus . the extracted toothbrush was 20 cm long . repeated upper gastrointestinal endoscopy was performed 4 h later and showed no evidence of mucosal lesion to the stomach or the esophagus . the patient was discharged home in excellent clinical condition after being observed for 6 hours . foreign bodies in the stomach will pass uneventfully through the gastrointestinal tract in 8090% of cases [ 1 , 2 , 3 ] . however , foreign objects longer than 10 cm , such as a toothbrush , can not negotiate the duodenal c - loop due to its fixed retroperitoneal position . in such cases , these objects should be removed as soon as possible to avoid pressure necrosis and gastric perforation [ 2 , 3 , 4 , 5 ] . removal of long foreign bodies from the stomach is influenced by the patient 's clinical condition and technical abilities of the endoscopist [ 2 , 3 , 4 , 5 ] . if endoscopic removal fails or there is evidence of obstruction or perforation , surgical gastrotomy should be performed . we had no need for conscious sedation since the patient was actively participating during the procedure . special attention has to be paid during the extraction of the toothbrush to its alongside alignment with the esophagus . otherwise , this most critical and demanding part of the extraction procedure may easily result in mucosal damage or foreign body impaction . the second important phase of the extraction procedure is when the foreign body reaches the oropharynx . the patient has to extend his head backwards and the endoscopist has to reach for the toothbrush with his hand and pull it out . this case report describes a rare case in whom a toothbrush was safely extracted from the stomach endoscopically by snare extraction . the procedure is brief , does not require conscious sedation , and the patient can be discharged from hospital after a few hours . early endoscopic retrieval of the toothbrush is critical for reducing morbidity and mortality . in cases when endoscopic removal fails

most ingested foreign bodies will pass uneventfully through the gastrointestinal tract . nevertheless , long and rigid foreign bodies are associated with an increased risk of gastrointestinal impaction , perforation and bleeding . moreover , there has been no case of spontaneous passage of a toothbrush reported . therefore , the prompt removal of such ingested foreign objects is recommended before complications develop . this case report describes a case of an 18-year - old woman who accidentally swallowed her toothbrush . the toothbrush was successfully removed via flexible endoscopy using a polypectomy snare . a swallowed toothbrush is a special clinical challenge . early endoscopic retrieval of the toothbrush is critical for reducing morbidity and mortality . in cases when endoscopic removal fails , a laparoscopic surgical approach may be an alternative .

Introduction Case Report Discussion Conclusion Disclosure Statement

stress cardiomyopathy ( scm ) is an acute cardiac syndrome resembling st - segment elevation myocardial infarction characterized by decreased wall motion in the apical and midportion of the left ventricle ( lv ) . these abnormalities extend beyond a single coronary distribution without significant obstructive coronary artery disease.1 case reports in the literature suggest that strong psychological or physical stress is associated with the occurrence of scm.2 we believe that the present case is the first report of scm following diffuse alveolar hemorrhage ( dah ) owing to aspiration of polyethylene glycol ( peg ) . a 37-year - old woman was transferred from a primary clinic to our emergency department because of chest pain and shortness of breath . she had undergone gastroscopy and colonoscopy in the left lateral decubitus position at the clinic that day to evaluate her complaint of constipation . during the examination , she aspirated some gastric contents with residual peg . although she received immediate first aid , her hypoxia did not resolve fully and her chest pain gradually worsened . at the time of her arrival at the emergency department , she complained of chest pain , shortness of breath , and blood - tinged sputum . her initial vital signs were as follows : blood pressure , 80/50 mm hg ; heart rate , 114 beats / minute ; respiration rate , 22 breaths / minute ; body temperature , 37.2 ; and saturation , 95% in fio2 0.32 by pulse oxymetry . laboratory tests revealed a white blood cell count of 13,280/l with 94.3% neutrophils and 0.3% eosinophils , hemoglobin of 13.0 g / dl , platelet count of 182,000/l , activated partial thromboplastin time of 30.9 s , prothrombin time of 14.2 s , c - reactive protein of 2.10 mg / dl , significantly elevated troponin - i of 3.07 ng / ml , and elevated creatine kinase - mb of 15.7 ng / ml . the total creatine kinase level was not elevated at 194 u / l , but the creatine kinase - mb / creatine kinase ratio was markedly elevated at 8% . the electrocardiogram revealed sinus tachycardia without significant st segment abnormality or t - wave inversion . chest computed tomography showed ground - glass opacities and multiple low attenuation areas in the bilateral dependent portions , especially in the left upper lobe ( fig . the transthoracic echocardiogram revealed global hypokinesia , especially in the basal and midportions of the lv ( fig . the lv ejection fraction was estimated to be approximately 30% by the a / l method , and there was no lv outflow tract obstruction , pulmonary hypertension , or mitral regurgitation . initial pharmacologic management included aspirin , clopidogrel , and antibiotics based on the suspicion of acute coronary syndrome with aspiration pneumonia . although left ventriculography and ergonovine provocation tests were not conducted , the typical echocardiography findings without significant coronary artery lesions were sufficient to confirm the diagnosis of scm . after the patient was transferred to the cardiac intensive care unit , her symptoms of hypoxia and dyspnea gradually worsened and fresh hemoptysis accompanied this worsening . while she was on 80% oxygen , her arterial blood gas measurements were as follows : ph , 7.306 ; pco2 , 26.7 mm hg ; po2 , 58.4 mm hg ; hco3 , 13.0 mmol / l ; and sao2 , 88.2% . ultimately , the patient was intubated , ventilator care was begun , and fiberoptic bronchoscopy was performed . no bronchial mucosal lesion was identified , but five sequential aliquots of bonchoalveolar lavage ( bal ) fluid with a progressively bloody appearance were observed in the airway of the left upper area . analysis of the bal fluid revealed a red blood cell count of 28,000/mm and a white blood cell count of 100/mm with 40% neutrophils and 44% macrophages , consistent with alveolar hemorrhage . the patient received ventilator care for 3 days and was treated with methylprednisolone ( starting dose of 1 mg / kg and tapering for 7 days to stop ) and antibiotics . seven days into her hospitalization , her symptoms of dyspnea , hemoptysis , and chest discomfort had fully resolved . follow - up measurements of creatine kinase and creatine kinase - mb were normalized , and troponin - i was decreased to 0.13 ng / ml . the haziness on her chest radiograph had also disappeared ( fig . six months after her discharge from the hospital , a follow - up transthoracic echocardiogram revealed a 60% improvement in lv ejection fraction by the a / l method without regional wall motion abnormality ( fig . the pathophysiologic mechanism of scm has not been clearly established , but a catecholamine - mediated mechanism has been recognized as the most reliable precipitating factor.3 dah - induced hypoxia and hypercapnia have a multiplicative effect on the output of carotid chemoreceptors , and sympathetic activation is substantially increased.4 the exaggerated sympathetic responsiveness in dah triggers myocardial stunning , multi - vessel epicardial spasm , microvascular spasm , and direct catecholamine - mediated myocyte injury , each of which has been reported to cause scm.1 dah - induced scm could be explained in the same manner , but it has not previously been reported in the literature . dah refers to pulmonary hemorrhage originating from the alveolar capillaries , arterioles , and venules and is defined by the clinical symptoms of hemoptysis and anemia , diffuse radiographic pulmonary infiltration , and hypoxemic respiratory failure.5 dah is characterized histologically by the presence of intraalveolar red blood cells , fibrin , and hemosiderin - laden macrophages , which may take up 48 to 72 hours to accumulate.5,6 although a surgical biopsy specimen is considered the gold standard for diagnosis , it is often impractical . bal is an accepted alternative for confirming the diagnosis when 20% hemosiderin - laden macrophages are present.7 according to one recent study , however , the percentage of hemosiderin - laden macrophages in bal fluid can be increased in patients with dah , but this may not be diagnostic.6 in our case , we performed bal , but prussian blue stained hemosiderin - laden macrophages were not present in the bal fluid . bronchoscopic examination was performed within 24 hours , which may have been too early in the course of the disease to detect the accumulation of hemosiderin - laden macrophages . the area of alveolar hemorrhage in this case was not extensive and the clinical course of our patient was relatively mild compared with other reported cases of dah . injury to the alveolar microcirculation is mainly associated with local lung injury or systemic disorders such as vasculitis or connective tissue disease.5 our patient did not demonstrate any clinical evidence of a systemic disorder . therefore , it seems more likely that local lung injury due to aspirated fluid followed by progression of acute respiratory distress syndrome was the cause of the dah . although a wide range of dah causes have been reported on a case - by - case basis , peg exposure is a very rare etiology . peg is a popular bowel cleansing agent because of the safety and efficacy of the solution ; however , complications such as nausea , vomiting , and abdominal bloating are frequent.8 some studies have reported esophageal rupture with vomiting or respiratory failure after peg aspiration.9 - 11 the pathophysiological mechanism of peg - induced lung injury remains unclear . according to some studies , the hyperosmotic properties of peg solution can induce intra - luminary fluid shifting within the alveolar and interstitial spaces , resulting in pulmonary edema.12,13 in this case , massive lavage could retrieve a sufficient amount of aspirated fluid , especially peg , preventing further deterioration of chemical pneumonitis and acute respiratory distress syndrome . however , peg aspiration - induced dah in young adults is very rare and only a few patients have recovered fully after retrieval of aspirated fluid with bal .

stress cardiomyopathy ( scm ) is usually precipitated by a physiologically or psychologically stressful event . although it occurs only rarely , hypoxia- and hypercapnia - induced sympathetic activation may also cause scm . we present the case of a 37-year - old woman affected with scm after a routine colonoscopy . during the procedure , she aspirated residual polyethylene glycol from her stomach . hypotension , resting dyspnea , and hemoptysis were subsequently observed . laboratory findings revealed elevated cardiac enzymes , and a transthoracic echocardiogram revealed left ventricular ( lv ) global hypokinesia . she was ultimately diagnosed with diffuse alveolar hemorrhage - associated scm . after successful treatment with a ventilator and corticosteroids , her lv systolic function and dimensions normalized and she was discharged without complications .

INTRODUCTION CASE REPORT DISCUSSION

solitary fibrous tumor ( sft ) is a rare neoplasm that was initially described in the visceral pleura and subsequently reported in extrapleural sites including the pericardium , peritoneum , mediastinum , lung , upper respiratory tract , thyroid , liver , testicle , nasal cavity , parotid , orbit , and meninges . sft development in the spine is an exceedingly rare event about which little is known . we present a unique case of an intradural extramedullary malignant sft having an extradural component with pulmonary metastases . to the best of our knowledge , this is the second case of malignant sft spine with distant metastases reported in literature . a 35-year - old female , with no comorbidities , presented with a 1 year history of progressive backache that was followed by lower extremity weakness for 4 months . neurological examination revealed paraparesis , hypertonia , increased deep tendon reflexes in the lower limbs , positive babinski sign , sustained bilateral ankle clonus and a sensory level of d12 with impaired temperature and proprioceptive sensation . contrast - enhanced magnetic resonance imaging ( mri ) of thoracolumbar spine revealed an intradural extramedullary mass ( 1.2 cm 1.25 cm ; which was predominantly hyperintense on t2 and hypointense on t1 images ) compressing and displacing cord at d10d11 level toward right side with extradural component ( 9.2 cm 5.2 cm ) extending into left neural foramen with widening at d10d11 level . contrast - enhanced computed tomography showed large well defined heterogeneously enhancing mass ( 9 cm 8.2 cm 6 cm ) in left paravertebral region extending from d10 to d12 vertebrae . the mass was seen passing through d10d12 neural foramina and causing its widening and extending into spinal canal causing rightward displacement of spinal cord [ figure 1c and d ] . she underwent d10d11 laminectomy with excision of intraspinal component of tumor by neurosurgeon from outside . histopathology examination ( hpe ) the tumor cells were positive for cd34 ( diffuse ) , cd99 , ema , bcl2 and negative for cd31 , s100 , cd117 , dog1 on immunohistochemistry ( ihc ) [ figure 2c and d ] . she was then referred to general surgery department of our institute where she was investigated for distant metastases which was negative , and then she underwent thoracotomy and excision of left paravertebral tumor . because of the incomplete surgical resection of the tumor and the high risk of locoregional and distant recurrence , adjuvant radiotherapy and chemotherapy was considered , but she went lost to follow - up for 5 months . when she turned up , an mri whole spine and positron emission tomography ( pet ) scan was performed for assessment of extent of disease . mri revealed a residual mass at the level of d11d12 neural foramina on left side ( 1.5 cm 1.2 cm 1.5 cm ) [ figure 3a and b ] with mild fluorodeoxyglucose uptake and multiple subcentimetric nodular lesions in bilateral lungs suggestive of metastases on pet scan . she was administered palliative radiotherapy of 20 gy in five fractions over 1 week to the primary residual site and planned for palliative chemotherapy with ifosfamide ( 1.4 g / m ; day 14 ) and epirubicin ( 60 mg / m ; day 12 ) . ( a ) sagittal view and ( b ) axial view of t2 weighted magnetic resonance imaging spine showing intradural extramedullary mass with extradural component . ( c ) axial view and ( d ) coronal view of contrast enhanced computed tomography scan showing left paravertebral mass ( a ) tumor is cellular and in diffuse growth pattern ( h and e , 100 ) . ( b ) tumor cells are round to oval with finely granular chromatin , indistinct cytoplasmic boundaries , and showing few mitosis ( h and e , 400 ) . ( d ) ki-67 proliferation index is ~5 - 7% ( a ) axial view and ( b ) sagittal view of t2 weighted magnetic resonance imaging of spine showing residual tumor sfts most often affect the pleura , but examples are increasingly being reported at other sites . spinal sfts are usually intramedullary ( 58% ) or intradural and extramedullary ( 24% ) . the majority of symptoms with which patients were presenting at time of admission were pain , hypoesthesia , paresis , urinary dysfunction , or a combination of these . similarly , as in our case , the symptoms consisted of a backache , paraparesis , and loss of sensation in lower extremities . 56% of sft patients are male , and most commonly it is seen on patients between 40 and 60 years old . two - thirds of sfts on mri were isointense on t1-weighted imaging with the remainder being either heterogeneous or hypointense . nearly , two - thirds of cases were hypointense on t2-weighted imaging with hyperintense being the next most common ( 17% ) . over three - quarters of cases demonstrated diffuse or homogeneous contrast enhancement with gadolinium administration ( 78% ) ; however , a significant portion demonstrated only partial or heterogeneous enhancement ( 21% ) . . typically , hpe features of sfts are spindle cells embedded in a fibrous matrix in a patternless architecture and alternating hypercellular and hypocellular areas with perivascular hyalinization or myxoid degeneration . overlapping histologic features , differentiation of sft from other soft tissue tumors may be difficult . typically , sfts are positive for cd34 , bcl-2 , and cd99 and negative for sma , desmin , pan - cytokeratin , and s-100 protein on ihc . findings such as nuclear atypia , increased cellularity , necrosis , and > 4 mitoses/10 hpfs , are suggestive of the malignant potential of sfts . in the current case , based on previous case reports , malignant sft showed rapid local recurrence and distant metastasis . at present , due to the rarity of the disease , standard therapies for malignant sft have not been well established . recurrence occurred commonly in cases involving incomplete excision , possibly caused by the level of difficulty in achieving complete resection . in these cases , . showed that conventional chemotherapy with anthracycline and ifosfamide is effective in controlling or stabilizing locally advanced and metastatic sfts . our patient being in metastatic setting , received palliative radiotherapy at local site and was considered for palliative chemotherapy with ifosfamide and epirubicin . the authors report here the second case of malignant sft of spine with distant metastases . although malignant sft is extremely rare , it should be considered in the differential diagnosis of spindle cell tumors in the spine .

solitary fibrous tumor ( sft ) usually originates from the pleura because of abnormal proliferation of fibroblast cells . it is extremely rare for the tumor to originate from the spine . here , we report the second case of malignant sft of thoracic spine with distant metastases in a 35-years - old female .

Introduction Case Report Discussion Conclusion Financial support and sponsorship Conflicts of interest

rufinamide ( 1-[(2,6-difluorophenyl)methyl]-1hydro-1,2,3-triazole-4 carboxamide ) is a novel antiepileptic drug ( aed ) that is structurally distinct from other aeds available on the market ( levy et al 2002a ) . an application was filed by eisai ltd in 2005 for european approval of this agent as an adjunctive treatment for seizures associated with lennox - gastaut syndrome ( lgs ) in children and adults ( eisai co ltd 2005 ) . lennox - gastaut syndrome is a catastrophic epileptic encephalopathy of childhood onset ( typically at age 35 years ) , for which effective treatment options are limited . this syndrome is associated with multiple types of generalized seizures , especially drop attacks and tonic seizures , delayed psychomotor development , behavioral and personality disorders , and a characteristic electroencephalogram pattern containing both generalized slow spike wave activity and paroxysms of generalized fast rhythmic activity during sleep which reflect excessive neocortical excitability ( markand 2003 ; nabbout and dulac 2003 ; guerrini 2006 ) . the long - term prognosis is very poor , with persistence of seizures in more than 75% of patients and severe mental retardation in more than 50% of patients ( markand 2003 ; dulac and engel 2003 ; schmidt and bourgeois 2000 ) . consequently , lgs is viewed as one of the most difficult epilepsies to treat . currently available treatment options for the epileptic encephalopathies in europe center around the use of valproate , with adjunctive benzodiazepines , topiramate , lamotrigine , or felbamate ( guerrini 2006 ) . however , a cochrane review has concluded that no single aed could be considered highly efficacious for lgs ( hancock and cross 2003 ) . indeed , the efficacy of valproate as a first - line treatment has been described as unimpressive , and its use in young children should be accompanied by great caution due to the risk of life - threatening hepatic toxicity ( schmidt and bourgeois 2000 ) . other first - generation aeds ( carbamazepine , phenytoin , and phenobarbital ) are not recommended for the treatment of lgs , due to the potential for aggravation of absence and myoclonic seizures by carbamazepine and phenytoin , and exacerbation of the behavioral problems seen in patients with lgs by barbiturates . among the benzodiazepines , clobazam may be viewed as the best tolerated , although there is no head - to - head comparison to confirm this . however , for this class of drugs , development of tolerance is well documented in the literature ( levy et al 2002b ) . the only aeds which have been evaluated in a randomized , controlled trial in lgs are lamotrigine , topiramate , and felbamate , the outcomes of which are discussed later in this paper . although felbamate is licenced for the treatment of lgs in the usa and some other countries ( eg , germany ) , its use has been limited following reports of serious toxicity ( borowicz et al 2004 ) . this has led to the recommendation that , in lgs , it should be used only in patients aged over 4 years who can not be treated satisfactorily with other aeds ( schmidt and bourgeois 2000 ) . thus , there is a clear need for new options in the management of this intractable condition . rufinamide was granted orphan drug status by the european medicines agency ( emea ) and the us food and drug administration ( fda ) in 2005 . this review will evaluate the relevant pharmacologic and clinical data for rufinamide , and explore its potential role in the future management of this severe form of epilepsy in the context of other products currently available for lgs . an extensive preclinical evaluation has suggested that the principal mechanism involved in the antiepileptic activity of rufinamide is its ability to modulate the activity of sodium channels , limiting high - frequency firing of neuronal action potentials over a broad range of concentrations , as demonstrated in vitro ( mclean et al 2005 ) . in radioligand binding studies , rufinamide does not interact with other neurotransmitter systems , including monoamine , adenosine , acetylcholine , histamine , glycine , -amino-3-hydroxy-5-methyl-4-isoxazole propionate ( ampa)/kainite , n - methyl - d - aspartate ( nmda ) , and -amino butyric acid ( gaba ) systems ( bialer et al 1996 ) . rufinamide has demonstrated broad spectrum anticonvulsant activity in animal models of epilepsy , elevating both electrically and chemically induced seizure thresholds and preventing seizure spread following oral and intraperitoneal administration in mice and rats ( white et al 2005 ) . furthermore , the protective index for rufinamide ( ratio of the concentration required for 50% anticonvulsant efficacy to that for neurotoxicity ) was superior to that of each of the comparator aeds examined ( phenytoin , phenobarbital , ethosuximide , and valproate ) . the pharmacokinetic properties of rufinamide have been established both in healthy volunteers and in patients with epilepsy . in a study of 3 healthy volunteers , an oral dose of 600 mg rufinamide demonstrated high absorption , and monoexponential elimination with a mean half - life ( t ) of 9 hours ( waldmeier et al 1996 ) . excretion was largely renal ( 85% ) and complete ( 98% ) within 7 days . following administration of 400 mg rufinamide as two 200-mg tablets or 400-mg oral suspension after a standard meal , mean maximum plasma concentrations ( cmax ) of 3.03 and 3.32 g / ml were reached after 6.6 and 7.2 hours , respectively , leading to elimination with a terminal t of 8.8 and 9.1 hours , respectively ( cheung et al 1995 ) . in a further study , the absorption of a single dose of 400 mg rufinamide was accelerated in fed subjects compared with fasting conditions , resulting in a 43% increase in exposure ( 96-hour exposure ) , without affecting the t ( cardot et al 1998 ) . however , no effect of food was observed with repeat dosing ( eisai , data on file ) , and no difference in rufinamide pharmacokinetics has been found between older ( aged 6677 years ) and younger ( 1840 years ) healthy subjects in either single- or multiple - dosing conditions ( chang et al 1998 ) . rufinamide pharmacokinetics in a study of 16 children aged between 2 and 17 years treated with 10 or 30 mg / kg daily rufinamide for 2 weeks were also comparable to those reported elsewhere for adult patients ( sachdeo et al 1998 ) . overall , evaluation of these data suggests that no age - related dose adjustments are likely to be required for either pediatric or elderly populations ( bialer et al 2001 ) . the pharmacokinetic parameters of single ascending doses of rufinamide ( 0 , 400 , 800 , 1200 mg ) have also been compared between 3 healthy subjects and 16 patients with epilepsy receiving enzyme - inducing aeds ( brunner et al 1994 ) . absorption rates were comparable , and both groups showed dose - related increases in cmax and exposure . rufinamide elimination was accelerated in aed - treated patients compared with healthy subjects , but t was independent of the dose administered in both groups . the pharmacokinetics of rufinamide have also been evaluated in 129 pediatric and adult patients with lgs ( critchley et al 2005 ) . rufinamide clearance was not affected by concomitant lamotrigine or topiramate , and was also unaffected by liver or kidney function . however , rufinamide concentrations were increased by concomitant valproate : 40% in children and 11% in adults . therefore , the rufinamide dosage may require adjustment during the addition or withdrawal of concomitant valproate therapy . rufinimade is extensively metabolized ( 2% excreted unchanged in urine and 2% in feces ) , predominantly via hydrolysis of the carboxylamide group to yield an inactive metabolite ( cgp-27292 ) , and is largely excreted in urine ( 84.7% of dose ) ( bialer et al 1999 ) . thus , rufinamide has a low propensity for drug drug interactions , based on its low protein binding ( approximately 34% ) and absence of metabolism via hepatic cytochrome p450 or inhibition of the major enzyme subclasses ( bialer et al 1996 ; kapeghian et al 1996 ) . this is supported by the absence of clinically relevant alterations of concomitant aed levels observed in a population pharmacokinetic drug this analysis was conducted using data from 5 double - blind studies involving pediatric and adult patients with inadequately controlled seizures despite previous treatment with up to 3 aeds . in total , 903 patients were receiving carbamazepine , 588 valproate , 200 lamotrigine , 299 phenytoin , 149 phenobarbital , and 69 topiramate . rufinamide co - administration did not affect the clearance of topiramate or valproate , but increased the clearance of carbamazepine and lamotrigine and decreased clearance of phenobarbital and phenytoin . however , all produced effects were small , were considered unlikely to be of clinical significance , and were independent of age . at a rufinamide plasma concentration of 15 g / ml , representing steady - state concentrations following doses of 45 mg / kg daily in children or 3200 mg / day in adults , changes in clearance conversely , in a similar study involving 471 patients , valproate co - administration decreased rufinamide clearance by approximately 22% , while any combination of phenytoin , phenobarbital , or primidone increased rufinamide clearance by approximately 25% , compared with patients not receiving these aeds ( bialer et al 2001 ) . rufinamide also resulted in a small increase in clearance of the oral contraceptive ortho - novum ( ortho - mcneil pharmaceutical , inc . raritan , nj , usa ) 1/35 in healthy female volunteers , though the clinical relevance of this effect is not known ( svendsen et al 1998 ) . the efficacy of rufinamide as an adjunctive therapy for treatment - resistant lgs has been evaluated in a multicenter , randomized , 12-week , double - blind , placebo - controlled , parallel - group study in 138 patients aged 437 years ( mean age 14.1 years ) , followed by an open - label extension phase ( glauser et al 2005a , 2005b ) . patients diagnosed with lgs , experiencing 90 seizures in the month prior to study entry , and receiving stable treatment with 13 concomitant aeds , were evaluated . after a 28-day baseline , patients randomized to twice - daily oral treatment with rufinamide ( n=74 ) or placebo ( n=64 ) entered a 14-day titration period ( rufinamide target dose 45 mg / kg daily ) , followed by 70 days of double - blind maintenance treatment . the reduction from baseline in the frequency of tonic - atonic seizures ( drop attacks ) per 28 days was significantly greater with rufinamide than placebo ( median change 42.5% vs + 1.4% , respectively , p<0.0001 ) . similarly , the reduction from baseline in total seizure frequency per 28 days was significantly greater with rufinamide than placebo treatment ( median change 32.7% vs 11.7% , respectively , p=0.0015 ) . these improvements were associated with significantly greater proportions of responders ( patients achieving 50% reduction in seizures per 28 days ) with rufinamide than placebo treatment , both for tonic - atonic seizures ( 42.5% vs 16.7% , p=0.0020 ) ( glauser et al 2005a ) and total seizure frequency ( 31.1% vs 10.9% , p=0.0045 ) ( kluger et al 2006 ) . following completion of the double - blind study , 123 patients continued to receive open - label treatment in the extension phase , at a median dose of 1800 mg / day ( range 1034865 mg / day ) for a median duration of 432 days ( range 101149 days ) ( glauser et al 2005b ) . the reduction in median total seizure frequency observed at 12 weeks was maintained , with some further improvement noted with continued treatment for up to 3 years ( see figure 1 ) . similarly , the responder rate for total seizures was maintained , with 36.9% of patients achieving a reduction in total seizures of at least 50% overall during the extension study . furthermore , 21.3% of patients achieved a 75% reduction in total seizures overall during the extension study . during the double - blind study of rufinamide in patients with lgs , the most common adverse events ( reported by 10% patients in either treatment group ) were : somnolence ( 24.3% rufinamide , 12.5% placebo ) , vomiting ( 21.6% rufinamide , 6.3% placebo ) , pyrexia ( 13.5% rufinamide , 17.2% placebo ) , and diarrhea ( 5.4% rufinamide , 10.9% placebo ) ( glauser et al 2005a ) . notably , rufinamide treatment was associated with a lower incidence of cognitive / psychiatric adverse events ( such as psychomotor hyperactivity and lethargy ) than placebo treatment ( 17.6% vs 23.4% , respectively ) . there was no clinically significant change in clinical laboratory tests , physical examinations or vital signs . during the subsequent open - label extension study , the most commonly reported all - causality adverse events were vomiting ( 30.6% ) , pyrexia ( 25.8% ) , upper respiratory tract infection ( 21.8% ) , and somnolence ( 21.0% ) ( glauser et al 2005b ) . there were two deaths , which were not considered to be related to rufinamide treatment . thus , long - term treatment of patients with lgs does not appear to be associated with any increase in central nervous system ( cns ) adverse events , with somnolence noted with somewhat lower frequency than during the double - blind phase . these tolerability and safety data in patients with lgs are further supported by a pooled analysis of rufinamide tolerability and safety in a large population of epilepsy patients , in which the data were evaluated separately for patients receiving short - term or long - term treatment ( krauss et al 2005 ) . the short - term safety population comprised 1240 patients treated with rufinamide ( mean age 31.7 years ; mean rufinamide dose 1373 mg / day ; mean duration of treatment 2.8 months ) and 635 placebo recipients ( mean age 28.6 years , mean duration of treatment 3.0 months ) . somnolence was reported somewhat less frequently in this population than in the lgs study , with an incidence of 11.8% for rufinamide and 9.1% among placebo recipients . the timing of events in this pooled analysis indicated a pattern of early onset and rapid resolution , and a tolerability profile not substantially different from placebo ; the incidence of the most frequently reported adverse events with rufinamide vs placebo were headache ( 22.9% vs 18.9% ) , dizziness ( 15.5% vs 9.4% ) , fatigue ( 13.6% vs 9.0% ) , somnolence ( 11.8% vs 9.1% ) , and nausea ( 11.4% vs 7.6% ) . the pooled tolerability and safety analysis included 1978 patients assessed for long - term tolerability and safety ( mean age 31.3 years , mean rufinamide dose 1700 mg / day , maximum dose 7200 mg / day ) , 47% of whom were treated with rufinamide for at least 12 months ; the most frequently reported adverse events in this population were headache ( 29.5% ) , dizziness ( 22.5% ) , and fatigue ( 17.7% ) ( krauss et al 2005 ) . few randomized , controlled clinical trials have been conducted in patients with lgs , none of which is a head - to - head comparison . consequently , the available treatments for lgs can not be compared directly , and only limited conclusions may be drawn from indirect comparisons . the most robust approaches for indirect comparisons include systematic reviews and meta - analyses , such as those conducted by the cochrane group . a systematic ( cochrane ) review of randomized , controlled trials in lgs ( hancock and cross 2003 ) retrieved only 5 evaluable studies : one each conducted with topiramate , lamotrigine , felbamate , cinromide , and a thyrotropin releasing hormone ( trh ) derivative , respectively ( anonymous 1989 , 1993 ; inanaga et al 1989 ; motte et al 1997 ; sachdeo et al 1999 ) . this review concluded that no single agent was found to be highly efficacious in lgs , and that of those evaluated only lamotrigine , topiramate and felbamate were considered potentially efficacious as add - on therapy ( hancock and cross 2003 ) . data derived from this cochrane analysis for each of the clinical trials are summarized in table 1 , together with data from the more recently reported rufinamide study ( glauser et al 2005a ) . although this provides an indirect comparison , it is of interest to note that rufinamide produced a particularly marked effect in the reduction in drop attacks ( tonic - atonic seizures ) , since this is also the parameter viewed as the primary outcome variable for lgs according to guidelines from the american academy of neurology ( french et al 2004 ) . the median reduction in tonic - atonic seizures was 42.5% with rufinamide , which compares well with rates observed in the separate studies with topiramate ( 14.8% reduction ) or lamotrigine ( 34% reduction ) ; the effect of felbamate on drop attacks was not reported . when corrected for the magnitude of placebo response , the median effect size for rufinamide on drop attacks was 41.1% , compared with 19.9% for topiramate and 14.8% for lamotrigine . similarly , the proportion of patients who could be considered responders to therapy ( achieving at least a 50% decrease in drop attacks ) was 42.5% with rufinamide , 45.6% with topiramate , and 37.3% with lamotrigine , or 25.8% , 25.6% , and 14.8% , respectively , when corrected for placebo response . as with drop attacks , the effects of these agents on total seizure frequency produced some interesting findings . the rufinamide , topiramate , and lamotrigine studies demonstrated 32.7% , 20.6% , and 32.0% median reductions in total seizures , and treatment effect sizes ( adjusted for placebo ) of 20.0% , 12.6% , and 17.6% , respectively . in the felbamate study , outcomes were reported as mean rather than median values , resulting in a 19% mean reduction in total seizures , and a 23% mean effect size . the number of patients achieving complete cessation of seizures has only been reported with felbamate , and although these results are favorable , can not be compared with the other aeds in the studies available to date . of course , the overall benefit of treatment comprises a balance between reduction in ( or cessation of ) seizures and tolerability / safety . the reported tolerability of treatment was broadly comparable among the studies described in table 1 , with relatively small differences in rates of discontinuations due to adverse events . few randomized , controlled clinical trials have been conducted in patients with lgs , none of which is a head - to - head comparison . consequently , the available treatments for lgs can not be compared directly , and only limited conclusions may be drawn from indirect comparisons . the most robust approaches for indirect comparisons include systematic reviews and meta - analyses , such as those conducted by the cochrane group . a systematic ( cochrane ) review of randomized , controlled trials in lgs ( hancock and cross 2003 ) retrieved only 5 evaluable studies : one each conducted with topiramate , lamotrigine , felbamate , cinromide , and a thyrotropin releasing hormone ( trh ) derivative , respectively ( anonymous 1989 , 1993 ; inanaga et al 1989 ; motte et al 1997 ; sachdeo et al 1999 ) . this review concluded that no single agent was found to be highly efficacious in lgs , and that of those evaluated only lamotrigine , topiramate and felbamate were considered potentially efficacious as add - on therapy ( hancock and cross 2003 ) . data derived from this cochrane analysis for each of the clinical trials are summarized in table 1 , together with data from the more recently reported rufinamide study ( glauser et al 2005a ) . although this provides an indirect comparison , it is of interest to note that rufinamide produced a particularly marked effect in the reduction in drop attacks ( tonic - atonic seizures ) , since this is also the parameter viewed as the primary outcome variable for lgs according to guidelines from the american academy of neurology ( french et al 2004 ) . the median reduction in tonic - atonic seizures was 42.5% with rufinamide , which compares well with rates observed in the separate studies with topiramate ( 14.8% reduction ) or lamotrigine ( 34% reduction ) ; the effect of felbamate on drop attacks was not reported . when corrected for the magnitude of placebo response , the median effect size for rufinamide on drop attacks was 41.1% , compared with 19.9% for topiramate and 14.8% for lamotrigine . similarly , the proportion of patients who could be considered responders to therapy ( achieving at least a 50% decrease in drop attacks ) was 42.5% with rufinamide , 45.6% with topiramate , and 37.3% with lamotrigine , or 25.8% , 25.6% , and 14.8% , respectively , when corrected for placebo response . as with drop attacks , the effects of these agents on total seizure frequency produced some interesting findings . the rufinamide , topiramate , and lamotrigine studies demonstrated 32.7% , 20.6% , and 32.0% median reductions in total seizures , and treatment effect sizes ( adjusted for placebo ) of 20.0% , 12.6% , and 17.6% , respectively . in the felbamate study , outcomes were reported as mean rather than median values , resulting in a 19% mean reduction in total seizures , and a 23% mean effect size . the number of patients achieving complete cessation of seizures has only been reported with felbamate , and although these results are favorable , can not be compared with the other aeds in the studies available to date . of course , the overall benefit of treatment comprises a balance between reduction in ( or cessation of ) seizures and tolerability / safety . the reported tolerability of treatment was broadly comparable among the studies described in table 1 , with relatively small differences in rates of discontinuations due to adverse events . each of these currently available agents has been associated with warnings relating to severe adverse events . the use of felbamate has been restricted in many countries following reports of idiosyncratic aplastic anemia and hepatic failure , which led to its initial withdrawal in the usa , although limited use was granted subsequently ( fda 1994 ) . similarly , the efficacy of lamotrigine must be balanced against the risk of serious skin reactions in susceptible individuals , for which boxed warnings or safety alerts have been issued ( committee on safety of medicines 1996 ; fda 1997 ) , while the use of topiramate is associated with warnings relating to oligohydrosis and hyperthermia in children , and also metabolic acidosis and glaucoma ( fda 2003 ; janssen - cilag 2006 ) . in addition , factors such as cognitive slowing and weight loss may be issues with the use of topiramate ( levy et al 2002c ) . the timing of these warnings , some time after the initial marketing of these aeds , illustrates the difficulties in identifying such events during clinical trials , and that further evaluation is required to confirm the long - term safety of rufinamide in comparison with the marketed drugs . indeed , increased qt interval dispersion has been demonstrated in children with epilepsy , potentially predisposing them to serious cardiac events ( akalin et al 2003 ) . rufinamide has demonstrated excellent cardiovascular tolerability , with no increase in qt intervals in a cardiac safety study conducted in 15 healthy volunteers treated with placebo or ascending doses of rufinamide ( from 800 to 7200 mg / day ) over 18 days ( marchand et al 2005 ) . on the contrary , lgs is a catastrophic and debilitating age - specific epileptic disorder , for which a substantial unmet need exists for highly effective treatment options . the data reviewed here indicate that adjunctive therapy with rufinamide , a new aed that is structurally unrelated to other currently available aeds , significantly reduces seizure frequency in patients with lgs , accompanied by good tolerability , both of which are maintained during long - term use . in particular , the reduction in drop attacks adds substantially to patient quality of life . the efficacy of rufinamide is most likely derived from its ability to block sodium channels , thereby limiting high - frequency action potential firing , a mechanism shared by some other aeds including phenytoin , carbamazepine , lamotrigine , and oxcarbazepine ( mclean et al 2005 ) . however , it is commonplace for patients with lgs to remain symptomatic despite optimal therapy , and it is notable that , in the studies reviewed , adjunctive rufinamide treatment was able to exert significant improvement in a treatment - resistant population receiving 13 other aeds at baseline . in an indirect comparison with randomized controlled trials examining other aeds , the results with rufinamide appear impressive : a median reduction in drop attacks of 42.5% with rufinamide , compared with 14.8% with topiramate and 34% for lamotrigine , representing treatment effect sizes , after adjustment for placebo response , of 41.1% , 19.9% , and 14.8% , respectively . this was associated with a median reduction in total seizures of 32.7% with rufinamide , compared with 20.6% in the topiramate study and 32% in the lamotrigine study . although the full safety data for the rufinamide study in lgs have yet to be reported , data from a pooled safety database suggest a good tolerability profile for both short- and long - term use of rufinamide , with a pattern of early onset and early resolution of events . cardiac safety profile , based on a dose - ranging study in healthy volunteers which included qt analysis . however , the full spectrum of efficacy as well as the safety profile of rufinamide will become clear with accumulation of long - term clinical experience and usage . overall , these findings suggest that rufinamide may be a valuable option for the adjunctive treatment of lgs .

rufinamide , a triazole derivative that is structurally distinct from currently marketed antiepileptic drugs ( aeds ) , is in development for the adjunctive treatment of lennox - gastaut syndrome ( lgs ) in children and adults . rufinamide is well absorbed after oral administration , demonstrates low protein binding , and is metabolized by enzymatic hydrolysis without involvement of cytochrome p450 enzymes , conferring a low drug interaction potential . in a randomized , double - blind trial involving 138 adult and pediatric patients with lgs , compared with placebo , rufinamide 45 mg / kg / day resulted in significantly superior reductions in drop attacks ( median change 42.5% vs + 1.4% with placebo ) and total seizures ( 32.1% vs 11.7% with placebo ) , accompanied by significantly higher responder rates . these results are comparable with findings reported for other aeds in randomized , controlled clinical trials in patients with lgs . rufinamide produced statistically significant seizure reduction which was maintained during long - term therapy and accompanied by good tolerability . the most frequently reported adverse events from a pooled safety database evaluating short- and long - term therapy were headache ( 22.9% and 29.5% ) , dizziness ( 15.5% and 22.5% ) and fatigue ( 13.6% and 17.7% ) . rufinamide therefore presents a favorable efficacy and tolerability profile and is a promising candidate for the adjunctive therapy of lgs .

Introduction Pharmacology and pharmacokinetics of rufinamide Rufinamide in Lennox-Gastaut syndrome Tolerability and safety of rufinamide Rufinamide in context Other randomized controlled trials in LGS Additional tolerability and safety considerations Conclusions

a 55-year - old man presented at our emergency department with sudden - onset sharp chest pain that had started two hours previously . he also complained of progressive swelling and bruising on his neck , and the development of dysphagia and dyspnea over the previous two hours . he had a history of admission with spontaneous hemothorax seven years previously , and at that time , despite several work - ups for hemothorax , its origin was not found . other than this incident , he had hypertension but no history of gastrointestinal or bleeding disorders . upon arrival at the emergency department extensive neck swelling was observed , as well as bruising on the neck and upper chest . during the examination , he suddenly vomited bright red blood with food material three times , and approximately a quarter - cup of blood was generated on each occasion . other laboratory tests , including cardiac markers and his coagulation profile , were within their normal ranges . 1b ) revealed extensive non - enhancing soft tissue lesions in the posterior mediastinum and neck , which were suspected to be hematomas . emergency gastroscopy showed many blood clots but no definite focus of gastrointestinal bleeding , such as an ulcer , mass , or perforation . instead , gastroscopy showed extrinsic esophageal compression . since the patient was hemodynamically stable , conservative management was adopted in the intensive care unit . followup ct on the third day post - admission showed a focal aneurysm in the right bronchointercostal trunk ( fig . subsequent selective bronchial arteriography revealed a 1-cm aneurysm in the right bronchial artery ( fig . since the aneurysm was considered to be the cause of the hematemesis and mediastinal hemorrhage , we decided to perform embolization to prevent recurrence . the right bronchial artery was subsequently embolized with four microcoils ( vortx-18 ; boston scientific co. , marlborough , ma , usa ) and 33% glue ( fig . 2c ) , and repeat ct showed that the mediastinal hematoma had nearly disappeared ( fig . the patient was discharged without complications . over the course of one year of follow - up spontaneous mediastinal hemorrhage can develop as a result of trauma , aortic dissection , the valsalva maneuver , or iatrogenic procedures . the rupture of a bronchial artery aneurysm ( baa ) is also known to result in mediastinal hemorrhage . when a baa ruptures into the mediastinum , most patients present with chest pain , hemothorax , or hemomediastinum . furthermore , if hematemesis is a prominent symptom of baa rupture , it can be confused with boerhaave s syndrome , variceal disease , or a perforated ulcer . to the best of our knowledge , only three cases of baa presenting with hematemesis have been reported in the literature [ 46 ] . in one case , a pinhole connection between the aneurysm and esophagus was found during an endoscopic examination , but in the present case , no evidence of communication was found between the aneurysm and the esophagus . we first suspected that the mediastinal hemorrhage and hematemesis were caused by the perforation of an esophageal ulcer , but repeated gastroscopy showed a normal esophagus . the only abnormal finding was the finding of a baa without evidence of extravasation three days after the onset of symptoms . after ruling out the possibility of mediastinal hematoma , the baa was the only remaining possible source of the mediastinal hemorrhage . since the baa was regarded as the source of the mediastinal hemorrhage and hematemesis , it was embolized to avoid recurrence . the treatment of a ruptured baa depends on the patient s hemodynamic status and the presence of cardiorespiratory compromise . if the patient is hemodynamically stable , endovascular embolization is considered to be the first - line management strategy , and surgery should only be considered when embolization is contraindicated , as in patients allergic to contrast medium or when a medullary artery is involved . in patients presenting at an emergency department with hematemesis and mediastinal hemorrhage ,

hematemesis is a rare manifestation of a ruptured bronchial artery aneurysm ( baa ) in the mediastinum . it is difficult to diagnose a ruptured baa presenting as hematemesis , because it can be confused with other diseases , such as boerhaave s syndrome , variceal disease , or a perforated ulcer . in this report , we describe a case of baa resulting in hematemesis and mediastinal hemorrhage .

CASE REPORT DISCUSSION

these include isolates from disease outbreaks in africa , europe , the caribbean , and , more recently , from the russian / trans - caucasian epizootic and attenuated variants . in brief , swine red bone marrow cell culture was used for asfv isolate amplification , and swine anti - asfv serum and erythrocytes were subsequently added to the culture . asfv isolates for which the hemadsorption phenomenon was inhibited by serum belonging to the same group within serogroups 18 were clustered into a homologous serogroup . only asfv hemadsorbing strains could be analyzed by this method , and some hemadsorbing asfv isolates could not be placed into existing serogroups because hai was not observed with available reference serum ( 8,9 ) . asfv isolates from the depository at vniivvim were also classified by using a standard asfv genotyping protocol previously published by bastos et al . , the variable part of the p72 ( b646l ) gene was amplified by conventional pcr , and the amplicons were directly sequenced by using a 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) according to manufacturer s recommendations . sequences determined at vniivvim were aligned with other publically available asfv sequences and analyzed by using minimum evolution ; a rooted tree was constructed with mega 5.0 software ( 14 ) and edited with figtree v1.4 ( http://tree.bio.ed.ac.uk/ ) ( figure 1 ) . phylogenetic tree of african swine fever virus ( asfv ) isolates maintained in a collection at the national research institute for veterinary virology and microbiology in okrov , russia ; the variable part of b646l gene relative to the 22 known p72 genotypes ( labeled i - xxii ) was used for analysis . the tree was reconstructed by using the minimum evolution method with 1,000 replicates . * the isolates were selected from the african swine fever virus ( asfv ) collection at the national research institute for veterinary virology and microbiology ( vniivvim ) in okrov , russia . asfv genotypes are assigned according to p72 ( b646l ) nucleotide sequencing and phylogenetic tree reconstruction . serogroups are defined on the basis of results of hemadsorption inhibition assay ( hai ) with reference serum ( serogroups 18 ) . democratic republic of congo ; , not applicable ; nk , not known ; nd , not defined . if a country name changed since the virus was isolated , the current name of the country is shown in parentheses . isolates bartlett , magadi , and davis could not be placed into an existing serogroup because no hai was observed with available reference serum , so exact serogroups for these isolates are not defined . isolate spenser does not belong to any of the 22 known genotypes and remains untyped . newly identified asfv genotypes and known serogroups were mapped together so their geographic distribution in africa and europe ( including the european part of the russian federation ) could be visualized . the results ( figure 2 ) show that genotypic and serogroup diversity are greatest in a relatively limited area , mainly in southeastern africa . in contrast , non - asfv endemic countries , where asf outbreaks were caused by asfv of a single genotype , exhibited low or no serogroup diversity . world distribution of african swine fever virus ( asfv ) isolates maintained in a collection at the national research institute for veterinary virology and microbiology in okrov , russia . results of p72 genotyping and hemadsorption inhibition assay of asfv isolates are summarized on the map . genotype ii of asfv isolates from lithuania , latvia , estonia , poland , and belarus was identified by cisa - inia ( animal health research center ; european union reference laboratory for african swine fever ) . asfv isolate o-77 , which was isolated in 1977 from odessa , ukraine ( at the time , part of the union of soviet socialist republics ) , was used in this study . on the basis of cisa - inia results , currently circulating isolates in ukraine belong to genotype ii . the oval with a 1 inside indicates switzerland ; the oval with a 2 inside indicates so tome and principe . country names are presented as 2-letter country codes as designated by the international organization for standardization country codes ( iso 3166 , http://www.iso.org/iso/country_codes.htm ) . single genotype clades of asfv were observed to contain viruses of multiple serogroups ( table ) . for example , asfv isolates belonging to serogroups 1 , 2 , and 4 were specifically clustered within genotype i , and did not group with other genotypes . we also found several serogroups of asf viruses within genotype x. the asfv isolates tsp80 ( serogroup 5 ) and ts-7 ( serogroup 6 ) were subsequently isolated from 1 field sample derived from a naturally infected pig in tanzania . of note , 1 serogroup 2 isolate ( spenser ) demonstrated a novel genotype within the p72 phylogenetic tree and relative to other serogroup 2 viruses ( figure 1 ) , indicating that the p72 genotype , in addition to lacking serotype resolution , has potential to be incongruous relative to serogroup . together , these data indicate that the antigenic heterogeneity of asfv strains is not fully captured by using the standard genotyping approach . the virus elements responsible for protective and cross - protective immune responses are not well known . given the structural and genetic complexity of asfv , it is likely that genes encoding different antigens will be more suited for virus typing . substantial genetic variability can exist between strains and predominate in specific genomic regions , and it is these regions that may provide improved targets for genotyping . our findings support that of a previous study that showed that geographic areas with asfv of high genotypic and serotypic diversity are located in countries where multiple mechanisms of asf transmission ( mixed sylvatic and domestic cycle ) are established ( 11 ) . hai serology provides a measure of asfv typing that , compared with p72 genotyping , better discriminates biologically pertinent phenotypes . viruses belonging to one p72 genotype may be serotypically heterogeneous : strains that are closely related genetically , even from a single isolate , may have different phenotypes and form homologous serogroups . our assessment of asfv genotyping relating to hai serotyping shows the serologic diversity within a p72 genotype . our results highlight the potential for using serogroup classification to understand issues of homologous cross - protection among asfv isolates and virus determinants that influence disease emergence . the key finding from our study is that p72 asfv phylogenetic analysis fails to accurately define asfv hai serogroups . thus , conventional asfv genotyping can not discriminate between viruses of different virulence or predict the efficacy of a specific asfv vaccine . this finding also suggests that for vaccination - based control of asf , it may be more important to determine serogroups rather than genotypes of asfv isolates .

african swine fever virus ( asfv ) causes highly lethal hemorrhagic disease among pigs , and asfv s extreme antigenic diversity hinders vaccine development . we show that p72 asfv phylogenetic analysis does not accurately define asfv hemadsorption inhibition assay serogroups . thus , conventional asfv genotyping can not discriminate between viruses of different virulence or predict efficacy of a specific asfv vaccine .

The Study Conclusions

entrapment of the ulnar nerve in the elbow region is the second most common compression neuropathy in the upper extremity . many procedures have been advocated for decompression of the ulnar nerve at the elbow , including anterior subcutaneous transposition , anterior intramuscular transposition , anterior submuscular transposition , medial epicondylectomy , simple decompression , neurolysis , and in situ decompression . the introduction of endoscopic release , the newest of all the surgical options for this problem has been described by several authors . the new approach to peripheral nerve surgery specifically in cubital tunnel syndrome is the introduction of the endoscopic procedure . since the advent of endoscopic methods to release the course of the ulnar nerve entrapment at the elbow site , there has been a flurry of interest and controversy about the efficacy and safety of this newest technique . endoscopic decompression of the ulnar nerve at the elbow was first described in 1995 by tsai et al . . there are different variations of endoscopic surgical technique , but the purpose and goals preserve the vascularity of the ulnar nerve at the elbow , release all possible compression sites , allow early mobilisation of the elbow , avoid extensive surgical exposure , and scar discomfort . it provides for limited soft tissue dissection , thereby allowing more rapid recovery with minimal scarring . potential ulnar nerve entrapment can occur at five sites around the elbow : the arcade of struthers , the medial intermuscular septum , the medial epicondyle , the cubital tunnel , and the deep flexor pronator aponeurosis . various surgical techniques for decompression of the ulnar nerve have been described in the literature , and a definitive gold standard does not exist . comparative studies have shown some short - term advantages to one or another technique , but overall results between the treatments have essentially been equivocal . a thorough preoperative diagnosis and workup will help guide us for the type of surgical technique . we report our experience with this newer technique in six patients , with special focus on the clinical and surgical outcome . the study was carried out in the department of orthopaedics and traumatology , division of hand and foot surgery , hong kong university medical centre at queen mary hospital from period of february 2008 to may 2010 . their data was retrieved from queen mary hospital and david trench rehabilitation centre . there were six cases with endoscopic cubital tunnel release . the inclusion criteria are failure of conservative treatment for at least 6 months , have progressive clinical findings of atrophy , elevated two - point sensory discrimination , weakness of the ulnar nerve distribution , and positive electrophysiological conformation studies . the exclusion criteria include the subluxation of the ulnar nerve if it was felt to subluxate over the medial epicondyle on passive elbow flexion , long - standing elbow contracture , mass or space - occupying lesions , history of significant trauma of the elbow , history of cervical neuropathy or the double crush syndrome , prior surgery , scarred , adherent nerve , history of fracture of the elbow malunion , and cubital valgus . all patients underwent conservative management with splintage , avoidance of provocative activities , and rehabilitation program . failure of conservative management and positive electrophysiological studies with clinical weakness of the ulnar nerve distribution and/or muscle atrophy is the criteria in surgical management . all six patients were offered endoscopic release as the alternative for standard open release procedure . a uniform endoscopic cubital tunnel release was done with a single senior orthopaedic hand surgeon with experience in endoscope release technique . six patients who had compressive cubital tunnel syndrome at the elbow were treated with endoscopic cutr at our institution after electrophysiological conformation of the diagnosis . the sensory conduction studies ( i.e. , amplitude of the sensory action potential and sensory conduction velocity of the ulnar nerve ) were considered prolonged in all cases ; the motor conduction studies ( i.e. , nerve site , onset , amplitude , segment , latency difference , distance , and conduction velocity ) were prolonged in all cases . the dellon 's scale score was used for rating the severity of the lesions and the postoperative outcome was assessed based on the modified bishop rating scale system . the final postoperative outcome was assessed 6 months after the surgery by subjective information based on modified bishop scoring classification system ( severity of residual system , improvement , work status , strength , and sensibility ) , this bishop score is defined as poor , 0 to 2 ; fair , 3 to 4 ; good , 5 to 7 ; excellent , 8 to 9 . surgery is performed in patient under ga in supine position with upper limb side table . the patient shoulder in abduction and external rotated and elbow slightly extended in position . a pneumatic tourniquet applied in the most proximal area of the arm , a clearly marked of anatomical portal and course of the ulnar nerve . a 1.5 cm3 cm transverse incision is made between the medial humeral epicondyle and the olecranon at the course of the ulnar nerve . the ulnar nerve has been identified , subcutaneous pouch developed and endoscope set inserted ( karl storz , tuttlingen , germany ) into subcutaneous tunnel and release under endoscopic guidance proximally . another distal pouch developed with the same entry site , and endoscope has been inserted . a distal release into two heads of flexor carpi ulnaris and the nerve branches of the fcu have been identified and protected . saline irrigation and drain was inserted and anchored in view of oozing from minor vessels and clot formation . a soft dressing is applied with long - arm backslab in slightly 45 degrees elbow extension as haemostasis for two days , and gentle active mobilisation exercise . our patient population in this series is six patients : two woman and four men with 1 : 2 being the ratio . the median average age was 55 years ( range , 3377 ) . a surgical endoscopic release was performed on the right side in four elbows and on the left side in two elbows . the main job profile of two patients is mainly table - top tasks , one works as dental hygienist , and three are nonworking in their retirement age , and all patients were not manual labourer . in five patients , the right side is dominant except one patient who was ambidextrous ( table 1 ) . the mean length of the surgery in the endoscopic release average 47 minutes ( range , 3262 minutes ) . the mean length of the skin incision was 2.25 cm ( range , 1.53 cm ) . retrospectively , no patient was classified as mild , three patients ( 50% ) were moderate , and three ( 50% ) were severe according to dellon 's classification in stages of ulnar nerve compression at the elbow ( table 2 ) . the postoperative outcome result is based on modified bishop rating system classification based on severity of residual symptoms , improvement of symptoms , work status , strength , and sensitivity which shows two ( 33% ) with excellent results , and four ( 66% ) have good results ( table 3 ) . the dental hygienist who has major risk factors identified sustained exposed to work related repetitive left elbow flexion more than 90 degrees constantly during the therapy session ( > 1 hour ) and sustained holding of hand tools . surgical site is not the dominant hand , then exposure to the vibratory tooth scaler machine is not a risk factor . there is no change of job to this patient , but with recommendations incorporate half - hour sessions of duties avoiding sustained gripping or sustaining affected elbow flexion > 90 degrees after every half - hour of therapy session and regular intermittent stretching of left elbow during the therapy session . three patients who are in retirement age with initial subjective evaluation base on modified bishop 's classification reach to score of good result without adding the subtype point score of work status of the patients for the reason , and this is not applicable to this group of patients ( table 4 ) . none of the patient was converted endoscopic release to in situ open due to any complication . no patients develop a postoperative infection , cutaneous nerve injury , hematoma , or painful surgical site . all patients improved symptom one day after the surgery with sensory loss improved and go back to their full activity in one month , and three patients previously went back to work after two months . the recovery and return to work was rapid and with a high patient satisfaction and no recurrence of symptom noted . none of the patients complained about scar discomfort , painful neuroma , burning sensation , superficial hypersensitivity , no elbow extension deficit , or ulnar nerve subluxation . sensory lost improved in all patients after the surgery and gradually improve after reevaluation at six - month subjective scale with good to excellent results . all preoperative electrophysiological studies were considered in all cases with abnormal results and with postoperative comparison which result findings five ( 83% ) residual changes but one patient ( 16% ) who has residual impaired because of the preoperative findings of evidence of axonal loss . this patient presented with severe preoperative compromise of the intrinsic musculature of the hand and subjective persistent numbness of the ulnar 1(1/2 ) side digits distribution , the postoperative ncv shows evidence of axonal loss but after 6 months postoperative ncs shows interval improvement with moderate prolonged . this patient improved the subjective parameters of modified bishop scoring system to good score result even when with persistent ulnar nerve distribution numbness and subjectively claimed that numbness decrease by 90% postoperatively , and objective parameter was satisfactory with overall clinical improvement . overall , these six cases were good to excellent subjective improvement of the result and also objective parameters in grip strength improvement . patients usually complain of sensory symptoms rather than muscle weakness so the result of the surgery was considered satisfactory by the patients , as their major complaints were related to the sensory symptoms . there were no complications in this series of six patients with good patient satisfaction and successful outcome without untoward complication . there is no gold standard in the surgical management of the cubital tunnel syndrome for the main reason that no single standard consensus of primary problem in nerve compression . in two groups of authors one believed that nerve compression is caused by overlying structure and that the syndrome is best treated by decompression of the ulnar nerve without removing it from its bed . other group of authors are citing evidence that the nerve is under tension with elbow flexion . that can only be relieved by placing the nerve anterior to the medial epicondyle . various surgical techniques for decompression of the ulnar nerve have been described in the literature , and a definitive gold standard does not exist . heithoff ( 1999 ) stated that all surgical techniques for cubital tunnel syndrome yielded similar results and that the choice of surgical technique should be based on simplicity . the endoscopic approach to in situ decompression in our series of six patients has a rapidity of postoperative improvement of symptoms compatible to the study of hoffmann and siemionow in 2006 . a completely new approach to surgery which enables to see and to do more through much smaller incision than those used by more traditional technique . it is a minimally invasive alternative for decompression of the ulnar nerve at the elbow , aiming to minimize the trauma to the tissues and improved postoperative recovery to the patients . its theoretical advantages over the classical open approach are the immediate well being of the patient , decreased invasiveness , minimal vascular complications , and less scar discomfort . in our view , like that patient with our inclusion criteria , 1992 , 1995 ; pavellza et al . , 2004 ; taniguchi et al , 2002 ; tsai et al . , 1999 ) is that the transposition of the ulnar nerve is not only unnecessary for the treatment of cubital tunnel syndrome , but that it may often be harmful and seriously disadvantageous , considering its potential complications ( heithofff , 1999 , mariani et al . , 1999 ) . the efficacy of simple decompression for the treatment of cubital tunnel syndrome was first reported by osborne ( 1957 ) . since then , many authors have reported good to excellent results with simple decompression ( chan et al . , 1980 ) . adelaar et al . , ( 1984 ) , bismmler and meyer ( 1996 ) , davies et al . , ( 1991 ) , and foster and edshage ( 1981 ) compared simple decompression with anterior transposition , and found no significant difference in clinical outcome . prospective randomized studies have shown results of simple decompression to be equal to those of anterior transposition [ 17 , 18 ] . in situ decompression also appears to have a low failure rate . but open decompression techniques require a longer incision , involve massive soft tissue dissection and are common associated with large postoperative scarring and wound tenderness . we can conclude that anterior transposition versus simple decompression has no significant difference in clinical outcome . a recent comparison between endoscopic techniques and in situ decompression demonstrated statistically significant less pain and greater satisfaction with the endoscopic technique . by minimally invasive with a direct visualisation to the ulnar nerve by endoscopic guidance can be visualised better more the potential compression sites of ulnar nerve entrapment , and all potential sites of nerve compression in the elbow region were released without damage to the macroscopically visible nerves . with endoscopy , a long portion of the nerve can be released without damage to cutaneous innervation . limited soft - tissue dissection with the preservation of the anatomy , especially vascularisation , minimises perineural fibrosis and enables rapid postoperative rehabilitation and can be safe and reliable with good functional and aesthetic result . according to the study by hoffmann and siemionow , the results of endoscopic release showed better functional recovery , lower morbidity and faster return to manual labour compared to conventional open method , and there were no serious complications . we conclude that endoscopic release is a safe procedure in the hands of the experienced surgeon with careful protection of the nerve and the branches and a complete decompression . the observed postoperative results demonstrated that this surgical technique to the ulnar nerve at the level of the elbow was very effective , and there was improvement in the clinical and electrophysiological outcomes in all the subjects who underwent the procedure . this procedure is a relatively alternative to the conventional open release technique in the uncomplicated cases . the short term has proven to be a safe and effective tool for the operative management of uncomplicated cases . the results showed better immediate functional recovery , lower morbidity , and faster and shorter rehabilitation time , and return to active activity was rapid or quicker return of the patients to their daily activity , acceptable aesthetic result and above all with a high patient satisfaction rate . endoscopic cubital tunnel release theoretically has better short - term outcome comparing to other technique in decompression but , however , to date , the number of studies reporting the case remains small .

cubital tunnel syndrome is one of the common upper extremity problem encountered . a mild syndrome can be often treated without surgery , but a failure of conservative treatment with constant symptoms or muscle atrophy and weakness requires surgical intervention . despite the fact that is the second most common nerve entrapment in the upper limb , there is no accepted gold standard in the surgical management . but with the new technique in minimally invasive surgery and available endoscope , it addresses all potential compression sites with good visualisation but with small surgical exposure . the procedure is safe and reliable way to address this problem .

1. Introduction 2. Method 3. Surgical Technique 4. Result 5. Discussion

as part of a 629-patient , randomized , double - blind , multicenter clinical trial , 89 patients seen at the foothills medical center ( calgary , canada ) receive a 10-day course of vancomycin 125 mg 4 times daily or 200 mg twice daily of fidaxomicin orally as treatment of first episodes or first recurrences of cdi . patients submitted fecal samples , > 10 g / sample , at study entry ; on days 4 , 10 , 14 , 21 , and 28 ; and on day 3842 days . with the exception of specimens collected on weekends , which were refrigerated at 2c8c for processing on the following monday , 1-g aliquots of the samples were processed on the collection date for quantitative counts of c. difficile by serial dilution ( 10 , 10 , 10 , and 10 ) and plated onto cycloserine cefoxitin fructose agar ( ccfa ) , modified by using fastidious anaerobe agar base ( labm , bury , united kingdom ) . an additional 1-g aliquot was subjected to 100% ethyl alcohol shock ( 1:1 vol / vol 1-hour exposure ) and plated onto taurocholate - ccfa with the same dilution scheme as that used for total c. difficile counts . c. difficile cytotoxin b in fecal filtrates was measured by cell cytotoxicity assay , using vero cells with neutralization of cytotoxicity ( techlab , blacksburg , va ) . the toxin titer end point was the concentration showing 50% cell rounding ; titration range was 1/201/32 000 . to avoid confounding by concomitant antibiotic therapy for other indications , ten subjects who were in good health and using no antibiotics or medications donated fecal samples to serve as normal flora controls . fecal samples from 10 vancomycin- and 10 fidaxomicin - treated patients who had received no prior treatment for cdi before entry into the study , had sustained clinical cures of cdi ( ie , cure with recurrence ) and provided all samples to day 28 and variably to day 3842 were selected for microbiome characterization . the state of the microbiome was evaluated by quantitative real - time polymerase chain reaction ( qpcr ) , using previously published primers ( table 1 ) and methods [ 2025 ] . bacterial dna from 250 mg of fecal sample was extracted using the qiaamp dna stool mini kit ( qiagen , mississauga , canada ) . following the addition of lysis buffer and 200 mg of zirconium beads ( diameter , 0.2 mm ) , purified genomic dna ( gdna ) was eluted off the columns with 200 l of molecular - grade water . the bacterial gdna was checked for purity and concentration , using the nanodrop 2000 spectrophotometer ( thermo scientific , wilmington , de ) . yield ( in nanograms of dna per milligram of stool ) was calculated in samples , after which they were stored at 20c for later analysis . qpcr was performed using iq5 and cfx96 detection systems ( biorad , mississauga , canada ) . amplification and detection were conducted in 96-well plates with sybr green 2 qpcr master mix ( biorad ) . each sample was run in duplicate in a final volume of 20 l containing a final concentration of 0.3 m of each primer and 5 l of 4-ng/l template gdna . amplifications were conducted using the 2-step template ramping profile : 1 cycle at 95c for 5 minutes , followed by 49 cycles at 95c for 30 seconds , 52c60c for 30 seconds , and 72c for 30 seconds . table 1.16s ribosomal rna probes used for quantitative real - time polymerase chain reaction to quantify shifts in major components of the fecal flora during and after treatment of clostridium difficile infectiongroupnamesequencereferencebacteroidesbac303fgaaggtcccccacattgbernhard et al , 2000bacteroidesbac708rcaatcggagttcttcgtgbernhard et al , 2000enterobacteriaceaeeco1457fcattgacgttacccgcagaagaagcbartosch et al , 2004enterobacteriaceaeeco1652ctctacgagactcaagcttgcbartosch et al , 2004clostridium leptum subgroupsg - clept - fgcacaagcagtggagtmatsuki et al , 2002c . difficileg - cdif - rccatcctgtactggctcacctrinttil et al , 2004desulfovibrio desulfuricans groupg - desulf - fggtaccttcaaaggaagcacrinttil et al , 2004d . desulfuricans groupg - desulf - rgggatttcacccctgacttarinttil et al , 2004enterococcus speciesg - enter - fcccttattgttagttgccatcattrinttil et al , 2004enterococcus speciesg - enter - ractcgttgtacttcccattgtrinttil et al , 2004veillonella speciesg - veill - fa(c / t)caacctgcccttcagarinttil et al , 2004veillonella speciesg - veill - rcgtcccgattaacagagcttrinttil et al , 2004prevotellacfb286fgtaggggttctgagaggaprobebase pb-00045prevotellacfb719ragctgccttcgcaatcggprobebase pb-00047lactobacillusuni331ftcctacgggaggcagcagtnadkarni et al , 2002lactobacilluslacto371rtgg aag att ccc tac tgcprobebase pb-00195bifidobacterium speciesbif551fcgcgtcyggtgtgaaagdelroisse et al , 2008bifidobacterium speciesbif794rccccacatccagcatccadelroisse et al , 2008 16s ribosomal rna probes used for quantitative real - time polymerase chain reaction to quantify shifts in major components of the fecal flora during and after treatment of clostridium difficile infection the specificity of the primers was determined by using purified template dna from reference strains , including bacteroides fragilis american type culture collection ( atcc ) 25825 , bacteroides thetaiotaomicron atcc 29741 , bacteroides distasonis atcc 8503 , bacteroides ovatus atcc 8483 , bacteroides vulgatus atcc 8482 , prevotella melaninogenica atcc 25845 , clostridium coccoides atcc 29236 , fusobacterium nucleatum subspecies nucleatum atcc 25586 , peptostreptococcus anaerobius atcc 27337 , bifidobacterium bifidum atcc15696 , and c. difficile atcc 43255 . melting curve analysis was performed to ensure the specificity of the primer sets and subsequent qpcr amplification reactions . the limit of detection for each assay was determined with concentrations of purified dna of the reference strains ranging from 0.0000220 ng . this range was also used to generate each standard curve using serial 10-fold dilutions . to estimate the amounts of target bacterial dna from fecal dna extractions , threshold cycle ( ct ) values were first converted into gdna copies using the standard curve generated from each qpcr run . for construction of standard curves , 10-fold - dilution series of 1010 target genomes from target species were prepared and run with samples . to calculate colony - forming units ( cfu ) per gram of stool , the target genomes calculated from 20 ng of gdna ( the amount used per qpcr reaction ) were extrapolated into cfu per gram of stool by multiplying the cfu per nanogram of gdna by the yield ( total nanograms of gdna extracted from 1 g of stool ) . the majority of the qpcr tests were designed to detect a wide range of related and diverse bacterial species that , most likely , did not have the same genome size or ribosomal dna copy numbers . therefore in addition to the foregoing , 5 normal control samples were analyzed pair wise by quantitative cultures and by qpcr for bacteroides group organisms , enterobacteriaceae , and enterococcus species . the quantitative bacterial counts of c. difficile and calculated cfu per gram of target organism group by real - time qpcr were log transformed , after which changes in each time of collection were compared between fidaxomicin and vancomycin treatments , using wilcoxon signed - rank tests for nonparametric data ( graphpad prism ; graphpad , san diego , ca ) . the specificity of the primers was determined by using purified template dna from reference strains , including bacteroides fragilis american type culture collection ( atcc ) 25825 , bacteroides thetaiotaomicron atcc 29741 , bacteroides distasonis atcc 8503 , bacteroides ovatus atcc 8483 , bacteroides vulgatus atcc 8482 , prevotella melaninogenica atcc 25845 , clostridium coccoides atcc 29236 , fusobacterium nucleatum subspecies nucleatum atcc 25586 , peptostreptococcus anaerobius atcc 27337 , bifidobacterium bifidum atcc15696 , and c. difficile atcc 43255 . melting curve analysis was performed to ensure the specificity of the primer sets and subsequent qpcr amplification reactions . the limit of detection for each assay was determined with concentrations of purified dna of the reference strains ranging from 0.0000220 ng . to estimate the amounts of target bacterial dna from fecal dna extractions , threshold cycle ( ct ) values were first converted into gdna copies using the standard curve generated from each qpcr run . for construction of standard curves , 10-fold - dilution series of 1010 target genomes from target species were prepared and run with samples . to calculate colony - forming units ( cfu ) per gram of stool , the target genomes calculated from 20 ng of gdna ( the amount used per qpcr reaction ) were extrapolated into cfu per gram of stool by multiplying the cfu per nanogram of gdna by the yield ( total nanograms of gdna extracted from 1 g of stool ) . the majority of the qpcr tests were designed to detect a wide range of related and diverse bacterial species that , most likely , did not have the same genome size or ribosomal dna copy numbers . in addition to the foregoing , 5 normal control samples were analyzed pair wise by quantitative cultures and by qpcr for bacteroides group organisms , enterobacteriaceae , and enterococcus species . the quantitative bacterial counts of c. difficile and calculated cfu per gram of target organism group by real - time qpcr were log transformed , after which changes in each time of collection were compared between fidaxomicin and vancomycin treatments , using wilcoxon signed - rank tests for nonparametric data ( graphpad prism ; graphpad , san diego , ca ) . of the 89 patients randomly assigned in the study , 44 vancomycin- and 45 fidaxomicin - treated patients responded to treatment . c. difficile strain typing showed that 13% of strains were ribotype 027 . recurrence was observed in 23% of vancomycin - treated patients ( 10 of 44 ) and in 11% of fidaxomicin - treated patients ( 5 of 44 ) . results of qpcr analysis of fecal samples obtained from healthy donors are summarized in table 2 . paired tests showed that qpcr results were approximately 1 log10 cfu / g lower than results by quantitative culture . table 2.results of quantitative real - time polymerase chain reaction detection of components of the microflora from 10 healthy control donors of stoolmean log10 cfu / g sdmean log10 cfu / g sdbacteroides9.66 0.28bifidobacterium6.78 0.86prevotella9.20 0.50veillonella5.80 0.90clostridium coccoides9.02 0.53desulfovibrio5.28 1.24clostridium leptum7.73 0.77enterobacteriaceae5.53 0.75lactobacillus7.59 0.51enterococcus species5.30 0.75abbreviations : cfu , colony - forming unit ; sd , standard deviation . results of quantitative real - time polymerase chain reaction detection of components of the microflora from 10 healthy control donors of stool abbreviations : cfu , colony - forming unit ; sd , standard deviation . figures 110 show counts of target organisms in individual patients over time after receipt of vancomycin or fidaxomicin , as well as mean values for 10 healthy controls . clinical trial site subject numbers have been removed , and subjects are listed sequentially by number to preserve confidentiality . mean counts of bacteroides species , prevotella species , c. coccoides , and clostridium leptum were approximately 2 log counts lower at study entry , compared with results from healthy control fecal samples . for samples collected on day 4 , a washout effect was noted on the basis of a transitory dip in cfu - per - gram for fidaxomicin - treated subjects , likely as a result of continued diarrhea , whereas the decrease in microbial counts was substantially greater for vancomycin - treated subjects , presumably because of both a washout effect plus microbial suppression / killing . as a result , the differences between the day-0 and day-10 samples at the end of treatment appear to be the best comparators of the effect of treatment on microbial counts . vancomycin markedly suppressed bacteroides , prevotella , c. coccoides , and c. leptum group organisms and suppressed bifidobacteria species to a lesser extent . fidaxomicin appeared to spare ( for most groups ) or act indifferently ( for bifidobacteria species ) on these groups . veillonella species counts increased during vancomycin treatment and decreased in the post - treatment phase , whereas counts were unchanged in fidaxomicin - treated patients , suggesting an opportunistic increase in counts during suppression of other organism groups during vancomycin treatment . vancomycin suppressed counts of non vancomycin - resistant enterococci during therapy , after which there was a rebound in counts and gradual reduction in numbers later in the follow - up period . counts of enterobacteriaceae were 23 logs higher than those in healthy controls at study entry and , for both treatments , appeared to drift downward in the posttreatment period . figure 1.bacteroides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 2.clostridium coccoides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . abbreviation : cfu , colony - forming units . figure 3.clostridium leptum microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 4.prevotella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 5.veillonella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 6.bifidobacterium microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 7.lactobacillus microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 8.desulfovibrio microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 9.enterobacteriaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 10.enterococcaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . bacteroides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . clostridium coccoides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . clostridium leptum microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . prevotella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . veillonella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . abbreviation : cfu , colony - forming units . bifidobacterium microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . lactobacillus microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . desulfovibrio microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . enterobacteriaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . enterococcaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . abbreviation : cfu , colony - forming units . figure 11a c and table 3 summarize the effect of vancomycin versus that of fidaxomicin during and following cdi treatment for 20 patients who achieved sustained cure . the day 3842 samples , which were collected in 34 patients in each group , made comparisons difficult at the end of the follow - up period . because most recurrences in the trial occurred within 23 weeks of completing therapy , the comparison of samples obtained at study entry to day-28 samples likely illustrates the differences between treatments . at study entry , for bacteroides / prevotella organisms , vancomycin markedly suppressed this organism group during therapy . even at day 28 , significant differences were noted between vancomycin and fidaxomicin . similar differences were observed for the c. coccoides and c. leptum groups , but suppression by vancomycin was transient , with recovery by day 21 . figure 11.levels of major cultivable and noncultivable genera of the normal fecal microbiota in 20 patients with clostridium difficile infection who were randomly assigned to receive treatment with fidaxomicin or vancomycin ( 10 patients / treatment arm ) . table 3.results of quantitative real - time polymerase chain reaction for major bacterial groups for 10 patients receiving vancomycin or fidaxomicin for 10 days , followed by 4 weeks of follow - upbacteroidesprevotellaclostridium coccoides groupclostridium leptum groupbifidobacteriaenterobacteriaceaelactobacillusenterococcaceaemean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sddayvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicin07.61 2.218.52 1.537.62 2.178.11 1.667.89 1.588.17 0.715.41 2.215.81 2.226.96 0.726.35 1.427.50 1.707.94 0.707.55 0.627.62 0.546.17 0.515.48 1.3544.26 0.887.81 1.545.30 0.817.84 1.614.73 0.536.91 0.942.04 0.393.53 5.085.30 0.815.30 1.338.52 1.468.81 0.877.90 0.457.10 0.785.23 0.315.75 1.18105.26 0.919.33 1.305.50 0.928.84 1.194.68 0.738.49 0.552.02 1.315.08 2.265.17 1.096.28 1.408.84 0.577.84 1.187.91 0.707.64 0.655.43 0.365.93 0.95145.99 2.109.52 1.195.43 1.309.06 1.046.74 2.218.65 0.444.03 1.835.29 2.376.56 1.436.18 1.758.01 1.047.98 0.808.11 0.437.66 0.736.26 0.856.07 1.22215.95 2.309.30 1.446.63 2.218.97 1.378.51 0.648.73 0.585.85 1.215.83 2.196.94 0.936.06 1.697.62 1.037.06 0.927.69 0.487.34 0.936.15 0.676.12 0.90286.95 2.879.08 1.166.83 2.548.93 0.858.66 1.118.07 1.456.18 1.015.82 1.355.89 1.196.10 1.357.33 1.185.95 2.147.65 0.557.23 1.095.77 0.725.79 0.73427.83 1.709.34 0.737.89 1.639.22 0.358.41 0.698.40 0.827.09 1.336.89 1.085.62 1.516.11 1.176.52 1.616.50 1.107.50 0.507.60 0.805.77 0.765.71 0.79data are cfu per gram of stool . results support data in figures 110.abbreviations : cfu , colony - forming unit ; sd , standard deviation . levels of major cultivable and noncultivable genera of the normal fecal microbiota in 20 patients with clostridium difficile infection who were randomly assigned to receive treatment with fidaxomicin or vancomycin ( 10 patients / treatment arm ) . results of quantitative real - time polymerase chain reaction for major bacterial groups for 10 patients receiving vancomycin or fidaxomicin for 10 days , followed by 4 weeks of follow - up data are cfu per gram of stool . table 4 summarizes the results of total quantitative cultures for c. difficile at study entry , day 4 , day 10 , and in the follow - up period , along with spore counts and c. difficile cytotoxin b titers . mean total and spore counts at study entry were similar between treatment arms . at days 4 and 10 , both treatments effectively reduced c. difficile counts to the lower limit of quantitation , after which there appeared to be a similar low - level rebound of c. difficile counts . toxin titers were similar at study entry , and all patients had no detectable toxin ( titer , < 1/20 ) on days 410 of treatment . however , vancomycin - treated patients were twice as likely to reexpress toxin in the follow - up period ( p = .03 ) , primarily within the first 2 weeks of treatment . in the total patient group of 89 patients , patients who achieved sustained cure , vancomycin - treated patients who experienced recurrence of cdi had significantly lower counts of bacteroides and prevotella organisms at the end of treatment ( table 5 ) . a trend toward lower counts of these organisms was also observed in fidaxomicin - treated patients who subsequently experienced recurrence of cdi . at diagnosis of recurrent cdi , toxin titers , pathogen counts , and microbiome profiles were similar to findings at study entry ( data not shown ) . table 4.quantitative cultures for clostridium difficile total counts , spore post alcohol shock counts , and c. difficile cytotoxin b titers in 89 patients randomly assigned to receive a 10-day course of vancomycin or fidaxomicin therapyday 0day 4day 10day 14day 21day 28day 42mean log10 cfu / g sd ( spore sd ) van6.2 2.12.2 0.92.0 0.03.1 2.24.5 2.73.9 2.24.5 2.4 fdx5.9 2.82.0 0.12.0 0.12.7 1.94.2 2.43.9 2.33.0 1.9mean log10 spore count ( sd ) van4.8 1.72.7 1.12.2 0.63.2 2.04.0 1.93.3 1.53.2 1.6 fdx4.7 1.82.0 0.02.1 0.72.3 1.23.3 1.83.1 1.42.7 1.3mean toxin b titer semday 0day 4day 10pooled for days 14 , 21 , 28 , 42 van2800 1250negativenegative1260 350 fdx2250 600negativenegative2400 1400a total of 44 patients received van , and 45 received fdx.abbreviations : cfu , colony forming units ; fdx , fidaxomicin ; sd , standard deviation ; sem , standard error of the mean ; van , vancomycin . several values were higher than total counts , reflecting several samples for which spore counts appeared higher than total counts , which could represent variation near the lower limit of detection and possibly less competition after alcohol shock treatment cultures . data are for positive titers only . for the van group , 7 of 30 specimens tested from day 14 , 15 of 30 from day 21 , 3 of 22 from day 28 , and 1 of 12 from day 42 were positive . for the fdx group , 1 of 23 specimens tested from day 14 , 6 of 27 from day 21 , 5 of 20 from day 28 , and 1 of 21 from day 42 were positive . the value was 1140 770 if the outlier toxin titer of 1/16 000 was excluded . table 5.comparison of changes in microflora among patients who achieved sustained cure of clostridium difficile infection with changes among patients who achieved cure but had subsequent recurrencelog10 cfu / g feces sdpatients with sustained curepatients with recurrenceday 0day 10day 0day 10bacteroides group vancomycin7.60 2.215.26 0.918.40 2.014.37 0.39 fidaxomicin8.52 1.539.33 1.305.76 1.947.00 2.33prevotella species vancomycin7.62 2.175.50 0.928.11 1.994.43 0.67 fidaxomicin8.11 1.668.84 1.195.26 1.966.57 2.20clostridium coccoides group vancomycin7.89 1.584.67 0.728.46 0.514.95 1.30 fidaxomicin8.17 0.718.49 0.557.21 1.776.80 2.77ten patients in the vancomycin group and 10 in the fidaxomicin achieved sustained cure , whereas 10 in the vancomycin group and 5 in the fidaxomicin group had recurrence . fidaxomicin - treated patients with recurrence appeared to have greater reductions in bacteroides counts at study entry.abbreviation : cfu , colony - forming unit . p < .03 , compared with day 10 values for patients with recurrence . p = .03 , compared with day 10 values for patients with recurrence . p = .08 , compared with day 10 values for patients with recurrence . p = .007 , compared with day 10 values for patients with recurrence . p = .11 , compared with day 10 values for patients with recurrence . quantitative cultures for clostridium difficile total counts , spore post alcohol shock counts , and c. difficile cytotoxin b titers in 89 patients randomly assigned to receive a 10-day course of vancomycin or fidaxomicin therapy a total of 44 patients received van , and 45 received fdx . abbreviations : cfu , colony forming units ; fdx , fidaxomicin ; sd , standard deviation ; sem , standard error of the mean ; van , vancomycin . several values were higher than total counts , reflecting several samples for which spore counts appeared higher than total counts , which could represent variation near the lower limit of detection and possibly less competition after alcohol shock treatment cultures . 7 of 30 specimens tested from day 14 , 15 of 30 from day 21 , 3 of 22 from day 28 , and 1 of 12 from day 42 were positive . for the fdx group , 1 of 23 specimens tested from day 14 , 6 of 27 from day 21 , 5 of 20 from day 28 , and 1 of 21 from day 42 were positive . the value was 1140 770 if the outlier toxin titer of 1/16 000 was excluded . comparison of changes in microflora among patients who achieved sustained cure of clostridium difficile infection with changes among patients who achieved cure but had subsequent recurrence ten patients in the vancomycin group and 10 in the fidaxomicin achieved sustained cure , whereas 10 in the vancomycin group and 5 in the fidaxomicin group had recurrence . fidaxomicin - treated patients with recurrence appeared to have greater reductions in bacteroides counts at study entry . abbreviation : cfu , colony - forming unit . p < .03 , compared with day 10 values for patients with recurrence . p = .03 , compared with day 10 values for patients with recurrence . p = .08 , compared with day 10 values for patients with recurrence . p = .007 , compared with day 10 values for patients with recurrence . p = .11 , compared with day 10 values for patients with recurrence . cdi is the most prominent clinical example of the consequence of impairment of the normal microbiota of the gut , resulting in loss of colonization resistance , pathogen proliferation , and diarrheal disease . measurement of the degree of impairment of the intestinal flora that is thought to occur with intestinal infections has awaited new technologies available only in the past decade . studies to date have shown depletion of bacteroides species and firmicutes in several patients experiencing multiple recurrences , floral shifts by denaturing gradient gel electrophoresis , and changes in bacteroidetes and firmicutes , as determined by microarray signaling , in a comparison of cohorts of patients with and control patients without cdi . a systematic prospective and quantitative study to evaluate microbiome shifts during fidaxomicin treatment of cdi is reported here . the extent and nature of impairment of the microbiome before the onset of cdi diarrheal symptoms ( equivalent to the concept of the latent period ) are unknown . fecal samples could also be regarded as an indirect measure of biological processes because events on the mucosal interface likely are more determinative in pathogenesis . nevertheless , given the technical challenges of obtaining and separating microbes in the fecal stream from those on surfaces , comparison of microbiome profiles in fecal samples from healthy controls with samples at the onset of cdi are a measure of the degree of impairment of the microbiome . findings in this study extend and support results of the phase ii clinical study that used conventional culture and fluorescence in situ hybridization . bacteroides and firmicutes groups ( c. coccoides plus c. leptum ) , which constitute the bulk of the microflora , appear to be reduced 10100-fold at the time of cdi diagnosis on the basis of qpcr testing . on the basis of paired culture versus qpcr results , using bacteroides as an indicator group , the mean reduction could be 1-log10 greater . there is substantial variation in the degree of suppression of the normal microbiota , with some patients showing bacteroides counts in the 10-cfu / g range . because diarrheal movements could have a washout effect on normal flora , it is likely that a decreased degree of perturbation of the microbiome can result in cdi . because the decrease in counts at day 4 appears to be a further washout effect due to diarrheal movements before disease resolution ( with or without antimicrobial suppression ) , differences in counts at study entry and at the end of treatment at day 10 have been used to quantify the effect of drug treatment on the residual microbiome . the results of microbiome profiling document the collateral damage to the microflora seen with vancomycin therapy , a further 23-log decrease in bacteroides group counts resulting in a 46-log decrease from healthy control counts at the end of vancomycin therapy . the observation that bacteroides organisms are more numerous on the mucosal surface compared with the feces itself suggests an important role in colonization resistance , perhaps by a process of bacterial interference versus intestinal pathogens . this decrease is associated with reappearance of c. difficile cytotoxin b in fecal filtrates at a frequency that is twice that after fidaxomicin therapy and , in this study population , was accompanied by twice the number of cdi recurrences . these findings are consistent with outcomes in the clinical trials as a whole [ 11 , 12 ] and underscore the important observation that the majority of recurrences eventuate during the 23-week interval after treatment . a reduction ( p = .001 ) in number of spores at day 4 of fidaxomicin treatment , compared with day 4 of vancomycin treatment ( table 4 ) , the c. difficile microbial profiles were not different between treatments and therefore support the alternate hypothesis that reduced recurrence is related to the health of the residual normal microflora . comparison of the pattern of suppression of members of the microbiome between the 2 treatments suggests that bacteroides species , prevotella species , the firmicutes , and bifidobacteria species play a beneficial role in maintaining a healthy microbiome and that high counts of coliforms and enterococci are a marker for a depleted gut microbiome . the observation that patients with recurrence of cdi have lower counts of bacteroides and prevotella organisms at the end of therapy is additional supportive evidence of a protective role of these microbes . in the final analysis , the unchanged counts seen in this study neither support nor refute a role for lactobacilli in preventing cdi recurrences . on the basis of the high efficacy of fecal microbiome transplantation in arresting recurrent cdi , it is likely that the dominant members of the microbiome are critical for preventing cdi . in summary , the microflora - sparing features of fidaxomicin shown in this report offer selective therapy for cdi , a more robust microbiome , and a decreased risk for recurrent disease .

the microflora - sparing properties of fidaxomicin were examined during the conduct of a randomized clinical trial comparing vancomycin 125 mg 4 times per day versus fidaxomicin 200 mg twice per day for 10 days as treatment of clostridium difficile infection ( cdi ) . fecal samples were obtained from 89 patients ( 45 received fidaxomicin , and 44 received vancomycin ) at study entry and on days 4 , 10 , 14 , 21 , 28 , and 38 for quantitative cultures for c. difficile and cytotoxin b fecal filtrate concentrations . additionally , samples from 10 patients , each receiving vancomycin or fidaxomicin , and 10 samples from healthy controls were analyzed by quantitative real - time polymerase chain reaction with multiple group - specific primers to evaluate the impact of antibiotic treatment on the microbiome . compared with controls , patients with cdi at study entry had counts of major microbiome components that were 23-log10 colony - forming units ( cfu)/g lower . in patients with cdi , fidaxomicin allowed the major components to persist , whereas vancomycin was associated with a further 24-log10 cfu reduction of bacteroides / prevotella group organisms , which persisted to day 28 of the study , and shorter term and temporary suppression of both clostridium coccoides and clostridium leptum group organisms . in the posttreatment period , c. difficile counts similarly persisted in both study populations , but reappearance of toxin in fecal filtrates was observed in 28% of vancomycin - treated patient samples ( 29 of 94 ) , compared with 14% of fidaxomicin - treated patient samples ( 13 of 91 ; p = .03 ) . similarly , 23% of vancomycin - treated patients ( 10 of 44 ) and 11% of fidaxomicin - treated patients ( 5 of 44 ) had recurrence of cdi . whereas vancomycin and fidaxomicin are equally effective in resolving cdi symptoms , preservation of the microflora by fidaxomicin is associated with a lower likelihood of cdi recurrence.clinical trials registration . ntc00314951 .

METHODS Determination of Specificity and Limit of Detection Quantification of Target Bacterial DNA in Fecal Samples by Real-Time qPCR Statistical Analyses RESULTS DISCUSSION

magnetic resonance imaging ( mri ) is a powerful noninvasive imaging technique , providing high - resolution anatomical images with excellent soft - tissue contrast . a large fraction of mri studies utilize a contrast agent , e.g. , more than half the clinical studies performed at our institution make use of a gadolinium(iii)-based contrast agent . these contrast agents are simple , water - soluble , ternary gadolinium(iii ) complexes with gadolinium(iii ) coordinated by an octadentate poly(aminocarboxylato ) ligand and an aqua coligand . gadolinium(iii ) is advantageous for use in mri contrast agents because of its large electron spin number ( s = /2 ) and long electronic relaxation time , which result in efficient nuclear relaxation of nearby solvent water molecules . contrast agents are characterized by their relaxivity , defined as the change in the solvent relaxation rate ( 1/t1 or 1/t2 ) normalized to the concentration of the contrast agent ( units of mm s ) . first - generation clinical contrast agents are monomeric gadolinium(iii ) complexes based on a [ gd(dota)(h2o ) ] or [ gd(dtpa)(h2o ) ] ( dtpa = diethylenetriaminepentaacetic acid ) framework and have relaxivities in the range 35 mm s. second - generation contrast agents such as gadofosveset ( ms-325 , ablavar ) used reversible protein binding to increase the relaxivity . protein binding slows the rotational tumbling rate of the complex , and as this rate approaches the proton larmor frequency , nuclear relaxation is enhanced . specific protein binding also serves to direct the contrast agent to specific pathologies , e.g. , thrombosis , as well as enhance the relaxivity . however , the increase in the relaxivity due to protein binding is greatest by far at low magnetic fields ( 1.5 t ) and falls off precipitously at higher fields . clinically , 3 t mri is becoming common , and the major equipment vendors all sell 7 t scanners . for small - animal imaging , even higher field strengths are common . at high fields ( 3 t ) , the proton larmor frequency is much faster than the tumbling rate of protein - bound complexes but still much slower than the tumbling rate of discrete monomeric complexes . we and others have reported multimeric contrast agents with increased high - field relaxivity due to a tumbling rate intermediate between that of protein - bound agents and small molecules . multiple gd(dotala ) moieties could be incorporated into oligopeptides of defined structure and size , resulting in relaxivities tuned for high - field applications . the modular nature of gd(dotala ) also lends itself to the design of serum albumin - targeted contrast agents . here , we report on the synthesis and properties of a modified derivative of the dotala ligand , dotalap . we sought to improve the properties of the dotala ligand by replacing one of the acetate moieties by methylenephosphonate . the increased charge of the phosphonate is expected to increase the solubility of the resultant gadolinium(iii ) complexes . gadolinium(iii ) complexes with phosphonate - containing ligands often yield higher relaxivities than complexes of the analogous carboxylate ligand due to an effect on water in the second coordination sphere , and this second - sphere effect persists at high fields . we also expected that inner - sphere water exchange should be faster for the phosphonate - substituted ligand . for gadolinium(iii ) complexes undergoing water exchange via a dissociative mechanism , substitution of a carboxylate with the bulkier phosphonate donor group results in faster water exchange as the energy difference between the 8-coordinate transition state and the 9-coordinate ground state becomes smaller . on the other hand , this steric crowding can also result in an 8-coordinate ground state with an inner - sphere water ligand eliminated , which would result in lower relaxivity . in this study , we sought to address the following questions : ( i ) how does substitution of one of the acetate donors in dotala for methylenephosphonate impact the hydration number and water exchange kinetics and , in turn , the relaxivity ? ( ii ) how does phosphonate substitution affect kinetic inertness with respect to gadolinium(iii ) dissociation ? ( iii ) what is the effect of amine versus carboxylate functionalization of the propionamine arm of dotalap on the hydration number , water exchange kinetics , relaxivity , and gadolinium(iii ) dissociation kinetics ? ( iv ) are any of these properties altered when a gd(dotalap ) derivative is bound to serum albumin ? ( v ) does phosphonate substitution confer enhanced high - field relaxivity properties and , if so , do these result in an increased in vivo mri signal ? to address these questions , we synthesized the dotalap ligand by altering the previously established dotala synthesis . additionally , we prepared four dotalap derivatives substituted at either the amine or carboxylate of the propionamine arm . the gadolinium(iii ) complexes were prepared and characterized with respect to variable - field , variable - temperature relaxivity , water exchange kinetics , albumin binding studies , and gadolinium(iii ) dissociation kinetics . europium(iii ) complexes were also synthesized , and luminescence lifetime measurements were performed to quantify the hydration number of these complexes . benzyl 2-[[(benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate represents an intermediate common to both dotala and dotalap , so we used the previously established synthetic steps to furnish this first intermediate ( scheme 1 ) . in order to introduce a protected phophonate arm onto the monoderivatized cyclen macrocycle , tri - tert - butyl phosphite was synthesized according to ref ( 22 ) and appended using a mannich - type reaction . the reaction was monitored for disappearance of the monofunctionalized macrocycle using liquid chromatography mass spectrometry ( lc ms ) , followed by filtration and aqueous workup of the filtrate . the introduction of these two chelator arms at this stage is key : because of their steric hindrance , it is not possible to incorporate them efficiently at a later stage . the crude intermediate containing the difunctionalized macrocycle was then redissolved in acetonitrile and subjected to alkylation with tert - butyl bromoacetate , followed by preparative high - performance liquid chromatography ( hplc ) to purify the protected intermediate compound 1 . orthogonal protection group chemistry provided us with a free and ready - to - couple carboxylate and primary amine on compound 2 after hydrogenation - mediated removal of the carboxybenzyl ( cbz ) and benzyl protecting groups of 1 . the overall number of steps starting from cbz - nh - ser - oh - obn is 5 , and compound 2 is obtained with an approximate 10% overall yield based on the protected serine . the lower yield is due to the formation of a greater number of side products compared to the dotala synthesis . one single phosphonate species ( 1,7-functionalized with respect to the aminopropanoate arm ) is obtained and isolated , as indicated to us by h nmr . compound 2 also represents the last common intermediate for the synthesis of compounds 35b , which all are furnished by activation of the carboxylate with 1-[bis(dimethylamino)methylene]-1h-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate ( hatu ) in the presence of diisopropylethylamine ( dipea ) as a base , followed by overnight amide coupling and subsequent hplc purification . the coupled , purified products still carry tert - butyl ester protective groups on the acetate groups and the phosphonate . these esters are then removed using an acid - catalyzed deprotection reaction with trifluoroacetic acid in dichloromethane , yielding the final ligands without the formation of side products . complexes were formed under standard conditions by mixing the ligand with an aqueous solution of the lanthanide trichloride salt . the ph of the solution was adjusted to 7 by the dropwise addition of a 0.1 m solution of naoh . complexation was followed by hplc in order to confirm that > 95% of ligand was complexed . we also used the xylenol orange test to further confirm that no free lanthanide was present . according to our hypothesis , gd(dotalap ) derivatives should display water exchange rates that exceed the previously reported water exchange rates of gd(dotala ) derivatives . we determined the water exchange rates of the gadolinium(iii ) complexes by variable - temperature measurement of the transverse relaxation time of h2o in the presence and absence of each gadolinium(iii ) complex at 11.7 t. in the case of gd(4a ) , gd(4b ) , and gd(5b ) , we observed only minor changes in the transverse relaxation rate when the complexes were added , indicating a lack of an inner - sphere water ligand for these complexes . for instance , the maximum o relaxivity , r2 , was 2.5 mm s for these three complexes , while the maximum r2 is typically > 15 mm s for q = 1 complexes at this field strength . on the other hand , gd(3 ) and gd(5a ) had much higher maximum r2 values consistent with an inner - sphere water ligand ( 18 and 16 mm s , respectively ) . figure 1 shows the reduced transverse relaxation rate r2r as a function of the reciprocal temperature . the crossover to exchange - limited r2r occurs at a low temperature for both complexes , which indicates the predicted , very fast water exchange kinetics . we used a four - parameter model described previously to fit the data and obtained water residency times at 37 c of m = 8.1 ns for gd(3 ) and m = 6.4 ns for gd(5a ) ; see table 1 . while these water residency times are considerably shorter than those seen for [ gd(dota)(h2o ) ] and [ gd(dotala)(h2o ) ] , they are similar to the water residency time for [ gd(do3ap)(h2o ) ] , a close structural analogue of dotalap ( table 1 ) . the coordination spheres of gd(3 ) and gd(5a ) differ in that gd(3 ) contains an anionic propionate oxygen donor and gd(5a ) contains a neutral propionamide oxygen donor but their water exchange kinetics are similar . the propionate to propionamide substitution does not result in different water exchange kinetics in other gadolinium(iii ) complexes . this is in contrast to the acetate to acetamide substitution , which is well established to reduce the water exchange rate . interestingly , substitution at the propionamine [ gd(4a ) and gd(4b ) ] gives a ligand with the same donor set as 3 , but these peripheral modifications now result in a gadolinium complex with no inner - sphere water ligand . similarly , changing the amide substituent from gd(5a ) to gd(5b ) results in the latter complex having no inner - sphere water ligand . for complexes with exceedingly fast water exchange kinetics , these results indicate that seemingly minor modifications to the periphery of the ligand can alter the hydration number of the complex . temperature dependence of the o nmr ( 11.7 t ) reduced transverse relaxation rates of gd(3 ) ( 6.73 mm , left ) and gd(5a ) in pbs ( 4.16 mm , right ) . the solid line represents a fit to the data to determine the mean water residency time m . determination of the relaxivity of the gadolinium(iii ) complexes also gives further indication of their hydration state . the longitudinal proton relaxivity ( r1 ) was measured in ph 7.4 pbs at 37 c at two larmor frequencies , 20 and 60 mhz . for small molecules with q > 0 , the relaxivity is dominated by the short rotational correlation time at these frequencies and the hydration state . additionally , phosphonate - containing gadolinium(iii ) chelates have been reported to result in increased second - sphere relaxivity . if all of the complexes were q = 1 , then we would expect the relaxivity to increase proportionally with the molecular weight , ranking them as follows : gd(3 ) < gd(4a ) gd(5a ) < gd(4b ) gd(5b ) . in fact , only gd(3 ) and gd(5a ) behave as expected with a relatively high , but for q = 1 phosphonato complexes typical , relaxivity . on the other hand , gd(4a ) , gd(4b ) , and gd(5b ) show relaxivities below 4 mm s , which is more typical for q = 0 complexes with elevated outer - sphere relaxivity ( table 2 ) . these relaxivity measurements are consistent with the o nmr measurements , which suggests the lack of an inner - sphere water ligand for gd(4a ) , gd(4b ) , and gd(5b ) . the hydration number of a gadolinium(iii ) complex can be estimated using a surrogate complex with the luminescent lanthanide ions eu or tb . this is done by measuring the rate constants for luminescence decay in d2o and h2o solutions . an empirical expression originally reported by horrocks and sudnick relates these rate constants to q ( table 3 ) . it is then assumed that gadolinium(iii ) has the same hydration number based on the similar ionic radii of these ions and their propensity for forming isostructural complexes . however , application of this method to phosphonate - containing complexes typically results in an overestimation of q by 0.30.6 . with this consideration in mind , we find that the q values obtained mirror the relaxometric behavior of the corresponding gadolinium complexes . the hydration numbers calculated are proportional to the obtained relaxivity values in pbs at 20 and 60 mhz , with eu(3 ) and eu(5a ) providing q values above 1 , while the other complexes show lower q values . figure s1 ( supporting information ) shows the correlation of the 20 mhz relaxivity values of the gadolinium(iii ) complex and the apparent hydration number of the corresponding europium(iii ) complex . nd = not determined . because of the decreased solubility of eu(5b ) compared to the other europium complexes described here , we were not able to generate a solution with high enough complex concentration required for this measurement . we had previously conjugated ibuprofen to dotala and found that a 0.1 mm solution of its gadolinium(iii ) complex [ compound gd(6 ) , figure 2 ] was 70% bound to 4.5% ( w / v ) hsa , and this complex also displayed high relaxivity ( at low fields ) in the presence of hsa . to understand how the phosphonate modification affects protein binding and relaxivity for a slow tumbling system , we synthesized the ibuprofen conjugates gd(4b ) and gd(5b ) shown in figure 2 . structures of ibuprofen conjugates gd(4b ) and gd(5b ) reported here , as well as previously reported compound gd(6 ) . compared to gd(6 ) , the affinity of gd(4b ) for hsa was increased 3-fold ( kd = 260 and 80 m , respectively ) . the reason for the higher percentage of bound complex in the case of gd(4b ) is likely the double negative charge , which may provide a stronger interaction with the lysine - rich , positively charged binding pocket of hsa . on the other hand , the zwitterionic gd(5b ) we also measured the relaxivity for these complexes in a 4.5% hsa solution ( table 4 ) . at 20 mhz , the relaxivity of gd(4b ) increased over 5-fold when hsa was added , whereas for gd(5b ) , the increase was less than 3-fold . such a difference could not be accounted for by the difference in the fraction bound to albumin between the two complexes . in the absence of albumin , the relaxivity of gd(4b ) is about 25% higher than that of gd(5b ) , but in the presence of albumin , the relaxivity of gd(4b ) is 120% and 80% higher than that of gd(5b ) at 20 and 60 mhz , respectively ( table 4 ) . in order to better understand this phenomenon , we performed additional r1 measurements at different temperatures and additional field strengths . we acquired relaxivity data for gd(4b ) and gd(5b ) in pbs and 4.5% ( w / v ) hsa at 20 , 30 , 60 , 100 , 200 , and 400 mhz and at 10 , 25 , 37 , and 50 c ( tables s1s4 in the supporting information , si ) . we also measured the fraction ( fb ) of gd(4b ) and gd(5b ) bound to hsa at each of these temperatures ( table s5 in the si ) . because we know the fraction bound to hsa for each complex and we measure the relaxivity of the unbound complex independently , we can calculate the relaxivity due to the hsa - bound species ( r1bound ) for both gd(4b ) and gd(5b ) ( eq 1).1 the magnitude and field dependences of these hsa - bound relaxivities are shown in figure 3 . the high relaxivity of gd(4b ) could not be well described by a pure second - sphere model , which assumes some adjustable number of long - lived water molecules in close proximity to the metal ion . instead , we reasoned that the difference in the hsa - bound relaxivities between gd(4b ) and gd(5b ) was due to the presence of an inner - sphere water ligand on some fraction of the gd(4b ) complexes ( eq 2 ) . we assumed that the difference in the hsa - bound relaxivity between gd(4b ) and gd(5b ) was the contribution from this inner - sphere water ( eq 3 ) . we then calculated this difference at each temperature and field and simultaneously fit the resultant data set using solomon bloembergen morgan theory ( figure 3 and eqs 28 ) . while the hsa - bound relaxivity of gd(4b ) was higher than expected , it was not as high as one would anticipate for a complex with one coordinated water , and so we left the hydration number q as a temperature - dependent adjustable parameter defined by an equilibrium constant . we found that this entire data set could be reasonably well described ( figure 3 ) by five adjustable parameters : q ( mole fraction of q = 1 species at 310 k ) , h ( enthalpy change for equilibrium between q = 0 and 1 species ) , m , h ( activation energy for water exchange ) , and [ square of the trace of zero - field - splitting ( zfs ) tensor ; eqs 38].2345678we found that the data were relatively insensitive to the rotational correlation time as long as this correlation time was large . this is reasonable because the very short water residency time should dominate the correlation time for relaxation . in addition , the data were insensitive to v , the correlation time for electronic relaxation , and so we fixed this to 20 ps , in line with other studies of similar complexes . the quality of the fit could likely be improved by the addition of more adjustable parameters , e.g. , lipari szabo - type treatment , but the conclusions are unlikely to change . relaxivity of hsa - bound gd(5b ) ( filled circles ) and gd(4b ) ( open circles ) at ph 7.4 , at ( a ) 10 c , ( b ) 25 c , ( c ) 37 c , and ( d ) 50 c . the difference between the solid and open circles is the inner - sphere contribution to the gd(4b ) relaxivity , and the solid line is the fit to the data described in the text . the analysis shows that there is a 15% fraction of gd(4b ) that exists as a q = 1 complex with a short residence time for the first sphere water molecule . for the equilibrium in eq 2 , the two reactions are the formation and dissociation of the q = 1 complex . in principle , there can also be water exchange on the q = 1 complex . because we only observe free water , we can not distinguish between these two reactions ( dissociation to a stable q = 0 complex versus water exchange at the q = 1 complex ) . therefore , we likely are measuring the formation and dissociation of water to the gadolinium complex ; i.e. , m , the residence time in the bound state , corresponds to 1/kd . the relatively high relaxivity of gd(4b ) in the presence of hsa , especially at high fields , prompted us to further explore the kinetic inertness of this complex with respect to gadolinium(iii ) dissociation . acetate to methylenephosphonate substitution in lanthanide(iii ) polyaminocarboxylates typically results in a more thermodynamically stable complex ( because of the increased basicity of the phosphonate ) , but at the same time , the complex can become more kinetically labile . we measured the half - life of gd(4b ) under different forcing conditions and compared it directly to the acetate analogue gd(6 ) measured under the same conditions . in the first experiment , we challenged each complex with 1 equiv of ms-325 ligand [ a dtpa derivative of higher thermodynamic stability than gd(dotala ) derivatives ] at ph 3 and 37 c and monitored transchelation by hplc . under these conditions , we found no measurable decomplexation of gd(4b ) , while gd(6 ) underwent complete transchelation to ms-325 with a half - life of 115 h. this result suggests that gd(4b ) is much more thermodynamically stable than ms-325 . in a second experiment , we challenged each complex with a 100-fold excess of citrate at ph 3 and 37 c . with a citrate challenge , we found the opposite behavior : transchelation proceeded to completion in both cases but over 1 order of magnitude more rapidly in the case of gd(4b ) ( t1/2 = 9.5 h ) than gd(6 ) ( t1/2 = 110 h ) ( figure 4 ) . dechelation of gd(4b ) ( filled circles ) and gd(6 ) ( open circles ) at ph 3 and 37 c in the presence of 10 mm citrate . this is in agreement with the previously reported kinetic data obtained for acyclic phosphonate complexes compared to their acetate analogues . we hypothesize that the phosphonate arm promotes the formation of ternary complexes with citrate molecules , which facilitates decomplexation in the case of gd(4b ) . although gd(4b ) was more labile than gd(6 ) , we note that acyclic complexes like [ gd(dtpa)(h2o ) ] or its derivatives , such as [ gd(bopta)(h2o ) ] ( table s5 in the si ) , are much more labile under these conditions , e.g. , reaching transchelation equilibrium with ms-325 ligand in under 10 min . because the high field relaxivity of gd(4b ) was 50% higher than that of the approved contrast agent ms-325 at 4.7 t , we decided to assess its relaxation properties in vivo . in addition , both gd(4b ) and ms-325 showed equivalent affinity to hsa , making this comparison more direct . a catheter was placed in the tail vein so the mouse could be injected while positioned in the scanner . we performed 3d t1-weighted imaging before and immediately after injection of each compound ( 0.1 mmol kg ) . immediately after injection , the blood vessel enhancement observed with gd(4b ) was 38 2% higher than that observed with ms-325 ( figure 5 ) consistent with the higher relaxivity of the former . preinjection , as well as 1 min postinjection , coronal images obtained with ms-325 ( second from left ) and gd(4b ) ( right ) . gd(4b ) shows visibly better contrast in the vena cava , which can be quantified as 38 2% better contrast ( vs muscle ) . the dose of agent , time point , and image windowing was the same for both studies . kidney ( ki ) and vena cava ( vc ) are denoted in the far right image . we have synthesized and evaluated the properties of five different derivatives of a new phosphonate analogue ( dotalap ) of the single amino acid chelate dotala . on the basis of literature examples , we anticipated and observed an increased relaxivity due to the established second - sphere effect of the phosphonate . however , for [ gd(dotala)(h2o ) ] and gd(dotala)-propionamide derivatives , water exchange is already very fast ( m = 9 and 17 ns , respectively ) . for the gd(dotalap ) complexes investigated here , we found that further acceleration of water exchange through crowding of the coordination sphere may instead lead to stabilization of the q = 0 , 8-coordinate state . the five complexes investigated here comprised two that existed primarily as 9-coordinate q = 1 complexes , while the other three appear to be primarily q = 0 , presumably 8-coordinate complexes . these conclusions are supported by o nmr and h relaxivity data on the gadolinium(iii ) complexes and by luminescence lifetime measurements on the europium(iii ) complexes . the energy difference between the 8- and 9-coordinate states is quite small , and peripheral changes to the macrocyclic ligand outside the inner coordination sphere can alter the coordination number in a nonpredictable manner . this delicate equilibrium between q = 0 and rapidly exchanging q = 1 underlines a challenge in the design of gadolinium(iii ) complexes with extremely fast water exchange rates . two of the investigated derivatives , gd(4b ) and gd(5b ) , were designed to bind hsa . the relaxivities of these compounds in the absence of hsa were similar and low , although r1 for gd(4b ) was about 25% higher than r1 for gd(5b ) . this small increase in the relaxivity could be due to the presence of a small amount of q = 1 species for gd(4b ) . however , in the presence of hsa , the relaxivity difference between the complexes is magnified , and this increased difference seen when the molecular tumbling rate is decreased is indicative of a small fraction of q = 1 species . variable - temperature nmrd allowed us to estimate that about 15% of gd(4b ) exists as a q = 1 species at 37 c , at least while it is bound to albumin . the presence of this q = 1 complex whose coordinated water has an extremely short residency time results in remarkably high relaxivities at high field for a complex bound to hsa . for instance , the relaxivity at 4.7 t was 50% higher than that for the albumin - targeted approved agent ms-325 . the nmrd results have profound consequences for the development of slow tumbling contrast agents for high - field applications . the variable - temperature data clearly indicate that the water residency time , m , is the dominant correlation time for modulating the relaxivity in this system . figure 6 shows data at two temperatures and two fields to illustrate this point . at 0.47 t ( 20 mhz ) , m at 2.4 ns is too short for optimal relaxivity , but as the temperature is lowered , m increases and the relaxivity goes up . on the other hand , at higher fields ( > 2 t ) , the optimal correlation time is in the 13 ns range . at 37 c , a m of 2.4 ns is ideal for relaxivity at 2.4 t ( 100 mhz ) . however , as the temperature is lowered and m increases , the correlation time becomes too long for optimizing high - field relaxivity . the very short m provides increased relaxivity at high fields and offers a strategy to increase the relaxivity of slow tumbling entities at high magnetic fields . we note that the data in figure 6 are for a complex that is only 25% ( at 10 c ) or 15% ( at 37 c ) for q = 1 . thus , the relaxivity gains for a single q = 1 species are 46-fold higher . inner - sphere relaxivity of gd(4b ) bound to hsa at two different temperatures ( 10 and 37 c ) and larmor frequencies . because the relaxivity of q = 0.15 gd(4b ) was still clearly enhanced compared to q = 1 ms-325 at higher fields , we considered its evaluation for high - field in vivo imaging . for this , we first tested the kinetic inertness of gd(4b ) under forcing conditions . we found the somewhat decreased kinetic inertness , when compared with the all - acetato analogue gd(6 ) , is a typical reflection of the results previously reported in conjunction of phosphonato compounds . this encouraged the in vivo evaluation and cross - comparison with the approved hsa binding compound ms-325 . the subsequent in vivo imaging experiment at 4.7 t was found to mirror the in vitro results well because we were able to obtain better angiographic contrast for gd(4b ) compared to ms-325 . an increased blood signal is clearly found in the increased relaxivity for this compound at intermediate magnetic fields . we consider this outcome a promising result that encourages us to further investigate m acceleration as a way to enhance the relaxivity at higher fields because an entirely q = 1 complex with such a fast exchange rate would have 5 times the relaxivity observed with gd(4b ) . however , the somewhat unpredictable nature of the compound behavior in terms of the inner - sphere hydration number is a clear challenge . in summary , we have successfully synthesized a monophosphonate analogue of the single amino acid chelator dotala , which we term dotalap . the gd(dotalap ) derivatives show enhanced second - sphere relaxivity compared to gd(dotala ) . in two derivatives , we found increased water exchange kinetics , and this combination of fast water exchange and increased second - sphere relaxivity results in improved relaxometric behavior . we also found , however , that functionalization of this single amino acid chelate of either the nitrogen or carbon terminus with different r groups can sometimes yield gadolinium complexes with no inner - sphere water ( q = 0 ) or a mixture of q = 0 and 1 complexes . this decrease in the hydration number appears as a side effect of accelerated water exchange and does not correlate with a specific r group or location of functionalization . for one derivative , gd(4b ) , where r was chosen such that the corresponding gadolinium complex had affinity to hsa , we found that the hydration number was 0.15 in the presence of hsa , i.e. , a 6:1 mixture of q = 0 to q = 1 . the corresponding mean water residency time obtained for the albumin - bound complex was shorter than the rotational correlation time , which , in this case , leads to m becoming the dominant correlation time for relaxation . because m is considerably shorter than r , higher inner - sphere relaxivity was achieved for this complex at intermediate and high magnetic fields than what can be obtained with the clinically approved hsa targeting agent ms-325 . we have demonstrated the effect of higher relaxivity also in vivo with higher blood vessel to muscle contrast for gd(4b ) when compared to ms-325 . future endeavors will include the identification of fast exchanging complexes that are exclusively q = 1 in order to maximize the relaxivity gains at high fields . h and c nmr spectra were recorded on a varian 11.7 t nmr system equipped with a 5-mm broad - band probe . hplc purification of intermediates was performed on a rainin , dynamax ( phenomenex c18 column : 250 mm 21.2 mm , 10 m ) using method a : 0.1% trifluoroacetic acid ( tfa ) in water with a gradient of 595% ( 0.1% tfa in mecn ) over 20 min with a 15 ml min flow rate . hplc purity analyses ( both uv and ms detection ) were carried out on an agilent 1100 system ( phenomenex luna c18(2 ) column : 100 mm 2 mm , 0.8 ml min flow rate ) with uv detection at 220 , 254 , and 280 nm and positive - mode electrospray ionization ( esi ) using the following methods . method b : solvent a = water , solvent b = mecn ; 260% b over 15 min . method c : solvent a = 10 mm nh4oac , solvent b = 10% 10 mm nh4oac , 90% mecn ; 595% b over 15 min . method d : solvent a = 10 mm nh4oac , solvent b = 10% 10 mm nh4oac , 90% mecn ; 535% b over 15 min . chemicals were supplied by aldrich chemical co. , inc . , and were used without further purification . solvents ( hplc grade ) were purchased from various commercial suppliers and used as received . the monoalkylated cyclen precursor to compound 1 tri - tert - butyl phosphite was synthesized according to a procedure reported by manning et al . amide coupling followed by deprotection of the tert - butyl protective groups was done by activation of the carboxylate with hatu ( 1.2 equiv ) and dipea ( 1.2 equiv ) for 5 min in n , n - dimethylformamide ( dmf ) , followed by the addition of amine ( 1 equiv ) dissolved in dmf and stirring at room temperature for 18 h. after confirmation of the presence of the amide - coupling product by lc ms ( method b ) , the intermediate was isolated by preparative hplc ( method a ) , eluting between 11 and 13 min . the clean fractions containing the desired product were pooled and lyophilized to afford the intermediate as an off - white powder in 1535% yield . redissolution of intermediates described above in a 1:1 mixture of dichloromethane and tfa , followed by stirring for 18 h , afforded the final ligand with quantitative yield . an amount of a stock solution containing lncl36h2o ( 0.95 equiv based on the ligand weight ) is added to the ligand solution under monitoring of the ph . the lightly cloudy solution is filtered and lyophilized to afford the corresponding lanthanide complex as an off - white powder . ms ; the presence of free lanthanides was excluded using the xylenol orange test . 2-[[(benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate ( 1.475 g , 3.05 mmol , 1 equiv ) and paraformaldehyde ( 0.34 g , 11.45 mmol , 3.75 equiv ) were dissolved in tetrahydrofuran and vigorously stirred under a dinitrogen atmosphere for 0.5 h. tri - tert - butyl phosphite ( 1.145 g , 4.5 mmol , 1.5 equiv ) was then added , and the reaction was stirred for 16 h. reaction monitoring by lc ms showed the product as [ m + h ] ( m / z 690.4 ) . the reaction mixture was then filtered , concentrated in vacuo , and redissolved in etoac ( 200 ml ) . the organic layer is washed with 50 ml of saturated na2co3 and 50 ml of brine , dried with sodium sulfate , and concentrated to afford the dialkylated intermediate ( 1.73 g , 2.5 mmol , 82% ) , which was used in the subsequent step without further characterization . k2co3 ( 0.68 g , 4.9 mmol , 2 equiv ) was added , and the mixture was stirred vigorously after the addition of tert - butyl bromoacetate ( 0.766 g , 0.575 ml , 3.94 mmol , 1.6 equiv ) . after 16 h , water ( 70 ml ) was added and most of the mecn was removed in vacuo . the oily residue was taken up in etoac ( 80 ml ) and washed with water and brine ( 100 ml each ) . the organic layer was separated , dried with sodium sulfate , and concentrated to afford the crude product , which was redissolved in mecn ( 5 ml ) and purified using preparative hplc ( method a ) . fractions containing the product are pooled and lyophilized to afford the product as a white powder ( 0.43 g , 0.46 mmol , 20% yield ) . h nmr ( cdcl3 , 500 mhz , ppm ) : 9.9 ( s , br , 2h ) , 7.367.34 ( m , 10h ) , 5.255.16 ( m , 4h ) , 4.3 ( m , 1h ) , 3.352.68 ( m , 21h ) , 2.04 ( s , 4h ) , 1.511.43 ( m , 36h ) . c nmr ( cdcl3 , 125.7 mhz , ppm ) : 175.4 , 170.6 , 170.3 , 135.0 , 131.1 , 129.4 , 128.6127.9 , 81.4 , 81.0 , 67.7 , 57.347.4 , 30.6 , 28.2 , 22.1 . calcd for c47h76n5o11p4 : m / z 917.5 . found : m / z 918.6 ( [ m + h ] ) . compound 1 ( 0.43 g , 0.47 mmol ) was dissolved in etoh ( 40 ml ) . pd / c [ 10% ( w / v ) , 0.215 g ] was added , and the reaction mixture was purged first with dinitrogen and then charged with dihydrogen ( 1 atm ) . the mixture was then stirred under a dihydrogen atmosphere for 3 h , after which the reaction was found to be complete . the pd / c was removed by filtration , and the solvent was removed in vacuo to afford the product as a colorless oil ( 0.32 g , 0.46 mmol , quantitative conversion ) . h nmr ( cd3od , 500 mhz , ppm ) : 5.15 ( s , br , 1h ) , 4.462.48 ( m , 24h ) , 1.631.47 ( m , 36h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 167.9 , 160.5 , 86.6 , 82.6 , 55.248.5 , 30.6 , 29.2 , 26.9 . calcd for c32h64n5o9p : m / z 693.4 . found : m / z 694.7 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 5.15 ( s , br , 2h ) , 4.262.46 ( m , 25h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 175.1 , 169.6 , 55.448.9 . calcd for c16h32n5o9p : m / z 469.2 found : m / z 470.1 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.387.23 ( m , 5h ) , 5.15 ( s , br , 1h ) , 4.76 ( m , 1h ) , 3.982.86 ( m , 24h ) , 1.47 ( t , 3h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 159.4 , 140.9 , 128.5126.8 , 116.7 , 114.4 , 53.3 , 49.5 , 46.1 , 17.1 . calcd for c25h40n5o10p : m / z 601.2 . found : m / z 602.4 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.28 ( d , 2h ) , 7.20 ( d , 2h ) , 4.73 ( s , br , 1h ) , 3.912.87 ( m , 24h ) , 2.45 ( m , 2h ) , 1.84 ( m , 1h ) , 1.481.29 ( m , 5h ) , 0.89 ( d , 6h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 172.8 , 169.6 , 140.5 , 138.2 , 129.5 , 127.0 , 54.748.2 , 30.0 , 21.3 , 17.1 . calcd for c28h48n5o9p : m / z 629.3 . found : m / z 630.5 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.397.26 ( m , 5h ) , 5.02 ( s , br , 1h ) , 4.152.46 ( m , 24h ) , 1.641.46 ( m , 3h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 169.7 , 166.3 , 160.4 , 142.7 , 128.5125.8 , 119.6 , 117.3 , 114.9 , 112.7 , 53.349.5 , 27.2 , 20.85 . calcd for c24h41n6o8p : m / z 573.3 . found : m / z 574.6 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.24 ( d , 2h ) , 7.10 ( d , 2h ) , 4.212.44 ( m , 30h ) , 1.941.29 ( m , 6h ) , 0.89 ( d , 6h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 175.4 , 169.8 , 167.4 , 140.2 , 138.8 , 128.9 , 126.7 , 54.648.1 , 38.2 , 30.1 , 21.3 , 17.1 . p nmr ( cd3od , 202.4 mhz , ppm ) : 6.1 . lc calcd for c31h54n7o9p : m / z 699.3 . found : m / z 700.6 ( [ m + h ] ) . calcd for c16h28gdn5o9p [ gd(3 ) ] : m / z 623.1 . found : m / z 625.1 ( [ m + 2h ] ) . . found : m / z 620.1 ( [ m + 2h ] ) . . found : m / z 757.2 ( [ m + 2h ] ) . calcd for c24h36eun5o9p [ eu(4a ) ] : m / z 750.1 . found : m / z 752.1 ( [ m + 2h ] ) . calcd for c29h44gdn5o10p [ gd(4b ) ] : m / z 811.2 . found : m / z 813.4 ( [ m + 2h ] ) . calcd for c29h44eun5o10p [ eu(4b ) ] : m / z 806.2 . found : m / z 808.4 ( [ m + 2h ] ) . calcd for c29h44tbn5o10p [ tb(4b ) ] : 812.2 . found : m / z 814.4 ( [ m + 2h ] ) . calcd for c24h38gdn6o8p [ gd(5a ) ] : m / z 727.2 . found : m / z 728.2 ( [ m + h ] ) . calcd for c24h38eun6o8p [ eu(5a ) ] : m / z 722.2 . found : m / z 723.3 ( [ m + h ] ) . calcd for c31h51gdn7o9p [ gd(5b ) ] : m / z 852.3 . found : m / z 853.5 ( [ m + h ] ) . calcd for c31h51eun7o9p [ eu(5b ) ] : m / z 849.3 . found : m / z 850.4 ( [ m + h ] ) . luminescence lifetime measurements of europium complexes in h2o and d2o were performed on a hitachi f-4500 fluorescence spectrophotometer . for the measurements in d2o , the complexes were first dissolved in d2o ( 99.98% d ) , lyophilized , and dissolved in d2o again to reduce the amount of residual hdo . measurements were taken with the following settings : excitation at 396 nm ( eu ) emission at 616 nm ( eu ) 30 replicates , 0.04 ms temporal resolution ( 020 ms ) , pmt voltage = 400 v. lifetimes were obtained from monoexponential fits of the data using igor pro software ( version 6.0 , wavemetrics , lake oswego , or ) . o nmr measurements of solutions were performed at 11.7 t on 350 l samples contained in 5 mm standard nmr tubes on a varian spectrometer . the temperature was regulated by an air flow controlled by a varian vt unit . the h2o transverse relaxation times of a > 4 mm solution of all gadolinium complexes ( ph 7.4 , 10 mm pbs buffer ) were measured using a cpmg sequence . the exact concentration of the sample was determined by inductively coupled plasma mass spectrometry ( icp - ms ) . reduced relaxation rates , 1/t2r , were calculated from the difference of 1/t2 between the sample and water blank and then divided by the mole fraction of coordinated water , assuming q = 1 . the temperature dependence of 1/t2r was fit to a four - parameter model as previously described . the gd o hyperfine coupling constant , a/ , was assumed to be 3.8 10 rad s. ultrafiltration measurements of protein binding solutions of a 0.1 mm gadolinium compound and 4.5% hsa ( 200 l total ) were added to a 5000 mw ultrafiltration unit ( regenerated cellulose membrane , millipore , bedford , ma ) . the samples were incubated at a set temperature for 30 min and subsequently centrifuged to filter off the nonprotein - bound components . the concentration of bound complex in the protein samples was determined by subtracting the gadolinium concentration ( unbound complex ) found in the filtrate from the gadolinium concentration of the initial solution . stock solutions of ms-325-l ( the ligand of the ms-325 complex ) , gd(4b ) , and gd(6 ) were prepared . for experiment 1 , ms-325-l was added to solutions of the gadolinium complexes in ph 3 water and incubated at 37 c . a 10 l aliquot was removed for hplc analysis and analyzed at time points 1 , 20 , 50 , 72 , 80 , 96 , 120,168 , 192 , 216 , 240 , 288 , 312 , 336 , 360 , and 480 h , while the remainder of the solution was incubated at 37 c . for experiment 2 , the gadolinium complexes were added to a 10 mm ph 3 citrate buffer , affording a 0.1 mm complex concentration , and incubated at 37 c . a 10 l aliquot was removed and analyzed at 0 , 0.75 , 1.5 , 2 , 3 , 4 , 5 , 6 , 24 , 72 , 96 , 120 , 168 , 192 , 216 , 240 , 288 312 , 336 , and 408 h. nu / nu mice were anesthetized with isoflurane ( 12% ) and placed in a specially designed cradle with the body temperature maintained at 37 c . the tail vein was cannulated for intravenous delivery of the contrast agent while the animal was positioned in the scanner . imaging was performed at 4.7 t , using a custom - built volume coil to acquire coronal t1-weighted images with a field of view that covered the major organs of the thorax and abdomen ( heart , stomach , liver , intestines , and kidney ) . t1-weighted images were acquired using a 3d fast low angle shot ( flash ) sequence : tr / te / flip angle = 15.3 ms/1.54 ms/40 , fov = 4.8 2.4 2.4 cm , matrix = 192 96 96 , 1 average .

here , we describe the synthesis of the single amino acid chelator dotalap and four of its derivatives . the corresponding gadolinium(iii ) complexes were investigated for their kinetic inertness , relaxometric properties at a range of fields and temperatures , water exchange rate , and interaction with human serum albumin ( hsa ) . derivatives with one inner - sphere water ( q = 1 ) were determined to have a mean water residency time between 8 and 6 ns in phoshate - buffered saline at 37 c . the corresponding europium complexes were also formed and used to obtain information on the hydration number of the corresponding coordination complexes . two complexes capable of binding hsa were also synthesized , of which one , gd(5b ) , contains no inner - sphere water , while the other derivative , gd(4b ) , is a mixture of ca . 15% q = 1 and 85% q = 0 . in the presence of hsa , the latter displayed a very short mean water residency time ( m310 = 2.4 ns ) and enhanced relaxivity at intermediate and high fields . the kinetic inertness of gd(4b ) with respect to complex dissociation was decreased compared to its dotala analogue but still 100-fold more inert than [ gd(bopta)(h2o)]2. magnetic resonance imaging in mice showed that gd(4b ) was able to provide 38% better vessel to muscle contrast compared to the clinically used hsa binding agent ms-325 .

Introduction Results Discussion Conclusions Experimental Procedures

in the previous issue of critical care , kmpers and colleagues demonstrated a direct correlation between increased peripheral blood levels of the vascular growth factor , angiopoietin ( angpt)-2 , and mortality in 43 critically ill adults with sepsis . endothelial injury is one of the main hallmarks of sepsis , leading to capillary leak , microcirculatory dysfunction , organ failure , and eventual death in many critically ill patients . angpt-1 and angpt-2 are two of the best - characterized members of a family of endothelial - derived vascular growth factors necessary for both normal and pathologic angiogenesis and vasculogenesis . both angpt-1 and angpt-2 appear to bind to the tyrosine kinase receptor , tie-2 , found primarily on the luminal surface of endothelial cells . recent studies have also shown that the tie-2 receptor may be found on certain populations of peripheral blood monocytes , although the function and role of the tie-2 receptor in the host innate immune response remain relatively unexplored . angpt-1 is a tie-2 agonist and promotes endothelial stabilization and quiescence , whereas angpt-2 is a tie-2 antagonist and promotes endothelial activation , destabilization , and inflammation . as such , the relative balance between angpt-2 and angpt-1 at the tie-2 receptor may be more relevant to the pathobiology of sepsis than the absolute levels of the individual growth factors . several studies have demonstrated increased peripheral blood levels of angpt-2 in critically ill patients with sepsis [ 5,7 - 9 ] , multiple trauma , acute lung injury ( ali ) , and cardiopulmonary bypass when compared with healthy controls . more importantly , increased angpt-2 levels appear to be associated with adverse outcomes [ 5,6,9 - 12 ] . for example , the study of kmpers and colleagues showed that increased peripheral blood angpt-2 levels correlated with surrogate markers of tissue hypoxia , disease severity , and mortality in 43 critically ill adults with sepsis . also of note , consistent with the opposing roles of angpt-2 and angpt-1 on the tie-2 receptor , peripheral blood levels of angpt-1 were significantly lower in the patients with sepsis compared with healthy controls . unfortunately , in the study of kmpers and colleagues , similar to the aforementioned studies , the temporal kinetics of angpt-1 and angpt-2 were not assessed as blood samples were collected upon the first day of admission to the intensive care unit only . angpt-2 is stored in the weibel - palade bodies within endothelial cells in a more or less prepackaged form . it is therefore not surprising that angpt-2 levels are increased early in response to endothelial activation or injury . whether angpt-2 levels remain increased in critically ill patients with sepsis has not been directly addressed and is a question for future investigation . it is certainly tempting to speculate that peripheral blood angpt-2 levels would be an ideal biomarker of early endothelial activation and injury . similarly , whether angpt-1 levels remain decreased in critically ill patients who eventually succumb to their illness is an interesting question . angpt-1 may be a biomarker of endothelial recovery ; however , given its purported anti - inflammatory role , angpt-1 would appear to be an attractive therapeutic target as well . to this end , several studies have suggested that manipulating the ratio of angpt-2 to angpt-1 by augmenting angpt-1 levels may represent an ideal therapeutic strategy for patients with sepsis and ali . important translational laboratory studies are necessary to show that increased angpt-2 levels in critically ill patients are more than just an epiphenomenon . the role of angpt-2 in the pathobiology of sepsis and ali needs to be further elucidated by using in vitro cell - based studies and animal models of critical illness . similarly , the presence of the tie-2 receptor on certain subpopulations of peripheral blood monocytes suggests a larger role for angpt-2 in the host innate immune response . finally , manipulation of the angpt / tie-2 system may be a rational therapeutic strategy for the management of critically ill patients with sepsis and ali . all of these questions remain an active focus in several laboratories , including our own . the authors ' research is funded by the national institutes of health ( bethesda , md , usa ) ( grant numbers 5ko8gm077432 and 1r03hd058246 ) .

the early recognition and management of sepsis remain the greatest challenges in the field of critical care medicine . endothelial injury is one of the hallmarks of sepsis , leading to capillary leak , microcirculatory dysfunction , organ failure , and eventual death in many critically ill patients . the angiogenic growth factors , angiopoietin ( angpt)-1 and angpt-2 , act upon the tie-2 receptor in opposing roles . angpt-2 has been found in abundance in septic patients when compared with healthy controls . in the study by kmpers and colleagues in the previous issue of critical care , angpt-2 levels correlated with markers of tissue hypoxia , disease severity , and mortality in septic adults . however , the temporal kinetics of the angiopoietins were not assessed . it remains to be seen whether angpt-2 levels will function solely as an early marker of sepsis or whether the manipulation of the angpt / tie-2 system will become a rational therapeutic target for the management of sepsis .

None Abbreviations Competing interests Acknowledgements

we compared monthly listeriosis data from england and wales with temperature records from 1989 through 2007 to determine the influence of various potential predictors on the number of listeriosis cases . uk health protection agency ( hpa ) data listing total monthly cases of human listeriosis in england and wales during 19902007 ( 4,5 ) are aggregate . all age categories and regions were included and were collated by the hpa centre for infections from voluntary reporting by microbiology laboratories and from referrals of cultures . these publicly available data were also validated by the hpa , and in our analysis we used revised figures based on that validation . our analysis covered the period from 1990 , when active surveillance of listeriosis began , through 2007 . undated cases that could not be assigned to a particular month were excluded from analysis . we used the uk met office mean monthly area temperature time series for 19892007 and 30-year means averaged during 19611990 ( 6 ) . exploratory linear regression analyses suggested a positive correlation between the number of listeriosis cases and the monthly mean uk ambient temperature , as well as suggesting a change in this relationship after 2000 ( p = 0.001 ; figure 1 , panel a ) . however , residual variability was not constant , and the monthly counts are likely to be overdispersed due to clustering of cases ( 3 ) . the data were fitted again by using a negative binomial generalized linear model with a logarithmic link function , a common model for time series of foodborne illness cases ( 7,8 ) . to separate seasonality of listeriosis rates from dependence on temperature , we considered the 30-year mean monthly temperatures from 19611990 , as well as monthly temperature anomalies ( observed mean temperature minus 30-year mean ) . to determine whether temperatures could have a delayed effect on listeriosis incidence , we also included mean and anomaly temperature variables lagged by 1 or more months . to allow for 2 break points at which the incidence of listeriosis may have suddenly changed , dummy variables were used to represent periods before , between , and after the months in which these increases might have occurred . a best - fit model was selected according to the corrected akaike information criterion ( aic ) ( 9 ) by using stepwise regression at all combinations of 2 break point months from january 1996 through december 2007 . to examine changing effects on incidence , interactions between break point indicators and other variables were considered , even if the main effects were not yet in the model . main effects were subsequently added to the final model if any interaction terms were included . a ) listeriosis cases compared with mean observed monthly uk temperatures , 19902000 ( triangles ) and 20012007 ( circles ) . shown are an increased overall incidence in 20012007 ( dashed line ) versus 19902000 ( solid line ) and a significant change in the linear dependence of incidence on temperature ( p = 0.001 ) . b ) best - fit pair of break points and other pairs of break points with support , according to the corrected akaike information criterion ( aic ) . the 2 break - point months are varied to find the lowest value ( black square ) . pairs of break points with good support relative to the best model ( corrected aic within 4 of the best fit ; dark gray squares ) or moderate to weak support ( corrected aic within 10 of the best fit ; light gray squares ) are also shown . pairs of break points with little or no support ( corrected aic > 10 greater than the best fit ; white area ) include those models for which only 1 break point exists ( squares along the dashed line ) . the best - fit model included 2 break points ( tables 1 , 2 ; figure 1 , panel b ; figure 2 ) . the overall rate of cases increased by 40.83% in april 2001 ( adjusted p = 0.001 , 95% confidence interval [ ci ] 17.29%68.93% ) ; a further increase of 47.76% occurred in march 2003 ( adjusted p<0.001 , 95% ci 27.92%71.13% ) . according to the corrected aic , there is statistical evidence of changes in the incidence of listeriosis in a range of months around these best - fit values , and weak support for a change in early 2005 , apparently due to low numbers of cases in the first months of that year and the following winter ( figure 1 , panel b ) . most of the seasonality in the number of cases was accounted for by the 30-year mean monthly temperatures . each extra degree celsius of mean monthly temperature corresponded to a 2.42% increase in cases in the current month ( adjusted p = 0.044 , 95% ci , 0.55%4.33% ) and a 4.09% increase in the following month ( adjusted p<0.001 , 95% ci 2.19%6.03% ) . the 1-month lag reflects delays between food production and consumption plus the known long incubation of listeria infections . a relationship also appears to exist between the rate of listeriosis and the 1-month lagged temperature anomaly starting in april 2001 , which corresponded to an additional 5.71% of listeriosis cases per degree celsius ( 95% ci 2.10%14.15% ) when compared with the main effect of temperature anomaly in 19902007 . this increase was not significant ( adjusted p = 0.312 ) , although the power of this test is only 15% at an adjusted significance level of 0.05 ( post hoc power analysis performed by computer simulation of the fitted model in the r statistical computing environment ) . the model also includes a slight negative trend in cases over time ( 0.13% per month , 95% ci 0.26%0.01% ) , but evidence for this trend is very weak ( adjusted p = 0.188 ) . * p values are for 2-sided tests of significance of the difference of each coefficient from zero ; adjusted p values are according to holm s method ( 10 ) . percentage changes in the main text can be derived from these coefficients by taking exponentials , subtracting 1 , and multiplying by 100 . the monthly number of listeriosis cases ( circles , lower plot ) is strongly seasonal , with a close relationship to the monthly mean temperature ( solid line , upper plot ) . overall listeriosis incidence per month underwent 2 sudden increases , at break points around april 2001 and march 2003 . our fitted statistical model ( dashed line , lower plot ) provides a close fit to the observed data ; the seasonally - adjusted fitted model ( solid line , lower plot ) shows the large magnitudes of the jumps in the rate of cases at the 2 break points . the changes in the incidence of listeriosis appear to have been quite sudden ( figure 2 ) . causative explanations based on gradual demographic or behavioral changes have previously been ruled out ( 3 ) , and those based on dynamic processes seem unlikely because no evidence exists for epidemiologic feedback between the source of infections and clinical cases . one possibility is that the increases are due to contamination of a small number of food products , as has been suggested as an explanation for an upsurge in listeriosis rates in the late 1980s . however , a restricted range of strains was responsible for most of the additional cases at that time ( 12 ) , and no evidence exists of such a pattern since 2001 ( 3 ) . an alternative possibility is that the phenomenon is a consequence of changes in government policy or business practices that have had more widespread effects on food processing , distribution , or preparation . we found notable coincidences between the dates of the increases in listeriosis infection rates and the dates of events associated with the 2001 foot and mouth disease ( fmd ) outbreak in the united kingdom . the april 2001 increase in listeriosis rates occurred shortly after the outbreak of fmd in february 2001 , allowing for a delay similar to the incubation period of listeria infections . the march 2003 increase in listeriosis rates occurred in the same month as the relaxation of movement restrictions on livestock instituted after the 2001 fmd outbreak ( 13 ) the diverse and widespread consequences of the 2001 fmd outbreak are well - documented ( 14,15 ) , and it seems plausible that such major disturbances to agricultural production could be the ultimate cause of the large increases in listeriosis rates that have been observed . the coincidence of these events raises the possibility that the change in listeriosis rates would be an unrecognized outcome of the 2001 uk fmd crisis , although we caution that our analysis of the of these cases does not demonstrate whether a causative link exists . listeriosis incidence in england and wales has increased notably since the beginning of 2001 , with 2 separate , sudden increases recorded in april 2001 and march 2003 . gillespie et al . argue that blame for these increases can not be ascribed to any of a variety of specific factors ( 3 ) . instead , more widespread changes affecting food production , processing , or consumption could be the root of the problem . incidence of this serious disease has risen substantially in england and wales , and an understanding of why will be important for management of listeriosis as a public health issue .

the monthly incidence of listeriosis infections in england and wales had 2 sudden increases during april 2001 ( 41% ) and march 2003 ( 48% ) . although no causative association is demonstrated , these increases correspond to key dates relating to the onset and aftermath of the 2001 foot and mouth disease outbreak in the united kingdom .

The Study Conclusions

attachment is a relatively stable emotional bond which is created between child and mother or those with whom an infant regularly interacts . parents responses to the signs of child 's attachment behavior and their availability in stressful situations , provides a safe place and condition for children , based on which , children organize their expectations from the environment . the attachment between child and primary caregiver ( usually mother ) would become internalized and later act as a mental model which is used by the adult person to use as a base for building friendship and romantic relationships ; it can affect the attitudes of people in their adulthood as well . adult attachment styles are subdivided into three categories : ( 1 ) secure : secure people are intimate and comfortable in making relationships , and they are sure that others would like them . ( 2 ) anxious - ambivalent : they have a strong desire for close relationships but also have many concerns of rejection . these people have a negative image of themselves , but a positive attitude toward others . ( 3 ) avoidance : for this group of people , self - reliance is the most valuable issue . hence , it can be said that attachment styles affect other aspects of one 's life and have an impact on persons relationships with other people after childhood . many researchers and authorities have shifted their focus toward the topics such as joy , happiness , life satisfaction , and positive emotions . according to many theories of emotions , one of the six great emotions is happiness ; the six great emotions include surprise , fear , anger , happiness , disgust , and worry . happiness is a type of conception about individual 's own life ; it includes items such as life satisfaction , positive emotions , and mood , lack of anxiety and depression and its different aspects of emotions . when people are satisfied with their living conditions and are frequently experiencing positive and less negative emotions , it is said that they are at high levels of mental health . increased levels of happiness is directly associated with the better status of health , appetite , sleep , memory , family relationships , friendships , family status , and ultimately mental health . the relationship between subjective well - being and emotion regulation with attachment styles in various studies has been explained . despite the important role of medical students in public health and the significance of their happiness which is related to their attachment styles , so far , this research was aimed to assess the relationship between attachment styles and happiness and demographic characteristics of medical students . this descriptive and analytical study was conducted on medical students in kurdistan university of medical sciences , in 2012 . as exclusion criteria , students who were unwilling to fill out a questionnaire and guest students since there were five independent variables in the study and it was needed to include 35 samples for each variable in the regression model , the calculated sample size was 175 people ; a total of 200 students were included in the study . samples were chosen through stratified sampling method ( different levels of education ) and each stratum was proportional to the size of each class . to collect the data , after obtaining permission from the ethics committee of kurdistan university of medical sciences , list of all medical students , which was classified by educational level , was obtained from education office . the samples were systematically selected from the list provided by education office ; they were selected in proportion to the number of students in each educational level ( physiopathology , extern , intern level ) . after taking their consent to participate in research and explaining the objectives , questionnaires were given to the participants . the questionnaires were filled out by the students and were collected the same day . before completing the questionnaire ( 47 questions ) , students were assured that all information will be confidential , and they were also asked to answer the questions accurately . they were allowed to ask their questions in case of facing any ambiguity in the questionnaire . this scale is developed by hazan and shaver ( 1987 ) and it has 15 items , with five items for each of the three types of secure attachment , ambivalent attachment , and avoidant attachment style . it is scored from never ( zero ) to almost always ( score = 4 ) . the score of each attachment subscale is obtained by calculating the mean of five items for each subscale . in various studies , the reliability of the questionnaire has been calculated from 0.78 to 0.81 ; moreover , its reliability in iranian culture was tested by boogar et al . , the obtained results for the entire test , the ambivalent , avoidant , and secure attachment styles were 0.75 , 0.83 , 0.81 , and 0.77 , respectively . to measure the happiness variable , the scale has 29 items which is scored on a range of zero to four ; it has five marks including life satisfaction with eight items , self - esteem with seven items , subjective well - being with five items , satisfaction with four items , and positive manner with three items . because two items have a correlation coefficients of < 35% with any of the five other components , they are not included in any of the components , but they are included in the total score . the reliability of this scale among iranian students has been reported to be 0.93 . the collected data were entered into spss version 16 ( ibm , chicago il , usa ) . quantitative data were described using the mean and standard deviation ( sd ) , and string variables were described using frequency and percentage . the correlation between happiness score and attachment style scores were assessed using pearson 's correlation coefficient . the difference between the happiness score and the scores of different attachment styles in each sex were compared using independent tests . the scores for different educational levels were compared using one - way anova . finally , using multiple regressions ( enter method ) , happiness variable as the dependent variable and the score of different attachment styles , gender , educational level , and grade point average ( gpa ) as the independent variables , if applicable , were entered into the model . this scale is developed by hazan and shaver ( 1987 ) and it has 15 items , with five items for each of the three types of secure attachment , ambivalent attachment , and avoidant attachment style . it is scored from never ( zero ) to almost always ( score = 4 ) . the score of each attachment subscale is obtained by calculating the mean of five items for each subscale . in various studies , the reliability of the questionnaire has been calculated from 0.78 to 0.81 ; moreover , its reliability in iranian culture was tested by boogar et al . , the obtained results for the entire test , the ambivalent , avoidant , and secure attachment styles were 0.75 , 0.83 , 0.81 , and 0.77 , respectively . to measure the happiness variable , the scale has 29 items which is scored on a range of zero to four ; it has five marks including life satisfaction with eight items , self - esteem with seven items , subjective well - being with five items , satisfaction with four items , and positive manner with three items . because two items have a correlation coefficients of < 35% with any of the five other components , they are not included in any of the components , but they are included in the total score . this scale is developed by hazan and shaver ( 1987 ) and it has 15 items , with five items for each of the three types of secure attachment , ambivalent attachment , and avoidant attachment style . it is scored from never ( zero ) to almost always ( score = 4 ) . the score of each attachment subscale is obtained by calculating the mean of five items for each subscale . in various studies , the reliability of the questionnaire has been calculated from 0.78 to 0.81 ; moreover , its reliability in iranian culture was tested by boogar et al . , the obtained results for the entire test , the ambivalent , avoidant , and secure attachment styles were 0.75 , 0.83 , 0.81 , and 0.77 , respectively . to measure the happiness variable , the revised oxford happiness inventory was used which had an overall reliability of 0.91 . the scale has 29 items which is scored on a range of zero to four ; it has five marks including life satisfaction with eight items , self - esteem with seven items , subjective well - being with five items , satisfaction with four items , and positive manner with three items . because two items have a correlation coefficients of < 35% with any of the five other components , they are not included in any of the components , but they are included in the total score . the collected data were entered into spss version 16 ( ibm , chicago il , usa ) . quantitative data were described using the mean and standard deviation ( sd ) , and string variables were described using frequency and percentage . the correlation between happiness score and attachment style scores were assessed using pearson 's correlation coefficient . the difference between the happiness score and the scores of different attachment styles in each sex were compared using independent tests . the scores for different educational levels were compared using one - way anova . finally , using multiple regressions ( enter method ) , happiness variable as the dependent variable and the score of different attachment styles , gender , educational level , and grade point average ( gpa ) as the independent variables , if applicable , were entered into the model . the mean ( sd ) of participants age was 22.42 ( 2.45 ) years . of all , 122 students ( 61% ) were female and 185 persons ( 92.5% ) were single . a total of 89 students ( 44.5% ) were in basic sciences educational level and the majority of participants , i.e. , 97 students ( 48.5% ) had gpa of 1517 [ table 1 ] . the distribution of demographic variables in studied subjects overall , the mean ( sd ) score of happiness was 62.71 ( 17.61 ) , secure attachment style was 11.46 ( 2.56 ) , avoidant attachment style was 9.34 ( 3.32 ) , and ambivalent attachment style was 7.93 ( 3.47 ) . there was no significant relationship between gender and attachment styles , however , the happiness score was 67.2 ( 17.2 ) in men and 59.9 ( 17.36 ) in women , and the difference was statistically significant ( p = 0.005 ) . the avoidant attachment style was 9.48 ( 3.34 ) in singles and 7.6 ( 2.66 ) in married people , and the difference was also statistically significant ( p = 0.03 ) [ table 2 ] . the relationship between gender and marital status of the studied subjects with attachment styles and happiness scores there was no significant relationship between the happiness score and educational level . the score of secure attachment style in students with gpa of 1720 was about 9.91 ( 2.9 ) , which was lower compared to those with lower gpas ( p = 0.051 ) . no significant relationship was observed between happiness score and other attachment styles with students gpas . age was not significantly correlated with happiness scores ( p = 0.797 , r = 0.019 ) . in the multivariate analysis , the relationship between attachment styles and happiness scores were compared and the results showed that after controlling for important factors , the variables of secure attachment style ( p = 0.001 ) , male gender ( p = 0.004 ) , and gpa ( p = 0.047 ) were associated with higher happiness scores ( r = 0.180 ) [ table 3 ] . comparison of the relationship between happiness scores and attachment style and other variables using multiple regression analysis the most common attachment style among students was secure attachment style that was consistent with the results of other studies . secure attachment style leads to activation of a system which bowlby calls the discovery system . this system allows a person to explore his / her environment and experience its own ability to control the condition . secure attachment gradually creates a sense of mastery and ability to handle frustration , and finally , in the context of a secure attachment relationship , then the person is enabled to reflect his / her emotions and positive beliefs about personal values and effectiveness . positive perfectionism , self - esteem , personal control , greater happiness in relationships better emotional management , less stress , and greater job satisfaction are among the specifications of secure attachment style ; these features may be a positive prognostic factor in medical students who usually endure much stress . in our study , the minimum frequency was observed in ambivalent attachment style ; our finding was similar to other studies . in asgharinejad et al.s study as well as ahadi et al.s study avoidant attachment style was the most common and secure attachment was second common style . due to differences in statistical samples and scales , which have been used in these two studies , these differences can be justified . attachment theory is focused on cognitive schema ; the schema affects the organization of individual 's relations with others and his / her perceptions of the world around . attachments formed in childhood can affect adulthood and the attachment between child and primary caregiver ( usually mother ) is internalized and serves as a mental model . according to the mentioned explanations , we can conclude that attachment styles are formed based on schemas and inner experiences , experiences which obtained through interaction with parents and others over time , the role of these factors is much stronger than the effect of gender alone . according to our results , there was a significant relationship between avoidant attachment style and marital status , and avoidant attachment style was more common among single people than married ; so , avoidant attachment could be a barrier to marriage . finney and noler believe that adults with avoidant attachment style have the same characteristics as those with dismissive attachment style ( self - positive model , others negative , with a low anxiety , and high avoidance ) . people with avoidant attachment styles have a negative attitude toward others and have difficulty in communicating with others and maintaining relationships ; they have a high sense of self - esteem and put low values on close relationships with others , which confirms our findings . the results showed no significant relationship between attachment style and gpa of individuals ; however , secure attachment style was less common in participants with high gpa . individuals with a secure attachment style are better able to interact with the environment , so they are expected to have better educational status , but the results of our study did not confirm this idea . it might be that struggling to get a higher score , sometimes help individual to compensate for a sense of frustration and low self - control . it is also possible that the educational system would create an unhealthy competitive environment and promote negative behaviors such as blind imitation without critical thinking . on the other hand , in our study , it was not determined to which educational level and age range each gpa belongs . in addition , the effects of other factors were not considered , and they have not even been considered in other studies as well , and this is one of the limitations of our study . in sheikhmoonesi et al.s study the average score of subjects in the happiness inventory was 41.23 and the average score of happiness in students of tehran university of medical sciences in 2010 was 47.13 . based on these results , our students had higher levels of happiness which could be due to facilities , the status of their field of study and university , their future career perspectives , and their inner attitudes . on the other hand , the statistical sample size , the age range , and demographic conditions can justify these differences . in our study , secure attachment style was associated with higher happiness scores and this finding was consistent with the findings of other studies . people with secure attachment style are successful in making relationships with others and have positive attitudes about self and others ; the mentioned items are effective in creating higher levels of happiness . researches also show that people with insecure attachment styles are more affected with emotional and psychological challenges and with increasing the feeling of helplessness in the marital relationship , they will be at lower levels of happiness . in a study , girls with secure attachment style , compared with girls with avoidant attachment style , were more satisfied with relationships with their fathers . as another results of our study , there was a significant relationship between happiness scores and gender ; accordingly , the happiness scores in boys was higher than that in girls . in keshavarz et al.s study , contrary to the results of our study , there was a positive relationship between female sex and happiness that could be due to differences in the studied populations . we studied students , while in keshavarz et al.s study , yazd population ( males and females ) were studied . study , no significant relationship was observed between sex and happiness . however , in solymani 's et al . study , men achieved higher scores in subscales of life satisfaction and self - esteem while men had higher scores in a positive manner and inner satisfaction . to interpret these differences , it can be said that working and educational condition , society 's attitudes toward gender , which is strongly influenced by cultural factors , can affect a person 's happiness . in our study , there was a negative correlation between age and happiness scores ; however , this relationship was not significant . in sheikhmoonesi et al.s study the happiness scores in people aged below 22 years were higher than that in people aged more than 22 years . to justify the consistency between the two studies , we can note the similarities in the field of study and age range . in keshavarz et al.s study , older age was associated with greater happiness which could be due to differences in population and age range . in boogar et al.s study , job satisfaction among younger nurses was higher than that in older people . in our studied population , individuals at different ages are not facing the same stressors and expectations ; indeed , the course materials , environmental conditions , and people whom they are communicating with ( professors , personals working in different wards , and patients ) are different at any stage . life satisfaction is not an objective and stable trait , rather it is sensitive to situational changes and is shaped based on individual 's perceptions and perspectives . in multiple regression analysis which was performed with the control of key factors , variables of secure attachment style , gender , and gpa were associated with higher happiness scores . such an analysis has not been carried out in other studies and is one of the strengths of our study . the higher gpa was associated with higher happiness scores and other studies have not addressed this issue . there was higher level of dissatisfaction and expectation among people with lower gpas ; on the other hand , students with higher gpas are dealing with more stress of keeping current situation and they have more competition with others . moreover , mediocre gpa did not indicate higher dissatisfaction , and it might even signify less competitive pressure and family expectations ; this greatly originates from individual 's attitudes and expectations . perfection - seeking individuals may excessively get higher scores , but they are less satisfied and happy . according to our results , the satisfaction score was not significantly associated with educational level which was consistent with the results of sheikhmonesi et al.s study . every educational level brings up different external conditions and stressors which may have different effects depending on the internal characteristics , student 's ability to cope with environment , and individual 's expectation , behavior , and social interaction with others . based on the findings of this study , the most common attachment style was secure attachment style , which could be a positive prognostic factor in medical students , helping them to manage stress . the frequency of avoidant attachment style among single persons was higher than that in married people , which is mainly due to their negative attitude toward others and failure to establish and maintain relationships with others . the variables of secure attachment style , male gender , and average gpa were associated with higher happiness scores these factors can be taken into account while planning for promoting happiness levels in students .

background : attachment theory is one of the most important achievements of contemporary psychology . role of medical students in the community health is important , so we need to know about the situation of happiness and attachment style in these students.objectives:this study was aimed to assess the relationship between medical students attachment styles and demographic characteristics.materials and methods : this cross - sectional study was conducted on randomly selected students of medical sciences in kurdistan university , in 2012 . to collect data , hazan and shaver 's attachment style measure and the oxford happiness questionnaire were used . the results were analyzed using the spss software version 16 ( ibm , chicago il , usa ) and statistical analysis was performed via t - test , chi - square test , and multiple regression tests.results:secure attachment style was the most common attachment style and the least common was ambivalent attachment style . avoidant attachment style was more common among single persons than married people ( p = 0.03 ) . no significant relationship was observed between attachment style and gender and grade point average of the studied people . the mean happiness score of students was 62.71 . in multivariate analysis , the variables of secure attachment style ( p = 0.001 ) , male gender ( p = 0.005 ) , and scholar achievement ( p = 0.047 ) were associated with higher happiness score.conclusion:the most common attachment style was secure attachment style , which can be a positive prognostic factor in medical students , helping them to manage stress . higher frequency of avoidant attachment style among single persons , compared with married people , is mainly due to their negative attitude toward others and failure to establish and maintain relationships with others .

Introduction Materials and Methods Data collection tools Hazan and Shaver's attachment style measure Oxford happiness inventory Data analysis Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

human acute radiation syndrome ( ars ) follows intense , acute whole - body or significant partial - body radiation , of doses > 1 gy , delivered at relatively high rates . clinical components of ars include the hematopoietic sub - syndrome ( h - ars , 2 6 gy ) , gastrointestinal sub - syndrome ( gis ; 6 8 gy ) and the cerebrovascular ( > 8 gy ) sub - syndrome . dividing ars into these sub - syndromes oversimplifies the clinical reality of ars as it often involves complex , concurrent and multi - organ dysfunctions . cerebrovascular sub - syndrome is considered incurable , whereas h - ars alone or in combination with gis , are more likely to be amenable to countermeasures ; therefore , the later two sub - syndromes are specific targets for the development of novel medical countermeasures ( mcm ) . there are several biologics at different developmental stages to be considered as mcm for ars ( figure 1 , tables 1 and 2 ) . a brief description and current status of promising biologics are provided in this article . schematic representation of the biological agents as radiation countermeasures under development . currently , there are three agents with fda ind status : entolimod , hemamax and neupogen . neupogen and leukine have been procured for sns availability and are expected to obtain fda eua in the near future . pegylated g - csf is not currently stocked in the sns but may also obtain fda eua approval once filgrastim is approved . ars : acute radiation syndrome ; eua : emergency use authorization ; fgf : fibroblast growth factor ; ind : investigational new drug ; sns : strategic national stockpile . ars : acute radiation syndrome ; eua : emergency use authorization ; fgf : fibroblast growth factor ; ind : investigational new drug ; sns : strategic national stockpile . biologics with us fda ind / procured for sns / close to fda approval . gi : gastrointestinal ; ind : investigational new drug ; nhp : nonhuman primate ; pahpra : pandemic and all hazards preparedness reauthorization act ; rhuil-12 : recombinant human il-12 ; sns : strategic national stockpile . we are unable to cite all relevant references because of limitation of total number of references for this article . ars : acute radiation syndrome ; fgf : fibroblast growth factor ; fgf - p : fibroblast growth factor - derived peptide ; gi : gastrointestinal ; gis : gastrointestinal sub - syndrome ; nhp : nonhuman primate . biologics are evaluated for marketing by the fda through the filing of a biologic license application ( bla ) , the equivalent to new drug application for other agents . the agents discussed here qualify for consideration by the fda under bla for approval . since conventional human clinical efficacy trials for ars mcm are not possible due to ethical reasons , these trials are substituted with animal efficacy rule , a very stringent and possibly more difficult fda approval pathway . the criteria required to move through this approval process include : well - characterized animal model(s ) that is predictive of human response , a good understanding of the mechanism of action of radiation injury and that of the mcm , study end point focused on prevention of mortality or major morbidity , as well as the good understanding of pharmacodynamics so that the effective human mcm doses can be determined . this pathway relies heavily on a large animal model for preclinical safety and efficacy studies . the majority of agents at advanced stages of development have received fda fast track and orphan drug statuses . the fda fast track approval process is designed to facilitate development and expedite the review and approval processes for new treatments of serious or life - threatening conditions . radiation mcm for ars are considered emergency need drugs . the us emergency use authorization ( eua ) program , established by project bioshield , is a critical tool for the medical and public health communities . it permits the fda to approve the emergency , off - label use of products approved for other indications or the use of drugs , devices and medical products that currently have no prior approval , clearance or licensing by fda . it is applicable to both civilian and military use , and it fills the need for timely medical treatment in emergency situations . the strategic national stockpile ( sns ) program ensures that such agents are appropriately pre - positioned so that they are readily available and easily accessible for state and local public health agency distribution in the event of a national emergency . as stated above , there are several biologics under development as radiation mcm for ars ( tables 1 and 2 , figure 1 ) . neupogen and two others , entolimod and hemamax , have considerable efficacy and safety profiles and have received fda investigational new drug status for clinical investigation . mechanistic studies have suggested that the various countermeasures for ars have different modes of action ( figure 2 ) . simplified representation of systemic biological effects due to radiation exposure , with promising biologics intervening at various steps . radiation induces free radical formation , dna damage and apoptosis , which can then lead to ars or death . various biologics are able to minimize the damaging effects of irradiation through different mechanisms of action . ars : acute radiation syndrome ; fgf - p : fibroblast growth factor - derived peptide ; mpc : myeloid progenitor cells . ars : acute radiation syndrome ; fgf - p : fibroblast growth factor - derived peptide ; mpc : myeloid progenitor cells . csf have high potential and well - documented therapeutic efficacy in countering myelosuppression and may receive full licensing approval from the fda in the future . filgrastim , sargramostim and pegylated filgrastim have already been used off - label for treating radiation accident victims . currently , there are four recombinant leukocyte growth factors with bla for related indications : bla 103353 ( neupogen ) , bla 125031 ( neulasta ) , bla 103362 ( leukine ) and bla 125294 ( tbo - filgrastim ) . g - csf / filgrastim has completed a good laboratory practice compliant study in a nonhuman primate ( nhp ) model . the use of growth factors to treat radiation - exposed victims has been rationalized based on three facts : i ) a large clinical database documenting consistent efficacy in mitigating chemotherapy - induced myelosuppression and that associated with stem - cell transplant conditioning regimens as well as consistent safety profile ; ii ) enhanced recovery from radiation - induced myelosuppression in four animal species and improved survival in sublethal and lethally irradiated animal models ; and iii ) demonstration of effective granulopoietic activity in a number of radiation - accident victims . additionally , the american society of clinical oncology extended their recommendation for use of recombinant human g - csf ( rhu g - csf ) and pegylated rhu g - csf to treat patients exposed to therapeutic doses of total - body radiation . radiation - accident reports show that csfs have been used to treat the victims of 16 radiological and nuclear accidents with observed benefits . in three accidents csfs were used within 48 h of accidents ( tokai - mura , soreq and nesvizh ) , but in others csf administration was initiated weeks after the incidence . the limited and anecdotal clinical data available regarding these growth factors validate their biological response ; however , the variable and delayed manner in which these agents were administered makes the csf s role in recovery difficult to determine concisely . during a recently conducted fda meeting , members overwhelmingly voted ( 17:1 ) to support that filgrastim will produce clinical benefits to humans who have been exposed to radiation with doses capable of inducing myelosuppression . the one committee member , who voted no , concluded that those who survive a radiological or nuclear incident will most likely not have received a radiation dose high enough to produce myelosuppressive effects and therefore would not benefit g - csf administration . its pharmacological action is based on its binding to toll - like receptor 5 and the activation of nf-b signaling . studies conducted with entolimod using rodent and nhp ( good laboratory practice ) models suggest that it will be a highly promising treatment for lethally irradiated humans , due to its ability to efficiently ameliorate h - ars and gis , as well as having an extended therapeutic time window after radiation exposure [ 6 - 8 ] . entolimod is currently in clinical development ; a human safety study indicated that it was well tolerated and the biomarker results correspond to data from animal models . ( buffalo , ny , usa ) is preparing a pre - eua application for entolimod . hemamax is recombinant human il-12 ( rhuil-12 ) cytokine and has been shown to increase mice survival when a single dose was administered , either 24 h before or within 1 h after total - body irradiation . allometrically equivalent doses of mouse and human hemamax had similar pharmacokinetics and significantly increased mouse and nhp survival , when administered 24 h post - irradiation , even when no antibiotics , fluids or blood products were administered . to demonstrate the safety of hemamax , neumedicines conducted a phase ib study where healthy volunteers were administered a single dose of hemamax that is predicted to be the effective dose for treating h - ars , based on nhp data ; this trial suggests rhuil-12 to be safe and well tolerated . phase ii equivalent data ( randomized , double - blinded , good laboratory practice ) showed that single administration of rhuil-12 to nhps significantly increased survival and reduced radiation - induced hematopoietic toxicity when administered 24 h post - irradiation . administration of rhuil-12 promotes multilineage hematopoietic recovery , immune functions and possibly , gi functions . neumedicines is developing rhuil-12 for the treatment of h - ars for bla submission to the fda under the animal efficacy rule . several additional biologics have been identified as potential countermeasures and have shown promise in murine and nhp models of ars . some of these agents have already been used off - label in radiological accident victims . hopefully , in the future , they will be fully developed agents to combat radiation injury . some of these potential agents are myeloid progenitors , anti - ceramide antibody , fibroblast growth factor-2 ( fgf-2 ) , its derived peptide ( fgf - p ) , igf-1 and various cytokines ( table 2 ) [ 16 - 18 ] . csf have high potential and well - documented therapeutic efficacy in countering myelosuppression and may receive full licensing approval from the fda in the future . filgrastim , sargramostim and pegylated filgrastim have already been used off - label for treating radiation accident victims . currently , there are four recombinant leukocyte growth factors with bla for related indications : bla 103353 ( neupogen ) , bla 125031 ( neulasta ) , bla 103362 ( leukine ) and bla 125294 ( tbo - filgrastim ) . g - csf / filgrastim has completed a good laboratory practice compliant study in a nonhuman primate ( nhp ) model . the use of growth factors to treat radiation - exposed victims has been rationalized based on three facts : i ) a large clinical database documenting consistent efficacy in mitigating chemotherapy - induced myelosuppression and that associated with stem - cell transplant conditioning regimens as well as consistent safety profile ; ii ) enhanced recovery from radiation - induced myelosuppression in four animal species and improved survival in sublethal and lethally irradiated animal models ; and iii ) demonstration of effective granulopoietic activity in a number of radiation - accident victims . additionally , the american society of clinical oncology extended their recommendation for use of recombinant human g - csf ( rhu g - csf ) and pegylated rhu g - csf to treat patients exposed to therapeutic doses of total - body radiation . radiation - accident reports show that csfs have been used to treat the victims of 16 radiological and nuclear accidents with observed benefits . in three accidents csfs were used within 48 h of accidents ( tokai - mura , soreq and nesvizh ) , but in others csf administration was initiated weeks after the incidence . the limited and anecdotal clinical data available regarding these growth factors validate their biological response ; however , the variable and delayed manner in which these agents were administered makes the csf s role in recovery difficult to determine concisely . during a recently conducted fda meeting , members overwhelmingly voted ( 17:1 ) to support that filgrastim will produce clinical benefits to humans who have been exposed to radiation with doses capable of inducing myelosuppression . the one committee member , who voted no , concluded that those who survive a radiological or nuclear incident will most likely not have received a radiation dose high enough to produce myelosuppressive effects and therefore would not benefit g - csf administration . its pharmacological action is based on its binding to toll - like receptor 5 and the activation of nf-b signaling . studies conducted with entolimod using rodent and nhp ( good laboratory practice ) models suggest that it will be a highly promising treatment for lethally irradiated humans , due to its ability to efficiently ameliorate h - ars and gis , as well as having an extended therapeutic time window after radiation exposure [ 6 - 8 ] . entolimod is currently in clinical development ; a human safety study indicated that it was well tolerated and the biomarker results correspond to data from animal models . ( buffalo , ny , usa ) is preparing a pre - eua application for entolimod . hemamax is recombinant human il-12 ( rhuil-12 ) cytokine and has been shown to increase mice survival when a single dose was administered , either 24 h before or within 1 h after total - body irradiation . allometrically equivalent doses of mouse and human hemamax had similar pharmacokinetics and significantly increased mouse and nhp survival , when administered 24 h post - irradiation , even when no antibiotics , fluids or blood products were administered . to demonstrate the safety of hemamax , neumedicines conducted a phase ib study where healthy volunteers were administered a single dose of hemamax that is predicted to be the effective dose for treating h - ars , based on nhp data ; this trial suggests rhuil-12 to be safe and well tolerated . phase ii equivalent data ( randomized , double - blinded , good laboratory practice ) showed that single administration of rhuil-12 to nhps significantly increased survival and reduced radiation - induced hematopoietic toxicity when administered 24 h post - irradiation . administration of rhuil-12 promotes multilineage hematopoietic recovery , immune functions and possibly , gi functions . additionally , there is a report of successful interspecies dose conversion . neumedicines is developing rhuil-12 for the treatment of h - ars for bla submission to the fda under the animal efficacy rule . several additional biologics have been identified as potential countermeasures and have shown promise in murine and nhp models of ars . some of these agents have already been used off - label in radiological accident victims . hopefully , in the future , they will be fully developed agents to combat radiation injury . some of these potential agents are myeloid progenitors , anti - ceramide antibody , fibroblast growth factor-2 ( fgf-2 ) , its derived peptide ( fgf - p ) , igf-1 and various cytokines ( table 2 ) [ 16 - 18 ] . since no fda - approved ars mcm exits , there is an urgent need to develop such agents . based on studies with large numbers of nhps , entolimod appears to be a promising radiation countermeasure for h - ars as well as for gis . entolimod s existing efficacy , safety data and animal - to - human dose conversion are enough to proceed with a pre - eua application to reduce the risk of death following radiation exposure . independent of the fda s approval and licensing process , the us federal government has procured filgrastim and sargramostim to be stockpiled in the sns under the pandemic and all - hazards preparedness reauthorization act . filgrastim and pegylated filgrastim have demonstrated efficacy in recently conducted nhp studies , and these data have been submitted to the study sponsor for submission to the fda for approval . in a recent study , g - csf failed to demonstrate efficacy in the nhp model ; however , this discrepancy may be due to the lack of supportive care . the center for disease control currently holds both investigational new drug and eua applications with the fda for the use of neupogen / g - csf in the event of a nuclear or radiological incident . however , the stability , the implementation plans in the radiological nuclear incident scenario and potential side effects are not fully understood and are of concern . a single injection of hemamax , without supportive care , significantly improved survival and promoted multilineage hematopoietic recovery in an nhp model of h - ars . each drugs sponsor have made clinical progress ; these drugs are moving forward to fill the need for mcm that has increased over the past decade due to increased terrorist threats . in our opinion , the above biologics hold the most promise in the future due to their limited side effects and would be safe and effective agents when approved .

despite significant scientific advances toward the development of a safe , nontoxic and effective radiation countermeasure for acute radiation syndrome ( ars ) over the past six decades , no drug has been approved by the us fda . several biologics are currently under development as radiation countermeasures for ars , of which three have received fda investigational new drug ( ind ) status for clinical investigation . presently , two of these agents , entolimod ( cblb502 ) and hemamax ( recombinant human il-12 ) are progressing with large animal studies and clinical trials . neupogen ( g - csf , filgrastim ) has recently been recommended for approval by an fda advisory committee . filgrastim , gm - csf ( leukine , sargramostim ) , and pegylated g - csf ( neulasta ) have high potential and well - documented therapeutic effects in countering myelosuppression and may receive full licensing approval by the fda in the future . the former two biologics are available in the us strategic national stockpile ( sns ) for use in the event of nuclear or radiological emergency . the emergency use authorization ( eau ) application for entolimod may be filed soon with the fda . biologics are attractive agents that are progressing along the path for fda approval , to fill the unmet need for ars countermeasures .

Introduction Regulatory issues of biologics approval for use as ARS countermeasures Biologics at advanced stages of development as radiation countermeasures CSF Entolimod HemaMax Additional potential biologics as countermeasures against ARS Expert opinion

neural plasticity refers to the capacity of our brain to change in response to internal demand and/or external experience . burgeoning research has corroborated that the neural plastic changes induced in our brains and behaviors are specific to the experiences ( e.g. , [ 27 ] ) . for instance , the london taxi drivers who have protracted experience in driving around the city with complex road infrastructure demonstrated significant increases in brain structural volume in the posterior hippocampus , which is implicated in storing spatial representation of the environment , suggesting that intensive experiences with spatial navigation can induce specific neural plastic changes in the corresponding brain region . similarly , lee et al . have identified distinct patterns of neural activation associated with different forms of meditation practice , namely , focused - attention meditation ( fam ) and loving - kindness meditation ( lkm ) , during the performance of the sustained attention task . while fam is a form of meditation practice with a heavy emphasis on focusing attention on a particular object , lkm stresses on the cultivation of a state of universal love and compassion as to relieve pain and suffering for the self and others . their findings revealed that fam practitioners ( both experts and novices ) showed significantly greater neural activation in the attention - related network when engaging in the sustained attention task , whereas similar neural activation was not observed in the lkm practitioners . the dissociable neural pattern associated with fam and lkm indicated that meditation , as a form of mental exercise , could induce domain - specific neural plastic changes that accord with the form of meditation practice . these findings offer an insight that perhaps a cognitive experience can be designed to specifically target triggering experience - dependent neural plastic changes in the cognitive domain of interest . yet , our brain , albeit malleable , inevitably undergoes certain degrees of age - associated cortical degeneration at the molecular level as age advances . for instance , significant reduction in global and regional gray matter volume , particularly in the prefrontal and medial temporal regions ( e.g. , [ 1013 ] ) , as well as age - related alterations in the functional connectivity of the default mode network ( e.g. , [ 14 , 15 ] ) have been consistently reported in older adults . substantial research has yielded evidence of age - related decrements in attention and working memory performance ( e.g. , [ 1620 ] ) . mani et al . observed that older adults committed significantly more errors than younger adults when performing a task on sustained attention , suggesting that the ability to sustain attention ( in terms of response accuracy ) was compromised during the natural aging process . it has been speculated that older adults , in order to compensate for their deficits in attentional performance , are required to recruit additional cognitive resources [ 20 , 22 ] . observed that the performance of older relative to younger adults declined with increased working memory load . in terms of long - term memory , profound changes in episodic memory have also been evidenced [ 24 , 25 ] , which can be attributed to the age - associated degeneration in the medial temporal regions , especially the hippocampus . findings from a six - year longitudinal study revealed that hippocampal atrophy and a reduction of neural activation in the hippocampus were only identified in older adults demonstrating declines in memory performance but not in those with intact memory function . research on neuroplasticity - based intervention for age - associated cognitive changes is timely . despite the deleterious effects of age on the brain and behavior , increasing research has shown that the neural plastic potential is preserved until late adulthood ( e.g. , ) . cabeza found that high - performing older adults could counteract age - associated neural decline by recruiting bilateral prefrontal regions when performing a cognitively demanding source memory task , as opposed to the right lateralization observed in both younger adults and low - performing older adults . the reduction in hemispheric asymmetry appears to compensate for the age - associated decline in cognitive performance by reorganizing the neural recruitment [ 28 , 29 ] . in another study , boyke et al . found that older adults demonstrated a similar extent of gray matter changes in the middle temporal area of the visual cortex ( hmt / v5 ) as did their younger counterparts , after acquiring the skill of juggling a three - ball cascade . although the juggling performance of older adults was less proficient than that of their younger counterparts , the observed brain structural changes in the visuomotor region bolstered up the claim that an aging brain is still capable of change in response to experience . further support was substantiated by colcombe et al . , which showed that older adults demonstrated significant increases in brain structural volume after completing aerobic exercise training , in comparison to that of the older adults in the stretching control group . considering that our brain retains its neural plastic potential until late adulthood , lately there has been a growing trend toward exploring any cost - effective intervention that can mitigate or even slow down the age - related declines in cognitive functions . such cognitive training or interventions are primarily targeted at training either general cognitive function or specific cognitive domains , such as attention and working memory , and promising findings have been received thus far ( e.g. , [ 3235 ] ) . building on the principle of neuroplasticity and the concept of the sensory deprivation model , smith et al . developed a self - administered cognitive training program that aimed at improving the auditory processing speed as well as its accuracy . their program consists of six computerized exercises that stress sharpening one 's ability to discriminate , judge , recognize , and match the sequences or pairs of confusable syllables ; to reconstruct the sequences of verbal instruction ; and to discern the details after listening to a story presented verbally . individuals are encouraged to perform cognitive exercises for one hour every day , five days per week , for a total of eight weeks ( equivalent to 40 sessions ) . it was found that participants demonstrated significantly greater improvement in auditory attention and memory after completing the brain fitness program , when compared with their peers in the active control group . recently , the feasibility and potential of implementing the cognitive training program to older adults at risk of cognitive decline have garnered increasing empirical attention and equivocal evidence has been obtained ( e.g. , [ 37 , 38 ] ) . it is important to gather evidence on whether the cognitive training can induce general or specific neural plastic changes in cognitive function among the older adults who experience greater than normal rate of age - related cognitive decline and predispose to greater risk of developing dementia or specifically ad ( e.g. , [ 39 , 40 ] ) . to bridge the aforementioned research gap , the current study was a longitudinal study that examined the specific cognitive effects of planned experience delivered in a cognitive training program . the program , modeled after the brain fitness program , aimed at providing the training for attention and working memory but not verbal or visual - spatial memory of older adults at risk of cognitive decline . based on the principle of experience - induced neural plasticity , it is hypothesized that participants from the cognitive training ( ct ) group , relative to their peers in the active control ( ac ) group , would demonstrate training - specific improvement in attention and working memory but not verbal and visual - spatial memory following 13 weeks of cognitive training . ethics approval of this study was granted by the institutional review board of the university of hong kong / hospital authority hong kong west cluster ( hku / ha hkw irb ) . informed consents for participation in this study were obtained from the 209 right - handed community - dwelling chinese older adults at risk of cognitive decline recruited via a local monthly newsletter , local elderly centers , and word of mouth . these older adults were considered at risk of cognitive decline because their montreal cognitive assessment ( moca ) scores fell into the range of 19 to 26 . other than meeting the moca 's cutoff score , the participants also fulfilled the following inclusion criteria : they ( 1 ) were 60 years old or above , ( 2 ) were literate , ( 3 ) had normal or corrected - to - normal vision and/or hearing , ( 4 ) were right - hand dominant as assessed by the lateral dominance test , and ( 5 ) had normal intelligence as measured by the test of nonverbal intelligence ( toni - iii ) . participants were excluded from this study if they met one of the criteria below : they ( 1 ) had current or a history of neurological or psychological disorders ( e.g. , head injuries , stroke , major depression , or generalized anxiety disorder ) , ( 2 ) had current or a history of substance abuse and/or alcoholism , ( 3 ) were on antidementia medication and/or ( 4 ) were diagnosed with thyroid dysfunction or vitamin b12 deficiency , and ( 5 ) scored in the moderate or severe range ( 11 ) in either subscales ( depression or anxiety ) of hospital anxiety and depression scale ( hads ) . the 209 participants were randomly assigned to the ct and ac groups by an experimenter blind to the cognitive status of the participants using computer - generated random sequences of numbers . specifically , each participant i d was paired with a random number and the order of the participants was rearranged based on the value of the assigned number ( from smallest to largest ) . results of significance test confirmed that the ct and ac groups did not differ in demographic characteristics , cognitive processing speed , and depression scores , variables that have been consistently reported to play a significant role in modulating the rate of cognitive decline ( e.g. , [ 4548 ] ) . the training protocol used in this study was modelled after the brain fitness program ( posit science , san francisco , california , glenn smith ) . it is a self - administered training program of three one - hour sessions per week for a total of thirteen weeks . older participants in the ct group were encouraged to practice four out of six cognitive exercises ( approximately 15 minutes each ) in each training session . in the following , a brief description of each of the six cognitive exercises was provided . in each trial , participants are presented with two sound sweeps . each sound sweep either begins low and rises upward or begins high and goes downward . participants are instructed to indicate the frequency of the two sound sweeps by clicking the corresponding arrows using the mouse . represents that the frequency of the sound sweep shifts from low to high , whereas the down - arrow means that the frequency of the sound sweep shifts from high to low . their task is to discriminate between the different sizes of the heptagons and to identify which heptagon ( left or right ) has a relatively larger size . to increase the level of difficulty , the size contrast decreases ( i.e. , the size difference is not that apparent ) as the number of trials advances . their reaction time and response accuracy for each trial are recorded . in each trial , eight cards are shown on a screen . each card is associated with a syllable . whenever the participants click on each card , their task is to pair up the same syllables that are randomly dispersed among the eight cards . participants can hear the sound as many times as they can but they are encouraged to complete the matching task using the minimal number of steps . their reaction times and response accuracy for each trial are recorded . in each trial , participants hear two pairs of rhythm and are asked to recognize whether the two pairs of rhythm are the same or different . the level of difficulty increases with increased length of the rhythm . the participants ' reaction times and response accuracy for each trial are recorded . in this exercise , participants are first shown three stars appearing in random locations on the screen in quick succession . their task is to repeat the sequence and location where the three stars were shown on the screen in the same order . if the participants ' performance reaches a certain level of accuracy , they would proceed with four - star and five - star conditions . participants are instructed to listen to a number of short narrative stories that comprise short conversations and answer the questions pertaining to the details of the conversation afterward . the purpose of the ac group was to ascertain whether the observed improvement in cognitive functioning can be attributed to factors ( e.g. , the training format ) other than the training content . to account for the confounding factors , the training duration , frequency , and format of the ac group were comparable to those of the ct group , except the content of the training . participants in the ac group did not work on the same kinds of cognitive exercises as did their peers in the ct group . instead , they were shown educational programs covering diverse topics ( e.g. , history , science , health information , and local social issues ) on a group basis . immediately after watching the video , they were instructed to answer several questions that were related to the video content . the current study adopted a longitudinal study design to evaluate the effects of a brain plasticity - based training program on enhancing attention and working memory in a group of chinese older adults at risk of cognitive decline ( figure 1 ) . all participants from the ct and ac groups were required to attend a total of 39 one - hour training sessions over thirteen weeks in groups of four to eight . for both the ct and ac groups , each participant was assigned a laptop , a headset , and a mouse that were used for performing the cognitive exercises . all the training sessions were conducted in a quiet and well - lit room in our laboratory . a research assistant was present in each training session to keep track of their attendance and address any questions pertaining to the task instruction raised by the participants . these research assistants were also responsible for conducting the posttraining assessments . to capture training - associated changes in cognitive function , several cognitive measures on sustained attention , working memory , and memory were administered to all the older adults before and after completing the cognitive training . the digit vigilance test is a measure of vigilance and sustained attention that requires participants to cross out a target number , as fast as they can , throughout the page of digits mixed with other numbers . the numbers are randomly dispersed throughout the page , which adds to the difficulty of identifying the target numbers . both the reaction time and number of errors are recorded . in our study , only the reaction time was included for statistical analysis . the seashore rhythm test taps an individual 's ability to discriminate between similar and dissimilar pairs of rhythms and demands auditory attention . participants listen to one pair of musical beats at a time and are asked to judge whether each pair of musical beats is the digit span test of the wechsler memory scale , third edition ( wms - iii ) , is a measure of auditory working memory . subjects are instructed to repeat the strings of digits in the same ( forward sequence ) and reverse ( backward sequence ) order that is verbally presented by the examiner . the level of difficulty increases as the length of the string of the digits increases . the wms - iii visual - spatial span test assesses the visual - spatial working memory and requires the subjects to touch or point at the blocks in the same ( forward sequence ) and reverse ( backward sequence ) sequences as demonstrated by the examiner . the logical memory subtests of the wms - iii were used to measure verbal memory . subjects are required to recall two stories that are verbally presented by the examiner immediately and after a delay . following the delayed recall trials , a forced - choice yes / no recognition test is also administered . the participants were shown four pictures , one at a time , and were asked to recall the details of each picture . prior to the statistical analyses , an independent t - test was conducted to examine if any between - group differences ( ct and ac ) in the demographic variables ( age , gender composition , and levels of education ) , general intellectual abilities ( scores on toni - iii ) , cognitive processing speed ( processing speed index on the wechsler adult intelligence scale , third edition ( chinese version ) ) , cognitive status ( scores on the cantonese version of moca ) , and depression ( scores on the geriatric depression scale ) were present . a 2 ( training groups : ct and ac ) 2 ( time - points : pretraining and posttraining ) repeated measures anova was used to examine the training effect on sustained attention , working memory , and memory , in chinese older adults at risk of cognitive decline . post hoc comparisons ( paired sample t - tests ) were carried out for each significant interaction effect to further clarify the effect of cognitive training on a specific outcome measure . our final sample consisted of 209 older adults aged 60 to 88 years ( 164 females and 45 males ; age : m = 70.1 years ; sd = 6.38 years ) who successfully completed the pre- and posttraining assessment , of which 109 older adults were randomly assigned to the ct group ( 87 females and 22 males ; age : m = 70.1 years ; sd = 6.21 years ) and 100 older adults were in the ac group ( 77 females and 23 males ; age : m = 70.0 years ; sd = 6.60 years ) ( table 1 ) . participants from the ct and ac groups were matched for their demographic characteristics ( age , gender composition , and years of education ) , cognitive processing speed , and depression scores ( all p > .05 ) . seashore rhythm test and digit vigilance test are the indicators of the auditory and visual attention . a 2 ( training groups : ct and ac ) 2 ( time - points : pre- and posttraining ) repeated measures anova only revealed significant interaction effect on the seashore rhythm test , f(1,207 ) = 5.054 , p = .026 , but not on the reaction time of the digit vigilance test , f(1,207 ) = .046 , p = .831 ( table 2 ) . post hoc comparison indicated that only the ct group demonstrated significant improvement in the seashore rhythm test following the training , t(108 ) = 3.707 , p < .000 , whereas the ac group did not show any significant change in their scores but simply maintained a similar level of performance on the seashore rhythm test upon completion of the training , t(99 ) = .796 , p = .428 . digit and visual - spatial span assess the verbal and visual - spatial attention and working memory , respectively . the two - way repeated measures anova only identified significant interaction effects on the total digit span , f(1,207 ) = 6.473 , p = .012 , as well as the total visual - spatial span , f(1,207 ) = 5.047 , p = .026 , which represent auditory and visual - spatial attention and working memory ( table 2 ) . post hoc comparisons showed that the ct group exhibited significant increases in the total digit span , t(108 ) = 4.119 , p = .000 , and total visual - spatial span , t(108 ) = 3.835 , p = .000 , after three months of cognitive training . in contrast , the ac group did not show any statistically significant increase or decrease in the total digit span , t(99 ) = .121 , p = .904 , and total visual - spatial span , t(99 ) = .552 , p = .582 . both immediate and delayed recall trials of wms - iii subtests of logical memory and family picture neither the main effects nor interaction effects on the immediate and delayed recall trials of wms - iii logical memory and family picture were significant ( all p > .05 ) ( table 2 ) . to explore whether levels of cognitive status would have been associated with different training outcomes , pearson correlational analyses were carried out between the baseline general cognitive statuses ( as measured by moca ) and the pre- versus posttraining changes in cognitive measures among the older participants from the ct group . none of the pre- versus posttraining changes in cognitive variables ( including attention , working memory , and cognitive processing speed ) was significantly correlated with the initial general cognitive statuses of the participants from the ct group ( all p > .05 ) . this study used a longitudinal design to examine the specific effect of planned experience on cognitive functioning . consistent with our a priori hypothesis , behavioral changes are specific to the types of experience induced to the brain . in this study , only attention and working memory , but not verbal and visual - spatial memory , showed an improvement after the 13-week systematic cognitive training of these cognitive domains . this pattern of finding is consistent with the prediction set forth by experience - dependent neural plasticity model . the specific improvement in attention and working memory can not be attributed to other demographic , cognitive , or emotional variables because the ct and ac groups were matched on these dimensions . these training - induced modality - specific improvements in attention and working memory performance have underscored the importance of designing a tailor - made experience as to induce experience - specific changes in cognitive functions ( e.g. , [ 27 , 49 , 50 ] ) . perhaps future research can consider exploring the potential of cognitive intervention that is targeted at training a specific cognitive domain or even a specific modality . for example , it is well - documented that atrophy of the medial temporal lobe ( especially in the hippocampus ) may explain the aging - related memory impairment [ 2426 ] . based on the findings of this study , planned cognitive training inducing learning and memory consolidation may lead to experience - specific neural plastic changes in the hippocampus leading to long - term memory enhancement . furthermore , while memory is the major cognitive domain being affected in people suffering from alzheimer 's disease , training targeted at verbal and visual - spatial memory may be beneficial in slowing the memory decline . the nonsignificant training effect on sustained attention , measured by the digit vigilance test , is inconsistent with previous findings . we speculated that the discrepant findings could relate to the use of different age cohorts between ours and previous studies . literature has pointed out that normal aging is associated with widespread neuronal and synaptic atrophy and physiological degradation , which contribute to age - related blunting of neuroplastic responses in an aging brain . on the ct group , our correlational analyses showed that their initial general cognitive status was not significantly associated with any of the pre- versus posttraining changes in cognitive variables of interests ( including attention , working memory , and memory ) , indicating that older adults who were considered to be at greater risk of cognitive decline could capitalize on the cognitive training to a similar extent and their initial cognitive status may not confine the neural plastic potential of their aging brains . considering that our study specifically targeted older adults who scored within the restricted range of moca ( i.e. , 19 to 26 ) , it remains unclear whether the significant correlation identified in our study can be generalized to the elderly population who scored in the normal range , so it is worthwhile to look into this unaddressed question in the future . the findings of this study could be complemented by neuroimaging data that can inform the neural changes associated with the observed behavioral improvement . future research could investigate whether cognitive training can trigger changes at both the behavioral and neural levels , which could definitely advance our understanding of the mechanisms underlying the training effects . future studies can probe into the temporal window during which the transition from the neural plastic changes to behavioral changes can be captured . previous reports have suggested the importance of understanding the generalization of the training effect to activities of daily living ( adl ) and other untrained cognitive domains . unfortunately , we were unable to test for the generalization of the training effects beyond the laboratory setting . it is worthwhile to devote the research effort to elucidate the mechanism underlying the training - transfer effects in order to augment the beneficial effect of cognitive training brought to older adults . while the current study did not intend to inspect the potential impact of educational background on the training effect and hence matched for older participants ' level of education , our previous study did report that the less educated older adults were more likely to gain from the cognitive training than their better educated peers . future investigation can gauge the potential role of educational background and possibly other factors as to maximize the extent to which the older adults can take advantage of the cognitive training . cortical degeneration accompanied by aging , coupled with cognitive deterioration , has placed a heavy socioeconomic burden on the health care system . increasing research has evidenced that our brain retains a capacity to change in response to experience until late adulthood . our findings shed insight into the potential of implementing cognitive training for older adults at risk of cognitive decline and provided substantial support that the neural plastic potential continues until older age . most importantly , this study has provided strong evidence for the potential application of the experience - induced neuroplasticity model to develop cost - effective strategies that can potentially slow down the rate of cognitive decline associated with aging .

increasing research has evidenced that our brain retains a capacity to change in response to experience until late adulthood . this implies that cognitive training can possibly ameliorate age - associated cognitive decline by inducing training - specific neural plastic changes at both neural and behavioral levels . this longitudinal study examined the behavioral effects of a systematic thirteen - week cognitive training program on attention and working memory of older adults who were at risk of cognitive decline . these older adults were randomly assigned to the cognitive training group ( n = 109 ) and the active control group ( n = 100 ) . findings clearly indicated that training induced improvement in auditory and visual - spatial attention and working memory . the training effect was specific to the experience provided because no significant difference in verbal and visual - spatial memory between the two groups was observed . this pattern of findings is consistent with the prediction and the principle of experience - dependent neuroplasticity . findings of our study provided further support to the notion that the neural plastic potential continues until older age . the baseline cognitive status did not correlate with pre- versus posttraining changes to any cognitive variables studied , suggesting that the initial cognitive status may not limit the neuroplastic potential of the brain at an old age .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusion

chemically , phloroglucinol ( 1,3,5-trihydroxybenzene , pg ) and its methylated derivative tri - o - methylphloroglucinol ( tmp ) , figure 1 , are established pharmaceutical agents inhibit the action of catechol - o - methyl transferase , inducing relaxation of smooth muscles , and decreasing glycerol - induced abdominal pain and are also characterized by a swift and strong spasmolytic activity , hence relieving pain . therefore , pg is often used in combination with trimethylphloroglucinol as an antispasmodic drug and is regarded to be effective in decreasing smooth muscle spasm . pg / tmp combination is recommended against biliary calculi , severe pain of urinary or gastrointestinal tract , pain of abdominal region , spastic conditions of the female genital system , and pain in gynecology [ 17 ] . literature survey reveals that some of the analytical methods for phloroglucinol are available including extraction and high - performance liquid chromatography ( hplc ) [ 810 ] , hplc - mass spectrometry [ 11 , 12 ] , gas chromatography - mass spectrometry , and spectrophotometry . other reported methods include titrimetry , spectrophotometry , paper chromatography , and flow injection analysis [ 1520 ] . however , there is no simple and sensitive method to be followed on industrial basis especially in general quality control laboratories . therefore , they can not be followed in the laboratories particularly those of third world countries . hence our group already developed a cost effective method for its fixed dose composition in tablet form , but still there was a need for an analytical method which would help to determine the active pharmaceutical ingredients ( apis ) in parenteral products and physiological fluid . accordingly , the purpose of this write - up is to suggest a systematic approach for the development of a validated simple , sensitive , and stability indicating rp - hplc method that should meet the current ich and regulatory requirements . the present method was designed to be easy to use , sensitive , and rapid . separation and quantification of pg and tmp in pharmaceutical drug formulations and blood were achieved with an isocratic elution and with dual wavelength technique . sil 10a autoinjector hplc system comprising of scl 10a system , controller , spd 20a prominence uv / vis detector , and shimadzu lc 20 at pump with lc solutions software , was used . separation was performed on a hibar bondapak ods c18 hplc column ( 4.6 250 mm ; 10 m bead size ) and maintained at 25c . a uv - visible shimadzu 1650 pc spectrophotometer with uv probe software , ultrasonic cleaner ( elmasoni e 60 h ) , jenway 3240 ph meter , and sartorius te2145 analytical balance were used in the research work . throughout the work only amber glass flasks were used to avoid light effect on the solution of pg and tmp standards and samples . trimethylphloroglucinol and phloroglucinol were kind gifts from a national pharmaceutical company , and sulphuric acid ( merck , germany ) and acetonitrile ( hplc grade ) were purchased from fisher scientific . the injectable containing pg and tmp were obtained from commercial source ( spasfon injection , spadix injection , furosinol injection , and anafortan plus injection ) labeled to contain 10 mg / ml and 0.01 mg / ml of pg and tmp , respectively . the compound was chromatographed isocratically with a mobile phase consisting of acetonitrile ( hplc grade ) : sodium n - heptane sulphonate ( 0.005 m ) : sulfuric acid 0.1 m ( 50 : 50 : 0.3 , v / v / v ) with the ph adjusted if required to 3.0 0.1 using 0.1 m sulfuric acid or 0.1 m naoh . while for sample and standard preparation diluent was prepared from acetonitrile ( hplc grade ) : distilled water ( 50 : 50 , v / v% ) . the mobile phase was filtered by passing through a 0.45 m membrane filter ( millipore , bedford , ma , usa ) and degassed via sonication . the flow rate was 1.0 ml / min , and the injected volume was 20 l . the effluent was monitored at dual wavelength of 266 and 205 nm . in a 100 ml volumetric flask , about 10 mg of tmp reference standard was weighed accurately and dissolved in diluent to have a stock solution of 100 g / ml . similarly in another 100 ml volumetric flask , accurately 500 mg of pg reference standard was weighed and dissolved in diluent , sonicated for 2 minutes , and let to cool to room temperature . then , 5 ml of tmp stock solution was added , and volume was made up to the mark with the same diluent . finally 2 ml of this solution was diluted in 100 ml of diluent to get a 100 g / ml of pg and 0.1 g / ml of tmp working standard solution . the standard was then filtered through 0.45 m filter paper and injected into the hplc system . for making a sample of 100 g / ml of pg and 0.1 g / ml of tmp , 10 ampoules were broken , and content was mixed to get an evenly homogenized stock sample . the sample volume was taken accurately equivalent to 10 mg of pg & 0.01 mg of tmp in 100 ml volumetric flask , and 50 ml of diluent was added . the sample was sonicated for 1 minute and then diluent was added up to the mark and placed on stirrer for 5 minutes . the sample was then filtered through 0.45 m filter paper and injected into the hplc system . blood samples were collected from healthy volunteers in evacuated glass tube through an indwelling cannula placed on forearm vein by a trained clinical laboratory technician . the volunteers were not involved in any medication , smoking , and strenuous activity . the blood was shaken and centrifuged at 6,000 rpm for 30 min to separate out plasma . 9 ml acetonitrile was added to 1 ml plasma and centrifuged at 6,000 rpm for 30 min to deproteinate it . the supernatant serum thus obtained was stored at 20c and filtered for subsequent analysis . for making a working sample solution of 12.5 ml , stock sample was taken in 50 ml flask followed by 30 ml of serum addition . the sample thus obtained was stirred for 10 minutes and then diluent was added up to the mark . further 2 ml of this solution was diluted in 50 ml of flask with the aid of diluent . all samples prepared were filtered through 0.45 m membrane filter and injected in triplicate into the hplc system . for this purpose 5 ml of the stock sample was diluted in 100 ml diluent to make a stock sample solution . for working purpose , 10 ml of stock sample solution was diluted in four individual 50 ml volumetric flasks , and 15 ml of degrading agent were added to each flask , with the exception of one to which only diluent was added ; these included 0.1 n hcl , 0.1 n naoh , and 30% h2o2 , and then to each flask diluent was added up to the mark . the samples were then filtered through 0.45 m membrane filter and injected into the hplc system . intentional degradation was attempted with stress conditions exposing the drugs to acid ( 0.1 n hcl ) , alkali ( 0.1 n naoh ) , hydrogen peroxide ( 30% ) , and heat ( 60c ) to evaluate the ability of the proposed method to separate drugs from its degradation products . for all conditions the temperature was kept constant at 60c for a period of one hour . for stability studies the commercially available injection ( parenteral ) samples in ampoules were placed at accelerated conditions of temperature that is at 40c with 75% relative humidity and at ambient conditions of 30c temperature with 65% relative humidity in environmental chamber for six months . the stability protocol in table 6 various procedures including specificity , linearity , range , accuracy , and intraday and interday precision were evaluated . to study linearity , twenty dilutions of standard were made to prepare standard solution in range from 10% to 200% , that is , from 10 g / ml to 200 g / ml of pg and from 0.01 g / ml to 0.2 g / ml of tmp , respectively , drugs content . the standard calibration curve was generated using regression analysis with online help ( http://wessa.net/ ) . for specificity commonly used excipients in injection preparation were spiked in a preweighed quantity of drugs to determine the effect and interference of excipients in quantification of the drugs . in order to find out the repeatability and reproducibility of the method , precision was studied to find out intra- and interday variations in the test method of pg and tmp in the concentration range of 80120 g / ml for pg and 0.080.12 g / ml for tmp , respectively . precision was determined by analyzing corresponding bulk sample daily for a period of three days and three times a day with an interval of 8 hours against a freshly prepared standard . for determining accuracy the pg and tmp reference standard were accurately weighed and spiked to the injection sample at three different concentration levels to gain 110% , 120% , and 130% of both apis . at each level , limit of detection and quantification ( lod and loq ) for the method were established by sequentially diluting the standard solutions at decreasing concentrations , in the range of 1001 pg / ml for pg and 101 pg / ml for tmp . the limit of detection was defined as the concentration for which a signal - to - noise ratio of 3 was obtained , and for quantification limit , a signal - to - noise ratio of 10 was considered . the standards were injected in lc system , and measured signals from the diluted standards were compared with those of blank samples . to study robustness , samples of injections were assayed with deliberate variation in the method parameters , such as in the chromatographic conditions , like mobile phase , flow rate , and temperature so forth . justification of system suitability was established by calculating % relative standard deviation of replicate injections and analyzing the symmetry , resolution of the standard peaks , and theoretical plates of the column . the hplc method development and its validation are the prioritized requirements for any drug available in the market to ensure the quality of the products . a few methods are available for determination of the apis as described earlier , but many of them are used only for certain definite objectives and lack generalization for simultaneous analytical applications in form of pharmaceutical products and serum . similarly none of them are as much sensitive as ours in terms of their precision , accuracy , limit of detection ( lod ) , and limit of quantification ( loq ) especially as compared to [ 7 , 9 , 12 , 14 ] whose lod or loq was in microgram range only , while ours method is sensitive enough to be used for pharmacokinetic studies . here , the lod and loq for both apis are 10 pg / ml and 33 pg / ml , respectively ; however considering their ratio in sample formulation , the lod and loq for pg are taken as 10 ng / ml and 33 ng / ml , respectively , and hence loq and lod for tmp can also be considered as the levels of sensitivity of the method . for developing an efficient method for the simultaneous analysis of pg and tmp , parameters such as detection wavelength , mobile phase composition , optimum ph , and concentration of the standard solutions both pg and tmp were diluted in dilution solvent and then run through uv spectrophotometer in uv range of 190 nm400 nm to get maximal wavelengths , figure 2 , where maximum absorbance was gained , that is , 266 nm for both apis . however considering the difference of concentration in both apis that is 10 to 0.01 mg / ml for pg and tmp , respectively , therefore 205 nm was used for tmp and 266 nm for pg . the chromatographic parameters were evaluated using a hibar bondapak ods c18 column the mobile phase composed of acetonitrile : buffer of given proportion promoted a short run time ( 10 min ) without any interference , so this condition was adopted in subsequent analysis . the literature survey also revealed that almost all the methods developed so far have utilized acetonitrile as a major component in mobile phases . acetonitrile is always preferred due to the supreme solubility properties and uv absorbance characteristics , and there is no counterpart substitute for acetonitrile in the reverse phase hplc and uv application . therefore , keeping in view the chromatography type and the detection wavelengths in use , acetonitrile was chosen for analysis , though in our previous work we used methanol for its cost effectiveness . the linearity ranges were found to be 10200 g / ml for pg and 0.010.2 g / ml for tmp . the assay was judged to be linear , as the correlation coefficient was 0.9999 and 0.9992 for pg and tmp , respectively , as calculated by the least - square method . a linear correlation was found between the peak areas and the concentrations of apis , in the assayed range . chromatogram shown in figure 3 proves specificity or selectivity of the assayed method , as the chromatograms in samples were found identical with standard chromatogram and no interference peak was observed in sample chromatogram . peak purities higher than 98.0% were obtained in the chromatograms of sample solutions , demonstrating that other compounds did not coelute with the major peaks . the chromatogram obtained with the mixture of the injection excipients proves that there is no interference from excipients and peak of interest that fulfills all the requirements of symmetrical peak , and hence , the specificity is confirmed . the precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous bulk . intraday precision of the method was evaluated at three different independent concentrations that are , 80% , 100% , and 120% for both drugs ( n = 3 ) by determining their assays . interday precision of the method was tested for 3 days at the same concentration levels . since the interday and intraday precision obtained % rsd were less than 2% it is assured that the proposed method is quite precise and reproducible , as shown in table 2 . the accuracy was investigated by spiking reference standards to a mixture of the injection excipients at three different concentration levels , that is , multiple level recovery studies , and subjected to the proposed hplc method . the obtained recovery ( n = 9 ) was 98.86100.96% ( rsd% = 0.488 ) for pg and 98.56%101.24% ( rsd% = 0.139 ) for tmp , demonstrating the accuracy of the method . percentage recoveries for marketed products were found to be within the limits , table 3 . the statistical analysis showed no significant difference between results obtained by employing the analytical conditions established for the method and those obtained in the experiments in which variations of some parameters were introduced . the parameters used in system suitability test were symmetry of peaks , tailing factor , resolution , and rsd% of peak area for replicate injections . thus , the method showed to be robust for changes in mobile phase acetonitrile proportion , mobile phase ph , flow rate , and column temperature , table 4 . during the degradation study , it was observed that upon treatment of pg and tmp with base ( 0.1 m naoh ) , acid ( 0.1 m hcl ) , hydrogen peroxide ( 30% ) , and heat , maximum degradation was observed in acid and h2o2 . it is a fact that phenolic compounds , such as phloroglucinol , are known to undergo ready oxidation in basic solutions ( due to dissolved oxygen ) and by agents such as hydrogen peroxide which increases the absorbance capacity of the molecules , causing increased peak area and hence % calculated amount . and the same is revealed by the data given that pg is more vulnerable to stress conditions especially alkaline and oxidation treatments as compared to tmp . degraded peaks are observed in case of acid hydrolysis , and peak had been broadened due to extra absorbance caused by oxidation of the pg , whereas no appreciable degradation was observed in heat treated sample ; hence the drug is stable under heat stress conditions . the purpose of stability testing is to provide evidence of how the quality of a drug substance or drug product varies with time under a variety of environmental conditions , like temperature , humidity , and light and enable recommendation of storage conditions , retest periods , and shelf life to be established . the two main aspects of drug product that play an important role in shelf - life determination are assay of the active drug and the degradation products generated during stability studies . the proposed assay method was applied to the stability study of commercially available injections , for which the samples were placed at 30c with relative humidity of 65% and at 40c with relative humidity of 75% . according to the results obtained both apis were found to be stable at applied conditions of temperature and relative humidity and were accurately analyzed with the proposed method . the proposed new hplc method described in this paper provides a simple , universal , convenient , and reproducible approach for the simultaneous identification and quantification of phloroglucinol and trimethylphloroglucinol in human serum and pharmaceutical formulations ( injectable ) with good separation and resolution . in addition , this method has the potential application to clinical research of drug combination . analytical results are accurate and precise with good recovery and lowest detection limit values . in short , the developed method is simple , sensitive , easy , and efficient having short chromatographic time and can be used for routine analysis in qc laboratory and therapeutic monitoring .

a reverse phase stability indicating hplc method for simultaneous determination of two antispasmodic drugs in pharmaceutical parenteral dosage forms ( injectable ) and in serum has been developed and validated . mobile phase ingredients consist of acetonitrile : buffer : sulfuric acid 0.1 m ( 50 : 50 : 0.3 v / v / v ) , at flow rate 1.0 ml / min using a hibar bondapak ods c18 column monitored at dual wavelength of 266 nm and 205 nm for phloroglucinol and trimethylphloroglucinol , respectively . the drugs were subjected to stress conditions of hydrolysis ( oxidation , base , acid , and thermal degradation ) . oxidation degraded the molecule drastically while there was not so much significant effect of other stress conditions . the calibration curve was linear with a correlation coefficient of 0.9999 and 0.9992 for pg and tmp , respectively . the drug recoveries fall in the range of 98.56% and 101.24% with 10 pg / ml and 33 pg / ml limit of detection and limit of quantification for both phloroglucinol and trimethylphloroglucinol . the method was validated in accordance with ich guidelines and was applied successfully to quantify the amount of trimethylphloroglucinol and phloroglucinol in bulk , injectable form and physiological fluid . forced degradation studies proved the stability indicating abilities of the method .

1. Introduction 2. Experimental 3. Analytical Procedure 4. Results and Discussion 5. Conclusions

fibromyalgia ( fm ) , which affects at least 10 million american adults , is a multisymptom condition resulting in not only widespread chronic pain , but also fatigue , sleep disturbances , and morning stiffness . in addition , many patients experience depression , anxiety , and dyscognition [ 2 , 3 ] . fm has a significant adverse impact on many individuals ' physical and mental health [ 4 , 5 ] and also leads to reduced workplace productivity and increased health care / disability expenses , with the estimated cost of fm on the us economy being reported to be 1214 billion dollars [ 1 , 6 ] . due to the lack of clarity for the underlying cause(s ) of fm , it poses a diagnostic challenge , often requiring multiple visits by specialists to render a diagnosis . the lack of understanding of the biological basis of this condition also confounds our ability to develop effective interventions and/or monitor disease progression . fm has been suggested to be a complex , multifactorial trait that is influenced by age , gender ( frequency is the highest in middle - aged females ) , and stress / trauma . despite showing a strong familial aggregation [ 810 ] , attempts to identify genetic factors associated with fm ( primarily through polymorphism association studies ) have yielded inconsistent results , with some investigators showing associations between fm and specific genes ( including , but not limited to , genes for catechol - o - methyltransferase [ 1113 ] , serotonin-2a receptor [ 14 , 15 ] , serotonin transporter gene regulatory region [ 16 , 17 ] , dopamine d4 receptor , -2 adrenergic receptor , gamma - aminobutyric acid receptor subunit beta-3 , trace amine - associated receptor 1 , interferon - induced guanylate - binding protein 1 , regulator of g protein signaling 4 , cannabinoid receptor type 1 , and glutamate receptor 4 ) , while others failed to identify a relationship [ 2125 ] . since a consistent , straightforward association with a gene(s ) has not yet been forthcoming , scientists have suggested that the familial influence on fm may more likely reflect a genetic susceptibility to environmental events [ 21 , 26 , 27 ] . for example , klengel and binder identified differential methylation for a glucocorticoid response element ( the fkbp5 gene ) that resulted from the presence of both an at - risk allele ( polymorphism ) and the occurrence of childhood trauma in subjects they studied who had posttraumatic stress disorder . epigenetics , which refers to the process that affects gene expression independent of actual dna sequence ( such as methylation changes , histone alterations , and micro - rna expression ) , has enabled scientists to conceptualize the impact of the environment upon one 's genes and one 's health . genes can be turned on or off and display variations in their level of expression , in part , due to epigenetic modifications . thus , epigenetics provides a biological means for understanding the molecular processes of complex biological networks that connect the brain , behavior , and health outcomes . given the overlap in symptoms and the medical / adverse social histories present in people who have fm , when compared to other conditions that have been shown to be impacted by somatic epigenetic and genetic alterations ( such as depression and stress ) , it is plausible that similar epigenetic mechanisms may underlie the individual variability in the outcome of genetic and emotional inputs for fm . knowing that histone and other epigenetic modifications play a key role in establishing and maintaining chromatin structure , it follows that changes in epigenetic profiles , as a consequence of initiating events ( such as stress / environmental exposure ) , could also lead to an increased frequency of somatic chromosomal changes . indeed , we have shown that stress levels can impact the frequency of acquired chromosomal abnormalities by demonstrating a significantly increased frequency of somatic cell chromosomal instability in adult women who experienced childhood sexual abuse when compared to their identical cotwins who did not experience childhood sexual abuse ( quantified using a micronucleus assay ) . further support that chromosomal instability could arise as a downstream effect following perturbations in methylation comes from studies of individuals who have immunodeficiency , centromeric region instability , and facial anomalies ( icf ) syndrome , which is an autosomal recessive condition resulting from a mutation in the methyltransferase gene b. people with this condition have an increased frequency of acquired chromosomal abnormalities . an efficient means for quantifying the frequency of acquired ( somatic ) chromosomal abnormalities is the cytokinesis block micronucleus ( cbmn ) assay , which provides information regarding the presence of chromosomal errors in somatic cells with minimal influences attributable to in vitro selective growth pressures . this technique is less labor intensive than conventional cytogenetic studies but provides results that are in close agreement to those obtained using the gold standard of metaphase chromosomal analyses . micronuclei , which are the primary cytological structures scored in the cbmn assay , are thought to contain chromatin ( from one or more chromosomes ) that was not incorporated ( lagging or lost ) into the daughter binucleates following nuclear division . micronuclei frequencies have been shown to increase with age and have been associated with a variety of health conditions [ 36 , 37 ] . however , to date , no investigators have reported the frequency of mn in women with fm . based on the symptomatology and comorbidities related to this condition , we hypothesized that women with fm will have an increased frequency of acquired epigenetic and chromosomal alterations . to test this hypothesis , we initiated a pilot study to quantify chromosomal instability levels and genome - wide methylation patterns in women having fm and to compare these genetic / epigenetic values to those present in comparably aged , healthy control women . data for this study were obtained from a subset of participants ( n = 10 ) , who were randomly recruited by mail from a larger , parent study sample of 64 women diagnosed with fibromyalgia ( vcu irb protocol number hm12211 ) ( table 1 ) . in the parent study , the participants completed a two - group randomized , controlled , clinical trial to examine the effect of guided imagery on self - efficacy , perceived stress , pain , fatigue , depression , and neuroendocrine / neuroimmune biomarkers in women with fibromyalgia syndrome . inclusion criteria for the women having fm were age ( at least 18 years old ) ; gender ( female ) ; receipt of a physician - confirmed diagnosis of fm based on the 1990 american college of rheumatology criteria ; an ability to understand and sign the consent form ; and an ability to understand / complete the study questionnaires . exclusion criteria for the women in the fm pilot group included the presence of other systemic rheumatologic conditions ; being immunocompromised ( e.g. , diagnosis of hiv / aids ) ; receiving corticosteroid treatments ; having a major psychiatric or neurological condition that would interfere with study participation , or being pregnant . each of the study subjects completed a self - report form to provide information regarding age , race / ethnicity , marital status , length of time since diagnosis of fibromyalgia , height and weight for calculating body mass index ( bmi ) , and lifestyle practices such as history of smoking and alcohol use . the healthy , comparatively aged control group of women for the mn studies ( n = 42 ) were ascertained through their participation in a study of acquired genetic changes associated with normal aging , the latter of which is a twin study ( vcu irb protocol number 179 ) . the inclusion criteria for this subset of control subjects were gender ( female ) and age ( range from 36 to 69 years old ) , with all people from the previous study who met the criteria being included as controls for the current study to avoid sampling biases . the control cohort of women included both cotwin pairs ( n = 30 women or 15 cotwin pairs ) and single twins , whose cotwin did not participate in this normal aging study ( n = 12 women ) . due to cost limitations , dna methylation studies were limited to a subset ( n = 8) of the control women . this subset of women was randomly selected and included 8 unrelated females ( no cotwins ) . all of the control women self - reported their age , race / ethnicity , and lifestyle practices , such as history of smoking and alcohol use . following the collection of the blood specimens from the patient and control women , the cells were processed to obtain binucleates for the micronuclei studies and dna for the methylation studies as described in the following section . lymphocytes were collected using histopaque-1077 ( sigma ) , stimulated with the mitogen phytohemagglutinin , established in culture , and blocked at cytokinesis according to the protocol of fenech . briefly , cytochalasin b ( 3.0 g / ml ; sigma , 14930 - 92 - 2 ) was added to the cells 44 hours after culture initiation . at 72 hours , binucleate interphase cells were harvested using standard techniques , which included a 10-minute incubation in hypotonic solution ( 0.075 m kcl ) and serial fixation ( three times using a 3 : 1 methanol : acetic acid solution ) . micronuclei were visualized following giemsa staining ( 4% harleco giemsa solution ) and identified according to the criteria established by fenech ( figure 1 ) . the frequencies of micronuclei observed in the cytochalasin - b - blocked binucleated cells of the women with fm and the healthy control women were calculated by averaging the values obtained from two replicate scores ( 1000 binucleates were evaluated from two independent areas of the slide ) for a total of 2000 binucleates that were evaluated per study participant . genomic dna was isolated from whole blood according to standard methods using the puregene dna isolation kit ( qiagen ) . an aliquot ( 500 ng per study participant ) of dna was then sent to hudson alpha institute for biotechnology for bisulfite conversion , using standard methods ( zymo research ez methylation kit ) and genome - wide methylation pattern assessment , using the 450 k humanmethylation chip , according to the manufacturer 's protocol ( illumina ) . intensity data from the scanned arrays were imported into illumina 's genomestudio software and the minfi bioconductor package in the r programming environment to obtain the values for each probe . to test the hypothesis that women with fm have an increased frequency of acquired chromosomal alterations , the frequency of mn given that a portion of the healthy controls were cotwins , a mixed effect model was used to control for familial relationships . mn frequency comparisons between cases and controls were adjusted for age since several studies have demonstrated increases in mn frequency with age [ 37 , 4042 ] . additional environmental effects were considered that have previously been shown to influence micronuclei frequencies . these included body mass index , alcohol use , and smoking in the women having fm . however , given that values for body mass index , alcohol use , and smoking were not available for the controls ( in a format comparable to those for cases ) , these variables were only assessed for the women with fm . because the 450 k humanmethylation assay includes both the infinium i design ( which includes two probes for interrogating a cpg site ) and the infinium ii design ( which includes only one probe ) , the gc content was plotted separately by infinium design type for the fully methylated sample , for which all cpg sites are expected to have consistently high values . based on these results , probes having a gc content greater than 40 were excluded from further analysis to ensure that the results would not be biased by the in addition , since the performance of probes containing single nucleotide polymorphisms ( snps ) can be variable , probes containing snps that were present within 10 bases of the target site were also excluded . because the expression value , , reported for each cpg site represents proportion methylated which is constrained to an interval value from 0 to 1 , where a of 1 indicates complete methylation and 0 indicates no methylation , the expression values were transformed using the logit transformation [ m = log(/(1 ) ] to promote normality . prior to the logit transformation , imputation was completed ( 0.001 for values that were 0 and 0.999 for values that were 1 ) to avoid nonexistent m values . to adjust for the observation that values from the infinium ii designed probes have a compressed range compared to the values from the infinium i design [ 43 , 45 , 46 ] , the peak - correction method was applied to the logit transformed values for the infinium ii designed probes . statistical analyses were then performed on the peak corrected logit transformed values from the patient and control samples . for each cpg site , differential methylation between specimens collected from women with fm and controls was tested using the moderated t - test in the limma bioconductor package [ 51 , 52 ] in the r programming environment . to adjust for the multiple hypothesis tests , the p values were used to estimate the false discovery rate ( fdr ) following benjamini and hochberg 's method . the david gene functional classification tool was used to identify biological relationships among the differentially methylated sites . as expected , the frequency of mn was correlated with age in both the women having fm ( r = 0.717 ; p = 0.021 ) and the healthy controls ( r = 0.579 ; p = 4.79 10 ) ( figure 1 ) . after controlling for the effect of age and cotwin status , a significantly increased frequency of mn was observed in the women having fm ( 51.4 ( 21.9 ) ( mean ( sd ) ) per 1000 binucleates ) compared to controls ( 15.8 ( 8.5 ) ( mean ( sd ) ) per 1000 binucleates ) ( = 45.6 ; df = 1 ; p = 1.49 10 ) ( figure 1 ) . the increased levels of mn in the women having fm were not significantly correlated with their body mass index ( range from 19.44 to 45.70 ; mean ( sd ) was 29.52 ( 7.21 ) ; p = 0.997 ) , smoking history ( 4 smokers ; 6 nonsmokers ; p = 0.75 ) , or alcohol use ( 6 consumers ; 4 nonconsumers ; p = 0.93 ) . to assess if there might be a cumulative biological effect associated with experiencing symptoms associated with fm , we compared mn frequencies for the case subjects ( n = 10 ) with the total number of years that had lapsed since these women received their diagnosis of fm . while there was a trend toward a positive correlation between a woman 's mn frequency and the number of years since she was diagnosed with fm ( ranged from 2 to 19 years ) , this relationship did not reach significance in this small pilot study ( p = 0.134 ) ( table 1 ) . after completion of the quality control assessments that were performed to remove any potential biases associated with probe sequence length , probe gc content , and inclusion of snps , a total of 381,989 cpg sites were retained . from these , a total of 69 sites were determined to be differentially methylated ( dm ) between the patients who have fm and the healthy controls , with 63 of these dm sites having higher values in the patients with fm and 6 having lower values ( figure 2 ) . these 69 dm sites included cpg islands ( 46.4% ) ; north shores ( 20.3% ) ; south shores ( 8.7% ) ; as well as north ( 4.3% ) and south ( 1.4% ) shelves and sites that were not annotated into the previously noted categories ( 18.8% ) . the dm sites were localized to 47 different genes ( table 2 ) , with 3 genes having multiple sites identified ( n - acetyltransferase 15 gene ( nat15 ) had 4 dm sites ; dnaj ( hsp40 ) homolog , subfamily c , member 17 ( dnajc17 ) had 2 dm sites ; and slc17a9 and 2 dm sites ) . an assessment of potential biologically related clusters of dm sites resulted in the recognition of 15 groups , including gene clusters involved in neuron differentiation and nervous system development ( table 3 ) . while the sample size in this pilot study is small , the mn frequency patterns of both the case and control women showed an age - related increase , which is a finding that is in agreement with the age - related increase that has consistently been reported in larger studies [ 40 , 41 ] . interestingly , the mean frequency of mn in the women with fm was 3.26-fold higher than the level seen in the healthy controls . in comparison , patients who have cancer have been noted to have 1.37- to 3.13-fold higher frequencies of mn when compared to healthy controls [ 57 , 58 ] . given that the risk for cancer has been shown to be predicted by mn levels [ 37 , 41 , 58 ] , the results of this preliminary data , if confirmed , suggest that mn frequency assessments may be useful for evaluating / diagnosing women with fm . indeed , recent assessments of mn frequencies in people evaluated from various areas of biobehavioral science have shown increased levels of acquired chromosomal instability ( assessed using mn frequencies ) in adult women who experienced childhood sexual abuse ; patients who have neurodegenerative conditions , such as alzheimer 's disease and parkinson 's disease ; and adults with type 2 diabetes and cardiovascular disease . the presence of acquired chromosomal instability , which could lead to somatic tissue mosaicism , has been conjectured to occur as a global biological process that affects many tissues and contributes to the development of several conditions , including ( but not limited to ) autism , schizophrenia , autoimmune diseases , and alzheimer 's disease . given that several of these conditions are age related , one could speculate that there may be a factors contributing to mn formation are multifold and include both genetic and environmental influences [ 37 , 40 , 41 ] . environmental exposures that have been shown to increase the frequency of mn include , but are not limited to , diet ( especially folate deficiency ) [ 41 , 62 , 63 ] , hormone levels , and exposures to substances / occupational hazards . the biological means whereby these genetic / environmental influences lead to acquired chromosomal instability are likely to be varied but have been noted to reflect the chromatin conformation of the chromosomes . one can speculate that alterations in chromatin conformation , which are likely to arise ( at least in part ) from epigenetic changes , may compromise the ability of the chromosomes to align , attach to mitotic spindle fibers , and/or separate , thereby leading to their increased frequency of abnormalities . in turn , the presence of acquired chromosomal abnormalities could lead to additional epigenetic alterations . while limited in number , studies performed to assess the effect of methylation on chromosome segregation have consistently shown an increase in the frequency of cytogenic abnormalities associated with perturbations in the methylation status of chromosomes . in this study , it is interesting to note that dm sites were identified for genes having a function related to chromatin compaction and/or segregation ( nat15 ; hdac4 ; uhrf1 ) . for example , dm sites were observed for the nat15 gene , which is a gene that has been identified to play an important role in normal chromosomal segregation during anaphase . while the results of the genome - wide methylation patterns evaluated in this study are preliminary , it is exciting to note that several of the sites that were dm between the women with fm and controls were localized to genes that have functional relevance to the symptoms seen in patients with fm . of particular interest was the observation of a significant difference in the methylation pattern of the bdnf gene between patients with fm and controls . the bdnf gene has been noted to play an important neuromodulatory role in pain transduction ( eliciting hyperalgesia ) [ 6971 ] and has also been recognized as a contributor to learning and memory [ 72 , 73 ] . a second gene of apparent relevance with which a dm site was associated was the protein kinase c , alpha gene ( prkca ) ( table 3 ) . this gene , which is involved in cell signaling pathways , has been associated with emotional memory formation , posttraumatic stress syndrome , cancer , and aging . a third gene of particular interest that had a dm site is reticulon 1 ( rtn1 ) . rtn1 has been associated with neurological diseases ( and cancer ) and is thought to influence membrane trafficking in neuroendocrine cells . overall , genes with which dm sites were associated include ( but are not limited to ) those having functions in chromatin compaction ( nat15 ; hdac4 ; uhrf1 ) ; dna damage / repair or chromosomal segregation ( sod3 ; uhrf1 ; nat15 ) ; muscle contraction ( nr4a3 ; hdac4 ; fez1 ; prkg1 ; kcnh7 ) ; axonal bundling / outgrowth ( fez1 ) ; cell signaling in muscle ( nr4a3 ; prkg1 ) ; neuronal excitability / synaptic transmission ( bdnf ; bzrap1 ; epha5 ; kcnh7 ) ; muscle maturation ( hdac4 ) ; response to oxidative stress ( sod3 ) ; and inflammatory processes ( axl ; sh2b2 ) . however , since two of the significant biological clusters that were identified ( table 3 ) were for polymorphisms and sequence variants , it is important to recognize that this is a pilot study and that a larger number of individuals will need to be evaluated to allow one to determine the extent , if consistently present , of dm on the development or severity of symptoms associated with fm . the results of genome - wide methylation studies have provided insight regarding the role of genes and environmental influences for a variety of conditions , with many of these investigations focusing on the areas of cancer and psychiatric conditions [ 74 , 75 ] . however , the epigenomes of diseases causing chronic pain have been less extensively evaluated . in their study of rheumatoid arthritis , nakano et al . observed several dm sites between patients who have rheumatoid arthritis and controls . they also identified distinct epigenomic signatures when comparing patterns from patients with rheumatoid arthritis and osteoarthritis . akin to the results of this pilot study , the findings of their investigation led to the recognition of perturbations in the methylation status of several genes having functions related to the symptoms associated with rheumatoid arthritis . thus , the use of genome - wide epigenetic assessment seems to be a promising tool for evaluating a broad spectrum of conditions , including those associated with chronic pain . in summary , the results of this pilot study suggest that chromosomal instability and alterations in methylation are present in women with fm . if these results can be confirmed , they could provide a basis for improving our understanding of the biological changes leading to the development of fm and may provide a basis for stratifying patients based on their epigenomic and symptom patterns . moreover , since epigenetic changes demonstrate plasticity , the recognition of consistent epigenetic alterations associated with fm could provide a means for developing future therapeutic approaches to reverse these changes , with a goal of alleviating symptoms in people who have fm .

fibromyalgia ( fm ) , characterized by chronic widespread pain , fatigue , and cognitive / mood disturbances , leads to reduced workplace productivity and increased healthcare expenses . to determine if acquired epigenetic / genetic changes are associated with fm , we compared the frequency of spontaneously occurring micronuclei ( mn ) and genome - wide methylation patterns in women with fm ( n = 10 ) to those seen in comparably aged healthy controls ( n = 42 ( mn ) ; n = 8 ( methylation ) ) . the mean ( sd ) mn frequency of women with fm ( 51.4 ( 21.9 ) ) was significantly higher than that of controls ( 15.8 ( 8.5 ) ) ( 2 = 45.552 ; df = 1 ; p = 1.49 1011 ) . significant differences ( n = 69 sites ) in methylation patterns were observed between cases and controls considering a 5% false discovery rate . the majority of differentially methylated ( dm ) sites ( 91% ) were attributable to increased values in the women with fm . the dm sites included significant biological clusters involved in neuron differentiation / nervous system development , skeletal / organ system development , and chromatin compaction . genes associated with dm sites whose function has particular relevance to fm included bdnf , nat15 , hdac4 , prkca , rtn1 , and prkg1 . results support the need for future research to further examine the potential role of epigenetic and acquired chromosomal alterations as a possible biological mechanism underlying fm .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

plx4032 was synthesized using the general procedures previously described.6 expression and purification of b - raf , structure determination , protein kinase activity measurements , and xenograft studies were carried out as previously described.6 clinical methods have also been recently described.5 melanoma patients were selected for study using previously described taqman methodology.8 semi - quantitative immunohistochemistry for perk and ki67 was performed on 5 m - thick formalin - fixed paraffin - embedded tumor biopsies following h&e staining to determine pathologic diagnosis and tissue morphology and integrity . the degree of phospho - erk staining in the nucleus and cytoplasm was interpreted semiquantitatively by assessing the intensity and extent of staining on the slides . for ki67 staining ,

b - raf is the most frequently mutated protein kinase in human cancers.1 the finding that oncogenic mutations in braf are common in melanoma2 followed by the demonstration that these tumors are dependent on the raf / mek / erk pathway3 offered hope that inhibition of b - raf kinase activity could benefit melanoma patients . herein , we describe the structure - guided discovery of plx4032 ( rg7204 ) , a potent inhibitor of oncogenic b - raf kinase activity . preclinical experiments demonstrated that plx4032 selectively blocked the raf / mek / erk pathway in braf mutant cells and caused regression of braf mutant xenografts.4 toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity , enabling phase 1 clinical trials using a crystalline formulation of plx4032.5 in a subset of melanoma patients , pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment . this analysis revealed substantial inhibition of erk phosphorylation , yet clinical evaluation did not show tumor regressions . at higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of erk phosphorylation in the tumors of patients correlated with clinical response . indeed , the phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 these data demonstrate that braf - mutant melanomas are highly dependent on b - raf kinase activity .

METHODS SUMMARY Supplementary Material

the localization and subsequent translation of mrna are important means of regulating gene expression with high spatial and temporal control . in neurons , targeting mrna to specific sites and synthesizing proteins where and when they are needed is particularly beneficial because neurons have long dendrites and axons extending from a few hundred microns to up to a meter from the cell body . the physiological roles of mrna localization in neurons have been implicated in various neuronal functions such as synaptic plasticity , axonal guidance and regeneration . however , there are many remaining questions regarding how mrnas navigate through the complex neuronal arbors , how they are captured in specific regions , and how local translation is regulated ( buxbaum et al . , 2015 ; holt and schuman , 2013 ; hutten et al . , 2014 ; xing and bassell , 2013 ) . observing the dynamics of mrna localization and translation will provide critical information to help understand how precise spatio - temporal regulation of gene expression is processed . most previous studies have used in situ hybridization ( ish ) methods ( lawrence and singer , 1985 ) to visualize rna molecules inside a cell . in particular , fluorescence in situ hybridization ( fish ) enables the detection of individual rna molecules with various signal amplification procedures in fixed cells . single - molecule fish ( smfish ) , which uses multiple fluorescent probes hybridized to a single mrna ( femino et al . , 1998 ; , 2008 ) , is widely used to profile transcription , localization and degradation of rna . however , fish experiments using fixed cells can not provide temporal information on rna regulation . recent innovations in live - cell imaging technologies have made it possible to observe the sequence of molecular events in real time , which is critical to our understanding of mrna dynamics ( moon et al . , 2016 ; spille and kubitscheck , 2015 ) . by imaging single mrna molecules in live cells , we can begin to understand the cause - and - effect relationship and to model the kinetics of rna regulation more quantitatively and predictably . in this review , we provide a brief overview of recent progress in studies on mrna localization and translation in neurons . in particular , we highlight several recent reports that have used single - molecule imaging techniques to contribute to our knowledge of mrna translocation in dendrites and axons . additionally , we review new technical developments for multicolor imaging of single mrnas and their translational activities . these approaches will provide a powerful toolkit to help understand the molecular mechanisms of rna localization and local translation in neurons , with unprecedented temporal and spatial resolution . since the discovery of polysomes at the base of dendritic spines ( steward and levy , 1982 ) , many lines of evidence have indicated that de novo protein synthesis in dendrites is required for long - term synaptic plasticity ( hanus and schuman , 2013 ; jung et al . , 2014 ) . deep rna sequencing revealed 2,550 mrnas that are present in dendrites and axons in the hippocampus ( cajigas et al . , 2012 ) . a large fraction of these mrnas encode synaptic proteins such as signaling molecules , scaffolds and receptors . among these mrnas , activity - regulated cytoskeleton - associated protein ( arc ) , the -subunit of calcium / calmodulin - dependent protein kinase ii ( camkii ) and -actin mrnas have been extensively studied to unravel their regulation in live neurons . to understand how these mrnas are sorted and transported to dendrites , it is crucial to track the movement of individual mrna molecules ( fig . single - mrna tracking has revealed the diverse and stochastic nature of mrna trafficking ( park et al . , 2010 ) . transport and localization of mrna depend on the interplay of cis - acting rna elements , rna binding proteins ( rbps ) , and motor proteins ( czaplinski , 2014 ) . various kinds of rbps bind to their target mrnas to form messenger ribonucleoprotein complexes ( mrnps ) . after recruiting motor proteins , mrnps are transported along cytoskeletal tracks . a cis - acting rna sequence that confers dendritic localization , which is called a dendritic targeting element ( dte ) , is typically located in the 3 untranslated region ( 3 utr ) of the mrna . , 2000 ) or the arc 3 utr ( dynes and steward , 2007 ; 2012 ) exhibit bidirectional transport in dendrites and localize at the base of dendritic spines . a 54-nucleotide - long zipcode in the -actin 3 utr ( kislauskis et al . , 1994 ) and zipcode binding protein 1 ( zbp1 ) are required for the localization of -actin mrna in dendritic filopodia and axonal growth cones ( eom et al . , 2003 ; zhang et al . , 2001 ) . microinjected camkii and septin7 mrnas colocalize in the same particles , while map2 mrnas reside in different particles , suggesting that some rnas that share a common dte can be sorted in the same complexes and transported to distal dendrites ( tubing et al . , for example , there have been conflicting results for defining the dte in the camkii 3 utr ( doyle and kiebler , 2011 ) . the cis - acting rna elements interact with a set of transacting factors or rbps to form mrnp complexes that are transported along microtubules by kinesin or dynein . tracking reporter rnas in fmr1 knock - out ( ko ) neurons revealed the important roles of fragile x mental retardation protein ( fmrp ) as an adaptor that links mrna to the kinesin light chain ( dictenberg et al . , 2008 ) and for mrna delivery to dendritic spines in response to synaptic stimulation ( kao et al . , 2010 ) . live imaging of neurons also showed that huntingtin ( htt ) , huntingtin - associated protein 1 ( hap1 ) , kif5a , and dynein intermediate chain are associated with -actin mrna during dendritic transport ( ma et al . , 2011 ) . knocking down the levels of these proteins reduced the transport of -actin mrna , suggesting that they have roles in mrna localization in neurons . an actin - based motor , myosin - va , contributes to the transport of tls ( translocated in liposarcoma)-containing mrnps in dendritic spines ( yoshimura et al . , 2006 ) . most previous mrna tracking studies have been performed using transfection or injection of exogenous reporter mrnas . however , reporter rnas usually do not contain all of the cisregulatory sequences and trans - acting rbps and thus may not represent the true characteristics of endogenous rnas . to label endogenous mrna , genetically engineered mice have been generated by inserting ms2 binding site ( mbs ) stemloops into a target rna and expressing ms2 capsid protein ( mcp)-gfp proteins ( lionnet et al . all the endogenous -actin mrnas were labeled with the ms2-gfp system in live mice ( park et al . , 2014 ) . tracking the labeled endogenous -actin mrnas revealed that mrna diffusion was much slower in neurons than in fibroblasts . while most of the -actin mrnas in fibroblasts showed diffusive motion , a small fraction of the -actin mrnas in neurons showed bidirectional motion with a mean speed of 1.3 m / s . recently , glutamate uncaging was used to demonstrate that -actin mrnas can localize in the stimulated dendritic region within 15 min of stimulation ( yoon et al . , 2016 ) . nmda receptors and zbp1 have important roles in the localization process . in neurons cultured from zbp1 ko mice , -actin mrnas were transported to the stimulated region ; however , they did not maintain their localization . the authors also showed that newly synthesized -actin proteins accumulated in the stimulated spine head and that this helped stabilize the expanded spines . taken together , tracking single - mrna movements enables us to reveal how rna transport and localization happen in live neurons . the current working model is called the sushi belt model , in which mrnps stroll along axons and dendrites like sushi plates on a conveyor belt to serve potential customers ( doyle and kiebler 2011 ) . those mrnas may be captured by synaptic tags that are relevant to the formation of long - term potentiation ( ltp ) and long - term depression ( ltd ) ( frey and morris , 1997 ) . further studies using new developments in genetic engineering and in systems biology approaches will elucidate the precise regulatory mechanisms of mrna localization in dendrites . while local translation in dendrites is well accepted , local protein synthesis in axons has been controversial over the years . although a few early studies reported the presence of ribosomes in axons ( bunge , 1973 ; tennyson , 1970 ; zelena , 1970 ) , the low density of axonal mrnas and ribosomes supported the traditional view that axonal proteins are synthesized in the cell body . later , however , two studies showed evidence for local translation in the axons . campbell and holt observed that growth cones of retinal ganglion neurons lose their ability to turn away from a source of sema3a when translation is inhibited ( campbell and holt , 2001 ) . zhang and poo showed that localized synaptic potentiation by bdnf requires presynaptic protein translation in xenopus nerve - muscle cultures ( zhang and poo , 2002 ) . since then , there have been many studies on mrna localization in axons and on the implications of those localized mrnas for axon guidance , survival and regeneration ( gumy et al . , 2014 ; jung et al . , bdnf- or netrin-1-induced growth cone turning is mediated by local translation of -actin mrna ( leung et al . , 2006 ; bdnf induces src - mediated zbp1 phosphorylation , which releases zbp1 from -actin mrna ( huttelmaier et al . , 2005 ) . after zbp1 is dissociated , -actin proteins are locally synthesized from the mrna and then direct growth cone turning . in a similar mechanism , map1b mrnas are also locally translated in response to sema3a ( li et al . , 2010 ) . it has been suggested that fmrp has a role in protein synthesis - dependent collapse of growth cones induced by sema3a ( fig . in addition , branching of sensory axons requires local protein synthesis and the stalling of mitochondria ( spillane et al . , 2013 ) . using a gfp reporter construct containing the 3 utr of -actin mrna , the authors found that hot spots of axonal translation occur at the base of branches that also contained mitochondria . zbp1 is colocalized with myosin - va in axons , and inhibition of myosin - va function increases the number of motile zbp1 particles ( nalavadi et al . , 2012 ) . furthermore , some rbps can bind to several species of mrnas . in dorsal root ganglion neurons , sfpq , a splicing factor , works as an rbp binding to lmnb2 , bcl2l2 , impa1 and creb1 mrnas ( cosker et al . , 2016 ) . in addition , sfpq plays a role in the axonal transport of these mrnas to promote axon survival . in central nervous system ( cns ) axons , studies on mrna localization have been facilitated by the use of microfluidic devices that separate axons from cell bodies and dendrites ( taylor et al . , 2005 ) . using a microfluidic device in conjunction with microarray analysis and fish , many species of mrnas have been detected in the axons of cns neurons as well as of peripheral nervous system ( pns ) neurons . studies of mature cortical axons have identified mrnas that are involved in the translation machinery , transport , cytoskeletal components and mitochondrial maintenance ( taylor et al . , 2009 ) . to date , there are only a few studies on live - cell imaging of axonal mrna transport . using the ms2-gfp system , bi et al . demonstrated that the 5 and 3 utrs of kor ( -opioid receptor ) mrna are required for its axonal transport in dorsal root ganglion ( drg ) neurons ( bi et al . , 2006 ) . more recently , a molecular beacon method was used to visualize nefl ( neurofilament - l ) mrna in axons of primary mouse cortical neurons and human motor neurons derived from ips cells ( alami et al . simultaneous imaging of nefl mrna and trans - active response dna - binding protein 43 ( tdp-43 ) , an rbp related to amyotrophic lateral sclerosis ( als ) , revealed that tdp-43 promotes anterograde transport of nefl mrnp granules . this result suggests that tdp-43 has a role in the delivery of target mrna to distal axons . with the development of microfluidic devices , rna labeling techniques , and high - resolution live - cell imaging , it is now more feasible to study mrna transport in axons . for instance , integrated microfluidic platforms are developed for investigating regeneration of injured axons ( kim et al . , neurons cultured from mcp mbs knock - in mice now allow us to image single endogenous mrnas without transfection steps ( park et al . , 2014 ) . because local translation plays a crucial role in axon development and regeneration , it is important to understand the molecular mechanisms that govern mrna trafficking and localization in axons . translation is a highly regulated , complex biological process in which mrna is decoded by a ribosome to produce a protein . several whole - genome techniques for studying translation have been developed based on rna sequencing ( rna - seq ) or mass spectrometry ( ms ) . ribosome profiling , for example , measures ribosome - protected regions of mrnas with next - generation sequencing ( ngs ) , providing position information on translating ribosomes throughout the cellular transcriptome ( ingolia et al . , 2009 ) . global , averaged measurements of translation initiation , elongation , and termination rates can be made by ribosome profiling ( ingolia et al . , 2011 ) . another sequencingbased technique called translating ribosome affinity purification ( trap ) ( heiman et al . , 2008 ) or ribotag ( sanz et al . , 2009 ) relies on pulling down gfp- or ha - tagged ribosomal proteins and then the identification of mrnas associated with them . ms - based methods such as pulsed stable isotope labeling by amino acids in cell culture ( silac ) ( schwanhausser et al . , 2009 ) and biorthogonal noncanonical amino acid tagging ( boncat ) ( dieterich et al . , 2006 ) have been used to identify newly synthesized proteins after adding labeled amino acids . because genome - wide methods can only detect the average behavior of many cells , imaging - based techniques have been developed to study the dynamics of translation in single cells ( chao et al . , 2012 ) . fluorescent proteins have been used to detect local protein synthesis ( aakalu et al . , 2001 ) ; however , the long maturation time of fluorescent proteins has been a major limitation of such methods . as an attempt to visualize translation from single mrnas , halstead et al . developed an rna biosensor termed translating rna imaging by coat protein knock - off ( trick ) that can distinguish untranslated mrnas from those that have undergone at least one round of translation ( halstead et al . orthogonal bacteriophage pp7 and ms2 stem - loops were used to label mrna with two different fluorescent proteins ( fig . . simultaneous expression of the pp7 capsid protein fused with green fluorescent protein ( pcp - gfp ) and the ms2 capsid protein fused with red fluorescent protein ( mcp - rfp ) resulted in yellow fluorescent signals from untranslated mrnas . during the first round of translation , pcp - gfp is removed from the mrna as a ribosome traverses the coding region that contains the pp7 stemloops . thus , translated mrnas are labeled only with mcp - rfp bound to the ms2 stem - loops in the 3 utr . while the trick technique provides information on the first round of mrna translation , recent studies by four independent groups ( morisaki et al . , 2016 ; wang et al . , 2016 ; wu et al . , 2016 ; yan et al . , 2016 ) have demonstrated two - color imaging of mrna and its nascent polypeptides . these four studies used the ms2 or pp7 systems to label mrna and the suntag ( tanenbaum et al . , 2014 ) or flag tag ( viswanathan et al . , 2015 ) fluorescence - tagged ms2 or pp7 capsid proteins were expressed in cells , providing a strong fluorescence signal from each mrna . the suntag and flag tag systems use fluorescently labeled antibody fragments that bind to multiple copies of a short epitope on each polypeptide . an mrna undergoing active translation can be identified by the colocalization of two fluorescent signals from the mrna and nascent proteins . wu et al . , 2016 ) used the suntag - ms2 technique not only in cell lines but also in hippocampal neurons . they used ornithine decarboxylase ( odc ) or auxin - induced degron ( aid ) to facilitate rapid degradation of the epitope array after translation . this allowed them to distinguish nascent polypeptides translated on the mrna from the background of freely diffusing polypeptides in the cytosol . wang et al . found that the arc 3 utr increased the number of translation sites in the dendrites of neurons . wu et al . showed that ~40% of the reporter mrnas were translated in the proximal dendrites ; however , only ~10% were translated in the distal dendrites . both studies this result contradicts the previous speculation that translation is repressed during the transport of mrna to localization sites . although these findings were obtained using exogenous reporters , the tagging method could be applied to endogenous genes with crispr / cas9 genome - editing techniques ( nelles et al . , 2016 ) . if we could track endogenous mrna while measuring its translational output in response to diverse signaling in live tissues , we would be able to uncover many interesting details regarding the dynamics of gene expression . further improvements in these imaging techniques ( pichon et al . , 2016 ) will certainly offer new insights into the complex regulation of translation at the single - molecule level in situ . the elongated morphology of neurons requires directed transport of mrna and local translation of proteins for efficient gene expression in dendrites and axons . because local protein synthesis has important roles in neuronal development and synaptic plasticity , it is crucial to understand how specific mrna molecules are targeted to the activated regions for local translation . recent advances in live - cell single - rna imaging techniques have significantly increased our knowledge on the dynamics of rna in neurons what are the cis - regulatory rna sequences for selective transport into dendrites and axons ? how are mrnps delivered and captured at the stimulated synapses ? understanding the molecular mechanisms behind rna localization , translation and degradation in neurons will provide clearer answers to questions about the physiological relationships between gene expression and brain functions .

local protein synthesis mediates precise spatio - temporal regulation of gene expression for neuronal functions such as long - term plasticity , axon guidance and regeneration . to reveal the underlying mechanisms of local translation , it is crucial to understand mrna transport , localization and translation in live neurons . among various techniques for mrna analysis , fluorescence microscopy has been widely used as the most direct method to study localization of mrna . live - cell imaging of single rna molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein ( mrnp ) complexes in neurons . here , we review recent advances in the study of mrna localization and translation in live neurons using novel techniques for single - rna imaging .

INTRODUCTION SINGLE-mRNA TRAFFICKING IN DENDRITES IMAGING mRNA LOCALIZATION IN AXONS MONITORING TRANSLATION FROM A SINGLE mRNA IN LIVE NEURONS CONCLUSION

the modern world is an interconnected mesh of networks satisfying a myriad of functions : transportation , electrical power , food distribution , finance , and health care to name a few . the interoperability of these networks developed as part of urban evolution over the past century such that these and other webs connect to national and/or global networks ( national research council of the national academies , 2005 ) . this is the engineered webbing of humanity , but there are comparable natural structures in the spheres of biology , ecology , sociology , and physiology . this modernity is manifest in the military through the development of network - centric warfare ( ncw ) which takes cognizance of human behavior in a networked environment of organized actions directed toward political , social , and military ends and is the basis of a new theory of war ( office of force transformation , 2004 ; garstka and alberts , unpublished ) . thus , ncw has at its core a shift in focus from military platforms such as ships and tanks to networks having platforms as members . army scientists need to understand the dynamics , controllability , and predictability of generic non - linear complex networks in order to realize their goal of supporting both society and the soldier through research and the development of new technologies . it is not only our external world that is cluttered with networks , but our internal world as well . the neuronal network carrying the brain s signal to the body s physiological networks is even more complex than the modern city or a typical ecological network . thus , the basic research into network science must span and encompass a multitude of disciplines ; understanding each sheds light on the others . the problem addressed within this program is to develop the basic research foundation of a science of networks that supports the linkage between the cognitive and social domains as they relate to decision making . this approach is not directed at the totality of developing a network science , but has the more modest goal of understanding the deeply interdependent human networks of crucial importance to society as a whole and to the army in particular . even such a restricted problem is a significant challenge due to the multiply interconnecting networks buttressing the common decision making objective . on the one hand , the military is proactive in that networked forces can operate in an agile manner to promote decision making superiority . on the other hand , the army is reactive in the need to respond to enemies who are also using the power of networks against united states interests . the research program provides insight to allow the army to anticipate the enemy s use of network strategy and thereby reduce the reactive mode of operation . in grigolini and west ( 2011 ) we reviewed what is presently known about complex networks , regardless of the disciplinary context and adapted that understanding to the decision making paradigm . moreover , the barriers to further understanding and to filling the gaps in knowledge of the linkages between social and human decision making networks were addressed . the research strategy of the arl program is based on theory , computation / simulation , and experiment / observation . this is a cyclic interactive process in which new theory stimulates unique simulations , yielding insight into parameter values and network configurations , which in turn suggests specific experiments , whose outcome guides the refinement and development of theory . this modern approach to scientific research is applied to the phenomenon of human decision making with a view for eventual application to ncw . the core group of army scientists is the focal point for external researchers requiring militarily relevant challenges and internal arl efforts . one of the mysteries of human social interaction is how agreements are reached and cooperative alliances are made . individuals become part of social groups or networks in a number of ways : choice , peer pressure , and subliminal seduction ; but always through a sequence of decisions , either conscious or not . network characteristics can not be deduced from the properties of individuals ; they emerge during the formation and growth of the network . consequently we need mathematics to quantify the strength of the interactions between the network components , as well as to describe how a network develops in time and responds to perturbations ( stimulation ) . this has been done through the construction of the decision making model ( dmm ) that for very weak coupling is much like the ising model of cooperative behavior , but for strong coupling can be very different ( turalska et al . , 2009 ) ubiquitous aspects of complex networks are the appearance of non - stationary , non - ergodic , and renewal statistical processes . law statistical distributions that not only challenge traditional understanding of complexity in physical networks , but require new strategies for understanding how information is exchanged between networks ( west et al . , 2008 ; west and grigolini , 2011 ) , as in the case of interest here among human networks including cognitive networks . the approach is to adapt the methods of non - equilibrium statistical physics that have been used to characterize the dynamics of complex phenomena and phase transitions . these methods were extended to the study of such social phenomena as linguistics , biofeedback techniques , and the brain s response to music ( bianco et al . , 2007 ) and to further develop them to model decision making with incomplete information in an uncertain environment . the mathematics of complex networks has been examined using the newly developed dmm to understand consensus ( turalska et al . , 2009 ) ; a psychophysical model of how individuals make decision and then irrationally change their minds shows agreement between theory and experiments ( west and grigolini , 2010a ) ; renewal statistics reveal how we habituate to the familiar ( west and grigolini , 2010b ) and forget the uninteresting ( west and grigolini , 2010c ) ; 1/f variability captures how the brain processes tasks of increasing complexity during decision making ( grigolini et al . , 2009 ) ; and finally we have determined how all these various pieces fit into the overall picture of exchanging information between complex networks ( west et al . , 2008 ; grigolini and west , 2011 ; west and grigolini , 2011 ) . the major accomplishment of the present research program has been the identification of the first universal principle in the science of networks , that being , the principle of complexity management ( pcm ) discussed in grigolini and west ( 2011 ) , west and grigolini ( 2011 ) . pcm states that the maximum information is exchanged between two complex networks when there is compatibility of the complexity of the two networks . a mathematical proof of this principle has been constructed over the past year ( aquino et al . , 2010 , 2011 ) . the mathematician norbert wiener speculated that the transfer of influence from a complex network high in information to one low in information even though the latter may be higher in energy represents a new kind of interaction ( wiener , 1985 ) , which we called wiener s rule ( grigolini and west , 2011 ) . his insight was vindicated a half century later ( aquino et al . , 2010 , 2011 ) and required the generalization of a number of concepts from statistical physics ( allegrini et al . , 2007 , 2011 ; aquino et al . , 2007 ; budini and grigolini , 2009 ) resulting in the pcm ( west et al . , 2008 ; west and grigolini , 2011 ) as we discussed last year ( grigolini and west , 2011 ) . one measure of the information content of a network is provided by the probability density most often used in the determination of the negative entropy of shannon and wiener . an apparently ubiquitous distribution in the description of empirical complex networks is the hyperbolic , having the survival probability : which asymptotically becomes an inverse power law . the average time between events in complex webs such as power grid blackouts , heartbeats , time between earthquakes ( west and grigolini , 2011 ) of a given magnitude can be determined using the probability density (t ) = d(t)/dt , to be it is interesting that when the power < 3 the distribution has a finite first moment and the statistics are ergodic , meaning that the time average and ensemble averages yield the same result . law index is < 2 there are no finite integer moments and the time and ensemble averages are not the same , that is , the process is non - ergodic . one measure of the information transfer between two complex networks is the cross - correlation between a complex network p and a complex network s being perturbed by p with the strength of the perturbation . for our purposes it is sufficient to apply the generalized linear response theory ( lrt ; aquino et al . , 2010 , 2011 ) we previously developed the normalized cross - correlation function : the perturbing complex network p is characterized by the non - stationary autocorrelation function p ( t , t ) , which depends separately on the time of the last perturbation t and time of the measurement t. the function rs(t ) is the rate of generating perturbing events at time t within the network being perturbed and is based on renewal theory ( west and grigolini , 2011 ) . the perturbed network s is characterized by the stationary autocorrelation function s ( t t ) , which depends only on the difference in times from the last perturbation to the measurement . in figure 1 the asymptotic cross - correlation function normalized to the strength of the perturbation is graphed as a function of the power the cube displays a number of remarkable properties : ( 1 ) when the power law indices are both equal to two there is an abrupt jump from zero correlation in region ii to perfect consensus in region iii ; ( 2 ) the upper plateau region iii indicates that when p is non - ergodic 1 < p < 2 and s is ergodic 2 < s < 3 there is an information response in which the perturbed network tracks the perturbing network exactly and the information transfer is maximal ; ( 3 ) when p is ergodic 2 < p < 3 and s is non - ergodic 1 < s < 2 there is no response asymptotically and the information transfer is minimal as shown in region ii . how a complex network responds to a perturbation by another complex network is determined by the kind of mismatch that exists in the complexity of the fluctuations in the two networks . the cross - correlation cube . the asymptotic cross - correlation function defined by eq . law indices of the perturbed network s and the perturbing network p. ( from west and grigolini , 2011 , with permission . ) wiener s rule describes the influence of the perturbing network outside the lower plateau region of the cross - correlation cube . in all regions except this one the weak perturbation significantly modifies the properties of the complex network being perturbed . in the upper plateau region the perturbation by network p actually dominates the properties of the perturbed network s and reorganizes it , just as wiener anticipated . the pcm embodied in the cross - correlation cube therefore subsumes wiener s rule as we reviewed in west et al . in addition we showed the application of pcm to the phenomenon of habituation and other activities involving the human brain . aquino et al . ( 2011 ) observe that the growing interest in the dynamics of complex networks is shifting research attention from the synchronization of two stochastic units ( pecora and carroll , 1990 ) to the synchronization of large numbers of units ( wang , 2002 ) , an interesting phenomenon that is closely related to the very popular model of kuramoto ( 1984 ) . the single units of the processes of chaos synchronization are chaotic and they surprisingly synchronize while maintaining the erratic dynamics that they have in isolation . although the single units of the kuramoto model are regular , it is becoming increasingly evident that the emergence of a global synchronization is a condition independent of whether the single units are regular or stochastic . ( 2011 ) are poisson processes and if one of them drives the other , they would obey the principle of aperiodic stochastic resonance ( lukovic et al . , 2008 ) . if the two units are bi - directionally coupled they are expected to undergo a condition of perfect synchronization if the coupling is sufficiently intense . when the number of interacting units is very large a phase transition occurs from the non - cooperative to the cooperative behavior ( bianco et al . , 2008 ; turalska et al . , 2011 ) . it is important to stress that at criticality no permanent consensus is reached , and the mean value of the global field vanishes . yet ( no ) state for an extended time before making a transition to the no ( yes ) state . it is surprising that the phase transition literature seems to have overlooked , with only a few exceptions ( contoyiannis and diakonos , 2000 ; bianco et al . , 2008 ; turalska et al . , 2011 ) , that the transitions from the yes in other words , the time interval between two consecutive transitions is derived from a pdf that has the asymptotic time structure of eq . < < 3 . some authors ( bianco et al . , 2008 ; turalska et al . , 2011 ) argue that = 1.5 and others ( frantsuzov et al . , 2009 ) , releasing the condition that all the units share the same poisson rate , generate a global condition with crucial events characterized by < 2 , but significantly departing from the value = 1.5 . ( 2009 ) , might yield the misleading impression that the crucial value of is a consequence of ordinary statistical physics . ( 2011 ) is the discovery of a promising road to settle the problem of information transmission from one to another complex network . in fact , if the inner synchronization corresponds to a criticality condition and criticality generates crucial events with a power < 3 , then a complex network at criticality is a generator of 1/f noise , with a power spectrum s(f ) 1/f . thus , the problem of information transmission from one to another complex network becomes equivalent to the phenomenon of 1/f resonance illustrated in aquino et al . the experiments ( onsager , 1944 ; silvestri et al . , 2009 ) support the dynamical rather than the phenomenological lrt . it is important to stress that phenomenological lrt is a natural consequence of adopting the asymptotic time perspective replacing the waiting - times pdf ( ) of eq . 1 with ( ) 1/. this way of proceeding , although generating the elegant mathematics of fractional derivatives , has as an ultimate effect the misleading discovery of the death of linear response . this is not a unique way of connecting the longtime to the short - time regime . however , whatever form we adopt we are convinced that there will be a parameter corresponding to the parameter t of eq . it is reasonable to assume that an external perturbation may perturb either t or , or both . however , the perturbation of is incompatible with the assumption of a weak stimulus . in fact , is a consequence of the cooperation among the units of the network , and a perturbation may affect only if its strength is large enough to influence the interaction among the units of the network . thus , an external weak perturbation can only have an effect on t , thereby making the dynamical lrt become the proper way to study the response of a complex network to a weak external stimulus , in accordance with the experimental results ( onsager , 1944 ; allegrini et al . , 2009 ; silvestri et al . , 2009 ) . for these reasons , we can conclude that figure 1 is an original , and important , result of this research program . we hope that the application of the pcm may open the door to solving the problem of information transmission from one complex network to another , a research topic that is still in its infancy . in order to better understand the transfer of information between complex networks last year we numerically analyzing networks consisting of a large number of non - linearly interacting nodes . ( 2009 ) using a master equation formalism ( turalska et al . , 2011 ) were discussed where each element of the network is a two state oscillator and is described by a master equation of the form and pj(t ) is the probability of being in the state j = 1 ; p1 + p2 = 1 . the coupling functions are time - independent for non - interacting members of the network . subsequently , we considered a network consisting of l discrete variables located at the nodes of a 2d square lattice . each unit si is a stochastic oscillator and can be found in either of the above two states . for the dynamic complex lattice each element si interacts with each of its nearest neighbors and is updated in an elementary time step with transition rate g : here m is the total number of nearest neighbors ; m + 1 and m 1 are the number nearest neighbors that have made the decision yes and no , respectively . the single individual changes opinion , and as a consequence these numbers are variables fluctuating in time , while the total number of elements is constant . ( no ) makes a transition to the state no ( yes ) faster or slower according to whether the majority of the elements are in the state no ( yes ) or yes ( no ) , respectively . 2011 ) do all calculations on a l l lattice with periodic boundary conditions . they characterize the network in terms of the global order parameter whose variability is not dichotomous . in figure 2 an example of the temporal evolution for a single unit is compared with that of the global order parameter . ( a ) temporal evolution of a single unit and ( b ) of the global order parameter for the dmm realized on a square lattice with l = 50 , g0 = 0.01 , and k = 1.70 . ) note that the amplitude of the global order parameter depends on the value of the coupling constant k. when k = 0 , single units of the network are independent . when k > 0 , single units are less and less independent , resulting in a non - zero average . the quantity kc is the critical value of the control parameter k , at which point a phase transition to a global majority state occurs . in numerical calculations they use the time average eq |(t)| as a measure of the global majority . more precisely after an initial million time steps , which is sufficient time to suppress any transients , an average is taken over the same number of consecutive time steps in the dmm . they find that in the special case when m is the same for all the nodes and g0 1 , that dmm generates the same kind of phase transition as is observed in the 2d ising model discussed in onsager s seminal paper ( onsager , 1944 ) . the phase transition for the global variable eq is indicated in figure 3 under various conditions . it is evident that the dmm phase transition on a lattice is only equivalent to the ising model under very restricted conditions . the apparent equivalence between the ising and dm models is merely formal , since the dmm does not have a hamiltonian origin and its elements are not in contact with a thermal bath ( turalska et al . , 2011 ) . these differences explain why the equivalence requires the transition rate to vanish , so as to freeze the dynamics of the single units , in the absence of cooperation . the phase diagram for the global variable eq . the thin solid line and dashed line are theoretical predictions for the fully connected and 2d regular networks , respectively . in both cases l = and the latter case is the onsager prediction for a 2d regular lattice . the thick solid line corresponds to the global states observed for a 2d regular lattice with l = 100 and g0 = 0.01 . when the transition rate assumes a finite value the equivalence between dmm and the ising model is lost . ( 2011 ) investigate the parameter phase space to determine the domain of phase transitions and find that they can occur for values of k below that of the theoretical kc . there is also a situation for relative high transition rates in which every unit is surrounded by nearest neighbors in the opposite state , yielding an update of its state at every time step and generating the condition in which the order parameter is exactly zero at all times . ( 2011 ) conjecture that the crossings of the origin by the global order parameter are the significant events to observe . as illustrated in figure 2 they interpret the time interval between two consecutive crossings as the time duration of a given decision , even if this decision may rest on a slight and fluctuating majority . they evaluate the distribution density of decision time duration , ( ) , and the corresponding survival probability ( ) , where although emerging from a simple regular lattice , that is , one with no structural complexity , the survival probability presented in figure 4 shows a scale - free property that extends over more than four orders of magnitude in time for k kc a further increase in the coupling strength does not affect the power law region . survival probability function ( ) for the global order parameter evaluated on a 2d lattice with l = 50 , g0 = 0.01 and increasing values of the coupling constant k. the straight line corresponds to a slope of 0.50 , namely = 1.5 since ()1/. ( from turalska et al . wiener s rule maintains that a network with high information can organize one with low information . for example a tightly coupled organization , with rules and policies to cover all contingencies , changes little over time and therefore is low in information . cpm quantifies wiener s rule by introducing a measure of complexity allowing us to compare the level of information in interacting complex networks . law index of the hyperbolic distribution and a generalization of lrt enabled us to construct the cross - correlation cube to determine the degree of asymptotic influence one network has on another . in this way the 1/f variability of stimuli is found to resonate with the human brain ( grigolini et al . , 2009 ) , as when we are entranced by music or irritated by a dripping faucet ( grigolini and west , 2011 ; west and grigolini , 2011 ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the army research laboratory program on the network science of human decision making brings together researchers from a variety of disciplines to work on a complex research problem that defies confinement within any single discipline . consequently , new and rewarding solutions have been obtained for a problem of importance to society and the army , that being , the human dimension of complex networks . this program investigates the basic research foundation of a science of networks supporting the linkage between the cognitive and social domains as they relate to human decision making . the research strategy extends recent methods of non - equilibrium statistical physics to non - stationary , renewal stochastic processes characteristic of the interactions among nodes in complex networks . the theoretical analyses of complex networks , although mathematically rigorous , often elude analytic solutions and require simulation and computation to analyze the underlying dynamic process . the information transfer between two complex networks is calculated using the principle of complexity management as well as direct numerical calculation of the decision making model developed within the project .

Introduction Principle of Complexity Management Synchronization and Information Exchange Decision Making Model and Phase Transitions Conclusion Conflict of Interest Statement

retinoblastoma ( rb ) is a primary intraocular tumor that is most commonly malignant in childhood ; this condition arises from primitive neural retinal cells destined to become photoreceptors and corresponds to 11% of all cancers in the first year of life . the incidence of rb worldwide is estimated at between 5,000 and 8,000 new cases per year , and although rb can occur at any age , this tumor occurs most often in preschool children ( 95% of cases diagnosed before 5 years of age ) [ 3 - 5 ] . the primary genetic event associated with rb is the inactivation of both alleles of the rb1 gene ( gene i d : 5925 , omim 614041 ) in the 13q14 locus by translocations , deletions , insertions , and point mutations . genetically , rb presents as hereditary ( 40% ) or non - hereditary ( 60% ) . in the inherited form , a constitutional mutated allele is always transmitted via germs ; meanwhile , in the non - hereditary form , both alleles are inactivated by somatic mutations . the tp53 gene ( gene i d : 7157 , omim 191170 ) is located at 17p13.1 and provides instructions for making the p53 tumor protein ( p53 ) ; this protein acts as a tumor suppressor and binds directly to dna . when p53 is damaged , other genes are activated to repair the damage . if the dna can not be repaired , this protein prevents the cell from dividing and sends signals for apoptosis ; thus , the protein has been nicknamed the guardian of the genome . genetic polymorphisms are variants of genome mutations that appear in certain individuals . these polymorphisms are transmitted to offspring and acquire a certain frequency in the population after many generations . the most common polymorphisms are single base changes , known as single nucleotide polymorphisms ( snps ) . the aim of this study was to determine the frequency and association between the snp rs9568036 ( the rb1 gene ) and the snp rs1042522 ( tp53 ) and clinical characteristics in a group of mexican children with rb . a total of 11 pediatric patients under 10 years of age with a diagnosis of rb confirmed by histopathological studies who presented at our hospital between march 2015 and august 2015 for close follow - up exams after completion of chemotherapy were recruited for polymorphism detection ( men 63.6% , mean diagnosis age=16 months , range 3 - 37 ; woman 36.4% , mean diagnosis age=20 months , range 4 - 42 ) . the mean duration of follow - up after diagnosis was 30 months ( range 4 - 87 ) . we excluded patients who had received blood transfusions in the previous 6 months or those who had received an organ donation . the study was approval by the local research ethics committee of high specialty medicine unit , specialty hospital ( umae ) no . 71 of mexican institute of social security ( imss ) with code registered r201550170 20 , and informed consent was obtained from the responsible caregiver for all cases . the study was conducted in accordance with the declaration of helsinki and in compliance with the laws and regulations of the mexican general law of health in research for health . fifteen microliters of venous blood of patients with rb were obtained , placed in 100 l of cell lysis solution , and immediately stored at 2 c . polymorphism analysis was performed by amplification of the polymorphic site with conventional pcr using gotaq master mix ( promega inc . , madison , wi ) to obtain similar amounts of amplification products from each sample . the 15 l reaction contained 7.5 l gotaq master mix , 5.7 l water , 0.3 l the each primer ( aag gga ggg ata gca tta gga ( sense ) , gaa tgt gca ggt ttg tta cat agg ( antisense ) for rb1(rs9568036 ) and cat cac acc ctc agc atc tc ( sense ) , gcc tgg tca gga gct tat tt ( antisense ) for tp53 ( rs1042522 ) ) and 1.2 l blood lysate . high - resolution melt ( hrm ) analysis , which is based on the characterization of the pcr products according the dissociation behavior of the dna strand , was performed because this method is sensitive even to a simple change of base . a melting temperature ( tm ) of 55 c to 95 c the conditions of the conventional pcr amplification were : initial denaturation at 94 c for 10 min , followed by an amplification of 55 cycles of 95 c / 45 s ; 54 c / 45 s ; 72 c / 45 s. the reaction mixture for each sample ( 15 l ) was prepared according to the manual accompanying the hrm type - it reagent ( qiagen inc . , germantown , md ) , and contained 7.5 l , 5.9 l water , 0.3 l sense primer , 0.3 l antisense primer , and 1 l pcr product ( table 1 ) . the conditions of a second amplification using real - time pcr were the same that conventional pcr , only that they used 20 cycles for both polymorphisms ; for the analysis of the melt curves a rotorgene thermocycler ( quiagen inc . dna melting curve analysis between 55 c and 90 c for both genes was used as an internal control to prevent false - negative or false - positive results ( figure 1 ) . as the temperature increased , the strands of the amplicon separate to form single - stranded dna , causing the fluorescent intercalating dye to dissociate from the dna and stop fluorescing . the snps rs9568036 ( rb1 ) and rs1042522 ( tp53 ) selected for the study were taken from the hapmap snp database and were genotyped by iselect hd bead - chip ( illumina , san diego , ca ) . statistical analysis included measures of central tendency and dispersion ( p values less than 0.05 were considered significant ) , and the chi - square and student t tests were used to determine the association between variables using spss version 19.0 software ( spss inc . , genotype and allele frequencies , as well as hardy weinberg equilibrium , were evaluated using guo and thompson s method . the frequencies of genotypes and alleles obtained for each of the examined snps are presented in table 2 . there was a statistically significant difference ( p<0.05 ) between the frequency of the genotypes and the expected genotypes in the population according to the hardy weinberg equilibrium ( table 3 ) . there was a statistically significant difference between the rb1-gg / rs9568036 polymorphism and tumor chemoresistance , as well as between the rb1-aa and ag / rs9568036 polymorphisms and tumor chemosensitivity ( table 4 ) . there was no statistically significant association ( p = 0.6576 ) between the rb1/rs9568036 polymorphism and any allelic variants with unilateral or bilateral disease presentation . there was a statistically significant relation between unilateral presentation of the disease and the polymorphism tp53-cc / rs1042522 ( p = 0.0386 ) , rate ratio ( rr ) = 2.0 ( 95% ci = 0.755.32 ) , but the allelic variants tp53-cg and gg / rs1042522 had no relation with the laterality of the disease ( p = 0.4611 ) . / rs9568036 and men ( p = 0.0386 ) , rr = 2.0 ( 95% ci = 0.765.32 ) , as well as between the rb1-aa and ag / rs9568036 polymorphisms and women ( p = 0.0027 ) , rr = 8.0 ( 95% ci = 1.2850.04 ) . the rb1 gene plays an important role in the negative control of cell cycling and tumor progression by translating the rb protein and inhibiting cell division . in the hypophosphorylated state , the rb protein is active and prevents the attachment of an e2 factor ( e2f ) transcription factor ( related to dna replication for division ) . being hyperphosphorylated , the rb protein receives kinases phosphate groups and is inactivated , allowing cells to move from the g1 to the s phase and proceed with division . the absence of rb protein activity , as a result of mutations that inactivate or deregulate its phosphorylated state , causes continuous cell division and leads to a large number of human cancers and metastasis , including cellular response to mitotic inhibition induced cell death during chemotherapy . to have equal concentrations of dna , the first amplification was performed with end - point pcr , after which the pcr - hrm technique was performed to analyze the polymorphisms . the pcr - hrm technique used to analyze the polymorphisms in this study has been used in different clinical applications and successful investigations even in the analysis of snps in different types of cancer . each double - stranded dna fragment presents a characteristic fusion pattern , which is determined by the content , length , and position of the cgs in the dna sequence , thus allowing the detection of minimal alterations in the sequences , making this method faster , simpler , and less expensive compared with other methods used for amplification and quantification of dna sequences . liu and colleagues studied some polymorphisms of the rb1 gene and found that in response to platinum- and taxane - based chemotherapy , patients with the polymorphism rs4151510 gg had better survival rates for squamous cell lung cancer , while patients with the polymorphisms rs4151510 , rs4151465 , and rs9568036 had increased survival rates for non - small lung cancer cells . in this study , we found that the polymorphism rb1/rs9568036 homozygous mutant gg was associated with twice the likelihood of rb in men refractory to conventional chemotherapy , while the snp rs9568036 homozygous wild aa and heterozygous ag were associated with tumor sensitivity to chemotherapy . found that in rb the polymorphisms rs1801270 c > a and rs1059234 c > t of the cdkn1a ( gene i d : 1026 , omim 116899 ) gene were associated with an increased risk of rb ( odds ratio [ or ] = 2.5 , 95% ci = 1.384.53 ) , whereas patients with the cc genotype for both polymorphisms had a lower risk of rb ( or = 0.43 , 95% ci = 0.250.74 ) . in this study , we found that the polymorphism rb1/rs9568036 homozygous wild aa and heterozygous ag were associated with an increased likelihood ( 8x ) of rb in women . kadam - pai and colleagues studied the tsp509i polymorphism of the rb1 gene in eight asian populations and found an ethnic variation prevalent in southeast asia that confers increased susceptibility to rb . in the present study of a northern mexican population , we found that the rb1/rs9568036 polymorphism and the allelic variants rb1-gg / rs9568036 , rb1-aa , and ag / rs9568036 were associated with an increased risk for rb . furthermore , most of the somatic mutations in the tp53 gene alter the individual amino acids in p53 leading to the production of an altered version of the protein that can not bind dna efficiently . this defective protein can accumulate in the nucleus of cells and prevent them from suffering apoptosis in response to dna damage . thus , the damaged cells continue to grow and divide an unregulated manner . in the rs1042522 polymorphism of the tp53 gene , the substitution g > c exists in codon 72 and exon 4 . han and colleagues found that the gg genotype of the snp rs1042522 of the tp53 gene showed increased resistance to first - line chemotherapy compared with those with the genotypes cg or cc ( 60% versus 27% , p = 0.014 ) in a study of 148 patients with lung cancer with non - small cell carcinomas . shabazz and colleagues found a correlation between an increased risk of breast cancer and allelic variants of the rs1042522 polymorphism in women from bangladesh ( p = 0.0053 , or = 1.69 ) . meanwhile , chen et al . reported in a chinese population that this polymorphism is associated with local tumor invasion . in this study , we found a statistically significant relation between the unilateral presentation of the disease and the same snp . the rs9568036 polymorphism in the rb1 gene and its allelic variants can be associated with the type of response to medical therapy and associated with male sex , while the allelic variant rs1042522 polymorphism in the tp53 gene is associated with the unilateral presentation of the disease in a group of mexican children with rb . the rs9568036 polymorphism in the rb1 gene and its allelic variants can be associated with the type of response to medical therapy and associated with male sex , while the allelic variant rs1042522 polymorphism in the tp53 gene is associated with the unilateral presentation of the disease in a group of mexican children with rb .

purposeto determine the frequency and association of polymorphisms in the tp53 and rb1 genes with clinical characteristics in a group of children with retinoblastoma ( rb ) in northern mexico.methodsa prospective , longitudinal , and analytical study of 11 patients diagnosed with rb was conducted . endpoint pcr and high - resolution real - time pcr were performed . chi - square and student t tests were used to evaluate associations between variables . allelic frequencies , as well as genotypic and hardy weinberg equilibriums , were evaluated using guo and thompson s method.resultswe found a statistically significant difference between the polymorphism rb1-gg / rs9568036 and tumor chemoresistance ( p<0.05 ) . the allelic variants rb1-aa and ag / rs9568036 were determined to be associated with tumor chemosensitivity ( p<0.05 ) . a statistically significant relation between the polymorphism rb1-gg / rs9568036 and males ( p = 0.0386 ) , rate ratio ( rr ) = 2.0 ( 95% confidence interval [ ci ] = 0.765.32 ) , as well as between the allelic variants rb1-aa and ag / rs9568036 and females ( p = 0.0027 ) , rr = 8.0 ( 95% ci = 1.2850.04 ) , was observed . we also observed a statistically significant association between the rs1042522 polymorphism in the tp53 gene and unilateral presentation of the disease.conclusionsthe rs9568036 polymorphism in the rb1 gene and the allelic variants can be associated with type of response to medical therapy and associated with male sex , while the allelic variant rs1042522 polymorphism in the tp53 gene is associated with the unilateral presentation of the disease in a group of mexican children with rb .

Introduction Methods Results Discussion Conclusion

the nose is a central structure of the face , so attempts to reconstruct ideal form and esthetics can be complicated . the majority of patients with cleft lip nose deformity require augmentation rhinoplasty for a more balanced nasal dorsum . traditionally , autogenous cartilages from various sites are used to correct cleft lip nose deformity . however , sometimes results of corrective rhinoplasty using auricular or septal cartilages are unsatisfactory due to the weakness of the graft material . in that case , rib cartilage could be a good material of choice for corrective rhinoplasty for cleft lip nasal deformity . however , the harvesting procedure leaves a cutaneous scar that is unacceptable , especially for a young female patient . so we need to find alternative surgical options to replace the autogenous rib cartilage . in 1998 , a new biomaterial called permacol ( tissue science laboratories plc , aldershot , uk ) was licensed in europe for permanent implantation in human body . permacol consists of acellular , cross - linked porcine dermal collagen that is resistant to degradation by collagenase . it had been widely used in general and gynecological surgery in the treatment of hernia and otolaryngology surgery such as surgical closure of nasal septal perforation . recently it was described in facial contour augmentation surgery including esthetic augmentation rhinoplasty and reconstruction of a post - traumatic nasal deformity . the author augmented nasal dorsum after reconstruction of the lower third of the nose during corrective rhinoplasty in patients with cleft lip nasal deformity . in this report we describe three cases of rhinoplasty procedures using autogenous auricular cartilage and synthetic porcine dermal collagen with highlights of how to use it . a 17-year - old female patient with secondary deformity due to unilateral incomplete cleft lip and palate was referred to the department of oral and maxillofacial surgery , gangneung - wonju national university dental hospital . five years ago , she underwent autogenous bone graft on her cleft alveolus . during this time , her chief complaint was the deformed nose , so we decided upon corrective rhinoplasty and minor revision surgery on her upper vermilion . after taking standard rhinoplasty photograms , formal consent for the use of xenograft material was obtained . the operation was performed under general anesthesia with additional anesthetics consisting of 2% lidocaine and 1:100,000 epinephrine in the planned dissection area . via an open rhinoplasty approach with intercartilaginous and stair - shaped transcolumellar incision ( fig . 1a ) , the lower lateral cartilage was dissected from surrounding soft tissues and prepared a subperiosteal pocket for the xenograft on nasal bone ( fig . the permacol sheet comes ready to use and can be cut to the desired size easily . we inserted two layers ( 3 mm ) of permacol under the dorsal skin of the nose , and fixed it to the underlying cartilages with 4 - 0 polydioxanone ( fig . after closing the surgical wounds , we applied external nasal stent and tape for two weeks , as well as a nasal conformer for six months at night postoperatively . esthetic results in visits at one month , six months , and one year were satisfactory for patients and surgeon ( fig . 2 ) . the dorsal implant was intact and did not show any resorption or displacement . a 17-year - old male patient with secondary deformity due to unilateral he had undergone several surgeries including distraction osteogenesis for collapsed maxilla , secondary alveolar bone grafting for cleft alveolus , and pharyngoplasty by the author . on this occasion ( january 25 , 2009 ) , corrective rhinoplasty was performed . under general anesthesia , open rhinoplasty was performed via an intercartilagenous incision , continuous with the transcolumellar incision on the line of previous scar ( fig . 3c ) , we applied it as a columellar strut and gull - wing type graft ( fig . 4 ) . a 14-year - old male patient with secondary deformity due to bilateral cleft of primary palate presented to the department . his mother s chief complaint was the visible scar on upper vermilion and the compressed nose , so we decided to perform a cheilorhinoplasty . under general anesthesia , open rhinoplasty was done via an intercartilagenous incision inside both nostrils and the incision for scar revision ( fig . , we applied it as a columellar strut and gull - wing type graft for prominent nasal dome ( fig . we then implanted two layers of permacol in the form of an onlay graft , and fixed it to the grafted auricular cartilages with monocryl sutures ( fig . a 17-year - old female patient with secondary deformity due to unilateral incomplete cleft lip and palate was referred to the department of oral and maxillofacial surgery , gangneung - wonju national university dental hospital . five years ago , she underwent autogenous bone graft on her cleft alveolus . during this time , her chief complaint was the deformed nose , so we decided upon corrective rhinoplasty and minor revision surgery on her upper vermilion . after taking standard rhinoplasty photograms , formal consent for the use of xenograft material was obtained . the operation was performed under general anesthesia with additional anesthetics consisting of 2% lidocaine and 1:100,000 epinephrine in the planned dissection area . via an open rhinoplasty approach with intercartilaginous and stair - shaped transcolumellar incision ( fig . 1a ) , the lower lateral cartilage was dissected from surrounding soft tissues and prepared a subperiosteal pocket for the xenograft on nasal bone ( fig . the permacol sheet comes ready to use and can be cut to the desired size easily . we inserted two layers ( 3 mm ) of permacol under the dorsal skin of the nose , and fixed it to the underlying cartilages with 4 - 0 polydioxanone ( fig . after closing the surgical wounds , we applied external nasal stent and tape for two weeks , as well as a nasal conformer for six months at night postoperatively . esthetic results in visits at one month , six months , and one year were satisfactory for patients and surgeon ( fig . 2 ) . the dorsal implant was intact and did not show any resorption or displacement . a 17-year - old male patient with secondary deformity due to unilateral complete cleft lip and palate was referred to our department . he had undergone several surgeries including distraction osteogenesis for collapsed maxilla , secondary alveolar bone grafting for cleft alveolus , and pharyngoplasty by the author . on this occasion ( january 25 , 2009 ) , corrective rhinoplasty was performed . under general anesthesia , open rhinoplasty was performed via an intercartilagenous incision , continuous with the transcolumellar incision on the line of previous scar ( fig . 3c ) , we applied it as a columellar strut and gull - wing type graft ( fig . a 14-year - old male patient with secondary deformity due to bilateral cleft of primary palate presented to the department . his mother s chief complaint was the visible scar on upper vermilion and the compressed nose , so we decided to perform a cheilorhinoplasty . under general anesthesia , open rhinoplasty was done via an intercartilagenous incision inside both nostrils and the incision for scar revision ( fig . , we applied it as a columellar strut and gull - wing type graft for prominent nasal dome ( fig . we then implanted two layers of permacol in the form of an onlay graft , and fixed it to the grafted auricular cartilages with monocryl sutures ( fig . there is a vast number of surgical techniques and grafting materials for cleft lip nose deformity reconstruction . in general , autogenous tissues remain the gold standard for reconstructive surgery of cleft lip nose deformity . especially for augmentation of the nasal dorsum however , autogenous grafting procedures require additional surgery and donor site morbidity , and sometimes graft material is limited . thus maxillofacial plastic and reconstructive surgeons always need to prepare allogeneic or alloplastic materials as an alternative . silicone , high - density porous polyethylene , expanded polytetrafluoroethylene , and human dermal collagen are used as alloplastic materials for augmentation rhinoplasty , and have both advantages and disadvantages . permacol can be a useful material for augmentation rhinoplasty because it is readily available in the required volume , it is easy to shape , and it seems to maintain the structural integrity of the nose . in this study , three patients underwent corrective rhinoplasty via external approach using autogenous auricular cartilage and permacol composite grafting . the original dimension of the permacol sheet used was 60301.5 mm , and two layers of permacol were inserted into the nasal dome with customized size for each patient . if layering is required , the material can be cut into separate sheets , creating edges rather than folding or rolling , probably due to possible colonization of microorganisms . , the auricular cartilage was covered with a double layer of permacol to form the ideal nasal contour . afterwards , the author felt two layers of permacol was aesthetically sufficient for the patient with thin nasal skin ( case 1 , 3 ) , but deficient for the patient with thick facial soft tissues ( case 2 ) . previous studies demonstrate that permacol , when implanted in human body , is readily surrounded by host tissues such as fibroblasts and vascular endothelial cells . hunter et al . observed a permacol explant 15 months post implantation , originally inserted for augmentation rhinoplasty . macroscopically , permacol was covered with a clear layer of fibrous tissue , characterized histologically by the irregular arrangement of collagen as well as the presence of some cells and elastin fibers . suggested that permacol acted as a matrix into which autogenous tissue could grow . from those studies and our own limited experiences with three patients , inserted fourty - five permacol implants for augmentation rhinoplasty over a 4-year period , with only one case of postoperative implant removal . they concluded that permacol seemed to be a biocompatible alternative to conventional grafting materials , with minimal morbidity . compared to the removal rate of silicone and the infection rate of gore - tex , pitkin s clinical results are encouraging . moreover , permacol can be safely used in a contaminated field . in our study , immediately after the operation , no patients complained of any foreign body sensations or discomfort at usual daily activities . . recommended long - term follow - up for the use of permacol because some graft absorption was noted in their patient , who had undergone nasal profile augmentation . the author followed three patients for more than five years , but there was no sign of graft absorption . hopkins et al . also report successful long - term use of permacol in rhinoplasty . in conclusion , we obtained pleasing results in reconstruction of cleft lip nose deformity with simple and safe surgical procedures . in our limited experience with three patients , the author concludes that the permacol can be a reliable surgical option for patients with cleft lip nose deformity .

esthetic reconstruction of cleft lip nose deformity is a challenging task in surgical management of patients with orofacial cleft . the author reconstructed cleft lip nose deformity effectively using autogenous auricular cartilage and a relatively new graft material of porcine dermal collagen , permacol. after correction of the deformed lower third of the nose with patient s auricular cartilage , we applied permacol to augment the entire nasal dorsum . three patients were treated and followed for up to five years . all patients improved in nose aesthetics without any inflammatory or immunogenic reaction . the author suggests that the use of permacol for nasal profile augmentation in the treatment of cleft lip nose deformity is an alternative surgical strategy with minimal surgical invasiveness . the author report long - term experience with combined use of auricular cartilage and permacol in nasal reconstruction for cleft lip nose deformity .

Introduction Case Report Case 1 Case 2 Case 3 Discussion

prostate cancer ( pca ) is a frequent cancer that can be cured if early diagnosed ( 1 ) . prostate specific antigen ( psa ) has become an important tool in pca screening ( 2 , 3 ) and men with serum psa greater than 4ng / ml are at higher risk of pca . these patients are usually referred for a prostate biopsy ( bxp ) . however , increased psa levels are also associated with conditions other than cancer ( 3 ) , such as benign prostatic hyperplasia ( bph ) and prostatitis ( 4 , 5 ) . chronic abacterial prostatitis is a common diagnosis in men of all ages , with widespread demographics , and it is a common reason for yearly visits to the doctor in the united states ( 3 ) . only a few studies have linked prostatitis to an increase in serum psa ( 6 - 9 ) . subclinical inflammation of the prostate could elevate serum psa in asymptomatic patients , confounding the use of psa values to indicate bxp ( 10 ) . in the majority of cases , prostatitis is an incidental pathological finding that has no clinical relevance . there has been investigation into ways to decrease the misleading diagnosis resulting from inflammation . repeat psa measurements after a period of observation in asymptomatic men can help to avoid unnecessary bxp ( 10 - 12 ) . it has been suggested that antibiotic therapy ( at ) can also avoid bxp in many patients with prostate inflammation , in the psa grey zone ( 4.0 to 10.0ng / ml ) ( 6 , 13 , 14 ) . currently , the indications of re - bxp in patients with a negative initial biopsy are few ; therefore , the first bxp must be precise . in an effort to improve the reliability of psa reduction as an indicator , and consequently avoid unnecessary prostate biopsy , we conducted a prospective , controlled , non - randomized study to evaluate the effect of at on psa levels in patients who have an initially mild psa elevation ( 4.0 - 10ng / ml ) . a prospective , controlled , non - randomized trial was carried out , with 106 asymptomatic men with total psa ( tpsa ) levels between 4.0 and 10.0ng / ml , who underwent routine evaluation from april 2007 to october 2011 . the criteria for inclusion in the trial were digital rectal examination ( dre ) with no suspicion of malignancy and no history of urologic instrumentation , use of antibiotics , and urinary infection or sexually transmitted disease in the previous 12 months . patients with diseases like diabetes or alcoholism , those describing the use of illegal drugs , those undergoing treatment for bladder outlet obstruction and patients with previous bxp ( positive and negative ) were excluded , as were those participants who did not use the at correctly or who did not carry out the follow - up correctly . after institutional review board ( ethical committee ) approval , all study participants provided informed written consent before enrolment . after the initial consultation , the serum levels of total psa ( tpsa ) and free psa ( fpsa ) were determined . the body mass index ( bmi ) , free psa fraction ( % fpsa ) , and psa density ( dpsa ) the participants were divided into two groups ( figure-1 ) according to their decision regarding the use of antibiotics . this decision was made after the authors explained about the lack of evidence for this treatment . group i patients received antibiotics , while group ii patients chose not to use at . in group i , 64 men used ciprofloxacin 500 mg twice a day for a period of three weeks . then , three weeks after the end of at , the serum psa , dpsa , and fpsa were again determined ( figure-1 ) . the results of these patients were compared with those of 42 participants with psa between 4.0 and 10.0ng / ml whose exams were repeated after 6 weeks , without antibiotics , at the participant s discretion . psa was considered to be normalized after treatment when the values returned to lower than 4ng / dl . all of the participants underwent bxp 2 - 4 weeks after the second psa determination . based on the pre- ( psapre ) and post - treatment ( psapost ) psa values , the variation ( psavar ) thus , the variation rate ( psavar ) can be calculated with psa = ( psavar / psapre).100% . the bxp was ultrasound - guided and a minimum of 12 cores were sampled with determination of prostate volume in cubic centimetres ( cc ) . for those participants whose biopsy did not show pca , dre : digital rectal examination ; tpsa : psa total ; fpsa : psa free . the data obtained were analysed by testing the difference between two proportions for incidence of pca in each group , and evaluation of the decrease in psa levels . the other data were evaluated using the student t test , citing the critical t for a significance level of 0.05 . the data obtained were analysed by testing the difference between two proportions for incidence of pca in each group , and evaluation of the decrease in psa levels . the other data were evaluated using the student t test , citing the critical t for a significance level of 0.05 . the age of the participants ranged from 47 to 78 years , with a mean age of 66.1 years and median age of 61.8 years . the mean age of g1 was 61.8 years and of g2 was 62.6 years ( p=0.60 ) . the prostate volume was 51.123.8 gr in g1 and 53.919.2 gr in g2 ( p=0.52 ) . the diagnosis of pca was confirmed in 25 of the 106 participants ( 23.6% ) ; in 9 out of 42 participants in g2 ( 21.4% ) and in 16 out of 64 in g1 ( 25% ) ( z=0.42 ; p>0.05 ) . group igroup iipwith atb meansdwithout atb meansdage ( years)61.817.8362.576.370.6bmi ( kg / m)27.863.5129.668.090.02volume ( cc)51.1523.8753.9719.280.527psapre6.821.666.761.680.429psapost5.291.85.382.160.409fpsapre1.250.471.310.400.233fpsapost1.030.441.060.480.383dpsapre0.150.050.140.060.261dpsapost0.110.060.110.070.369values expressed in mean standard variation . student t test there was a more than 10% psa decrease in 72 out of 106 patients ( 67.9% ) ; this rate was 65.6% and 71% in groups 1 and 2 , respectively ( z=-0.63 ; p>0.05 ) . of the 42 participants from g1 in which the psa decreased after at , 6 ( 14.3% ) had a positive and 36 ( 85.7% ) a negative biopsy . in g2 , 30 participants had a psa reduction , with 4 ( 13.3% ) with a positive and 26 ( 86.7% ) a negative biopsy . after the use of at , the psa level decreased to < 4ng / ml in 26 participants ( 24.5% ) ; in 15 ( 23.4% ) of those who used at , and in 11 ( 26% ) from g2 ( z=-0.032 ; p>0.05 ) . the percentage of participants who had a value below 2.5ng / ml in the second psa was 9.4% for g1 , and 7.1% for g2 ( z=0.40 ; p>0.05 ) . the initial and final values for psa , fpsa , % fpsa and dpsa decreased significantly in both groups ( table-2 ) . table 2comparison of initial and final psa values between groups.group igroup ii pre - atbpost - atbppsa initialpsa finalptpsa6.821.665.291.8<0.0016.761.685.382.16<0.0001fpsa1.250.471.030.440.00021.310.41.060.480.0003%fpsa18.265.0820.136.350.001719.915.5621.086.950.072dpsa0.150.050.110.06<0.00010.140.040.110.070.0003values expressed in meanstandard variation . student t test the mean total psa in g1 was 6.821.66 and 5.291.8ng / ml before and after treatment , respectively ( p<0.001 ) ; this represents a psavar of -1.531.93ng / ml . in g2 , the mean psa before and after antibiotics was 6.761.68 and 5.382.16ng / ml , respectively , with a psavar of -1.381.87 ( p<0.001 ) . the psa in g1 was 25.56 , and in g2 was 28.7 ( p=0.429 ) . the differences between g1 and g2 are shown in table-2 , according to the initial and final fpsa and dpsa . there was no statistical difference between these values . in relation to dpsa , for g1 , the initial mean was 0.150.05ng / ml / gr of prostate , which decreased to 0.110.06ng / ml / gr ( p<0.0001 ) . in g2 , these figures were 0.140.04ng / ml / gr and 0.110.07ng / ml / gr , respectively ( p=0.0003 ) . these same comparisons , analysing the cases with and without pca , are shown in table-3 . in the participants diagnosed with pca , there was no statistical difference in relation to variation in psa , fpsa , % fpsa or dpsa . however , in the participants with negative bxp , there was a significant reduction in psa , fpsa and dpsa , but not in % fpsa . table 3comparison of psa values between patients with and without prostate cancer.benignprostate cancer psa initialpsa finalppsa initialpsa finalptpsa6.851.724.961.980.0016.631.496.521.190.2fpsa1.350.441.060.470.0011.020.370.970.400.077%fpsa19.965.0522.205.890.12415.524.7515.015.690.641psad0.140.060.100.06<0.000010.150.020.150.070.61values expressed in meanstandard variation . student t test the sensitivity to a decrease greater than 10% in psa after the use of at to a diagnosis of prostate cancer was 31% , with a specificity of 23% ; the positive predictive value ( ppv ) was 12% and the negative predictive value ( npv ) was 23% . regarding to the possibility of reducing unnecessary bxp after at , it should be emphasized that none of the 25 participants with pca had a final psa below 4ng / ml . the present study analysed the effect of at on psa ( tpsa , fpsa , % fpsa and dpsa ) and investigated whether a relevant psa reduction induced by at could be related to a decreased cancer detection rate at biopsy . physiological conditions other than cancer can cause an increase in serum psa levels that lead to potentially unnecessary biopsy procedures , increasing inconvenience for the patient , and causing over - diagnosis , over - treatment and elevated medical costs ( 15 ) . prostate cancer is determined in only 34% of biopsies performed on the basis of psa elevation ( 1 ) , and in 20 - 30% in patients with normal dre and psa values of between 4 and 10ng / ml . therefore , there is a high level of unnecessary biopsies , particularly in this group ( 16 , 17 ) . the literature demonstrates a relationship between acute and chronic inflammation with elevated psa , but there have been recent studies that suggest the effects and benefits of chronic prostatitis treatment on psa ( 18 - 21 ) . in our study , pca was diagnosed more frequently in patients treated with at ( 25% versus 21.4% , p>0.05 ) , but without statistical relevance . scardino ( 22 ) suggested that the changes in psa with at were similar to the random variations found in healthy men . also , potts ( 23 ) demonstrated no significant differences in bacterial cultures before or after at between psa responders and non - responders . okada ( 24 ) and schatteman ( 25 ) concluded that subclinical inflammation could cause psa elevation , and emphasized the fact that nearly half of all clinically asymptomatic men with elevated psa levels have laboratory signs of prostatitis . they suggest that the use of antibiotics would result in a decrease in psa levels in almost 50% of patients , thereby avoiding bxp . this approach , however , requires careful follow - up , especially for patients whose psa levels fail to decrease to within the normal range ( 26 , 27 ) . kaygisiz ( 1 ) and del rosso ( 19 ) suggested that at should be administered for 3 weeks , regardless of the presence of inflammation when psa levels are in the grey zone , before making a decision regarding whether or not to carry out a biopsy . on the other hand , serretta et al . ( 28 ) found no cancer present if psa levels decreased to below 4ng / ml , or more than 70% , and postulated that biopsy can be postponed , with only a small risk of failing to detect cancer . in multivariate analysis with other clinical variables , although this was not a randomized trial , it was prospective , assessing asymptomatic males without a clinical indication of prostatitis ; the study demonstrated that a large reduction in psa following antibiotics may help to avoid biopsy in selected patients in whom the psa elevation is probably due to inflammation / infection . our data show that those patients who received at and those who did not had the same rate of normalization of psa ( < 4ng / ml ) . , psa returned to normal levels in 23.4% of patients , compared with 26% patients in the non - treatment group . the reason for this is still unclear , although psa normalization does not rule out pca diagnosis , and biopsy must still be considered . magri et al . ( 29 ) , showed that the presence of bph may prevent the reduction of psa induced by combination pharmacological therapy , and suggest that care must be taken in the adoption of psa as a marker of therapeutic efficacy in the presence of confounding factors like bph . according to these authors , psa should be used as a significant component of a strategy that integrates multiple diagnostic approaches . ( 11 ) showed a psa reduction in 63% of patients following at , with psa returning to normal values in 9% of cases , thus avoiding prostate biopsy . ( 23 ) documented psa normalization in 42% of patients , and brett et al . these studies did not perform bxp in all patients to exclude the diagnosis of pca after treatment . however , our study performed biopsy in all patients ( treated and not - treated ) , comparing the pca incidence between groups . however , the entry of participants was not random , but based on the decision made by each participant ; this may represent a selection bias . moreover , the shared decision with the patient is one of the commonalities in international guidelines . approximately 10 - 15% of men will have a psa level > 4ng / ml in any given round of screening . however , the level will return to normal in the subsequent test in 26 - 37% , and will become normal with the next testing in 40 - 55% . ( 31 ) showed that psa levels tend to fall when repeated after 45 days , regardless of at . once normalized , 65 - 83% of men have normal psa levels for several years without therapy ( 32 ) . if psa levels do not fall , the probability of finding cancer is higher than if levels decrease . this occurs because pca is more likely to occur in men with sustained psa elevation than in those with a randomly variable psa that is temporarily elevated ( 24 ) . our study shows a lower , but not significant , cancer detection rate in patients with decreased psa , fpsa and dpsa after antibiotic therapy , demonstrating a correlation between psa normalization and prostatitis or negative biopsy . a psa reduction rate of 10% occurred in 58.5% of patients ; however , it was lower in patients who received antibiotics than in those who received no treatment ( 65.6% versus 71% ) , but there was not significant difference ( p>0.05 ) . the sensitivity , specificity and accuracy of a psa reduction rate of 10% were 31% , 23% and 25% , respectively . this level is therefore not recommended as a cut - off point for clinical decision - making . ( 28 ) showed a significantly lower cancer detection rate in patients with decreased psa after antibiotic therapy , demonstrating a correlation between psa reduction and negative biopsy , with an odds ratio varying from 1.2 to 3.9 for reduction percentages of between 10 and 90% . they suggested that a psa reduction rate of 50% can be adopted and 11% of biopsies avoided until a further psa increase occurs . ( 33 ) reported that the f / t psa ratio appears to be more suggestive of pca than psa in these cases . it should be emphasized , however , that a long follow - up time is needed to determine whether any of these men will have prostate cancer in the near future , and wider studies are required to identify the optimal psa reduction level at which biopsy can be postponed . ( 35 ) claimed that bph and prostatitis appear to be more frequent causes of psa elevation . scardino ( 22 ) recommended that asymptomatic men presenting with a modestly elevated psa level ( < 10ng / ml ) and a normal digital rectal examination could be reassured and then the psa level could be repeated once or twice ; if the levels remained elevated , this would be an indication of the need to perform a biopsy . ( 36 ) , in a prospective randomized and double - blind trial with placebo , demonstrated that psa reduction occurred after antibiotic and placebo application , and suggested that a decrease in psa does not indicate the absence of pca . recently , faydaci et al . ( 37 ) demonstrated that at given to patients with psa levels higher than the threshold value has not led to a significant change in prostate needle biopsy decisions , and suggested that bxp should be considered without the use of at in patients with high psa values if a suspicion of prostatitis does not exist . the literature does not support the evidence that antibiotics alter psa levels except in the presence of bacterial prostatitis , which is an uncommon condition . we should wait for a second psa assessment before prostate biopsy in asymptomatic male patients once the psa will spontaneously reduce in a quarter of cases ; and that antibiotic use has no role in this clinic scenario ( 36 ) . there are several implications in the use of empiric at for patients with elevated psa levels . scardino ( 22 ) emphasized some disadvantages of this approach , such as cost , toxicity , and the fact that it can cause complications of infection . moreover , a decrease in psa after at does not absolutely exclude the presence of pca , even if the psa decreases to very low levels . in addition , there is concern that the indiscriminate use of empiric antibiotics could lead to the development of resistant bacterial species and thereby expose the patient to more resistant and aggressive sepsis , should a biopsy eventually be performed ( 38 - 40 ) . empirical antibiotic therapy in asymptomatic male patients is not related to psa reduction and that psa reduction after antibiotic can not postpone prostate biopsy . based in our findings additionally , it is not possible to define a safe rate reduction and further studies stratifying the relative values of reduction and cancer risk are needed .

purpose we investigated the effect of antibiotics on psa in asymptomatic patients with mild psa elevation.materials and methods we prospectively evaluated , in a non - randomized design , 106 asymptomatic patients with psa of 4 - 10ng / ml , with a negative digital rectal examination and with no urinary tract infection evidence for 2 years . patients were divided into two groups : those treated with antibiotics for 3 weeks ( g1 ) and those who were not treated ( g2 ) . psa was taken six weeks after and prostate biopsy was performed in all patients.results pca was diagnosed in 25 of 106 patients ( 23.6% ) : 16 ( 25.0% ) in g1 and 9 ( 21.4% ) in g2 ( p>0.05 ) . psa normalization was experienced in 24.5% . in g1 , psa returned to < 4ng / ml in 15 ( 23.4% ) patients compared to 11 ( 26% ) patients in g2 . in the patients with a positive biopsy , no significant variation was noted in psa , fpsa , % fpsa and dpsa after antibiotic treatment . a significantly lower cancer detection rate was noted with decreased psa , fpsa , and dpsa after antibiotic use . a psa reduction rate of 10% occurred in 58.5% , and this was similar in both g1 and g2 groups . the sensibility , specificity and accuracy of psa reduction of 10% were 31% , 23% and 25% , respectively.conclusion empirical antibiotic therapy in asymptomatic male patients is not related to psa reduction . the greater than 10% psa reduction after antibiotic in this population can not postpone prostate biopsy .

INTRODUCTION MATERIAL AND METHODS Statistical analysis RESULTS DISCUSSION CONCLUSIONS

chronic myeloproliferative neoplasms ( mpns ) are rare hematologic diseases characterized by the clonal proliferation of mature blood elements from several myeloid lineages , associated in certain cases with bone marrow fibrosis , splenomegaly , and/or hepatomegaly . they include chronic myelogenous leukemia ( cml ) , three related entities named polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and primary myelofibrosis ( pmf ) ( called philadelphia chromosome - negative ( phi - negative ) mpns ) , chronic eosinophilic leukaemia , mastocytosis , and unclassifiable mpns . cml and other mpns are classified based on the presence or the absence of the bcr - abl fusion gene which is the hallmark of cml . three types of molecular markers are associated with phi - negative mpns : activating mutations in the jak2 gene ( jak2-v617f being the most frequent mutation , present in all subtypes of mpns ) ; activating mutations in the mpl gene ( mpl - w515l / k mostly ) ; and alterations of calr , the gene coding calreticulin ( calr ) , detected in et and in pmf [ 311 ] . a small percentage of mpn patients ( < 15% ) do not carry mutations in the jak2 , mpl , or calr genes . the exact roles played by jak2 , mpl , and calr mutations in the pathogenesis , phenotype , and complications of the three mpn subtypes are not fully elucidated . none of the jak2-v617f , mpl - w515l / k , or calr mutations is specific of a particular mpn subtype . they are detected in patients with very different phenotype and disease evolution , and therefore their presence alone is not sufficient to explain the clinical presentation and complications observed in mpn patients . moreover , for subsets of patients , the jak2-v617f mutation has been shown to be a rather late event , sometimes recurrent , which indicates that other genetic events are responsible for clonality in these patients [ 1418 ] . interestingly , some of the clinical symptoms and complications appear to be linked to the chronic inflammation which almost always accompanies mpn disease , and reduction of symptoms linked to inflammation is beneficial to patients [ 19 , 20 ] . presently it is unclear whether the inflammation - related biological markers and clinical symptoms observed in mpn patients are consecutive or reactive to , or perhaps even precede , the main mutations harbored by mpn clones . obviously , a better understanding of the mechanisms that underlie inflammation in the different mpn subtypes should have a significant impact on the design of future protocols tested for the therapy of mpns . to help address this issue , the present review describes the role played by somatic as well as germline genetic defects in the increased production of inflammatory cytokines and other inflammation markers in mpns ; potential nongenetic causes of chronic inflammation are also discussed . inflammation is a pathological process typically triggered by an external aggression , which may be a physical or chemical injury , irradiation , or infection . in addition , chronic hypoxia ( e.g. , when cells accumulate in a solid tumor or in the bone marrow in the context of blood malignancy or in any type of tissue in case of venous or arterial thrombosis ) can also lead to inflammation [ 2123 ] . chronic inflammation is characterized by the prolonged stimulation of the production of immune blood cells from the lymphoid and myeloid lineages and the release of various mediators , notably inflammatory cytokines , in blood vessels and in tissues . myelopoiesis is stimulated during inflammation so as to produce sufficient quantities of polyclonal granulocytes , monocytes , and macrophages to ensure the destruction of damaged cells , tissues , or infectious pathogens , adequate phagocytosis , and presentation of antigens to lymphocytes . the production of polyclonal megakaryocytes and platelets is frequently increased , to ensure thrombus formation and hemostasis in case of damaged blood vessels in inflamed tissues . chronic inflammation may lead to hypoxia of variable severity in the damaged tissues and , accordingly , to increased production of polyclonal erythroid progenitors and red blood cells in an effort to improve cell and tissue oxygenation . conversely , hypoxia can lead to increased production of inflammatory cytokines : individuals with mountain sickness present with elevated levels of inflammatory cytokines in peripheral blood , and healthy volunteers exposed to a hypoxic environment ( three nights in high altitude above 3400 meters ) presented with a high level of interleukin- ( il- ) 6 [ 24 , 25 ] . patients with chuvash polycythemia associated with homozygous germline mutation in the von hippel - lindau ( vhl ) gene , a major actor of the hypoxia sensing pathway , present with elevated levels of tumor necrosis factor- ( tnf- ) and interferon- ( ifn- ) . inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis also provide evidence of cross talk between hypoxia and inflammation . in rheumatoid arthritis , hypoxia - inducible factor- ( hif- ) 2 is the hif isoform that plays a major role in inflammation , notably by inducing expression of il-6 and tnf- . importantly , hif-1 plays an essential role in survival and function of myeloid cells during inflammation . if the initial injury persists , the inflammation response and associated chronic stimulation of hematopoiesis are prolonged , and the risk of dna alteration increases in cells from the damaged tissues or / and in overstimulated hematopoietic progenitors . over time the acquisition of genetic defects in the inflamed tissues or / and hematopoietic progenitors may eventually lead to the development of solid cancer or / and clonal hematopoiesis and hematological malignancy ( figure 1 ) . in fact , all types of solid and blood cancers , including mpns , are accompanied by some degree of chronic inflammation [ 21 , 22 ] chronic inflammation is an early event in many types of cancers and in certain lymphoma but in mpns , the possibility that chronic inflammation precedes the acquisition of the main mpn mutations is a new subject of research . whatever its chronology , chronic inflammation facilitates further dna alteration in cancer and adjacent cells , and targeting inflammation and its causes should offer new opportunities of cancer treatment and also help reduce complications [ 2123 ] . in the context of solid cancer , chronic inflammation may be reactive to a persistent tissue injury ( exposure to toxics or to infectious agents ) or / and to the tumor itself ; it may also be a consequence of tumor - associated mutations or of treatment ( radiotherapy or chemotherapy ) ( figure 2 ) . thus inflammation may precede or / and accompany malignancy , and polyclonal hematopoietic cells of the myeloid and lymphoid lineages participate in the inflammation process . whatever the cause(s ) of inflammation , sustained stimulation of the proliferation of lymphoid or myeloid cells to maintain inflammation over months or years increases the risk of dna alteration in these cells and the subsequent emergence of a mutated clone ( initiation of malignancy ) or of additional mutated clones ( during or after radio- or chemotherapy ) . figure 2 represents progression from chronic inflammation and stimulation of polyclonal myelopoiesis to clonal myelopoiesis , expansion of a mutated myeloid clone , and myeloid malignancy . in mpns , cells from all myeloid lineages may belong to the malignant clone : erythroid cells , megakaryocytes , neutrophils , and monocytes ; b - lymphocytes or / and t - lymphocytes may be mutated too , but only rarely and usually in pmf . in contrast to patients with solid cancer , for whom myelopoiesis is normal and polyclonal , the immune response in patients with mpns includes the mobilization and activity of mutated ( clonal ) myeloid cells as well as of healthy myeloid and lymphoid cells . depending on the small or large size of the mpn clone , the myeloid part of immune and inflammatory responses may be partially or mostly clonal and subsequently mildly or severely defective . this side of myeloid malignancy is often neglected but likely important in the pathogenesis and complications of mpns . one cause of chronic inflammation recognized as increasing the risk of malignant transformation of affected cells and tissues is chronic infection . indeed it is now well established that latent infection can be associated with various types of solid cancer or / and with lymphoid malignancy [ 3137 ] . in blood malignancies , two main transforming mechanisms may be at play : direct cell infection and transformation by oncogenic molecules or indirect transformation via chronic antigen stimulation and cell proliferation resulting in increased risk of acquisition of genetic defects . during inflammation , cytokines are released which signal cells such as t - lymphocytes and monocytes - macrophages to travel to the site of injury . in turn , activated immune cells increase their production of inflammatory cytokines , chemokines , hematopoietic cytokines , and other growth factors , hereby stimulating numerous cell types from their environment ( fibroblasts and endothelial cells ) , which further increases the production of inflammatory cytokines . in this context , the nuclear factor kappa - b ( nf-b ) and jak1/stat1 pathways are the two main molecular pathways activated to enhance the production of inflammation cytokines ( figures 3 and 4 ) [ 12 , 21 , 38 ] . in case of inflammation linked to hypoxia , which may occur after thrombosis or because of cell accumulation , the production of inflammatory cytokines and growth factors by the cells exposed to hypoxia is upregulated via the hif-1 pathway [ 39 , 40 ] . as shown in figure 3 , the nf-b , hif-1 , and jak / stat pathways interact closely . they act in synergy , nf-b activating the hif-1 pathway , which in turn leads to increased activation of several signaling pathways , including jak2/stat5 ( via the production of erythropoietin ( epo ) ) , stat3 ( via inflammation cytokines from the il-6 family or via epo , hepatocyte growth factor ( hgf ) , platelet - derived growth factor ( pdgf ) , and vascular endothelial growth factor ( vegf ) ) , and stat1 ( via type i and type ii inflammatory cytokines ) ( figure 4 ) . moreover , the level of jak activity affects the expression of transcription factors hif-1 and hif-3 [ 13 , 41 ] . in the context of malignancy , the genetic mutations associated with the tumor may or may not induce the production of inflammation cytokines in mutated cells . this aspect is particularly important in the context of blood cancers since the mutated cells are involved in the immune response or / and are major sources of production of inflammatory cytokines . situations where chronic inflammation results from more than one cause are not rare : physical injury and infection , thrombosis and hypoxia , solid cancer and infection , jak2-mutated mpn and thrombosis , and so forth . the degree of activation and overall synergistic action of the three main pathways which control the production of inflammatory cytokines may vary widely , which allows for infinite qualitative and quantitative differences ( figure 4 ) . thus the cytokine profile and degree of overproduction of inflammatory cytokines and other mediators of inflammation are expected to vary from patient to patient , according to the cause(s ) of inflammation , the cell types being stimulated , and the molecular pathways involved . cytokines may be divided into four groups on the basis of their biological functions : ( i ) natural immunity , for tnf- , il-1 , il-6 , il-5 , il-8 , and chemokines ; ( ii ) lymphocyte activation , growth , and differentiation , for il-2 , il-4 , and transforming growth factor- ( tgf- ) ; ( iii ) regulation of inflammation , also for il-4 , tgf- , and il-1 , il-10 , ifn- , and granulocyte macrophage - colony stimulating factor ( gm - csf ) ; and ( iv ) stimulation of leucocyte growth and differentiation , for il-1 , il-3 , il-5 , il-6 , granulocyte - csf ( g - csf ) , macrophage - csf ( m - csf ) , and gm - csf . cytokines are also classified as th1 ( proinflammatory ) cytokines ( il-1 , il-2 , il-12 , tnf- , and ifn- ) and th2 ( anti - inflammatory ) cytokines ( il-4 and il-10 , notably ) . th1 cytokines cause stimulation of cd8-positive cytolytic t - lymphocytes , leading , for instance , to viral clearance . hence the cytokines produced during chronic inflammation vary according to the cause of inflammation and the cell types involved . the cytokines produced in large quantities during inflammation may also vary according to the molecular pathways that are being activated ( due to the acquisition of mutation(s ) , infection , hypoxia , etc . ) . the cytokines produced following activation of the nf-b and jak1/stat1 pathways include il-1 , il-6 , il-8 , il-10 , il-11 , il-12 , il-13 , il-15 , il-22 , vascular endothelial growth factor ( vegf ) , tnf- , tgf- , platelet - derived growth factor- ( pdgf- ) bb , b - fibroblast growth factor ( fgf ) , g - csf , gm - csf , ifn- , macrophage inflammatory protein- ( mip- ) 1 , mip-1 , mip-3 , hgf , ifn--inducible protein 10 ( ip-10 ) , monocyte chemotactic protein- ( mcp- ) 1 , monokine induced by ifn- ( mig ) , and regulated on activation , normal t - cell expressed and secreted ( rantes ) [ 13 , 40 , 41 ] . in case of hypoxia , increased hif-1 expression leads to the upregulation of the production of epo , vegf , insulin growth factor 2 ( igf-2 ) , tnf- , tgf- , pdgf , fibroblast growth factor ( fgf ) 2 , il-6 , hgf , and its receptor met ( list not exclusive ) . most inflammation cytokines activate the jak1/stat3 pathway , thus ensuring enhanced survival of many cell types , including fibroblasts , endothelial cells , and hematopoietic progenitors . certain cytokines and growth factors activate other molecular pathways , such as the smad proteins for tgf- , jak1/stat1 for ifn , or the jak2/stat5 pathways for epo and g - csf , which stimulate the production of red blood cells and granulocytes , respectively [ 4346 ] . in mpns , several inflammatory cytokines and growth factors ( il-6 , il-8 , gm - csf , hgf , vegf , b - fgf , and tgf- ) are found to be significantly overproduced in all subtypes , yet with a large variability in quantity ( table 1 ) . of note , tgf-1 inhibits normal hematopoiesis in humans via its receptor ii ( tgf-rii ) . in cancer cells , a reduced expression of tgf-rii is frequent , which suggests that malignant mpn progenitors may also acquire resistance to tgf-1 by downregulating tgf-rii expression [ 47 , 48 ] . for certain cytokines , excess production of il-4 , il-10 , and tnf- has been reported in et ; elevation of il-11 levels has been described only in pv ; and in pmf , many cytokines , growth factors , and chemokines are produced at high levels but ifn- levels are usually low ( table 1 ) [ 45 , 4954 ] . the cellular sources of production of inflammation cytokines , chemokines , and growth factors are many and of course vary depending on the mpn subtype and associated complications ( thrombosis and bone marrow fibrosis ) . however , they usually include most of the cell types which constitute the bone marrow microenvironment or hematopoietic niche , fibroblasts , macrophages , t - lymphocytes , and endothelial cells , as well as healthy or mutated ( clonal ) hematopoietic progenitors and mature blood elements , platelets , neutrophils , monocytes , and macrophages [ 5559 ] . macrophages may present with a m1 phenotype , where they produce large amounts of tnf- and il-12 ( both elevated in pv and pmf ) as well as il-23 . macrophages of the m2 phenotype secrete il-4 or il-10 ( both elevated in et ) . in mpns the presence of the activating jak2-v617f mutation in > 95% of pv cases likely compensates for the low epo production by rendering erythroid progenitors highly responsive to low doses of epo , to result in polycythemia . this explanation is likely also valid for the 5060% of et and pmf which are jak2-v617f - mutated . intriguingly , blood levels of other cytokines which also activate the jak2/stat5 pathways ( tpo and g - csf ) or the jak2/stat3 pathways ( gm - csf , il-12 , il-33 , and cytokines of the il-6 family : il-6 , il-11 , and oncostatin m ( osm ) ) are often normal or elevated in mpns ( table 2 ) . several of these cytokines are produced by nonhematopoietic cells and also by myeloid progenitors , and they promote the survival and proliferation of both clonal and nonclonal myeloid progenitor cells . this is the case notably for tpo , il-6 , il-8 , il-11 , il-33 , gm - csf , hgf , and tnf- and several of these cytokines have been proven to contribute to the expansion of the jak2-v617f - mutated cells [ 5153 , 59 , 61 ] . regarding tpo , it is important to note that the low surface expression of mpl ( the tpo receptor ) observed in mpn progenitors and platelets likely limits the effect of high circulating levels of tpo . the reasons for the low expression of mpl in mpn patients are not fully understood . on one hand , jak2-v617f is thought to be less efficient than wild type jak2 to bring mpl receptors to the cell surface and possibly to increase mpl destruction . on the other hand , a high tpo level and activating jak2-v617f or mpl - w515l / k mutations may be ways of counteracting mpl repression in progenitor cells . il-33 is an alarmin known to help fight viral infection that is implicated in autoimmunity , and an increased risk of autoimmune disease has been reported in mpn patients [ 61 , 62 ] . chronic stimulation by the above cytokines also facilitates the survival and expansion of fibroblasts and fibrosis ( il-6 and b - fgf ) , monocytes - macrophages ( il-6 and gm - gsf ) , and platelet production ( il-6 ) and neoangiogenesis ( vegf ) , whereas il-12 and il-33 activate t - lymphocytes and natural killer ( nk ) cells . in addition , mpn cells accumulate in the bone marrow , which leads to some degree of hypoxia and subsequent activation of the hif-1 pathway , with upregulation of stat3 expression and production of cytokines which further promote cell survival ( igf-2 , hgf , and il-6 ) , fibrosis ( pdgf , fgf2 , and il-6 ) , and neoangiogenesis ( vegf ) [ 42 , 63 ] . altogether , the qualitative and quantitative differences found in cytokine production in the three mpns subtypes hint that the causes and mechanisms of chronic inflammation likely differ in et , pv , and pmf . the jak2-v617f , mpl - w515l / k , and calr mutations likely influence clinical symptoms but do not explain differences in inflammation . for instance , jak2-v617f , mpl , and calr mutations are detected at similar levels of expression in et ( associated with mild or very mild inflammation ) and in pmf ( characterized by severe inflammation ) . thus it is important to investigate and understand the mechanisms of inflammation at play in each mpn subtype , including those independent of jak2 , mpl , or calr mutations . the main clinical symptoms observed in mpns which are linked to an increased production of inflammation cytokines are fatigue , fever , itching , night sweats , weight loss , and , to some extent , splenomegaly . these symptoms are frequent in pmf ; they occur in pv but are mostly absent in et , which is a good reflection of the degree of production of inflammation cytokines characteristic of pmf ( high or very high ) , pv ( moderate to high ) , and et ( mild ) . the main biological parameters routinely assessed which are affected in case of inflammation include blood cell counts ( in particular leukocyte , neutrophil , and platelet counts ) , iron levels , and several proteins : the c - reactive protein ( crp ) , haptoglobin , alpha-1 acid glycoprotein ( orosomucoid ) , ferritin and fibrinogen ( increased ) , and albumin and transferrin ( decreased ) . these inflammatory proteins present with different kinetics : inflammatory positive markers which are increased early include crp , haptoglobin , and alpha-1 acid glycoprotein , whereas fibrinogen , ferritin , and transferrin are late - acting inflammatory proteins . elevation of the leukocyte and platelet counts is typical of mpns and thus does not allow distinguishing between inflammation and mpn . crp is elevated in mpns , particularly in pmf , and pentraxin 3 has been reported to decrease in mpns . a high crp and low pentraxin 3 were linked to a high risk of thrombotic events in pv and in et , and a high crp was associated with shortened leukemia - free survival in mpn patients with myelofibrosis [ 64 , 65 ] . the level of il-6 in serum is almost constantly increased in case of mpn but il-6 levels ( and other inflammatory cytokines ) are not measured in routine laboratory practice . mpns are characterized by the activating jak2-v617f and mpl - w515l / k mutations , the calr mutations , and also high levels of total jak2 ( wild type and v617f - mutated ) in neutrophils and platelets [ 310 ] . the effect of jak2-v617f mutation is to activate primarily the stat5 pathways but the stat3 pathways are also activated ( figure 4 ) . the mpl - w515l / k mutations presumably stabilize mpl , the jak2-coupled dimeric receptor for tpo . tpo is known to stimulate the jak / stat pathways and also pi3k / akt , erk , p38 , nf-b , and hif [ 6769 ] . accordingly , in transfected cells expression of mpl - w515l / k mutants resulted in increased activation of erk ( extracellular signal - regulated kinases ) 1 and erk 2 ( erk1/2 ) and akt ( protein kinase b ) in absence and in presence of tpo [ 5 , unpublished observations ] . to our knowledge , the effect of mpl - w515l / k mutations on nf-b and hif has not been studied . in any case , the jak2-v617f mutation activates stat3 and the mpl - w515l / k mutations activate stat1 and stat3 , which implies that they may stimulate the production of inflammatory cytokines ( figure 4 ) . however , in mpn progenitor cells and platelets , the expression of mpl receptors at the membrane surface is often very low , which likely attenuates the effect of tpo stimulation and mpl - w515l / k mutation . calreticulin is a calcium - binding protein chaperone normally located in the endoplasmic reticulum ( er ) ; the calr mutations associated with mpns all result in c - terminal truncated forms of calreticulin located in the cytosol . thus it is presumed but not formally demonstrated that calr mutants may affect intracellular calcium flux as well as the trafficking and secretion of glycoproteins , which could potentially lead to altered expression and activation of cytokines , receptors , jak2 , and other signaling molecules . consistently , the initial papers reported an activation of the jak2/stat5 pathway in transfected cells which expressed calr exon 9 mutants [ 8 , 9 ] . however , the precise molecular mechanisms linking calr mutants and the jak2/stat5 pathway have not been identified . more rarely , in et or pmf the driving mutation may be a loss - of - function mutation in the lnk gene or in the cbl gene [ 7074 ] . lnk is an adaptor protein which acts as a negative regulator of tpo / mpl - mediated activation of jak2 . expression of lnk loss - of - function mutants also results in enhanced activation of the jak2/stat5 pathway . cbl codes for an e3-ubiquitin ligase which promotes the ubiquitination of signaling molecules , including tyrosine kinases . the cbl mutations detected in mpns cause the loss of e3-ubiquitin ligase activity , thus resulting in increased signaling and cell proliferation . so far there is no evidence that lnk or cbl mutations induce the production of inflammatory cytokines , but they may alter their signaling . mutations in the tet2 , idh1 , idh2 , ezh2 , aslx1 , or dnmt3a genes may also be found in mpns . they are not specific of mpns : they are found also in other blood and solid malignancies . tet2 and idh1/2 mutants impair the hydroxylation of methylcytosine and thus affect dna methylation . more precisely , tet gene products catalyze the conversion of 5-methylcytosine to 5-hydroxy - methylcytosine ( 5-oh - mec ) , a reaction that depends in part on iron and oxygen [ 80 , 81 ] . ezh2 ( enhancer of zeste homolog 2 ) gene codes for a histone methyl transferase , and asxl1 ( additional sex combs like transcriptional regulator 1 ) gene product belongs to the polycomb group of proteins and thus is thought to disrupt chromatin and alter gene transcription . dnmt3a codes for a dna methyltransferase and mutations presumably alter the epigenetic regulation of gene expression . thus one can not exclude that these mutations may alter the expression of genes coding for inflammatory cytokines or receptors . several groups have studied the production of inflammation cytokines in jak2-v617f - mutated cells or in murine jak2-v617f - driven mpn models . so far published reports concluded that , in vitro , jak2-v617f can increase the production of il-6 , il-8 , il-9 , osm , ccl3 , ccl4 , and tnf- [ 53 , 59 , 84 , 85 ] . however , in mpn patients there is no correlation between the jak2-v617f burden and the blood or serum levels of these cytokines . in fact , it is highly probable that only a fraction of these cytokines is under the control of jak2-v617f . firstly , il-6 , il-8 , and osm are abundantly produced by nonhematopoietic ( nonclonal and nonmutated ) cells [ 5153 ] . secondly , certain molecules produced under the control of jak2-v617f , such as osm , in turn stimulate the production of other inflammatory cytokines in a jak2-v617f - independent manner . thirdly , in the jak2-v617f hel cell line , anti - jak2 mirna experiments had only a partial inhibiting effect on il-6 mrna expression ; in these experiments , anti - jak2 mirna experiments had no effect on the expression of il-11 and hgf . thus in jak2-v617f - mutated cells , major inflammatory cytokines may be controlled partially ( il-6 ) or totally ( il-11 and hgf ) by molecular pathways not regulated by jak2-v617f . regarding mpl - w515 mutations , only one group reported the analysis of inflammation cytokines produced in mpl - w515l - mutated cells , in a murine bone marrow transplantation model : expression of mpl - w515l was associated with a significant increase in the production of il-6 , il-10 , il-12 ( p40 ) , tnf- , csf3 , and chemokines ccl2 , cxcl9 , and cxcl10 . again , mpl - w515l - mutated cells were not the sole source of production of these cytokines . regarding the calr exon 9 mutations associated with mpns , their effect on cytokine expression is not known . it is interesting to note that soluble calreticulin has been reported associated with increased production of il-6 and tnf- . regarding tet2 , idh1/2 , and asxl1 mutations , it was reported that mutated forms of idh1/2 were associated with specific dna hypermethylation profiles , and the list of genes found to be differentially methylated includes several genes linked to inflammation , particularly the il11-r and tgf-ri receptors . interestingly , il-11 and tgf- are secreted at high levels in case of inflammation and both alter myelopoiesis . hypermethylation of the genes encoding il11-r and tgf-ri receptors would presumably lower their expression and subsequently make clonal progenitor cells less sensitive to the inhibiting action of tgf- and anti - inflammatory action of il-11 . since tet2 and idh1/2 mutations are mostly found in pmf , it is possible that these mutants play a role in the aggravation of inflammation observed in severe forms of pmf [ 87 , 88 ] . in myelodysplastic syndromes , asxl1 mutations combined with setbp1 mutations were reported to repress the tgf- pathways . however no study of cytokine or receptor protein expression in relation to asxl1 mutation in mpns has been published . germline defects , variants , or haplotypes can affect , directly or indirectly , the expression or signaling of inflammatory cytokines and receptors , thus potentially attenuating or aggravating chronic inflammation . the 46/1 ( jak2 ggcc ) haplotype and single - nucleotide polymorphisms ( snp ) in jak2 , in the telomerase reverse transcriptase ( tert ) , in the mds1 and evi1 complex locus ( mecom ) , or in hbs1l - myb have been reported to be associated with a predisposition to mutation in the jak2 gene on the same allele ( jak2 ggcc haplotype ) or a predisposition to the development of a mpn ( mecom , tert , jak2 , and hbs1l - myb variants ) [ 9094 ] . to this day it remains unclear how these hereditary genetic variants act to facilitate the development of mpns , but alteration of the transcription of the concerned genes is possible . regarding germline jak2 variation , inappropriate expression of jak2 would clearly disturb myelopoiesis and alter the contribution of myeloid cells to inflammation responses . consistently , the jak2 ggcc haplotype was reported to be associated with a defective response to cytokine stimulation , increased risk of inflammation , and impaired defense against infection [ 95 , 96 ] . in cml , cells with short telomere length were found to express a specific telomere - associated cytokine and chemokine secretory phenotype . little is known on the functional effects of mecom variants on cytokine production but yasui et al . recently reported that the evi1 oncoprotein could alter tgf- signaling and tgf--mediated growth inhibition [ 98 , 99 ] . it is established that variations due to snps in the promoter region of genes coding for inflammatory cytokines and receptors potentially affect their production . many groups have published snps associated with an altered production of a cytokine or a cytokine receptor , and such snps concern all the main cytokines involved in inflammation : il-1 , il-1r , il-2r , il-6 , il-8 , il-10 , il-12 , il-33 , tnf- , hgf , and mcp1/ccl2 [ 100115 ] . snps have been shown to control the expression of these cytokines in vitro and individuals who carry the snp are described as high or low producers [ 116118 ] . cytokine polymorphisms have been studied in association with specific diseases , response to infectious agents , or immune response to inflammation . chronic inflammation associated with mpn may have several causes , and their recognition should allow offering improved and individualized treatment to mpn patients . as described above , part of the inflammation is clonal since mpn clonal cells produce inflammatory cytokines ( il-6 , il-8 , il-9 , il-11 , osm , tnf- , ccl3 ( mip-1 ) , and ccl4 ( mip-1 ) ) ; the eventual acquisition of idh1/2 or tet2 mutations may aggravate clonal inflammation by altering the expression of certain receptors ( il-11r , tgf-r1 ) . the consequences are multiple : ( i ) enhanced survival and growth of clonal cells ( il-6 , il-8 , il-11 , and tnf- ) ; ( ii ) increased production of inflammation cytokines that target bone marrow stromal cells as well as hematopoietic progenitors , via the action of osm , il-11 , and il-6 ; ( iii ) resistance of clonal cells to growth inhibitors , via a reduced expression of il-11r or tgf-r1 [ 53 , 59 , 61 , 63 , 119 , 120 ] . in addition , clonal cells can recruit and activate neutrophils , monocytes , and natural killer cells via the production of ccl3 and ccl4 ; the neutrophils and monocytes potentially recruited may be clonal or non - clonal . any malignant process induces nonclonal immune responses which aim to restrict and eventually destroy the malignant cells . in case of advanced disease , nonclonal inflammation may also be reactive to treatment . in mpns , the mature myeloid cells which participate in the antitumoral or hypoxia - induced or therapy - related reactive inflammation response may be clonal or nonclonal . depending on the mpn subtype , the size of the clonal population is likely to be large ( pv and pmf ) or moderate or small ( et ) , implying that the clonal part of reactive inflammation is probably more significant in pmf and pv than in et . this should not be overlooked because clonal cells likely mount a less efficient immune response than healthy cells , meaning that the inflammation / immune response could be rather inefficient in pv and pmf . the observation that major inflammatory cytokines are produced independently of mpn - associated mutations and the demonstration that jak2-v617f can be a late event in mpn development are consistent with the hypothesis that chronic stimulation of myelopoiesis ( via inflammation ) may precede the acquisition of mutation in the jak2 ( mpl and calr ? ) a frequent objection is the lack of evidence of inflammation or myeloid stimulation prior to the diagnosis of a mpn . however , it is not rare that routine blood tests of patients , especially older patients , reveal a slight elevation of leukocyte or platelet counts , or hematocrit , with or without biological evidence of mild inflammation . there are dozens of reasons for mild alterations of blood counts , ranging from smoking , stress , obesity , and diverse latent infections to mild forms of chronic inflammatory diseases ( intestinal , rheumatoid , skin , type 2 diabetes , atherosclerosis , etc . ) . such patients are simply observed ; investigation begins when blood counts rise significantly ( reach at least one of the who criteria of mpn ) or when patients present clinical symptoms related to mpn or to the underlying cause of chronic myeloid stimulation or inflammation . also , it is not rare to detect lymphoid infiltrates in the bone marrow of mpn patients and monocytosis or lymphopenia in peripheral blood , sometimes prior to the diagnosis of mpn ; these observations may be considered as evidence of a disturbed immune response . thorough investigation of the stages preceding the diagnosis of overt mpn , similar , for instance , to the studies that established monoclonal gammopathy of undetermined significance ( mgus ) as the precancerous stage of multiple myeloma , is needed in mpns to validate the hypothesis of chronic ( antigen - mediated or not ) stimulation of myelopoiesis preceding the acquisition of jak2 , mpl , or calr mutation . the chronic inflammation and myeloid stimulation hypothesis is attractive , because it can explain several if not all of the mysteries that persist in mpns . for instance , chronic myeloid stimulation allows the recurrent acquisition of jak2-v617f , multiple jak2 mutations , and combinations of jak2 , mpl , or calr mutations regularly reported in mpns . early chronic inflammation and myeloid stimulation would explain that jak2-v617f burden and clinical symptoms and disease severity are not correlated . the recent reports that patients under treatment with jak inhibitors may develop or reactivate viral infection , possibly due to impaired nk cell function , are also consistent with chronic infection contributing to the inflammation associated with mpns . importantly , the chronic stimulation hypothesis allows for multiple causes of inflammation , infectious or not , some mild ( as observed in et ) and some severe ( as typical of pmf ) . last but not least , the chronic myeloid stimulation hypothesis allows for many different initial causes and thus would explain why the jak2-v617f mutation and to a lesser degree the mpl exon 10 and calr exon 9 mutations are associated with very different diseases ( et , pv , pmf , rars - t , and splanchnic thrombosis for jak2-v617f ; et , pmf , and rars - t for mpl and calr mutations ) . for all these reasons , chronic myeloid stimulation and inflammation , and notably latent infection , deserve investigation as initial , early , or complicating events of mpns . indeed chronic exposure to various toxic compounds or to infectious pathogens and subsequent chronic inflammation frequently precedes malignant cell transformation in the context of solid cancers ; importantly , the same toxics or infectious agents associated with solid cancer may also lead to lymphoid malignancy ( table 2 ) [ 3137 ] . normal immune responses following infection include the stimulation of myelopoiesis ( granulocytes and monocytes ) . the production of b - lymphocytes and plasma cells is stimulated to produce polyclonal ig . if infection becomes chronic , a focusing of the ig response from polyclonal to monoclonal ( mc ) immunoglobulins ( ig ) may occur , and that will persist as long as the infection . epstein - barr virus ( ebv ) , hepatitis c virus ( hcv ) , hepatitis b virus ( hbv ) , or helicobacter pylori ( h. pylori ) stimulate polyclonal b - cell proliferation , and these pathogens are implicated in several b - cell malignancies ( burkitt , hodgkin , non - hodgkin lymphoma , and chronic lymphocytic leukemia ) via cell infection and direct transformation ( ebv and hcv ) , via antigen - driven stimulation ( h. pylori ) , or both ( ebv and hcv ) [ 3237 ] . moreover , ebv , cytomegalovirus ( cmv ) , hhv-8 , and hhv-6 can induce a chronic monoclonal ig response [ 123126 ] . in support of the hypothesis that infection may predispose to chronic hematological malignancy , we showed that , for about 25% of patients with multiple myeloma , the purified mc ig specifically recognizes an antigen from hcv , ebv , or h. pylori [ 124127 ] . these important findings suggest that infectious agents may also initiate multiple myeloma , not just certain types of lymphoma , which opens new possibilities of curative treatment , as demonstrated recently by the regression of one case of hcv - associated myeloma following treatment by ifn- . antigen - driven proliferation as a facilitator of dna mutation acquisition and cell transformation is rarely investigated in the context of myeloid malignancies but since chronic antigen stimulation also concerns myeloid cells , latent infection as a cause of inflammation in chronic myeloid disorders should not be excluded . thus a promising research approach for chronic myeloid disorders is to propose that , for subsets of patients , malignancy may result from chronic , polyclonal abnormal immune response by myeloid cells , eventually facilitating excessive myeloid proliferation , acquisition of genetic alterations in genes that are critical for myelopoiesis ( jak2 and mpl ; calr ? ) , and transformation of progenitor cells from the most stimulated lineage(s ) and then expansion of a malignant clone . logically , the jak2-v617f mutation rapidly became the main target of treatment in mpns after its discovery in 2005 . in contrast , chronic inflammation has so far been neglected in the treatment of these diseases . recognition of the importance of inflammation in the pathogenesis of mpns offers great opportunities to improve therapy . the jak inhibitor trials showed that blocking jak2 function significantly reduced inflammatory cytokine levels and other markers of inflammation in pmf patients , resulting in improved clinical symptoms . patients benefited from jak inhibitors even when the jak2-v617f mutant burden was not reduced or when their disease was not associated with jak2 mutation . although the comprehension of the causes and mechanisms of inflammation in mpns is still very incomplete , accumulated knowledge indicates that nf-b and jak1 are major pathways for the production or / and signaling of inflammatory cytokines . in certain cases , the three pathways are closely linked ( figures 3 and 4 ) , and used alone , inhibitors of the jak / stat pathways ( or inhibitors of nf-b ) can not be expected to completely block cytokine production and signalling in mpns . in a murine model of jak2-v617f - mutated mpn , in fact , alteration of stat3 function ( deletion or hyperactivation ) is known to lead to altered myelopoiesis and increased expression of stat1 and inflammatory cytokines , notably il-6 , a strong stimulant of platelet production , fibroblast proliferation , and inflammatory acute phase protein production [ 130132 ] . in support of this mechanism , grisouard et al . reported increased expression of stat1 and stat1 target genes in jak2-v617f mice after stat3 deletion ; il-6 and other inflammatory cytokines were not measured in this study . ideally to cure a mpn , one should aim to reduce the effects of the jak2 , mpl , or calr mutant carried by the mpn clone , as well as the production and signaling of the main inflammation cytokines produced by the patient . this can be achieved by blocking the three main pathways responsible for cytokine production ( these include the jak1/jak2 pathways ) and by suppressing the cause(s ) of mpn mutation , when identified . used alone , jak1/2 inhibitors have the capacity to block the jak2-v617f and mpl - w515l / k mutations and a large fraction of the production and signaling of inflammatory cytokines . but for complete treatment of inflammation and mutations in mpns , the addition of nf-b and hif-1 inhibitor drugs should benefit patients [ 133138 ] . this contrasts with myeloma , a disease not driven by the activation of the jak2/stat pathways where nf-b and hif-1 inhibitors used alone can reduce both disease and inflammatory cytokines . another advantage of such combination therapies would allow lowering the dose of each drug and hopefully reduce toxicity . of note , one reason why ifn- is able to induce a complete clinical , biological , and molecular remission ( jak2-v617f - negativation ) in pv and in et patients is that ifn- acts on several jak / stat pathways as well as on other pathways critical for the production of inflammatory cytokines [ 140 , 141 ] . in short , as represented in figure 5 , the ideal mpn therapy may combine the following : ( 1 ) inhibition of the jak1 pathway and jak2-v617f , mpl - w515l / k , or calr mutations with a jak1/2 inhibitor and ( 2 ) nf-b and hif inhibitors ( note that ( 1 ) and ( 2 ) may be achieved with ifn- ) . whenever an early cause of chronic inflammation is identified , adequate treatment should be added : for instance , antibiotics or antiviral treatment in case of latent infection . the complexity of inflammation in mpns should not discourage attempts to define it biologically at the time of diagnosis , prior to therapeutic decisions , and during treatment monitoring . knowing the precise inflammation status of each mpn patient would greatly help improve his / her treatment . as described earlier , the inflammation status and cytokine profile of a mpn patient are expected to vary according to the mpn subtype , presence of jak2 , mpl , or calr mutation , eventual cause of inflammation preceding mpn - driving mutation , and personal genetic background . yet what matters for therapy is the resulting cytokine profile of the patient , and nowadays establishing the inflammation cytokine profile of an individual is technically simple and not overly expansive and requires only a blood sample . knowing that a patient is a strong producer of il-6 , hgf , or tnf- , for instance , would allow focusing treatment on the target cytokine(s ) , perhaps by adding to the patient 's combination therapy one of the existing antagonist drugs or neutralizing antibodies that specifically block these cytokines or receptors [ 119 , 142144 ] . last but not least , extensive genetic studies and murine models have not succeeded to fully explain most of the chronic hematological malignancies , including mpns . this suggests that genetic aberrations , although crucial , are probably not sufficient for a lymphoid or myeloid malignancy to develop , and more attention is now given to the hematopoietic niche and cytokine production , the human microbiome and oncogenic infectious pathogens , and the host 's immune response [ 145 , 146 ] . there is no reason to limit these important pathogenic mechanisms to lymphoid malignancies and solid cancer , and perhaps the next major research effort in the mpn field should be to investigate the validity of the hypothesis of chronic inflammation / myeloid stimulation preceding mutation acquisition . more specifically , a systematic search for latent infection in mpn patients is feasible and simple , thanks to various tests based on the multiplexed antigen or peptide microarray technology ; these assays require only a small blood sample [ 127 , 147 ] . obviously the identification of an infectious cause of inflammation in subsets of mpn patients would offer additional possibilities of combined treatment ( with antibiotics or antiviral therapy ) ( figure 5 ) . regarding research and animal models of mpns , it is possible to develop new murine models of chronic myeloid stimulation , antigen - mediated or not . in conclusion , inflammation is very complex yet there are relatively simple laboratory tools to diagnose and characterize inflammation in patients . several predictive inflammation markers are already identified in mpns , and potent drugs that target the molecular pathways of inflammation or the inflammatory cytokines detected in excess in patients already exist . designing new , more complete , and individualized combination treatments that include drugs that block mpn mutations as well as

myeloproliferative neoplasms ( mpns ) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages . recent advances in the biology of mpns have greatly facilitated their molecular diagnosis since most patients present with mutation(s ) in the jak2 , mpl , or calr genes . yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of mpns are not fully elucidated . importantly , chronic inflammation has long been associated with mpn disease and some of the symptoms and complications can be linked to inflammation . moreover , the jak inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the jak2-v617f mutant load . these observations suggested that part of the inflammation observed in patients with jak2-mutated mpns may not be the consequence of jak2 mutation . the aim of this paper is to review the different aspects of inflammation in mpns , the molecular mechanisms involved , the role of specific genetic defects , and the evidence that increased production of certain cytokines depends or not on mpn - associated mutations , and to discuss possible nongenetic causes of inflammation .

1. Introduction 2. Chronic Inflammation, including Inflammation Associated with Solid Cancer or MPNs 3. Activation of the Molecular Pathways of Inflammation by MPN-Associated Mutations 4. Inflammatory Cytokines Produced as a Consequence of MPN-Associated Mutations 5. Inflammatory Cytokines Produced as a Consequence of Germline Genetic Defects 6. Clonal and Nonclonal Chronic Inflammation in MPNs 7. Consequences for the Treatment of MPNs

charcot neuropathic osteoarthropathy ( cn ) is a chronic , progressive condition of bones , joints , and soft tissues , most commonly occurring in the area of the foot and ankle as a result of peripheral neuropathy . it is characterized by a local inflammatory process in the early stages and gradual development of bone loss , joint dislocation , and fixed deformities . these deformities diabetes mellitus , together with neuropathy , is currently considered the main cause of cn . data indicating the prevalence and incidence of the condition suggest that it often goes undiagnosed among sufferers of diabetes , with figures ranging from 0.4 to 13% among diabetics . however , changes diagnosed by x - ray and corresponding with cn are detected in up to 29% of diabetics . bilateral disability has been observed in numbers ranging from 9 to 39% of patients . when mri is used as a diagnostic method , the common issue is an early diagnosis and an appropriate treatment , in case of an acute phase where it is difficult to differentiate an acute osteomyelitis . even though the treatment of cn is mostly conservative however , the crucial question is when , where , and how a surgical therapy has to be used . a pubmed search was done with the key word charcot foot , neuropathic arthropathy , charcot arthropathy . we could trace about 400 up - to - date papers on the subject . electronic database was systematically searched for literature discussing the history , pathophysiology , assessment , imaging methods , diagnosis including osteomyelitis , classification , and management of cn . reasons for exclusion of articles based on title or abstract were ( 1 ) nonoriginal data ( e.g. , editorials , guidelines , and comments ) , ( 2 ) nonclinical articles ( e.g. , technical or animal studies ) , ( 3 ) case reports , and ( 4 ) articles not written in english language . all authors independently chose the most up - to - date papers with regard to target topics resulting in the identification of 59 most pertinent articles . together we discussed and compared the relevant information from all these sources with our clinical practice and included them in this review . later , mitchell supposed the relation between spinal lesion and rheumatism of lower extremities in 1831 . charcot described the neuropathic aspect of the condition in detail in 1868 and detected spinal damage resulting from tabes dorsalis as a cause and his brilliant presentation , demonstration of arthropathic affections of locomotor ataxy , at the 7th international medical congress ( 1881 ) , established this disease as a distinct pathological entity . much later , in 1936 , jordan revealed diabetes mellitus to be a possible cause of neuropathic osteoarthropathy . nevertheless , the etiology , diagnostics , and treatment of this condition have to this day yet to be fully addressed . two main theories concerning the origin of the condition have been discussed in the past . the neurotraumatic theory is based upon damage to sensory feedback resulting from progressive destruction of bones and joints brought about by repeated trauma . the neurovascular theory highlights the changes in blood supply caused by neuropathy , most of all lesions in the sympathetic nerves which affect bone resorption . the current accepted theory of cn origin states that , in susceptible individuals with peripheral neuropathy , an unregulated inflammatory process is triggered which leads to an increased expression of the polypeptide receptor activator of nuclear factor kappa - b ligand ( rankl ) . rankl triggers the synthesis of the nuclear transcription factor , nuclear factor- ( nf- ) , and this in turn stimulates the maturation of osteoclasts from osteoclast precursor cells . at the same time , nf- stimulates the production of the glycopeptide osteoprotegerin ( opg ) from osteoblasts . a repetitive trauma with the loss of pain sensation results in continual production of proinflammatory cytokines , rankl , nf- , and osteoclasts , which in turn leads to continuing local osteolysis . another possible cause is decreased secretion of the calcitonin gene related peptide ( cgrp ) from damaged nervous endings . under physiological circumstances this peptide works as an antagonist of the rankl synthesis and at the same time the powerful bone anabolic wnt/-catenin pathway plays a critical role in remodeling as well as preservation of the foot skeleton in the acute and chronic stages of the disease . in general , diabetes may predispose to cn occurrence through a number of mechanisms . apart from the presence of neuropathy and possible osteopenia , these include the effects of advanced glycation end products , reactive oxygen species , and oxidized lipids , which may all enhance the expression of rankl in diabetes . the diagnosis is based on patient 's history , clinical examination , and imaging methods . patients are quite often not aware of any injury as a result of their lowered perception of pain . trigger factors of development of the cn can also be infections ( osteomyelitis precedes cn ) and revascularization . a necessary condition for the emergence of cn is the presence of peripheral neuropathy , in particular , diabetic distal sensitive polyneuropathy . the most common is the semmes - weinstein 5.07/10 g monofilament . next is the pinprick test or even the more sensitive neurometer test . a small fiber - predominant neuropathy is an early manifestation of diabetic neuropathy and it can progress to distal symmetric polyneuropathy . it can be difficult to diagnose because the examination ( decreased reflexes , impaired vibration , and weakness ) and electrodiagnostic testing can be normal . autonomic neuropathy ( a type of small fiber neuropathy ) is also common and plays a key role in the development of the cn . symptoms include gastroparesis , constipation , urinary retention , erectile dysfunction , and cardiac arrhythmias . signs of inflammation are an important sign since inflammation plays a key role in the pathophysiology of cn . oedema , erythema , warmth , and more than a 2c difference in local temperature in comparison to the contralateral extremity are typical symptoms of active cn and it could be difficult to differentiate them from phlegmon with osteomyelitis or from an acute attack of gout , especially since pain occurs in only 50% of neuropathy cases . in this stage the vascular supply to the foot is still maintained though it could be difficult to palpate an arterial pulse due to prominent swelling . the eventual fracture and joint dislocation can lead to deformities , typically to rocker bottom foot with possible ulceration . during this time skin temperature measurement is the most widely used method in the assessment of the activities of the cn . with the use of a surface - sensing temperature device ( infrared thermometer ) , temperatures are recorded in the most affected area of the foot and compared with the same areas of the contralateral foot . for further prognosis and therapy instability of the forefoot can be assessed according to assal and stern : the pressure on the foot in the sagittal plane dorsally when the ankle joint is locked in dorsiflexion . relatively common in cn is the contraction of the triceps surae muscle which is involved in plantar inclination of the calcaneus ( figure 1 ) . primarily it must be emphasized that the changes on the x - ray are typically delayed and have low sensitivity . a basic examination is an x - ray of the talus and the weight bearing foot in the anteroposterior and dorsoplantar lateral projection . during the initial stage the x - ray finding can be negative or only minor bone infractions and joint incongruence are present . in a developed stage fractures and subluxations or luxations the x - ray finding depends on the specific type of cn . in a typical rocker bottom deformity this deformity arises due to deformed midtarsal bones and the shortening of the achilles tendon , which loses its elasticity during glycosylation . another finding on the lateral projection due to deformity of the middle part of the tarsus is a negative angle between the axis of talus and i metatarsus ( figure 2 ) . the dorsoplantar projection shows changes of the position in the lisfranc as well as chopart joint , with resulting abduction or adduction deformity of the foot . examination using magnetic resonance imaging is a very valuable method for the early stages of the illness when x - ray imaging alone results in practically normal findings . an important finding is oedema of the bone marrow of two or more bones , oedema of the adjacent soft tissues , and fluid in several joints or cortical fractures . certain methods of nuclear medicine can be helpful not only as an alternative diagnostic method , for example , in revealing the presence of osteomyelitis , but also for monitoring the progress of the treatment . three- or four - phase bone scintigraphy ( tc - mdp ) is highly sensitive but with a low specificity . scintigraphy with labelled leucocytes ( tc - wbc nebo in - wbc ) is highly sensitive and very specific for diagnosing the infection but it is difficult to differentiate soft tissue from bone . that is why either a combination of both the abovementioned methods or fdg - pet / ct is recommended . the diagnostics of osteomyelitis in cn is difficult primarily in the active stage of the disease when clinical symptoms are practically the same both in osteomyelitis and in cn . on the contrary , in the chronic stage symptoms in osteomyelitis due to ischaemia and immunodeficiency diagnosing osteomyelitis is not possible on the basis of one examination alone . in general , for the differential diagnosis of osteomyelitis and cn , an important role plays a complex view of inflammatory markers and clinical manifestations of infection . the origin of osteomyelitis is in most cases caused by spread of infection from the soft tissues . history of ulceration or presence of ulceration and/or previous amputation in the area of the foot are possible factors to weigh when suspecting osteomyelitis . a high predictive value for the presence of osteomyelitis is ulcerations bigger than two cm and deeper than three mm . the basis of the test is whether a fine blunt steel probe can penetrate the ulceration to the bone . laboratory readings in cn do not show higher markers of inflammation ( practically normal numbers of leucocytes , crp , procalcitonin , and fw ) . due to the chronicity of infection in osteomyelitis we can find particularly higher fw ( most commonly > 70 mm / hour ) . these changes are visible within two to three weeks of the onset of the infection . the abovementioned methods of nuclear medicine ( four - phase bone scintigraphy , marked leucocytes , f fdg - pet , or ga spect / ct ) can also be used to diagnose osteomyelitis , and the range of osteomyelitic changes is also highly visible on the mri . also tc - wbc spect / ct hybrid image is a useful tool in addition to mri . the most reliable examination is a bone biopsy , either preoperative percutaneous or peroperative , made with a biopsy needle after disinfection of the ulceration . swabs are often contaminated with normal skin flora or colonizers , and their use may result in failure to identify deep tissue pathogens . for identification of pathogens , the international working group on diabetic foot ( iwgdf ) however , swab cultures are less invasive than tissue biopsy or curetted tissue and swab culturing ( including a vacuum transport container ) may be reliable for identification of pathogens in superficial diabetic foot wounds . typical ischaemic symptoms like claudication and pains at rest , which would normally appear in the history , might be unrecognised because of the presence of neuropathy . during a physical examination pulsation an example of a noninvasive diagnostic method that can be also used would be measuring blood pressure in the ankle using a doppler probe ( it carries a higher risk of artificially higher pressures in the case of mediocalcinosis ) . in our department we use measure pressure on the big toes or transcutaneous oxygen tension ( this method carries a risk of artificially lower pressures in the case of oedema ) . if angiography is performed postoperatively , there could be a risk of activating cn after prospective revascularization . examination of the vascular supply must be repetitive even after surgical reconstruction of cn when a higher risk of ischaemia exists ( oedema , thrombus formation of vessels ) . based upon our experience , we evaluate this risk as the most significant from the point of view of possible postoperative complications . the most commonly used classification according to eichenholtz was published in 1966 ( table 1 ) . currently , in spite of the quite widespread usage of this classification , it is necessary to consider and search for new alternative methods of imaging . eichenholtz evaluated x - ray images in 68 patients ( altogether 94 joints ) of whom only 12 were diabetic . the development of examination methods of nuclear medicine and magnetic resonance have shown that there are already noticeable and recordable changes of cn even when x - ray images are negative , and starting treatment at such an early stage can prevent deformities . this stage has been labelled as stage 0 and has been added to the original classification . alternatively , stage 1 has been divided into 1a and 1b . with regard to the fact that the initial change in cn is an inflammatory reaction , which corresponds with oedema of bone marrow , a classification of cn based upon mr imaging has been suggested . it recognises two stages of the disease active and inactive according to the presence or absence of oedema of the bone marrow and distinguishes two grades0 and 1according to the presence or absence of cortical fractures . these fractures represent a worse prognosis from the point of view of developing deformities ( table 2 ) . this method is based on immobilisation and the complete absence of weight bearing for the affected extremity in the active stage . there are various opinions concerning the type of immobilisation and the period of nonweight bearing for the foot . the most common immobilisation used is a total contact cast ( tcc ) changed three days after the initial application and then every week . alternatively , it is possible to use charcot restraint orthotic walker ( crow ) prefab orthoses . the period of fixation depends on the reduction in oedema and a drop in skin temperature below 2c compared to the contralateral extremity . the recommended length of fixation varies from six weeks to three months followed by a change of orthosis . similarly , the recommended period without any weight bearing varies starting from weight bearing during application of tcc to the usage of a wheelchair as a preventive means against overloading the other extremity . recommend six to eight weeks of tcc and a wheelchair with subsequent change for individual orthosis fixing the affected segment and at the same time preventing tibial rotation , thus enabling only axial weight bearing ( a so - called frame orthosis ) . in the chronic stage of the condition , a deformed foot in plantigrade position capable of weight bearing in shoes or an orthosis without increasing deformity is suitable for conservative treatment . the type of the prosthetic equipment depends on the gravity of the deformity and on the eventual presence of ulceration . it is possible to use various types of walkers , ankle - foot orthoses , orthotic shoes , or adjusted regular shoes . in general we prefer conservative treatment in both stages by a multidisciplinary team . only after the failure of conservative management does a patient this treatment is beneficial in cn refractory to off - loading and immobilisation or in the case of recalcitrant ulcers . a separate question is the problem of weight bearing in the case of conservatively treated or operated cn . we have the same experience as koller et al . , most of the patients suffering from peripheral neuropathy are not able to reduce weight loading of the foot in a controlled manner with the help of crutches , and there is a risk of overloading the contralateral limb and a risk of fall . therefore we recommend full weight bearing wherein we gradually prolong the time and speed of the walk . to support healing some medicaments have been used , bisphosphonates , which inhibit osteoplastic bone resorption , and intranasal calcitonin , which has had fewer complications . nevertheless , beneficial effect of pharmacological treatment ( improvement of markers of resorption versus an absence of clinical marks of healing and side effects of the therapy ) as well as physical stimulation of the bone growth is yet to be fully demonstrated . besides conservative treatment , the possibilities of surgical treatment have also been looked into and the benefits and risks of such treatment have been considered . saltzman et al . evaluated retrospectively conservative treatment of 127 extremities in 115 patients over a period of 20 years . the study found that the annual rate of amputations was 2.7% , 47% of patients used an orthosis for a period longer than 18 months , and the risk of ulceration appeared in 40% of patients . ulceration is often accompanied by a high risk of amputation . at present , specific methods for the surgical treatment of cn to save the extremity or delay major amputation are still being developed . foot reconstruction , resection of bony prominences , and major amputations are considered for the surgical treatment ( table 3 ) . major amputations in cn ( generally we prefer the below - the - knee amputation ) are still the current solution . if carried out properly , if the healing is complete , and if the patient is equipped in the prosthetic and rehabilitation department with a suitable prosthesis and has an adequate walking regime as part of the rehabilitation , then we know from experience that these patients , though initially perhaps unwilling to undergo surgery , are more satisfied compared to those who use orthosis for a long time , who required constant dressings of ulcerations and repeated visits to hospital . based on our personal experiences , we use transcutaneous oxygen tension more than 35 mmhg as a predictive factor for successful healing of below - knee amputation . in dialysis patients we deal with problem of a suitable prosthesis after major amputation due to changes of extremity volume between dialyses . bone resections are done as a separate intervention in isolated bone prominences , mostly in cases of a high bony pressure that can not be accommodated with orthotic and prosthetic means and in stable plantigrade foot . in some cases , a strayer procedure or achilles tendon prolongation such intervention carries a risk of instable foot in the case of larger bone resection . a bone resection is also done as preparation for reconstruction of the foot in case an infected ulceration is present or if there is suspicion of osteomyelitis . with regard to poor bone quality and the presence of neuropathy in long - term healing , the so - called superconstruction principles for reconstruction operations have been set up : ( a ) extending arthrodesis beyond the affected area on neighbouring joints , ( b ) resection of the bone for mild shortening of the foot enabling adequate repositioning of the deformity without excessive tension of soft tissues , thus helping prevent secondary ischaemisation , ( c ) usage of the strongest possible implant which can be tolerated , and ( d ) introduction of an implant that can maximally increase mechanic stability , which is the main goal . in general we can use different types of external fixators or internal fixators according to the type of deformity and preference of the surgeon . in the case of external fixators , the most suitable enabling gradual correction seems to be ankle - foot fixators of the ilizarov type or the taylor spatial frame . mostly , a three - plane fixation that combines common types of external fixators is used . an advantage of this method is the absence of internal implant that may increase the risk of infection and the possibility of earlier weight bearing on the foot . as for the internal fixations , plates are recommended if the implantation is from the plantar side , although nowadays angle stable plates are used more often . they enable good stability in an osteoporotic bone and greater variability from the point of view of plate placement . the disadvantage is the need for a wider surgical approach and problematic healing with the exposed implant . in our department when the resection is done and the reposition is finished we use them to fix intramedullary both the medial and lateral columns and subsequently apply plaster cast fixation . by using such a technique we eliminate the disadvantages of using the plates as mentioned above and we did not observe any osseous healing failure reporting by some authors [ 43 , 44 ] . most of the earlier operations have been carried out only in the chronic , inactive stage . in the active stage , an inflammatory reaction with oedema and osteoporosis are present , thus increasing the risk of complicated healing . on the other hand , this stage enables easier corrections than in the fixed deformity as it is possible to use the remodeling capacity of the bone . indications for surgery in the active stage are heavy instability , progression of the deformity , prevention of the dislocation of fragments by muscle contraction , and the general failure of conservative treatment . the position of the foot is gradually corrected by this external fixator into the correct plantigrade position , then an arthrodesis of joints is carried out , and the fixator is left in place for at least three more months . nevertheless , to date no sufficient relevant studies have been published demonstrating the success rate of the surgeries carried out during this active stage . in our department sanders and frykberg classified individual localizations of cn on the foot ( table 4 ) . in this case prevailing resorptive changes were creating deformities of the metatarsal bones of the so - called candy bar type ( figure 3 ) . popelka recommends a fixation with the use of two screws as sufficient for this surgery . on the basis of our experience we recommend using plates in cn in neuropathic terrain . in the case of heavy deformities or it is necessary to distinguish cn and osteomyelitis as mistaking cn for osteomyelitis may often lead to transmetatarsal amputation . translation of metatarsi medially or laterally is usually associated with lowering the medial column and valgus heel . a frequent consequence is abduction of the forefoot , which includes a perinavicular affection with the talus and navicular bone in plantar flection while the cuneiform bone is dislocated dorsally with the i metatarsus . the contraction of the tibialis anterior muscle worsens the deformity and practically excludes successful conservative treatment . this type of cn is very often combined with the following type iii , which is why surgical treatment of both these types will be described together . moreover , a normal position of the hindfoot is a prerequisite for the correction of the forefoot . this plantar bone prominence causes chronic ulcerations which do not respond to conservative therapy ( figure 4 ) . it is necessary to do reconstruction and stabilisation of the medial and lateral column and , in case of persistent instability , subtalar arthrodesis as well . one advantage is regional anaesthesia and minimal usage of a tourniquet . in the case of pes equinus , the first phase , according to the preoperative assessment , involves either the strayer procedure or , more frequently , prolongation of the achilles tendon , which can be carried out using either a z - plasty or a technique of three mini - incisions three cm apart from each other and up to one - half of the tendon diameter ( most frequently a lateral - medial - lateral incision ) . the position of the sole is corrected up to 90 degrees in respect to the long axis of the fibula , with the knee in full extension . this procedure restores the positive inclination of the calcaneus and facilitates the reconstruction of the medial part of the tarsus . care must be taken to avoid pes calcaneus by overextended prolongation ; this position causes heel ulcerations that do not heal , leading to the necessity for below - knee amputation . we temporarily fix the corrected position of the hindfoot with the help of the kirschner wire from the calcaneus to the tibia . in the next phase we make a slightly s - shaped incision on the medial side of the foot from the talus to the base of the i metatarsus , where we identify individual joint dislocations . earlier we used the approach to the lateral column according to ollier which led to a higher percent of secondary healing . that is why , in a deformity where the cuboid bone is the lowest bone , the callus is excised from a plantar approach . reconstruction of the lisfranc joint follows , and we correct abduction or adduction deformity performing osteotomy using the previously introduced kirschner wires . we temporarily fix the corrected position with the help of kirschner wire . in the third phase the navicular and cuboid bones are usually plantarly dislocated , with the goal being resectional talonavicular , naviculocuneiform , and calcaneocuboid arthrodesis in the corrected position , which is again temporarily fixed by the kirschner wires . a final fixation with a midfoot fusion bolt of 6.5 mm ( depuy / synthes ) is the last step . we introduce the implant medially over the head of the i metatarsus according to the deformity of the hallux from a dorsal or plantar approach up to the talus bone . laterally we introduce the implant from the mini - incision from the area of base of the iv metatarsus through the cuboid bone to the calcaneus . to date we did not have to address residual instability between the talus and calcaneus , for which subtalar arthrodesis with the same implant is recommended . we use resected bones as local autografts , reinsert tendon attachments of the medial column , and insert redon drains and the wounds are sutured . finally , we apply a padded plaster cast according to the type of intervention ( figure 5 ) . in this type the ankle and frequently a subtalar joint are most affected . because of the instability the deformity progresses and calluses and ulcerations emerge . we indicate arthrodesis of the ankle and the subtalar joint along with an external fixation ( figure 6 ) . healing complications have been observed by us in these cases . in the case of severe deformities is healed it is necessary to use an orthosis for several months ( figure 7 ) . this is the least frequent type affecting the calcaneus ( figure 8) . with regard to the poor quality of bone and thus to the retention of osteosynthetic material and the risk of infection surgical treatment is considered in the case of progressing deformity when a dislocation of fragments occurs as a result of contracting achilles tendon . the primary goal is the stability of the hindfoot and preventing the formation of ulcerations . postoperative care depends on the type of corrected deformity , the implant used ( internal or external fixation ) , the course of healing , whether or not the contralateral extremity is affected , and the ability of nonweight bearing on the operated extremity . in general a great deal of attention must be paid to the appropriate off - loading in the early and subacute postoperative stage and in the case of chronic cn in terms of localization . the advantage of external fixation is the possibility of earlier weight bearing ; internal fixation is supplemented by a plaster cast usually for three to four months . after the external fixator or plaster cast fixation is removed , individual orthoses are applied , and weight bearing is gradually increased by reduction in limitation of walking time and speed . a suitable time for using the orthosis is up to one year . after this period a lifelong follow - up including diabetes , nutrition , and infection control by antibiotic treatment if necessary is essential . thanks to new findings from the aetiopathogenesis of the condition and its biomechanics it is possible , in indicated cases , to supplement cn treatment with reconstructive procedures along with suitable implants , thus avoiding major amputation . nevertheless , to evaluate the benefits and risks of these procedures further evidence - based studies will be necessary .

charcot neuropathic osteoarthropathy of the foot is a relatively common complication of diabetic neuropathy . incorrect diagnosis and improper treatment often result in the extremity having to be amputated . this paper summarises the current view on the etiology , diagnostics , and treatment of diabetic charcot neuropathic osteoarthropathy , with particular focus on preserving the extremity through surgical intervention from our own experiences .

1. Introduction 2. Materials and Methods 3. Surgical Treatment in Individual Localizations according to Sanders and Frykberg 4. Postoperative Care 5. Conclusion

packaging is a link connecting production with marketing whereby the goods reach from the production center to the consumers in a safe and sound condition with a minimum overall cost . packaging can also be defined as the coordinated system which can enclose or protect the products for distribution , storage , preservation , transportation , information , and sales . the pharmaceutical brands are most vulnerable due to their higher market share , ease of production , and greater profit margins . products sidetracked from their proper distribution channel , or sold past their expiry date , or by modification of the package are associated with the problem of counterfeiting . counterfeits are unauthorized reproductions of a trademarked brand , which are closely similar or identical to genuine articles . the first international meeting on counterfeit medicines was held during april 13 , 1992 , at world health organization ( who ) in geneva and the following definition was accepted : a counterfeit medicine is one which is purposely and falsely mislabeled in accordance to identity and/or source . counterfeiting can pertain mutually to both branded as well as generic products comprising of either correct or incorrect ingredients , lacking active ingredients , or with forged packaging . later on , the concept was modified by the nigerian national agency for food and drug administration and control ( nafdac ) as those medicines with the same quantity of active ingredient as that of genuine brand , insufficient or no active ingredients , medicines which are post expiry date , herbal preparations that are toxic or ineffective and medicines which do not bear the name and address of the manufacturer are counterfeit . counterfeiting is a high - volume , high - profit business which causes the infringement of intellectual property rights , medicine legislations , and other aspects of criminal law . counterfeiting and piracy are in term the same since they are both the reproduction of identical copies of the genuine product . the most common counterfeit drugs in industrialized or developed countries are so - called lifestyle drugs . counterfeit drugs are the major cause of morbidity , mortality , and loss of confidence in the healthcare system . in india , the consumers reported a wider use of counterfeit and pirated products through reuse , repair , and refill of products . repackaging is one of the sources of fake drugs in europe and the united states . estimates put the total loss of life to counterfeit pharmaceuticals between 500,000 and 1,000,000 people per year . according to the bcc research report , the anticounterfeit packaging technology in the united states increased to approximately $ 34.2 billion in 2006 . sales are likely to reach $ 43 billion in 2012 , viewing a compound annual growth rate ( cagr ) of 3.2% over the tenure of 5 years from 2007 to 2012 , while as per the statement released by the organization for economic co - operation and development ( oecd ) , three - fourths of forged drugs supplied world over have some origin in india , followed by 7% and 6% from egypt and china , respectively . as of 2005 , oecd estimates the international trade of counterfeit goods to be $ 200 billion per year . china ranks first for the counterfeit products , the united states is at the second and india at the fourth rank globally . who estimates that up to 1% of medicines available in the developed world are likely to be counterfeits . this figure rises to 10% globally , although in some developing countries the estimation is about one - third for the counterfeit medicines . medicines have been the center of attraction for the counterfeiters due to the number of reasons listed in table 1 , and the factors responsible for the counterfeiting are shown in figure 1 . the medicines counterfeited worldwide with their outcomes are mentioned in the table 2.[2432 ] medicine as a target for counterfeiting drivers of counterfeiting counterfeit medicines globally authentication is of utmost importance because the use of counterfeit medicines can be harmful to the health and wellbeing of the patients . overt features are expected to assist the users to confirm the genuineness of a pack . the process can be untidy and does not always provide the print quality necessary for creating small codes , which must stay clear for weeks or months . barcodes are high - density linear or two - dimensional codes incorporated onto the product package , which are scanned and sent to the central database as shown in figure 2 . users must make sure that there is a sufficient print contrast between light and dark bars to produce a legible representation . package showing two - dimensional barcodes , scanned and sent to the central database holography is well known for its capacity to produce striking three - dimensional images , which are difficult to get through with the conventional photography . a major benefit of this process is that they can be reformed under white light . holograms are generated from the interference patterns obtained through the contact of laser beams by either angular image or laser technology . such high - definition holograms are used as a security feature on the product bottle as shown in figure 3 . the complexity of the hologram varies from the traditional three - dimensional images to computer - generated two - dimensional diffraction patterns . holograms are now widely available in variety of formats such as holographic shrink sleeves , blister packaging aluminum foil , holographic induction cap seals , polyester - based tamper evident labels , and holographic hot stamping foil . but still it is reported that more than half the sales of the artesunate drug in south east asia is forged , despite the presence of the hologram.[4253739 ] the overall advantages and disadvantages of overt technologies are described in table 3 . bottle with a hologram as a security feature advantages and disadvantages of overt and covert technologies the rationale of a covert feature is to aid the brand owner to recognize a counterfeited product . the general public will not be aware of its presence nor will have the resources to confirm it . a covert feature should be difficult to sense or copy without the specialist knowledge . the highly appreciated though expensive technology used presently is the radio frequency identification device ( rfid ) . , rfid includes batch information which can be interrogated at a distance without requiring the line of sight . the rfid allows the recognition of objects through wireless communications in a set frequency band . three vital components in any rfid system are the tag , the reader , and the software . the tag is an integrated circuit containing an exclusive tracking verifier , called an electronic product code ( epc ) , which is transmitted via electromagnetic waves in the radio spectrum . the reader captures the transmitted signal and provides the network connectivity . for their track and trace usage , the diverse rfid tags that are used . the high cost of a rfid tag ( 2050 cents as opposed to a 2 cent barcode ) might forbid the companies from implementing it . the biometric fingerprints introduced by the bayer group utilize 1-mw diode lasers to analyze the innate surface makeup of each item . microscopic irregularities which are caused by the setting of paper fibers or plastic result in spreading of the laser beam by a laser speckle event . by measuring the dispersal of the light at various angles , surface uniqueness is recorded . overt features are expected to assist the users to confirm the genuineness of a pack . the process can be untidy and does not always provide the print quality necessary for creating small codes , which must stay clear for weeks or months . barcodes are high - density linear or two - dimensional codes incorporated onto the product package , which are scanned and sent to the central database as shown in figure 2 . users must make sure that there is a sufficient print contrast between light and dark bars to produce a legible representation . package showing two - dimensional barcodes , scanned and sent to the central database holography is well known for its capacity to produce striking three - dimensional images , which are difficult to get through with the conventional photography . a major benefit of this process is that they can be reformed under white light . holograms are generated from the interference patterns obtained through the contact of laser beams by either angular image or laser technology . such high - definition holograms are used as a security feature on the product bottle as shown in figure 3 . the complexity of the hologram varies from the traditional three - dimensional images to computer - generated two - dimensional diffraction patterns . holograms are now widely available in variety of formats such as holographic shrink sleeves , blister packaging aluminum foil , holographic induction cap seals , polyester - based tamper evident labels , and holographic hot stamping foil . but still it is reported that more than half the sales of the artesunate drug in south east asia is forged , despite the presence of the hologram.[4253739 ] the overall advantages and disadvantages of overt technologies are described in table 3 . bottle with a hologram as a security feature advantages and disadvantages of overt and covert technologies the rationale of a covert feature is to aid the brand owner to recognize a counterfeited product . the general public will not be aware of its presence nor will have the resources to confirm it . a covert feature should be difficult to sense or copy without the specialist knowledge . the highly appreciated though expensive technology used presently is the radio frequency identification device ( rfid ) . , rfid includes batch information which can be interrogated at a distance without requiring the line of sight . the rfid allows the recognition of objects through wireless communications in a set frequency band . three vital components in any rfid system are the tag , the reader , and the software . the tag is an integrated circuit containing an exclusive tracking verifier , called an electronic product code ( epc ) , which is transmitted via electromagnetic waves in the radio spectrum . the reader captures the transmitted signal and provides the network connectivity . for their track and trace usage , the diverse rfid tags that are used the high cost of a rfid tag ( 2050 cents as opposed to a 2 cent barcode ) might forbid the companies from implementing it . the biometric fingerprints introduced by the bayer group utilize 1-mw diode lasers to analyze the innate surface makeup of each item . microscopic irregularities which are caused by the setting of paper fibers or plastic result in spreading of the laser beam by a laser speckle event . by measuring the dispersal of the light at various angles , surface uniqueness is recorded . authentication in packaging utilizes mainly the concept of sealing which has been significantly demonstrated by american bank note holographics . holoseal is a patented pressure - sensitive , tamper apparent holographic security label that features a customized tamper apparent fracture pattern , black light verification system , and machine - readable entrenched code . for the tracking purpose , holoseal can be numbered or personalized to give an account of region , plant , or product . holocap comprises several different heat - sealable films which are attached directly to the containers using the induction seal technology . these different films unite to form a single holographic inner seal that provides a higher level of defense to containers or bottles . package sealing tapes the trend of utilizing sealing in packaging for the development of anticounterfeiting techniques changes with the upcoming concept of lasers and forensic approaches . holospot is a discrete forgery - proof information carrier that can be attached to any product . it uses laser encryption of computer - generated lithograms into small polymeric data carriers and offers multiple overt and covert security features , whereas on the other hand ident seal is a visible text or high - contrast barcode inscribed by lasers . the anticounterfeit label forge guard of fujifilm corporation can visualize full color images or text clearly with a special viewer . this label regulates light wavelengths on the nano - optic level , so those with the viewer can see hidden full color images . still the need was for the invisible substrates to enhance the security and the research led to the development of forensic markers . these forensic markers include physical , chemical , and biological taggants . a merck product , esan features numerous hidden and forensic security features on the pack from its securalic product line . the security features are built into the ivy leaf design element printed on the folded box . other systems include a tamper evident packaging system , film wrappers , and breakable caps . the tamper evident feature helps to maintain the integrity of the drug product by preventing the profit of counterfeiters by either repacking or reselling of the pharmaceutical products as shown in figure 5 . a film wrapper is a transparent film with a characteristic design covered securely around a product or product container . the film must be cut or torn to open the container and remove the product . substrate options include ultradestructible films , solvent - sensitive papers , and voidable films that provide images when removed as shown in figure 6 . breakable caps , as the name suggests , break when an effort to open is made . these caps provide external tamper evidence and can also be combined with the internal seals , thereby assisting with double security . the overall choice of anticounterfeit techniques at various levels of packaging has been described in table 4 . external and internal tamper evident feature label that leaves a void mark on tampering choice of anticounterfeit solutions at different packaging levels medicines save lives and prevent diseases and epidemics only if they are safe , efficacious , of good quality , and are rationally used . the use of unsafe , substandard , ineffective , and counterfeit medicines can be harmful to the health and wellbeing of the patients . governments must establish the national medicine regulatory authority ( nmra ) to monitor the quality of medicines in the market to detect and prevent any substandard and counterfeit medicines from reaching the public , by working closely with national law enforcement agencies such as the police and custom officers . the nmra must ensure that all medicine manufacturing , import , export , and distribution activities are carried out in premises approved by the nmra , and that individuals and companies engaged must have licenses to operate such activities . the counterfeit pharmaceuticals initiative ( cpi ) was launched by the icc in 2003 for the creation of a counterfeit pharmaceutical database with online search facility;construction of a dedicated cpi website;liaising with regulators;providing assistance to members by lobbying and investigation;special projects and surveys , e.g. , internet pharmacies ; andthe implementation of anticounterfeiting technologies . the creation of a counterfeit pharmaceutical database with online search facility ; construction of a dedicated cpi website ; liaising with regulators ; providing assistance to members by lobbying and investigation ; special projects and surveys , e.g. , internet pharmacies ; and the implementation of anticounterfeiting technologies . the implementation of overt and covert techniques by the industries can raise the bar for the counterfeiters , but the final awareness must be at the consumer level . the consumers should buy medicines only from licensed pharmacies and medicine outlets , be suspicious of heavily discounted medicines , and check if the packaging indicates the batch number , manufacturing date , expiry date , and the manufacturer 's name . counterfeit drugs not only affect the sick and innocent consumers but also the general public , and deserve more attention .

packaging is the coordinated system that encloses and protects the dosage form . counterfeit drugs are the major cause of morbidity , mortality , and failure of public interest in the healthcare system . high price and well - known brands make the pharma market most vulnerable , which accounts for top priority cardiovascular , obesity , and antihyperlipidemic drugs and drugs like sildenafil . packaging includes overt and covert technologies like barcodes , holograms , sealing tapes , and radio frequency identification devices to preserve the integrity of the pharmaceutical product . but till date all the available techniques are synthetic and although provide considerable protection against counterfeiting , have certain limitations which can be overcome by the application of natural approaches and utilization of the principles of nanotechnology .

INTRODUCTION MARKET SCENARIO AND DRUG PERSPECTIVES AUTHENTICATION TECHNIQUES Overt (Visible) Features Covert (Hidden) Features SECURITY-ENFORCING PACKAGE COMPONENTS CONCLUSION

chemotherapeutic drugs , alone or as an adjuvant therapy to surgery and radiation , are a vital part in cancer treatment . however , their effectiveness is often offset by severe side effects caused by poor selectivity and toxicity to normal cells . in recent years , the rapid advance in the fields of bioinformatics and genomics facilitated the identification of numerous target molecules that are uniquely or overly expressed in cancer cells , including certain receptors and enzymes . these discoveries were successfully utilized for the specific delivery of chemotherapeutic drugs to tumor cells , with the double goal of increasing their therapeutic efficacy while decreasing their toxicity to normal cells . the enzyme dipeptidyl peptidase iv ( dppiv ) ( ec 3.4.14.5 ) , also known as cd26 ( t - cell surface activation antigen ) or adenosine deaminase ( ada ) , was first identified in 1966 . dppiv is a member of the prolyl oligopeptidase ( pop ) family of serine proteases and specifically hydrolyzes the peptide bond c - terminal to proline or alanine in the penultimate p1 position . it is normally present on the apical membrane surface of several cell types , including t cells and b cells , natural killer ( nk ) cells , epithelial cells of kidney , intestine brush border membranes , plasma , and endothelial cells of blood vessels , and is involved in many diverse physiological processes . in addition , it was recently discovered that dppiv is overly expressed in human renal cell carcinoma tissues , and it is thought to play a role in the pathogenesis of other various human cancers as well . moreover , proline - containing substrates are hydrolyzed exclusively by proline - specific enzymes and are less susceptible to nonspecific peptidases and proteases . thus , a dppiv - cleavable prodrug containing a proline dipeptide conjugated to a cytotoxic chemotherapeutic agent may limit nonspecific activation . the purpose of this study was to investigate the feasibility of exploiting dppiv as a prodrug activating enzyme , to allow specific targeting of chemotherapeutic agents to cancer cells . to that end , the expression profiles of dppiv in 60 cancer cell lines ( nci 60 ) were obtained , and 7 cell lines , which represent the spectrum of dppiv expression , were selected . a candidate prodrug was then synthesized by linking the cytotoxic drug melphalan ( figure 1a ) to a proline dipeptide , creating glycyl - prolyl - melphalan prodrug ( gp - mel ; figure 1b ) with expected dppiv affinity . functional activity studies of dppiv with the prodrug , in the absence vs presence of inhibitor , confirmed that the prodrug is a specific dppiv substrate . finally , hydrolysis and cell proliferation studies were performed in high and low dppiv expression cancer cell lines . overall , this work indicates that dppiv may be exploited as a prodrug activating enzyme for efficient chemotherapeutic drug targeting . chemical structures of ( a ) melphalan and ( b ) its gly - pro dipeptide prodrug , gp - mel . melphalan , porcine kidney dipeptidyl peptidase ( porcine dppiv ) , glycyl - prolyl - p - nitroanilide ( gp - pna ) , glycyl - phenylalanyl - p - nitroanilide ( gf - pna ) , glycyl - arginyl - p - nitroanilide ( gr - pna ) , p - nitroaniline ( pna ) , diprotin a ( ile - pro - ile ) , xtt , pms ( n - methyldibenzopyrazine methyl sulfate ) , and ladder sybr green were purchased from sigma chemical co. ( st . louis , mo ) . n , n - dimethylformamide ( dmf ) , piperidine , triisopropylsilane ( tis ) , triethanolamine ( tea ) , and trifluoroacetic acid ( tfa ) were purchased from aldrich chemical co. ( milwaukee , wi ) . boc - l - gly - l - pro , 1-hydroxybenzotriazole anhydrous ( hobt ) , 2-(1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) , triisopropylsilane ( tis ) , n-(9-fluorenylmethoxycarbonyloxy)succinimide ( fmoc - osu ) , and wang resin ( 100200 mesh ) were obtained from calbiochem - novabiochem ( san diego , ca ) . access rt - pcr kit , dna blue / orange loading dye , 1 kb ladder and 100 bp ladder were obtained from promega ( madison , wi ) . 420% tbe gels , trizol reagent , and custom - ordered dppiv sense and antisense primers were from invitrogen life technologies ( carlsbad , ca ) . the cancer cell lines igrovi ( ovarian ) , pc-3 ( prostate ) , 786 - 0 ( renal ) , sk - mel-5 ( melanoma ) , sk - ov-3 ( ovarian ) , caco-2 ( colon ) , and hepg2 ( liver ) were obtained from national cancer institute or atcc . dulbecco s modified eagle s medium ( dmem ) , rpmi-1640 , fetal bovine serum ( fbs ) , fetal calf serum ( fcs ) , phosphate buffered saline ( pbs ) , and trypsin - edta were purchased from gibco brl ( grand island , ny ) . minimal essential medium ( mem ) was obtained from atcc . all hplc grade solvents ( acetonitrile , dmf , ethyl acetate , and piperidine ) used for peptide synthesis or hplc analysis trifluoroacetic acid for hplc analysis was obtained from pierce ( rockville , il ) . the solid - phase synthesis of the l - glycyl - l - prolyl prodrug of melphalan was carried out in a stepwise fashion with wang resin as described below and is summarized in the schematic shown in figure 2 . fmoc - l - melphalan synthesis : fmoc - osu ( 300 mg , 0.89 mmol ) was added to an ice - cold solution of melphalan , 1 ( 200 mg , 0.82 mmol ) , in a mixture of acetone ( 20 ml ) , distilled water ( 10 ml ) , and nahco3 ( 300 mg ) . the mixture was stirred for 1 h at 0 c and then reacted at room temperature for 16 h. after the reaction was complete , the mixture was concentrated . 15 ml of ethyl acetate and 15 ml of distilled water were added . the aqueous phase was then extracted with ethyl acetate ( 3 15 ml ) . the combined organic phases were washed with distilled water and brine and dried over mgso4 . the filtrate was concentrated under vacuum to yield fmoc - l - melphalan , 2 ( 330 mg , yield 98% ) . fmoc - l - melphalan ( 330 mg ) and wang resin ( 0.1 mmol ) were added into a 100 ml round reaction bottle with 20 ml of dmf . hobt ( 0.3 mmol ) , hbtu ( 0.3 mmol ) , and tea ( 0.3 mmol ) were then added to the resin mixture and stirred overnight at room temperature . the wang resin , 3 , was washed 3 times with 10 ml of dmf after filtration and then washed 3 times for 10 min each in 10 ml of 20% piperidine in dmf . boc - l - glycyl - l - proline ( 0.3 mmol ) and wang resin 3 were added to 20 ml of dmf in a 100 ml round reaction bottle . hobt ( 0.3 mmol ) , hbtu ( 0.3 mmol ) , and tea ( 0.3 mmol ) were added and stirred overnight at room temperature . the resultant wang resin , 4 , was washed 3 times with 10 ml of dmf after filtration and then washed 3 times for 10 min each in 10 ml of 20% piperidine in dmf . wang resin 4 was then added to 10 ml of cleavage buffer ( tfa : tis : h2o , 95:2.5:2.5 ) and stirred for 1 h at room temperature . after filtration , the solution was concentrated and cold ether ( 10 ml ) was added to precipitate out the prodrug 5 . after removing ether , the residues were reconstituted with 5 ml of distilled water and lyophilized . the purity of the melphalan prodrug was determined by hplc to be 95% . electrospray ionization mass spectra ( esi - ms ) were obtained on a thermoquest lcq electrospray ionization mass spectrometer . the observed molecular weight of the prodrug was found to be consistent with that required by its structure . the structural identity of the prodrug was then confirmed using proton nuclear magnetic resonance spectra ( h nmr ) . schematic of solid - phase synthesis of l - glycyl - l - prolyl - melphalan prodrug . l - glycyl - l - prolyl - melphalan : yield 13% ; percent purity 95% ; h nmr ( dmso - d6 ) 1.952.20 ( m , 4h , ch2 on proline ) , 3.053.35 ( m , 12h , ch2 ) , 4.444.80 ( m , 5h , ch , ch2 ) , 6.506.90 ( m , 4h , phenol h ) ; esi - ms 459.4 ( m + h ) . the microarray data and the u95av2 affymetrix data were downloaded from the dtp database ( https://wiki.nci.nih.gov/display/ncidtpdata/molecular+target+data ) . the programming language perl was used to sift through more than 26,000 genes to find all enzymes with names that end with the suffix ase . the enzyme text file was then used to separate the enzymes into different classes , such as hydrolases , peptidases , and esterases as described earlier . visual basic was then used to arrange the data retrieved so that the expression of each enzyme gene in the 60 cancer cell lines could be easily visualized using visual tools such as cluster and treeview programs . the arranged data from peptidases was normalized using a pool of 12 cell lines ( nci - h226 , colo 205 , mcf-7 , hs 578 t , ovcar-3 , ovcar-4 , k-562 , hl-60 ( tb ) , caki-1 , lox imvi , pc-3 , snb-19 ) as reference and clustered according to the origin of the cell lines . the sorted expression data for the 60 cancer cell lines was clustered using hierarchical clustering , and a file was created ( .cdt ) such that the expression of the genes in 60 cancer cell lines could be visualized using treeview . the potential enzyme target , dppiv , was selected on the basis of the differential expression in various cell lines or tissues with emphasis on high expression in a particular tissue as compared to other tissues and high substrate specificity of the enzyme . candidate cancer cell lines were selected from the nci 60 cell lines based on the expression levels of dppiv , doubling time , and growth requirements . thus , the cancer cell lines selected , igrovi , pc-3 , 786 - 0 , sk - ov-3 , and sk - mel-5 , represent cells with high , medium , and low expression of dppiv , reasonably short doubling times , and standard growth requirements . in addition , caco-2 as well as hepg2 cell lines were also used in the studies . total rna was lysed and purified from each cancer cell line with trizol reagent according to the manufacturer s protocol . isolated rna ( 0.5 g ) was reverse transcribed to cdna with amv reverse transcriptase , oligo ( dt ) , and access rt - pcr kit following the standard protocol . each of the 20 cycles of amplification consisted of denaturation ( 94 c for 25 s ) , annealing ( 50 c for 25 s ) , and extension ( 68 c for 40 s ) . the primer set used for dppiv amplification was the sense primer ( 5-ccttctactctgatgagtcactgc-3 ) and the antisense primer ( 5-gtgccactaagcagttccatcttc-3 ) . pcr product was identified by electrophoresis in 420% tbe gels followed by sybr green staining . the gel was then visualized with uv light , and the relative intensities of the rt - pcr product bands were measured using metamorph software . porcine kidney dppiv was used in the studies due to its commercial availability and its close alignment with human dppiv ( 88% homology ) . stock solutions of porcine dppiv were prepared by reconstituting lyophilized powder ( 0.87 unit , 23 units / mg ) in 1 ml of 0.1 m tris - hcl ( ph 8.0 ) and stored at 80 c . solutions of the various substrates glycyl - prolyl - p - nitroanilide ( gp - pna ) , glycyl - phenylalaninyl - p - nitroanilide ( gf - pna ) , or glycyl - argininyl - p - nitroanilide ( gr - pna ) at 0.4 mm were mixed with appropriate amounts of diluted dppiv solution ( final dppiv concentration in mixture , 0.473 g / ml ) and incubated at 37 c for 1530 min in flat - bottom 96-well plates . the hydrolysis of the substrates was monitored by measurement of absorbance at 405 nm ( generation of p - nitroanilide ) using a precision microplate reader ( emax , molecular devices ) . the competitive inhibition of hydrolysis of the substrates by porcine kidney dppiv in the presence of the inhibitor diprotin a was determined by including 0.4 mm diprotin a in the diluted dppiv mixture prior to incubation . initial hydrolysis rates were determined in all hydrolysis experiments by assaying the amounts of pna released . dppiv activity in the selected cancer cells was determined using the standard substrate gp - pna . briefly , cells were grown to 90% confluence in 150 mm culture plates , washed carefully with 0.15 m nacl , and collected by scraping . the cells were resuspended in 0.15 m nacl and then centrifuged at 3,000 rpm for 5 min . the cell pellet was resuspended in phosphate buffered saline ( pbs , ph 7.4 ) , and sonicated for 5 s two times on ice . the sonicated cell suspension was centrifuged at 18000 g for 30 min at 4 c . the supernatant was then used in hydrolysis studies , and to determine protein content ( bio - rad dc protein assay ) . the protein content was adjusted to approximately 1000 g / ml by appropriate dilutions before use in hydrolysis studies . hydrolysis studies were conducted in a 96-well microplate containing the cell homogenate suspensions at 37 c . the reactions were initiated by the addition of gp - pna ( final concentration 1 mm ) to the suspensions . the absorbance was monitored every minute for a total of 3060 min using a precision microplate reader ( emax , molecular devices ) at 405 nm . in the competitive inhibition studies , diprotin a at 1 mm was included along with 1 mm gp - pna , and the mixture was added to the cell homogenate suspensions and absorbance monitored as described above . dppiv activity was expressed as the amount of pna ( micromoles ) released per minute , normalized to the amount of protein . hydrolysis of the melphalan prodrug by porcine kidney dppiv and the effect of the competitive inhibitor diprotin a were assayed using 96-well microplates . porcine dppiv enzyme solution ( 230 l at a final concentration 4.4 g / ml ) was placed in triplicate wells maintained at 37 c . the reactions were initiated by the addition of gp - mel ( 1 mm final concentration in mixture ) , and 40 l aliquots were removed at predetermined time points and added to two volumes of 10% ice - cold tfa to quench the reaction and precipitate protein . in the inhibition studies , diprotin a and gp - mel were both added ( 1 mm final concentration ) to the enzyme solution , incubated at 37 c , and sampled as described above . the quenched precipitated samples were then filtered through a 0.45 m filter plate and centrifuged at 1800 g and 4 c for 20 min . the extent of hydrolysis of gp - mel in caco-2 and sk - mel-5 cell homogenates was determined as follows . caco-2 and sk - mel-5 cells and cell homogenates were prepared as described earlier . the hydrolysis reactions were carried out in 96-well plates ( corning , corning , ny ) . 230 l of the cell suspensions ( 1000 g / ml protein ) were placed in triplicate wells and the reactions initiated by the addition of gp - mel ( final concentration 1 mm in mixture ) and incubated at 37 c . at predetermined time points 40 l aliquots were removed and added to two volumes of 10% ice - cold tfa to quench the reaction and precipitate protein . in the inhibition studies , diprotin a and gp - mel were both added ( 1 mm final concentration ) to the cell suspensions , incubated at 37 c , and sampled as described above . the quenched precipitated samples were then filtered through a 0.45 m filter plate and centrifuged at 1800 g and 4 c for 20 min . dppiv activity was expressed as the amount ( picomoles ) of melphalan released per minute , normalized to the amount of protein . the concentrations of gp - mel and melphalan were determined on a waters hplc system ( waters inc . , the hplc system consisted of two waters pumps ( model 515 ) , a waters autosampler ( wisp model 712 ) , and a waters uv detector ( 996 photodiode array detector ) . the system was controlled by waters millennium 32 software ( version 3.0.1 ) . samples were injected onto a waters xterra c18 reversed phase column ( 5 m , 4.6 250 mm ) equipped with a guard column . the flow rate was 1 ml / min , and the mobile phase was 70:30 ( % standard curves generated for prodrug and parent drug were utilized for quantitation of integrated area under peaks . cell proliferation assays were conducted to determine the cytotoxic activities of the prodrug gp - mel and the parent melphalan . the assays were carried out with caco-2 and sk - mel-5 cells since the expression of dppiv was found to be highest and lowest , respectively , in these cells , based on rt - pcr expression results . caco-2 and skmel-5 cells were plated overnight in a 96-well cell culture plate at a density of 5,000 cells / well per 0.1 ml . stock solutions ( 1 mm ) of gp - mel and melphalan were prepared in rpmi-1640 phenol red free medium supplemented with fbs . stock solutions were serially diluted to obtain a total of six drug concentrations , 1 mm , 0.5 mm , 0.25 mm , 0.125 mm , 0.0625 mm , and 0.03125 mm , for cell proliferation studies . after 24 h , the medium in the 96-well plate was aspirated and replaced with drug solutions in the medium . the cells were then incubated at 37 c and 5% co2 for 48 h. after 48 h , 50 l of xtt labeling mixture ( 5 ml of 1 mg / ml xtt in rpmi-1640 phenol red free medium mixed with 100 l of 0.383 mg / ml pms in phosphate buffered saline ) was added to each well . the color development , due to formation of formazan dye by metabolically active cells , was monitored for 4 h , after which the plates were read at 490 nm ( 805 nm as the reference wavelength ) with a precision microplate reader ( emax , molecular devices ) . the percent cell viability , at different drug and prodrug concentrations , relative to control was then plotted as a function of drug / prodrug concentration to compute the gi50 values . after the genes had been sorted as hydrolases , peptidases , and esterases , they were clustered and their expression in the 60 cancer cell lines was visualized as described previously . the expression patterns of proline - specific peptidases such as prolinases / peptidase , prolyl carboxypeptidase , prolyl oligopeptidase / prolyl endopeptidase , dppiv / cd26 , aminopeptidase p , and prolidase / peptidase d , were selected for further assessment . dppiv was overexpressed in most renal carcinoma cell lines ( uo-31 , tk-10 , sn12c , rxf 393 , caki-1 , achn , a498 , and 786 - 0 ) compared to cancer cell lines derived from other tissues . the relatively higher dppiv expression in 786 - 0 renal carcinoma cell line compared to its expression in sk - ov-3 ovarian carcinoma or sk - mel-5 melanoma cell lines was consistent with affymetrix expression data . thus , in addition to the selection of igrovi ( ovarian cancer cell line ) , pc-3 ( prostate cancer cell line ) , 786 - 0 ( renal cancer cell line ) , sk - ov-3 ( ovarian cancer cell line ) , and sk - mel-5 ( melanoma ) based on the u95av2 affymetrix gene expression of dppiv ( table 1 ) , hepg2 and caco-2 cells were also selected for characterization of dppiv activity with standard substrates and rt - pcr determinations of dppiv expression . rt - pcr of extracellular mrna in the seven selected human cancer cell lines was performed with primers specific for dppiv . the expression profiles of dppiv in the seven cancer cell lines evidenced by the band at 315 bp are shown in figure 3 . the results of semiquantitative rt - pcr analysis ( table 1 ) indicated that dppiv was expressed at high levels in caco-2 and hepg2 cells , and at very low levels in sk - mel-5 cells . thus , the relative rt - pcr expression ( expression in sk - mel-5 set at unity ) in the seven cell lines listed in table 1 were caco-2 ( 13.49 ) , hepg2 ( 12.71 ) , igrovi ( 7.92 ) , pc-3 ( 5.51 ) , 786 - 0 ( 2.81 ) , sk - ov-3 ( 1.89 ) , and sk - mel-5 cells ( 1.0 ) . the relative rt - pcr expression in the cancer cell lines exhibited an excellent linear correlation ( r = 0.94 ) with u95av2 affymetrix microarray data that was available for five of the seven cell lines ( figure 4 ) . expression profiles of dppiv ( 315 bp product ) in 7 cancer cell lines determined by semiquantitative rt - pcr : 100 base pair dna ladder ( lane 1 ) , caco-2 ( lane 2 ) , hepg2 ( lane 3 ) , igrovi ( lane 4 ) , pc-3 ( lane 5 ) , 786 - 0 ( lane 6 ) , sk - ov-3 ( lane 7 ) , and sk - mel-5 ( lane 8) . linear correlation of affymetrix genechip expression of dppiv with dppiv expression determined using rt - pcr in 5 cancer cell lines . the specific activity of a standard substrate gp - pna for porcine dppiv was determined by the release of p - nitroanilide and the resultant effect on the clarity of the solution . the baseline activity was 14.02 0.90 nmol / min/g dppiv . in the presence of the specific inhibitor diprotin a , it decreased substantially to 0.15 nmol / min/g dppiv . the kinetic parameters km and vmax obtained from a plot of v0 versus [ s ] ( figure 5 ) were 231 33 m and 0.650 0.037 mol / min / mu dppiv , respectively ( fit estimate sd ; n = 3 ) . the kcat value determined from vmax and dppiv concentration ( e0 ) was 36 s. in contrast , gf - pna or gr - pna produced no detectable release of p - nitroanilide ( negligible absorbance at 405 nm ) , indicating that these two molecules were resistant to cleavage by porcine dppiv . gp - pna hydrolysis kinetic profile with porcine dppiv ( mean sd , n = 3 ) . the activity of dppiv in various cancer cell homogenates was assessed using the standard substrate gp - pna . the results shown in figure 6 indicate that dppiv activity was in the order hepg2 caco-2 igrovi pc-3 786 - 0 sk - ov-3 sk - mel-5 cells . caco-2 and hepg2 cells exhibited high dppiv activity ( around 20 pmol / min/g protein ) whereas sk - mel-5 cells showed the lowest activity ( 1.3 pmol / min/g protein ) among the cells tested ( figure 6 ) . these results are in excellent correlation ( r = 0.96 ) with the relative rt - pcr expression of dppiv in the cell lines ( figure 7 ) . the hydrolysis of gp - pna in the presence of diprotin a was significantly inhibited in all cancer cell homogenates examined ( figure 6 ) , with an average inhibition of 95% ( range : 85100% ) . specific activity of dppiv against gp - pna ( pmol / min/g protein ) in 7 cell homogenates , in the absence vs presence of diprotin a ( mean sd , n = 3 ) . linear correlation of specific activity of dppiv against gp - pna ( pmol / min/g protein ) in 7 cancer cell homogenates with dppiv expression determined using rt - pcr . the specific activity of pure porcine enzyme against gp - mel was 1.2 0.1 nmol / min/g protein . the activity was substantially lowered to 100 pmol / min/g protein , a 93% inhibition , in the presence of diprotin a. dppiv activity against the prodrug determined in caco-2 and sk - mel-5 homogenates was 5.14 0.01 and 0.68 0.03 pmol / min/g protein , respectively , which was significantly inhibited by an average of 85% in the presence of diprotin a ( figure 8) . specific activity of dppiv against gp - mel ( pmol / min/g protein ) in caco-2 and sk - mel-5 cell homogenates , in the absence vs presence of diprotin a ( mean sd , n = 3 ) . the antiproliferative activity of gp - mel was determined in caco-2 and sk - mel-5 cancer cells and compared with that obtained with the parent drug melphalan . the percent cell viability profiles as a function of drug or prodrug concentration are shown in figure 9 . the profiles indicate that the antiproliferative action of melphalan was similar in caco-2 and sk - mel-5 cells with gi50 values of 34.9 m and 56.6 m , respectively . the gi50 value for gp - mel in caco-2 cells ( 261.3 m ) , however , was significantly lower than that obtained with sk - mel-5 cells ( 806.7 m ) . the cytotoxic activity of gp - mel in caco-2 and sk - mel-5 cells was consistent with the expression levels of dppiv in these cells . cell proliferation assay of melphalan and gp - mel after 48 h incubation with caco-2 and sk - mel-5 cells . data are expressed as mean sd ; n = 3 for each treatment at each concentration . in the past three decades , the science of molecular biology was revolutionized by the rapid advancement of the complementing fields of genomics and bioinformatics . recently , a number of enzymes from the peptidase / protease class were found to be highly expressed in several types of tumors , and to play an important role in the pathophysiology of tumor cells . dppiv has been previously associated with the onset and progression of several cancer types ; inamoto et al . demonstrated that the blockage of ddpiv reduced several cancer - related processes in the human renal carcinoma cell line caki-2 . it also reduced the tumor size and increased the survival of mice in a xenograft model . furthermore , in patients with clear cell renal cell carcinoma ( ccrcc ) , higher dppiv activity was linked with a significant decrease in patients 5 year survival rates . dppiv was also shown to be overexpressed in several human colon cancer tissues and in human colon cancer cell lines , and its inhibition reduced carcinogenesis in a rat model . in the prostate , dppiv activity in cancerous versus benign prostatic hyperplasia was increased 2-fold . an elevation of dppiv activity was also found in the prostatic secretions and the peripheral zone of the prostate , where most prostate cancers arise . higher dppiv levels in cancerous versus normal prostate tissue was correlated with psa level , tumor residue , cancer stage , and tumor size in humans . in addition to its expression and involvement in cancer , dppiv is one of the few proline - specific proteases that is able to cleave proline - associated peptide bonds , as the unique cyclic structure of proline serves as a structure regulation element which limits the susceptibility for nonspecific enzymatic degradation . thus , a high level of expression in cancer cells , combined with high substrate specificity , indicated that dppiv may be a potential target molecule for the delivery of chemotherapeutic drugs , and sparked our interest in developing a dppiv - cleavable anticancer prodrug . the functional activity of pure porcine dppiv was assessed against three gly - x dipeptide chromogenic compounds , gp - pna , a well - known standard dppiv substrate , as well as gf - pna and gr - pna , two dipeptide analogues that do not contain proline in the p1 position . the kinetic parameters obtained in this study were consistent with those previously reported for gp - pna with porcine dppiv . the negligible activity observed with gf - pna and gr - pna is consistent with dppiv substrate requirements , and eliminates the possibility of nonspecific degradation . similarly , in the seven investigated cancer cell lines , gp - pna exhibited an excellent linear correlation ( r = 0.96 ) with rt - pcr dppiv expression ( figure 7 ) , and dppiv activity was significantly inhibited in the presence of the specific inhibitor diprotin a ( average inhibition 95% ) . the similarity of the extent of inhibition in cell homogenates with that observed with porcine dppiv underscores the similarity of human and porcine dppiv . melphalan , phenylalanine mustard ( l - pam ) , is an established anticancer agent that was originally approved for the treatment of multiple myeloma . recently it has been suggested as a therapeutic agent in the treatment of a variety of cancers such as ovarian cancer , breast cancer , colorectal cancer , and melanoma . however , its use has been limited due to severe side effects such as bone marrow suppression , leukopenia , and thrombocytopenia , largely due to lack of selectivity for cancer cells , and so it was selected as the model drug for investigating our approach . a proline dipeptide prodrug of melphalan was designed to follow dppiv substrate specificity requirements . additionally , the peptide bond between p1 and p1 requires a trans - configuration . the release of melphalan from the gp - mel prodrug by pure porcine dppiv was extensive , and was 93% reduced in the presence of diprotin a , indicating that the activation of gp - mel was dppiv - dependent . this was further corroborated in caco-2 and sk - mel-5 homogenates when the prodrug activation was significantly inhibited by diprotin a ( figure 8) . the 8-fold greater activation of the prodrug in caco-2 compared to sk - mel-5 homogenates ( figure 8) was consistent with the 14-fold higher rt - pcr dppiv expression levels , and the 15-fold higher dppiv activity against gp - pna in caco-2 cells compared to sk - mel-5 cells ( figure 7 ) . as a preliminary assessment of gp - mel potential to serve as an anticancer prodrug , we have investigated the antiproliferative activity of gp - mel in caco-2 and sk - mel-5 cell lines . the antiproliferative activity of the prodrug was shown to be dependent on dppiv expression level in the cells ; the cytotoxicity ( represented by gi50 ) of gp - mel in caco-2 cells was 3-fold higher ( 260 m ) than that in sk - mel-5 cells ( 800 m ) ( figure 9 ) . in contrast , for the free parent drug melphalan , similar gi50 values were obtained in caco-2 and in sk - mel-5 cells ( 35 and 44 m , respectively ) , demonstrating the nonselective cytotoxic action of melphalan ( figure 9 ) . the cytotoxic effect of gp - mel on both cell lines was significantly lower in comparison to melphalan ; the prodrug failed to show significant cytotoxic effect in concentrations equimolar to those required for maximal growth inhibition by melphalan . this result may indicate that the gp - mel prodrug is not likely to be cytotoxic by itself , and further validates the key role of dppiv in the activation of the gp - mel prodrug . on the other hand , it may indicate insufficient activation , resulting in too low free drug levels . overall , our results demonstrate the potential to exploit dppiv as a prodrug activating enzyme for efficient chemotherapeutic drug targeting . in conclusion , dppiv was identified as a potential prodrug target due to its differential expression levels in tumor and normal tissues and relatively strict substrate specificity . a gly - pro dipeptide prodrug of melphalan , gp - mel , was designed and synthesized , based on the highly specific substrate requirements of dppiv . the finding that the activation and antiproliferative activity of gp - mel in cells were highly dependent on dppiv expression levels confirmed our hypothesis that dppiv is a feasible functional prodrug target for effective and selective chemotherapeutic action .

the efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells . recently , the enzyme dipeptidyl peptidase iv ( dppiv ) was considered as a potential target for the delivery of chemotherapeutic drugs . the purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to dppiv as a strategy to enhance their specificity . the expression profile of dppiv was obtained for seven cancer cell lines using dna microarray data from the dtp database , and was validated by rt - pcr . a prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline - glycine dipeptide moiety , followed by hydrolysis studies in the seven cell lines with a standard substrate , as well as the glycyl - prolyl - melphalan ( gp - mel ) . lastly , cell proliferation studies were carried out to demonstrate enzyme - dependent activation of the candidate prodrug . the relative rt - pcr expression levels of dppiv in the cancer cell lines exhibited linear correlation with u95av2 affymetrix data ( r2 = 0.94 ) , and with specific activity of a standard substrate , glycine - proline - p - nitroanilide ( r2 = 0.96 ) . the significantly higher antiproliferative activity of gp - mel in caco-2 cells ( gi50 = 261 m ) compared to that in sk - mel-5 cells ( gi50 = 807 m ) was consistent with the 9-fold higher specific activity of the prodrug in caco-2 cells ( 5.14 pmol / min/g protein ) compared to sk - mel-5 cells ( 0.68 pmol / min/g protein ) and with dppiv expression levels in these cells . our results demonstrate the great potential to exploit dppiv as a prodrug activating enzyme for efficient chemotherapeutic drug targeting .

Introduction Materials and Methods Results Discussion Conclusions

about 28 million people live in areas at risk of chagas disease , 1114.5 million of whom are affected worldwide . trypanosoma cruzi , the pathogen that causes chagas disease , is found in most south american countries , representing an important cause of heart damage among the economically active population . after a successful chemical control of triatoma infestans ( klugi , 1834 ) , the other main vectors of chagas causing agent , panstrongylus megistus burmeister , 1835 , rhodnius prolixus stal , 1859 , and triatoma brasiliensis sensu lato neiva 1911 . triatoma brasiliensis sensu lato ( s.l . ) , found in anthropogenic habitats and considered the main vector in northeast brazil [ 2 , 3 ] , was recently found to be a species complex that includes t. b. brasiliensis , t. b. macromelasoma galvo , 1956 , t. juazeirensis costa & felix , 2007 , t. sherlocki papa , jurberg , carcavallo , cerqueira & barata , 2002 , and t. melanica costa et al . these taxa exhibit wide phenotypic and morphological variability , displaying specific ecological requirements and chromatic patterns . in this respect , the systematic of triatominae species is based on morphological characters of the adult exoskeleton and male phallic structures . however , insects captured during vector monitoring and control or received for identification and notification are often immature . although their characteristics are similar to those of adult individuals , they are difficult to distinguish . the morphology of triatominae species is not well described ; with studies on the immature forms performed for only 40 species , eggs and nymphs described for only 20 species , a key to identify nymphs to the species level has yet to be developed . available keys are useful and partially applicable to other stages , but specific identification of all live forms remains unresolved . members of the t. brasiliensis complex have been distinguished by analyzing isoenzymes , mitochondrial dna sequences , and random amplification of polymorphic dna - rapd . in the present study we analyzed the barcoding co1 sequences of nine t. brasiliensis s.l . populations from northeastern brazil ( states of pernambuco , paraba , and rio grande do norte ) in order to identify their genetic relationships . we also conducted a pcr - rflp assay to distinguish between t. b. macromelasoma and t. b. brasiliensis subspecies . triatominae were collected by surveillance technicians during active inspections in anthropogenic environments ( domestic and peridomestic habitats ) . live specimens were collected in nine localities in northeast brazil ( table 1 , figure 1 ) using tweezers , flashlights , and pirisa . housed in plastic boxes ( 7 cm diameter 8 cm high ) lined with folded filter paper , the bugs were transported in coolers to the culicid and triatomine laboratory of the department of epidemiology , faculty of public health / usp . adults were identified as t. b. brasiliensis according to the key by lent and wygodzinsky . nymphs were assumed to be t. brasiliensis s.l . because , in addition to the difficulty in distinguishing immature triatominae based on chromatic and morphological characters , comparative material or a key for nymph identification to species level are not available . after morphological identification , dna was extracted from individuals from the nine localities and sequenced the 520 bp barcode portion of the co1 gene ( table 1 ) . specimens collected in pernambuco state ( even nymphs ) were assumed to be t. b. macromelasoma , according to costa et al . and costa et al . . genomic dna was extracted from the legs of 10 individual samples of each population using the qiagen dneasy blood and tissue kit ( qiagen , crawley , united kingdom ) following the manufacturer 's protocol . the co1 barcode region was amplified from whole genomic dna using primers lcoi 1490 ( 5-ggt caa caa atc ata aag ata ttg g-3 ) and hcoi 2198 ( 5-taa act tca ggg tga cca aaa aat-3 ) . pcr amplification was carried out in a final volume of 50 l containing pcr buffer , 0.2 mm of each dntp , 2.5 mm mgcl2 , and 1.25 units of taq polymerase . initial pcr denaturation was at 94c for 5 min , followed by 40 cycles of denaturation ( 1 min ) at 94c , annealing ( 2 min ) at 50c , and extension ( 2 min ) at 72c . amplicons were sequenced in both forward and reverse directions using abi prism bigdye terminator cycle sequencing ready reaction kits ( perkin elmer , foster city , ca ) on an abi prism 3100 genetic analyzer / hitachi . one or two representative haplotypes for each population was chosen because of the low variation within populations . phylogenetic reconstructions were performed by neighbor joining and maximum likelihood methods ( both using the kimura-3-parameter distance model k81 ) in mega 5.0 , and a divergence matrix was constructed under kimura two parameters ( k2p ) ( table 2 ) . phylogenetic analyses included 1000 bootstrap replicates and a triatoma sordida co1 sequence ( genbank acc . co1 sequences were also analyzed using nebcutter version 2.0 to select appropriate endonuclease enzymes . individual co1 sequences were pcr - amplified using the above parameters and digested in a 10 l reaction with styi ( promega ) and hincii ( new england biolabs , ipswich , ma ) enzymes . the reaction contained 1 l of 10x buffer , 4 l deionized water , 4 l of amplification product , and 1 unit of restriction enzyme . the digestion mixture was incubated at 37c for 2 h and then resolved on 2.0% agarose gel . phylogenetic trees derived from the neighbor joining , maximum likelihood , and parsimony methods showed similar topologies ( data shown for ml tree in figure 2 ) . since most of co1 sequences were identical into and among the populations , the ml tree was constructed using only two samples of each one . this ml tree and the nucleotide distance matrix ( table 2 ) indicated sequence divergence of up to 4% between the two main clades . the basal clade , with about 4% divergence from the other populations , consisted of the salgueiro population ( 15a / b ) . this population was considered to be t. b. macromelasoma due to its high sequence divergence compared to the other populations and because it was collected in its type locality . the second clade , with pairwise distances up to 1% , showed that pernambuco populations are more basal , although those from serra talhada clustered with paraba populations likely because of the city 's proximity to the pernambuco - paraba border . paraba populations formed a large cluster that also included the rio grande do norte population . based on sequence analysis , a pcr - rflp assay was performed to differentiate between the subspecies t. b. macromelasoma and t. b. brasiliensis . pcr fragment digestion using the styi enzyme produced two restriction fragments ( 342 bp and 192 bp ) in co1 sequences from salgueiro samples and only one fragment in samples of the other eight populations . conversely , the hincii enzyme yielded two fragments ( 297 bp and 240 bp ) in all population samples ( n = 10 samples of each population ) except that from salgueiro ( figure 3 ) . styi and hincii enzymes therefore produced distinct fingerprints for t. b. macromelasoma and t. b. brasiliensis , suggesting that they are different subspecies . the molecular protocols described above are a useful tool in the study of populations and cryptic species , contributing to the identification of insect vectors . the identification of adult triatominae based on morphological and chromatic pattern is considered relatively easy for most species ; however , this is commonly misguided owing to the wide phenotypic variability within this subfamily . for instance , triatoma maculata erichson , 1848 and triatoma pseudomaculata corra & espnola , 1964 which were first treated as members of a same species complex due to morphological similarities thereafter proved to be genetically distant [ 1719 ] . panstrongylus herreri wygodzinsky , 1948 and panstrongylus lignarius walker , 1873 in turn , were considered to be separate species until marcilla et al . and crossa et al . demonstrated that they are the same species , cytogenetically identical with regard to the second internal transcribed spacer . ( light brown tones ) and rhodnius nasutus stl , 1859 ( pinkish tones ) seems to be associated with the color of the palm trees they colonize , but others such as triatoma rubrovaria blanchard , 1843 , exhibit well - known 4 chromatic morphotypes . other studies report the occurrence of natural homoploid hybrids between t. infestans and triatoma platensis neiva , 1913 , t. infestans and triatoma rubrovaria , and sympatric species of phyllosoma complex and species of the t. brasiliensis complex . this interspecific crossing can be decisive in originating and diversifying wild species , resulting in important epidemiological consequences due to differential competence and the capacity of hybrid vectors [ 3 , 10 , 11 ] . therefore , the characterization ( or identification ) of triatominae specimens based only on morphological and chromatic patterns , the most common identification method , is more complex than previously believed . studies on immature stages are crucial for group systematics . however , literature reports on immature forms of certain groups are scarce , difficult to use , or nonexistent . many species undergo changes in color , structure , and morphology during their development , hindering their identification [ 25 , 26 ] . , molecular analyses are successfully used to characterize morphotypes of species complexes such as t. brasiliensis sp . , which exhibits wide chromatic and morphological variation [ 7 , 8 ] . marked differences in color pattern and ecological features among species from the triatoma brasiliensis complex were detected by microsatellites , mitochondrial 12s , and cytochrome b genes , reinforcing species diagnosis [ 7 , 16 ] . however , individuals from subspecies t. b. brasiliensis and t. b. macromelasoma might be clustered within the same co1 clade , since earlier studies have shown that some cytochrome b haplotypes of t. b. macromelasoma are similar to those of t. b. brasiliensis . moreover , the basal clade of the maximum likelihood tree ( figure 1 ) was identified as subspecies t. b. macromelasoma because it is highly divergent ( 6% , as shown in the nucleotide distance matrix , table 2 ) from the other populations and was collected in its type locality . on the other hand , however , interpopulation divergence values ( < 1% ) suggest that t. b. brasiliensis is still diversifying and/or exhibiting ongoing gene flow , probably due to human - assisted dispersal . based on wing morphometry , costa et al . recently formulated a hypothesis that t. b. macromelasoma is the result of homoploidal hybridization between t. b. brasiliensis and t. juazeirensis in the state of pernambuco , and that this is a form of speciation in sympatric populations . in northeastern brazil , the epidemiological importance of triatominae bugs is mainly defined by their high rate of natural t. cruzi infection and ability to adapt to multiple ecotopes . control measures therefore require a precise identification of which species of the t. brasiliensis complex is being targeted . moreover , it is important to understand the ecoepidemiology of triatominae since these vectors are found in large numbers in their natural habitat . in this respect , the pcr - rflp protocol described here is suggested as rapid , relatively simple , and economical assay to distinguish triatoma b. macromelasoma from triatoma b. brasiliensis subspecies . even at small geographic scales , domestic populations are genetically structured by ecological parameters , thereby exhibiting small differences from the wild counterparts from which they are derived . the present study highlights the effectiveness of the co1 gene in identifying subspecies of the t. brasiliensis complex and its contribution to classic taxonomy .

triatoma brasiliensis sensu lato ( s.l . ) , the main vector of chagas disease in northeastern brazil , is a species complex comprising four species , one with two subspecies ( t. brasiliensis brasiliensis , t. brasiliensis macromelasoma , t. juazeirensis , t. sherlocki , and t. melanica ) , and each taxon displaying distinct ecological requirements . in order to evaluate the genetic relationships among nine t. brasiliensis s.l . populations from northeastern brazil , we analyzed their mitochondrial cytochrome c oxidase subunit 1 sequences and suggested a pcr - rflp assay to distinguish between t. b. macromelasoma and t. b. brasiliensis subspecies . all the specimens were morphologically identified as t. b. brasiliensis . the resulting phylogenies identified two major clades that are congruent with the geographical populations studied . based on collection sites and in accordance with type - location , one clade was identified as the subspecies t. b. macromelasoma . the second clade grouped t. b. brasiliensis populations . restriction endonuclease sites were observed in the sequences and used in pcr - rflp assays , producing distinct fingerprints for t. b. macromelasoma and t. b. brasiliensis populations . the results suggest that these are different species and that gene flow occurs only among t. b. brasiliensis populations , possibly associated with human activity in the area .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

ablation of the cavotricuspid isthmus ( cti ) for the treatment of atrial flutter ( afl ) has become standard practice . most of the procedures are performed using radiofrequency energy ( rf ) [ 15 ] . high chronic success rates are described but the majority of the data comes from a relatively short term follow - up [ 113 ] . a recent study by chinitz et al . reported some interesting data regarding the long term follow - up of 80 patients with common type afl who underwent cti ablation using rf . they found a 12.5% ( ten patients ) recurrence rate at an average of 21 months after the procedure with most patients having a recurrence after the first year post ablation . our prior experience using cryothermy in a limited number of patients also showed that afl may recur 1 year after cti cryoablation . the purpose of this study was to evaluate the long term outcome of cti cryoablation in a large patient population with common type afl in a single center . one hundred and eighty patients with sustained symptomatic common type afl referred for cti ablation were enrolled prospectively from july 2001 to july 2006 in our institution . signed written consent , approved by the local ethics committee , was obtained from all participants . before cti cryoablation , anticoagulation with warfarin aiming a therapeutic international normalized ratio of 2 to 3 was kept for at least 3 weeks . we focused our study on the clinical aspects and long term follow - up of patients with afl who were submitted to cti cryoablation . briefly , our methodology was as follows : under local anesthesia and via the femoral route , a decapolar catheter is positioned in the distal coronary sinus ( for evaluation of left atrial activation ) , a duodecapolar catheter ( 2-mm interelectrode spacing , halo catheter , biosense webster , baldwin park , ca ) for mapping the right atrial lateral wall and a quadripolar catheter in the his bundle area . a deflectable , 10.5 f , 6.5 mm tip cryoablation catheter ( cryocor inc . , san diego , ca ) is inserted into the right atrium through a 12f , 65-cm - long sheath ( daig , st . entrainment to confirm the isthmus dependence of the afl circuit was performed in every patient in whom afl was present or could be induced at the start of the procedure , according to previously published techniques [ 2 , 10 , 15 , 16 , 20 , 21 ] . if af , requiring cardioversion , was present or developed during stimulation or if we were unable , even under isoproterenol infusion , to induce afl , cti ablation was performed during sinus rhythm . linear lesions were created by use of a point - by - point technique with gradual pullback of the cryocatheter in a ventricular atrial fashion . the first application was delivered at the ventricular insertion of the isthmus and applications were continued for an average of 3 min . in patients in whom ablation of the posterior isthmus proved insufficient , an attempt was made to ablate the septal isthmus ( four patients ) . after documentation of bidirectional isthmus conduction block , the atrial pacing ( from the proximal coronary sinus ) protocol ( up to three atrial extrastimuli at three pacing cycle lengths and incremental atrial pacing ) was performed without and with isoproterenol infusion ( 1 to 3 g / min ) . acute success was defined as bidirectional isthmus conduction block , 30 min after the last application without and with isoproterenol infusion . those presenting in af while on the catheterization table were converted to sinus rhythm by internal or external cardioversion . during the procedure intravenous heparin was given as a 100-iu / kg bolus dose after the venous sheaths were inserted . the 12-lead ecg and intracardiac electrograms were recorded and stored by the bard labsystem pro . all patients were monitored in hospital for 24 h and oral anticoagulation was started the day of ablation . antiarrhythmic drugs ( aad ) were stopped after the procedure in patients without a history of af ; in those with af / afl the same aad were continued . subsequently , the need for chronic anticoagulation was assessed by the amount of recurrences of afl / af and the presence of risk factors for thromboembolic events . all patients had a 12-lead ecg and a 24 h holter recording at discharge and during each clinic visit ( 1 month , 3 , 6 , 9 , 12 months and yearly thereafter ) or earlier if they had symptoms . due to the logistics of the maastricht area and also the presence of a dedicated research nurse ( s. p. ) who was available to address patients concerns and questions at any time we were able to follow every patient on an individual basis . continuous variables are presented as mean sd , where appropriate . in cases of a non - gaussian distribution , categorical variables are expressed as numbers and percentages of patients . statistical analysis was performed using the student t test for unpaired data . we focused our study on the clinical aspects and long term follow - up of patients with afl who were submitted to cti cryoablation . briefly , our methodology was as follows : under local anesthesia and via the femoral route , a decapolar catheter is positioned in the distal coronary sinus ( for evaluation of left atrial activation ) , a duodecapolar catheter ( 2-mm interelectrode spacing , halo catheter , biosense webster , baldwin park , ca ) for mapping the right atrial lateral wall and a quadripolar catheter in the his bundle area . a deflectable , 10.5 f , 6.5 mm tip cryoablation catheter ( cryocor inc . , san diego , ca ) is inserted into the right atrium through a 12f , 65-cm - long sheath ( daig , st . entrainment to confirm the isthmus dependence of the afl circuit was performed in every patient in whom afl was present or could be induced at the start of the procedure , according to previously published techniques [ 2 , 10 , 15 , 16 , 20 , 21 ] . if af , requiring cardioversion , was present or developed during stimulation or if we were unable , even under isoproterenol infusion , to induce afl , cti ablation was performed during sinus rhythm . linear lesions were created by use of a point - by - point technique with gradual pullback of the cryocatheter in a ventricular atrial fashion . the first application was delivered at the ventricular insertion of the isthmus and applications were continued for an average of 3 min . in patients in whom ablation of the posterior isthmus proved insufficient , an attempt was made to ablate the septal isthmus ( four patients ) . after documentation of bidirectional isthmus conduction block , the atrial pacing ( from the proximal coronary sinus ) protocol ( up to three atrial extrastimuli at three pacing cycle lengths and incremental atrial pacing ) was performed without and with isoproterenol infusion ( 1 to 3 g / min ) . acute success was defined as bidirectional isthmus conduction block , 30 min after the last application without and with isoproterenol infusion . those presenting in af while on the catheterization table were converted to sinus rhythm by internal or external cardioversion . during the procedure intravenous heparin was given as a 100-iu / kg bolus dose after the venous sheaths were inserted . the 12-lead ecg and intracardiac electrograms were recorded and stored by the bard labsystem pro . all patients were monitored in hospital for 24 h and oral anticoagulation was started the day of ablation . antiarrhythmic drugs ( aad ) were stopped after the procedure in patients without a history of af ; in those with af / afl the same aad were continued . subsequently , the need for chronic anticoagulation was assessed by the amount of recurrences of afl / af and the presence of risk factors for thromboembolic events . all patients had a 12-lead ecg and a 24 h holter recording at discharge and during each clinic visit ( 1 month , 3 , 6 , 9 , 12 months and yearly thereafter ) or earlier if they had symptoms . due to the logistics of the maastricht area and also the presence of a dedicated research nurse ( s. p. ) who was available to address patients concerns and questions at any time we were able to follow every patient on an individual basis . continuous variables are presented as mean sd , where appropriate . in cases of a non - gaussian distribution , of the 180 enrolled patients , 39 patients ( 22% ) were women with a mean age of 58 ( from 18 to 80 ) years . more than half of the patients ( 92 patients , 52% ) had structural heart disease : arterial hypertension : 57 patients , coronary artery disease : 22 patients , valvular heart disease : 13 patients , congenital heart disease : 11 patients , idiopathic cardiomyopathy : 18 patients . counterclockwise afl was documented in 91% ( 164 ) of the patients and clockwise afl in 9% ( 16 patients ) . the mean left atrial diameter and the mean left ventricular ejection fraction were 44 7 mm and 57 7% , respectively . a prior history of af was present in 123 ( 69% ) of the patients . the clinical characteristics of the patients , related to the presence or absence of af before ablation , are described in table 1 . note that af during follow - up is significantly higher in the group with a prior history of this arrhythmia . table 1characteristics of the 180 patients with atrial flutter referred for cti cryoablation related to the presence or absence of atrial fibrillation af before ablation af / afl patients ( 123 patients ) , 69%afl only ( 57 patients ) , 31%p valueage ( year)57 1358 13nswomen19% ( 23 patients)28% ( 16 patients)nsno shd55% ( 68 patients)32% ( 18 patients ) < 0.05lad ( cm)4.44.5nslvef ( % ) 5855nsacute failures5% ( 6 patients)5% ( 3 patients)nsaf in long term follow up69% ( 85 patients)35% ( 20 patients ) < 0.05patients in whom cti cryoablation did not result in bidirectional block ( failed procedure).af atrial fibrillation , afl atrial flutter , cti cavotricuspid isthmus , lad left atrium diameter , lvef left ventricular ejection fraction , shd structural heart disease characteristics of the 180 patients with atrial flutter referred for cti cryoablation related to the presence or absence of atrial fibrillation af before ablation patients in whom cti cryoablation did not result in bidirectional block ( failed procedure ) . af atrial fibrillation , afl atrial flutter , cti cavotricuspid isthmus , lad left atrium diameter , lvef left ventricular ejection fraction , shd structural heart disease total fluoroscopic mean of 30 18 min ( range , 12 to 152 min)and procedure times mean of 2.6 1.1 h ( range , 1 to 6.5 h)decreased throughout our study with a long duration of a procedure and fluoroscopy being attributed mainly to the learning curve of a new technology . an average of 7 ( 3 to 20 ) applications per patient were delivered with a mean temperature and duration of 88c and 3 min , respectively . the acute success rate for cryoablation of the cti was 95% ( 171 patients ) . of the nine patients in whom bidirectional cti block was not achieved , three underwent a successful reablation . the other six patients had much improvement of their symptoms ( despite an incomplete line ) and preferred not to have another procedure . after a mean follow - up of 27 17 ( range from 12 to 60 ) months , recurrences of symptomatic afl occurred in 15 patients ( 9% ) resulting in a 91% chronic success rate . those recurrences occurred in six patients within the first 3 months post ablation , in four patients from 3 to 6 months post ablation , in four patients from 6 to 9 months post ablation and in one patient at 14 months post ablation ( fig . 1 ) . the other five had not only reablation of the isthmus but also pulmonary vein cryoisolation ( pvi ) for af during the second procedure . 1percentage of patients ( 171 successfully ablated ) free of common type atrial flutter over time percentage of patients ( 171 successfully ablated ) free of common type atrial flutter over time despite the success as far as afl was concerned , af was still present in 85 patients ( 69% ) with a prior history of this arrhythmia . those patients were treated by aad ( 69 patients , 81% ) , pvi ( 14 patients , 16% ) or av nodal ablation with pacemaker implantation ( two patients , 3% ) . new episodes of af developed in 20 ( 35% ) of those 57 patients without documented af prior to cti cryoablation and were all controlled by aad . our current study showed a 91% chronic success rate of cti cryoablation in a large population ( 180 patients ) with afl followed for a long period of time ( 1 to 5 years , mean of 27 months ) . according to the most recent acc / aha / esc guidelines for the management of supraventricular arrhythmias cti ablation is the only therapy with a class i indication for the long term management of afl . the majority of those ablations are done using rf energy . concerning the follow - up length of those procedures , most of the literature available reports on a relatively short period , the majority of them being less than 2 years . however chinitz et al . published a study of 80 patients with afl submitted to cti ablation using rf that had up to 6 years follow - up . interestingly , they found a 12.5% recurrence of afl that occurred on an average of 21 months post ablation , ratifying the need of data with a longer follow - up . it is important to keep in mind though , that arrhythmias after ablation do not always correlate with symptoms [ 23 , 24 ] and one could question if those patients with such a late recurrence had those episodes much earlier than what is reported . that might be one reason why our results , where most of our recurrences happened within 1 year , are discrepant with those from their study . the intrinsic characteristics of our hospital , our clinical follow - up and the population of maastricht could be responsible for our ability to find those recurrences earlier . regarding cryothermy as energy source , a 9 months follow - up study in 39 patients undergoing cti cryoablation with a different system was reported . they achieved a chronic success rate of 100% in the cryoablated group , despite documenting reconduction through the isthmus in 31% of patients during a 3 month follow - up electrophysiologic study . the craaft trial presented the results in 32 patients with afl submitted to either cryo- or rf ablation of the cti . they report an 84% success rate after a follow - up of 14 months . those two small studies , with a relatively short follow - up , reported similar success rates with rf and cryothermy for afl ablation . the results from our study which included a significant larger patient population ( 180 ) with a longer follow - up ( 1 to 5 years , mean of 27 months)reinforces the effectiveness of cryothermy for the treatment of afl , being the outcomes comparable to those reported in the literature using rf ( where most outcome data also comes from non invasive follow - up ) [ 1 , 47 , 11 , 12 , 27 , 28 ] . therefore , cryothermy can be considered as an efficient energy source for the treatment of common type afl . the close relation between af and afl our data showed a high prevalence of af ( 123 out of 180 patients , 69% ) in patients with predominant afl referred for cti ablation . a recent study by ellis and colleagues strengthened even more this association . they followed 363 patients with lone afl who underwent cti rf ablation during a mean follow - up of 39 months and reported an 82% incidence of drug refractory af in their patient population . the new development of af in our patient population without a prior history of af preceding afl ablation may be a sign of an already present electrical and morphological change in the right and left atria . if in addition , a functional or anatomical line of block between the two venae cavae ( or elsewhere ) occurs , atypical afl(s ) may develop either in the right ( because the cti is already ablated ) or in the left atrium [ 11 , 14 , 21 , 31 , 32 , 3739 ] . our recurrence data rely mostly on the subjective assessment by the patient , like the great majority of rf data [ 1 , 4 , 68 , 12 ] . only an objective measurement ( such as a repeat electrophysiological study with documented bidirectional block ) will determine the long lasting effect of cti ablation for the treatment of common type afl . our current study showed a 91% chronic success rate of cti cryoablation in a large population ( 180 patients ) with afl followed for a long period of time ( 1 to 5 years , mean of 27 months ) . according to the most recent acc / aha / esc guidelines for the management of supraventricular arrhythmias cti ablation is the only therapy with a class i indication for the long term management of afl . the majority of those ablations are done using rf energy . concerning the follow - up length of those procedures , most of the literature available reports on a relatively short period , the majority of them being less than 2 years . however chinitz et al . published a study of 80 patients with afl submitted to cti ablation using rf that had up to 6 years follow - up . interestingly , they found a 12.5% recurrence of afl that occurred on an average of 21 months post ablation , ratifying the need of data with a longer follow - up . it is important to keep in mind though , that arrhythmias after ablation do not always correlate with symptoms [ 23 , 24 ] and one could question if those patients with such a late recurrence had those episodes much earlier than what is reported . that might be one reason why our results , where most of our recurrences happened within 1 year , are discrepant with those from their study . the intrinsic characteristics of our hospital , our clinical follow - up and the population of maastricht could be responsible for our ability to find those recurrences earlier . regarding cryothermy as energy source , a 9 months follow - up study in 39 patients undergoing cti cryoablation with a different system was reported . they achieved a chronic success rate of 100% in the cryoablated group , despite documenting reconduction through the isthmus in 31% of patients during a 3 month follow - up electrophysiologic study . the craaft trial presented the results in 32 patients with afl submitted to either cryo- or rf ablation of the cti . they report an 84% success rate after a follow - up of 14 months . those two small studies , with a relatively short follow - up , reported similar success rates with rf and cryothermy for afl ablation . the results from our study which included a significant larger patient population ( 180 ) with a longer follow - up ( 1 to 5 years , mean of 27 months)reinforces the effectiveness of cryothermy for the treatment of afl , being the outcomes comparable to those reported in the literature using rf ( where most outcome data also comes from non invasive follow - up ) [ 1 , 47 , 11 , 12 , 27 , 28 ] . therefore , cryothermy can be considered as an efficient energy source for the treatment of common type afl . the close relation between af and afl is well described [ 1 , 514 , 21 , 2939 ] . our data showed a high prevalence of af ( 123 out of 180 patients , 69% ) in patients with predominant afl referred for cti ablation . a recent study by ellis and colleagues strengthened even more this association . they followed 363 patients with lone afl who underwent cti rf ablation during a mean follow - up of 39 months and reported an 82% incidence of drug refractory af in their patient population . the new development of af in our patient population without a prior history of af preceding afl ablation may be a sign of an already present electrical and morphological change in the right and left atria . if in addition , a functional or anatomical line of block between the two venae cavae ( or elsewhere ) occurs , atypical afl(s ) may develop either in the right ( because the cti is already ablated ) or in the left atrium [ 11 , 14 , 21 , 31 , 32 , 3739 ] . our recurrence data rely mostly on the subjective assessment by the patient , like the great majority of rf data [ 1 , 4 , 68 , 12 ] . only an objective measurement ( such as a repeat electrophysiological study with documented bidirectional block ) will determine the long lasting effect of cti ablation for the treatment of common type afl . in this prospectively studied large population of patients with common type afl , cryoablation of the cti has a 91% chronic success rate during long term follow - up ( range 1 to 5 years , mean of 27 months ) . these results are similar to those obtained with rf , validating cryothermy as an efficient alternative energy source .

objectiverecent literature has shown that common type atrial flutter ( afl ) can recur late after cavotricuspid isthmus ( cti ) catheter ablation using radiofrequency energy ( rf ) . we report the long term outcome of a large group of patients undergoing cti ablation using cryothermy for afl in a single center.methodspatients with afl referred for cti ablation were recruited prospectively from july 2001 to july 2006 . cryoablation was performed using a deflectable , 10.5 f , 6.5 mm tip catheter . cti block was reassessed 30 min after the last application during isoproterenol infusion . recurrences were evaluated by 12-lead ecg and 24 h holter recording every clinic visit ( 1/3/6/9 and 12 months after the procedure and yearly thereafter ) or if symptoms developed.resultsthe 180 enrolled patients had the following characteristics : 39 women ( 22% ) , mean age 58 years , no structural heart disease in 86 patients ( 48% ) , mean left atrium diameter 44 7 mm and mean left ventricular ejection fraction 57 7% . the average number of applications per patient was 7 ( 3 to 20 ) with a mean temperature and duration of 88c and 3 min , respectively . acute success was achieved in 95% ( 171 ) of the patients . there were no complications . after a mean follow - up of 27 17 ( from 12 to 60 ) months , the chronic success rate was 91% . the majority of the recurrences occurred within the first year post ablation . one hundred and twenty three patients had a history of atrial fibrillation ( af ) prior to cti ablation and 85 ( 69% ) of those remained having af after cryoablation . in 20 of 57 ( 35% ) patients without a history of af prior to cti ablation , af occurred during follow-up.conclusionsthis prospective study showed a 91% chronic success rate ( range 12 to 60 months ) for cryoablation of the cti in patients with common type afl and ratified the frequent association of af with afl .

Introduction Methods Electrophysiologic study and ablation Post ablation management Statistical analysis Results Discussion Main findings The relation of AF with AFL Study limitations Conclusions

an anal fistula is a chronic phase of anorectal sepsis and is characterized by chronic purulent drainage or cyclical pain associated with abscess formation , followed by intermittent spontaneous decompression [ 1 , 2 ] . the goals in the treatment of an anal fistula are to eliminate the primary fistula opening , any associated tracts , and any secondary openings without a change in continence . most anal fistulae are simple and can be treated using a fistulotomy , which has a low recurrence rate and an acceptable rate of morbidity [ 3 - 6 ] . however , the treatment of a complex anal fistula , which is defined as a fistula whose treatment poses an increased risk for a change in continence , still represent a challenge [ 7 - 9 ] . the recurrence rate for a complex anal fistula managed with a cutting seton is reported to be 0 to 8% , with minor and major incontinence being reported in 34 to 63% and 2 to 26% of patients , respectively [ 10 - 14 ] . advancement flap is still considered to be the gold standard of treatment for a complex anal fistula . successful healing of the fistula has been demonstrated in 55 to 98% of patients [ 8 , 10 , 15 - 17 ] . however , this procedure is technically demanding , and although the sphincter mechanism is not divided during advancement flap repair of the fistula , minor incontinence has been found in up to 31% patients and major incontinence in up to 12% of patients [ 10 , 18 , 19 ] . because of the risk of a change in continence with these conventional techniques , sphincter - preserving techniques for the management of complex anal fistulae have been evaluated . glue was easy to apply , but probably not ideal for fistula treatment because of its liquid consistency . a failure of the formed glue clot in a properly sealed tract , the inability to securely fix the material within the tract and the uncertainty of tissue in growth into the glue may all explain the possible causes of the poor outcomes . this article aims to review the literature and to identify the new sphincter - preserving techniques , such as the anal fistula plug , the ligation of intersphincteric fistula tract ( lift ) procedure and the cell therapy , used in the management of anal fistulae . the small intestinal submucosa is a natural biomaterial harvested from porcine small intestine and fabricated into a biomedical product of various shapes and thickness . the fact that it has been demonstrably useful as a bioprosthetic material in infected fields makes its application in fistula surgery quite reasonable . 1 ) . the idea is to bridge the defect of the fistula with a biocompatible material that would act as a scaffold for the patient 's own fibroblasts to come in and promote wound healing in the fistula tract [ 23 , 24 ] . the technique of plug deployment is as follows : the tract is explored , probed , and irrigated gently with hydrogen peroxide . then , the apex of the plug is tied to the probe from the internal opening , and the plug is dragged through to the external opening . it is cut to fit and is secured in the internal opening by using a figure - of - eight suture , incorporating it with the mucosa of the anorectum to close the internal opening ( fig . the anal fistula plug has been used in a number of cases with widely varying results ( table 1 ) [ 21 , 24 , 26 - 33 ] . in an early prospective series of 46 patients reported by champagne et al . , after a median follow - up of 12 months ( range , 6 to 24 months ) , 17% of fistulae recurred . johnson et al . published a series comparing two prospective cohort groups of patients undergoing plug closure versus patients undergoing fibrin glue closure . they reported an 87% closure rate for the plug group versus a 40% closure rate for the glue group . others have had less favorable results with fistula recurrence rates as high as 80% [ 21 , 28 - 31 ] . on review of these publications , possible explanations for this discrepancy include differences in patient selection and technical problems . in an attempt to standardize the indications for use of the bioprosthetic anal fistula plug and techniques for its placement , first , the group stressed that the use of the fistula plug could only be recommended for use in transsphincteric anal fistulae without acute inflammation or infection . the group felt that dislodgement of the anal fistula plug was a problem related to issues in the technique of plug placement . the group also stressed the importance of the external fistula opening being patent to allow for drainage . although most fistula recurrences occur within the first 3 months after treatment , a significant number of recurrences are known to occur after this time . a minimum one - year follow - up is generally accepted as being needed to justify the results of sphincter - preserving surgery . reported that posterior fistula location , tobacco smoking , and a history of previous failure of the plug were independent predictors of plug failure . the main limitation of application of the anal fistula plug in the treatment of anal fistulae in korea is the high cost . in korea , the cost of an anal fistula plug is not reimbursed by the national insurance system . therefore , the cost of using a plug must be paid by the patients themselves . it is interesting to note that the anal fistula plug procedure would probably be suitable for a low transsphincteric fistula . in a study from the university of minnesota , however , it was 65% for fistulae that involved less than one - third of the sphincter and 22% for those involving two - thirds or more . recent reports from chulalongkorn university , bangkok , thailand , have described a novel technique called lift for the treatment of anal fistulae . a success rate of 94.4% was reported in the treatment of 18 patients [ 34 , 35 ] . this technique prevents the entry of fecal material into the fistula tract and eliminates the formation of a septic nidus in the intersphincteric space to allow healing of the anal fistula . the location of internal opening is identified by injection of hydrogen peroxide or water through the external opening or by gently probing the fistula tract . a 1.5 to 2.0 cm curvilinear incision is made at the intersphincteric groove overlying the fistula tract . the dissection is kept close to the external sphincter to avoid cutting through the internal sphincter and breaching the anal mucosa . after the intersphincteric tract has been identified and dissected out , the tract is ligated close to the internal sphincter . . the tract next to the suture site is divided , and the rest of intersphincteric tract is excised . after removal of the correct fistulous tract has been confirmed , infected granulation tissues in the rest of the tract and cavity are thoroughly removed with curettage . the lift procedure has been used in five case series with promising early results ( table 2 ) [ 34 , 36 - 39 ] . bleier et al . reported that successful fistula closure was achieved in 57% of the 35 patients . the advantages of the lift procedure may include preservation of the anal sphincter , minimal tissue injury , shorter healing time , and its being a procedure that is relatively easy to perform . additionally , even if the fistula is not healed successfully , the lift procedure may convert a difficult - to - treat transsphincteric fistula into an easier - to - manage intersphincteric fistula . first , the published results are the only case series of a heterogeneous population , and the good results may come from selection bias . second , there were some variations in the manner in which the fistula was ligated and in the coring out or curettage of the external tract . third , a longer and objective follow - up would be a better indicator of the durability , and no standardized questionnaires were used pre- or postoperatively with respect to fecal incontinence and quality of life . additionally , as is the anal fistula plug , the indication for the lift procedure seems to be limited to the transsphincteric fistula . interestingly , another intersphincteric approach for the treatment of a complex anal fistula has been described by matos et al . . first , the ligation of the fistula tract is more secure than over sewing , and second , removal of infected granulation tissue by curettage is more practical and less time - consuming than total excision of the tract and primary repair . apart from the difficulty in the lift procedure for a high transsphincteric or a suprasphincteric fistula , the identification of the intersphincteric tract through the intersphincteric approach seems to require more advanced technical skills than the removal of the intersphincteric tract and infected granulation tissue in a conventional technique does . neal ellis published outcomes with the biolift technique for the management of transsphincteric fistulae . the biolift technique is a variation of the lift technique in which a bioprosthetic is placed in the intersphincteric plane to reinforce the closure of the fistula tract . the bioprosthetic graft acts as a physical barrier in the intersphincteric space . actually , the biolift technique utilized a transection of the intersphincteric tract and closure of the fistula opening in the internal sphincter , which is similar to the procedure described by matos et al . , instead of ligating the intersphincteric tract . when compared to the lift , the biolift technique has two potential disadvantages . first , it requires a more extensive dissection in the intersphincteric space because the bioprosthetic must overlap the closure of the fistula tract by at least 1 to 2 cm in all directions . mesenchymal adult stem cells extracted from certain tissues , such as adipose tissue , can differentiate into various types of cell . [ 43 , 44 ] chose adipose tissue as the source of stem cells because of their two biologic properties : their ability to suppress inflammation and their differentiation potential . moreover , enough fat can be obtained with liposuction , and the fat cells can be harvested with minimal adverse effects on the patient . their phase i clinical trials show that adipose - derived stem cells are safe for the treatment of a fistula in crohn 's disease , and they published a phase ii multicenter , randomized controlled trial to further investigate the effectiveness and safety of ascs in the treatment of complex anal fistulae compared with the fibrin glue group . the treatment procedures are as follows : 1 ) tract identification , with special emphasis on the identification of the internal opening ; 2 ) tract curettage , with special emphasis on the intersphincteric tract ; 3 ) closure of the internal opening ; 4 ) injection of a cell suspension through a long fine needle into the tract walls ; 5 ) sealing of the tract with fibrin glue . they reported that fistula healing was observed in 71% of 24 patients who received ascs in addition of fibrin glue compared with 16% of 25 patients who received fibrin glue alone ( relative risk for healing , 4.43 ; confidence interval , 1.74 to 11.27 ; p < 0.001 ) . the proportion of patients with healing was similar between the crohn 's and the non - crohn 's subgroups . an advantage of using stem cells to treat an anal fistula is that , because tract resection is not required , the treatment does not injure the anal sphincter ( fig . 4 ) . there are , nonetheless , some limitations of the ascs - based therapy . first , the cost of treatment with ascs is difficult to evaluate in detail at present . second , technically , secure closure of the internal opening and complete injection of the cell suspension over the tract may be complicated procedures . finally , as yet , there are no reports on the long - term effects of using ascs . in the meantime , a pilot study on autologous fibroblast - based therapy for the treatment of anal fistulae is in progress in the united kingdom . in brief , the authors suggest that the addition of autologous fibroblasts to the filling material ( collage paste ) provides the best histologic healing . to the colorectal surgeon , the complex anal fistula remains a challenging condition to manage despite the best of technologic advances . therefore , the colorectal surgeon should become familiar with various new techniques for treating an anal fistula and remember that conventional fistula surgery , such as cutting seton and advancement flap , has an important role . especially , the advancement flap is still the gold standard of treatment for complex anal fistulae . definitive evidence of the advantage of the new sphincter - preserving techniques compared with the traditional intervention requires randomized control studies , which are now being conducted .

surgery for an anal fistula may result in recurrence or impairment of continence . the ideal treatment for an anal fistula should be associated with low recurrence rates , minimal incontinence and good quality of life . because of the risk of a change in continence with conventional techniques , sphincter - preserving techniques for the management complex anal fistulae have been evaluated . first , the anal fistula plug is made of lyophilized porcine intestinal submucosa . the anal fistula plug is expected to provide a collagen scaffold to promote tissue in growth and fistula healing . another addition to the sphincter - preserving options is the ligation of intersphincteric fistula tract procedure . this technique is based on the concept of secure closure of the internal opening and concomitant removal of infected cryptoglandular tissue in the intersphincteric plane . recently , cell therapy for an anal fistula has been described . adipose - derived stem cells have two biologic properties , namely , ability to suppress inflammation and differentiation potential . these properties are useful for the regeneration or the repair of damaged tissues . this article discusses the rationales for , the estimated efficacies of , and the limitations of new sphincter - preserving techniques for the treatment of anal fistulae .

INTRODUCTION THE ANAL FISTULA PLUG LIFT PROCEDURE BIOLIFT PROCEDURE EXPANDED ADIPOSE-DERIVED STEM CELLS (ASCs) CONCLUSION

a growing body of evidence demonstrates that susceptibility and progression of both acute and chronic central nervous system ( cns ) disease is closely associated with an innate immune response that can manifest from either direct infection and/or infection - triggered damage . a common feature of these diseases is the systemic activation of inflammatory mediators , which via the blood can disrupt the blood - brain barrier , affect the circumventricular organs in the brain ( which lack a blood - brain barrier ) , or interact with the brain endothelium , thereby eliciting brain inflammation . furthermore , the presence of activated inflammatory cells derived from systemic circulation or from dormant brain resident populations is a key feature of many cns diseases . more recently , the importance of innate immune receptors in cns injury , the so - called toll - like receptors ( tlrs ) , has also been emphasized . in this paper we will focus on how neonatal sepsis and tlr - mediated inflammation increase the vulnerability of the newborn brain . infants with sepsis have an increased incidence of cerebral palsy and white matter abnormalities [ 511 ] . in a large study of 6093 extremely low birth weight ( < 1000 g ) infants , those who were infected ( including early - onset sepsis , suspected sepsis ( culture negative ) , and had necrotizing enterocolitis ( nec ) ) were more likely to have cerebral palsy than children who did not have a neonatal infection . in another recent large sample - size study involving 1155 infants born at 23 to 27 weeks gestation , it was found that children who had both late bacteremia ( positive blood culture result after the first postnatal week ) and surgical nec were at increased risk of diparetic cerebral palsy compared with children who had neither . moreover , by comparing outcomes of 150 infants with periventricular leukomalacia ( pvl ) with controls matched for gestational age , it was found that infants with bacterial sepsis were twice as likely to develop pvl , and those with meningitis were almost four times as likely to develop white matter disease . similar findings were noted in a smaller case - control study , where associations between cerebral palsy , clinical chorioamnionitis and sepsis were demonstrated . moreover , there was an increased incidence of gram - negative bacterial and fungal infections in a very low birth weight population , and these infants were at significantly increased risk for moderate to severe cerebral palsy and neurodevelopmental impairment at 18 months of age . escherichia coli is one of the main pathogens causing early - onset infections in preterm neonates , accounting for up to 40% of the cases of bacteremia among very low birth weight preterm infants ( < 1,500 g ) . cerebral white matter injury has been found by mri following escherichia coli meningitis in human newborn infants . furthermore , escherichia coli induce brain damage in a number of antenatal rabbit and rodent models [ 1926 ] . also , in a recent study , white matter injury was demonstrated in an animal model of neonatal escherichia coli sepsis in 5-day - old rat pups . experimental studies show that early - life escherichia coli exposure can also have long - term effects , influencing the vulnerability to other factors in adulthood , for example , age - related cognitive decline as well as attenuated glial and cytokine responses to amphetamine challenge . in recent years , coagulase - negative staphylococci ( cons ) have emerged as the most prevalent and important neonatal pathogens , responsible for approximately 50% of all episodes of late - onset neonatal sepsis in neonatal intensive care units around the world [ 3033 ] . cons cause significant morbidity , mortality , and healthcare costs worldwide in preterm newborns , especially in very low birth weight infants [ 3438 ] . the vulnerability of preterm infants to cons infection has been suggested to be due to the special characteristics of the premature infant 's innate immunity . although there is no direct evidence of cons causing perinatal brain injury , the presence of cons in the chorioamnion space at delivery is associated with increased risk for the development of cerebral palsy in preterm infants [ 40 , 41 ] . further , in children with an established diagnosis of cerebral palsy , who are admitted to pediatric intensive care , there is a high rate of carriage of abnormal bacteria , including cons . in very low birth weight preterm infants with early onset neonatal sepsis , the rate of group b streptococcal ( gbs ) infections is relatively low in comparison with e. coli infections . there is no direct evidence of gbs sepsis playing a role in cerebral palsy ; however , nearly half of all infants who survive an episode of gbs meningitis suffer from long - term neurodevelopmental sequelae . further , extensive cortical neuronal injury was found in gbs - infected neonatal rats , which was mediated through reactive oxygen intermediates [ 44 , 45 ] . toll - like receptors ( tlrs ) play a central role in primary recognition of infectious and viral pathogens . the presence of all 13 known tlrs has been demonstrated in the brain [ 4648 ] . tlr4 mediates cellular activation in response to lps derived from escherichia coli , while cons and gbs infections are , at least partly , believed to be mediated by tlr2 . interestingly , the role of tlrs in nonbacterial - induced brain injury has also recently been highlighted . tlrs signal through the recruitment of intracellular adaptor proteins , followed by activation of protein kinases and transcription factors that induce the production of inflammatory mediators ( figure 1 ) . the adaptor protein myd88 is used by most tlrs , except tlr3 , while the trif adaptor protein is used only by tlr3 and tlr4 . lps - induced activation of tlr4 elicits , via both myd88 and trif , a broad inflammatory response in tissues , including the immature brain . there is relatively little information regarding the expression of tlrs in the developing brain . during embryonic life , protein expression of both tlr-3 and -8 has been identified [ 53 , 54 ] , while tlr-2 expression is relatively low before birth and increases during the first two weeks of life . we have shown that mrna for tlr1 - 9 is expressed in the neonatal mouse brain . it appears that some of the tlrs may play important roles during normal brain development , as tlr2 inhibits neural progenitor cell proliferation during the embryonic period , and tlr3 deficiency increases proliferation of neural progenitor cells , while tlr8 stimulation inhibits neurite outgrowth [ 5355 ] . in support , we , and others , have shown that systemic administration of lps results in brain injury in both fetal and newborn animals [ 5860 ] . these injuries appear , both histologically and by mri analysis , to be very similar to those found in preterm infants . furthermore , it is now well established that pre - exposure to lps can increase the vulnerability of the immature brain to hypoxia - ischemia ( hi ) , in both rats [ 62 , 63 ] and mice . these effects are tlr4 and myd88 dependent [ 64 , 66 ] . in a recent study , it was also shown that a very low dose of lps , specifically increased the vulnerability of the immature white matter . low - dose lps ( 0.05 mg / kg ) sensitized hi injury in p2 rat pups by selectively reducing myelin basic protein expression and the number of oligodendrocytes while increasing neuroinflammation and blood - brain barrier damage in the white matter . rat pups subjected to lps / hi at p1 responded with weak cytokine response , while there was a prominent upregulation of cytokines in p12 pups subjected to the same insult . interestingly , il-1 was upregulated at both ages ; il-1 injections sensitize the newborn brain to excitotoxicity and repeated il-1 exposure during the neonatal period induces preterm like brain injury in mice . although it has clearly been demonstrated that lps can increase the vulnerability to hi , under certain circumstances lps can also induce tolerance to brain injury . we have shown that the time interval between lps exposure and the subsequent hi is imperative to the outcome [ 71 , 72 ] , where a 24 h interval seems to induce a tolerant state that makes the brain less vulnerable . this has been confirmed by others who have implicated several possible mechanisms , including upregulation of corticosterone , which is further supported by the fact that administration of dexamethasone prevents learning impairment following lps / hi in neonatal rats . furthermore , akt - mediated enos upregulation in neurons and vascular endothelial cells have been implicated in lps - induced preconditioning . the importance of the time interval between lps and other insults seems to be a generalized phenomenon . we have recently demonstrated in an in vitro model that conditioned medium from lps - activated microglia affects the antioxidant nrf2 system and cell survival in astrocytes in a time - dependent manner . lps - induced inflammation had dual , time - dependent , effects on the nrf2 system in that sustained activation ( 72 h ) of gsk3beta and p38 downregulated the nrf2 system , possibly via the activation of histone deacetylases , changes that were not observed with a 24 h ( tolerance ) interval [ 76 , 77 ] . these studies support our previous report demonstrating that reductions in antioxidants were more pronounced when hi was preceded by lps injection in 8-day rats 3 days prior to the hi insult . compared to tlr4 , much less as mentioned above , tlr2 , tlr3 , and tlr8 can affect normal brain development [ 5355 ] . activation of tlr2 in neonatal mice decreases volume of cerebral gray matter , white matter in the forebrain , and cerebellar molecular layer . further , we have recently demonstrated the expression of both tlr1 and tlr2 in the neonatal mouse brain following hi . in these studies , tlr2 deficiency resulted in reduced infarct volume after hi , while tlr-1-deficient mice were not protected . maternal viral immune activation is believed to increase the risk of psychiatric disorders such as schizophrenia in offspring , and in order to examine this relationship , several authors have investigated the vulnerability of the fetal brain to synthetic double - stranded rna , polyriboinosinic - polyribocytidilic acid ( poly i : c ) , a tlr3 agonist . maternal injection with poly i : c towards the end of gestation ( g15 ) causes sensorimotor gating deficits in the adult offspring in mice and increased sensitivity to the locomotor - stimulating effects of mk-801 . maternal poly i : c injection on gd9 , but not gd17 , significantly impaired sensorimotor gating and reduced prefrontal dopamine d1 receptors in adulthood , whereas prenatal immune activation in late gestation impaired working memory , potentiated the locomotor reaction to a nmda - receptor antagonist , and reduced hippocampal nmda - receptor subunit 1 expression . in particular , poly i : c injections early during rodent pregnancy affect structural brain development , such as a transient decrease of myelin basic protein in the neonatal offspring and cerebellar pathology . e. coli infections are common in preterm neonates , and considerable evidence suggests that e. coli - induced inflammation play a role in the development of white matter damage in preterm infants . there is much less data available concerning the importance of two other common neonatal pathogens , cons and gbs , in perinatal brain injury . furthermore , it is becoming clear that tlrs have important roles during development and may be involved in both pathogen - induced damage as well as so called sterile hi - induced inflammation . in order to better understand the underlying causes of perinatal brain injury , the interaction between common neonatal pathogens and tlrs in the newborn brain deserves further investigation .

a growing body of evidence demonstrates that susceptibility and progression of both acute and chronic central nervous system disease in the newborn is closely associated with an innate immune response that can manifest from either direct infection and/or infection - triggered damage . a common feature of many of these diseases is the systemic exposure of the neonate to bacterial infections that elicit brain inflammation . in recent years , the importance of innate immune receptors in newborn brain injury , the so - called toll - like receptors , has been demonstrated . in this paper we will discuss how neonatal sepsis , with particular emphasis on escherichia coli , coagulase - negative staphylococci , and group b streptococcal infections in preterm infants , and toll - like receptor - mediated inflammation can increase the vulnerability of the newborn brain to injury .

1. Introduction 2. Neonatal Sepsis and Brain Injury 3. Toll-Like Receptor-Mediated Vulnerability of the Immature Brain 4. Conclusion

assessing the degree of walking independence among inpatients is one of the important roles of physical therapists . overestimation of a patient s ability to walk can result in falls , whereas underestimation can lead to disuse syndrome due to a decrease in physical activity . preliminary research investigating cutoff values for parameters of walking independence in patients with stroke has reported the efficacy of physical performance examinations such as assessments of walking speed1 . however , preliminary studies often exclude stroke patients with cognitive disorders in order to increase the rate of identifying patients with walking independence and improve the reliability of examinations . stroke patients with cognitive disorders are often examined in the clinical setting2 , 3 , where the cutoff value for excluding stroke patients with cognitive disorders in preliminary research can not be used to evaluate those with cognitive disorders . as a result , walking independence in stroke patients with cognitive disorders is presently being assessed on the basis of subjective assessment by a physical therapist , and no clear protocol exists regarding this issue . therefore , determination of the cutoff values for parameters of walking independence in patients with stroke and cognitive disorders is necessary . but there have been few studies so far that have evaluated the reference values associated with walking independence in stroke patients with a cognitive impairment4 . the purpose of this study was to determine the threshold for classifying walking independence in stroke patients with and without cognitive disorders . the subjects included 130 patients with initial stroke hemiplegia admitted to the rehabilitation unit at koseinenkin kochi rehabilitation hospital in japan from april 2007 through december 2012 . patients were eligible for inclusion if they met the following criteria : ( 1 ) age 40 years ; ( 2 ) more than one month had passed since stroke onset ; ( 3 ) ability to complete the mini - mental state examination ( mmse ) ( total score : > 1)5 ; ( 4 ) first stroke , i.e. , no previous history of stroke ; ( 5 ) ability to walk a minimum of 10 m using mobility aids such as a cane or a brace ; and ( 6 ) a history of independent activities of daily living before the onset of stroke . we investigated basic information in addition to walking status , physical function , and performance of the patients during physical therapy based on their medical records . the survey items included gender , age , side of paralysis , functional independence measure ( fim ) for walking6 , mmse score , lower limb brunnstrom stage7 , one - leg standing time on the paralytic side , one - leg standing time on the non - paralytic side , and 10-m walking speed . for measuring the one - leg standing time , we measured the duration for which the patients were able to maintain one leg in the air without touching the floor8 , 9 . we excluded the measurements of one - leg standing time in the following cases : swaying while balancing , using the arms for balancing , hopping to maintain balance , and putting the foot down on the floor . the maximum measurement time was 60 s. the time before falling over was measured , and the maximum time of two repetitions was determined . the 10-m walking speed was used to quantify the ambulatory status in terms of a fast gait speed10 . the time from the starting line to a line marking 10 m was measured using a stopwatch . the participants performed two trials at each speed , and the calculated walking speed ( m / min ) was recorded . we conducted the statistical analysis according to the presence or absence of cognitive disorders . we classified the patients with mmse scores of 24 points into the high - score group and those with mmse scores of 23 points into the low - score group11 . the mmse measures various domains of cognitive functioning including memory , orientation to place and time , naming , reading , visuospatial orientation / construction ability , writing , and the ability to follow a 3-stage command . it is difficult to diagnose inattention and accomplishment dysfunction in patients with stroke using the mmse . therefore , patients with a deteriorated cognitive function may have been included in the high - score group . the statistical analysis was conducted to investigate the main factors associated with walking independence using a logistic regression analysis . the cutoff levels for the main factors used to distinguish walking independence were determined according to the compatibility of the regression models using the area under the curve ( auc ) of the receiver operating characteristic ( roc ) curve and the youden index12 . we calculated test diagnostics ( sensitivity , specificity , positive predictive value , negative predictive value , positive likelihood ratio , and negative likelihood ratio ) for a series of cutoff points . subjects were included after obtaining informed consent , and the study protocol was approved by the ethics committee of seirei christopher university . the high - score group included 69 subjects ( 53.1% ) , and the low - score group included 61 subjects ( 46.9% ) ( table 1table 1.characteristics of the high - score group and low - score groupvariablewalking statusdependentindependenthigh - score groupno . of patients2940age ( years)72.4 ( 11.8 ) 68.9 ( 11.3)female ( % ) 62.140.0left - sided hemiplegia ( % ) 58.652.5time after stroke onset to measurement ( m)3.5 ( 2.4)2.4 ( 2.2)brunnstrom stage ( 16)0.0.11.3.10.50.0.0.2.5.33*one - leg standing time on the paralytic side(s)0.4 ( 1.3)10.4 ( 15.4)*one - leg standing time on the non - paralytic side(s)2.3 ( 5.3)13.6 ( 18.2)*10-m walking speed ( m / min)23.9 ( 21.5)82.2 ( 47.5)*low - score groupno . of patients4021age ( years)79.4 ( 6.9)76.4 ( 7.9)female ( % ) 62.533.3*left - sided hemiplegia ( % ) 50.019.0*time after stroke onset to measurement ( m)3.1 ( 0.9)2.3 ( 0.7)*brunnstrom stage ( 16)0.0.3.6.16.150.0.0.1.1.19*one - leg standing time on the paralytic side(s)0.38 ( 0.74)3.8 ( 3.5)*one - leg standing time on the non - paralytic side(s)0.9 ( 1.7)4.8 ( 3.3)*10-m walking speed ( m / min)24.5 ( 17.6)71.7 ( 21.0)**p<0.05 the incidence of walking independence was 40 of 69 ( 58.0% ) patients with maintained cognitive function . according to the logistic regression analysis , the main factor associated with walking independence in the high - score group was the 10-m walking speed ( odds ratio , 1.076 ; 95% confidence interval , 1.0421.111 ) ( table 2table 2.results of the logistic regression analysisvariablebodds ratio95% confidence intervalhigh - score group10-m walking speed*0.0731.076 1.0421.111constant3.044identification rate87.0low - score group10-m walking speed*0.1071.1131.0451.186one - leg standing time on the non - paralytic side0.4951.6400.9692.777constant7.146identification rate88.5dependent variable : walking status ( dependent , 0 ; independent , 1 ) independent variable : 10-m walking speed and one - leg standing time on the non - paralytic side . odds ratio for a 1-second increase ) . as for the compatibility of the regression models of the roc curve , the auc was 0.921 . using a cutoff level for the 10-m walking speed of 41.4 m / min resulted in a sensitivity of 87.5% , specificity of 86.2% , positive predictive value of 90.0% , negative predictive value of 83.3% , positive likelihood ratio of 6.3 , and negative likelihood ratio of 0.2 ( fig . 1 . the auc was 0.921 in the high - score group . using a cutoff level for the 10-m walking speed of 41.4 the auc was 0.953 in the low - score group . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a sensitivity of 95.2% and a specificity of 87.5% . ) . mann - whitney test dependent variable : walking status ( dependent , 0 ; independent , 1 ) independent variable : 10-m walking speed and one - leg standing time on the non - paralytic side . * odds ratio for a 1 m / min increase . odds ratio for a 1-second increase the auc was 0.921 in the high - score group . using a cutoff level for the 10-m walking speed of 41.4 the auc was 0.953 in the low - score group . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a sensitivity of 95.2% and a specificity of 87.5% . the incidence of walking independence in the low - score group was 21 of 61 ( 34.4% ) patients with impaired cognitive function . according to the logistic regression analysis , the main factor associated with walking independence in the low - score group was the 10-m walking speed ( odds ratio , 1.113 ; 95% confidence interval , 1.0451.186 ) ( table 2 ) . with regard to the compatibility of the regression models of the roc curve , the auc was 0.953 . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a sensitivity of 95.2% , specificity of 87.5% , positive predictive value of 89.5% , negative predictive value of 80.6% , positive likelihood ratio of 7.6 , and negative likelihood ratio of 0.1 ( fig . the patients in the low - score group based on the examinations for the presence of cognitive disorders accounted for 50% of all subjects . the subjects of this study included patients with initial stroke hemiplegia who had been treated in the rehabilitation unit at koseinenkin kochi rehabilitation hospital . on the basis of preliminary research it has become clear that patients with a history of several strokes can not be evaluated using the cutoff values employed in preliminary research that excludes patients with cognitive disorders . the results of this study showed that the primary factor associated with walking independence in the high - score group of stroke patients was the 10-m walking time . preliminary studies have found that the walking speed can be used to classify patients according to the degree of walking independence in the community15 . investigated the cutoff value of walking speed for community walkers with stroke and reported a positive predictive value of 93% and a negative predictive value of 57% for a cutoff value of 0.66 m / s , with an auc of 0.8515 . it is clear from these results that there is a standard value for classifying walking independence with walking speed . this study demonstrated the 10-m walking speed to be an effective parameter for identifying walking independence in the hospital . in the present study , the main factor associated with walking independence in the low - score group of stroke patients was the 10-m walking time . therefore , the 10-m walking speed is an effective parameter for classifying stroke patients with cognitive disorders according to the degree of walking independence in the hospital . examinations using complicated instructions decrease the reliability of assessments of physical function in patients with deteriorated cognitive function . it is therefore necessary to use simple examinations in patients with cognitive function disorders . we used the 10-m maximal walking speed to examine the walking independence status in this study . cognitive impairment in patients with stroke results in significant inattention and accomplishment dysfunction16 . patients with cognitive function disorders have difficulty in maintaining attention during long examinations and performing the required movements in assessments with complicated instructions17 . measuring the 10-m walking speed is an effective laboratory procedure due to its simplicity and allows for the determination of walking independence in patients evaluated with the mmse .

[ purpose ] the aim of this study was to determine the threshold for classifying walking independence in stroke patients with and without cognitive disorders . [ subjects ] the subjects were 130 patients with initial stroke hemiplegia . [ methods ] the following factors were analyzed for associations with walking independence : brunnstrom stage , one - leg standing time on the paralytic side , one - leg standing time on the non - paralytic side , and 10-m walking speed . we classified the patients with mini - mental state examination ( mmse ) scores 24 points into the high - score group and those with mmse scores of 23 points into the low - score group and examined the main factors and cutoff values associated with walking independence in each group . [ results ] the high - score group included 69 subjects ( 53.1% ) , and the low - score group included 61 subjects ( 46.9% ) . the primary factor associated with high mmse scores among the stroke patients was the 10-m walking time . using a cutoff level for the 10-m walking speed of 41.4 m / min resulted in a positive likelihood ratio of 6.3 . the primary factor associated with low mmse scores among the stroke patients was the 10-m walking time . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a positive likelihood ratio of 7.6 . [ conclusion ] the cutoff value for the 10-m walking speed can be used to evaluate walking independence in patients with stroke among patients with high or low mmse scores .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

data were obtained through an internet survey of u.s . adults self - identified as taking insulin to treat type 1 or type 2 diabetes ; the survey was conducted 13 june to 7 july 2008 by harris interactive , a contract research organization . patients were recruited by email if they had diabetes and currently used a syringe or insulin pen to deliver insulin . institutional review board approval for the study protocol was obtained from the human subject research committee of loyola university maryland . data collected from participants included the following : 1 ) basic demographic information ; 2 ) disease type , duration , complications , and treatment ; 3 ) perceived burden of insulin injections ; 4 ) the experience of injections ; 5 ) negative affect toward insulin injections ; and 6 ) frequency of skipping insulin injections . respondents reported whether they had ever been diagnosed with type 1 or type 2 diabetes , depression , obesity , or cardiovascular disease ( high blood pressure or heart disease ) and whether they treat their diabetes with diet , exercise , and medications . interference with eating and exercise was measured as the mean of two items asking , how much does the way you inject insulin interfere with eating / exercising when you want ? ( response options : 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal ) . interference with activities of daily living was measured as a count of the affirmative responses to the question , do your insulin injections have a negative effect on : social activities , recreational activities , sexual activity , work / career , family care - giving ? another measure of interference was whether the respondent plans daily activities around insulin injections ( 1 = yes , 0 = no ) . there were five measures dissatisfaction with injection time needed , ease of use , pain , inflammation / bruising , and embarrassment each measured by a single item ( response options : 1 = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied ) . i dread insulin injections ; injecting myself with insulin is the hardest part of managing my diabetes ; i have to mentally prepare myself before each injection ( response options : 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree ) . worry about hypoglycemia was measured by a single item ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . the dependent variable in this study was the response to the question , how often do you skip insulin injections that you know you should take ? ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . multiple regression analysis was used to assess independent relationships with frequency of skipping insulin injections . control variables ( demographic and disease characteristics ) were entered first , and then injection - related experience and attitudes were entered using stepwise criteria ( p < 0.05 ) . separate analyses were performed in the type 1 and type 2 diabetic populations to see whether associations differed between populations . data collected from participants included the following : 1 ) basic demographic information ; 2 ) disease type , duration , complications , and treatment ; 3 ) perceived burden of insulin injections ; 4 ) the experience of injections ; 5 ) negative affect toward insulin injections ; and 6 ) frequency of skipping insulin injections . respondents reported whether they had ever been diagnosed with type 1 or type 2 diabetes , depression , obesity , or cardiovascular disease ( high blood pressure or heart disease ) and whether they treat their diabetes with diet , exercise , and medications . interference with eating and exercise was measured as the mean of two items asking , how much does the way you inject insulin interfere with eating / exercising when you want ? ( response options : 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal ) . interference with activities of daily living was measured as a count of the affirmative responses to the question , do your insulin injections have a negative effect on : social activities , recreational activities , sexual activity , work / career , family care - giving ? ( possible range = 05 ) . another measure of interference was whether the respondent plans daily activities around insulin injections ( 1 = yes , 0 = no ) . there were five measures dissatisfaction with injection time needed , ease of use , pain , inflammation / bruising , and embarrassment each measured by a single item ( response options : 1 = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied ) . i dread insulin injections ; injecting myself with insulin is the hardest part of managing my diabetes ; i have to mentally prepare myself before each injection ( response options : 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree ) . worry about hypoglycemia was measured by a single item ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . the dependent variable in this study was the response to the question , how often do you skip insulin injections that you know you should take ? ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . interference with eating and exercise was measured as the mean of two items asking , how much does the way you inject insulin interfere with eating / exercising when you want ? ( response options : 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal ) . interference with activities of daily living was measured as a count of the affirmative responses to the question , do your insulin injections have a negative effect on : social activities , recreational activities , sexual activity , work / career , family care - giving ? another measure of interference was whether the respondent plans daily activities around insulin injections ( 1 = yes , 0 = no ) . there were five measures dissatisfaction with injection time needed , ease of use , pain , inflammation / bruising , and embarrassment each measured by a single item ( response options : 1 = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied ) . i dread insulin injections ; injecting myself with insulin is the hardest part of managing my diabetes ; i have to mentally prepare myself before each injection ( response options : 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree ) . worry about hypoglycemia was measured by a single item ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . the dependent variable in this study was the response to the question , how often do you skip insulin injections that you know you should take ? ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . multiple regression analysis was used to assess independent relationships with frequency of skipping insulin injections . control variables ( demographic and disease characteristics ) were entered first , and then injection - related experience and attitudes were entered using stepwise criteria ( p < 0.05 ) . separate analyses were performed in the type 1 and type 2 diabetic populations to see whether associations differed between populations . the sample ( n = 502 ) was 55% male , 73% white , 11% hispanic , 11% african american , and 5% other race / ethnicity , with a mean age of 55 years ( table 1 ) . only about one - third ( 38% ) were presently employed , and those who were not employed included 8% students and 8% disabled ; the remainder were mostly retired or nonworking spouses . * 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal . = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied . 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree . 1 = never , 2 = rarely , 3 = sometimes , 4 = often . approximately one - third ( 32% ) reported having been diagnosed by a health care professional as having depression . a total of 77% of the sample said they had type 2 diabetes , and the rest said they had type 1 diabetes ; patients reported having diabetes for an average of almost 15 years . a total of 61% of the patient sample identified a primary care physician as their primary diabetes health care provider , whereas 28% named an endocrinologist and 11% named another ( nonphysician ) health care provider . of the sample , 39% reported engaging in physical activity and 55% said they followed a healthy diet . a total of 70% of patients surveyed said they took insulin using a syringe and 30% said they used a pen ; most ( 56% ) changed their needle with each injection . patients reported taking an average of 2.7 injections a day ( maximum of five recorded ) . a substantial minority of respondents ( 22% ) said they planned their daily activities around their insulin injections , and similar proportions reported that insulin injections interfered with their lives : 23% said insulin injections interfered with their eating / exercising schedule more than a little , and 25% said that insulin injections had a negative effect on one or more activity of daily living . further , a substantial minority of respondents ( 22% ) reported they had to mentally prepare themselves before each injection , and 33% identified they had some level of dread associated with taking their daily injections . attitudinal measures tended to fall below the halfway point of the response options ( i.e. , < 2.5 ) . respondents reported moderate levels of satisfaction with the pain and the inflammation and bruising associated with insulin injections ( the scores for pain and inflammation / bruising were significantly higher than those for embarrassment , time needed , and ease of use , p < 0.001 ) . a quarter ( 24% ) of respondents had a score representing negative affect toward injections ( they scored above the midpoint on the scale ) , and 21% reported often worrying about hypoglycemia . over half ( 57% ) of respondents reported skipping insulin injections they knew they should take ; 20% report skipping them sometimes or often . table 2 shows the results of the regression analysis of intentional insulin omission frequency . control variables ( demographic and diabetes characteristics ) accounted for 26% of the variance in intentional insulin omission . older respondents , those who were disabled , those with higher household income , and those who followed a healthy diet were significantly less likely to skip injections , whereas subjects who were students and had the highest education or type 2 diabetes and subjects who took more injections were significantly more likely to skip injections . multiple regression analysis of frequency of insulin injection omission * reference category = male . four measures of injection burden and experience had significant independent associations with higher levels of intentional insulin omission and accounted for an additional 10% of the variance . these included two aspects of interference ( planning one 's activities around insulin injections and injections interfering with activities of daily living ) and two aspects of the injection process itself ( pain and embarrassment ) . respondents who planned daily activities around insulin injections , those who said that taking injections interfered with activities of daily living , and those who reported injection - related pain or embarrassment intentionally skipped insulin injections more often . separate analyses using the variables listed in table 2 were performed among subjects with type 1 diabetes and subjects with type 2 diabetes ( results not shown ) . because there were more participants with type 2 diabetes , the overall model most closely resembled that for type 2 diabetes . only two variables significant in the overall model were not significant in the type 2 model being a student and following a healthy diet . only three variables were significant in the type 1 model following a healthy diet , number of daily insulin injections , and interference with activities of daily living ( being a student had a p value of 0.056 ) . the sample ( n = 502 ) was 55% male , 73% white , 11% hispanic , 11% african american , and 5% other race / ethnicity , with a mean age of 55 years ( table 1 ) . only about one - third ( 38% ) were presently employed , and those who were not employed included 8% students and 8% disabled ; the remainder were mostly retired or nonworking spouses . * 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal . = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied . 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree . 1 = never , 2 = rarely , 3 = sometimes , 4 = often . approximately one - third ( 32% ) reported having been diagnosed by a health care professional as having depression . a total of 77% of the sample said they had type 2 diabetes , and the rest said they had type 1 diabetes ; patients reported having diabetes for an average of almost 15 years . a total of 61% of the patient sample identified a primary care physician as their primary diabetes health care provider , whereas 28% named an endocrinologist and 11% named another ( nonphysician ) health care provider . of the sample , 39% reported engaging in physical activity and 55% said they followed a healthy diet . a total of 70% of patients surveyed said they took insulin using a syringe and 30% said they used a pen ; most ( 56% ) changed their needle with each injection . patients reported taking an average of 2.7 injections a day ( maximum of five recorded ) . a substantial minority of respondents ( 22% ) said they planned their daily activities around their insulin injections , and similar proportions reported that insulin injections interfered with their lives : 23% said insulin injections interfered with their eating / exercising schedule more than a little , and 25% said that insulin injections had a negative effect on one or more activity of daily living . further , a substantial minority of respondents ( 22% ) reported they had to mentally prepare themselves before each injection , and 33% identified they had some level of dread associated with taking their daily injections . attitudinal measures tended to fall below the halfway point of the response options ( i.e. , < 2.5 ) . respondents reported moderate levels of satisfaction with the pain and the inflammation and bruising associated with insulin injections ( the scores for pain and inflammation / bruising were significantly higher than those for embarrassment , time needed , and ease of use , p < 0.001 ) . a quarter ( 24% ) of respondents had a score representing negative affect toward injections ( they scored above the midpoint on the scale ) , and 21% reported often worrying about hypoglycemia . over half ( 57% ) of respondents reported skipping insulin injections they knew they should take ; 20% report skipping them sometimes or often . table 2 shows the results of the regression analysis of intentional insulin omission frequency . control variables ( demographic and diabetes characteristics ) accounted for 26% of the variance in intentional insulin omission . older respondents , those who were disabled , those with higher household income , and those who followed a healthy diet were significantly less likely to skip injections , whereas subjects who were students and had the highest education or type 2 diabetes and subjects who took more injections were significantly more likely to skip injections . multiple regression analysis of frequency of insulin injection omission * reference category = male . reference category = nonwhite . four measures of injection burden and experience had significant independent associations with higher levels of intentional insulin omission and accounted for an additional 10% of the variance . these included two aspects of interference ( planning one 's activities around insulin injections and injections interfering with activities of daily living ) and two aspects of the injection process itself ( pain and embarrassment ) . respondents who planned daily activities around insulin injections , those who said that taking injections interfered with activities of daily living , and those who reported injection - related pain or embarrassment intentionally skipped insulin injections more often . separate analyses using the variables listed in table 2 were performed among subjects with type 1 diabetes and subjects with type 2 diabetes ( results not shown ) . because there were more participants with type 2 diabetes , the overall model most closely resembled that for type 2 diabetes . only two variables significant in the overall model were not significant in the type 2 model being a student and following a healthy diet . only three variables were significant in the type 1 model following a healthy diet , number of daily insulin injections , and interference with activities of daily living ( being a student had a p value of 0.056 ) . this study suggests that intentional omission of insulin injections that should be taken occurs in the majority of adults using insulin to treat their diabetes and is common in 20% of these individuals . it also is associated with indicators of perceived burden and the experience of injections as painful and embarrassing . we found that respondents with higher household income , but not individuals with more education , were less likely to skip insulin injections they knew they should take . this may reflect easier access to medications and supplies among individuals with higher income , but it is also likely that higher socioeconomic status is associated with more access to diabetes education , higher health literacy , greater control over one 's daily routines , and better problem - solving skills ( 19 ) . our study appears to be among the first to identify an association between socioeconomic status and insulin omission . future research should seek to identify potential mediators of this relationship , i.e. , what links lower socioeconomic status to insulin omission . contrary to earlier reports ( 3,4 ) , we found no racial / ethnic differences in intentional insulin omission . this may be because we did not have enough nonwhite respondents to examine the different racial / ethnic groups separately . alternatively , this may be due to our controlling for income and education in the analysis , thereby eliminating the confounding of race / ethnicity with socioeconomic status . much prior research has suggested that intentional insulin omission is common among female adolescents with type 1 diabetes , serving as a weight control strategy and sometimes linked to eating disorders ( 10 ) . we found that students ( who were younger than nonstudents ) were more likely to skip injections they knew they should take , but this behavior was not more common among women than it was among men . we found no overall association between age and intentional insulin injection omission among patients with type 1 diabetes , suggesting that patients with type 1 diabetes age - out of this behavior by early adulthood , when they complete their education . ascertaining the validity of this interpretation would require following youth with type 1 diabetes as they age into adulthood to determine change in rate of insulin omission . our finding that , among individuals with type 2 diabetes , older respondents were less likely to skip insulin injections is consistent with earlier studies ( 3,4 ) . this suggests that there are parallel aging - out processes among individuals with type 1 and type 2 diabetes , but in type 2 diabetes , this process takes place later in the life course ( almost all people with type 2 diabetes are diagnosed as adults ) . ascertaining the validity of this interpretation would require following adults with type 2 diabetes as they age to determine change in rate of insulin omission . having type 2 diabetes was itself associated with higher levels of intentional omission of insulin injections . the beta for this variable ( 0.226 ) was approximately twice the size of the unadjusted eta ( 0.095 ) , reflecting the fact that controlling for confounding factors ( such as age and number of daily injections ) revealed a stronger underlying association . the independent association of type 2 diabetes with increased insulin omission may reflect the fact that patients with type 2 diabetes have a residual insulin response , reducing the immediate consequences of omitting an injection . thus , these individuals may feel less vulnerable to the effects of skipping insulin injections they know they should take . interestingly , whereas duration of diabetes was associated with the frequency of insulin injection omission , regression analysis revealed that duration of diabetes did not make an independent contribution to this behavior . that is , although insulin omission may be less common among individuals with longer duration of diabetes , this is likely a function of other factors such as age and type of diabetes rather than of duration per se . surprisingly , history of depression was not associated with insulin omission ; this contradicts findings from studies of general adherence ( 9,20 ) and of insulin omission among adolescents ( 10 ) . however , because current depression was not assessed , any concurrent association was likely to be lost . others have shown that depression symptom scores fluctuate substantially over relatively short periods of time ; individuals with elevated depression symptoms at a given point in time are likely to not report elevated symptoms 6 months later ( 21,22 ) . in addition , depression symptoms across the whole range of severity symptoms have been shown to predict regimen adherence more powerfully than diagnosed depression ( 23 ) . being disabled was associated with less insulin omission ; this may be due to a variety of reasons , including their receiving more assistance with care , or making a greater effort to compensate for poor health . two aspects of patients ' treatment regimens were associated with increased insulin omission respondents who took more injections each day and those who did not follow a healthy diet were more likely to skip injections . that dietary nonadherence is associated with insulin nonadherence more frequent injection omission among individuals taking more injections could reflect the frequently reported finding that more complex regimens are associated with lower levels of adherence ( 6 ) . it might also be that the impact of skipping a shot is reduced among individuals who take more shots . our study suggests that insulin omission is affected by the perceived burden of insulin therapy ( i.e. , having to plan one 's life around insulin injections and feeling that the insulin regimen interferes with activities of daily living such as social activities , work - related activities , and family care - giving responsibilities ) . we offer one caveat regarding our findings ; we do not believe that the behavior of planning one 's day around insulin injections actually increases the level of insulin injection omission , but we do believe that feeling that one has to plan around one 's injections is associated with higher frequency of skipping insulin injections one should take . that is , when there is a conflict between scheduling of treatment and life activities , one can either plan one 's activities in a way that reduces this conflict or deal with the conflict by ignoring treatment needs . reducing the perceived burden of insulin injections may require more effort from health care providers . as we have suggested elsewhere , providers must find out what the specific issues are for each patient and work with that patient to develop solutions that will work for him or her ( 24 ) . we note that the measure of interference with eating and exercise was significantly associated with insulin injection omission until interference with activities of daily living was entered into the model ( results not shown ) . thus , while interference with eating and exercise might be part of the burden of insulin therapy , interference with other aspects of daily living had a more substantial association with insulin omission . our study suggests that insulin omission may be affected by the immediate experience of injecting insulin as painful and embarrassing ( but not dissatisfaction with time needed , ease of use , or skin inflammation / bruising ) . there are numerous device - related strategies for reducing pain and embarrassment , including insulin pens , finer gauge needles , injection ports , needleless injectors , and other injection assistance devices . however , we have found that patients do not feel that their health care providers are giving them adequate assistance in managing these problems , even when they raise the issue with their providers ( 18 ) . we note that the measure of negative affect toward injections was significantly associated with insulin omission until dissatisfaction with injection embarrassment and pain were entered into the model ( results not shown ) . this suggests that addressing pain and embarrassment may reduce not only insulin omission , but also the emotional burden of injections , thereby enhancing psychological well - being . it is interesting that worry about hypoglycemia did not predict intentional omission of insulin injections , even though worry about hypoglycemia was high in the study population . this suggests that patients may address this worry by eating more or lowering insulin doses rather than by skipping injections altogether . strengths of the study include the large sample of diabetic patients drawn from a general population and the fact that the sample was weighted to be nationally representative . however , patients volunteer for the panel from which respondents were drawn and may not be representative of all patients ( e.g. , they may be more adherent with their treatment regimens ) . limitations of the study include the fact that there was no objective measure of insulin use ( e.g. , pharmacy records ) . moreover , while our measure of insulin omission was very specific ( i.e. , skipping injections that respondents knew they should take ) , it is possible that some respondents included injections they did not skip intentionally , but rather simply forgot to take . respondents might also have included scheduled injections that were appropriately skipped because a meal was not eaten or blood glucose levels were very low . this could explain ( at least in part ) the association between following a healthy diet and fewer skipped injections . more disciplined eating behavior reduces meal skipping , which is an often - cited reason for skipping insulin injections . this is consistent with the fact that the relationship between our diet measure and insulin omission is present only among individuals with type 1 diabetes where closer matching of food and insulin is required . finally , our study probably underestimated the level of insulin nonadherence in this population because it did not capture instances in which patients took an injection but gave less than the amount of insulin they knew was needed for optimal glucose control . our findings make clear that while most patients did not report regular omission of insulin injections , a substantial number did . thus , our findings suggest that it is important to identify patients who omit insulin and to be aware of the potential risk factors identified here . although much attention has been focused on insulin omission among adolescents with type 1 diabetes , our findings suggest that among adults , individuals with type 2 diabetes are at higher risk . patients who are not adhering to other elements of the treatment regimen , especially diet , also may be at risk for insulin omission . for patients who report injection - related problems ( interference with daily activities , injection pain , and embarrassment ) , providers should consider recommending strategies and tools for addressing these problems to prevent insulin omission . we found that respondents with higher household income , but not individuals with more education , were less likely to skip insulin injections they knew they should take . this may reflect easier access to medications and supplies among individuals with higher income , but it is also likely that higher socioeconomic status is associated with more access to diabetes education , higher health literacy , greater control over one 's daily routines , and better problem - solving skills ( 19 ) . our study appears to be among the first to identify an association between socioeconomic status and insulin omission . future research should seek to identify potential mediators of this relationship , i.e. , what links lower socioeconomic status to insulin omission . contrary to earlier reports ( 3,4 ) , we found no racial / ethnic differences in intentional insulin omission . this may be because we did not have enough nonwhite respondents to examine the different racial / ethnic groups separately . alternatively , this may be due to our controlling for income and education in the analysis , thereby eliminating the confounding of race / ethnicity with socioeconomic status . much prior research has suggested that intentional insulin omission is common among female adolescents with type 1 diabetes , serving as a weight control strategy and sometimes linked to eating disorders ( 10 ) . we found that students ( who were younger than nonstudents ) were more likely to skip injections they knew they should take , but this behavior was not more common among women than it was among men . we found no overall association between age and intentional insulin injection omission among patients with type 1 diabetes , suggesting that patients with type 1 diabetes age - out of this behavior by early adulthood , when they complete their education . ascertaining the validity of this interpretation would require following youth with type 1 diabetes as they age into adulthood to determine change in rate of insulin omission . our finding that , among individuals with type 2 diabetes , older respondents were less likely to skip insulin injections is consistent with earlier studies ( 3,4 ) . this suggests that there are parallel aging - out processes among individuals with type 1 and type 2 diabetes , but in type 2 diabetes , this process takes place later in the life course ( almost all people with type 2 diabetes are diagnosed as adults ) . ascertaining the validity of this interpretation would require following adults with type 2 diabetes as they age to determine change in rate of insulin omission . having type 2 diabetes was itself associated with higher levels of intentional omission of insulin injections . the beta for this variable ( 0.226 ) was approximately twice the size of the unadjusted eta ( 0.095 ) , reflecting the fact that controlling for confounding factors ( such as age and number of daily injections ) revealed a stronger underlying association . the independent association of type 2 diabetes with increased insulin omission may reflect the fact that patients with type 2 diabetes have a residual insulin response , reducing the immediate consequences of omitting an injection . thus , these individuals may feel less vulnerable to the effects of skipping insulin injections they know they should take . interestingly , whereas duration of diabetes was associated with the frequency of insulin injection omission , regression analysis revealed that duration of diabetes did not make an independent contribution to this behavior . that is , although insulin omission may be less common among individuals with longer duration of diabetes , this is likely a function of other factors such as age and type of diabetes rather than of duration per se . surprisingly , history of depression was not associated with insulin omission ; this contradicts findings from studies of general adherence ( 9,20 ) and of insulin omission among adolescents ( 10 ) . however , because current depression was not assessed , any concurrent association was likely to be lost . others have shown that depression symptom scores fluctuate substantially over relatively short periods of time ; individuals with elevated depression symptoms at a given point in time are likely to not report elevated symptoms 6 months later ( 21,22 ) . in addition , depression symptoms across the whole range of severity symptoms have been shown to predict regimen adherence more powerfully than diagnosed depression ( 23 ) . being disabled was associated with less insulin omission ; this may be due to a variety of reasons , including their receiving more assistance with care , or making a greater effort to compensate for poor health . two aspects of patients ' treatment regimens were associated with increased insulin omission respondents who took more injections each day and those who did not follow a healthy diet were more likely to skip injections . that dietary nonadherence is associated with insulin nonadherence more frequent injection omission among individuals taking more injections could reflect the frequently reported finding that more complex regimens are associated with lower levels of adherence ( 6 ) . it might also be that the impact of skipping a shot is reduced among individuals who take more shots . our study suggests that insulin omission is affected by the perceived burden of insulin therapy ( i.e. , having to plan one 's life around insulin injections and feeling that the insulin regimen interferes with activities of daily living such as social activities , work - related activities , and family care - giving responsibilities ) . we offer one caveat regarding our findings ; we do not believe that the behavior of planning one 's day around insulin injections actually increases the level of insulin injection omission , but we do believe that feeling that one has to plan around one 's injections is associated with higher frequency of skipping insulin injections one should take . that is , when there is a conflict between scheduling of treatment and life activities , one can either plan one 's activities in a way that reduces this conflict or deal with the conflict by ignoring treatment needs . reducing the perceived burden of insulin injections may require more effort from health care providers . as we have suggested elsewhere , providers must find out what the specific issues are for each patient and work with that patient to develop solutions that will work for him or her ( 24 ) . we note that the measure of interference with eating and exercise was significantly associated with insulin injection omission until interference with activities of daily living was entered into the model ( results not shown ) . thus , while interference with eating and exercise might be part of the burden of insulin therapy , interference with other aspects of daily living had a more substantial association with insulin omission . our study suggests that insulin omission may be affected by the immediate experience of injecting insulin as painful and embarrassing ( but not dissatisfaction with time needed , ease of use , or skin inflammation / bruising ) . there are numerous device - related strategies for reducing pain and embarrassment , including insulin pens , finer gauge needles , injection ports , needleless injectors , and other injection assistance devices . however , we have found that patients do not feel that their health care providers are giving them adequate assistance in managing these problems , even when they raise the issue with their providers ( 18 ) . we note that the measure of negative affect toward injections was significantly associated with insulin omission until dissatisfaction with injection embarrassment and pain were entered into the model ( results not shown ) . this suggests that addressing pain and embarrassment may reduce not only insulin omission , but also the emotional burden of injections , thereby enhancing psychological well - being . it is interesting that worry about hypoglycemia did not predict intentional omission of insulin injections , even though worry about hypoglycemia was high in the study population . this suggests that patients may address this worry by eating more or lowering insulin doses rather than by skipping injections altogether . strengths of the study include the large sample of diabetic patients drawn from a general population and the fact that the sample was weighted to be nationally representative . however , patients volunteer for the panel from which respondents were drawn and may not be representative of all patients ( e.g. , they may be more adherent with their treatment regimens ) . limitations of the study include the fact that there was no objective measure of insulin use ( e.g. , pharmacy records ) . moreover , while our measure of insulin omission was very specific ( i.e. , skipping injections that respondents knew they should take ) , it is possible that some respondents included injections they did not skip intentionally , but rather simply forgot to take . respondents might also have included scheduled injections that were appropriately skipped because a meal was not eaten or blood glucose levels were very low . this could explain ( at least in part ) the association between following a healthy diet and fewer skipped injections . more disciplined eating behavior reduces meal skipping , which is an often - cited reason for skipping insulin injections . this is consistent with the fact that the relationship between our diet measure and insulin omission is present only among individuals with type 1 diabetes where closer matching of food and insulin is required . finally , our study probably underestimated the level of insulin nonadherence in this population because it did not capture instances in which patients took an injection but gave less than the amount of insulin they knew was needed for optimal glucose control . our findings make clear that while most patients did not report regular omission of insulin injections , a substantial number did . thus , our findings suggest that it is important to identify patients who omit insulin and to be aware of the potential risk factors identified here . although much attention has been focused on insulin omission among adolescents with type 1 diabetes , our findings suggest that among adults , individuals with type 2 diabetes are at higher risk . patients who are not adhering to other elements of the treatment regimen , especially diet , also may be at risk for insulin omission . for patients who report injection - related problems ( interference with daily activities , injection pain , and embarrassment ) , providers should consider recommending strategies and tools for addressing these problems to prevent insulin omission .

objectivethe purpose of this study was to assess factors associated with patient frequency of intentionally skipping insulin injections.research design and methodsdata were obtained through an internet survey of 502 u.s . adults self - identified as taking insulin by injection to treat type 1 or type 2 diabetes . multiple regression analysis assessed independent associations of various demographic , disease , and injection - specific factors with insulin omission.resultsintentional insulin omission was reported by more than half of respondents ; regular omission was reported by 20% . significant independent risk factors for insulin omission were younger age , lower income and higher education , type 2 diabetes , not following a healthy diet , taking more daily injections , interference of injections with daily activities , and injection pain and embarrassment . risk factors differed between type 1 and type 2 diabetic patients , with diet nonadherence more prominent in type 1 diabetes and age , education , income , pain , and embarrassment more prominent in type 2 diabetes.conclusionswhereas most patients did not report regular intentional omission of insulin injections , a substantial number did . our findings suggest that it is important to identify patients who intentionally omit insulin and be aware of the potential risk factors identified here . for patients who report injection - related problems ( interference with daily activities , injection pain , and embarrassment ) , providers should consider recommending strategies and tools for addressing these problems to increase adherence to prescribed insulin regimens . this could improve clinical outcomes .

RESEARCH DESIGN AND METHODS Measures Burden of injections. Experience of injections. Frequency of intentional insulin omission. Statistical analysis RESULTS Sample profile Regression analysis CONCLUSIONS Demographic and disease factors Insulin and injection-related factors Study strengths and limitations Clinical implications

otosclerosis is a primary , hereditary , localized , metabolic bone disease of bone derived only from the bony otic capsule . it is characterized by alternating phases of abnormal removal of mature bone of the otic capsule by osteoclasts , and replacement with new woven bone of greater thickness , cellularity , and vascularity . because it has predilection for the fissula ante fenestrum , involvement of the stapedial footplate may result in its fixation and an asymmetric conductive hearing loss ( stapedial otosclerosis ) whereby sounds partly fail to reach the inner ear ( cochlea ) [ 1 , 2 ] . involvement of other parts of the otic capsule ( cochlear otosclerosis ) may result more often in cochlear symptoms ( especially a high - frequency sensorineural hearing loss , which usually manifests late in the disease , and 75% tinnitus ) and vestibular symptoms ( in 25% of cases , most commonly a sense of disequilibrium , occasionally attacks of positional and motion - related vertigo with rotatory nystagmus ) , or a combination of these 2 symptoms ( combined otosclerosis with mixed hearing loss ) . diagnosis of otosclerosis is usually based on family history and clinical and audiologic examination [ 46 ] . stapedectomy / stapedotomy is the surgery of choice ; placing a piston prosthesis into the vestibule through the oval window fenestra provides mobile continuity between the inner ear and ossicular chain . testing the vestibular function in patients with otosclerosis is of particular interest . the bithermal caloric test ( bct ) is representative physiological test of the function of the lateral semicircular canal ( lscc ) and its afferents , and vemps mainly reflects the function of the saccule and its afferents . it is an objective , quick , and non - invasive measure of saccule function that , along with the utricle , primarily respond to linear acceleration in any direction . the saccule , which is the lower of the 2 otolithic organs , is most sensitive to gravity because it is in the vertical plane . it also has a slight sound sensitivity , which is thought to be a remnant from the function of the saccule as an organ of hearing in lower animals . the acoustic sensitivity of the saccule has been attributed to its proximity to the footplate of the stapes , which leads to its mechanical stimulation during stapedial motion in response to sound and can be measured . this is the basis of the vemp test , which is almost entirely saccular in origin . it has been widely used since 1992 after colebatch and halmagy described it as it is known in modern days . they selected the scm as their standard recording site of vestibular and myogenic response to sound [ 911 ] . vemp testing utilizes sophisticated computerized equipment to interpret waveforms on a computer screen for each ear , each type of stimulus used , and each intensity level . the purpose of the air - conducted vemp is to determine if the saccule stimulated by the sound , the activated inferior vestibular nerve ( ivn ) , central connections in the brainstem [ lateral vestibular nucleus ( deiters s nucleus ) , accessory nerve nucleus ] , the 11 nerve , and the sternocleidomastoid muscle ( scm , mostly ipsilaterally ) via the medial vestibulospinal tract ( mvst ) are intact and working normally . bone - conducted vemp is a useful measure of vestibular function , especially in the presence of conductive hearing loss , such as in otosclerosis , where the bone - conducted sound activates the vestibular apparatus more effectively than air - conducted sound [ 1317 ] . the bone - conducted sound can stimulate the saccule and other parts of the vestibular end organs on both sides as the utricle . the objectives of the current study were to : ( a ) investigate the audio - vestibular profile of stapedectomy candidates by analyzing the audiogram , vemp , and calorics results ; ( b ) evaluate the cause of vestibular dysfunction in patients with otosclerosis ; and ( c ) to determine the clinical utility of vemps in the differential diagnosis of otosclerosis and otologic diseases that can cause pseudo - conductive hearing loss such as scds and lvas . seventy - four caucasian ( 51 female , 23 male ) patients were seen in our academic tertiary referral centre otolaryngology department of hippokration hospital , university of athens , and were prospectively assessed as candidates for stapes surgery for otosclerosis ( mean age 38.09 years , range 2360 years).we selected an age range of 2360 years because there is evidence that the vemp response rate decreases after age 60 [ 1820 ] . no patient had a positive history for chronic otitis media , skull trauma , superior canal dehiscence syndrome ( scds ) , or large vestibular aqueduct syndrome ( lvas ) . tinnitus was present in 62 ( 83.7% ) subjects , 24 ( 32.4% ) patients reported dizziness , with 1 case of left - sided bppv treated with epley manoeuvre , and another 2 presented with first - degree right - beating spontaneous nystagmus . all the patients enrolled in this study were evaluated prior to stapedectomy using a standard diagnostic protocol that included audiological assessment consisting of pure tone and speech audiometry , tympanometry with stapedial reflexes , eng with bithermal caloric testing ( bct ) , and vemp test . of these patients , 1 underwent high - resolution computed tomography ( hrct ) for further evaluation of the disease . in all patients , the mean of 4-frequency pure tone hearing thresholds ( 4-pta at 500 , 1000 , 2000 , and 4000 hz ) was measured for both air and bone conduction and consequently the abg ( air - bone gap ) . vemps were recorded with a gn otometrics ( taastrup , denmark ) ics chartr ep version 5.2 analyser with a 2-channel averaging capacity . vemps were recorded while patients were seated in an upright position and instructed to maintain their heads turned contralaterally to the stimulated ear to achieve intense and constant sternocleidomastoid muscular effort during the whole recording period of each trial .the skin was scrubbed , and the impedance of the recording electrodes was maintained below 5 kohms . the 2 active recording electrodes were placed symmetrically at the middle third of each scm , the reference electrode was placed on the upper forehead , and the ground electrode was placed at the middle of the forehead . the acoustic stimulus ( loud , short tone bursts ) intensity range was 95102 db hl for ac - vemps and 4265 db hl for bc - vemps [ 500 hz tone bursts were given at 95 db hl for ac - vemps and 42 db hl for bc - vemps ; 1000hz tone bursts were given at 102 db hl for ac - vemps and 65 db hl for bc - vemps ; rate 5.1/second , ramp 1 millisecond ( msec ) , plateau 0 ms ] . stimuli were delivered monaurally via headphones ( tdh-40 , telephonics , new york , usa ) for elicitation of vemps evoked by air - conducted signal ( ac - vemps ) and the bone oscillator b71 placed at the ipsilateral mastoid for evocation of responses by bone - conducted stimuli ( bc - vemps ) with no contralateral masking , and the myogenic potential was recorded ipsilaterally by surface electrodes . the electromyographic ( emg ) activity of the ipsilateral sternocleidomastoid muscle was recorded , and every trial of 150 stimuli was averaged and repeated twice to verify the reproducibility of the waveform , and to provide the final vestibular evoked myogenic potential waveform . the emg signal from each side was amplified and bandpass - filtered ( high - pass 2 hz , low - pass 500 hz ) . the peak latencies of the first positive - negative component of the vestibular evoked myogenic potential response ( p1 and n1 ) were measured for each patient . the vestibular evoked myogenic potential response was considered to be absent when there were no recognizable or reproducible biphasic waveforms , or when the amplitude of the potential was less than 20 mv . electronystagmography recordings ( eng ) were performed with a life - tech model 3002 electronystagmograph ( houston , texas , usa ) . a hortmann airmatic ( neurootometrie , gn otometrics , taastrup , denmark ) air irrigator any caloric asymmetry of more than 22% was defined as canal paresis . the statistical package for the social sciences version 11.0 software student s t - test , anova and chi square testing were used to compare different values . the criterion for statistical significance was set at a p - value of 0.05 ( 2-tailed ) . for comparison , 35 healthy volunteers adults ( 17 men and 18 women ; mean age 37.3 years)for ac - vemps and 13 healthy adults ( 6 men and 7 female ; mean age 37.76 years ) for bc - vemps , without previous hearing , vestibular , or neurological disorders , were also enrolled in this study . all volunteers had a normal otoscopic examination and a normal pure tone audiometric ( pta ) threshold . ac vestibular evoked myogenic potential waveforms were obtained on both sides in all of the 35 healthy volunteers ( 70 ears ) and bc vestibular evoked myogenic potential waveforms in all of the 13 healthy volunteers ( 26 ears ) . the mean ac p1 value was 16.26 ms ( sd 1.32 ) and the mean ac n1 value was 24.42 ( sd 2.52 ) . the mean bc p1 value was 14.09 ( sd 2.70 ) and the mean bc n1 value was 22.30 ( sd 3.91).the p1 and n1 delay was defined as any value greater than the mean plus 2 sds of the normal population ( i.e. , any p1 value greater than 18.49 ms and any n1 value greater than 30.12 ms ) was considered delayed . this study was conducted with the understanding and the consent of all subjects , patients , and volunteers before inclusion . pre - operatively , ac - vemp was performed in 126 ears and ac - vemp and bc - vemp were performed in 109 ears each . ac - vemp was present in 34 ears , while bc - vemp was recordable in 48 ears . the mean ac 4-pta , mean bc 4-pta , and mean abg ( air - bone gap ) were compared between the group of ears with ac - vemp presence and ac - vemp absence , respectively . the same was done between the group of ears with bc - vemp presence and bc - vemp absence . the results appear in table 1 . statistically significant differences ( student s t - test ) were found in all 3 parameters between the group with ac - vemp(+ ) ( present ) and ac - vemp( ) ( absent ) . in contrast , the comparisons between the ears with presence and absence of bc - vemp were not significant . table 2 shows the combination of the 2 tests ( ac - vemp and bc - vemp ) in a total of 109 ears . the response rates of present ac - vemps and present bc - vemps were 29.36% ( 32 ears ) and 44.03% ( 48 ears ) respectively . chi - square revealed a strong correlation between these 2 investigations ; when ac - vemp was present , it tended to be the bc - vemp ; but when ac - vemp was absent , so was the bc - vemp . the percentage of vemp presence in healthy subjects was shown to be about 100% in controls without history of ear disease or head trauma . table 3 shows no significant statistical difference between the means of the latencies p1 , ( nor of n1 ) of the normal ears and the ears affected with otosclerosis with recordable ac - vemp . on the contrary , a significant statistical difference was noted between the means of latencies p1(and n1 ) of normal ears and ears affected by otosclerosis with elicited bc - vemp . mean abg in each 1 of the 4 groups formed by the combination of ac and bc - vemp appears in table 4 . no significant effect of the factor group was found , meaning that no statistical differences existed among the abg means ( one - way between - subjects anova ) . on the contrary , anova showed statistically significant differences among the means of ac 4-pta between the group of ears with presence and absence of both air- and bone - conducted vemp . [ the mean of the group with both vemp present ( 48.55 db ) differs statistically from the mean of the group with both vemp absent ( 59.66 db ) ] . however , no statistical differences were found among the bc 4-pta in the same 4 groups . preoperatively , the results of caloric responses were assessed in relation with the results of ac and bc - vemp ( chi - square ) in 109 otosclerotic ears . table 5 shows no correlation either between canal paresis ( cp ) occurrence and air - conducted vemp presence ( p=0.461 ) , or with canal paresis and bone - conducted vemp presence ( p=0.366 ) . vestibular symptoms occur in approximately 2037% of patients affected by otosclerosis , but their pathogenesis remains unclear . in our study , 24 ( 32.4% ) of 74 patients presented with symptoms of dizziness in the form of vertigo or disequilibrium ( after carefully completing a medical questionnaire , only patients with symptoms compatible with labyrinthine origin were found in the dizziness group ) . to study the relationship between dizziness , means of ac 4-pta , bc 4-pta , ac - vemp , bc - vemp and calorics in patients affected by otosclerosis , pre - operatively , the following groups of patients were defined as : group d ( patients who complained of dizziness ) and group nd ( patients with no complaint of dizziness ) [ 3133 ] . the d group was composed of 42 ears and the nd group was composed of 84 otosclerotic ears . the mean of ac 4-pta thresholds in groups d and nd were 57.63(sd 16.81 ) and 53.22(sd 16.65 ) db , respectively ( table 6 ) . there were no significant differences between the 2 groups ( student s t test , p=0.167 ) . mean bc 4-pta thresholds in groups d and nd were 30.78(sd 9.88 ) and 27.65 ( sd 11.36 ) db , respectively . no significant difference was observed between the 2 groups ( student s t - test , p=0.131 ) ( table 6 ) . for caloric testing , 19 of 42 ( 45.2% ) ears showed abnormal results in d group with 11 unilateral cp ( 1 presented with left - sided bppv ) and 4 bilateral cp ( 1 presented with first - degree right - beating spontaneous nystagmus ) . in the nd group , 24 out of 84 ( 28.6% ) ears showed abnormal calorics , with 16 unilateral and 4 bilateral cp . no statistical relationship was found between the calorics results and the appearance of dizziness ( , p=0.06 ) ( table 6 ) . ( , p=0.06 for ac - vemp , p=0.598 for bc - vemp ) ( table 6 ) . to assess the possible relation of the factors of dizziness and caloric results in the measurements of ac 4-pta , bc 4-pta and abg , a 2-way anova was carried out with dizziness having 2 levels [ i.e. , yes ( dizziness present ) and no ( no dizziness ) ] and caloric results having 3 levels ( i.e. , cp at the ipsilateral ear [ the ear in which pta and air - bone gap(abg ) are measured and enter the study ] , cp at the contralateral ear ( so the ear measured has normal calorics ) , and normal calorics ( referring to cases with no canal paresis to either ear ) . for cases with bilateral cp in both ears were counted as ipsilateral cp . concerning ac and bc 4-pta , the anova did not show a significant main effect either for dizziness or for calorics , meaning that there were no significant differences of the means of the above measurements , neither between the dizzy and non - dizzy group , nor between the ears with cp and the ears with normal calorics . however , a statistically significant interaction was found between the 2 factors ( dizziness and calorics ) in relation to ac and bc - pta thresholds . in the dizziness group ( d ) , ears with cp showed much higher means of ac and bc 4-pta , whereas in the non dizziness ( nd ) group the contralateral ear ( with normal calorics ) showed higher ac and bc 4-pta means ( figures 1 and 2 ) . concerning the abg , no significances were found ( p=0.364 for the interactions , p=0.971 , p=0.522 for the main effects of dizziness and calorics , respectively ) . pre - operatively , ac - vemp was performed in 126 ears and ac - vemp and bc - vemp were performed in 109 ears each . ac - vemp was present in 34 ears , while bc - vemp was recordable in 48 ears . the mean ac 4-pta , mean bc 4-pta , and mean abg ( air - bone gap ) were compared between the group of ears with ac - vemp presence and ac - vemp absence , respectively . the same was done between the group of ears with bc - vemp presence and bc - vemp absence . the results appear in table 1 . statistically significant differences ( student s t - test ) were found in all 3 parameters between the group with ac - vemp(+ ) ( present ) and ac - vemp( ) ( absent ) . in contrast , the comparisons between the ears with presence and absence of bc - vemp were not significant . table 2 shows the combination of the 2 tests ( ac - vemp and bc - vemp ) in a total of 109 ears . the response rates of present ac - vemps and present bc - vemps were 29.36% ( 32 ears ) and 44.03% ( 48 ears ) respectively . chi - square revealed a strong correlation between these 2 investigations ; when ac - vemp was present , it tended to be the bc - vemp ; but when ac - vemp was absent , so was the bc - vemp . the percentage of vemp presence in healthy subjects was shown to be about 100% in controls without history of ear disease or head trauma . table 3 shows no significant statistical difference between the means of the latencies p1 , ( nor of n1 ) of the normal ears and the ears affected with otosclerosis with recordable ac - vemp . on the contrary , a significant statistical difference was noted between the means of latencies p1(and n1 ) of normal ears and ears affected by otosclerosis with elicited bc - vemp . mean abg in each 1 of the 4 groups formed by the combination of ac and bc - vemp appears in table 4 . no significant effect of the factor group was found , meaning that no statistical differences existed among the abg means ( one - way between - subjects anova ) . on the contrary , anova showed statistically significant differences among the means of ac 4-pta between the group of ears with presence and absence of both air- and bone - conducted vemp . [ the mean of the group with both vemp present ( 48.55 db ) differs statistically from the mean of the group with both vemp absent ( 59.66 db ) ] . however , no statistical differences were found among the bc 4-pta in the same 4 groups . preoperatively , the results of caloric responses were assessed in relation with the results of ac and bc - vemp ( chi - square ) in 109 otosclerotic ears . table 5 shows no correlation either between canal paresis ( cp ) occurrence and air - conducted vemp presence ( p=0.461 ) , or with canal paresis and bone - conducted vemp presence ( p=0.366 ) . vestibular symptoms occur in approximately 2037% of patients affected by otosclerosis , but their pathogenesis remains unclear . in our study , 24 ( 32.4% ) of 74 patients presented with symptoms of dizziness in the form of vertigo or disequilibrium ( after carefully completing a medical questionnaire , only patients with symptoms compatible with labyrinthine origin were found in the dizziness group ) . to study the relationship between dizziness , means of ac 4-pta , bc 4-pta , ac - vemp , bc - vemp and calorics in patients affected by otosclerosis , pre - operatively , the following groups of patients were defined as : group d ( patients who complained of dizziness ) and group nd ( patients with no complaint of dizziness ) [ 3133 ] . the d group was composed of 42 ears and the nd group was composed of 84 otosclerotic ears . the mean of ac 4-pta thresholds in groups d and nd were 57.63(sd 16.81 ) and 53.22(sd 16.65 ) db , respectively ( table 6 ) . there were no significant differences between the 2 groups ( student s t test , p=0.167 ) . mean bc 4-pta thresholds in groups d and nd were 30.78(sd 9.88 ) and 27.65 ( sd 11.36 ) db , respectively . no significant difference was observed between the 2 groups ( student s t - test , p=0.131 ) ( table 6 ) . for caloric testing , 19 of 42 ( 45.2% ) ears showed abnormal results in d group with 11 unilateral cp ( 1 presented with left - sided bppv ) and 4 bilateral cp ( 1 presented with first - degree right - beating spontaneous nystagmus ) . in the nd group , 24 out of 84 ( 28.6% ) ears showed abnormal calorics , with 16 unilateral and 4 bilateral cp . no statistical relationship was found between the calorics results and the appearance of dizziness ( , p=0.06 ) ( table 6 ) . ( , p=0.06 for ac - vemp , p=0.598 for bc - vemp ) ( table 6 ) . to assess the possible relation of the factors of dizziness and caloric results in the measurements of ac 4-pta , bc 4-pta and abg , a 2-way anova was carried out with dizziness having 2 levels [ i.e. , yes ( dizziness present ) and no ( no dizziness ) ] and caloric results having 3 levels ( i.e. , cp at the ipsilateral ear [ the ear in which pta and air - bone gap(abg ) are measured and enter the study ] , cp at the contralateral ear ( so the ear measured has normal calorics ) , and normal calorics ( referring to cases with no canal paresis to either ear ) . for cases with bilateral cp in both ears were counted as ipsilateral cp . concerning ac and bc 4-pta , the anova did not show a significant main effect either for dizziness or for calorics , meaning that there were no significant differences of the means of the above measurements , neither between the dizzy and non - dizzy group , nor between the ears with cp and the ears with normal calorics . however , a statistically significant interaction was found between the 2 factors ( dizziness and calorics ) in relation to ac and bc - pta thresholds . in the dizziness group ( d ) , ears with cp showed much higher means of ac and bc 4-pta , whereas in the non dizziness ( nd ) group the contralateral ear ( with normal calorics ) showed higher ac and bc 4-pta means ( figures 1 and 2 ) . concerning the abg , no significances were found ( p=0.364 for the interactions , p=0.971 , p=0.522 for the main effects of dizziness and calorics , respectively ) . nevertheless , few published studies have attempted to investigate inner ear function and impairment of vemps in otosclerosis . our results , by using ac and bc tone - burst vemps in combination with audiologic findings , are in agreement with the fact that ac - vemp is more vulnerable to conductive hearing loss , whereas bc vemp is not . the magnitude of mean abg , ac 4-pta , and bc 4-pta is likely responsible for the pre - operative difference in elicitation of ac - vemps , confirmed by the significantly lower pre - operative abg in patients with recordable ac - vemps in comparison to those with absent ac - vemps ( 22.23 vs. 27.27 db hl , table 1 ) , lower mean of ac 4-pta ( 47.67 vs. 57.28 , table 1 ) and bc 4-pta ( 25.64 vs. 30.01 , table 1 ) . the magnitude of mean abg , ac , and bc 4-pta appears to have no effect on the pre - operative difference in elicitability of bc - vemps . complementary to this is the finding that the latencies p1 and n1 of bc - vemps in otosclerotic ears are statistically longer than those of normal ears ( table 3 ) , whereas the latencies of ac - vemp are not . this can be explained by the persistence of bc - vemp appearance in ears with advanced otosclerosis , where ac - vemp has already vanished . the low presence rate of bc - vemps pre - operatively ( 44.03% ) could be caused by inner ear damage due to an ototoxic effect of substances produced by otosclerotic foci of the otic capsule on saccular receptors . the fact that the mean of abg differs considerably between ears with present and absent ac - vemp , whereas in ears with bc - vemp it does not ( table 1 ) , reveals a different behavior of the 2 types of vemps in relation to abg values . the different trends concerning the abg means when considering each vemp measurement separately , can explain the lack of statistical significance in the means of abg among the 4 groups formed by the combination of ac and bc - vemp . the same is true for bc 4-pta and ac 4-pta , with the exception that the latter shows a statistically significant difference between the group of ac / bc - vemp both present and ac / bcvemp both absent ( table 4 ) . the small group of 5 ears with present ac - vemp and absent bc - vemp could be explained by the fact that the bone conduction stimulus in the present study was relatively low ( 65 db hl ) , so these could be patients with otolithic organ disease needing a stronger stimulus to produce bc - vemp , whereas the much stronger air - conducted stimulus could do it . in fact , these are ears with relatively low abg means ( 18 db , table 4 ) , so the transition of air - conducted stimuli was not severely hindered . a possible relationship of vertigo and otosclerosis is an understandable assumption , because otosclerosis is a disease affecting structures proximal to the vestibule , but until now , sufficient and concrete evidence supporting this has been lacking . assessment of the functional state of the labyrinth as a total by using a combination of vemp with calorics provided us with interesting observations . firstly , when vertigo / dizziness as a symptom was compared with caloric responses ( not taking otosclerosis into account ) , results were borderline insignificant ( p=0.06 , table 6 ) . any vertiginous patient can have a normally functioning labyrinth with symptoms due to incomplete central compensation or migrainous vertigo . no relation could be found between vemp results and calorics , neither for air- nor for bone - conducted vemps ( table 5 ) . this is an interesting finding , supporting the notion that caloric deficiencies are rather irrelevant to vemp deficiencies in patients with otosclerosis , so it encourages the assumption that vemp deficiencies are due to otosclerosis and are not caused by a history of vestibular neuritis . ac and bc - vemp also did not show any relationship with dizziness either ( table 6 ) , although the percentage of ears with present ac - vemp in the non - dizziness population ( 32.1% ) was at the borders of statistical significance compared with the percentage of ears with present ac - vemp in the dizziness population ( 16.7% ) . there are authors who assume that the balance problems associated with otosclerosis are caused by saccular dysfunction originating from endolymphatic or saccular hydrops [ 3639 ] or direct invasion of the otosclerotic focus to the saccular macula or saccular afferents . a 2012 study by saka et al . found that balance problems in otosclerosis were associated with abnormal results for bc - vemp . conversely , the present analysis of 74 patients with otosclerosis , relying on the assessment of a higher clinical sample than other prospective studies , could not support this theory based on vemp only . a possible involvement of otolithic organs in vertiginous symptoms ( otolithic vertigo ) needs further investigation ( subjective visual vertical and horizontal ) as well as a more detailed questionnaire dealing with the specifics of otolithic vertigo symptoms . anova assessed the relation of vertigo and caloric results , each separately , as well as a combination , with abg and pta measurements ( i.e. , the severity of otosclerosis ) . the results showed no main effect of these 2 factors on the severity of otosclerosis ( abg ) , meaning that the degree of hearing loss was not relevant to either dizziness or caloric results . this is understandable , considering that the semicircular canals are relatively spared from the disease . bppv could be a clinical entity connecting ( not immediately ) saccular involvement with a semicircular canal , but just 1 patient is not enough to support any possible attribution of bppv to otosclerosis . however , anova showed a significant interaction of vertigo and calorics in relation to pta , both air - conducted ( ac 4-pta ) and bone - conducted ( bc 4-pta ) . this means that canal paresis ( cp ) influences pta means differently in the 2 groups of patients . in the group of vertiginous patients ( d ) , ears with canal paresis showed higher pta thresholds ; whereas in the non - vertiginous group ( nd ) , the ears with canal paresis showed lower pta thresholds than ears with normal calorics ( figures 1 and 2 ) . one possible explanation is that although otosclerosis is not a cause of canal paresis or vertigo , if vestibular neuritis ( or hydrops or any other vestibular cause of canal paresis ) affects an ear with advanced otosclerosis , ( i.e. , high threshold of ac and/or bc - pta ) , central compensation is delayed and vertiginous symptoms persist ; whereas in cases of less advanced otosclerosis , compensation is expected to be faster . the principal assumption of the present study is that the mere presence of vemp does not exclude otosclerosis . despite the conductive hearing loss , vemp can still be elicited in a number of preoperative otosclerotic ears . in our study , the response rate of the ac - vemps was only 29.36% , but the response rate of bc - vemps was 44.03% . therefore , a possible suggestion is to proceed to vemp threshold measurements in case of doubt . specifically , positive ac - vemp with pathologically decreased thresholds ( less than 70 db to tone - burst testing at 500 hz , while thresholds for evoking vemp using air - conducted tone - burst in our study were ranging between 95 and 102 db ) excludes middle ear pathology such as otosclerosis and provides a reference for further imaging examination to exclude the alternative diagnosis of scds ( superior canal dehiscence syndrome ) and lvas ( large vestibular aqueduct syndrome ) . therefore , when it is not possible to make an accurate diagnosis based solely on the audiogram and immittance testing results , vemp testing can be added in the diagnostic work - up of otosclerosis to provide additional confirmation . thus , vemp testing outcomes may provide the basis not only for better preoperative counselling , but also for safety issues regarding stapedectomy candidates . the presence of vemp does not exclude otosclerosis . despite the conductive hearing loss , vemp can be elicited in a small number of ears with otosclerosis . the ac - vemp is more vulnerable to conductive hearing loss , whereas bc - vemp is not ; however , the low presence rate of bc - vemps could be caused by inner ear damage . the evaluation of vemp thresholds , by using air - conducted tone - burst , can be added in the diagnostic work - up of suspected otosclerosis in case of doubt , providing additional confirmation , narrowing down the differential diagnosis in patients with pseudo - conductive components , and reducing medical cost by preventing unnecessary radiation exposure and unsuitable middle ear surgery . vertiginous symptoms should not be attributed to otosclerosis itself , but to causes affecting the semicircular canals . perhaps further investigations into otolithic disturbances ( such as subjective visual vertical and horizontal ) and a questionnaire more oriented to otolithic vertigo symptoms could reveal possible clinical impacts of utricular and saccular involvement in the disease .

backgroundthe aim of this prospective clinical study was to evaluate the clinical importance of vestibular - evoked myogenic potentials ( vemps ) in the assessment and differential diagnosis of otosclerosis and otologic diseases characterized by pseudo - conductive components . we also investigated the clinical appearance of balance disorders in patients with otosclerosis by correlating vemp results with the findings of caloric testing and pure tone audiometry(pta).material / methodsair - conducted(ac ) 4-pta , bone - conducted(bc ) 4-pta , air - bone gap(abg ) , ac , bc tone burst evoked vemp , and calorics were measured preoperatively in 126 otosclerotic ears.resultsthe response rate of the ac - vemps and bc - vemps was 29.36% and 44.03% , respectively . statistical differences were found between the means of abg , ac 4-pta , and bc 4-pta in the otosclerotic ears in relation to ac - vemp elicitability . about one - third of patients presented with disequilibrium . a statistically significant interaction was found between calorics and dizziness in relation to pta thresholds . no relationship was found between calorics and dizziness with vemps responses.conclusionsac and bc vemps can be elicited in ears with otosclerosis . ac - vemp is more vulnerable to conductive hearing loss . evaluation of ac - vemp thresholds can be added in the diagnostic work - up of otosclerosis in case of doubt , enhancing differential diagnosis in patients with air - bone gaps . otosclerosis is not a cause of canal paresis or vertigo .

Background Material and Methods Results AC-VEMPs and BC-VEMPs Incidence of AC-VEMPs and BC-VEMPs Comparison of mean latencies of peaks P1 and N1 between healthy (controls) and otosclerotic ears Comparison of the mean ABG, AC 4-PTA, and BC 4-PTA among the 4 groups formed by the combination of AC and BC-VEMP Caloric responses in otosclerosis Dizziness in patients with otosclerosis Discussion Conclusions

osteoma is a benign osteogenic lesion , which was first described as a specific entity by jaffe in 1935 . in the course of their slow but steady increase in size , osteomas of the maxillofacial bones remain asymptomatic until they attain sufficient sizes as to cause disfigurement and/or direct interference with the normal function of their anatomic location . osteomas are bone lesions with different onset and slow growth that may be divided into : ( 1 ) cranial and mandibular exophytic osteomas ( or eburnean exostoses ) ; ( 2 ) paranasal sinuses , facial bones , and orbit osteomas ( orbital cavity osteoma ) ; ( 3 ) exostoses or bone islands ; and ( 4 ) long bone superficial osteomas ( juxtacortical ) . the etiology seems to be unknown ; however , it may be developmental , infectious , or of traumatic origin . solitary osteomas of the facial skeleton are quite rare , although multiple osteomas of the jaws have been reported as part of gardner 's syndrome . little information is available about the gender predilection ; however , some authors suggest that there is a higher incidence of osteomas in male , especially in between second and fourth decade of life . a 45-year - old male patient was referred to the department of periodontics , krishnadevaraya college of dental sciences and hospital , bangalore , for the evaluation and treatment of a palatal bony swelling [ figures 1 and 2 ] . patient complained of a slow growing , painless mass in the left palatal maxillary area , which was present since six months . he also gave a history of foreign body ( fish bone ) lodgement in 25 and 26 region seven months ago ; however , no treatment was taken for the same . he was systemically healthy with no relevant medical history , and was asymptomatic except for the discomfort in that region and difficulty with mastication . on oral examination , a firm , lobulated , painless , well - circumscribed , exophytic , mass 1.51 cm in diameter was observed on the maxillary posterior left palatal area in relation to 25 and 26 regions . periodontal probing revealed a 9 millimeter deep periodontal pocket associated with the mid palatal area of 26 which prompted us to intervene and surgically excise the bony mass . clinically , the tooth was firm and not associated with any mobility [ figure 3 ] . bony lesion on palate nine millimeter pocket depth a region of well defined opacity was seen in the palatal regions of 25 and 26 [ figure 4 ] , when seen on a maxillary occlusal radiograph . the radio - opacity showed a well defined medial convex margin ; laterally , the margins were indistinct with rest of the structures . region of well defined radio - opacity in the region of 25 and 26 under local anesthesia , buccal and palatal flaps were raised , giving crevicular incisions in 24 , 25 , and 26 regions [ figures 5 and 6 ] . a second vertical incision was placed on the distal of the first premolar and a full thickness flap was raised to allow for adequate exposure of the surgical site . the bony mass was excised with the help of a kirkland chisel which has one end that is a back - action chisel and the other end is a straight chisel [ figures 7 and 8 ] . care was taken to excise the lesion keeping a periphery of healthy tissue around the lesion . the straight end of the kirkland chisel was used to refine the bone margin of the surgical site and to round off any sharp edges . thorough scaling and root planing coupled with removal of the granulation tissue followed by irrigation of the surgical site with saline were performed . margins of the flap were trimmed to achieve good approximation and the flap was sutured with black silk sutures [ figure 9 ] . the specimen was put in a bottle containing 10% formalin solution and sent for histopathological examination . the healing was uneventful , and the sutures were removed after 10 days [ figure 10 ] . the case was followed - up for a period of one year , and there was no sign of recurrence [ figures 11 and 12 ] . sulcular incision given flap raised using vertical incision excision of bony lesion with kirkland chisel section showed parakeratinized stratified squamous epithelium ; sub epithelially a rim of normal connective tissue was seen around the rete ridges . deeper connective tissue showed plump mesenchymal cells with prominent nuclei leading to formation of bony trabeculae . the lesional tissue showed numerous delicately arranged trabeculae with the osteocytes , but was devoid of osteoblastic rimming . the inter - trabecular tissue showed marrow spaces pre - dominantly made up of adipose tissue , presence of few arterioles and capillaries [ figures 13 and 14 ] . histopathologically , the lesion was diagnosed as peripheral ( mature ) osteoma , which is also referred to as ivory osteoma . ( b ) adipose cells ( a ) parakeratinized stratified squamous epithelium ( b ) connective tissue a region of well defined opacity was seen in the palatal regions of 25 and 26 [ figure 4 ] , when seen on a maxillary occlusal radiograph . the radio - opacity showed a well defined medial convex margin ; laterally , the margins were indistinct with rest of the structures . under local anesthesia , buccal and palatal flaps were raised , giving crevicular incisions in 24 , 25 , and 26 regions [ figures 5 and 6 ] . a second vertical incision was placed on the distal of the first premolar and a full thickness flap was raised to allow for adequate exposure of the surgical site . the bony mass was excised with the help of a kirkland chisel which has one end that is a back - action chisel and the other end is a straight chisel [ figures 7 and 8 ] . care was taken to excise the lesion keeping a periphery of healthy tissue around the lesion . the straight end of the kirkland chisel was used to refine the bone margin of the surgical site and to round off any sharp edges . thorough scaling and root planing coupled with removal of the granulation tissue followed by irrigation of the surgical site with saline were performed . margins of the flap were trimmed to achieve good approximation and the flap was sutured with black silk sutures [ figure 9 ] . the specimen was put in a bottle containing 10% formalin solution and sent for histopathological examination . the healing was uneventful , and the sutures were removed after 10 days [ figure 10 ] . the case was followed - up for a period of one year , and there was no sign of recurrence [ figures 11 and 12 ] . section showed parakeratinized stratified squamous epithelium ; sub epithelially a rim of normal connective tissue was seen around the rete ridges . deeper connective tissue showed plump mesenchymal cells with prominent nuclei leading to formation of bony trabeculae . the lesional tissue showed numerous delicately arranged trabeculae with the osteocytes , but was devoid of osteoblastic rimming . the inter - trabecular tissue showed marrow spaces pre - dominantly made up of adipose tissue , presence of few arterioles and capillaries [ figures 13 and 14 ] . histopathologically , the lesion was diagnosed as peripheral ( mature ) osteoma , which is also referred to as ivory osteoma . ( a ) trabeculae with osteocytes ( b ) adipose cells ( a ) parakeratinized stratified squamous epithelium ( b ) connective tissue in gnathic bones , osteoma can appear in bone 's surface , as mushroom - like masses pedunculated or sessile ( periosteum osteoma ) , or located in medullary space ( endosteum osteoma ) . these lesions are usually asymptomatic , but may proliferate in medullary bone ( endosteal ) or on the bone surface as a polypoid or sessile mass ( periosteal ) . reactive bone growth from infection , trauma , or vascular malformation may produce lesions simulating an osteoma . these lesions may arise at any age but with a distinct predilection for elderly adults and they are twice more common in male than in female . as a pathologic entity , osteoma is difficult to differentiate radiographically from other lesions with pronounced ossifications . exostosis , which is the term generally applied for well demarcated and extremely opaque bony lesion , osteosclerosis and similar alterations of the bone are always discussed alongside osteomas . the other differential diagnosis include enostosis , cementoma , complex odontome , fibrous dysplasia , osteochondroma , paget 's disease , peripheral ossifying fibroma , a reactive focal lesion ; periosteal osteoblastoma , osteoid osteoma , and paraosteal osteosarcoma that present as painful masses of rapid growth.[79 ] though the exact etiology is not known , it may be suggested , in this case that trauma from foreign body lodgment as afore mentioned might have been the trigger for this proliferative response . osteomas are slow growing benign osteogenic lesions arising from the proliferation of either cancellous or compact bone . they are usually asymptomatic and have a low recurrence rate . simple excision of the lesion in its entirety is the treatment of choice in symptomatic lesions . the association of osteoma with gardner 's syndrome must always be kept in mind before deciding the treatment plan .

osteomas are benign slow growing , osteogenic lesions which may arise from proliferation of either cancellous or compact bone . they are usually sessile tumours composed of dense sclerotic , well formed bone projecting out from the cortical surface , most often of the skull and facial bones . this paper reports a case of a peripheral osteoma in the hard palate of a 45-year - old man , which was treated by periodontal flap surgery with surgical excision of the bony lesion . peripheral osteomas of jaw bone are uncommon and usually associated with gardner 's syndrome . histological examination confirmed the clinical impression of a peripheral osteoma . patient was reviewed after one year and was asymptomatic with no recurrence of the lesion .

INTRODUCTION CASE REPORT Radiographic examination Surgical procedure Histopathology DISCUSSION CONCLUSION

fig fruit represents an important constituent of the diet , because of their nutritional and medicinal values . this type of diet is considered one of the healthiest and is associated with longevity . figs are an excellent source of minerals , vitamins , polyphenols , and dietary fiber ; they are fat and cholesterol - free and have a high antioxidant activity . figs have recently attracted a great deal of attention and are widespread throughout the world . the world produces over one million tons of figs yearly , of which 82% are produced in mediterranean countries . in tunisia , the production is about 29 000 tons ; it represents 3% of total world production . the fig is an economically important fruit species which is cultivated extensively in southeast regions . fig nursery plants production is mainly based on cuttings taken from the mother tree and placed in a rooting , which will eventually produce roots and shoots [ 68 ] . the traditional method for growing fig consisted in taking cuttings from one- or two - year - old shoots and directly planting them in soil to obtain new trees . however , the investigation on ways to propagate fig tree material and produce vigorous and healthy fig plants under field conditions becomes necessary in order to develop fig sector . the objective of this study was to characterize and compare fig nursery plants of some local fig cultivars by using hardwood cuttings under field conditions . in this way , five cultivars and six cuttings sizes were compared in order to optimize the propagation method of fig in tunisia . in this research , hardwood cuttings of 4 uniferous ( one production per year ) fig cultivars ( bayoudhi , jemaoui , ragoubi , and zidi ) , one biferous ( two productions per year ) ( bither ) , and one caprifig ( male fig ) ( bouharrag ) were used as plant material . fig tree is a species having high level of adaptation to different soil types and dry conditions . these cultivars are frequently cultivated for producing fresh and sun drying fig in arid region of tunisia ( 100200 mm annual precipitation ) . these ones are widely grown in southern tunisia and were described in a previous work [ 10 , 11 ] . hardwood cuttings were taken at the beginning of february 2009 and february 2010 , from adult fig trees of the germplasm collection of arid land institute ( ira)medenine , located in el gordhab tataouine ( tunisia ) . it consists in comparing the ability of cutting of 5 cultivars ( bayoudhi , jemaoui , ragoubi , zidi , and bouharrag ) . ten cuttings per replicate and per cultivar were used , with a total of 150 cuttings . it consists in comparing the ability of cutting of 5 cultivars ( bayoudhi , jemaoui , ragoubi , zidi , and bouharrag ) . ten cuttings per replicate and per cultivar were used , with a total of 150 cuttings . it was involved on the cultivar bither with 6 cuttings types as described below : type 1 : cuttings of one - year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 2 : cuttings of one - year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 3 : cuttings of one - year - old shoot , length ( 60 cm ) , and diameter ( < 1.5 cm ) ; type 4 : cuttings of 2-year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 5 : cuttings of 2-year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 6 : cuttings of 2-year - old shoots , length ( 60 cm ) , and diameter ( > 2 cm ) . it was involved on the cultivar bither with 6 cuttings types as described below : type 1 : cuttings of one - year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 2 : cuttings of one - year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 3 : cuttings of one - year - old shoot , length ( 60 cm ) , and diameter ( < 1.5 cm ) ; type 4 : cuttings of 2-year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 5 : cuttings of 2-year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 6 : cuttings of 2-year - old shoots , length ( 60 cm ) , and diameter ( > 2 cm ) . type 1 : cuttings of one - year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 2 : cuttings of one - year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 3 : cuttings of one - year - old shoot , length ( 60 cm ) , and diameter ( < 1.5 cm ) ; type 4 : cuttings of 2-year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 5 : cuttings of 2-year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 6 : cuttings of 2-year - old shoots , length ( 60 cm ) , and diameter ( > 2 cm ) . ten cuttings per replicate and per cutting types were used , with a total of 180 cuttings . during this research , cuttings were planted directly in rooting units ( 2.5 0.65 m ) under field conditions . cuttings were planted in an inclined position ( angle of 45 ) on sandy soils , where 3/4 of cutting is sinking into the soil at the professional agricultural training center ( cfpa ) el gordhab tataouine ( tunisia ) . all plants were irrigated by submersion technique with 40 l / rooting unit/15 days on the early morning during the course of the experiment in the three seasons . maintenance works such as weed control and hoeing were carried out during the whole vegetation period . root emergence percentage , number of emerged buds / nursery plant , length of nursery plant from the soil surface , and diameter of nursery plant were measured on the section 5 cm higher than soil surface and number of leaves / nursery plant . at the drawing out of young plants , cumulative growth ( cutting length + new growth ) of nursery plant and number of roots / nursery plant were determined [ 12 , 14 ] . all parameters were measured at the end of the vegetation period ( almost 8 months after plantation ) . emerged buds , leaves , and roots of each plant were counted manually . data were subjected to analysis of variance ( anova ) , using statbox version 6.40 . in case of significant cultivars and cutting type 's effects , comparison of values was performed by means of the least significant difference ( lsd ) test at a significance level of 0.05 , as the percentage root emergence ( re % ) was calculated by the means of three replications . the descriptive data analysis revealed a high variability among all the morphological characters of young plant fig cultivars studied under the field conditions . the most widely varied characters were root emergence percentage ( 10%90% ) and cumulative growth of nursery plant ( 32112 cm ) . considerable range of variation was also noticed in length of nursery plant ( 4 to 70 cm ) and number of leaves / nursery plant ; it varied from 3 leaves to 63 leaves , whereas very low variation was noted in number of emerged buds / nursery plant , diameter of nursery plant , and number of roots / nursery plant ( table 2 ) . the mean values of all morphological characteristics observed were statically different at a 5% level ( table 3 ) . this indicates a certain variation within cultivars , especially for length , diameter of nursery plants , number of leaves , and cumulative growth of nursery plants . root emergence percentage ( % re ) had the highest values ( 65% ) in jemaoui and rogabi cultivars and the lowest values ( 15% ) in bayoudhi . nursery plants characteristics like length of nursery plant ( lp ) , diameter of nursery plant ( dp ) , cumulative growth of nursery plant ( ag ) , and number of roots / nursery plant ( nr ) exhibit the highest values in cultivars bayoudhi . finally , the number of leaves / nursery plant ( nl ) was significantly high in cultivars bouharrag ( 37 leaves ) and bayoudhi ( 27 leaves ) . the result of this part can provide that the cultivars choice is very important to produce sufficient amounts of vigorous nursery plants . cultivar is very adapted to agroecological conditions , which can be eventually developed by farmers . moreover , the length of nursery plant ( lp ) , diameter of nursery plant ( dp ) , and number of roots ( nr ) are very important quality characters for selection of nursery plants of fig cultivars . results of the descriptive data analysis recorded high morphological variation in the plant material derived from the 6 fig cutting types of the cultivar bither , for all the traits . the most widely varied parameters were root emergence percentage ( 090% ) , length of nursery plant ( 377 cm ) , and number of roots / nursery plant ( 029 roots ) ( table 4 ) . results of 6 cutting types for the cultivar bither analysis showed a significant difference at a level of 5% in all the studied characters ( table 5 ) . the length of nursery plant ( lp ) , diameter of nursery plant ( dp ) , cumulative growth of nursery plant ( cg ) , and number of roots / nursery plant ( nr ) showed high significant difference . results revealed that root emergence percentage ( re % ) had the highest value in cutting type 3 and the lowest value in type 6 . also , type 3 exhibits the highest means of number of roots / nursery plant ( nr ) , length of nursery plant ( lp ) , and number of leaves / nursery plant ( nl ) . the cutting type 5 was significantly high in the cumulative growth trait in nursery plant ( cg ) . finally , the number of roots / nursery plant ( nr ) presents the highest value in cutting type 5 . the findings of the study put forth that cutting characteristics ( length , diameter , and age ) affected the growth and quality of fig plants . moreover , cutting length affected the length ( lp ) and cumulative growth ( cg ) of fig nursery plant . the use of long cuttings ( 4060 cm ) is recommended to produce vigorous fig nursery plants . the results achieved in this study showed that propagation of fig plant material under field conditions can be applied and should not require sophisticated equipment [ 6 , 12 ] . the values of characters measured on nursery fig plants in the present study were compared with those from other countries , namely , morocco [ 12 , 15 ] , turkey [ 14 , 16 ] , iran , thailand , and chile . similar percentage of rooted cuttings on some moroccan varieties propagated by using hardwood stem cutting under field conditions was found . chaari , aqounaq el hmam , ghouddane , ournaki , and kadota presented high values of % root emergence ; 89 , 98 , 94 , 87 , and 88% , respectively . cultivar sarilop from turkey has revealed high similarity in morphological characteristics of nursery plants with bayoudhi and jemaoui cultivars from the present study . in turkey , the fig cultivar calimyrna sarilop is used and propagated extensively by cuttings and calimyrna is the most popular and best cultivar in the world because it dries well . nevertheless , rooting ratio % , length of nursery plant , and diameter of nursery plant reported lower values than those of tunisian cultivars studied . the iranian cultivar sabz represents the large majority of fig trees present in estahban area . obtained values on root emergence percentage , number of emergence buds , length of nursery plant , and number of leaves / nursery plant variables are similar to our results . brown turkey , dauphine , and lisa cultivars from thailand showed variables values of nursery fig plants comparable with those of this work . however , root emergence percentage revealed lower values ( 12.5039.29% ) than that found in the present survey ( 1583.33% ) , whereas three cultivars showed higher values for the number of emerged buds and number of roots / nursery plant . moreover , the high variation was recorded among the % roots emergences and the other morphological traits within the 5 cultivars ( bayoudhi , jemaoui , ragoubi , zidi , and bouharrag ) and between the 6 different types of cuttings ( age , length , and diameters ) of the cultivar bither . these variations were influenced by ability of cultivar for rooting and characteristics of cuttings ( length , diameter , and age ) . furthermore , practices of farming ( soil preparation , irrigation , harrowing , etc . ) and climate conditions were cited to have an effect on the quality of fig plants in the nursery [ 13 , 18 ] . several factors seem to be involved in the improvement of fig hardwood cutting like the use of rooting hormonal treatment and the use of plastic tunnels in fig propagation . the present research showed that the hardwood cuttings of local cultivars of figs can be used to propagate material in field conditions . furthermore , it is possible to obtain vigorous young plants ( length , number of leaves , and cumulative growth ) from cuttings of cultivars bouharrag ( caprifig ) , bayoudhi ( common fig ) , rogabi , and zidi ( smyrna fig ) . bither 's rooting ability of the 6 cutting types studied revealed that the cutting type of one - year old characterized by 60 cm of length and diameter of < 1.5 cm gave the best percentage of rooting emergence . also , the best cumulative growth of plant was obtained with the cutting type ( 2 years old shoots , length ( 40 cm ) , and diameter ( < 1.5 cm ) ) . the positive aspects of this experiment have demonstrated the suitability of the hardwood cuttings method for vegetative propagation , since the production of fig plants in nursery can be realized and seems to be cheap . this work is supposed to be a preliminary positive contribution in the propagation of fig in nursery which can have practical application for fig horticulturists in the southeast of tunisia .

this research was carried out in southeast of tunisia in 2009 and 2010 , in order to study the propagation of six ( ficus carica l. ) cultivars by using hardwood cuttings under the field conditions . the effect of the cultivars and the type of buds , shoots age , shoots length , and shoots diameter were recorded . ten cuttings per cultivar and/or cutting types with three replications were planted in rooting unit . percentage of root emergence and six morphological parameters of young fig plants were measured . results showed that the responses of cuttings as fig nursery plants presented a high variability among the five cultivars . the most widely varied characters were % root emergence ( re ) and cumulative growth of young plant ( cg ) . the first one ranged from 10% to 90% , the second varied within 32 and 112 cm . concerning the bither cultivar , 6 cutting types with different age , length , and diameter were evaluated . results showed a great variation in % of root emergence ( 090% ) , length of nursery plant ( 377 cm ) , and number of roots / nursery plant ( 029 roots ) . the present research showed that the hardwood cutting of local fig cultivars can be propagated under field conditions in southeast of tunisia .

1. Introduction 2. Material and Methods 3. Results 4. Discussion and Conclusions

unintentional durotomy is a frequent complication of spinal surgical procedures , with a rate as high as 17% ( 1 ) . a small percentage of patients with unintentional durotomy sustain intracranial haemorrhage , likely due to an alteration in intradural hydrostatic dynamics caused by cerebrospinal fluid hypovolaemia . despite the gravity of this complication , to our knowledge , only a few cases have been previously documented . to our knowledge a case of new onset epilepsy has never been reported in the literature . the purpose of this report is to describe a case in which new - onset tonic - clonic seizures occurred following an unintentional durotomy during lumbar discectomy and to discuss the importance of our findings in relation to the possible pathophysiological mechanisms . a 62 year old female presented to a spinal orthopaedic clinic complaining of pain in the anterior aspect of her right thigh for at least a year . she could only walk for 15 minutes , thus this limited the patient s activities of daily living . medical history was unremarkable but for bipolar depression which was treated by electroconvulsive therapy and anti - psychiatric medication . 14 ) showed l2/3 disc prolapse impinging on the right exit canal and causing l2 and l3 compression , thus she was listed for l2/3 lumbar discectomy and decompression . examination of the right l2/l3 dermatomes revealed severe paresthesia and mild grade 4/5 weakness ; reflexes were all normal . preoperative sagittal t1 weighted image showing disc bulge at l2/3 preoperative sagittal t2 weighted image showing disc bulge at l2/3 elective bilateral l2/l3 lumbar discectomy and l2/l3 decompression was performed . preoperative axial t2 weighted image showing disc bulge at l2/3 to the right . during dissection , this was primarily sutured and covered with duragen ( integra lifesciences , plainsboro , new jersey ) . post - operatively the patient was well ; she experienced a few headaches but still had no neurological deficit . the patient was recovering well when suddenly , 48 hours post - operatively , the patient experienced four generalized tonic - clonic seizures , and was deeply post - ictal following the seizures . a diagnosis of tonic - clonic seizures secondary to low intracranial pressure was made . all routine blood investigations as well as blood amisulpride and olanzapine levels carried out on all post - operative days were found to be within normal limits . a post - operative head / brain computed tomographic ( ct ) scan showed no infarct , haemorrhage or mass lesions . the patient was started on oral phenytoin 3mg / kg daily and had no further episodes of seizures . she never reported any problems during the duration of follow - up and was successfully discharged back to the community . dural tears during spinal surgery are not uncommon , with reported incidence rates of 0.3% to 17% ( 1 , 2 ) . problems arising from this complication are rare , but devastating and require astute recognition of the early signs of neurological impairment . here we are attempting to make an association between the csf hypotension caused by an unintentional durotomy at lumbar surgery and the new onset of epilepsy which might have been propagated by the patients pre - operative anti - psychiatric medication . the phenomenon of intracranial haemorrhage remote from a surgical site has been described in the literature , and several authors have attempted to explain the pathophysiology ( 1 , 2 ) . although intracranial haemorrhage did not occur in this case , the same pathophysiology is thought to have contributed to provoke the new onset epilepsy . the so - called sag model postulates that haemorrhage results from altered cerebrospinal fluid hydrodynamics , causing caudal sagging of the cerebellum with stretching of cerebellar vermian veins ( 1 ) . taking this pathophysiology into account this was satisfactorily repaired intra - operatively , however , the csf leak was substantial and the dura may have been weakened and prone to later leak . we hypothesize that this may have resulted in delayed intracranial hypotension , causing primary tonic - clonic seizures and not an intra - cranial bleed . thus , we believe that the hypotensive insult coupled with an already lowered seizure threshold , due to the patient s long term anti - psychiatric medical history , gave rise to these new onset tonic - clonic seizures . we are unaware of any cases of new onset epilepsy post - durotomy . although dural rupture during surgery and csf hypovolemia are thought to be the main contributing factors to the above , we have great reservations about the types of chronic medications the patient was having . one may only postulate about the association between both olanzapine and amisulpride and their epileptogenic side effects . in the british national formulary a case report published by woolley and smith ( 3 ) shows that atypical neuroleptics like olanzapine and amisulpride lower seizure threshold . manufacturer 's trials gave a seizure rate , similar to other antipsychotics , of 0.88% patients ( product data sheet , eli lilly ) . in this article it is stated that the patient in question represents the strongest case to date implicating olanzapine alone in lowering seizure threshold , with objective eeg support . the manufactures data for both drugs issue warnings in their literature stating that both amisulpride and olanzapine can lower seizure threshold and therefore should be used with caution in patients previously diagnosed with epilepsy . however , they do not state that in cases where the seizure threshold is already lowered for example by csf hypotension , new onset epilepsy might be more common ( 4 ) . in conclusion , new onset seizures following dural tears might appear in patients on anti - psychiatric medication . we strongly recommend that patients on potentially epileptogenic medication are either changed to other forms of anti - psychiatric non - epileptogenic medication or are closely monitored post - operatively in the event of this rare complication developing .

we would like to present a rare case report describing a case in which new - onset tonic - clonic seizures occurred following an unintentional durotomy during lumbar discectomy and decompression . unintentional durotomy is a frequent complication of spinal surgical procedures , with a rate as high as 17% . to our knowledge a case of new onset epilepsy has never been reported in the literature . although dural tears during surgery and csf hypovolaemia are thought to be the main contributing factors , one postulates on the effects of anti - psychiatric medication with epileptogenic properties . amisulpride and olanzapine can lower seizure threshold and should be used with caution in patients previously diagnosed with epilepsy . however manufacturers do not state that in cases where the seizure threshold is already lowered by csf hypotension , new onset epilepsy might be commoner . finally , strong caution and aggressive post - operative monitoring is advised for patients with csf hypotension in combination with possible epileptogenic medication .

INTRODUCTION CASE REPORT DISCUSSION

it seems that complications of vitamin d deficiency have been completely removed by having enriched food by vitamin d , but rachitism is the top of iceberg of vitamin d deficiency . vitamin d deficiency can also causes osteopenia , osteoporosis , and muscle weakness and increases risk of hip fracture in adults . the most important effect of vitamin d is on parathyroid and bone and also plays a key role on the calcium absorption from the gut . the discovery of vitamin d receptor in most tissues and organs has developed a new perception from effects of vitamin d. for instance , vitamin d can decrease risk of many chronic diseases , cancers , autoimmune disease , infectious , and cardiovascular disease . it has been estimated that one billion people are vitamin d deficient in the world . this deficiency is common in all different cities with the same level of the sea or cities with lower geographic latitude . 20 ng / ml is the level of vitamin d deficiency and 20 < 25 ( oh ) d < 30 ng / ml is recognized as a vitamin d insufficiency . there are different ways to detect cut - off points for vitamin d deficiency . for instance , based on inverse relation between 25 ( oh ) d and pth , a direct relation between 25 ( oh ) d and 1 , 25 ( oh ) d and effect of vitamin d on fracture risk . the level of 25 ( oh ) d that would be enough for pth suppression , probably , is a suitable criterion for bone health and cut - off point for vitamin d deficiency . the aim of this study was detection of prevalence of vitamin d deficiency in secondary students in arak , a centrally located city in iran , and also detection of cut - off points for vitamin d deficiency . in a cross - sectional study in november 2010 , 420 students including 220 girls and 200 boys from secondary students from 10 to 16 years old were selected by a multistage sampling . first , all of the secondary schools were divided into two areas and then 21 schools including 11 girls schools and 10 boys , schools were selected by a cluster sampling . finally , the samples were picked up by a systematic random procedure based on the student 's initials . all the students and their parents were informed about the study and signed off the consent form . inclusion criteria were lack of hepatic , renal , bone disease and cancer , no history of intake of anticonvulsant drugs , calcium , vitamin d , and antineoplastic drugs . laboratory tests including hepatic , renal , calcium , phosphoros , alp ( alkaline phosphatase ) , albumin , pth , 25 ( oh ) d. 25 ( oh ) d levels by ria and pth by irma were measured ( made by bio source europe sa , belgium ) and its intensity categorized into three levels . mild deficiency : 15 25 ( oh ) d < 20 ng / ml.moderate deficiency : 8 25 ( oh ) < 15 ng / ml.severe deficiency : 25 ( oh ) < 8 ng / ml . mild deficiency : 15 25 ( oh ) d < 20 ng / ml . moderate deficiency : 8 25 ( oh ) < 15 ng / ml . severe deficiency : 25 ( oh ) < 8 ng / ml . the relation between vitamin d and pth was assessed by the scatter chart . the level of vitamin d that pth was increasing , defined as cut - off points for vitamin d deficiency . the prevalence of vitamin d deficiency in different intensities was assessed by spss 15 and variables compared between girls and boys , and also between deficient and sufficient vitamin d groups by student 's test or mann -- whitney u based on normal distribution based on k -- s ( kolmogrov -- smirnov ) test . the pearson test was used to identify the relation between 25 ( oh ) d and age , weight , and bmi . a total of 420 students including 46.9% boys and 53.1% girls participated in this study . mean age for boys and girls was 12.5 1 and 12.7 1 years old respectively p = 0.1 [ table 1 ] . mean characteristic of students ( mean sd ) mean age of vitamin d - deficient and nondeficient groups based on cut - off 25 ( oh ) d < 20 ng / ml were 12.6 1 and 12.6 2 years old respectively ( p = 0.5 ) . but mean bmi in two groups were 20 3.5 and 19 2.1 respectively ( p = 0.02 ) . there was no meaningful relation between 25 ( oh ) d and age ( p = 0.5 ) but there was an inverse relation between 25 ( oh ) d level and bmi ( r = -0.17 ) ( p = 0.01 ) , weight ( r = -0.14 ) ( p = 0.05 ) , pth ( r = -0.262 ) ( p = 0.001 ) . there was also a meaningful relation between vitamin d deficiency and sex ( p = 0.001 ) . based on the logistic regression analysis , the girls have 54.4 times more chance than boys for vitamin d deficiency ( or 54.4 ) ( p < 0.001 ) . totally 84% of the students had vitamin d deficiency [ table 2 ] . three cut - off points 33 , 21 , and 13 ng / ml were defined for mild , moderate , and severe vitamin d deficiency [ figure 1 ] using the scatter chart . the prevalence of vitamin d deficiency based on different intensities was evaluated in both genders [ table 3 ] . prevalence of vitamin d deficiency in students ( number/% ) mean of pth of different cut - off points for vitamin d deficiency prevalence of vitamin d deficiency based on local cut - off points ( % ) our findings in arak , a centrally located city of iran , showed a high prevalence of vitamin d deficiency of 84% among the students . the results showed that females were more likely to be vitamin d - deficient than males . the present study suggests three local cut - off points for vitamin d deficiency based on 25 ( oh ) d < 13 , 21 , and 33 ng / ml . vitamin d deficiency is a common medical problem throughout the world . it has been predicted that one billion people throughout the world are vitamin d deficient . prevalence of vitamin d deficiency in the countries where food is enriched by vitamin d ( scandinavian countries ) is 1.614.8% . in other european countries , among middle - aged and elderly people , 14 - -59.6 in tunessia 47.6% and finally among teenagers in boston was 24.1% . a total of 30 - -50% of people in india , lebanon , and turkey and also 45.2% of females in china were vitamin d deficient . several studies in different parts of iran and among different age groups have shown a high prevalence of vitamin d deficiency.[1114 ] prevalence of vitamin d deficiency in tehran ( the capital city of iran ) was 60% in males and 91% in females . the prevalence of vitamin d deficiency in isfahan , another central city of iran , was 46.2% among students including 72.1% girls and 18.3% boys and is similar to our findings in tabriz , another city in the north west of iran , which was 64.2% among females ( mild 33.7% , moderate 15.5% , severe 15.1% ) that in comparison with the present study is lower . another study in different cities of iran , with different climate , showed that 27.2% of females and 37.25% of males had a mild vitamin d deficiency , and also 47.85% of females and 34.75% of males had moderate to severe vitamin d deficiency that confirms the present study . different etiologies , such as insufficient sun exposure , clothing habits , and insufficient intake of vitamin d , may explain high prevalence of vitamin d deficiency . it has been shown that 2-years intake of calcium and vitamin d , increased serum 25 ( oh ) d level up to 28% . serum 25 ( oh ) d levels were lowest in blacks and highest in white people . although some studies found that sun expose and clothing habits were not different between sufficient and deficient groups . decreased calcium intake lead to increased serum pth level and increased catabolism of 25 ( oh ) d. awumey et al . showed higher activity of 24-hydroxylase in fibroblasts that lead to increased catabolism of 25 ( oh ) d of indian americans . the darker skin of the asian immigrants to european countries is another risk for vitamin d deficiency . found that 61% of the turkish group ( in turkey ) and 55% of the turkish immigrant group in germany had bone and muscle pain , while it was 15% within the german group with higher serum 25 ( oh ) d levels . our findings showed that the prevalence of vitamin d deficiency is much more in girls than boys ( girls 99.1% , 66.5% boys ) ( p < 0.001 ) . another study in iran , isfahan , also found that the prevalence of vitamin d deficiency was four times more in girls than boys and severe vitamin d deficiency in girls and boys were 14.5% and 0.6% respectively . another study in iran also found that moderate - to - severe vitamin d deficiency was much more in females than males . differences according to gender and age group maybe because of biological differences but might also due to behavioral differences . there might be gender - specific clothing differences ( necessarily not based on a culture religious ) . there also might be that females spend less time outside , resulting in less exposure to the sun . another study in the united states among female soldiers found that vitamin d levels decrease during military training in the summer due to the type of clothing worn during training , coupled with inadequate vitamin d intake . our study found that vitamin d deficiency was not associated with the age , while most studies found higher prevalence of vitamin d deficiency among elderly , probably due to lower capacity of the skin to produce vitamin d after sun exposure or less vitamin d intake in elderly , although a few studies reported higher prevalence of vitamin d deficiency in young people . our study shows a significant inverse association between bmi ( body mass index ) and weight in one hand with vitamin d deficiency in another hand , same as the tabriz study and against the isfahan study . considering the role of fat tissue as a depot of vitamin d , obese have increased storage capacity of 25 ( oh ) d , that leads to lower serum vitamin d levels . we determined three cut - off points for vitamin d deficiency based on 25 ( oh ) d < 33 , 21 , and 13 ng / ml for mild , moderate , and severe vitamin d deficiency , using scatter chart , versus 12.5 , 25 , and 40 ng / ml in another study in iran and 32 ng / ml in isfahan . high prevalence of vitamin d deficiency probably could be the result of above - mentioned combination . we recommend that some efforts could be made to encourage people to intake vitamin d and calcium supplements and also have more exposure to the sunlight .

introduction : vitamin d has a basic role in bone growth and metabolism and has been noticed for its important role in many diseases , such as diabetes , depression , hypertension , and cardiovascular disease . regarding some studies , detection of vitamin d deficiency in different places has important implication for health . this study determined prevalence of vitamin d deficiency in arak , a centrally located city in iran.materials and methods : based upon a cross - sectional study in 2010 , 420 students 10 - -16 years old including 220 girls and 200 boys , studied at arak secondary schools , were selected by a multistage sampling . the level of 25 ( oh ) d and pth ( parathormone ) was measured and also the prevalence of vitamin d deficiency in different intensities was evaluated and compared between girls and boys by the student t - test . vitamin d deficiency for the students was categorized into three intensities based on three levels of 25 ( oh ) d : mild deficiency - 15 25 ( oh ) d < 20 ng / ml , moderate deficiency -- 8 25 ( oh ) < 15 ng / ml , severe deficiency - 25 ( oh ) < 8 ng / ml . the relationship between the 25 ( oh ) d and pth was assessed by scatter chart to define cut - off points for vitamin d deficiency.results:based on 25 ( oh ) d < 20 ng / ml , 84% of the students had vitamin d deficiency including 60.7% mild , 22.4% moderate , and 0.7% severe . three local cut - off points including 13 , 21 , and 33 ng / ml were defined in the scatter chart . the results showed that the deficiency of vitamin d in girls is much more than that in boys ( p = 0.05).conclusion : the high prevalence of vitamin d deficiency could be probably a result of different etiologies . we recommended that people should be encouraged to take vitamin d and calcium supplements and also they should have more exposure to the sunlight .

I M R D C

prior to the availability of potent antiretroviral ( arv ) therapy , human immunodeficiency virus type 1 ( hiv-1 ) infection was notable for the inevitable progression to acquired immunodeficiency syndrome ( aids)-defining events and death . with the advent of mono- and dual - nucleoside reverse transcriptase inhibitor ( nrti ) therapy , there was recognition of diverse drug - related adverse events . the introduction of combination arv therapy with dual nrtis plus protease inhibitors ( pis ) was associated with a dramatic decline in aids and mortality , along with an increased appreciation for the development of diverse metabolic complications , such as insulin resistance and dyslipidemia , as well as more recent reports actually showing evidence of premature cardiovascular disease . in addition , co - morbid conditions such as renal and hepatic disease increasingly influenced the quality of the lives of hiv-1-infected individuals . over the ensuing years research has attempted to define the potential role that select arv agents and hiv-1 infection itself have on hepatic , renal , and cardiovascular disease . numerous factors are known to be associated with increased risk of atherosclerotic disease , such as diabetes mellitus , hypertension , smoking , family history and dyslipidemia , all of which occur with variable frequency in those with hiv-1 infection . as combination arv therapy allowed hiv-1-infected individuals to live longer , moreover , concerns are enhanced by the association between select arv agents and insulin resistance , dyslipidemia and fat maldistribution , along with case reports of premature cardiovascular disease . while there is little evidence clearly demonstrating an association between any specific drug and visceral adiposity , lipoatrophy does appear to be a hiv-1-specific condition primarily linked to the use of thymidine analogues such as zidovudine and stavudine . similarly , it is now clear that lipid abnormalities have been seen with select nrtis , non - nrtis , and pis . in fact , there are specific guidelines for the management of dyslipidemia in hiv-1-infected individuals . while several studies have reported a relationship between arv use and cardiovascular events , the d : a : d ( data collection on adverse events of anti - hiv drugs ) cohort is the largest longitudinal study specifically designed to address this question . this study has provided the best evidence that there is an independent relationship between duration of combination arv therapy and cardiovascular events , with most of the effect initially being linked to the use of pis . more recent analyses of this cohort have addressed the relationship between nrti use and cardiovascular disease . here they showed that the recent use of abacavir and , to a lesser extent , didanosine was associated with increased risk of cardiac events when compared to regimens that did not use these drugs . notably the absolute risk was small for all groups and was most prominent amongst those with other cardiovascular risk factors . a follow - up analysis presented at the 16 conference on retroviruses and opportunistic infections showed that there was also an independent association between cardiovascular events and the use of lopinavir / ritonavir and indinavir , while no such relationship was seen with tenofovir disoproxil fumarate ( tenofovir df ) or the non - nrtis efavirenz and nevirapine . it is important to note that while cohorts can include large numbers of longitudinally followed patients , they are not randomized controlled trials and therefore have numerous limitations . that being said , a similar association between abacavir use and cardiovascular events was recently reported from the smart ( strategies for management of anti - retroviral therapy ) study and a case control study of patients in the anrs ( national agency for aids research ) french hospital cohort . nevertheless , questions remain since there has been no definitive biological explanation given for these findings and other studies have not demonstrated such associations . intriguing data have recently emerged to suggest a relationship between ongoing viremia and cardiovascular disease . data from the smart study , designed to assess whether treatment interruption in those with higher levels of cd4 t cells could minimize some of the toxicity associated with arv therapy , actually showed overall increased risk of several non - aids events , including cardiovascular , renal and hepatic disease , amongst those that interrupted treatment . further analyses from the smart study have demonstrated that markers of inflammation , such as interleukin-6 ( il-6 ) , and coagulation , such as d - dimers , are increased in those off arv therapy , all of these markers having been shown in other populations to be associated with increased risk of cardiovascular events . stored samples from the smart study have further been used to link these changes in markers of inflammation with abacavir use . in a cross - sectional analysis of patients either on or off abacavir at baseline , the investigators showed increased levels of high sensitivity c - reactive protein and il-6 in those on abacavir compared to those not taking this drug . however , another study showed different results , demonstrating that related markers declined to a similar extent in both arms in a randomized controlled trial comparing tenofovir df / emtricitabine to abacavir / lamivudine . these differences may reflect the difficulty in observing a potentially modest relationship between select arv agents and markers of inflammation in the face of the overwhelming influence that suppressing plasma hiv-1 rna has on these same measures . while results are inconclusive , other potential markers associated with increased cardiovascular disease have also been evaluated as explanations for the possible relationship between select arv agents and cardiovascular events , such as endothelial function and platelet aggregation . while there remains considerable uncertainty as to how these various data sets should be interpreted , these observations have led to new research exploring the relationship between hiv-1 replication and potential pathogenic mechanisms for select end - organ events . renal disease is common amongst those with hiv-1 infection and is often multifactorial ; possible factors include hiv-1-associated nephropathy and co - morbid conditions such as diabetes mellitus and hypertension , as well as co - infection with hepatitis b and c . certain arv drugs have also been associated with nephrotoxicity , such as indinavir , a drug rarely used in the current era that frequently caused nephrolithiasis and occasionally interstitial nephritis . in addition , tenofovir df has been linked to the development of proximal renal tubular dysfunction . it is clearly recommended that routine monitoring of renal function should occur in all hiv-1-infected patients , with particular attention given to those with co - morbid conditions or taking nephrotoxic drugs . one recent study attempted to define the relationship between renal function , tenofovir df use , and the degree of plasma hiv-1 rna suppression . this was a relatively small cohort study showing that those with complete viral suppression on a tenofovir df - containing regimen actually experienced an increase in glomerular filtration rate . in contrast , there was a small but significant decline in renal function amongst those on tenofovir df that did not achieve full virologic suppression . the authors hypothesize that ongoing viremia , and perhaps the associated increase in inflammation , could be contributing to these adverse outcomes . other studies have shown a similar relationship between hiv-1 replication and progression of renal disease . the overwhelming burden of hepatic disease in hiv-1-infected individuals is related to co - infection with hepatitis b and c . hiv-1 and hepatitis co - infection treatment guidelines provide detailed information about relevant interactions between these chronic viral infections and how co - infection influences the management of each pathogen . there is also an increased risk of hepatic steatosis that may be associated with hyperlipidemia , insulin resistance and select arv agents . several arvs have also been shown to result in hepatotoxicity , the strongest links being with high - dose ritonavir , rarely used in the current treatment era , and tipranavir and nevirapine , the latter being in association with immunologic reactions that can be minimized by avoiding use in men with > 400 cd4 t - cells/l and women with > 250 cd4 t cells/l . there have been a few case reports of significant hepatotoxicity with the ccr5 antagonist maraviroc , but this has not been clearly seen in the pivotal randomized controlled trials . while there was some increased risk of hepatotoxicity associated with treatment interruption in the smart study , there are currently much fewer data linking ongoing hiv-1 replication to liver abnormalities than what has been described for cardiovascular and renal disease . numerous factors are known to be associated with increased risk of atherosclerotic disease , such as diabetes mellitus , hypertension , smoking , family history and dyslipidemia , all of which occur with variable frequency in those with hiv-1 infection . as combination arv therapy allowed hiv-1-infected individuals to live longer , moreover , concerns are enhanced by the association between select arv agents and insulin resistance , dyslipidemia and fat maldistribution , along with case reports of premature cardiovascular disease . while there is little evidence clearly demonstrating an association between any specific drug and visceral adiposity , lipoatrophy does appear to be a hiv-1-specific condition primarily linked to the use of thymidine analogues such as zidovudine and stavudine . similarly , it is now clear that lipid abnormalities have been seen with select nrtis , non - nrtis , and pis . in fact , there are specific guidelines for the management of dyslipidemia in hiv-1-infected individuals . while several studies have reported a relationship between arv use and cardiovascular events , the d : a : d ( data collection on adverse events of anti - hiv drugs ) cohort is the largest longitudinal study specifically designed to address this question . this study has provided the best evidence that there is an independent relationship between duration of combination arv therapy and cardiovascular events , with most of the effect initially being linked to the use of pis . more recent analyses of this cohort have addressed the relationship between nrti use and cardiovascular disease . here they showed that the recent use of abacavir and , to a lesser extent , didanosine was associated with increased risk of cardiac events when compared to regimens that did not use these drugs . notably the absolute risk was small for all groups and was most prominent amongst those with other cardiovascular risk factors . a follow - up analysis presented at the 16 conference on retroviruses and opportunistic infections showed that there was also an independent association between cardiovascular events and the use of lopinavir / ritonavir and indinavir , while no such relationship was seen with tenofovir disoproxil fumarate ( tenofovir df ) or the non - nrtis efavirenz and nevirapine . it is important to note that while cohorts can include large numbers of longitudinally followed patients , they are not randomized controlled trials and therefore have numerous limitations . that being said , a similar association between abacavir use and cardiovascular events was recently reported from the smart ( strategies for management of anti - retroviral therapy ) study and a case control study of patients in the anrs ( national agency for aids research ) french hospital cohort . nevertheless , questions remain since there has been no definitive biological explanation given for these findings and other studies have not demonstrated such associations . intriguing data have recently emerged to suggest a relationship between ongoing viremia and cardiovascular disease . data from the smart study , designed to assess whether treatment interruption in those with higher levels of cd4 t cells could minimize some of the toxicity associated with arv therapy , actually showed overall increased risk of several non - aids events , including cardiovascular , renal and hepatic disease , amongst those that interrupted treatment . further analyses from the smart study have demonstrated that markers of inflammation , such as interleukin-6 ( il-6 ) , and coagulation , such as d - dimers , are increased in those off arv therapy , all of these markers having been shown in other populations to be associated with increased risk of cardiovascular events . stored samples from the smart study have further been used to link these changes in markers of inflammation with abacavir use . in a cross - sectional analysis of patients either on or off abacavir at baseline , the investigators showed increased levels of high sensitivity c - reactive protein and il-6 in those on abacavir compared to those not taking this drug . however , another study showed different results , demonstrating that related markers declined to a similar extent in both arms in a randomized controlled trial comparing tenofovir df / emtricitabine to abacavir / lamivudine . these differences may reflect the difficulty in observing a potentially modest relationship between select arv agents and markers of inflammation in the face of the overwhelming influence that suppressing plasma hiv-1 rna has on these same measures . while results are inconclusive , other potential markers associated with increased cardiovascular disease have also been evaluated as explanations for the possible relationship between select arv agents and cardiovascular events , such as endothelial function and platelet aggregation . while there remains considerable uncertainty as to how these various data sets should be interpreted , these observations have led to new research exploring the relationship between hiv-1 replication and potential pathogenic mechanisms for select end - organ events . renal disease is common amongst those with hiv-1 infection and is often multifactorial ; possible factors include hiv-1-associated nephropathy and co - morbid conditions such as diabetes mellitus and hypertension , as well as co - infection with hepatitis b and c . certain arv drugs have also been associated with nephrotoxicity , such as indinavir , a drug rarely used in the current era that frequently caused nephrolithiasis and occasionally interstitial nephritis . in addition , tenofovir df has been linked to the development of proximal renal tubular dysfunction . it is clearly recommended that routine monitoring of renal function should occur in all hiv-1-infected patients , with particular attention given to those with co - morbid conditions or taking nephrotoxic drugs . one recent study attempted to define the relationship between renal function , tenofovir df use , and the degree of plasma hiv-1 rna suppression . this was a relatively small cohort study showing that those with complete viral suppression on a tenofovir df - containing regimen actually experienced an increase in glomerular filtration rate . in contrast , there was a small but significant decline in renal function amongst those on tenofovir df that did not achieve full virologic suppression . the authors hypothesize that ongoing viremia , and perhaps the associated increase in inflammation , could be contributing to these adverse outcomes . other studies have shown a similar relationship between hiv-1 replication and progression of renal disease . the overwhelming burden of hepatic disease in hiv-1-infected individuals is related to co - infection with hepatitis b and c . hiv-1 and hepatitis co - infection treatment guidelines provide detailed information about relevant interactions between these chronic viral infections and how co - infection influences the management of each pathogen . there is also an increased risk of hepatic steatosis that may be associated with hyperlipidemia , insulin resistance and select arv agents . several arvs have also been shown to result in hepatotoxicity , the strongest links being with high - dose ritonavir , rarely used in the current treatment era , and tipranavir and nevirapine , the latter being in association with immunologic reactions that can be minimized by avoiding use in men with > 400 cd4 t - cells/l and women with > 250 cd4 t cells/l . there have been a few case reports of significant hepatotoxicity with the ccr5 antagonist maraviroc , but this has not been clearly seen in the pivotal randomized controlled trials . while there was some increased risk of hepatotoxicity associated with treatment interruption in the smart study , there are currently much fewer data linking ongoing hiv-1 replication to liver abnormalities than what has been described for cardiovascular and renal disease . a thorough understanding of the relationship between different drugs and various adverse events is critical to the optimal management of such patients . the first step towards safely using any medication is to know what conditions any given individual is predisposed to , to understand the safety profile of each drug , and to monitor for adverse events . in the case of cardiovascular disease it is important to emphasize efforts to modify known risk factors and to monitor and manage dyslipidemia . when lipid abnormalities are present , clinicians should be aware of how different medications may be contributing to these problems and should consider changes in therapy when appropriate . the emerging data linking select drugs to cardiovascular disease , while not definitive , should also be considered when making any clinical decision , particularly in those with other risk factors . similar considerations apply to underlying renal disease , where control of traditional risk factors such as diabetes mellitus and hypertension should be prioritized along with careful monitoring and avoidance of nephrotoxic drugs in those at greatest risk . with regards to liver disease , the best strategy is to diligently screen for hepatitis b and c co - infection , minimize use of hepatotoxic agents and to provide immunization against viral hepatitis when appropriate . in addition , all patients should be carefully monitored for liver disease , with a particular focus on those taking hepatotoxic agents and who are hepatitis b and/or c co - infected . the recent data linking ongoing hiv-1 replication with cardiovascular disease , and possibly renal disease , are provocative , and the association remains an area of increasing investigation . clinicians should be aware of these studies , their potential implications with regards to the use of arv therapy , and how these findings might support the earlier initiation of treatment in a given individual . however , this needs to be balanced by the fact that the studies remain preliminary and the results thus far have been mostly hypothesis generating . furthermore , decisions regarding the timing of treatment must be made in the context of the specific patient to be treated as well as the overall costs and known risks associated with the use of arv therapy . he is a scientific consultant for abbott , bristol myers squibb , gilead , glaxosmithkline , merck , pfizer , schering plough , pathway , tibotec and serves on a data safety monitoring board for ardea biosciences .

the first 15 years of the human immunodeficiency virus type 1 epidemic was characterized by patients progressing to clinical acquired immunodeficiency syndrome and death . the availability of potent antiretrovirals led to the recognition of unique adverse events associated with select drugs . more recent data suggest that end - organ damage may be associated with ongoing viremia . further understanding of the potential role different drugs and the virus itself has on various organs can enhance the clinician 's ability to manage patients in the clinic .

Introduction and context Recent advances Cardiovascular disease Renal disease Hepatic disease Implications for clinical practice Competing interests

surgical instruments and materials continue to be retained in the peritoneal cavity despite precautionary measures . even though uncommon it is also under - reported and carries serious medico - legal consequences . gauzes and sponges ( gossypiboma ) are the most commonly retained materials and intra - abdominal retained artery forceps are much rarer but when they do occur lead to chronic abdominal pain and can be a rare cause of intestinal obstruction or strangulation with significant morbidity and mortality . we present a case of intraabdominal retained artery forceps in a 70-years - old lady who underwent laparotomy with splenectomy for a large spleen in a peripheral hospital . upon discharge 12 months later she presented to us with an acute ( surgical ) abdomen requiring another laparotomy . at laparotomy she had strangulated / gangrenous lower jejunual and upper ileal bowel loops , the small bowel mesentery of this area being tightly trapped between the jaws of the retained artery forceps retained instruments in intra - abdominal surgery can cause serious complication and should be treated surgically . high index of suspicion and appropriate investigations like plain abdominal x - ray , abdominal ultrasound and ct and mri scans should be instituted in patients who develop chronic abdominal symptoms following laparotomy . preventive measures against retained instruments the rarity of reported cases may be due to medico - legal implications or the surgeon 's unwillingness to advertise his errors . the incidence of retained foreign body in literature is 1 per 1000 to 1500 laparotomies . locally here , the incidence is not documented but it is suspected that the incidence is of a higher magnitude bearing in mind the level of socio - economic development in our environment . with increasing awareness of the public to their rights and privileges , this medico - legal problem will in the near future retained foreign body in peritoneal cavity is one of the known but avoidable complications of abdominal surgery . it is more liable to occur when surgery is done in a non - ideal environment and negligence or ignorance on part of the medical personnel . it is the responsibility of the operating surgeon and the theatre staff to ensure that no foreign body is left inside the peritoneal cavity before closure of the abdominal cavity . retained foreign bodies are of various types and include sponges / gauzes ( gossypiboma or texilloma ) , artery forceps , other metal instruments , surgical needles , surgical drains , and rubber tubes . the surgical sponge / gauze ( gossiypiboma ) constitutes the most frequently encountered object because of its common usage , small size and amorphous structure . exudative reaction will give rise to abscess formation resulting in peritonitis , fistula formation , tender abdominal mass and gut perforation . in contrast retained artery forceps remain inert and induce their effects through mechanical means giving rise to pressure effects resulting in abdominal pain or direct tissue / viscus injury leading to tissue strangulation or perforation of viscus leading to for example peritonitis or vascular injury resulting in hemorrhage . the clinical presentation of retained intra - abdominal foreign material may be acute or delayed . such patient present in the surgical units with increasing abdominal pain , abdominal mass , discharging sinus , intra - abdominal abscesses and acute or sub - acute intestinal obstruction . whilst many patients will present within days or few weeks after the initial surgery and have a re - laparotomy for retrieval of the foreign body , some patients with intra - abdominal foreign material may go unnoticed for years or even decades . however many of this subset of patients will have chronic abdominal symptoms such as abdominal pain or discomfort . the pre - operative diagnosis is based on history , clinical examination , plain abdominal radiographs , ultrasound , ct and mri scans . yet generalized peritonitis , systemic complications and presentation of acute intestinal obstruction are associated with high mortality . we present a case of retained artery forceps causing intestinal strangulation managed in our centre and review the possible factors that led to the retained artery forceps and advice on possible preventive measures . a 70yr old woman ( e.n ) presented to our accident and emergency unit as a referral from a cottage ( peripheral ) hospital with a complaint of sudden exacerbation of her chronic abdominal pain , associated now with abdominal distension , vomiting , absolute constipation and fever of four days duration . 12 months prior to presentation she had laparotomy with splenectomy for large spleen suspected to be due to tropical splenomegaly syndrome ( tss ) . few weeks following the splenectomy , she started complaining of intermittent abdominal pains of worsening severity which culminated in her present complaint . she had paid severe visits to the surgeon who did her splenectomy and had analgesis and antispasmodics prescribed and was always reassured that the abdominal pain will abate with time . examination revealed an acutely ill looking , toxic , pale and dehydrated elderly lady in respiratory distress , blood pressure of 80/50mmhg , pulse rate of 120/min and low volume , respiratory rate 40/min and a temperature of 38.4c . abdominal examination revealed a distended abdomen with a midline scar from the epigastrum to the symphysis pubis . chest examination revealed severe dyspnoea and bilateral basal rales . a clinical diagnosis of generalized peritonitis complicating intestinal obstruction , septic shock and acute respiratory distress syndrome ( ards ) were made . resuscitation was started with iv saline infusions , iv ceftriazone ( third generation cephalosporins ) , iv metronidazole , nasogastric tube suction , and urethral catheterization ( for urinary output monitoring ) . investigation showed packed cell volume of 22% , urea of 11.5 mmol / l , normal electrolytes and urinalysis . plain chest and abdominal ( erect and supine ) x - ray were ordered but were not done because the poor state of patient prevented her movement to the x - ray suit . however , she had abdominal pelvic ultrasound , which showed dilated bowel loops and large volume of intraperitoneal fluid . the jaws of a large size artery forceps was seen tightly trapping the small bowel mesentery from its distal extremity to half way up towards the duodeno - jejunal flexure with the tips of forceps pointing toward the duodeno - jejunal junction and the handle located in the right iliac fossa . this led to ischemic gangrene of about 65 cm of the small bowel involving the distal third of jejunum and upper two thirds of the ileum . there were no pulsations on the ileal and jejunal arteries distal to the trapping artery forceps ( figures 1 and 2 ) . the jaws of the artery is seen trapping the small bowel mesentery with the handle in the right iliac fossa and the tip towards the duodeno - jejunal junction . loops of gangrenous small bowel are held up by an assistant above the artery forceps showing artery forceps across small bowel mesentery . the patient had a turbulent post- operative phase and was managed in our critical care unit ( ccu ) . however , 12-hours post - operatively she died most probably from continuing sepsis . despite the awareness of the dangers of and the precautionary measures aimed to prevent them retained instruments continue to occur especially in the peritoneal cavity . retained foreign body in a patient after surgery is an uncommon but dangerous error and it is grossly under estimated . the rarity of the reported cases may be due to medico - legal reasons or the surgeon 's unwillingness to advertise his errors or complacency of his colleagues in exposing him , in order not to jeopardize his professional life or to protect him from medico - legal issues that might ensure . in literature the incidence of retained foreign body is quoted as 1 : per 1000 to 1500 laparotomies . here in our environment however , given the level of our socio - economic and manpower development in health care , it is strongly suggested that the incidence in our environment is much high than quoted in the literature . in our environment people are gradually beginning to appreciate their rights and privileges and the authors suspect that in the years ahead the medico legal issues associated with retained foreign bodies following surgery will be in the front burner . the most common retained foreign body is abdominal sponge ( gossypiboma ) because of its common usage and amorphous structure . whilst gossypiboma elicits two types of responses - asceptic fibrinous and exudative reaction , retained metals like artery forceps are inert . while many retained foreign materials are identified and retrieved immediately or shortly after surgery some may remain unidentified for years or decades . qamar , champ and nasir found one case each of retained artery forceps in their reviews of eleven , fifteen and ten cases respectively of retained foreign bodies in the abdominal cavity . retained metal like artery forceps are inert and exert their effect through mechanical means like direct tissue damage causing ischemia , pressure effect giving rise to pain and discomfort , or forming an axis on which loops of bowel entangle leading to intestinal obstruction and strangulation or a loop of bowel going through the eye of the retained artery forceps and getting trapped leading to strangulation . they may also penetrate hollow organs for example gut leading to peritonitis or blood vessels leading to hemorrhage . in our case , the artery forceps through its clinching jaws caused ischemia ( direct tissue damage ) leading to strangulation and gangrene of small bowel loops . metal retained materials are said to usually give rise to acute symptoms months or years after the operation . fadiora and godara noted in their cases that their patients presented with acute symptoms after 2 years and 2 years respectively of their initial surgical operations . our patient presented 12 months after surgery . however , in all cases of retained artery forceps in the abdominal cavity sited in literature , the patients had chronic recurrent abdominal pains or colic 's months or years before onset of acute ( surgical ) abdomen . our patient had months of recurrent abdominal pain before the acute ( surgical ) abdomen . in the literature most of the reported cases of retained artery forceps were operated in the peripheral hospitals . our patient had surgery at which retained forceps occurred in a peripheral hospital . in most of the peripheral hospitals in developing country like ours , qualified medical personnel are few and auxiliaries most times inexperienced are called in to help in the operating theatre . therefore , any surgeon working in a peripheral hospital who decides to undertake any major surgery must assume full responsibility of preventing retained instruments and must involve his assistants and auxiliaries in his anxiety . also in most of hospital theatres , adequate equipment and facilities are lacking , improvisation is key to many procedures and with this disasters are common including retained instruments . a recent retrospective study found that retained instruments and sponges following an operation occurred more frequently in emergency surgery , especially where there was an unplanned change in the operative procedure , in patients with high mean body mass index . others noted that huge intra - abdominal tumors or mass and severe intra- operative hemorrhage are risk factors for retained instrument . other risk factors are fatigued surgical team , staff changes during operation , and failure to account for all sponges and instruments , leaving closure of abdominal wound to a junior , sometimes inexperienced . the surgeon remains the head of the surgical team and must assume full responsibility for what he puts on the patient even though the general custom and practice is to delegate the task of counting sponges and other instruments to the nurses . the responsibility of the surgeon is even more important and pertinent in peripheral hospitals where trained peri - operative nurses are very scarce , and the surgeon uses nurse auxiliaries- who are most times untrained the following precautionary measures are likely to reduce the incidence of retained instruments : double sponge / instrument count , before and after surgery.number of sponges and instruments are clearly written in the theatre black board provided for this purpose.all large sponges ( abdominal mops ) applied into the peritoneal cavity should have long ribbon attached to the sponge and the ribbon must remain outside the main laparotomy wound.avoid use of small gauze inside the abdominal cavity ; only use large abdominal mops ( sponges with long ribbon attached).the surgeon must develop the attitude of thorough final sweep(visual and manual ) of the abdominal cavity to ensure no instruments or sponges are left inside the abdominal cavity before commencement of the wound closure.uses of sponges with radio - opaque markers are advised but these materials are not easily available to us here . also advocated is radiographic screening of high risk operative procedures before patients leave the operating room , even when counts are documented as correct . number of sponges and instruments are clearly written in the theatre black board provided for this purpose . all large sponges ( abdominal mops ) applied into the peritoneal cavity should have long ribbon attached to the sponge and the ribbon must remain outside the main laparotomy wound . avoid use of small gauze inside the abdominal cavity ; only use large abdominal mops ( sponges with long ribbon attached ) . the surgeon must develop the attitude of thorough final sweep(visual and manual ) of the abdominal cavity to ensure no instruments or sponges are left inside the abdominal cavity before commencement of the wound closure . uses of sponges with radio - opaque markers are advised but these materials are not easily available to us here . also advocated is radiographic screening of high risk operative procedures before patients leave the operating room , even when counts are documented as correct . this is ideal but not feasible in a developing world setting . in our environment and especially for surgeon operating in peripheral hospitals where the incidence of retained materials are much more commoner , he must rely on his own ability of making absolutely sure that all the procedures adopted for prevention of retained materials in wounds are carried out . no reliance should be placed on other theatre staffing since the short comings of these staff are obvious . these measures are even more pertinent when operating on a high risk patient for retained instruments . exploratory laparotomy remains the mainstay of treatment to retrieve retained intra - abdominal foreign material . other methods include laparoscopic removal and ultrasound assisted retrieval rarely small sponges and gauzes can discharge via the rectum spontaneously . it is advocated that patient who present with chronic abdominal discomfort , pain or colic 's , after laparotomy should be subjected to thorough history taking , clinical examination and should have plain abdominal x - rays , supplemented by ultrasound scan , and perhaps ct scan and possibly mri scan . adoption of this procedure will lead to early diagnosis of retained foreign materials , before the patient develops acute ( surgical ) abdomen . retained foreign body is avoidable and could be a costly error as our case illustrates . the need for the surgeon ( especially the rural surgeon ) who operates in the abdominal cavity to assume the captain of ship doctrine can not be over emphasized . all preventive protocols must be observed under the strict guidance and full responsibility of the surgeon especially the rural surgeon . the need to investigate properly a patient who complains of chronic abdominal symptoms following laparotomy should be encouraged as this will aid in making early diagnosis of retained foreign material before more serious complications of intestinal obstruction or strangulation , or peritonitis , intra - abdominal abscesses or entero - cutanous fistula supervenes .

context : surgical instruments and materials continue to be retained in the peritoneal cavity despite precautionary measures . even though uncommon it is also under - reported and carries serious medico - legal consequences . gauzes and sponges ( gossypiboma ) are the most commonly retained materials and intra - abdominal retained artery forceps are much rarer but when they do occur lead to chronic abdominal pain and can be a rare cause of intestinal obstruction or strangulation with significant morbidity and mortality.case report : we present a case of intraabdominal retained artery forceps in a 70-years - old lady who underwent laparotomy with splenectomy for a large spleen in a peripheral hospital . upon discharge she continued to complain of intermittent abdominal pain of increasing severity . 12 months later she presented to us with an acute ( surgical ) abdomen requiring another laparotomy . at laparotomy she had strangulated / gangrenous lower jejunual and upper ileal bowel loops , the small bowel mesentery of this area being tightly trapped between the jaws of the retained artery forceps . she had gut resection and enteroanastomosis . unfortunately she died from continuing sepsis on the second post - operative day.conclusion:retained instruments in intra - abdominal surgery can cause serious complication and should be treated surgically . high index of suspicion and appropriate investigations like plain abdominal x - ray , abdominal ultrasound and ct and mri scans should be instituted in patients who develop chronic abdominal symptoms following laparotomy . preventive measures against retained instruments must follow strict laid down protocols for surgical instruments handling in theatre .

Context: Case Report: Conclusion: Introduction Case Report Discussion Conclusion

like many developed nations and even some developing ones , jamaica is able to boast of its notable achievement in progress made toward advancing the health status of its populace , during the twentieth century which are expressed in the postponement of death , lowered fertility , high nutrition and sanitation and more importantly the increasing life expectancy . life expectancy which is an indicator of health status revealed that the country 's health status is reasonably good , as the life span for jamaicans was similar to those in some first world societies interestingly , the biological science highlighted that the ageing process comes with changes in physical functioning . according to the merck manual of health and aging older cells function less well . also , in some organs , cells die and are not replaced , so the number of cells decreases , indicates not only the decline functionality of aged body but also the role of diseases in this regards . the oldest - old categorization is said to be the least physical functioning compared to the other classification in chronological ageing . the young - old , on the other hand , are more likely to be the most functioning as the organism is just beginning the transition into the aged arena . a study conducted by costa , using secondary data drawn from the records of the union army ( ua ) pension programme that covered some 85% of all ua , showed that there was an association between chronic conditions and functional limitation which include difficulty walking , bending , blindness in at least one eye and deafness . those functional activities are classified as adl ( activities of daily living ) or ( i ) adl ( instrumental activities for daily living ) . these include activities such as feeding oneself ; bathing , dressing , grooming , work , homemaking and leisure . the ( i ) adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . within the context of ageing and the reality of having chronic diseases , ones adl and ( i ) adl will be hampered somewhat . some illnesses , such as huntington 's disease , hypertension , heart diseases , diabetes mellitus , cancer , cataract , and stroke , result in a gradual loss of the ability to provide self - care and some result in an immediate dependence or lowered functional capacity and sometimes even mortality . hence , if ageing were associated with no ailments , it still comes with reduced functional capacity . according to eldemire the majority of jamaican older persons are physically and mentally well and living in family units , suggesting that illnesses are eroding some of functional capacity of elderly jamaica which is synonymous to ageing . despite eldemire 's findings , a study on the elderly published in the caribbean food and nutrition institute 's magazine ( i.e. cajanus ) found that 70% of individuals who were patients within different typologies of health services were senior citizens , suggesting that elderly jamaicans were not only spending more time utilizing health care services than other age cohorts but that they were experiencing lowered functional capacity . the aforementioned health literature has shown that diseases positively influence functionality , and kim et al . they found that mci ( i.e. mild cognitive impairment ) patients performed significantly worse on four out of a total 15 items ( i.e. telephone , transportation , finances and household appliances ) of elderly 60 + year old . another descriptive study conducted by natividad and zimmer when further than kim et al . in 2000 , they found that 11.5% were having difficulties walking in the house ; 8.0% difficulty bathing ; 6.3% dressing themselves ; and 4.6% eating . on the issue of ( i)adl for natividad and zimmer 's work , for 2000 , 18.5% had difficulty with using transportation ; 17.6% shopping ; 13.8% preparing meals ; 13.8% light housework ; and 9.4% manage their money . in the same aforementioned study , using logistic regression , they went further and found that age was the significant factor that determine adl ( or=1.08 , p < 0.05 ) ; while age ( or = 1.07 , p < 0.05 ) and area of residence ( i.e. rural or=0.66 , furthermore , they found that marital status , education , and gender were not statistical significant determinants of adl or ( i ) adl . this study is timely as it aimed to examine adl , ( i ) adl and sought to investigate those determinants of functional capacity of older men in jamaica . using data for 2007 on 2,000 jamaican men 55 + years , the current study evaluated activities of daily living ( adl ) , instrumental activities ( ( i)adl ) , self - reported health status and using logistic regression , determined those factors that explain functional capacity of older men . the current study therefore will not only provide information upon which public health policies can be fashioned ; but it also will aid an understanding of older men and how they perceived health and determined their adl , ( i ) adl and those factors that influence their functional abilities and not assume that what obtains in other locality equally applies to jamaican men 55 + years . the study used primary cross - sectional survey data on men 55 years and older from the parish of st . the survey was submitted and approved by the university of the west indies medical faculty 's ethics committee . stratified multistage probability sampling technique was used to draw the sample ( 2,000 respondents ) . the instrument was sub - divided into general demographic profile of the sample ; past and current health status ; health - seeking behavior ; retirement status ; social and functional status . data was stored , retrieved and analyzed , using spss for windows ( 16.0 ) ( spss inc ; chicago , il , usa ) . the statistical institute of jamaica ( statin ) maintains a list of enumeration districts ( ed ) or census tracts . catherine is divided into a number of constituencies made up of a number of enumeration districts ( ed ) . the enumeration districts were listed and numbered sequentially and selection of clusters was arrived by the use of a sampling interval . forty enumeration districts ( clusters ) were subsequently selected with the probability of selection being proportional to population size ( table 1 ) . proportion of survey ( sample ) vs. proportion of population the sample population does not only speak to the parish of st . the sampling frame was men fifty - five years and older in the parish of st catherine . catherine was chosen as previous data and surveys[1113 ] suggested that it has the mix of demographic characteristics ( urban , rural and age - composition ) which typify jamaica . for the current study descriptive status employed to provide background information on the sample ; and chi - square was used to examine non - metric variables . level of significance was p - value < 0.05 and the only exclusion criterion was if more than 20% of the cases of a variable were missing . this variable is measured using people 's self - rate of their overall health status , which ranges from excellent to poor health status . the question was how would you rate your health today? ( 1 ) excellent ; ( 2 ) good ; ( 3 ) fair and ( 4 ) poor . what is [ your ] highest level of [ education ] attained ? the options were ( 1 ) no formal education ; ( 2 ) basic school ; ( 3 ) primary school / all age ; ( 4 ) secondary / high / technical school ; ( 5 ) vocational ( i.e. apprenticeship / trade ) ; ( 6 ) diploma ; ( 7 ) undergraduate degree ; ( 8) post - graduate degree do you take time out for regular exercise? ( 1 ) yes and ( 2 ) no . type of physical exercise . ( 1 ) yes , ( 2 ) no . if the response to either question was yes , this was coded as poor childhood health status and if the response was no in both cases it was coded a good health status in childhood . these are ( 1 ) ages 55 to 64 years ; ( 2 ) ages 65 to 74 years ; and ( 3 ) ages 75 years and older ( i.e. 75 + years ) . performance of activities of daily living ( adl ) is used to describe the functional status of a person . it is used to determine a baseline level of functioning and to monitor improvement in activities of daily living ( adl ) overtime . scoring the adl findings ( katz ) independence on a given function received a score of 1 point while if dependent , 0 point was given . there were 6 items ( eating refers to feeding oneself ; dressing denotes getting clothes and getting dressed , including typing shoes ; transferring means to get in and out of bed as well as in and out of a chair ; using toilet refers to going to the toilet and cleaning afterwards ; bathing denotes to sponge bath , shower , tub bath , or washing body with a wet towel ; continence denotes to control of urination and bowel movement ) . total scores thus could range from 0 to 6 with lower scores indicating low independence ( i.e. high dependence ) and larger scores indicating higher independence . if there was a score of 0 to 2 ( i.e. none to 2 of the six adl activities was chosen ) , the older person was classified as low independence ; if 3 to 4 of the activities were selected , the older man was classified as moderately independent and if 5 to 6 items were selected the older was classified as highly independent . instrumental activities of daily living ( ( i ) adl ) . the instrumental activities of daily living tool was the basis for assessing participants difficulty with ( i ) adl . ( i)adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . hence , ( i ) adl for older men in this study used the 8-item choices as is used for women . these are preparing meals ; shopping ; management medication ; money management ; transportation ; telephone and laundry . scoring the ( i ) adl . ( i)adl scores reflect the number of areas of impairment i.e. the number of skills / domains in which subjects are dependent . scores range from 0 to 8 , with higher scores indicating higher dependence and lower scores greater independence ( i.e. low dependence ) . if none to 3 activities were selected , the older person was classified as high dependence ; if 4 to 6 activities were selected the elder was classified as moderately dependent and if 7 to 8 items were selected the elder was classified as highly dependent . the cronbach alpha for the 8 item scales was 0.648 . in order to examine the effect of many variables on a single dependent variable , the researcher used multivariate analysis to test a single hypothesis ( physical functioning is determined by current health status , happiness , area of residence see equation . natividad and zimmer had used logistic regression to examine factors that determined adl , ( i ) adl and self - reported of older filipinos . using the literature , the current study investigated the correlates of functional status of older jamaicans within the context of the available data . the proposed model that this research seeks to evaluate is displayed ( eqn1 ) : fi = 0 + 1hti + 2happi + 3lsi + 4chi + 5ari + 6ai + 7ssi + 8cai + 9edi + 10hhi + 11msi + 12pi + 13hai + 14tmi + 15di + i where fi ( physical functionality ) is a function of some current health status , ht ; happiness , happi ; life satisfaction , lsi ; children , ci ; area of residence , ari ; age group of respondent , ai ; social support , ssi ; church attendance , cai ; educational level , edi ; head of household , hhi ; marital status , msi ; number of person in household , pi ; health status in childhood , hai ; employment status , emi ; depression , di ; taking medication , tmi ; health advise , hai . all the variables were identified from the literature . using the principle of parsimony , only those explanatory variables that are statistically significant ( p < 0.05 ) were used in the final model to determine f1 of older men in jamaica . this final model identified the correlates of fi of older men in jamaica , ( eqn2 ) . fi = 0 + 1ari + 2ai + 3ssi + 4cai + 5pii furthermore , the variables used in this study are based on ( 1 ) literature review which shows that these are likely to correlate with the particular dependent variable , and 2 ) the correlation matrix was examined in order to ascertain if autocorrelation ( or multicollinearity ) existed between independent variables . based on bryman and cramer , correlation can be low ( weak ) from 0 to 0.39 ; moderate any of the independent variables which had moderate to high correlation was excluded from the model . the correlation between life satisfaction and happiness was 0.633 ; happiness and social networking ( correlation coefficient = 0.12 , p = 0.003 ) ; happiness and marital status ( correlation coefficient = 0.107 , p = 0.026 ) ; marital status and income category ( correlation coefficient = 0.193 , p < 0.001 ) ; social networking and marital status ( r=0.205 , p < 0.001 ) ; social networking and age group ( correlation coefficient = 0.188 , p < 0.001 ) ; social networking and occupation ( correlation coefficient = 0.320 , p < 0.001 ) ; social networking educational category ( correlation coefficient = 0.420 , p < 0.001 ) ; adl and age cohort ( correlation coefficient = -0.813 , p = 0.032 ) ; income and occupation ( correlation coefficient = 0.7775 , p < 0.001 ) ; and , income and education ( correlation coefficient = 0.356 , p<0.001 ) ; employment and education category ( correlation coefficient = 0.283 , p < 0.001 ) , and depression and life satisfaction ( correlation coefficient = 0.160 , p < 0.001 ) . however , there was no correlation between happiness and present occupation ( p = 0.761 ) ; happiness and income ( p = 0.233 ) ; happiness and employment status ( p = 0.516 ) ; health status and depression ( p=0.876 ) as well as life satisfaction and employment status ( p = 0.261 ) . hence , life satisfaction and happiness ; occupation and income category will not be simultaneously used as explanatory variables . this variable is measured using people 's self - rate of their overall health status , which ranges from excellent to poor health status . the question was how would you rate your health today? ( 1 ) excellent ; ( 2 ) good ; ( 3 ) fair and ( 4 ) poor . what is [ your ] highest level of [ education ] attained ? the options were ( 1 ) no formal education ; ( 2 ) basic school ; ( 3 ) primary school / all age ; ( 4 ) secondary / high / technical school ; ( 5 ) vocational ( i.e. apprenticeship / trade ) ; ( 6 ) diploma ; ( 7 ) undergraduate degree ; ( 8) post - graduate degree do you take time out for regular exercise? ( 1 ) yes and ( 2 ) no . type of physical exercise . ( 1 ) yes , ( 2 ) no . and , were you frequently ill as a child ? ( 1 ) yes , ( 2 ) no . if the response to either question was yes , this was coded as poor childhood health status and if the response was no in both cases it was coded a good health status in childhood . these are ( 1 ) ages 55 to 64 years ; ( 2 ) ages 65 to 74 years ; and ( 3 ) ages 75 years and older ( i.e. 75 + years ) . performance of activities of daily living ( adl ) is used to describe the functional status of a person . it is used to determine a baseline level of functioning and to monitor improvement in activities of daily living ( adl ) overtime . scoring the adl findings ( katz ) independence on a given function received a score of 1 point while if dependent , 0 point was given . there were 6 items ( eating refers to feeding oneself ; dressing denotes getting clothes and getting dressed , including typing shoes ; transferring means to get in and out of bed as well as in and out of a chair ; using toilet refers to going to the toilet and cleaning afterwards ; bathing denotes to sponge bath , shower , tub bath , or washing body with a wet towel ; continence denotes to control of urination and bowel movement ) . total scores thus could range from 0 to 6 with lower scores indicating low independence ( i.e. high dependence ) and larger scores indicating higher independence . if there was a score of 0 to 2 ( i.e. none to 2 of the six adl activities was chosen ) , the older person was classified as low independence ; if 3 to 4 of the activities were selected , the older man was classified as moderately independent and if 5 to 6 items were selected the older was classified as highly independent . the instrumental activities of daily living tool was the basis for assessing participants difficulty with ( i ) adl . ( i)adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . hence , ( i ) adl for older men in this study used the 8-item choices as is used for women . these are preparing meals ; shopping ; management medication ; money management ; transportation ; telephone and laundry . scoring the ( i ) adl . ( i)adl scores reflect the number of areas of impairment i.e. the number of skills / domains in which subjects are dependent . scores range from 0 to 8 , with higher scores indicating higher dependence and lower scores greater independence ( i.e. low dependence ) . if none to 3 activities were selected , the older person was classified as high dependence ; if 4 to 6 activities were selected the elder was classified as moderately dependent and if 7 to 8 items were selected the elder was classified as highly dependent . the cronbach alpha for the 8 item scales was 0.648 . in order to examine the effect of many variables on a single dependent variable , the researcher used multivariate analysis to test a single hypothesis ( physical functioning is determined by current health status , happiness , area of residence natividad and zimmer had used logistic regression to examine factors that determined adl , ( i ) adl and self - reported of older filipinos . using the literature , the current study investigated the correlates of functional status of older jamaicans within the context of the available data . the proposed model that this research seeks to evaluate is displayed ( eqn1 ) : fi = 0 + 1hti + 2happi + 3lsi + 4chi + 5ari + 6ai + 7ssi + 8cai + 9edi + 10hhi + 11msi + 12pi + 13hai + 14tmi + 15di + i where fi ( physical functionality ) is a function of some current health status , ht ; happiness , happi ; life satisfaction , lsi ; children , ci ; area of residence , ari ; age group of respondent , ai ; social support , ssi ; church attendance , cai ; educational level , edi ; head of household , hhi ; marital status , msi ; number of person in household , pi ; health status in childhood , hai ; employment status , emi ; depression , di ; taking medication , tmi ; health advise , hai . all the variables were identified from the literature . using the principle of parsimony , only those explanatory variables that are statistically significant ( p < 0.05 ) were used in the final model to determine f1 of older men in jamaica . this final model identified the correlates of fi of older men in jamaica , ( eqn2 ) . fi = 0 + 1ari + 2ai + 3ssi + 4cai + 5pii furthermore , the variables used in this study are based on ( 1 ) literature review which shows that these are likely to correlate with the particular dependent variable , and 2 ) the correlation matrix was examined in order to ascertain if autocorrelation ( or multicollinearity ) existed between independent variables . based on bryman and cramer , correlation can be low ( weak ) from 0 to 0.39 ; moderate any of the independent variables which had moderate to high correlation was excluded from the model . the correlation between life satisfaction and happiness was 0.633 ; happiness and social networking ( correlation coefficient = 0.12 , p = 0.003 ) ; happiness and marital status ( correlation coefficient = 0.107 , p = 0.026 ) ; marital status and income category ( correlation coefficient = 0.193 , p < 0.001 ) ; social networking and marital status ( r=0.205 , p < 0.001 ) ; social networking and age group ( correlation coefficient = 0.188 , p < 0.001 ) ; social networking and occupation ( correlation coefficient = 0.320 , p < 0.001 ) ; social networking educational category ( correlation coefficient = 0.420 , p < 0.001 ) ; adl and age cohort ( correlation coefficient = -0.813 , p = 0.032 ) ; income and occupation ( correlation coefficient = 0.7775 , p < 0.001 ) ; and , income and education ( correlation coefficient = 0.356 , p<0.001 ) ; employment and education category ( correlation coefficient = 0.283 , p < 0.001 ) , and depression and life satisfaction ( correlation coefficient = 0.160 , p < 0.001 ) . however , there was no correlation between happiness and present occupation ( p = 0.761 ) ; happiness and income ( p = 0.233 ) ; happiness and employment status ( p = 0.516 ) ; health status and depression ( p=0.876 ) as well as life satisfaction and employment status ( p = 0.261 ) . hence , life satisfaction and happiness ; occupation and income category will not be simultaneously used as explanatory variables . this variable is measured using people 's self - rate of their overall health status , which ranges from excellent to poor health status . the question was how would you rate your health today? ( 1 ) excellent ; ( 2 ) good ; ( 3 ) fair and ( 4 ) poor . what is [ your ] highest level of [ education ] attained ? the options were ( 1 ) no formal education ; ( 2 ) basic school ; ( 3 ) primary school / all age ; ( 4 ) secondary / high / technical school ; ( 5 ) vocational ( i.e. apprenticeship / trade ) ; ( 6 ) diploma ; ( 7 ) undergraduate degree ; ( 8) post - graduate degree do you take time out for regular exercise? ( 1 ) yes and ( 2 ) no . type of physical exercise . ( 1 ) yes , ( 2 ) no . and , were you frequently ill as a child ? ( 1 ) yes , ( 2 ) no . if the response to either question was yes , this was coded as poor childhood health status and if the response was no in both cases it was coded a good health status in childhood . these are ( 1 ) ages 55 to 64 years ; ( 2 ) ages 65 to 74 years ; and ( 3 ) ages 75 years and older ( i.e. 75 + years ) . performance of activities of daily living ( adl ) is used to describe the functional status of a person . it is used to determine a baseline level of functioning and to monitor improvement in activities of daily living ( adl ) overtime . scoring the adl findings ( katz ) independence on a given function received a score of 1 point while if dependent , 0 point was given . there were 6 items ( eating refers to feeding oneself ; dressing denotes getting clothes and getting dressed , including typing shoes ; transferring means to get in and out of bed as well as in and out of a chair ; using toilet refers to going to the toilet and cleaning afterwards ; bathing denotes to sponge bath , shower , tub bath , or washing body with a wet towel ; continence denotes to control of urination and bowel movement ) . total scores thus could range from 0 to 6 with lower scores indicating low independence ( i.e. high dependence ) and larger scores indicating higher independence . if there was a score of 0 to 2 ( i.e. none to 2 of the six adl activities was chosen ) , the older person was classified as low independence ; if 3 to 4 of the activities were selected , the older man was classified as moderately independent and if 5 to 6 items were selected the older was classified as highly independent . the instrumental activities of daily living tool was the basis for assessing participants difficulty with ( i ) adl . ( i)adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . hence , ( i ) adl for older men in this study used the 8-item choices as is used for women . these are preparing meals ; shopping ; management medication ; money management ; transportation ; telephone and laundry . scoring the ( i ) adl . ( i)adl scores reflect the number of areas of impairment i.e. the number of skills / domains in which subjects are dependent . scores range from 0 to 8 , with higher scores indicating higher dependence and lower scores greater independence ( i.e. low dependence ) . if none to 3 activities were selected , the older person was classified as high dependence ; if 4 to 6 activities were selected the elder was classified as moderately dependent and if 7 to 8 items were selected the elder was classified as highly dependent . in order to examine the effect of many variables on a single dependent variable , the researcher used multivariate analysis to test a single hypothesis ( physical functioning is determined by current health status , happiness , area of residence natividad and zimmer had used logistic regression to examine factors that determined adl , ( i ) adl and self - reported of older filipinos . using the literature , the current study investigated the correlates of functional status of older jamaicans within the context of the available data . the proposed model that this research seeks to evaluate is displayed ( eqn1 ) : fi = 0 + 1hti + 2happi + 3lsi + 4chi + 5ari + 6ai + 7ssi + 8cai + 9edi + 10hhi + 11msi + 12pi + 13hai + 14tmi + 15di + i where fi ( physical functionality ) is a function of some current health status , ht ; happiness , happi ; life satisfaction , lsi ; children , ci ; area of residence , ari ; age group of respondent , ai ; social support , ssi ; church attendance , cai ; educational level , edi ; head of household , hhi ; marital status , msi ; number of person in household , pi ; health status in childhood , hai ; employment status , emi ; depression , di ; taking medication , tmi ; health advise , hai . all the variables were identified from the literature . using the principle of parsimony , only those explanatory variables that are statistically significant ( p < 0.05 ) were used in the final model to determine f1 of older men in jamaica . this final model identified the correlates of fi of older men in jamaica , ( eqn2 ) . fi = 0 + 1ari + 2ai + 3ssi + 4cai + 5pii furthermore , the variables used in this study are based on ( 1 ) literature review which shows that these are likely to correlate with the particular dependent variable , and 2 ) the correlation matrix was examined in order to ascertain if autocorrelation ( or multicollinearity ) existed between independent variables . based on bryman and cramer , correlation can be low ( weak ) from 0 to 0.39 ; moderate any of the independent variables which had moderate to high correlation was excluded from the model . the correlation between life satisfaction and happiness was 0.633 ; happiness and social networking ( correlation coefficient = 0.12 , p = 0.003 ) ; happiness and marital status ( correlation coefficient = 0.107 , p = 0.026 ) ; marital status and income category ( correlation coefficient = 0.193 , p < 0.001 ) ; social networking and marital status ( r=0.205 , p < 0.001 ) ; social networking and age group ( correlation coefficient = 0.188 , p < 0.001 ) ; social networking and occupation ( correlation coefficient = 0.320 , p < 0.001 ) ; social networking educational category ( correlation coefficient = 0.420 , p < 0.001 ) ; adl and age cohort ( correlation coefficient = -0.813 , p = 0.032 ) ; income and occupation ( correlation coefficient = 0.7775 , p < 0.001 ) ; and , income and education ( correlation coefficient = 0.356 , p<0.001 ) ; employment and education category ( correlation coefficient = 0.283 , p < 0.001 ) , and depression and life satisfaction ( correlation coefficient = 0.160 , p < 0.001 ) . however , there was no correlation between happiness and present occupation ( p = 0.761 ) ; happiness and income ( p = 0.233 ) ; happiness and employment status ( p = 0.516 ) ; health status and depression ( p=0.876 ) as well as life satisfaction and employment status ( p = 0.261 ) . hence , life satisfaction and happiness ; occupation and income category will not be simultaneously used as explanatory variables . most of the sample was lowly dependent ( 77.1% ) ; 55.4% reported a moderate health status and 63.6% indicated satisfied with life sometimes ( table 2 ) . demographic characteristics of sample when functional capacity was disaggregated into adl and ( i ) adl , the following disparities were observed in the findings . of the sample , however , with regards to ( i ) adl , 1.9% had low , 18.6% moderate and 79.6% high scores . of the sample , 43.1% reported that they were suffering from depression compared to 56.9% who stated no to the question of being depressed in the survey period . on examining depression and age cohort , no significant statistical association was between both variables ( p = 0.102 ) . one half of the sample indicated that they spent ja.$100 ( us $ 1.45 ) monthly for medical expenditure ; 34% of the respondents bought their prescribed medication ; 17.1% reported that they have been hospitalized since their sixth birthday and 65.8% reported that they took no medication . of those who mentioned that they were ill during childhood ( 17.5% , n=350 ) , 34.9% said that the illness was measles or chicken pox , 26.3% mentioned asthma , 10.0% pneumonic fever , 8.9% polio , 6.6% accident , 4.6% jaundice , 1.7% hernia , and 5.1% indicated gastroenteritis . twenty four percent of elderly men indicated that they were rarely happy , 40.5% said sometimes , 31.0% mentioned often and only 4.5% reported always . furthermore , 17.7% of the sample reported that they were seriously ill as children . the findings revealed that no statistical correlation existed between adl and age cohort of the sample ( p=0.205 ) . however , a relationship was found between ( i ) adl and age group of the sample ( 2 ( df = 4 ) = 16.011 ; p=0.003 ) ( table 3 ) . on further examination , it was revealed that as an older male increases in age from 55 - 64 years to 65 - 74 years and 75 + years , his high independence falls and while his moderate dependence increases . of those who were 55 - 64 years , 83.0% of them high independence compared to 78.9% of those 65 - 74 years and 73.9% for those 75 + years ( table 3 ) . adl and ( i ) adl by age group of the sample , 41.8% of older men were health literate or have been advised on medical relates conditions , causes , prognosis and precautions compared to 58.3% who were not aware or have been advice by a health care practitioners ( include a pharmacists , community aide ; nurse , or medical technologists ) . the study revealed that no statistical correlation was found between functional capacity of older men in jamaica and health advice ( or health literacy ) p = 0.845 . however , a weak statistical relationship existed between educational level and health literacy ( 2 ( df = 1 ) = 110.165 , p < 0.001 , correlation coefficient = 0.235 ) ( table 4 ) . health literacy by high level of education attained table 5 revealed that adl for men 55 + years was very high for each activity , with 88.5% for continence being the lowest level of independence . for ( i)adl , 56.7% of sample was still able to perform heavy duty housework , 62.7% were still performing their laundry , 98.1% managed their money and 77.8% were still shopping , and 70.2% prepared their own meals . disaggregating adl and ( i ) adl of sample no statistical correlation was found between health status and age cohort of respondents ( p=0.051 ) , and between life satisfaction and age cohort ( p = 0.430 ) as well as health status and area of residence ( p = 0.190 ) . a significant statistical difference between life satisfaction of urban and rural older men in jamaica ( df = 3 ) = 13.910 , p = 0.003 . however that the correlation was a weak one ( correlation coefficient = 0.083 ) . on further examination , 35.9% of older rural men revealed that they were rarely satisfied with life compared to 29.8% of urban older men . concurrently , 37.4% of urban older men reported that they were sometimes satisfied with life compared to 30.2% of rural men . twenty - nine percent of urban men indicated that were satisfied with life most times compared to 30.5% of rural men . a cross tabulation between life satisfaction and happiness revealed a significant statistical correlation - 2 ( df = 9 ) = 1334.448 , p < 0.001 . the association was a relatively strong one ( correlation coefficient = 0.663 ) table 6 . seventy - three percent of those older men who were rarely happy were rarely satisfied with life compared to 17.8% who indicated being always happy that were rarely satisfied with life . forty - seven percentages of those who were always satisfied with life were always happy . further investigation revealed that 2.1% of those who were always satisfied with life were rarely happy . life satisfaction by happiness based on table 7 , the model ( eqn . ) is a good fit for the data f ( 19 , 1855 ) = 6.492 , p < 0.00 . continuing , 36.2% of the variance can be explained by age of respondents ; social support ; church attendance ; area of residence ; the number of people in the household and depression . using beta weights , church attendance was the most significant predictor of functional capacity ( =0.245 ; 95% ci : 0.264 , 1.291 ) followed by social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and furthermore , urban older men in jamaica had a low functional capacity than rural men ; and that the older men becomes their functional capacity falls - elderly ( ages 64 to 74 years , =-0.051 ; 95%ci : - 0.427 , -0.009 ) ; elderly ( ages 75 years and older , =-0.054 , 95%ci=-0.523 , -0.013 ) . multiple regression of functional status by some explanatory variables , n=1,875 most of the sample was lowly dependent ( 77.1% ) ; 55.4% reported a moderate health status and 63.6% indicated satisfied with life sometimes ( table 2 ) . demographic characteristics of sample when functional capacity was disaggregated into adl and ( i ) adl , the following disparities were observed in the findings . of the sample , however , with regards to ( i ) adl , 1.9% had low , 18.6% moderate and 79.6% high scores . of the sample , 43.1% reported that they were suffering from depression compared to 56.9% who stated no to the question of being depressed in the survey period . on examining depression and age cohort , no significant statistical association was between both variables ( p = 0.102 ) . one half of the sample indicated that they spent ja.$100 ( us $ 1.45 ) monthly for medical expenditure ; 34% of the respondents bought their prescribed medication ; 17.1% reported that they have been hospitalized since their sixth birthday and 65.8% reported that they took no medication . of those who mentioned that they were ill during childhood ( 17.5% , n=350 ) , 34.9% said that the illness was measles or chicken pox , 26.3% mentioned asthma , 10.0% pneumonic fever , 8.9% polio , 6.6% accident , 4.6% jaundice , 1.7% hernia , and 5.1% indicated gastroenteritis . twenty four percent of elderly men indicated that they were rarely happy , 40.5% said sometimes , 31.0% mentioned often and only 4.5% reported always . furthermore , 17.7% of the sample reported that they were seriously ill as children . the findings revealed that no statistical correlation existed between adl and age cohort of the sample ( p=0.205 ) . however , a relationship was found between ( i ) adl and age group of the sample ( 2 ( df = 4 ) = 16.011 ; p=0.003 ) ( table 3 ) . on further examination , it was revealed that as an older male increases in age from 55 - 64 years to 65 - 74 years and 75 + years , his high independence falls and while his moderate dependence increases . of those who were 55 - 64 years , 83.0% of them high independence compared to 78.9% of those 65 - 74 years and 73.9% for those 75 + years ( table 3 ) . adl and ( i ) adl by age group of the sample , 41.8% of older men were health literate or have been advised on medical relates conditions , causes , prognosis and precautions compared to 58.3% who were not aware or have been advice by a health care practitioners ( include a pharmacists , community aide ; nurse , or medical technologists ) . the study revealed that no statistical correlation was found between functional capacity of older men in jamaica and health advice ( or health literacy ) p = 0.845 . however , a weak statistical relationship existed between educational level and health literacy ( 2 ( df = 1 ) = 110.165 , p < 0.001 , correlation coefficient = 0.235 ) ( table 4 ) . health literacy by high level of education attained table 5 revealed that adl for men 55 + years was very high for each activity , with 88.5% for continence being the lowest level of independence . for ( i)adl , 56.7% of sample was still able to perform heavy duty housework , 62.7% were still performing their laundry , 98.1% managed their money and 77.8% were still shopping , and 70.2% prepared their own meals . disaggregating adl and ( i ) adl of sample no statistical correlation was found between health status and age cohort of respondents ( p=0.051 ) , and between life satisfaction and age cohort ( p = 0.430 ) as well as health status and area of residence ( p = 0.190 ) . a significant statistical difference between life satisfaction of urban and rural older men in jamaica ( df = 3 ) = 13.910 , p = 0.003 . however that the correlation was a weak one ( correlation coefficient = 0.083 ) . on further examination , 35.9% of older rural men revealed that they were rarely satisfied with life compared to 29.8% of urban older men . concurrently , 37.4% of urban older men reported that they were sometimes satisfied with life compared to 30.2% of rural men . twenty - nine percent of urban men indicated that were satisfied with life most times compared to 30.5% of rural men . a cross tabulation between life satisfaction and happiness revealed a significant statistical correlation - 2 ( df = 9 ) = 1334.448 , p < 0.001 . the association was a relatively strong one ( correlation coefficient = 0.663 ) table 6 . seventy - three percent of those older men who were rarely happy were rarely satisfied with life compared to 17.8% who indicated being always happy that were rarely satisfied with life . forty - seven percentages of those who were always satisfied with life were always happy . further investigation revealed that 2.1% of those who were always satisfied with life were rarely happy . life satisfaction by happiness based on table 7 , the model ( eqn . ) is a good fit for the data f ( 19 , 1855 ) = 6.492 , p < 0.00 . continuing , 36.2% of the variance can be explained by age of respondents ; social support ; church attendance ; area of residence ; the number of people in the household and depression . using beta weights , church attendance was the most significant predictor of functional capacity ( =0.245 ; 95% ci : 0.264 , 1.291 ) followed by social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and furthermore , urban older men in jamaica had a low functional capacity than rural men ; and that the older men becomes their functional capacity falls - elderly ( ages 64 to 74 years , =-0.051 ; 95%ci : - 0.427 , -0.009 ) ; elderly ( ages 75 years and older , =-0.054 , 95%ci=-0.523 , -0.013 ) . multiple regression of functional status by some explanatory variables , n=1,875 most of the sample was lowly dependent ( 77.1% ) ; 55.4% reported a moderate health status and 63.6% indicated satisfied with life sometimes ( table 2 ) . demographic characteristics of sample when functional capacity was disaggregated into adl and ( i ) adl , the following disparities were observed in the findings . of the sample , however , with regards to ( i ) adl , 1.9% had low , 18.6% moderate and 79.6% high scores . of the sample , 43.1% reported that they were suffering from depression compared to 56.9% who stated no to the question of being depressed in the survey period . on examining depression and age cohort , no significant statistical association was between both variables ( p = 0.102 ) . one half of the sample indicated that they spent ja.$100 ( us $ 1.45 ) monthly for medical expenditure ; 34% of the respondents bought their prescribed medication ; 17.1% reported that they have been hospitalized since their sixth birthday and 65.8% reported that they took no medication . of those who mentioned that they were ill during childhood ( 17.5% , n=350 ) , 34.9% said that the illness was measles or chicken pox , 26.3% mentioned asthma , 10.0% pneumonic fever , 8.9% polio , 6.6% accident , 4.6% jaundice , 1.7% hernia , and 5.1% indicated gastroenteritis . twenty four percent of elderly men indicated that they were rarely happy , 40.5% said sometimes , 31.0% mentioned often and only 4.5% reported always . furthermore , 17.7% of the sample reported that they were seriously ill as children . the findings revealed that no statistical correlation existed between adl and age cohort of the sample ( p=0.205 ) . however , a relationship was found between ( i ) adl and age group of the sample ( 2 ( df = 4 ) = 16.011 ; p=0.003 ) ( table 3 ) . on further examination , it was revealed that as an older male increases in age from 55 - 64 years to 65 - 74 years and 75 + years , his high independence falls and while his moderate dependence increases . of those who were 55 - 64 years , 83.0% of them high independence compared to 78.9% of those 65 - 74 years and 73.9% for those 75 + years ( table 3 ) . adl and ( i ) adl by age group of the sample , 41.8% of older men were health literate or have been advised on medical relates conditions , causes , prognosis and precautions compared to 58.3% who were not aware or have been advice by a health care practitioners ( include a pharmacists , community aide ; nurse , or medical technologists ) . the study revealed that no statistical correlation was found between functional capacity of older men in jamaica and health advice ( or health literacy ) p = 0.845 . however , a weak statistical relationship existed between educational level and health literacy ( 2 ( df = 1 ) = 110.165 , p < 0.001 , correlation coefficient = 0.235 ) ( table 4 ) . health literacy by high level of education attained table 5 revealed that adl for men 55 + years was very high for each activity , with 88.5% for continence being the lowest level of independence . for ( i)adl , 56.7% of sample was still able to perform heavy duty housework , 62.7% were still performing their laundry , 98.1% managed their money and 77.8% were still shopping , and 70.2% prepared their own meals . disaggregating adl and ( i ) adl of sample no statistical correlation was found between health status and age cohort of respondents ( p=0.051 ) , and between life satisfaction and age cohort ( p = 0.430 ) as well as health status and area of residence ( p = 0.190 ) . a significant statistical difference between life satisfaction of urban and rural older men in jamaica ( df = 3 ) = 13.910 , p = 0.003 . however that the correlation was a weak one ( correlation coefficient = 0.083 ) . on further examination , 35.9% of older rural men revealed that they were rarely satisfied with life compared to 29.8% of urban older men . concurrently , 37.4% of urban older men reported that they were sometimes satisfied with life compared to 30.2% of rural men . twenty - nine percent of urban men indicated that were satisfied with life most times compared to 30.5% of rural men . a cross tabulation between life satisfaction and happiness revealed a significant statistical correlation - 2 ( df = 9 ) = 1334.448 , p < 0.001 . the association was a relatively strong one ( correlation coefficient = 0.663 ) table 6 . seventy - three percent of those older men who were rarely happy were rarely satisfied with life compared to 17.8% who indicated being always happy that were rarely satisfied with life . forty - seven percentages of those who were always satisfied with life were always happy . further investigation revealed that 2.1% of those who were always satisfied with life were rarely happy . based on table 7 , the model ( eqn . ) is a good fit for the data f ( 19 , 1855 ) = 6.492 , p < 0.00 . continuing , 36.2% of the variance can be explained by age of respondents ; social support ; church attendance ; area of residence ; the number of people in the household and depression . using beta weights , church attendance was the most significant predictor of functional capacity ( =0.245 ; 95% ci : 0.264 , 1.291 ) followed by social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and lastly by age of respondents . furthermore , urban older men in jamaica had a low functional capacity than rural men ; and that the older men becomes their functional capacity falls - elderly ( ages 64 to 74 years , =-0.051 ; 95%ci : - 0.427 , -0.009 ) ; elderly ( ages 75 years and older , =-0.054 , 95%ci=-0.523 , -0.013 ) . functional capacity of older men in jamaica was very high as 77 out of every 100 men 55 + years had a high independence ; 22 out of every 100 a moderate independence , and 1 out of every 100 had a low independence . this is in somewhat lower eldemire 's earlier work that showed that 93.5% percentage of elderly jamaicans were actively involved in daily management of the household ; 88.5% were physically functional , and 85.9% were mentally competent . comparatively though , the functional capacity of elder men with that of elder jamaicans showed that physically functionality of the men 55 + years had fallen by 11.5% in 12 years . on average , the physical functional capacity of older men has been declining by 1% each year since 1995 . using self - reported depression as in indicator of mental functioning , the current research revealed that 4 out of every 10 older men were suffering from depression , suggesting that there is also a decline in mental competency of older men based on eldemire 's earlier work on elders in jamaica . furthermore , older men were predominantly satisfied with life some ; having attained at most primary level education and had good health status . thirty - four out of every 100 older men in jamaica was rarely satisfied with life , with there being more unsatisfied older rural men than urban older men . on disaggregating the current study 's findings , using adl , it was revealed that most of men 55 + years were able to bathe , feed , use toilet , and dress themselves with minimal assistance . however , using ( i)adl , which measures activities that people can continue accomplish independently in their residence in the community , it was found that 79.6% needed minimal assistance ( high independence ) compared to 18.6% moderate and 1.9% low independence . there are some similarities and differences between older filipinos [ ] , and older men in jamaica . with respect to transportation 18.5% of filipinos had low independence compared to 1.9% of jamaicans older men ; 17.6% were having low independence with shopping compared to 22.3% of older men in jamaica ; 6.3% of filipinos needed assistance dressing themselves compared to 2.2% of elderly jamaican ; 11.5% of filipinos had difficulties walking in the house compared to 2.3% in this study ; and 8.0% of filipinos had difficulties bathing themselves compared to 2.5% in this research . on the matter of self - reported health status , for the current study no older jamaican men reported poor or very good health status , while 17.5% and 5.1% of filipinos reported poor and very good health status respectively . nineteen percentage of older men in sample indicated excellent health compared to 1.0% of older filipinos . with regards to good health status , 55.4% percent of current sample and this was 31.5% of filipinos ; and for fair health status , 25.6% of the current study and 45.0% of filipinos . like natividad and zimmer , the current study found that as older jamaican ages , their high independence in ( i ) adl falls . however natividad and zimmer found a similar result for adl , but this research found that there is not statistical difference between ageing for men 55 + years and adl . generally , there is a high degree of independence of older men in jamaican and older filipinos . unlike the filipinos study that did not examine life satisfaction , the current study found that only 4 out of every 100 men 55 + years were generally satisfied with life most times ; 64 out of every 100 reported sometimes and 33 out of 100 indicated that they were rarely satisfied with life . in this research , 18 out of every 100 men indicated that their health status has a child was poor . ten percent in both elder men in jamaica and older filipinos ( 10.1% ) had no formal education ; 57.1% of latter group lived in urban zones compared to 49% in the current research . another similarity between both studies is the use of 55 + years to collect the data from , which is used to examine a functional model . for the current work , the model can explain 36.2% of the variability in functional capacity of older men in jamaica . although natividad and zimmer 's work did not provide such information , age and area of residence were found to be common predictors of functional capacity in both studies ; however , in natividad and zimmer , an older filipinos was 0.34 times less likely to reported better ( i)adl than an urban elderly . in this study , this was not the case as it was revealed that urban older men 55 + years were less likely to reported better functional capacity than rural older men . the current work when further than natividad and zimmer 's study , by adding some more variables such as depression , number of people living in household ; social support , and church attendance . depression was found to be inversely associated with functional capacity as well as number of people in household . like natividad and zimmer , this study found that marital status and education were not statistically significant determinants of functional capacity ( i.e. adl or ( i)adl ) . however , the significant statistical correlation between church attendance and functional capacity is embedded in the ability to walk or the use of limb functions[1820 ] . hence , the findings is not supporting any perspective that church attendees were healthier ; but that they were highly probable to higher functional independence than non - attendees , and this also the case for those who attend other social institutions . the researcher needs to make the aforementioned distinction as the current research did not seek to investigate when those who attended church were healthier ; but that whether they were more likely to be functionally independent than non - attendees or for that matter those who attend other social networks . hence , the lowered functional capacity of those who aged explain not only reduced activities outside of the home ; but speaks volume about those who are able to attend outside activities ( including church functions ) that they are of higher independence . depression can be used to measure cognitive functionality , and so the negative correlation between depression and functional capacity concurs with the findings in other study that reported the strong correlation between cognitive functions and functional capacity . the matter of depression affects 4 in every 10 older men in jamaica , and with the inverse association between it and functional capacity , there is expected to be a decline in functional capacity of this cohort . although depression and life satisfaction are weakly correlated in this sample , the reality is it is further depleting the quality of life lived by men 55 + years in jamaica , and so offers some insight into the further decline in functional capacity of this cohort . while depression is permanent and to some extent it is fluid , rate of depression in the current older men in jamaica is too high , and offers another explanation for the high mortality of men in the elderly compared to older women . in a study conducted by yi and vaupel of 8,805 elder ages 80 - 105 years in china , self - reported health status was found to be significantly correlated to functioning and morality of older people , which was also found in earlier studies . in spite of those findings , the current study did not concur with those results as it was revealed herein that childhood health status or current health status was not significantly associated with functional capacity . this research also concurs with yi and vaupel 's work that there was no statistical difference between urban - rural residents in current health status . although no statistical correlated was found between the two aforementioned variables , only a minimal number of elder men in jamaica had high dependence on others ( 2 out of every 100 ) and none indicated poor health status . chevannes 's work begins the explanation of the cultural health care - seeking behavior of males therein a broader context of culturalization of boys . chevannes provided the explanation for this behavior by men , that it is embedded with social learning theory . in which the young imitate the roles of society members through role modeling of what constitute acceptable and good roles which is supported by reinforcement . the gender role of sexes is not limited to jamaica or the caribbean but a study carried out by ali and de muynck of street children in pakistan found a similar gender stereotype in that nation . it was a descriptive cross - sectional study carried out during september and october 2000 , of 40 school - aged street children ( 8 - 14 years ) . the sample was substantially males ( 80% ) , with a mean age of 9 years ( 2 years ) . the methods of data collection were ( i ) semi - structured interviews , and ( ii ) a few focus group discussions . ali and de muynck found that the sampled population would seek medical care based on severity of illnesses and financial situation . the reason being that mild ailment is not severe enough to barr them from physical functioning , which mean that they are okay ; and so some morbidity are not for - hospital , which was so the case in nairobi slums . therefore , like the cases in pakistan and nairobi , the jamaican men are equally not reporting illness and seeking health - care substantially because of their socialization . this is tied to the macho culture with which they are grown as pointed out by chevannes that they should suppress response to pain and similarly displayed in ali and muynck and taff and chepngeno works . the current study revealed that a miniscule percentage of older men in jamaica were satisfied with life most - to - always ; many of them were lowly dependent ; few indicated fair health status and no significant statistical correlation was found between adl and age cohort although one existed between ( i ) adl and age groups . the findings revealed that as men age ( i.e. from 55 years ) ; there is deterioration between ageing and ( i ) adl , suggesting the challenges of ageing and some aspects of functional capacity . concurrently , six factors explain functional capacity of older men in jamaica ( area of residence ; age ; social support ; church attendance ; number of people in household and depression ) . more rural men were rarely satisfied with life ; but more of them had a greater functional capacity than urban men . depression was found to negatively relate to functional capacity , and church attendees had a greater functional status than non - attendees .

background : an extensive search of the literature found no studies that have examined functional capacity [ activities of daily living ( adl ) and instrumental activities for daily living ( i ) adl ) ] of jamaican older men as well as factors that determine their functional capacity.aims:the current study examines 1 ) adl , 2 ) ( i ) adl ) , 3 ) self - reported health status , 4 ) functional capacity , and 5 ) factors that determine functional capacity of older men.methods and method : stratified multistage probability sampling technique was used to draw a sample of 2,000 55 + year men . a132-item questionnaire was used to collect the data . descriptive statistics provide background information on the sample , cross tabulations were used to examine non - metric variables and logistic regression provides a model of predictors of functional capacity.result:fifty-five percent of sample indicated good current health status . four percent was mostly satisfied with life ; 21.7% had moderate dependence ; 77.1% had high dependence ( i.e. independence ) ; 1.2% had low dependence ; 21.9% were ages 75 years and older ; 35.6% were ages 65 to 74 years and 42.6% reported ages 55 to 64 years . functional capacity can be determined by church attendance ( =0.245 ; 95% ci : 0.264 , 1.291 ) ; social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and lastly by age of respondents.conclusion:ageing in explains deterioration in their ( i ) adl , suggesting the challenges of ageing men 's independence . more rural men were rarely satisfied with life ; but more of them had a greater functional capacity than urban men . depression was found to negatively relate to functional capacity , and church attendees had a greater functional status than non - attendees .

Introduction Materials and Methods None Measure Model Results None Demographic Characteristics of Sample Multivariate Analysis Discussion Conclusions

they were randomized to placebo , metformin ( 850 mg twice daily ) , or a lifestyle intervention . the participants mean age was 51 years and mean bmi was 34.0 kg / m , 68% were women , and 45% belonged to u.s . the lifestyle and metformin interventions reduced the incidence of diabetes by 58 and 31% , respectively , versus placebo ( 7 ) . in total , 2,994 participants ( 988 on metformin ) consented to genetic investigation . demographic characteristics of the dpp cohort by treatment arm and genotype at rs11212617 we genotyped rs11212617 on a sequenom iplex platform as previously described ( 8,9 ) . diabetes incidence was determined by semiannual measurements of fasting glucose and an annual oral glucose tolerance test . the principal study outcome was the development of diabetes by american diabetes association criteria , including confirmation . besides diabetes incidence as a categorical outcome , we selected the insulin sensitivity index ( isi ) , fasting glucose , a1c , weight , and oral disposition index ( dio ) as indices of metformin response . we calculated the isi as 22.5/([fasting insulin fasting glucose]/18.01 ) ( or 1/homeostasis model assessment of insulin resistance ) ( 10 ) , and the dio as 1/fasting insulin insulinogenic index ( insulin/glucose over the first 30 min of the oral glucose tolerance test ) ( 11 ) . we tested the additive effect of genotype at rs11212617 on diabetes incidence by cox proportional hazards regression models with genotype and intervention and their interactions as the independent variables predicting time to diabetes over mean 3.2 years follow - up , adjusted for sex , ethnicity , treatment arm , baseline age , and waist circumference . we included all three treatment arms and an interaction test to simultaneously rule out a main effect of this variant on diabetes incidence independent of metformin , or under the action of a lifestyle intervention . we used generalized mixed models to test additive effect of genotype on baseline log - transformed traits and , to model change under metformin action , on the same traits after 1 year of intervention adjusted for the baseline value of the respective trait , age , sex , ethnicity , treatment arm , and waist circumference . post hoc power calculations ( which should be interpreted with caution ) show that the sample size in the dpp metformin arm has > 99% power to detect the change in a1c of 0.61% that was reported in the ukpds ( 5 ) . to control for the potential effect of ethnicity , we performed sensitivity analyses in the largest race / ethnic group ( white participants ) , which is most closely related to the populations examined in the original report and whom we have previously shown to be essentially free of non - european admixture ( 12 ) . they were randomized to placebo , metformin ( 850 mg twice daily ) , or a lifestyle intervention . the participants mean age was 51 years and mean bmi was 34.0 kg / m , 68% were women , and 45% belonged to u.s . the lifestyle and metformin interventions reduced the incidence of diabetes by 58 and 31% , respectively , versus placebo ( 7 ) . in total , 2,994 participants ( 988 on metformin ) consented to genetic investigation . diabetes incidence was determined by semiannual measurements of fasting glucose and an annual oral glucose tolerance test . the principal study outcome was the development of diabetes by american diabetes association criteria , including confirmation . besides diabetes incidence as a categorical outcome , we selected the insulin sensitivity index ( isi ) , fasting glucose , a1c , weight , and oral disposition index ( dio ) as indices of metformin response . we calculated the isi as 22.5/([fasting insulin fasting glucose]/18.01 ) ( or 1/homeostasis model assessment of insulin resistance ) ( 10 ) , and the dio as 1/fasting insulin insulinogenic index ( insulin/glucose over the first 30 min of the oral glucose tolerance test ) ( 11 ) . we tested the additive effect of genotype at rs11212617 on diabetes incidence by cox proportional hazards regression models with genotype and intervention and their interactions as the independent variables predicting time to diabetes over mean 3.2 years follow - up , adjusted for sex , ethnicity , treatment arm , baseline age , and waist circumference . we included all three treatment arms and an interaction test to simultaneously rule out a main effect of this variant on diabetes incidence independent of metformin , or under the action of a lifestyle intervention . we used generalized mixed models to test additive effect of genotype on baseline log - transformed traits and , to model change under metformin action , on the same traits after 1 year of intervention adjusted for the baseline value of the respective trait , age , sex , ethnicity , treatment arm , and waist circumference . post hoc power calculations ( which should be interpreted with caution ) show that the sample size in the dpp metformin arm has > 99% power to detect the change in a1c of 0.61% that was reported in the ukpds ( 5 ) . to control for the potential effect of ethnicity , we performed sensitivity analyses in the largest race / ethnic group ( white participants ) , which is most closely related to the populations examined in the original report and whom we have previously shown to be essentially free of non - european admixture ( 12 ) . the frequency of the c ( metformin - responsive ) allele was 42.4 , 72.4 , 40.1 , 51.6 , and 41.5% in 1,669 white , 609 african american , 497 hispanic , 127 asian / pacific islander , and 82 american indian participants , respectively ( table 1 ) . we found no association of genotype with diabetes incidence in all arms combined , either in unadjusted analyses ( hazard ratio [ hr ] per copy of the c allele 0.98 [ 95% ci 0.881.10 ] , p = 0.76 ) or after adjusting for age , sex , ethnicity , and treatment arm ( hr per copy of the c allele 0.95 [ 0.851.07 ] , p = 0.42 ) ; further adjustment for waist circumference produced indistinguishable results ( table 2 ) . though there was a nominal snp metformin interaction in the fully adjusted model ( p = 0.04 ) , the observed trend was in the opposite direction from the expected prevention effect ; the c allele conferred no detectable advantage on metformin recipients in diabetes prevention but was associated with a nonsignificant trend toward increased risk of diabetes ( hr per copy of the c allele 1.17 [ 0.961.42 ] , p = 0.13 ) . we found no significant associations of genotype with relevant quantitative glycemic traits at baseline ; similarly , there were no significant differences across genotype groups in change in isi , fasting glucose , a1c , or dio after 1 year of metformin ( table 3 ) . the c allele was associated with greater weight gain in the metformin arm . in this arm , there were no statistically significant interactions between the c allele and bmi or waist circumference on diabetes incidence . analyses stratified by ethnic group failed to show any ethnic - specific beneficial effects of the c allele with regard to diabetes incidence on metformin - treated participants ( table 4 ) . diabetes incidence in the dpp by genotype at rs11212617 , treatment arm , and self - reported ethnicity association of rs11212617 with quantitative glycemic traits at 1 year ethnic - specific effects of rs11212617 on diabetes incidence in the dpp in the dpp , the effect of metformin to prevent diabetes or improve relevant glycemic traits was not magnified among carriers of the c allele at rs11212617 in the atm gene . our findings do not support the previously reported association of this allele with improved metformin action on glycemic control . the original association was consistent in three different datasets ( the discovery sample and two follow - up cohorts ) and has been reported recently in other clinical cohorts similarly ascertained ( 13 ) . first , metformin response is defined differently in a prediabetic cohort ( impact on diabetes incidence or quantitative glycemic traits ) than it is in a disease cohort ( ability to reach a1c 7% under treatment ) . second , metformin may be more effective in individuals with a higher a1c at baseline , and thereby the effects of genotype on response might be easier to detect in the disease setting . third , the reported effect might be confined to populations of european descent , e.g. , if rs11212617 tags a low - frequency variant unique to white populations , further diminishing statistical power in the dpp multiethnic cohort . and fourth , the previously reported gwas was based on a retrospective evaluation of clinical records , where potential confounders ( e.g. , if genotype were to influence comorbidities that affect patient adherence , continuity of care , or frequency of a1c measurements ) are harder to control than in a clinical trial . to address potential ethnic differences in the genomic architecture of this region that might explain our negative results , we examined the haplotype structure around this locus in the hapmap european ( ceu ) and west african ( yri ) datasets . the full atm gene and the rs11212617 variant share a segment of tight linkage disequilibrium in both the ceu and yri populations ; there is a recombination hot spot downstream from rs11212617 , beyond which snps display equally low correlations with rs11212617 in ceu and yri , indicating that major differences in linkage disequilibrium patterns would be unlikely to account for potentially discrepant findings in europeans and africans . furthermore , the region distal to this hot spot was well captured by the original gwas array , suggesting that a true signal emerging from this region ( and which might have explained a stronger association in europeans than in africans ) would have also been detected by the original gwas . more generally , a better - powered genome - wide assessment of pharmacogenetic responses in t2d is needed ; whether genetic information will prove useful in diabetes prevention or therapeutics must be tested in prospective clinical trials .

objectivethe c allele at the rs11212617 polymorphism in the ataxia - telangiectasia mutated ( atm ) gene has been associated with greater clinical response to metformin in people with type 2 diabetes . we tested whether this variant modified the effect of metformin in the diabetes prevention program ( dpp ) , in which metformin reduced diabetes incidence by 31% in volunteers with impaired glucose tolerance.research design and methodswe genotyped rs11212617 in 2,994 dpp participants and analyzed its effects on diabetes incidence and related traits.resultscontrary to expectations , c carriers enjoyed no preventive advantage on metformin ; their hazard ratio , compared with a carriers , was 1.17 ( [ 95% ci 0.961.42 ] , p = 0.13 ) under metformin . there were no significant differences by genotype in metformin s effects on insulin sensitivity , fasting glucose , glycated hemoglobin , or disposition index.conclusionsthe reported association of rs11212617 with metformin response was not confirmed for diabetes prevention or for effects on relevant physiologic parameters in the dpp .

RESEARCH DESIGN AND METHODS Participants Genotyping Measurements Statistical analysis RESULTS CONCLUSIONS

pentalogy of cantrell is a rare multiple congenital malformation which occurs worldwide with a reported incidence of 5.5 per million live births.1 the exact cause is not known but is mainly thought to be sporadic , though its being associated with some chromosomal disorders like trisomy 1823 and deletion on locus at xq25 - 26 has been described in some cases.4 it was first described by cantrell et al . , in 19585 with the pentad consisting of a giant omphalocoele and a supra - umbilical anterior abdominal wall midline defect , sternal cleft , ectopia cordis , intracardiac congenital malformations like ventricular septal defect , atrial septal defect and tetralogy of fallot , ventral midline diaphragmatic defect with defect of the diaphragmatic pericardim.5 other associated defects include cranial and facial anomalies , clubfeet , malrotation of the colon , hydrocephalus and anencephaly.67 pentalogy of cantrell often have a poor outcome which is dependent on the severity of the malformations.8 though some cases of pentalogy of cantrell had been reported in nigeria , its true prevalence is not known and none of those reported had been proven to completely fulfill the five main components of the pentad.91011 we , therefore , report a case of pentalogy of cantrell in a 9-month - old boy who completely fulfilled the diagnostic criteria . a 9-month - old boy was first seen at the age of 5 months being delivered with multiple congenital malformations involving the anterior abdominal and chest walls . the child was delivered at term gestation and at home ; the pregnancy was not adversely eventful and she had no antenatal care . however , the child has defect on the anterior abdominal wall with a huge swelling extending from the lower anterior abdomen to the lower anterior chest wall , which was pulsatile and covered by a thin membrane which was initially reddish at birth but later became thickened and darker following daily cleaning and dressing . there was no history of vomiting or constipation , child sucked directly from the breast , though interrupted occasionally to catch his breath , there was no history of difficulty with breathing and no darkening of the lips or mucous membrane . the mother was a 25-year - old stay at home mother with four other children in a non - consanguineous marriage ; she neither smoked cigarette nor drank alcohol and had no adverse medical record . , there was a big pendulous and pulsatile mass extending from slightly above the symphysis pubis to the epigastrum measuring 12 10 cm [ figure 1 ] with an epithelised membranous covering ; it had visible peristaltic waves , it was pundunculated , with cardiac pulsation felt in the epigastric region [ figure 2 ] . bowel and heart sounds were heard over the mass below the level of the diaphragm . he was not tachypneic or tachycardic and had first and second heart sounds with a systolic murmur . his chest x - ray showed a midline heart with inferior displacement [ figure 3 ] , abdominal ultrasound showed the swelling to contain loops of bowl and echocardiography revealed a 6 mm secundum atrial septal defect . the child is currently being followed up awaiting staged repairs of the defects . a bulge in the region of the epigastrium and lower sternum x - ray showing loops of bowl in the swelling with displacement of cardiac shadow pentalogy of cantrell is commoner in boys and less than 60 cases have been documented so far worldwide as at 2007.8 the exact mechanism is not completely understood , however , the proposed pathogenesis involves a defect in embryogenesis between 14 and 18 days after conception due to failure of the in folding of the lateral mesoderm . while most affected children die shortly after birth due to the severity of their defect ( especially the associated intra - cardiac defect and risk of infection in open defects ) , it has been reported in a 32-year - old - man.12 our patient was 9-month - old and remained active despite the associated atrial septal defect and has better prospect with adequately staged surgical intervention . our case had all the major features of the pentad , though with a relatively favourable lesion because the membrane of the giant omphalocoele was completely epithelised [ figure 4 ] and the peduncle covered with skin , which helped in preventing infection ; furthermore , the atrial septal defect was of moderate size and the child had remained stable , though he stands the risk of trauma to the barely covered heart , but with proper counselling the child has survived up till this age . the mass covered by thickened , hyperpigmented keratinised membrane pentalogy of cantrell can be diagnosed prenatally but that has not been the case in those reported from nigeria so far because lack of accessibility to a proper antenatal healthcare and for the fact that most of those diagnostic tools are not readily accessible to these low income parents . patients with complete expression of pentalogy of cantrell are rare and few may survive to older age depending on the severity of the associated lesions , more especially the intracardiac defect . furthermore , we advised that patients with giant omphalocoele should be thoroughly evaluated , because they may have an incomplete expression of the syndrome .

pentalogy of cantrell is a rare congenital malformation whose exact cause is not completely understood ; it is characterized by defects in the anterior abdominal and thoracic walls consisting of omphalocoele , diaphragmatic defect , ectopia cordis , intracardiac defects and sternal clefts . the complex has variable clinical expression with complete and incomplete expressions reported . we , therefore , report a case of complete manifestation of the pentad in a 9-month - old boy .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

pustulotic arthro - osteitis ( pao ) was first described by sonozaki.it is a relatively rare disease a 53-year - old woman was admitted to our department in february 2007 with a pustular rash affecting the palms and soles . inflammation and swelling of the small joints and right - sided low back pain developed during hospitalization . the patient had no history of psoriasis and there was no history or clinical evidence of psoriasis in her family . on physical examination , sharply demarcated erythematosquamous lesions with groups of sterile pustules were seen on the palms and soles [ figures 1 and 2 ] . there was diffuse swelling and erythema over the right sternoclavicular , the right wrist and the left ankle joints . pustular lesions on the right palm pustular lesions on the medial sides of feet laboratory findings showed an erythrocyte sedimentation rate ( esr ) of 120 mm / h ( normal < 20 mm / h ) and c - reactive protein ( crp ) of 6.44 mg / dl ( normal<0.8 mg / dl ) . serum was negative for rheumatoid factor and antinuclear factor , and anti - dsdna , anti - ro , anti - la , and anti - sm antibodies were not present . a technetium-99 m diphosphonate - labeled scintigram of bones revealed abnormal focal increases in tracer uptake in the right sternoclavicular and left ankle joints [ figure 3 ] although x - ray of the wrist and the sternocostoclavicular region revealed soft tissue swelling , there was no ossification of the sternocostoclavicular region . a computed tomography scan showed sclerosis of the sacroiliac joints whole - body bone scintigram skin biopsy from lesions on the left hand showed spongiosis and dyskeratotic cells in the basal layer , with infiltration of polymorphonuclear cells under the orthokeratotic epidermis . a dense dermal neutrophilic infiltrate was also present . on the basis of these clinical and histopathologic features and the scintigram findings we made the diagnosis of pao . after about 1 week of treatment , the swelling and pain in the affected joints had decreased , and the prednisone dose was tapered to 10 mg per day . during that time , new palmoplantar pustulotic lesions appeared . the patient is now healthy after 6 months follow - up and no maintenance treatment is required . palmoplantar pustulosis is a skin disease characterized by recurrent eruptions of sterile pustules , with erythema and exfoliation . the lesions are situated exclusively , and often symmetrically , on the palms and/or soles.[13 ] the age of onset is usually 3060 years , and males and females are affected equally . pao is a rheumatic syndrome of unknown etiology and is characterized by an inflamatory osteitis of the sternoclavicular joint along with palmoplantar pustulosis . pao or sonozaki syndrome occurs in about 10%30% of patients with palmoplantar pustulosis . until a few years ago , this syndrome was considered to be a manifestation of psoriatic arthritis and was then erroneously classified by rheumatologists in the sapho ( synovitis , acne , pustulosis , hyperostosis , osteitis ) syndrome . similar clinical features have been observed in patients with sternocostoclavicular hyperostosis ( scch ) and sapho . scch is a rare condition characterized radiologically by progressive hyperostosis of the medial ends of the clavicles , upper ribs , and sternum , together with soft tissue ossification between the upper ribs and clavicles . clinically , the patients present with painful swelling of the sternum , clavicles , and upper ribs . sapho presents with anterior chest wall involvement , axial skeletal lesions , and osteitis of appendicular bones . pao is now classified as a member of the seronegative spondyloarthritis group of disease ( which includes ankylosing spondylitis , reiter 's syndrome , and psoriatic arthritis ) ; however , it is not associated with hla - b27 . the principal lesion in pao is sternoclavicular involvement , with lesions also occurring in the spinal column and the sacroiliac and peripheral joints . our patient had swelling of the sternoclavicular , right wrist , and left ankle joints , as well as low back pain . although our patient had osteoarthritis affecting the sternoclavicular region , right wrist , and left ankle , as shown by the bone scan , there were no obvious radiological changes . arthritis of the sternoclavicular joint is most frequent in patients with pao , but occurs in less than 15% of patients with ankylosing spondylitis , reiter 's disease , and psoriatic arthropathy.[68 ] in the majority of the cases , the arthritis is of seronegative mono- or oligo - arthritic type , as in our patient who had oligoarticular arthritis . in pao , the most commonly affected peripheral joints are the metacarpophalangeal joints , the proximal interphalangeal joints of fingers , and the elbow and knee joints . other joints such as the hip , the ankle , and the wrist joints have also been involved rarely . our patient had ankle and wrist arthritis , but the knee joint was not involved . pao is characteristically non - erosive and transient and does not induce contracture deformities , and this was true in our patient also . the interval between the onset of skin eruptions to the onset of arthro - osteitis is about 2 years in more than 70% of cases . in our patient , the cause of these abnormalities , like the cause of the underlying disease process , remains unclear . recently cultured propionibacterium acnes from biopsy specimens of patients with this syndrome in approximately 50% . this agrees with the experience of other workers , who have also failed to isolate any specific microorganism from the bones or skin lesions of affected patients . in pao , the arthropathy is not associated with hla - b27 , and this was true in our patient also . to the best of our knowledge , hla - b27 is related to generalized pustular psoriasis , psoriatic arthritis , ankylosing spondylitis , and reiter 's disease . x - ray findings have been consistently reported to be negative , except for soft tissue swellings in acute phases , as in our patient also . the presenting symptoms and findings in our patient , including palmoplantar pustulosis and arthro - osteitis , were compatible with sonozaki syndrome or pao . treatment is usually symptomatic and consists mainly of analgesics and nonsteroidal anti - inflammatory drugs such as phenylbutazone and indomethacin . corticosteroids , colchicine , sulfasalazine , methotrexate , and cyclosporine have been used with variable success . topical steroids , etretinate , and topical puva have been used for the pustular lesions on the palms and soles . if the anterior chest wall symptoms are so severe as to interfere with the patient 's daily activities , surgical resection of the clavicle is recommended . presented here , dapsone treatment was used successfully to treat the cutaneous manifestations and it also maintained the prolonged remission . to the best of our knowledge , this is the first report of pao from turkey . radiological studies must be performed in a patient with palmoplantar pustulosis and swelling of joints to avoid missing the diagnosis .

pustulotic arthro - osteitis , first described by sonozaki , is a relatively rare disorder . its prevalence is however probably underestimated in dermatological literature . early recognition of the signs can prevent misdiagnosis . we describe a turkish patient who presented with palmoplantar pustulosis and involvement of the sternoclavicular joint and peripheral oligoarthritis .

Introduction Case Report Discussion

shingles , also called herpes zoster ( hz ) , is a common viral disease . psoriasis is another common , chronic relapsing and remitting inflammatory disease that involves the skin and joints with an overall prevalence of 2% to 3% of the world 's population . koebner phenomenon ( kp ) , also called isomorphic response , is initially referred to the formation of psoriasiform lesions after cutaneous trauma on healthy skin areas of psoriatic patients , and now is extended to the instances that the people who had pre - existing dermatosis develop lesions after trauma or injury . only a few of psoriatic kp following shingles have been reported in literature , and the kp lesions always occur at healing or healed hz eruptions with a latent period of 1 week to 4 months from the occurrence of shingles in this condition . herein , we report a psoriatic patient in whom kp is occurring at the site of developing hz lesions , and to our knowledge , no similar description has been reported before . a 30-year - old man was referred with 3-day history of clustered scaly papules on the right chest wall . the patient had episodes of groups of erythematous papules with shooting pain distributed on his right side of chest alone in the dermatome 9 days ago , which quickly turned into vesicles within 2 days . he was diagnosed as hz and started on treatment of valaciclovir on the 4 day after the appearance of painful papules , resulting in rapid pain relief after being treated for 2 days . on the 6 day of the course of disease , the patient found that the vesicles began subsiding and became scaly papules rapidly in situ . the patient had 7-month history of scalp psoriasis showing good response to topical application of calcipotriol ointment , but he used the medicine irregularly resulting in recurrent lesions sometimes . he never had kp lesions , and any other associations , since the onset of psoriasis . cutaneous examination revealed that scaly erythematous papules and plaques located on the scalp and forehead ( figure 1a ) , and groups of clustered erythematous papules with silver scales in the dermatome distributed on the right side of chest wall where the prior hz lesions occurred ( figure 1b , c ) . no other kinds of lesions were observed elsewhere including limbs , nails , and the trunk except the site where the prior hz eruptions involved . the patient was diagnosed as kp of psoriasis , and was treated with topical application of clobetasol propionate and calcipotriol compounds , which resulted in excellent response after a week of treatment ( figure 1d ) . on 1 year of follow - up , the patient had occasional recurrence of psoriasis when the treatment was stopped ; no new zosteriform eruptions reoccurred . interestingly , the recurrent lesions occurred only on the scalp and the areas of prior shingles each time ( figure 1e , f ) . histology from thoracic lesion showed regular epidermal hyperplasia with test - tube - shaped rete ridges , thinning over some derma papilae , acanthosis , parakeratosis , and lack of granular layer ( figure 2 ) . ( b , c ) groups of clustered papules with silver scales in the dermatome distributed on the right side of chest wall where the previous developing hz involved . ( d ) the scaly papules relived markedly after 1 week of topical application of clobetasol propionate and calcipotriol compounds . ( e , f ) the recurrent psoriatic lesions on the areas of the previous shingle on 7 months of follow - up . histology shows regular epidermal hyperplasia with test - tube - shaped rete ridges , thinning over some derma papilae , acanthosis , parakeratosis , and lack of granular layer . the present patient had groups of painful papules and vesicles distributed in the dermatome on unilateral chest wall ; hz can be diagnosed clinically although without laboratory support . generally , the natural history of hz is that its initial lesions appear as erythematous papules , which turn into vesicle within 12 to 24 hours . the vesicles of hz progress into pustules in about 3 days and form scabs over the next 7 to 10 days . the inflammatory lesions within the involved dermatome after hz have been rarely described , but the lesions are present as flat topped lesions or annular papules , and usually appear within a month with the absence of scales . it was found in the present patient that his blisters became scaly papules on the 6 day during the course ; we considered that the scaly papules formed during developing stage of hz rather than recovering time . based on the psoriasis history and the present clinical features : the developing painful papules and vesicles became scaly papules with underlying bleeding points , which showed excellent response to topical application of clobetasol propionate and calcipotriol compounds , we considered that the present scaly papules are kp of psoriasis subsequent to developing hz rather than to healing hz lesions themselves , although slight scarring may occur in patient with hz at the site the blisters have been . the diseases tending to koebnerization include psoriasis , vitiligo , lichen planus , darier disease , bullous dermatoses , and so on . the provoking factors for kp include trauma , burns , friction , insect bites , surgical incision , as well as dermatoses and therapeutics , such as hz , syphilis , and ultraviolet b treatment , and even the cupping therapy , a kind of traditional chinese medicine . season , disease severity , trauma on skin , scar tissue , and emotional stress can also trigger kp in patient with psoriasis . the duration from injury to occurrence of kp may range from 3 days to years , but in general , it is between 10 and 20 days . only a few cases have described their kp lesions of psoriasis at the site of healed or healing hz as well as varicella involved . in the present patient , kp eruptions occurred at the areas of developing hz lesions with only 6-day interval from the painful chest and 4 days from the appearance of vesicles , which , to our knowledge , has never been reported before . other interesting entity is that , on a year of follow - up , the recurrent psoriatic lesions located only on the scalp and the healed hz areas . as the recurrent thoracic lesions occurred synchronously with the scalp 's each time , we considered them to be relapsing psoriasis rather than recurrently psoriatic kp on healed hz . however , the reason why the recurrent psoriasis was localized to the areas mentioned above was unknown . although kp is a common condition , its pathogenesis is not fully understood yet , but may involve cytokines , stress proteins , adhesion molecules , and autoantigens . it is considered that nerve growth factor , tumor necrosis factor- , substance p as well as interleukin-1 play important roles in the pathomechanism of koebnerization in psoriasis . the occurrence of psoriatic koebnerization at the site of shingles and varicella was considered to be mediated by neuropeptides such as substance p. substance p , an endogenous neuropeptide being potentiated by viral infection and acting as a chemomediator of nociceptive impulse from periphery to central nervous system , plays an important role in the hz - associated pain . viral infection can potentiate the effect of substance p by decreasing the degradation of its breakdown enzyme . we speculate that substance p may play an important role in the present kp , although the exact mechanisms remain unknown . interestingly , we soon met another typical shingles patient with prior severe psoriatic lesions over his whole body , but neither his prior shingles involved areas nor other areas occurred kp during 6 months of follow - up . it suggests that kp occurs at the site of hz lesions in not all the psoriatic patients with shingles . as a matter of fact , although both psoriasis and shingles are common diseases , few of psoriatic kp subsequent to hz have been reported . koebner lesions should be treated in the same way as the associated dermatosis , as our present patient did . the present patient was notable because the psoriatic kp lesions occurred at the site of developing hz , and the recurrent psoriasis localized only on the primarily areas and healed shingles areas . both psoriasis and shingles are common cutaneous diseases . about 25% of the patients with psoriasis not only healing and healed shingles can cause kp in psoriasis , but also developing shingles can cause such a condition at the site of hz lesions . the authors thank professor rashmi sarkar , md , department of dermatology , maulana azad medical college and lnjp hospital , new delhi , india , for her assistance in revising the manuscript ; and also thank the patient participating in the present study .

abstractboth shingles and psoriasis are common cutaneous diseases . about 25% of the psoriatic patients develop koebner phenomenon ( kp ) after various injuries , and in rare instance , kp may occur at the site of healed or healing shingles.we report a 30-year - old man with 7-month history of scalp psoriasis who developed kp at the areas of developing shingles . cutaneous examination revealed scaly erythematous papules and plaques located on the scalp and forehead , and groups of clustered erythematous papules with silver scales in the dermatome distributed on the right side of chest wall the prior herpes zoster lesions involved . after removal of the scales on the papules , underlying bleeding points were present.the lesions on chest had good response to anti - psoriatic therapies , as the lesions on scalp did . after a year of follow - up , recurrent psoriasis occurred , but the lesions were located only on the scalp , and the areas of prior occurrence of shingles , because of which we considered diagnosis of recurrent psoriasis rather than relapsing kp for the chest lesions.not only the healing and healed shingles can trigger kp in psoriasis , but also the developing shingles can cause psoriatic kp at the site of herpes zoster lesions .

INTRODUCTION CASE PRESENTATION DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS

subtilosin is a ribosomally - synthesized cyclopeptide produced by bacillus subtilis atcc 6633 and a recently identified natural isolate of bacillus amyloliquefaciens [ 1 , 2 ] . this protein has a unique structure among bacteriocins , and possesses antimicrobial activity against a variety of pathogenic organisms , including gardnerella vaginalis , listeria monocytogenes , and streptococcus agalactiae ( group b streptococcus ) . the ability to inhibit the growth of g. vaginalis is of particular importance as it is one of the primary causative agents for bacterial vaginosis ( bv ) , a common condition found in up to 30% of women in north america . bv is characterized by the replacement of normal vaginal lactobacilli with anaerobic bacteria ( e.g. , prevotella , bacteroides , and mobiluncus ) and mycoplasmas , as well as a several log increase in overall bacterial growth [ 57 ] . bv is asymptomatic in approximately one half of all cases , but is associated with a wide variety of symptoms and problems ranging from complications with pregnancies ( i.e. , preterm births , low fetal birth weight ) to the development of pelvic inflammatory disorder [ 711 ] . recent studies have estimated that nearly one in three women in the united states ( 29.2% ) suffer from bv , with varying prevalence according to ethnicity and education levels . one of the most troubling aspects of bv infection is the association with an increased risk of several sexually transmitted diseases ( stds ) , including chlamydia , herpes , gonorrhea , trichomoniasis , and hiv / aids [ 1216 ] . the treatments recommended by the centers for disease control ( cdc ) are clindamycin and metronidazole administered either orally or intravaginally . following these guidelines successfully treats 60% of bv cases , but 20% of these cases return with highly developed antibiotic resistances [ 1820 ] . in such cases , it would be extremely desirable to have an alternative form of treatment that could fully eradicate the infection . bacteriocins are typically divided into two major , yet diverse , classes : class i , or lantibiotics , contain unusual amino acids and are subject to extensive posttranslational modifications ; and class ii , the heat stable nonlantibiotics . subtilosin has a unique posttranslational structure that is unmatched among bacteriocins , which has led to speculation that it may belong in a distinct and separate class [ 2 , 3 ] . bacteriocins have been widely considered for use in medicinal and pharmaceutical applications , particularly for their bactericidal activity against multidrug resistant organisms . they are especially appealing alternatives since their cost of production is so comparatively low to conventional pharmaceutical treatments . much attention has recently been focused on the spermicidal activity of bacteriocins due to their targeted antibacterial activity and lack of effect on human tissues . nisin a , a well - studied class i bacteriocin produced by lactococcus lactis subsp . lactis , has human spermicidal activity [ 24 , 25 ] , and subtilosin has spermicidal activity against boar , bovine , horse , and rat spermatozoa . the results of these previous studies suggest that subtilosin may also have spermicidal activity against human spermatozoa , prompting our investigation . since subtilosin has proven antimicrobial activity against the pathogen largely responsible for bv , its toxicity to human tissues was assayed to determine its potential as a safe alternative remedy . for the human in vitro study , the epivaginal ectocervical tissue model ( mattek corporation , ashland , mass , usa ) was employed to examine the relationship between prolonged exposure to subtilosin and cell viability . spermicidal evaluations were conducted to investigate the ability of the peptide to restrict or eliminate sperm mobility , leading to its classification as a spermicidal agent . subtilosin was prepared as previously described . to prepare a cell - free supernatant ( cfs ) , cells were removed by centrifugation ( hermle z400k ; labnet , woodbridge , nj , usa ) for 25 minutes at 4500 g and 4c . the supernatant was filter sterilized using 0.45 m filters ( fisher , pittsburgh , pa , usa ) . the protein of interest was precipitated from the supernatant by adding 30% ammonium sulfate ( w / v ) while stirring overnight at 4c and was resuspended in 20 ml of double distilled h2o . the column chromatography method described by sutyak et al . was used to purify subtilosin from the cfs , producing a near - pure isolate in the 90% methanol eluate . the antimicrobial activity of all samples was confirmed by the well - diffusion assay according to the protocol of cintas et al . with additional modifications . the active fraction was concentrated to dryness using a savant sc110 speed vac and uvs400 universal vacuum system ( savant instruments , farmingdale , ny , usa ) , then resuspended in 1.5 ml ddh2o . the concentration of subtilosin in the column - purified fraction was determined using the micro bca protein assay kit according to the manufacturer 's protocol ( pierce , rockford , ill , usa ) . in brief , the assay measures the reduction of cu to cu by colorimetric detection of cu by bicinchoninic acid . bovine serum albumin ( bsa ) was used to develop a standard curve with concentrations ranging from 0.5 to 20 g / ml ; the concentration of subtilosin was calculated using the r value from the trendline of the standard curve graph . the concentration of subtilosin in the cfs was not measurable with the micro bca protein assay due to the high level of background proteins in the solvent ( mrs medium ) . as an alternative , the protein concentration was calculated by comparing the antimicrobial activity of known concentrations of column - purified protein to equal volumes of cfs . five two - fold dilutions were made from the stock samples of both the cfs and the column - purified fraction . well diffusion assays were performed using 50 l of each dilution against micrococcus luteus atcc 10420 , which is commonly used as a reference microorganism for the determination of a bacteriocin 's biological activity . as reported previously , the concentration of lactic acid in the cfs was measured to assess its potential effects on antimicrobial activity and cell viability . the quantity of each form of the acid in the sample was measured using a commercially available d - lactic acid / l - lactic acid kit ( roche boehringer , mannheim , germany ) , according to the manufacturer 's instructions . the epivaginal ( vec-100 ) ectocervical tissue model ( mattek corporation , ashland , mass , usa ) was used and maintained as fully described by dover et al . . the tissues were exposed to 83 l of subtilosin cfs ( 136 g / ml ) for 4 , 24 , and 48 hours . for exposure times over 24 hours , the tissues were aerated by placing them on two metal washers ( mattek corporation , ashland , mass , usa ) and fed with 5 ml of the assay medium . double - distilled water ( ddh2o ) was used as a negative control , and was applied to cells after 6 , 24 , and 48 hours . a spermicidal product containing 4% nonoxynol-9 ( ortho options conceptrol vaginal contraception gel , advanced care products , skillman , nj , usa ) was used as a positive control based on its documented cytotoxic properties [ 30 , 31 ] . a cream ( monistat-3 , ortho mcneil pharmaceutical , inc . , raritan , nj , usa ) containing 4% of the nontoxic , bv - active compound miconazole nitrate [ 30 , 31 ] , was used as a negative control . following the designated exposure times , the mtt ( 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ) assay was used to determine overall cell viability . the data were used to approximate an effective time ( et ) that would reduce cell viability to 50% ( et-50 ) . the mtt assay was carried out according to the protocol outlined by dover et al . . briefly , the viability of ectocervical cells after exposure to subtilosin was measured as a direct proportion of the breakdown of the yellow compound tetrazolium to the purple compound formazan , since only living cells can cause this reaction to occur . tissues were exposed to subtilosin and the two controls for several designated time points ; at the conclusion of each , the liquid in the plate wells was combined with the liquid from the tissue inserts . this mixture was then assayed spectrophotometrically using a 96 well - plate reader ( mrx revelation , dynex technologies , va , usa ) to determine the level of tetrazolium degradation . the viability ( % ) of the treated tissue inserts was calculated according to an equation provided by the manufacturer : % viability = od570 ( treated tissue)/od570 ( negative control tissue ) . the exposure time that reduced tissue viability by 50% ( et-50 ) was calculated according to [ v = a + b * log(t ) ] described by ayehunie et al . , where v = % viability , t = time in minutes , and a and b are constants representing the viability data from the time points preceding and following 50% viability . on the whole , there is a direct relationship between the length of the et-50 and the toxicity of the tested application ( i.e. , a shorter et-50 corresponds to a more harmful compound ) . the cfs gathered from a b. amyloliquefaciens culture was used to test the effect of subtilosin exposure on the motility of human spermatozoa . initially , the cfs was diluted with normal saline ( 0.9% ) so that 200 l of the final material was equivalent to 50 l , 100 l , or 200 l of undiluted cfs . each sample was collected by self - masturbation in a polypropylene specimen container ( fisher ) prior to transport to the laboratory . within 1 hour of collection , total sperm count was calculated using bright field light microscopy ( olympus bx50 ; 400x ) after dilution ( 1:50 ) of the semen in normal saline . the initial percentage of motile sperm was calculated prior to testing with a neubauer hemacytometer . the determination of motile sperm % was made using randomly selected field views ( 400x ) from a count of between 104201 cells . any visibly moving spermatozoa ( directional or stationary ) were counted as motile cells . the percentage of forward progressing spermatozoa was subjectively determined based on the assumption that 70% of the sperm in a normal sample would behave in such a manner . a modified sander - cramer test was used to determine the effect of column - purified subtilosin on human spermatozoa motility . this measured the effect of subtilosin after 30-second exposure times of 5 volumes ( 200 l ) of the solution at each dilution ( 25% and 50% in normal saline , and 100% ) with one volume ( 40 l ) of whole semen . the motilities of cells from random high - magnification fields ( 400x ) of the sample were determined in duplicate as described above . statmost32 ( version 4.1 ) statistical software ( datamost corporation , sandy , utah , usa ) was used to calculate all statistical parameters . any differences between treatment groups were assessed by the newman - keuls multiple range test . the concentration of subtilosin in the column - purified sample was estimated at 135.7 g / ml . the cfs and column - purified sample produced identical zones of inhibition at each dilution ( data not shown ) ; therefore , the concentrations of protein in both solutions were assumed to be equivalent . while it is improbable that a 100% yield would be attained from column chromatography , previous work has shown that protein concentrations can be precisely calculated based on the comparisons we conducted . due to the difficulty in measuring the cfs protein concentration via other assays , the chosen method was deemed the most accurate and reproducible . after 48 hours of exposure to subtilosin , the epivaginal ectocervical tissues retained a high level of viability compared to the positive control , nonoxynol-9 , and the negative control , miconazole nitrate ( table 1 ) . due to the lack of toxicity of the antimicrobial , the et-50 value for subtilosin could not be established since the total cell viability did not drop below 50% at any of the given time points . however , a projection of the et-50 value is possible by an extrapolation of the data . data presented in table 1 can be fit to a curve described by ln(v ) = a + bt , where a = 4.605995356 and b = 0.00014151 ( coefficient of determination , or r , = 0.9998 ) , from which the et-50 is estimated at 70 hours . subtilosin reduces human sperm motility in a dose - dependent manner ( figure 1 ) . the motility of the treated spermatozoa ranged from 0 to 88% of control motility levels over the four - fold range of subtilosin concentrations . all of the subtilosin concentrations tested reduced motility compared to the control samples . the differences in the proportion of motile spermatozoa in all samples ( 28.3 , 56.7 , and 113.3 g / ml protein equivalents ) were found to be significant ( p < .05 ) according to the newman - keuls multiple range test . tablecurve 2d ( ver 5.0 ) curve - fitting software ( spss scientific software , chicago , ill , usa ) was used to fit the data to a dose - response curve described by ln ( % motility ) = a + b [ subtilosin a]3 , where a = 4.20781 ; b = 2.5814e 06 ; and [ subtilosin a ] is expressed as g / ml protein equivalents . the ic50 value , or the amount of subtilosin required to reduce the motility of spermatozoa in whole semen by 50% , was calculated to be 64.5 g / ml . similar to motility , forward progression of spermatozoa is reduced in a dose - dependent fashion by subtilosin . in control samples , 70% of sperm exhibited forward progression ; in the presence of 50 l subtilosin this decreased to 5070% , while 100 l caused a decline to only 10% forward progression . all forward progression was eliminated after treatment with 200 l subtilosin , with most sperm tails becoming coiled . the b. amyloliquefaciens - produced bacteriocin subtilosin has proven antimicrobial activity against the vaginal pathogen g. vaginalis , but was not harmful to the normal and healthy lactobacillus vaginal microbiota . data from human vaginal cell viability assays convincingly demonstrated the safety of subtilosin for human applications in comparison to other accepted and available products , indicating it could be safely incorporated into personal care applications aimed at the treatment of bacterial vaginosis . prior research in our laboratory that involved similar studies with the epivaginal model was also carried out in conjunction with in vivo testing of the rabbit vaginal irritation ( rvi ) system , which doubly confirmed the safety of another antimicrobial peptide , lactocin 160 . therefore , we are confident that using the epivaginal model instead of animal testing to demonstrate the safety of subtilosin has provided reliable and valid results . subtilosin was also found to significantly reduce the motility of human spermatozoa in a concentration - dependent manner for all concentrations tested . the effect of subtilosin on the forward progression of spermatozoa was also observed to be a dose - dependent interaction . serial dilutions showed a steady decline in forward progression , with all progression halted at the highest concentration tested . it was also noted that at the highest concentration , the tails of the sperm cells were curved or coiled , indicating the cells were damaged beyond a simple restriction of movement . coiling of the cells is considered to be a sperm abnormality , and may indicate damage to the plasma membrane . tail coiling has been observed after in vitro exposure of monkey spermatozoa to methyl mercury . it is worth noting that nisin , a bacteriocin given generally recognized as safe ( gras ) status by the food and drug administration ( fda ) , was also shown to have impressive spermicidal activity . however , the allure of these results is diminished due to the fact that it also has potent antimicrobial activity against healthy vaginal microbiota ( chikindas et al . , unpublished data ) , a strong detraction from its commercial applicability . therefore , subtilosin 's spermicidal activity , combined with its overall safety to both human tissues and healthy human microbiota , make it a highly recommendable compound for inclusion in topical bv treatments and human contraceptive products . to facilitate the process of its incorporation into contraceptive treatments , additional analysis will be done to determine the reversibility of damage done to the spermatozoa , as well as a time course assay to further elucidate the exact changes effected by subtilosin .

bacterial vaginosis ( bv ) , a condition affecting millions of women each year , is primarily caused by the gram - variable organism gardnerella vaginalis . a number of organisms associated with bv cases have been reported to develop multidrug resistance , leading to the need for alternative therapies . previously , we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against g. vaginalis , but not against the tested healthy vaginal microbiota of lactobacilli . after conducting tissue sensitivity assays using an ectocervical tissue model , we determined that human cells remained viable after prolonged exposures to partially - purified subtilosin , indicating the compound is safe for human use . subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose - dependent manner , and can therefore be considered a general spermicidal agent . these results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and bv prophylaxis and treatment .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION

lymphangioma is a developmental malformation of lymphatic tissue , characterized by submucosal tumors covered with normal mucosa.1 these benign tumors are commonly found in children , and although they may develop anywhere , the tumors are usually found in the head , neck , and axillary regions , and rarely in the gastrointestinal tract.2 most reported cases of lymphangioma of the colon are solitary ; few cases involve multiple lymphangioma , also known as colonic lymphangiomatosis.1 colonic lymphangiomas are typically asymptomatic , are discovered only incidentally , and usually do not require treatment.3,4,5 however , in some cases , they may cause abdominal pain , bleeding , intussusception , or protein - losing enteropathy , requiring surgical or endoscopic resection.1,6 a 38-year - old man visited our comprehensive medical center for a general check - up . he had no previous medical problems other than diabetes mellitus and essential hypertension , which were diagnosed 1 year prior at another hospital . laboratory test results , including the levels of tumor markers carcinoembryonic antigen and carbohydrate antigen , were all within normal ranges , except the levels of triglycerides at 206 mg / dl ( normal , 10 to 180 ) and hemoglobin a1c at 9.3% ( normal , 4.0 to 6.0 ) . esophagogastroduodenoscopy showed hyperemic mucosal change in the antrum and body of the stomach , with multiple shallow erosions in the se - cond portion of the duodenum . an initial colonoscopy revealed multiple , variably sized cystic mass lesions in the mid - portion of the ascending colon up to the proximal portion of the transverse colon ( fig . computed tomography ( ct ) revealed no abnormalities other than a fatty liver ( fig . a solution composed of hypertonic saline , epinephrine , and methylene blue was injected around some of the cystic masses and biopsy specimens were taken with snare electrocautery ( fig . incision on one of the masses using a dual knife caused clear yellow fluid to flow out from the mass , after which the mass began to shrink . the biopsied tissue revealed normal colonic mucosa and markedly dilated lymphatic vessels in the submucosa . immunohistochemical staining for cd34 and d2 - 40 showed a positive reaction along the lining cells of the dilated lymphatic channels ( fig . cd34 is an endothelial cell marker in different vascular beds in human tissues,7 and the monoclonal antibody d2 - 40 is a marker of lymphatic endothelium.8 d2 - 40 does not stain vascular endothelium . eight months later , the patient underwent a third colonoscopic examination that showed normal colonic mucosa without the protruded lymphangiomatosis previously been observed in the ascending and transverse colon . no scarring or trace of the former lesions lymphangioma is regarded as a developmental malformation of lymphatic tissue ; obstruction or agenesis of lymphatic tissue causes abnormal dilatation and proliferation of the lymphatic channel , leading to the formation of a cystic mass.3,4 secondary lymphangioma formation can be caused by abdominal trauma , lymphatic obstruction , inflammatory processes , cancer , surgery , or radiation . in the present case , colonic lymphangiomatosis was discovered in adulthood , suggesting that the lesion developed secondary to a local disturbance of the lymphatic circulation . however , the patient had no specific history related to colonic lymphangiomatosis , and abdominal ct showed no comorbid disease ; thus , it would be more rational to conclude that lymphangioma developed primarily in the fetal period in this case , and was silent until detection on medical examination . lymphangiomas rarely occur in the abdomen , and the mesentery , omentum , mesocolon , and retroperitoneum are the most common sites.9 the incidence of lymphangioma in the intestinal wall is very low , and few cases of multiple colonic lymphangioma have been reported.1 in the present case , multiple lymphangiomas of the colon , known as colonic lymphangiomatosis , were observed , and had resolved by the time of the third colonoscopic examination . it is considered benign ; no cases of malignant transformation have been reported.1 trials have been conducted to explain how lymphangiomas may be resolved . several investigators have suggested that increased pressure in the lymphatic system may overcome incomplete obstructions . others have hypothesized that resolution may occur through the establishment of alternative routes of lymphatic drainage.10 the latter theory seems to fit well with the present case . it is possible that in the course of making incisions and taking excisional biopsies of the colonic lymphangiomatosis , conduits that drain lymphatic outflow toward the colonic lumen were formed , leading to the complete resolution of the lymphangiomatosis . there is no standard approach to the treatment of intra - abdominal or intraluminal lymphangiomas . historically , both were treated by surgical resection due to uncertainty regarding the nature of the lesion . however , since lymphangioma is a benign tumor , and most intra - abdominal or intestinal lymphangiomas are asymptomatic , they usually do not require treatment.3,4,5 kochman et al.5 suggested that asymptomatic lymphangiomas should be observed rather than treated . large lymphangiomas may produce bowel obstruction , intussusception , bleeding , anemia , and protein - losing enteropathy , in addition to symptoms such as abdominal pain , vomiting , and diarrhea.6 complete surgical excision is considered the treatment of choice for these large , symptomatic lymphangiomas.1,6 for pedunculated tumors < 2 cm , recent reports indicate a trend towards the use of endoscopic polypectomy.11 in the present case , no treatment other than several incisions and excisional biopsies was attempted because the observed tumors were all small , sessile , and did not exhibit symptoms or cause complications . in a follow - up study after the incisions and excisional biopsies , we found that such tumors were completely resolved . our assumption is that conduits were made at some sites of incisions or excisions , and lymphatic drainage through those conduits from the lymphatic channel to the colonic lumen brought about the resolution of the colonic lymphangiomatosis . during 8 months of surveillance after the procedure , no complications such as bleeding or protein - losing enteropathy were noticed . this is a case report of a patient in whom dozens of lymphangiomas were found in the ascending colon up to the transverse colon , which was observed and confirmed through colonoscopy and biopsy . the colonic lymphangiomatosis was observed to be completely resolved in follow - up studies , without any treatment .

lymphangioma is an uncommon malformation of the lymphatic system that involves a benign proliferation of the lymphatics , with no established treatment method . multiple colonic lymphangioma , or colonic lymphangiomatosis , is an extremely rare condition . we report a case of colonic lymphangiomatosis that was detected during a colonoscopic examination conducted as part of a general health check - up . the lesion completely resolved after excisional biopsy .

INTRODUCTION CASE REPORT DISCUSSION

drawings were collected and analysed as evidence for specific kinds of mental activity in the context of early child psychology , beginning in the 1870s . researchers distinguished children s image production from subjective artistic expression , establishing drawing as a material trace of universal patterns of cognitive development . of course , scribbles , doodles and sketches populate the margins of countless documents belonging to everyone from eighteenth - century schoolboys to us presidents ; in examining scientific uses of drawing , it becomes particularly important to underscore how the simple act of putting pen to paper is historically circumscribed . both in science and in art , changing norms of observation and representation have influenced the way that people draw . in early nineteenth - century europe and america , representational drawing ability belonged to the set of socially valued skills cultivated in elite schools . by the 1820s , a market for drawing instruction developed among the upwardly mobile middle classes , reflected in an explosion of popular how - to books such as samuel prout s easy lessons in landscape drawing ( figure 4 ) . drawing instruction was soon incorporated into the curricula of state - sponsored educational institutions , both for its salubrious influence on character and for its potential industrial applications . source : r. ackermann , easy lessons in landscape drawing ( london : r. ackerman , 1820 ) . source : r. ackermann , easy lessons in landscape drawing ( london : r. ackerman , 1820 ) . as a component of primary education regardless of the strategies that instructors adopted , their efforts established representational drawing as an increasingly standardised visual language . in this vein , david maclagan points out that drawings are linked in different ways to conventional communicative modes , reflecting the established pictorial codes of a particular time and place . however , maclagan also notes that the unconscious reproduction of these codes was understood , starting in the late nineteenth century , as a subliminal kind of pictorial lingua franca revealing the underlying essence of the human mind . indeed , it was precisely in this context a society where most primary school students learned to evaluate the correctness of outline , proportion and shading that drawings could become a taken - for - granted indicator of cognitive development and mental soundness . pioneers in the emerging field of child study turned the pedagogical goals of drawing instructors inside out by asking how the rules of visual language were acquired and what mental architectures they revealed . they documented and classified the process of learning to draw as a proxy for learning to think , based on the proposition that images provided unique access to the developing mind . g. stanley hall was , in large part , responsible for bringing quantitative , developmentally oriented child study to the united states from germany , where the growth of state - sponsored education had produced a vigorous field of pedagogical research by the 1870s . in 1880 , hall asked hundreds of boston school children to produce drawings of everyday objects as part of his inventory of the child mind. the inventory was , quite literally , a laundry - list of things that children knew : animals , places and household objects . the list was generated through one - on - one interviews conducted by elementary school teachers using standardised response forms . most of the prompts were verbal , but hall expressed reservations about this methodology , acknowledging that children s vocabulary did not necessarily correspond with their functional understanding . he thus included drawing tasks in the inventory , claiming that [ d]rawings reveal the child s psychic life in an extraordinary way and show its motor development [ and ] the directions of its interests ... . hall s main interest lay in amassing observations on a scale large enough to reveal generic patterns of the child mind. although hall soon moved on from child study to other endeavours in the emerging constellation of the social sciences , his followers attempted to systematise his methods and build theories from the data they gathered . pedagogical seminary , the journal that hall founded in 1891 , regularly published analyses of children s drawings , written by a growing class of psychologists and educators who had begun to specialise in this area . among the more prolific was herman t. lukens , who served as an honorary fellow under hall at clark university from 1894 to 1895 , and returned as a docent in pedagogy from 1899 to 1900 . lukens collected 3400 drawings from children between two and sixteen years of age , publishing his findings in 1896 . such vast accumulations had become a trend in child study ; some projects netted many thousands of drawings , and requested still more samples from the journal s readers , with the promise that treasured artworks would be duplicated and returned . unlike other traces of the child mind that had to be abstracted from observed behaviour into tables and charts , drawings were printed directly in professional journals utilising a visual rhetoric of immediacy . lukens presented series of drawings done by the same children over the course of many years to illustrate their linear development ; the gradual complete clarification of the mental image into a picture on the page . figure 5:drawing by lillian l. at four years , three months old . source : lukens , a study of children s drawings in the early years , pedagogical seminary 4 , 1 ( 1896 ) 102 . source : lukens , a study of children s drawings in the early years , pedagogical seminary 4 , 1 ( 1896 ) 102 . because the children in lukens s study were so young , he saw their drawings as an unmediated glimpse into their mental architecture , a subjective rather than an objective picture of the world as perceived and understood . a certain level of expertise , however , was required to interpret these often jumbled glimpses . the tangle of lines over the legs is not in any case to be interpreted as shading , he asserted of the child s drawing in figure 5 . such an interpretation would violate the accepted developmental chronology , attributing faculties to the child that experts believed that he or she could not possess . his text framed the drawings with a narrative that included statements of his subjects about their drawing processes and their intentions : the child drew one perpendicular line which she called a leg , then a parallel line which she called the other leg. he also provided reassurances about his control over the experimental setting . although the collection of drawings did not take place in a psychological laboratory , and parents or teachers were often present , lukens inserted narrative asides such as , her mother meanwhile looked on in silence , giving no suggestions , asserting the impartiality of the arrangement . when an adult intervened , lukens was careful to justify the action and minimise its influence on the results . barbara wittmann has emphasised the drastic stripping away of context in early italian and german studies of child art ; later researchers , such as lukens , recognised the need for some context , but maintained tight control over what information framed the material evidence . although lukens spoke of children s drawings as direct projections from their minds , motor development had an undeniable impact on the images . lukens incorporated the relative manual dexterity of children at different ages into his assessment . this opened up the possibility that very young children were , in fact , visualising objects the drawings of lillian l. at the age of two show the utter lack of any apparent connection between a mental picture in consciousness and the movements made by the hand and fingers in attempting to draw it. once lillian l.s drawing ability had improved to the point where she could capture contours , however , lukens readily asserted a correspondence between the acuity of her mental imagery and the accuracy of her drawing : it probably corresponds to a clarification of the mental image in the child s mind by a gradually greater discrimination on her part between the motor elements and the strictly visual elements ... . this clarification of the image by eliminating noise produced drawings of true scientific value . thus , although young children were considered ideal subjects for research into the primitive mind , in practice , they were not so useful during their most primitive stages . the theories of child study experts presupposed a true hand that could accurately relay the mind s messages . they focused on subjects within a particular developmental window who made optimal self - registering instruments . like hall , lukens used children s drawings to argue for a recapitulation theory of human mental development : both saw striking analogies in the pictorial evolution of man between the child and the race. influenced by widespread scientific enthusiasm for ernst haeckel s biogenetic law , they equated child art with the simplistic images produced by primitive cultures , which progressed to the mastery of realism and linear perspective indicative of modern civilisation . lukens , however , sought to nuance this conventional and generic classification of children s drawings . in his work , we see a turn away from hall s statistical abstractions and towards narrative interpretation . although he would not go as far as freud or later promoters of art therapy , lukens swerved into a grey area between the survey style , which considered children s affective states only in so far as they interfered with the registration of accurate responses , and the analytic style which saw drawings as manifestations of a non - verbalised subjectivity . the little child draws from his own consciousness and thus puts his own consciousness into the picture , lukens claimed , meaning that the drawing could not be a pure scientific object in the way that some recapitulationists had supposed . although he considered child art analogous to the pictographic stage in the race development [ that ] has preceded and led up to the alphabetic stage , it also usefully encoded the particulars of a child s experiences , likes and dislikes . it was , paradoxically , a more objective modality than speech for getting at the unique thinking of each child , since words are often repeated meaninglessly , while drawings are pure form synonyms for thoughts. again , lukens asserted that the mind hand relay of the ideal drawing child was free from interference by contextual factors beyond those that he identified as relevant to the investigation of children s interiority . while child study gave way to laboratory - centred developmental psychology in the early twentieth century , the use of drawings to assess children s cognitive ability was formalised in much the way that hall had envisioned . within the first decade after 1900 , drawing tasks as a measure of development had become standard , with systems proposed by frederic l. burke , james mark baldwin , james sully , william stern and many others . in 1926 , draw - a - man test , establishing a widely adopted rubric for correlating children s drawings with their intelligence as measured by the stanford - binet intelligence scale . also in the 1920s , jean piaget developed his influential four - stage theory of artistic development in children , which posited a progression from visual realism. recapitulation fell away as an explicit foundation for these rubrics , although informal comparisons between children , primitive races and even primates and animals persisted . instead , the post - world war i professionalisation of american psychology led to a narrowing of scope from the domain of cognitive process to that of cognitive performance and ability. psychometric tests with industrial and military applications proliferated , often employing drawing tasks that extended child study techniques for circumscribing subjectivity to adult test takers . the manual nature of these tests , the inscriptions they produced and the systems of evaluation that psychologists developed helped to naturalise a set of cultural assumptions about correct ways of drawing , making them a proxy for healthy ways of thinking . of course , the possibility of individualised meaning and interpretation was not excluded from all areas of psychology that employed drawing as a technique . many practitioners understood patient art as a pathway to the otherwise inaccessible interior of troubled psyches . patient art figured prominently in the case studies of freud , jung and followers of psychodynamic psychology . this analytical approach to drawing filtered into american popular culture as the cult of the doodle that arose in the 1920s and 1930s , wherein armchair analysts collected the doodles of the rich and famous and subjected them to psychobiographical interpretation . whether freudian or popular , this interpretive school linked drawings to individual subjects and specific sociocultural settings . practitioners still , however , assumed that the relay between mind and hand bypassed the subject s conscious mediation a mechanism that allowed subliminal desires to be manifested in the idle margin scrawls of patients , presidents and movie stars . this was the same assumption that allowed drawing to serve child study so well . it became embedded in the technique , even when that technique served differing ends . this gives us one history of the emergence of drawing as a material research practice around the turn of the twentieth century . child study understood drawing as revealing the workings of the mind on a stratum both evolutionarily and developmentally prior to that of language . primitiveness helped it stand as objective evidence within a recapitulationist framework , obscuring the larger cultural context of art education and the specific context of individual subjects . hall s concept of the child mind was a taxonomic one , and thus drawings were taken as quasi - archaeological artefacts that captured normalised developmental stages . such a concept lent itself to application on a mass scale , as the evaluation of images was based on formal criteria correspondence with the conventions of representational drawing rather than on subjective content . the position of psychology in the early twentieth century was such that practical applications in psychometry and intelligence testing were highly valued as routes to professional legitimacy and funding . on the other hand , lukens and those who leaned towards interpretation believed that children s drawings give us one of the surest ways with which to reach the contents of their minds ; the meaning of images lay in the subjectivity of their makers . this inclination to interpret rather than evaluate , and to read drawings as expressions of complex subjectivity , saw its realisation in freud s wolf man drawings , the art therapy of margaret naumburg and melanie klein s child psychoanalysis in which drawing served as a paper tool. although psychoanalysis approached the mind from a very different perspective to psychometry , both envisioned the drawing child as a circuit through which the mind s contents flowed relatively unencumbered , an assumption facilitated by the earlier work of child study . by the early decades of the twentieth century , a variety of practitioners embraced the notion that drawings reveal hidden information about the mind , but disagreed as to whether that information pertained to the mind s contents or its function . the second and third cases presented here follow the functional strain of this divergence , showing how the exclusion of subjective content became especially important for researchers making new and controversial knowledge claims in psychology . at the same time as child study took up drawings as psychological data , they became central to another fledgling field devoted to the investigation of the human mind . this field , psychical research , developed a different experimental methodology around the same techniques of pencil and paper . like the practitioners of child study , psychical researchers reproduced drawings as material proof of an elusive type of mental activity , in this case , telepathic communication . just as drawings could reveal a developmental resonance between the mind of an individual and the collective mind of a race , they could also demonstrate psychic resonance between two minds . psychical researchers who studied telepathic phenomena employed drawings as evidence , portraying them as thought - pictures that manifested , in objective material form , the otherwise - concealed mental processes of mediums . even critics aiming to debunk telepathy used drawing to prove alternate explanations of the phenomenon ; they argued for the prevalence of certain images in an unconscious community of thought , the contours of which they empirically determined by collecting drawings from the population . child study emerged concurrently with the first major surge of interest in psychical research , and thus , my comparison is not meant to suggest a chronological relationship in which drawing tests were passed from one to the other . both fields sought to establish drawing as a straightforward instrument that accessed and recorded the contents of the mind . both understood drawings as material evidence of thought processes . and both attempted to exclude the individualised , subjective meaning of images from their analyses by placing particular kinds of subjects into controlled experimental settings . i position child study first in this essay because of its direct connection to the mid - nineteenth - century theories of pedagogy that established drawing as a universal human faculty with objective standards . mandatory art instruction established drawing as a conventional communicative mode shared by scientists , subjects and the public at large . a professionalisation story would proceed from psychical research on the margins of science to more respectable child study , and thence to scientific neuropsychiatry . i have attempted to tell a story not about professionalisation , per se , but about changing attitudes towards subjects and subjectivity . starting with child study sets the stage for a process of circumscription that did not proceed linearly or uniformly across the discipline . further , this process generated popular offshoots that held intense interest for the public and continually smuggled subjectivity back into the psychological interpretation of drawings . interest in mind reading and thought transference surged in anglo - american culture of the 1870s , in an environment suffused with spiritualist sances , mesmeric stage performances and other supernormal manifestations . the public was caught up in the fascination of the unusual to the great concern of sceptical scientists , who were often called upon to expose the tricks of psychic charlatans . historians assign this occult revival a dual meaning : it reflected an anti - materialist backlash against secular science , which threatened cherished beliefs about the immortality of the soul . at the same time , it proclaimed the progress of science in conquering the realm of the invisible . the many proposed naturalistic explanations for telepathy worked by analogy to the transmission of light , magnetism and electricity . if those intangibles could be measured and quantified with the new instruments of physics , it seemed plausible that human thought , too , might have its own yet - to - be - discovered wavelength and might travel from mind to mind like a telegraph message . in an age when communication technology had broken down the barriers of time and distance , and scientists were working towards the wireless telegraph and radio , such expectations were hardly far fetched . indeed , the psychical researcher and outspoken spiritualist oliver lodge performed what many claim was the first public demonstration of wireless telegraphy at a meeting of the british association for the advancement of science in 1894 , proving that ideas , in the form of electrical impulses , could leap through space . psychical researchers hoped to determine whether impressions from the minds of those about us [ can reach ] our own minds by channels distinct from those of the senses. this was the widely adopted working definition of the phenomenon alternately named thought transference or telepathy . although they saw their project as empirical , researchers faced the challenge of studying people who claimed that their powers of mediumship or clairvoyance appeared only under very particular conditions . peter lamont has pointed out that psychic practitioners and sceptical inquirers almost never agreed on the evidential criteria for psychic phenomena ; their disagreements , however , were quite nuanced , as both claimed to be working in an empirical , evidence - driven paradigm . the production of evidence in settings that blended the scientific , social and performative made for minute wrangling over the limits of observation and self - report . both public mediums , who received payment for stage shows , and private mediums , who demonstrated in parlours and sitting rooms for small circles of invited guests , combined popular spiritualism in the form of channelling and automatic writing with parlour games such as guessing cards or locating hidden objects . impartial scientific observers , but they shared a social context that made certain behaviours unacceptable . for instance , spiritualist publications criticised harvard psychologist hugo mnsterberg for an incident in which he took credit for grabbing the foot of the medium eusapia palladino to catch her cheating . palladino s supporters argued that such breeches of conduct endangered the medium , who was already in a fragile mental state from her exertions . psychical researchers regularly exposed performers tricks , but they did so within a set of practices that positioned the medium as a particular kind of subject at the mercy of forces , whether spiritual or physiological , that he or she did not control . researchers attempted to use drawing , a technology of direct inscription , to bypass the epistemological tensions that marked their relationships with their subjects . such investigations first took an institutional form in 1882 with the founding of the british society for psychical research ( spr ) by physicist , inventor and professed spiritualist william fletcher barrett . the spr placed mediums and sensitives under experimental conditions aiming to disentangle fraud from genuine phenomena . one of their foundational experiments involved the brighton duo of george albert smith and douglas blackburn , whose successful transmission of images became a pillar of the spr s argument in favour of telepathy . the spr needed a more rigorous method to pre - empt deception , and they settled on drawing . in august 1882 , blackburn , a local newspaper editor , wrote to the spiritualist periodical light , claiming that george albert smith could read his thoughts with an accuracy that approaches the miraculous. smith , only eighteen years old at the time , had recently begun performing as a medium in brighton . he and blackburn colluded to promote his act , with blackburn serving as a confederate onstage at the same time as he published glowing reviews in his paper . blackburn would later claim that , as a service to science and to the public , he and smith decided to reveal how easy a matter it was to take in scientific observers and credulous spiritualists. the spr s committee on thought - transference , led by barrett , edmund gurney and frederic myers , responded to blackburn s letter with interest . myers and gurney were cambridge educated and independently wealthy ; they turned their philosophical and scientific training to establishing psychical research as an area of inquiry . during the autumn of 1882 , the three committee members conducted a series of tests to verify smith s powers . they began with the bread - and - butter demonstrations of thought reading : guessing of numbers , names and colours chosen by the experimenters , shown to blackburn and transmitted from blackburn to smith . these transmissions occurred , the spr promised , with no sound or movement of the lips of any one ; smith and blackburn were , however , allowed to hold hands . when the two men were placed in separate rooms their run of successes came to an abrupt halt , and blackburn complained of neuralgia caused by the strain of concentration . gurney and his colleagues allowed them to resume contact , and they produced more successful thought transferences . although the spr investigators appreciated that hand - holding could be a means of sending a coded message , their subjects asserted that it was also the channel through which their extra - sensory thought transference worked . in the interest of fairness , the investigators decided to alter the content transmitted , rather than altering the experimental set - up , to exclude the possibility of coded communication . gurney framed this methodological development with the question , how far do impressions of drawings or geometrical figures , inexpressible in descriptive words , admit of being transferred? the activity of drawing would become an instrument for fraud - proof thought transference in the eyes of the spr . on the second day of experiments , gurney , myers and barrett drew some image at random , the figure being of such a character that its shape could not easily be conveyed in words ... in order to meet the assumption that some code such as the morse alphabet was used by s. and b. they showed this image to blackburn , blackburn grasped smith s hands and smith sat down and reproduced the image on paper , sometimes while wearing a blindfold . smith s drawings , about as like the original as a child s blindfold drawing of a pig is like a pig , were nonetheless recognisable as intended to represent the original figure. this comparison with the rough , unpolished appearance of child art supported the spr s assertion of naivet on the part of the medium . as with child study , an objective concept was discernible through the garbled communication medium of the scrawl. as with child study , close attention to the signal s distortion figure 6:(a ) the committee s drawing ; and ( b ) smith s reproduction . ( third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 213 . ( a ) the committee s drawing ; and ( b ) smith s reproduction . ( third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 213 . the spr took smith s drawings as authoritative evidence of direct communication between minds , asserting that the burden of explaining these results rests upon those who deny the possibility of thought - transference. they made a point of reproducing the drawings in their journal , the proceedings of the society for psychical research , with the assurance that the whole series of figures ( nine in number ) are given in the accompanying plates , which are engraved from photographic reproductions , on the wood blocks , of the original drawings ( emphasis original ) . the technology of photographic wood - block engraving guaranteed that no foul play had intervened ; the direct , unmediated proof was plainly visible , and circulated in the persuasive form of a scientific journal . wood - block engraving , the illustration technique that generated the vast quantity of images demanded by nineteenth - century mass print , had transformed in the second half of the century from an interpretive art to a facsimile process . an engraver in the 1840s would have drawn or traced a copy of smith s figures onto the block , potentially smoothing over the rough , unsteady lines to distil a more correct representational image . by 1882 , smith s figures could be photographed , the film printed directly onto a wood block and this photographic block carved by a technician trained in mechanical exactitude . engraving became harnessed to photographic technologies whose rhetoric of verisimilitude made it possible for thousands of people all over the world to examine the same piece of evidence . photo - engraving enabled the reproduction of specific images from individual research subjects like smith : it preserved the errors and idiosyncrasies that constituted the actual proof of smith s telepathic power . as engravings went from idealised representations to exact , unmediated copies , readers could use figures in scientific journals such as the spr s proceedings and hall s pedagogical seminary in a new way . gurney , myers and barrett described the act of telepathic drawing with terms like deliberate and continuous that suggested the automatic nature of the medium s activity ; smith worked as if copying a drawing that is seen. the resulting artefacts could be used to reconstruct the process that produced them . this method was attractive because the standardised objects cards , colours , numbers that more statistically inclined psychical investigators hoped to employ for scientific data collection offered little insight into the cause or nature of thought transference . were thoughts transmitted in the form of words , images or as pure concepts ( what william barrett termed smith s success in reproducing drawings inspired the spr to move forward with these interesting questions of how and why , using visual specimens to make their arguments to colleagues and readers . figure 7:(a ) the original drawing shown to blackburn ; ( b ) smith s first attempt at reproducing the drawing ; and ( c ) smith s second attempt at reproducing the drawing , after blackburn looked at the original again ( source : the third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 207 ) . ( a ) the original drawing shown to blackburn ; ( b ) smith s first attempt at reproducing the drawing ; and ( c ) smith s second attempt at reproducing the drawing , after blackburn looked at the original again ( source : the third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 207 ) . how of thought transference , the spr would have to disentangle perception from cognition and determine whether thought travelled as image or idea . after an apparently unsuccessful thirteenth trial , the spr investigators asked blackburn , the agent , to reproduce the original image that he had just seen and communicated telepathically to smith ( figure 7(a ) . blackburn s reproduction revealed that he had misremembered the original image , and communicated that incorrect image to smith ( figure 7(b ) . for the previous two decades , physiologists had debated whether visual perception was more dependent on processes within the eye or on psychical researchers saw an opportunity to weigh in on this question by accessing the mind as it pivoted between perception and cogitation . the main errors of mr smith s drawings existed already in mr blackburn s recollection of the drawing , they reported , demonstrating that smith received the inaccurate image from blackburn s flawed memory rather than as a purely visual sense impression . when the investigators showed blackburn the image again , and allowed smith to redraw it ( after grasping blackburn s hand ) , they attributed the improvement in smith s accuracy to blackburn s improved memory ( figure 7(c ) . these very specific uses of drawing accessed the workings of two minds that of the agent , blackburn , and the percipient , smith as they supposedly communicated a visual image . the spr s gurney , myers and barrett weighed the possibility of fraud and ruled it prohibitively difficult due to the inexpressible nature of the images . let our readers , who may be familiar with the morse or other code of signals , try in some such way to convey a description of one of our drawings , to a friend who is blindfolded and has not seen the original. drawing was such a useful instrument for the spr because it seemed to capture the irreducible nature of thought in a material form that could be scrutinised for evidence of mental processes . descriptions of the smith and blackburn experiments arrived in the united states in the winter of 18823 . american subscribers to the spr s proceedings , including william james , g. stanley hall , george s. fullerton of the university of pennsylvania and henry p. bowditch of harvard medical school , would have seen the report , and it received favourable notice in the scientific press as an eliminate[d ] every possible element of charlatanism. by the following year , with william james leading the charge for an autonomous american version of the spr , the prospective leaders of this group began critically re - evaluating the british society s findings . although the american society represented itself as a sceptical counterweight to the spiritualist leanings of the british , their reviews of the smith and blackburn report embraced the use of drawing as an experimental methodology . in the published text of his first presidential address to the american society for psychical research ( aspr ) , circulated in the proceedings of july , 1886 , simon newcomb focused his critique on the spr s inconsistent procedures . in the experiments with smith and blackburn , smith was sometimes blindfolded , sometimes not ; sometimes smith and blackburn spoke to each other , conditions changed constantly as the experimenters thought of new scenarios they wanted to test . whereas gurney , myers and barret asserted that our experiments derive much strength and coherence from their very multitude and variety , newcomb regarded these conditions as unacceptably haphazard : without rigid controls , we have no right to attribute the result to one cause [ ie . , telepathy ] rather than another. this tension between the rigid planning and record keeping of the laboratory and the curiosity - driven exploration of the gentleman or amateur investigator would characterise the aspr s relationships with many of its own constituents , as well as with its british counterpart . the actual drawings that smith produced represented the most stable , scientifically valid element of the entire undertaking in newcomb s assessment . even in less - than - ideal conditions , these copies of drawings have a great advantage over verbal descriptions , newcomb noted , in that the record can be made the subject of future study. the permanent , reproducible nature of images was an antidote to the anecdotal style that newcomb and his colleagues found most galling in the spr s work . interestingly , newcomb s published endorsement of the smith drawings represented a change of course from the more suspicious note that he sounded in his actual presidential speech , on 12 january 1886 . in the speech itself , he deemed smith s drawings fraudulent . what changed his mind was a new set of sketches that he exchanged with william james in the months after his speech , through which james demonstrated the possibility of non - supernatural blindfolded draughtsmanship . your drawings seem to show that ... the [ smith ] drawings in question were at least possible , newcomb wrote to james on 16 february . their correspondence reveals the extent to which the practice and material product of drawing served as a persuasive tool for leaders of the aspr . while newcomb doubted the circumstances of the british society s procedure , a trustworthy colleague such as william james could produce corroborating evidence from his own hand . thanks to an efficient postal service and the portable nature of drawings , newcomb could then inspect the evidence first hand . their disagreement was resolved through the mail over the course of a mere ten days , and newcomb edited the text of his speech to reflect his confidence in drawings as evidence . the aspr further endorsed drawing as a methodology in 1885 , urging its members to attempt the experiments which have attracted so much attention from the english society. this represented an exciting expansion of their research program beyond the repetitive experiments requested in the society s previous circulars , such as the guessing of numbers , colours and cards . the society acknowledged the heightened appeal of personalised and infinitely variable drawing tasks : as the experiment of free - drawing may prove more interesting to some persons , it is hoped that more elaborate figures may be tried , and the results forwarded. figure 8:illustration from thought - transference by means of pictures. source : proceedings of the american society for psychical research , 1 ( 1885 ) , 44 . illustration from thought - transference by means of pictures. source : proceedings of the american society for psychical research , 1 ( 1885 ) , 44 . william henry pickering , a harvard astronomer and founding member of the aspr , published the results of such an experiment as a model accompanying this call for participation . pickering s drawings were made on one or two evenings as an amusement among friends , with a female acquaintance acting as the sensitive , so called. like most psychical experiments , these were a parlour activity , but one with greater entertainment value and greater power as evidence than the mundane and easily fudged guessing tasks . the graphic summary of pickering s experiment is particularly striking : all fifty - two pairs of drawings were reduced to thumbnail size and condensed into one page . this public presentation of a complete run of experiments asserted the scientific transparency of the aspr in contradistinction to the spr , which mainly printed examples of its successful results . the miniature images conserved valuable pages , but also conveyed the impression of a complete formal taxonomy rather than an informal parlour game . an aggregate view de - emphasised the idiosyncrasies of subject and circumstance that the spr used to justify the failures and fine tuning of their procedures . such serial presentation had become an important tool of visual rhetoric for nineteenth - century sciences ranging from physics to embryology , including pickering s discipline of astronomy , where debate over the nebular hypothesis in the 1830s had centred around the arrangement of successively brighter nebulae into an order that suggested temporal progression from diffuse clouds of matter to stars . pickering s grid of images , lacking any apparent order or progression , subtly asserted the absence of theory - driven manipulation and modelled the randomness necessary to ensure impartial psychical experimentation . drawings and diagrams featured regularly in the first year of the aspr s proceedings , but , for sceptics within the organisation , this visual evidence soon lost the lustre of the early smith and blackburn successes or rather , it was appropriated by a different camp within psychical research as proof against , rather than for , the reality of thought transference . gurney , myers and barrett of the spr interpreted resemblances among drawings as evidence of communication demonstrating direct transmission of content between minds . when simon newcomb embraced drawings as evidence , he cited the possibility for reinterpretation as their main advantage : they could be made the subject of future study. as the aspr set about gathering drawings from its own far - flung correspondents , some began to view the mass of responses as indicating unconscious habits or statistical probability rather than telepathy . such complex explanations were ultimately not necessary to account for the smith and blackburn phenomenon that had established telepathic drawing as a preferred methodology in psychical research . in a confession published in the popular magazine john bull more than twenty years after his experiments with smith , blackburn explained that he had used a numbered ten - by - ten grid to reproduce the transferred images , while smith indicated coordinates on the grid through hand squeezes , breathing or tapping the carpet . this method yielded drawings with an overall resemblance in shape , although smith often failed to recognise ( or intentionally botched ) the referential content of the original picture . interestingly , the use of a grid for proportion and perspective drawing was also part of art pedagogy , but faced criticism precisely on the grounds that it created superficial likeness without teaching the skills of active , synthetic observation . the spr s committee on thought - transference had acknowledged the possibility that smith and blackburn were using a secret code , but expressed confidence in their assessment of the men s characters and in the difficulty of cheating . it is probably no exaggeration to say that several scores , if not hundreds , of precise signs would be required to convey an idea as exact as that conveyed in many of mr smith s representations. indeed , a widely accessible popular literature for aspiring thought - readers provided systems for organising and communicating hundreds of precise signs . it is likely that smith and blackburn were familiar with such pamphlets , often attributed to famous stage performers such as w. irving bishop and robert houdin . figure 9:(a ) an alphabetical grid for sending images in the form of a code ; and ( b ) calostro gives an example of how to use the grid to transmit the image of a house . source : calostro ( pseudonym ) , the radio vision mind - reading code ( closter , nj : calostro publications , 1930 ) , 32 . ( a ) an alphabetical grid for sending images in the form of a code ; and ( b ) calostro gives an example of how to use the grid to transmit the image of a house . source : calostro ( pseudonym ) , the radio vision mind - reading code ( closter , nj : calostro publications , 1930 ) , 32 . the spr quietly dropped the smith and blackburn case from their canon of evidence , but the efforts of both the british and american societies to elicit telepathic drawings from the public had by then established this methodology among amateur researchers who understood it on their own terms , and adapted it to their changing interests . from 1904 until the 1940s , french parapsychologist ren warcollier worked with a method similar to the spr s , in which an agent observed a target image and distant percipients tried to draw it . warcollier s remote viewing experiments were remarkable for their scale and global scope , involving much like the spr investigators , he was not searching for perfect matches , nor for insight into his subjects psyches . he believed that analysing discrepancies and resemblances between agent and percipient drawings would reveal the mechanisms of mind - to - mind communication . by 1930 , when the novelist upton sinclair published a book of his telepathic drawing experiments entitled mental radio , the popularisation of freudian theory had opened up a wide range of new interpretive possibilities for mental imagery . yet sinclair largely hewed to the mode of analysis represented by the spr and warcollier , comparing the visual appearance of drawings and sometimes taking into account their manifest content ; only in a few isolated cases did he note the potential impact of the subject s history or emotions on a transmission . psychical researchers were engaged with the surface level of drawings , looking at their geometric and linear components and the explicit objects or ideas that they represented . like the collectors of children s drawings , they seemed increasingly nave in their approach from the perspective of psychologists who were either unpacking or working to contain the problem of subjectivity . after the deflation of the spr s case for telepathy in the first decade of the twentieth century , sceptical psychologists came to expect that , like smith and blackburn and any number of supposed psychic mediums , adults brought complex motives and resources to bear on drawing tasks . understanding these motives would require more art than science , and thus was not particularly useful in the goal of gaining scientific legitimacy for psychical research or for laboratory psychology in general . psychoanalysis threatened the integrity of drawings as automatic traces of mental processes by throwing open the gates of interpretation . however , there remained a special kind of subject whose neuropathologies produced characteristic changes in cognitive function that the act of drawing could make visible . simple black - and - white line drawings floating on a page of scientific text , labelled with a figure number and caption , helped to define a new area of neuropsychology in the mid - twentieth century . drawings became evidence of the internal workings of cognitive mechanisms not accessible through more holistic methods of testing and self - report . neurologists had used patients drawings to study the mechanics of cognition since the 1870s , slowly embracing the notion that these drawings , if elicited under controlled conditions and analysed by the appropriate experts , reveal how localised damage impacts specific pathways in the brain . neuropsychological literature has typically depicted patients with brain injuries in terms of their cognitive functions , or lack thereof , and , within this literature , patient art is presented as the output of damaged circuits rather than as subjective expression , in much the same way that child study and psychical research carefully circumscribed the agency of their drawing subjects , preferring to depict them as self - recording devices . the german physiologist herman munk is credited with coining the term psychic blindness or mind - blindness , in 1881 based on experiments in which he cauterised specific areas of the cortex in dogs . dogs with posterior occipital lesions were unable to identify their food bowls or recognise their handlers , although their vision was intact . in 1888 , the neurologist heinrich lissauer examined an eighty - year - old patient , mr gottlieb l. , suffering from a strange array of perceptual problems similar to those displayed by munk s dogs : the patient could see things but could not recognise them , unless allowed to examine them by touch . he could draw simple copies of pictures shown to him , but could not name the objects represented . lissauer used drawings to probe his patient s visual processing deficits because verbalization may not be accompanied by visual imagery ... one can get much further by asking people to draw from memory the same reasoning given by lukens in studying children s conceptual knowledge . based on his extensive work with gottleib l. , lissauer postulated that psychic blindness resulted from damage to the occipital areas of both hemispheres that prevented the association of visual perceptions with knowledge stored in memory , a condition that sigmund freud , still practising neurology in 1891 , termed agnosia , or loss of knowledge. the concept of agnosia that damage along its processing and integration streams can prevent the brain from connecting sensory input with meaning raised the possibility of physiologically divorcing vision from comprehension . a patient with this class of disorder a canny diagnostician could use the machine s output to locate the point at which normal visual processing had broken down . interest in cognitive deficits caused by localised neurological injury , specifically in the disorders of language processing called aphasias , surged in the 1850s , as neurologists such as paul broca and carl wernicke pursued the localisation of functions by correlating patient histories with autopsy findings . broca , wernicke and their peers debated the philosophical implications of language localisation ; aphasia became a way of probing what defines the human and what constitutes a conscious self . these high - profile aphasia debates are well studied , but few historians of science have engaged with the more recent history of agnosias , beginning with lissauer in the 1880s . unlike aphasia , which was manifested overtly in verbal communication , agnosias were slippery and difficult to observe in the clinical settings of the late nineteenth century . as lissauer noted , accessing patients perceptions required a technique not dependent on language or self - report . historians have , perhaps , been attracted to nineteenth - century aphasiology because its disruption of the correspondence between language and reality resonates strongly with late twentieth - century disruptions in the discipline of history itself . agnosia engages more recent historiographic trends , allowing us to extend the narrative of how science has mobilised visual evidence to depict brain functions untethered from human subjects . while aphasias are specific to the processing and production of language , agnosias encompass broader sensory deficits ranging from impaired face recognition ( prosopagnosia ) to impaired sensory perception of one s own sensory perception disorder ( anosognosia ) . classification of these disorders has , as researchers inevitably note in their literature reviews on the subject , depended on the availability of patients with highly specific lesions , a rare occurrence given the diffuse damage normally caused by head injury or stroke . historically , a contingent of neuropsychologists have made the case that categorisation is a useless exercise due to the individualised nature of each patient s difficulties , and that particular classes of agnosia do not exist as clinical entities . in this debate , drawing once again became a decisive tool and evidentiary form . it helped researchers develop an etiology of agnosias ; the examples given here centre on visual agnosia , but drawing became almost ubiquitous in assessments for a wide range of interrelated sensory perception and processing disorders . figure 10:a drawing task with original images on the top line and the patient s copies below . source : w. russell brain , visual object - agnosia with special reference to the gestalt theory. brain 64 ( 1941 ) , 48 . a drawing task with original images on the top line and the patient s copies below . source : w. russell brain , visual object - agnosia with special reference to the gestalt theory. brain 64 ( 1941 ) , 48 . tests like that shown in figure 10 , in which a patient was asked to copy three geometric forms , seem to project the subject s faulty perceptual function directly onto the page in a way that requires little interpretation . however , such images are rich with theoretical assumptions and rhetorical strategies that reinforce the immediacy of the drawing as a materialisation of mental processes . a set of characteristic drawing tasks emerged in european and american clinical neurology in the early twentieth century that relied on a shared visual repertoire of mundane objects , clocks , maps , geometry and perspectival rules . some newly diagnosable cognitive disturbances were subtle enough that patients compensated for them in everyday life without overt signs of difficulty . dysfunction was present , however , if a patient was quietly deviating from the normal pathways of image recognition and production built into the cultural practice of drawing . british neurologist henry head , in his authoritative 1926 work on aphasia , developed four drawing tasks for evaluating the nature of a patient s cognitive disability : drawing from a model , drawing from command ( head generally requested an elephant ) , drawing the ground plan of a familiar room and drawing anything that [ comes ] to mind. each of these tasks assumed a shared visual grammar rooted in western art and art education . jacyna , in his biography of head , underscores the importance of head s position at the empire hospital in vincent square during world war i , where he treated young , well - educated officers who had suffered acute head trauma . their social backgrounds and the specific nature of their battle wounds made these patients ideal for isolating pure forms of aphasia and agnosia . whereas their presumed naivet made the contents of children s minds easy to capture in drawings , the presumed intellectual soundness of head s patients prior to injury meant that the injury itself was the thing captured in diagnostic drawing tests . head reproduced their drawings as the only illustrations , aside from x - rays , in his two - volume tome . source : henry head , aphasia and kindred disorders of speech ; ( cambridge university press , 1926 ) , 131 ; and ( b ) patient no . source : henry head , aphasia and kindred disorders of speech ; ( cambridge university press , 1926 ) , 131 ; and ( b ) patient no . 25 s head is a prominent example of how neurologists in clinical settings developed ad hoc drawing tasks for the evaluation of patients with agnosic symptoms , using logic to connect errors in representation with localised brain damage . head attempted to create a more generalised set of evaluation criteria , applying a larger number of tests to a larger number of patients than previous researchers had . until this point , most agnosia research , like the smith blackburn experiments , was performed on isolated patients under test conditions that varied depending on the patients responsiveness and abilities . despite head s attempt to impose a standardised diagnostic framework , there was no professional consensus on the disease categories that various drawing tasks identified until the 1970s . in the 1940s , edinburgh neurologists oliver zangwill and andrew patterson developed a visual testing battery for patients suffering from spatial disorientation . by asking patients to bisect lines , copy drawings and mark different times of day on a clock face , they connected the recognised phenomenon of unilateral neglect with specific disruptions in processing caused by unilateral parietal lesions . their testing battery was widely adopted , and their explanation of unilateral neglect supported a modular view of brain function that would gain increasing acceptance in subsequent decades , building on this model of matching a lesion to a deficit revealed through drawing . drawing tasks helped researchers distinguish the discrete cognitive operations contributing to patients broader deficits , and refined these cognitive operations into successively narrower categories . throughout the 1950s and 1960s , a group of neuropsychologists argued that cases classed as agnosias did not reflect a specific disorder , but rather resulted from generalised brain damage and decreased cognitive functioning . to my knowledge of the literature , there does not exist a single case of agnosia without elementary sensory disorders and without mental deterioration , claimed german neurologist eberhard bay . bay and his supporters insisted that there is no need to search for any mystical gnostic activity to be disturbed , a point that sometimes led to questioning the intelligence or integrity of agnosic patients such as schn , a world war i veteran whom bay accused of exaggerating his symptoms . debates over the reality of this syndrome were particularly fraught because clinicians also played an advocacy role for their patients , certifying the need for pensions or disability support . advocates for visual agnosia as an etiologically distinct syndrome used drawings as evidence for a new theory of visual processing , modelling it as a stream that flowed from lower - level perception to stored perceptual knowledge to stored conceptual knowledge . along with a battery of other visual tasks , drawings helped to delineate a disorder which gained widespread acceptance with the work of george ettlinger , glyn humphreys and jane riddoch from the late 1960s onwards . dementia is a convenient label to apply when aspects of behavior are not fully understood , wrote humphreys and riddoch ; for agnosic patients , having a problem identified and labeled can be a reassurance. drawings captured patients subtle and disorienting errors in spatial representation , assembling parts into wholes , or recalling the forms of common objects , fixing in material form specific deficits that had once seemed diffuse and intractable . drawing tests mobilise an arsenal of familiar objects depicted in outline from a conventional point of view , in part , as an accommodation to the perceptual difficulties of neurological patients . pictures are much more difficult [ to recognise ] if the artist has used shade , reflection , or the skills of impressionism , reported john , the patient studied by humphreys and riddoch . this visual vocabulary also dispenses with the subjective problem of artistic expression , aiming , instead , for a conventional sense of accuracy shared by patients and researchers . i never had much drawing ability or the simplest comprehension of how to show a third dimension in scribbling [but ] i can comprehend enough of my own handiwork to know if it is a reasonable representation of what i had in mind. john s ability to produce a drawing and then reflect on its correspondence with an objective reference point acts as an assurance that he has no special training or expressive intentions he is a subject appropriately constrained in his use of drawing as an extension of pure cognitive function . batteries of drawing tests helped clinicians order lesions by location and construct a theory of the processing stream , in the same way that ordering children s drawings along a developmental sequence enabled psychologists to construct an evaluative framework based on that sequence . throughout these debates , neurologists recognised that deficits and lesions rarely match in a simple one - to - one correspondence because injuries are rarely limited to a single - function area of the brain ; patients often suffer from a combination of difficulties following an injury . however , once the disorder agnosia emerged as a medical category , neurologists could use the techniques developed in establishing the diagnosis to disaggregate symptoms and locate damaged points on the visual processing stream . this systematic use of patient drawings in an experimental context to illuminate fundamental cognitive processes rests on the same material substrate as the spr s experiments with smith and blackburn and g. stanley hall s catalogue of the child mind . though not an intentional continuation of these practices , it reveals how the epistemological assumptions built into supposedly basic psychomotor tasks have shaped different areas of psychology in similar ways . drawing seemed a reliable means to capture the mental processes of particular kinds of subject while bypassing the unruliness of subjectivity . in each of the examples above , researchers identified subjects who they believed could function as object - instruments capable of faithfully recording the desired mental phenomena . nave children , blindfolded mediums and head trauma victims , equipped with pencil and paper , could act as their own soul catchers in the sense of a material assemblage that fixes an ephemeral quality of the mind in a physical form . however , the goal of this process for researchers was to take what is conventionally understood as the soul out of the picture , leaving a pure mental function traced in black and white . much of the literature on scientific illustration and objectivity focuses on the production of images by experts , whether scientists , technicians or artists , disciplined to act as transparent recording media for the phenomena they observed . the subjectivity of the image maker is not intended to be part of the image s scientific meaning , although , of course , it is integral to the social and cultural construction of that meaning . on the other hand , certain image - making practices in psychology are understood to externalise important clues about the patient s subjectivity that can be interpreted for purposes of diagnosis and therapy . the images of mental function and dysfunction discussed above occupy a strange intermediate space . they did not illustrate observed phenomena , but rather manifested hidden mental phenomena in visible form . nor were they interpreted for emotional content rather , their scientific validity depended on the exclusion of meaning , history and individual voice . researchers in vastly different contexts elicited these drawings aiming to make an objective study of subjectivity and to access the mind through the hand .

researchers in the mind sciences often look to the production and analysis of drawings to reveal the mental processes of their subjects . this essay presents three episodes that trace the emergence of drawing as an instrumental practice in the study of the mind . between 1880 and 1930 , drawings gained currency as a form of scientific evidence as stable , reproducible signals from a hidden interior . i begin with the use of drawings as data in the child study movement , move to the telepathic transmission of drawings in psychical research and conclude with the development of drawing as an experimental and diagnostic tool for studying neurological impairment . despite significant shifts in the theoretical and disciplinary organisation of the mind sciences in the early twentieth century , researchers attempted to stabilise the use of subject - generated drawings as evidence by controlling the contexts in which drawings were produced and reproduced , and crafting subjects whose interiority could be effectively circumscribed . while movements such as psychoanalysis and art therapy would embrace the narrative interpretation of patient art , neuropsychology continued to utilise drawings as material traces of cognitive functions .

Child Drawings Telepathic Drawings Neuropathological Drawings Conclusion

the nuclear receptors comprise a family of transcriptional regulators involved in a wide variety of biological processes such as embryonic development , differentiation and homeostasis [ 1 , 2 ] . the family includes ligand - dependent zinc - finger transcription factors for steroid hormones , estrogens , thyroid hormones , retinoids , vitamin d and other hydrophobic molecules . in addition , several family members are ' orphan receptors ' for which ligands have yet to be identified . nuclear receptors have been assigned to six subfamilies on the basis of evolutionary studies . as the first member of the sixth subfamily , gcnf is also known by its systematic name nr6a1 . on the basis of homology and expression profile , the receptor has been given the alternative name of retinoic acid receptor - related testis - associated receptor ( rtr ) . transfection experiments reveal that gcnf can act as a constitutive repressor when binding as a homodimer to promoters containing a direct repeat dna element 5'-aggtcaaggtca-3 ' ( dro ) [ 7 , 8 , 9 , 10 ] . the mouse receptor ( mgcnf ) is highly expressed in the developing embryonic nervous system and the labyrinthine layer of the placenta [ 12 , 13 ] . in the adult , high transcript levels are restricted to the developing germ cells [ 5 , 14 , 15 , 16 ] . northern analysis reveals a transcript of 7.5 kilobases ( kb ) in somatic cells and an additional message of approximately 2.4 kb in male germ cells . this size difference is at least partially due to different polyadenylation sites , and it is therefore assumed that both transcripts code for identical proteins of 495 amino acids . the protein sequence is encoded by 11 exons . when differentiation of p19 embryonal carcinoma cells is triggered by retinoic acid , the transcript and the protein are temporarily upregulated and then downregulated . isolation of a human cdna coding for a protein ( hgcnf ) with an identity to the mouse protein of 98.7% , similar regulation in mouse p19 cells and in the human embryonal carcinoma cell line nt2/d1 , together with the presence of two mrnas of approximately 7.5 and 2.2 kb in human testis , suggested similar functions for mouse and human gcnf [ 18 , 19 , 20 ] . the cloning of human cdnas that give rise to different hgcnf isoforms , however , suggests a higher complexity in humans . currently , four different hgcnf cdnas have been isolated that code for isoforms ranging in size from 454 to 480 amino acids . we have investigated the genomic structure of mammalian gcnf to determine how the different gcnf isoforms are generated . here we compare the exon / intron structure of the previously characterized mouse gene with the human ortholog . our study shows that alternative splicing generates at least three of the different gcnf isoforms . to understand how the different human gcnf mrna isoforms are generated , we have identified all human protein - coding exons . the alignment of the full - length human gcnf cdna ( genbank accession number s83009 ) with the genome sequencing data at the ncbi localized the first protein - coding exon on chromosome-9-derived working draft sequence element nt_008491 . the genomic sequence was aligned with the previously identified mouse exon 1 containing the putative translational start site . the comparison was extended up to position -100 with respect to the mouse cdna reaching farthest in the furthest 5 ' direction . in addition , 100 base - pairs ( bp ) of the first identified intron were included ( figure 1 ) . while the transcriptional start sites of gcnf are still elusive , the 5 ' ends of the first protein - coding exons can not be defined . with respect to the sequence in figure 1 , the furthest 5'-reaching human cdna ( s83309 ) starts with nucleotide 171 . the putative translational start codons are in positions 346 and 350 of the mouse and human sequences , respectively . these start codons are present in all full - length mammalian gcnf cdnas characterized so far , suggesting a common amino terminus for the different gcnf isoforms . the comparison of the mouse 5'-untranslated sequence with the human genomic dna reveals high conservation with identical sequence elements of up to 50 nucleotides . the presence of 18 cg dinucleotides conserved between human and mouse is suggestive of a regulatory function of the untranslated sequence . five different human cdnas with alternative 5 ' ends have , however , been reported to genbank ( s83309 , u80802 , af004291 , nm001489/u64876 , x99975 ) . a comparison with the genomic sequence ( nt_008491 ) shows the sequence variation ( figure 2 ) . single - nucleotide polymorphism among cdnas isolated from different human libraries may reflect variants in the human population . two cdnas ( u64876/nm001489 , x99975 ) differ in their untranslated region with respect to the genomic sequence . therefore , it can not be ruled out that these cdna ends may have been generated during the cloning process . in addition , one of the cloned cdnas , coding for hgcnf-3 ( af004291 ) , has a deletion in the coding region of the first exon , giving rise to an open reading frame of 454 amino acids . the 5 ' part of hgcnf-3 has been isolated by the polymerase chain reaction , suggesting that this deletion may have been generated during the synthesis . the isolation of additional cdnas may give a clue as to which variants are true gcnf isoforms . the functional significance of the different isoforms is , at present , unknown but may lead to different transcriptional properties of gcnf isoforms . the comparison of the genomic sequences of exons 2 to 11 was extended by 100 bp of intronic sequence in both directions ( figure 3 ) . during the preparation of this manuscript all sequence information was made available by the international human genome project collaborators at the ncbi database and included in the contig nt_008491 . sequences of the 5'-untranslated region and of exon 7 obtained with a genome walking approach did not diverge from the sequence at the ncbi . two short exons of 42 bp and 45 bp , respectively , follow the first protein - coding exon in the mouse . short exons are relatively rare in mammalian genomes . the structure of the second protein - coding exon is conserved ( figure 3a ) . interestingly , on the basis of the genomic cdna , the third exon is highly conserved as well ( figure 3b ) . the human splicing apparatus preferentially , or exclusively , skips this putative exon , however . as splicing is highly regulated , a splice enhancer present in the mouse genome may not be present in the human genome . consequently , all known human gcnf isoforms lack the amino acids encoded by the putative third exon . of the 243 bp exon 4 that encodes the core of the dna - binding domain one of the reported sequences ( u64876/nm_001489 ) has a c to a transversion , however , which changes a codon for asparagine to one for lysine . splicing of exon 2 to exon 4 at the position characterized in the mouse results in isoform hgcnf-2 . in addition to this splice acceptor position , a splice acceptor site located 12 nucleotides further downstream is used to generate hgcnf-1 . two hgcnf-2 variants , hgcnf-2a and hgcnf-2b , which differ by a single amino acid , have been isolated . as speculated , alternative splicing generates the isoform 2b with a deletion of a serine residue . splicing to an acceptor site of exon 7 located three nucleotides the sequence and structure of exons 8 to 11 are also highly conserved ( figure 3g , h , i , j ) . the comparison of the 11th exon was extended up to the end of the human cdna sequence of s88309 . highly conserved sequence elements of up to 91 identical nucleotides indicate a regulatory function of the 3'-untranslated sequence following the translational stop codon . the aceview analysis at the ncbi based on the draft sequence and a blast search with s83309 of the celera genomics freely accessible whole - genome sequence data gave mostly similar intron sizes . both analyses revealed a large first intron of 37,652 bp in the public sequence data , and 37,157 bp in the private data . the size of the second intron separating exon 2 and exon 4 was only available in the ncbi database ( 14,869 bp ) . according to the ncbi and celera databases , introns 3 to 9 have sizes of 10,486 ( ncbi ) ( 10,471 , celera ) bp , 3629 ( 3615 ) bp , 190,321 ( 1708 ) bp , 1963 ( 1960 ) bp , 2716 ( 9019 ) bp , 1905 ( 1912 ) bp , and 1927 ( 1928 ) bp , respectively . the comparison of both analyses shows that the deduced sizes of two of the human introns differs greatly . it seems likely that these inconsistencies will be corrected in the final assembly of the human genome . to understand how the different human gcnf mrna isoforms are generated , we have identified all human protein - coding exons . the alignment of the full - length human gcnf cdna ( genbank accession number s83009 ) with the genome sequencing data at the ncbi localized the first protein - coding exon on chromosome-9-derived working draft sequence element nt_008491 . the genomic sequence was aligned with the previously identified mouse exon 1 containing the putative translational start site . the comparison was extended up to position -100 with respect to the mouse cdna reaching farthest in the furthest 5 ' direction . in addition , 100 base - pairs ( bp ) of the first identified intron were included ( figure 1 ) . while the transcriptional start sites of gcnf are still elusive , the 5 ' ends of the first protein - coding exons can not be defined . with respect to the sequence in figure 1 , the furthest 5'-reaching human cdna ( s83309 ) starts with nucleotide 171 . the putative translational start codons are in positions 346 and 350 of the mouse and human sequences , respectively . these start codons are present in all full - length mammalian gcnf cdnas characterized so far , suggesting a common amino terminus for the different gcnf isoforms . the comparison of the mouse 5'-untranslated sequence with the human genomic dna reveals high conservation with identical sequence elements of up to 50 nucleotides . the presence of 18 cg dinucleotides conserved between human and mouse is suggestive of a regulatory function of the untranslated sequence . five different human cdnas with alternative 5 ' ends have , however , been reported to genbank ( s83309 , u80802 , af004291 , nm001489/u64876 , x99975 ) . a comparison with the genomic sequence ( nt_008491 ) shows the sequence variation ( figure 2 ) . single - nucleotide polymorphism among cdnas isolated from different human libraries may reflect variants in the human population . two cdnas ( u64876/nm001489 , x99975 ) differ in their untranslated region with respect to the genomic sequence . therefore , it can not be ruled out that these cdna ends may have been generated during the cloning process . in addition , one of the cloned cdnas , coding for hgcnf-3 ( af004291 ) , has a deletion in the coding region of the first exon , giving rise to an open reading frame of 454 amino acids . the 5 ' part of hgcnf-3 has been isolated by the polymerase chain reaction , suggesting that this deletion may have been generated during the synthesis . the isolation of additional cdnas may give a clue as to which variants are true gcnf isoforms . the functional significance of the different isoforms is , at present , unknown but may lead to different transcriptional properties of gcnf isoforms . the comparison of the genomic sequences of exons 2 to 11 was extended by 100 bp of intronic sequence in both directions ( figure 3 ) . during the preparation of this manuscript all sequence information was made available by the international human genome project collaborators at the ncbi database and included in the contig nt_008491 . sequences of the 5'-untranslated region and of exon 7 obtained with a genome walking approach did not diverge from the sequence at the ncbi . two short exons of 42 bp and 45 bp , respectively , follow the first protein - coding exon in the mouse . short exons are relatively rare in mammalian genomes . the structure of the second protein - coding exon is conserved ( figure 3a ) . interestingly , on the basis of the genomic cdna , the third exon is highly conserved as well ( figure 3b ) . the human splicing apparatus preferentially , or exclusively , skips this putative exon , however . as splicing is highly regulated , a splice enhancer present in the mouse genome may not be present in the human genome . consequently , all known human gcnf isoforms lack the amino acids encoded by the putative third exon . of the 243 bp exon 4 that encodes the core of the dna - binding domain , 225 bp are identical in both species ( figure 3c ) . one of the reported sequences ( u64876/nm_001489 ) has a c to a transversion , however , which changes a codon for asparagine to one for lysine . splicing of exon 2 to exon 4 at the position characterized in the mouse results in isoform hgcnf-2 . in addition to this splice acceptor position , a splice acceptor site located 12 nucleotides further downstream is used to generate hgcnf-1 . two hgcnf-2 variants , hgcnf-2a and hgcnf-2b , which differ by a single amino acid , have been isolated . as speculated , alternative splicing generates the isoform 2b with a deletion of a serine residue . splicing to an acceptor site of exon 7 located three nucleotides the sequence and structure of exons 8 to 11 are also highly conserved ( figure 3g , h , i , j ) . the comparison of the 11th exon was extended up to the end of the human cdna sequence of s88309 . highly conserved sequence elements of up to 91 identical nucleotides indicate a regulatory function of the 3'-untranslated sequence following the translational stop codon . the aceview analysis at the ncbi based on the draft sequence and a blast search with s83309 of the celera genomics freely accessible whole - genome sequence data gave mostly similar intron sizes . both analyses revealed a large first intron of 37,652 bp in the public sequence data , and 37,157 bp in the private data . the size of the second intron separating exon 2 and exon 4 was only available in the ncbi database ( 14,869 bp ) . according to the ncbi and celera databases , introns 3 to 9 have sizes of 10,486 ( ncbi ) ( 10,471 , celera ) bp , 3629 ( 3615 ) bp , 190,321 ( 1708 ) bp , 1963 ( 1960 ) bp , 2716 ( 9019 ) bp , 1905 ( 1912 ) bp , and 1927 ( 1928 ) bp , respectively . the comparison of both analyses shows that the deduced sizes of two of the human introns differs greatly . it seems likely that these inconsistencies will be corrected in the final assembly of the human genome . in summary , our analysis reveals a conserved structure for gcnf , allows the verification and systematic analysis of splice variants , and may be the basis of a better understanding of gcnf . the conservation of the intron - exon boundaries is consistent with the extremely high degree of amino - acid conservation between the human and the mouse proteins . the generation of the proteins hgcnf-1 , hgcnf-2a and hgcnf-2b can be explained by alternative splicing of the rna . the sequence of the third coding mouse exon , including the splice sites , is highly conserved ; however , at present no human cdna has been isolated containing this putative exon . alternative splicing provides a plausible means for generating diversity and may contribute to a higher instructive complexity in human gcnf . exons of gcnf were identified by a blast search with the human gcnf cdna sequence ( s83009 ) in the " unfinished high throughput genomic sequences " and in the homo sapiens genomic contig sequences at the ncbi [ 24 , 25 ] . intron sizes given by the aceview analysis were compared with the numbers obtained by a blast search of celera 's assembled sequence of the human genome . the putative human gcnf exon 3 was identified by a blast search with the sequence of the third mouse exon . sequences were aligned using the wisconsin package version 10.0 of the genetics computer group ( gcg ) , madison , wisconsin . exons of gcnf were identified by a blast search with the human gcnf cdna sequence ( s83009 ) in the " unfinished high throughput genomic sequences " and in the homo sapiens genomic contig sequences at the ncbi [ 24 , 25 ] . intron sizes given by the aceview analysis were compared with the numbers obtained by a blast search of celera 's assembled sequence of the human genome . the putative human gcnf exon 3 was identified by a blast search with the sequence of the third mouse exon . sequences were aligned using the wisconsin package version 10.0 of the genetics computer group ( gcg ) , madison , wisconsin . the mouse - derived ( m ) dna sequence ( upper line , genbank accession number af254575 ) of the first protein - coding exon and flanking sequences are compared with the human - derived ( h ) sequence ( lower line , s83309 for the coding , and nt_008491 for the flanking sequences ) . identical nucleotides are highlighted in the human sequence by bold letters , gaps in the alignment are shown as dots . the deduced amino - acid sequence of the human protein is shown in the single - letter code . the bold p ( proline ) marks the position of an arginine in the mouse protein . sequence comparison of the 5 ' ends of human gcnf cdnas coding for a full - length protein with the genomic dna sequence . human cdna sequences have been aligned omitting the cloning sites at the 5 ' ends . one cytosine and five adenosines in the genomic sequence not found in any of the cdnas are underlined . the deduced amino - acid sequences ( aab50876 , aac52054 ) are given in the single - letter code . the upper line of nucleotide sequence shows the murine protein - coding exons and their flanking sequences ( af254815-af254821 ) . mgcnf indicates the deduced mouse protein sequence ; hgcnf indicates the deduced human protein sequences ; gcnf indicates identical protein sequences . forty - one out of 42 nucleotides are identical and the flanking splicing signals are conserved . a homologous sequence coding for identical amino acids was found in the human genomic sequence . the splice donor site shows the typical pyrimidine - rich sequence followed by the sequence 5'-ncag in both sequences , but the comparison reveals several base transitions . ( c ) a single splice donor site in the fourth exon coding for the dna - binding domain is used in all mouse - derived cdnas described so far . for the human isoform gcnf-2 , the corresponding splice site is used , giving rise to a protein containing the sequence isvsdd instead of the vsvpdd in mouse . usage of an alternative splice site located 12 bp further downstream gives rise to the shorter isoform gcnf-1 . an asparagine ( n ) is underlined because one of the human cdna clones codes for a lysine in this position ( u64876/nm_001489 ) . ( d ) sequences of the fifth exon coding for the carboxy - terminal extension of the dna - binding domain are highly conserved . an arginine in hgcnf-2b instead of serine results from alternative splice donor sites of the seventh exon . ( f ) the comparison of the seventh exon reveals three positions where the mouse and the human isoforms diverge . isoform hgcnf-2b is generated by using a splice donor site located three nucleotides further downstream . the exons coding for the putative -helices 3 to 6 ( g ) , 7 and 8 ( h ) , 9 and 10 ( i ) , 11 and 12 ( j ) in the ligand - binding domain are highly conserved . the comparison of the last coding exon in ( j ) was extended up to the end of the human cdna sequence of s88309 .

background : germ - cell nuclear factor ( gcnf , nr6ai ) is an orphan nuclear receptor . its expression pattern suggests it functions during embryogenesis , in the placenta and in germ - cell development . mouse gcnf cdna codes for a protein of 495 amino acids , whereas the four reported human cdna variants code for proteins of 454 to 480 amino acids . apart from this size difference , there is sequence conservation of up to 98.7% . to elucidate the genomic structure that gives rise to the different human gcnf mrnas , the sequence information of the human gcnf locus is compared to the previously reported structure of the mouse locus.results:the genomic structures of the mouse and human gcnf genes are highly conserved . the comparison reveals that the shorter human protein results from skipping the 45 base - pair third exon . three different human isoforms - gcnf-1 , gcnf-2a and gcnf-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon.conclusion:by homology with the mouse gene , 11 gcnf coding exons can be defined on human chromosome 9 . all human gcnf cdnas identified so far are , however , derived from mrnas generated by splicing the fourth to the second exon . although the genomic sequence is highly conserved , the analysis suggests that alternative splicing generates a higher complexity of human gcnf isoforms compared with the situation in the mouse .

Background Results and discussion Structure of the first coding exon Conserved structure of exons 2 to 11 Conclusion Materials and methods Database search Sequence analysis Figures and Tables

we report involvement of unusual site in ilio - acetabular region with breach in articular cartilage . intra - operatively breach in acetabular roof was seen which was missed in the scan . this case report emphasizes about the rarity of the lesion in this location and the rare chances of breach in articular cartilage of the joint . bone desmoid tumor was an unknown entity until when jaffe hl reported a case in 1958 . it is a locally aggressive benign tumor reported commonly in mandible and meta - diaphyseal region of long bones . we report a case of desmoid tumor of ilio - acetabular region in a 40 year old female . a 40 year old female came with complaint of pain in the left hip region which aggravated on squatting and climbing stairs . radiological evaluation showed a lytic lesion in the left ilio - acetabular region , surrounded by a sclerotic margin superiorly . mri scan was done which showed a well defined homogenous lytic lesion with no break in the cortex and no soft tissue involvement ( fig . plain radiograph of pelvis with both the hips which shows a well defined lytic lesion over the left ilio - acetabular region . there appears no obvious breach in the cortex or the articular margin a ct scan of the pelvis was taken to rule out any breach in the cortex . there appears no obvious discontinuity through an ilio - inguinal incision the lesion was approached through inner table of ilium . a defect on the roof of the acetabulum was found which was missed by the scan ( fig . thorough curettage was done and the roof defect was reconstructed with a cortical graft harvested from the inner table of the ilium . there appears a hollow curetted lesion within which a breach in the roof of the acetabulum is seen histopathological finding showed multiple spindle shaped fibroblast cells with small and elongated nuclei in the background of dense collagen fibres . histopathology specimen picture showing spindle shaped fibroblast cells with small and elongated nuclei over a background of collagen fibers . there appears no nuclear atypia or mitotic activity patient was kept non weight bearing for 3 months with gradual return to full weight bearing walking . at follow up of 20 months patient is symptom free and xray showing no evidence of lytic lesion ( fig 5 ) . however , on ct scan there appears a persistent lytic area which we suspect to be a recurrence ( fig 6 ) . hence patient is kept on regular follow - up . a 20 month post operative x ray picture showing well maintained articular margin and the joint space . the lytic lesion seems filled up ct scan of the hip taken at 20 month post op , however shows lytic lesion anteriorly which seems to be a recurrence few cases have been reported since then and common occurrence being in mandible and meta - diaphyseal region of the long bone . recognition of desmoplastic fibroma is important because on radiology and histology , the lesion may be mistaken for an indolent , benign fibrous lesion or more aggressive spindle - cell sarcomas . it commonly occurs in the age group between the adolescent to 40 years with no specific sex predominance . patients commonly come with complaint of pain ; however there are few reported cases of pathological fracture especially those involving tibia and femur . the lesions are well defined with no sclerosis or periosteal reaction except if there is associated pathological fracture . if a soft tissue lesion is invading onto the bone , they are eccentric in position with sclerosis due to endosteal bone formation . significant t2 shortening of a non - sclerotic fibro - osseous lesion should place desmoplastic fibroma high among the diagnostic considerations . radiologically they have to be differentiated from giant cell tumor , fibrous dysplasia ( long bone lesions ) , fibrosarcoma , brown tumors , and chondromyxoid fibroma and hence a tissue diagnoses is must . the differentiation from a low grade fibrosarcoma is difficult though . as these are rare tumors , there is no standard protocol for treatment of these lesions . all kinds of procedures like intralesional , marginal or wide resection are reported as a treatment . but if wide resection would result a major functional deficit , an attempt of intralesional curettage with bone grafting seems warranted . desmoid fibroma of ilio - acetabular region is very rare hence a great sense of suspicion is required for the appropriate management . this case report emphasizes about the rarity of the lesion in this location and the chances of breach in articular cartilage of the joint . desmoid tumor , though rare , should be kept as one of the important differentials for the lytic lesion around pelvis . one should also define whether there is any associated cortical or articular margin breach ( as seen in our case report ) and then decide further management regarding reconstruction .

introduction : desmoid tumor of bone is a rare benign tumor . it is reported commonly in mandibular and meta - diaphyseal region of long bones . we report involvement of unusual site in ilio - acetabular region with breach in articular cartilage.case report : a 40 year old female presented with pain in the left hip . radiologically , a lytic lesion at ilio - acetabular region was seen . intra - operatively breach in acetabular roof was seen which was missed in the scan . curettage and defect reconstruction was done . histopathology reported as desmoid tumor . 20 months post - operatively patient was symptom free.conclusion:desmoid tumor is a rare bone tumor . this case report emphasizes about the rarity of the lesion in this location and the rare chances of breach in articular cartilage of the joint . the chances of recurrences are high with intralesional curettage .

Introduction: Case Report: Conclusion: Introduction Case Report Discussion Conclusion

uterine malformations can cause amenorrhea , infertility , recurrent pregnancy loss and pain and can also be normally functioning , depending on the nature of the defect [ 13,5 ] . the prevalence of uterine malformation is estimated to be 6.7% in the general population , slightly higher ( 7.3% ) in the infertile population , and significantly higher in a population of women with a history of recurrent miscarriages ( 16% ) . until the sixth week of life , the male and female genital systems are identical . there are two pairs of symmetrical genital ducts , the mesonephric ( wolffian ) ducts and the paramesonephric ( mullerian ) ducts . in the female embryo , the mullerian ducts grow caudally and become enclosed in the peritoneal folds , which later give rise to the broad ligaments of the uterus to which the ovaries , fallopian tubes and uterus are attached . the mullerian ducts approach each other and begin to fuse . at 9 weeks gestation , mullerian tract anomalies result from incomplete bilateral duct elongation , fusion , canalization , or septal resorption of the mullerian ducts . an accessory cavitated uterine mass ( acum ) is associated with an otherwise normal uterus . acum can be difficult to differentiate from true cavitated adenomyomas and cavitated rudimentary uterine horns . an accessory uterine mass can be caused by duplication and persistence of the ductal mllerian tissue in a critical area at the attachment level of the round ligament , possibly related to gubernaculum dysfunction . for the differential diagnosis of acum and cavitated non - communicating rudimentary uterine horns , hysterosalpingography showing a normal eutopic uterine cavity is helpful . an old classification had been put by jone h.w jr . in 1953 for the congenital anomalies of the female urogenital system : 1.uterus : ( a ) single uterus . a recent eshre / esge classification of female genital tract anomalies ( 1)uterine anomalies :- u0 . ( b ) without rudimentary cavity ( bi- or unilateral uterine remnants / aplasia ) . a 20-year - old female married for 2 years had undergone a full - term pregnancy with normal vaginal delivery , one year prior to this case report . the patient presented with 3 missed periods with lower abdominal pain , and a pelvic ultrasound examination revealed an intrauterine pregnancy of twins that had died at 13 weeks of gestation ( miscarriage ) ( fig . 1 ) . induction of abortion by oxytocin drip had been attempted twice but failed . the gynecologist proceeded to perform cervical dilatation and curettage , but the uterus was found to be empty . the patient was seen by another gynecologist who repeated the curettage and also did not find the dead twins , and an ultrasound after the procedure still showed the dead twin fetuses . the patient was then seen at our clinic , and we submitted the patient to another curettage . perforation of the uterus was expected ; thus , we opened the abdomen and found a slightly enlarged normally shaped and positioned uterus with 2 fallopian tubes and 2 ovaries . there was a uterine perforation at the fundus ; thus , the omentum was pulled in through the perforation , drawn out of the uterus , transfixed , ligated by suture and trimmed . the perforation of the fundus was repaired with 3 stitches using one zero vicryl sutures , no other visceral injury was found . on exploration , we found a large pear - shaped mass emerging from the right broad ligament with a thick pedicle that ended near the uterine cervix . because we did not obtain a detailed gynecological history of the patient , we assumed the mass was a broad ligament cyst . the mass was easily excised by clamping , transfixing , ligating and cutting the pedicle . the mass is pear - shaped and 10 13 cm in size . the wide upper portion of the mass has 2 small appendages at either corner that are each 1 cm long ( fig . the narrow lower end of the mass was firm in consistency ( arrow in fig . 2 ) . we opened the mass and observed amniotic fluid and two dead fetuses , each 6 cm in length ( fig . the mass wall was thin in the upper segment and thick in the lower segment . the portion of the mass representing the cervix was solid and did not have an internal opening or cervical canal , as confirmed using a fistula probe . tissue samples taken from the upper and lower portions of the specimen for histopathological analysis both exhibited the smooth muscle fiber arrangement of uterine tissue . one year later , a hysterosalpingogram showed a normal uterine cavity and two patent fallopian tubes ( fig . a few months later , she became pregnant , and she had a full term pregnancy with normal vaginal delivery . she had four subsequent pregnancies over 9 years that all reached full term and all resulted in normal vaginal deliveries . there was a lack of a full medical history and poor communication between the gynecologist and the ultrasonographist . thus , despite the proficiency of the ultrasonographist , during two examinations , he did not thoroughly examine all pelvic organs once he detected a twin miscarriage in what appeared to be a uterine cavity . if he had examined all pelvic organs , he should have detected the empty normal uterus just beside the gravid abnormal uterus as it is clear in the post - operative hystersalpingography ( fig . hysteroscopy could have been useful in this case , but unfortunately , the only available hysteroscope in the hospital was broken - down . most of the congenital anomalies of the uterus are due to a fault in one or more step in the fusion process of the mullerian ducts . in this case , however , the accessory uterus most probably developed as a growth from the right mullerian duct generating an incompletely developed separate accessory uterus ( non - communicating , cavitated accessory uterus ) acum . most probably , the ovum entered the accessory uterus through the small appendages on the sides of the dome , which represent rudimentary fallopian tubes . the ovum likely became fertilized by sperms passing through the normal cervical canal of the normal uterus , uterine cavity , fallopian tube , and the peritoneal cavity , or the sperms may have travelled through the lymphatic vessels of the accessory uterus to fertilize the ovum inside it . 1.a deficient case history , lack of communication between the clinicians and the ultrasonographist and repeated shifting of the patient from one doctor to another , all led to the misdiagnosis and mismanagement of this patient.2.conception can occur in acum , however , it is very rare and may be difficult to diagnose . a deficient case history , lack of communication between the clinicians and the ultrasonographist and repeated shifting of the patient from one doctor to another , all led to the misdiagnosis and mismanagement of this patient . conception can occur in acum , however , it is very rare and may be difficult to diagnose . harith m. alkhateeb : surgery for the patient , writing of original text , communication with the editorial team . enas m. yaseen : the last gynecologist dealing with the patient , periodical clinical examinations and follow up of the patient . a copy of the written consents are available for review by the editor - in - chief of this journal .

highlightsobtaining a detailed medical history is important , and we should not depend on examination tools when dealing with patients.unexplained findings should raise suspicions regarding other possible diagnoses , and the same non - conclusive procedures should not be repeated.following this advice can prevent unnecessary operative procedures and the complication of perforation of the uterus .

Introduction The case Surgical pathology Discussion Conclusions Competing interests Funding Ethical Approval Authors contributions Consents Guarantor

a 19-year - old female with no significant past medical history or medication use presented to our hospital in april 2010 with severe nephrotic syndrome characterized by marked fluid retention , heavy proteinuria ( protein : creatinine ratio of 1244 mg / mmol ) , hypoalbuminaemia ( 13 g / l ) and elevated total serum cholesterol ( 9.3 mmol / l ) . renal function was preserved with a serum creatinine of 42 mol / l , and there was no haematuria evident on urine microscopy . lupus and vasculitis markers , hepatitis serology and serum electrophoresis were negative . a renal biopsy was performed which revealed normal glomeruli , tubules and interstitium . the patient was commenced on prednisolone 50 mg daily ( 1 mg / kg ) in addition to ramipril 5 mg daily , simvastatin 10 mg daily and warfarin therapy . complete remission was achieved within 2 months and prednisolone dose was reduced to 25 mg daily . soon after , and against medical advice , the patient abruptly ceased her prednisolone and within 72 h had a relapse of disease with recurrence of gross peripheral oedema , hypoalbuminaemia of 13 g / l ) and proteinuria of 1030 mg / mmol ( from 0.06 mg / mmol ) . despite recommencing high - dose steroids ( 60 mg / day ) , the disease thereafter remained refractory to steroid therapy with persistent gross oedema , nephrotic - range proteinuria and hypoalbuminaemia ( albumin of 9 g / l ) . after failing to respond to 3 months of high - dose steroids , the patient was commenced on cyclophosphamide . in light of the patient 's history of non - compliance and in an effort to reduce drug exposure and toxicity , pulse intravenous cyclophosphamide was administered in incremental doses of 0.5 , 0.75 and 1.0 g / m 1 month apart . during this period two months after the third pulse of cyclophosphamide was administered , there was no evidence of disease response with ongoing gross oedema , hypoalbuminaemia ( albumin 14 g / l ) and heavy proteinuria ( 1476 mg / mmol ) . at this point , it was decided to trial the monoclonal anti - cd20 antibody rituximab ( rtx ) , with two doses of 500 mg given 2 weeks apart . complete remission was rapidly induced within 1 month of rtx therapy with resolution of oedema , rising albumin to 31 g / l and absent proteinuria ( figure 1 ) . 1.changes in serum albumin ( g / l ) and urine protein : creatinine ratio ( mg / mmol ) over time , relative to treatment modality . changes in serum albumin ( g / l ) and urine protein : creatinine ratio ( mg / mmol ) over time , relative to treatment modality . the patient has remained in complete remission for more than 32 months since the two doses of rtx were administered in february 2011 . this is despite recovery of cd19 counts back to normal levels 24 months after treatment . mcd is the most common cause of nephrotic syndrome in children and accounts for 1025% of cases in adults . close to 75% of cases will respond initially to a course of high - dose glucocorticosteroids but relapse commonly occurs . steroid - sparing agents like calcineurin inhibitors and cyclophosphamide are often used for frequently relapsing or steroid - dependent mcd . besides the significant adverse side effects of these agents , a large proportion of patients relapse after cessation of these medications . while there was some suggestion that oral cyclophosphamide may be more successful than iv cyclophosphamide in achieving disease remission in refractory mcd , larger clinical trials have suggested that both forms of therapy are equivocal with respect to relapse rates and toxicity . there are an increasing number of reports of success of rtx therapy for steroid- and cyclosporine ( csa)-dependent nephrotic syndrome in paediatric populations [ 4 , 5 ] . a single rtx infusion also appeared to improve response to csa therapy and had significant steroid - sparing effects in children with steroid-dependent nephrotic syndrome ( sdns ) who had developed secondary resistance to csa . in a long - term follow - up study of 37 children who received rtx for sdns , 26 patients remained in remission after 12 months and 7 remained in remission after 24 months without further maintenance immunosuppression . however , response to rtx has been mixed in cases of steroid-resistant nephrotic syndrome ( srns ) in children . reported the successful use of rituximab in five patients with srns , two of which had mcd . . carried out a multi - centre questionnaire - based study in which the response rate in children with srns was as high as 44% . in another case series of four children with srns , including one case of mcd , all children failed to achieve sustained remission after a single dose of rituximab , despite complete b - cell depletion . in adults , the first case report of the efficacy of rtx in multi - relapsing mcd was published in 2007 . since then there have been a number of reports of success with rituximab in steroid - dependent mcd [ 12 , 13 ] . munyentwali et al . analysed the outcome of 17 adult patients who were treated with rtx for steroid - dependent mcd over a mean follow - up period of 29.5 months . after the first course of rtx , 11 patients ( 65% ) did not relapse after a mean follow - up of 26.7 months . twelve of the 17 patients ( 70% ) were free of any immunosuppressant drugs at last follow - up . there are minimal data available regarding the use of rtx in steroid - resistant mcd in adults . the first case report , published in 2008 , describes an adult patient with steroid- and mycophenolate mofetil - resistant mcd who remained in complete remission 1 year after initiation of rtx therapy . a further case report was of a 23-year - old patient with steroid- and csa - resistant mcd treated with two doses of rxt with sustained complete remission at the end of 1 year . the second dose of rtx was given at 6 months in response to a recovery of b - cell counts . a long - term follow - up study was published recently by bruchfeld et al . on 16 adult patients with biopsy - proven mcd of which 13 were steroid dependent , 2 multi - relapsing and 1 steroid and multi - drug resistant . in this study , the rtx dose varied from 1000 to 2800 mg . complete remission was achieved after initial rtx treatment in 13 patients . in the remaining cases , partial remission was achieved in all except the patient with steroid- and multi - drug - resistant disease . relapses can often occur after the reappearance of cd19 cells , although this is not always the case [ 12 , 14 ] . guigonis et al . found a consistent association between relapses ( 3 of 22 paediatric patients ) and an increase in cd19 cells which occurred at a median time of 11 months ( range 839 months ) . sellier - leclerc et al . evaluated disease outcome after a minimum cd19 depletion period of 15 months obtained by repeated rtx infusion and found that it induced long - term remission even after definitive cd19 recovery in almost two thirds of the paediatric patients without the use of maintenance oral immunosuppressive drugs . the rtx dosing has varied greatly in various reports including a single dose , doses of 500 or 1000 mg given at one or two time - points and 375 mg / m once weekly for 4 weeks . in the study by munyentwali et al . we report a unique case of steroid- and cyclophosphamide - resistant mcd responding rapidly to two doses of rtx at 500 mg each , with sustained complete remission even after cd19 recovery . as discussed , rtx has been shown to be efficacious in a growing number of small case series in steroid - sensitive and steroid - dependent mcd ; however , reported success of its use in multi - drug - resistant disease , particularly in adults , remains limited . in our case , clearly , a well - conducted multi - centred clinical trial of rtx for mcd is warranted either as a first - line agent or in cases of steroid - dependent and/or -resistant disease . the results presented in this paper have not been published previously in whole or part , except in abstract format .

we report a case of steroid- and cyclophosphamide - resistant nephrotic syndrome secondary to minimal - change disease occurring in an otherwise healthy 19-year - old female , responding rapidly to two doses of rituximab therapy . complete disease remission has been sustained up to last follow - up ( 32 months ) despite cd19 recovery . literature review suggests emerging evidence that rituximab may have a role to play in recurrent and/or refractory minimal - change disease .

Case report Discussion Conflict of interest statement

among all emergency cases the ratio of incarcerated / strangulated hernias differs across the countries . but it is well known that hernia is the common cause of small bowel obstruction , and mortality and morbidity rates increase in case of strangulation . during acute ischemic conditions , metal binding capacity of albumin this change is quantifiable and commonly known as ischemia modified albumin ( i m a ) . recently , i m a measurement has been proposed as a sensitive marker for the diagnosis of myocardial ischemia presenting with typical acute chest pain . however , i m a levels are also known to increase in other ischemic conditions , as well as to be an indicator of oxidative stress , and may not be specific for cardiac ischemia . recently , turedi et al . showed that the level of i m a may be of use in the diagnosis of pulmonary embolism , another ischemic condition . incarceration is the state of an external hernia which can not be reduced into the abdomen . roughly 5% of all patients having an operation for external hernia are explored as an emergency . strangulated external hernia is a relatively common and serious surgical emergency and accounts for most of the deaths from this condition . clinical validation of any test for ischemia is difficult as there is no accepted diagnostic gold standard . and the preoperative diagnosis or prediction of ischemia is important for surgeons because ischemia changes the operative strategy . thus we planned this study and aimed to show relation between the intestinal ischemia and i m a . our study was initiated after anakkale onsekiz mart university ( comu ) animal ethics committee approval ( approval number 2010/11.02 ) and took place in comu laboratories . twenty wistar albino rats were used weighing 153207 g and were housed in optimum conditions . in this study rats , they were kept for two weeks in cages with five animals each and 12 : 12-hour artificial light to dark cycles . we aimed to mimic incarceration by short clamping ( 45 minutes ) and strangulation by 6 hours clamping . ketamine hcl ( ketalar , pfizer , turkey ) 35 mg / kg and xylazine ( basilazin flacon % 2 , turkey ) 5 mg / kg were administered intraperitoneally as anesthetic agents . after midline laparotomy , mesenteric vascular structures of small intestine were clamped by a vascular clamp . then blood samples were taken [ for i m a ( ima1 ) and lactate dehydrogenase ( ldh ) ] and midline incision was closed separately by polypropylene sutures . after midline laparotomy , mesenteric vascular structures of small intestine were ligated by 2/0 polypropylene sutures and midline incision was closed separately by polypropylene sutures . midline laparotomy was done again at 6th hour , intestinal necrosis were seen , and blood samples were taken [ for i m a ( ima2 ) and ldh ] . after midline laparotomy then blood samples were taken [ for i m a ( ima1 ) and ldh ] and midline incision was closed separately by polypropylene sutures . but incarceration is a known ischemia reperfusion process and total vascular clamping is a widely used method to investigate ischemia reperfusion in rats . so we used this method to simulate incarceration in group i. also total occlusion of all vascular support of the small intestine is used in group ii to provide the smaller amounts of ischemic / necrotic tissue in group i and group ii and to prevent the potential effect of ischemic / necrotic tissue amount on i m a levels . reduced cobalt to albumin binding capacity ( i m a levels ) was analyzed using the rapid and colorimetric method developed by bar et al . . results higher than 0,4 absu were estimated as ischemia and lower than 0,4 absu were estimated as normal . kruskal - wallis test was used to compare the groups and probability ( p ) values less than 0.05 were considered significant . the significance of the parameters in kruskal - wallis test was confirmed by mann - whitney u test and p values less than 0,016 were considered significant . kruskal - wallis test is the standard variance analysis test to compare the means of 3 or more groups . kruskal - wallis test gives us the difference between the groups , but it does not give any idea about which group is the cause of this difference . we used mann - whitney u test with bonferroni correction ( the p value for mann - whitney u test by bonferroni correction is found to be 0,05/number of groups = 0,016 for this study ) . mean ima1 levels of group i , group ii , and group iii were 0,41 0,01 ( range 0,400,45 ) , 0,46 0,16 ( range 0,440,48 ) , and 0,34 0,03 ( range 0,280,39 ) , respectively mean ldh levels were 819,71 34,53 ( range 766874 ) , 808,57 24,69 ( range 774841 ) , and 514,14 19,92 ( range 487545 ) for groups i , ii , and iii , respectively . the difference of i m a levels between group i and group ii was not significant at 6th hour ( ima2 ) ( p : 0,71 ) during acute myocardial ischemia , the ability of binding ions such as copper , zinc , and cobalt is decreased ; therefore a form of albumin is produced , which is described as ischemia modified albumin ( i m a ) . the i m a was registered by the united states food and drug administration as a marker of myocardial ischemia . the test is based on the decreased ability of albumin to bind cobalt due to the structural change of nh2 which develops in the ischemic environment . it is important to emphasise that the decreased ability of albumins to bind cobalt occurs in hypoxia , acidosis , sodium and calcium pump malfunctions , and tissue damage caused by free radicals [ 7 , 8 ] . it has been shown that the concentration of i m a increases within a couple of minutes from the onset of ischemia , remains elevated until 6 to 12 hours , and returns to its normal range after 24 hours . but recent studies showed that i m a increases in mesenteric ischemia , stroke , and cerebrovascular accidents as well [ 1 , 10 ] . found significantly higher i m a levels in superior mesenteric artery thromboembolism and they concluded that i m a can be used in the diagnose of mesenteric ischemia . patients requiring emergency repair tend to be a member of an elderly and unfit population [ 1315 ] . ge et al . concluded that the main causes of high incarceration rates are economic problems and lack of insurance . morbidity and mortality risks of incarcerated hernias are increased by longer duration of symptoms [ 1315 , 19 , 20 ] , type of hernia ( femoral rather than inguinal hernia ) , a high american society of anesthesiology ( asa ) score [ 14 , 17 , 19 , 21 ] , the presence of comorbidities [ 14 , 15 ] , and the presence of bowel necrosis [ 14 , 17 , 19 ] . this study aimed to predict necrosis by i m a levels and explore the impact of ldh . we can evaluate asa score , duration of symptoms , and the presence of comorbidities preoperatively , but the need for an intestinal resection is unclear until surgery . this has been reported to be the case even for experienced surgeons where the sensitivity has been reported to be , at best , 48% . maeda et al . reported that the serum creatine phosphokinase ( cpk ) level was a useful marker for the diagnosis of a strangulated hernia , and ohira et al . found that cpk , lactate dehydrogenase , and metabolic acidosis , in addition to ultrasound and enhanced computed tomography , were of value in diagnosing strangulation of the intestine in some cases . a definitive diagnosis of strangulation of the intestine , however , can only be made during a surgical exploration [ 5 , 25 ] . the current series showed a significant increase in the need for a resection of the small intestine when the time from onset of symptoms to surgery was more than 12 hours ; the rate of resection of the small intestine in patients presenting within 12 hours was 14.3% , but it increased to 50% in patients presenting after 12 hours or more ( p = 0.0392 ) . reported that the resection rate in the bowel operated on within 24 hours was 7% , but when the time increased to 48 hours or more , the resection rate increased up to 27% . 's results.ldh was elevated in both incarceration and strangulation groups but it was failed to predict necrosis . in this study we aimed to predict intestinal necrosis by i it is important to predict necrosis , because incarcerated hernias are more frequently seen in elderly patients , and their prevalence in the over-60-year - old population ( added comorbidities and anesthetic risks ) has been reported to be 9.8% compared with 1.8% for younger patients . besides it is known that general and spinal anesthesia are both associated with higher rates of serious postoperative complications . the prediction of necrosis can give both the surgeon and the patient the chance of local anesthesia . unfortunately the disadvantage of this study is the lack of evaluating other predictive values like cpk levels and since this study is an animal study , pospective human studies must be performed using i m a to be able to suggest that i m a is a gold standard to predict strangulation in the hernia sac . i m a is a new marker of ischemia and its place in general surgery is not well known yet . further studies should be done to predict intestinal necrosis in the hernia sac , to be able to protect old and unfit patients from the anesthesia associated mortality and morbidities .

purpose . to evaluate the predictive effect of i m a in incarcerated hernias . methods . three groups ( n = 7 ) of rats were operated . group i aimed to mimic incarceration , group ii aimed the strangulation , and group iii was the sham group . i m a and ldh measurements were made . results . i m a levels were significantly higher in strangulation mimicking group and i m a levels were normal at postoperative 6th hour in incarceration mimicking group . ldh levels were significantly higher in both incarceration and strangulation mimicking groups . conclusion . i m a seems to be an effective marker in incarcerated hernias to predict necrosis . but we need further studies to generalise this hypothesis .

1. Introduction 2. Results 3. Discussion 4. Conclusion

iran is in the thalassemic belt with a frequency of about 6 -10% in the northern and southern provinces ( 1 ) . cancer antigen 15.3 ( ca15.3 ) , a product of muc1 gene ; is an epithelial mucin and its high level is associated with breast cancer . in patients with breast cancer , evaluation of ca15.3 has not been recommended by national comprehensive cancer network guidelines for surveillance purposes , but its monitoring suggested to detect recurrences of cancer evaluating therapeutic response of advanced disease and the assessment of prognosis in patients with early breast cancer ( 2,3 ) . also it may be high in nonmalignant conditions such as inflammatory processes and some hematologic disorders like sickle cell anemia and thalassemia major ( 4).improving management of thalassemia major has led to increased life expectancy and subsequently increased frequency of age related conditions such as malignancies ( 5 - 7 ) . as a result , some investigators have opted to check serum tumor markers in these patients . however , they showed an increased frequency of serum ca15.3 independent from the presence of malignancy in these patients . the suggestive mechanism is erythroid hyperplasia which could result in high level of muc1 expression on bone marrow - appoptosing progenitor cells ( 5 , 8) . up to our knowledge , evaluation of serum ca15.3 in patients with thalassemia minor has not been conducted . our hypothesis was that patients with beta - thalassemia minor has a higher serum level of ca15.3 compared to healthy individuals and patients with thalassemia major but with a milder degree . present study was designed to measure the serum level of ca15.3 in patients with beta - thalassemia minor compared to healthy individuals and to patients with cancer . also we aimed to determine the frequency of beta - thalassemia minor in a population of young patients with malignancies . in this cross - sectional study , 32 carriers of beta - thalassemia , 25 healthy individuals , and 49 patients with cancer were recruited from february to december 2011 . carriers of beta - thalassemia were non - randomly selected from the volunteers referred for premarital screening in an outpatient clinic affiliated to shiraz university of medical sciences , southern iran . all patients with cancer who referred to pediatric hematology - oncology clinic of shiraz university of medical sciences in the study period were recruited . healthy controls were randomly selected from healthy individuals who referred to pediatric hematology - oncology clinic for checkup examination . patients with cancer were subdivided to 2 groups of with and without beta - thalassemia minor . in all participants , the ca 15.3 was checked by canag ca 15 - 3 eia kit , sweden in all participants . the protocol for this study was approved by the ethics committee of shiraz university of medical sciences . written informed consent was obtained from all participants or their parents . test of normality was done by kolmogorov - smirnov test ( ca15.3 , p=0.2 and age , p=0.156 ) . comparison of serum level of ca15.3 level and age among various groups were done by anova test . to find the statistically significant difference among different groups , post hoc tukey hsd test was performed . sex distribution was compared among the studied groups using chi - square test , and p value less than 0.05 was considered statistically significant . participants were divided to 4 groups including : group 1 , beta - thalassemia minor ; group 2 : patients with cancer and without beta - thalassemia minor ; group 3 : patients with cancer and beta - thalassemia minor , and group4 : healthy individuals . comparisons of the mean age and sex ratio as well as mean serum ca 15.3 level among the four groups of participants are shown in table 1 . * statistically significant , ca ag 15 - 3 , cancer antigen 15.3 carriers of beta - thalassemia were significantly older than the other groups ( p<0.0001 for comparison of this group with each of the other three groups based on post hoc tukey hsd test ) . male to female ratio was not significantly different among the studied groups ( p=0.804 ) . the serum levels of ca 15.3 in all participants were in the normal range ( < 35 u / ml ) and its concentrations shown no significant difference among various groups of the participants ( p=0.723 ) . age was not significantly correlated with the serum level of ca 15.3 ( r= 0.039 , p=0.702 ) . as shown in table 2 , of 49 patients with cancer , 96% had hematologic malignancies with the highest frequency for acute lymphoblastic leukemia ( 37 patients ) . acute lymphoblastic leukemia : ( all ) , acute myeloblastic leukemia : ( aml ) , hodgkin lymphoma : ( hl ) , non hodgkin lymphoma : ( nhl ) of 49 patients with malignancies 11 ( 22.4% ) diagnosed as thalssemia minor by their cbc and hemoglobin electrophoresis results . we evaluated serum level of ca 15.3 in carriers of beta - thalassemia by comparing them to the three other groups including patients with cancer and those with or without beta - thalassemia minor as well as healthy individuals . we did not find any significant difference with regard to the ca 15.3 concentrations among the groups . moreover , we expected that the higher serum level of ca 15.3 in patients with cancer compared to the reference range . this is in contrast with the results of other reports which showed increased levels of ca 15.3 among patients with solid tumors ( 9 ) . we could not find any publication that has investigated the serum levels of ca 15.3 in patients with hematologic malignancies . as we mentioned most of our patients had hematologic malignancies and mostly acute lymphoblastic leukemia . this result suggests that the ca 15.3 may not be associated with hematologic malignancies and thus it can not be used as a tumor marker . we found a frequency of 22.4% ( 11 out of 49 ) of beta - thalassemia minor in the group of patients with malignancies . compared to the prevalence rate of about 10% in the general population therefore the suggestive mechanism could be shorter life span of red blood cells , and theoretically erythroid hyperplasia secondary to ineffective erythropoiesis which could cause errors in cell division leading to hematologic malignancies . these findings did not support the results of the previous study in our center which showed protective effect of minor thalassemia against childhood malignancies ( 1 ) . our study was limited due to lack of matched matched of carriers for beta - thalassemia with other groups because most of participants in this group selected from persons who were screened for marriage . but other groups were selected from younger children and persons referred to pediatric hematology - oncology clinic . however , we did not find a significant correlation between age and serum ca 15.3 concentration in our participant . moreover our research was a cross - sectional study and we can not conclude strongly about the relationship between cancer and beta - thalassemia minor . to document the accurate correlation we need a cohort study for this matter . moreover , during the course of our study , most of our patients in the cancer group had hematologic malignancies . the values of ca 15.3 serum levels were in the normal range for both in carriers of beta - thalassemia and in patients with malignancies . no correlation was found between ca 15.3 serum level with thalssemia minor or with childhood malignancies . compared to normal population , a high proportion ( 22.4% ) of thalssemia minor was observed in patients with cancer in our study . future prospective studies are needed to evaluate the relationship between cancer and beta - thalassemia minor accurately .

background : high serum level of cancer antigen 15.3 ( ca15.3 ) has been reported in some malignant and nonmalignant conditions including thalassemia major which could have been resulted from ineffective erythropoiesis . we aimed to evaluate the serum level of ca15.3 in carriers of beta - thalassemia by comparing them with cancer patients and healthy individuals . methods : this cross - sectional study was done from february to december 2011 in southern iran . participants consisted of 32 subjects with beta - thalassemia minor , 49 with cancer and 25 healthy individuals . the serum levels of ca15.3 were measured and compared in different groups . results : the serum levels of ca 15.3 in all participants were in the normal range ( < 35 u / ml ) . also it did not significantly differ among various groups of the participants ( p=0.723 ) . age was not significantly correlated with the serum level of ca 15.3 ( r= 0.039 , p=0.702 ) . the most frequent cancer in the group of patients with malignancies was hematologic malignancies ( 96% ) with the highest frequency for acute lymphoblastic leukemia ( 37 patients ) . frequency of thalassemia minor in patients with cancer was 11 ( 22.4% ) . conclusion : no correlation was found between ca 15.3 serum level with beta - thalssemia minor or with childhood malignancies . compared to general population , a high proportion of beta - thalssemia minor was observed in patients with cancer in our study . future prospective studies are needed to evaluate the relationship between cancer and beta - thalassemia minor accurately .

Introduction Methods Results Discussion Conclusion

the accelerated emergence of antibiotic resistance among the prevalent pathogens is of global health concern . enterococcus , is one such pathogen which is the leading cause of nosocomial bacteremia , urinary tract infections ( uti ) , and surgical site infections . enterococcus resistance to antimicrobial agents to which the genus streptococcus are generally susceptible and its ability to transfer the drug resistance genes from vancomycin - resistant strains to staphylococcus aureus is of concern . the therapeutic challenge of multiple - drug resistant ( mdr ) enterococci , identifies them as important nosocomial pathogens . enterococci infections have traditionally been treated with cell wall inhibitor agents in combination with an aminoglycoside . reduced susceptibility to -lactam antibiotics and vancomycin ; in combination with a high level aminoglycoside resistance ( hlar ) interferes with the penetration of the aminoglycoside into the bacterial cytoplasm , thus making the antibiotic synergism ineffective . hence , this study was designed to identify the magnitude of enterococcal infections and their antibiotic susceptibility pattern in a tertiary care hospital in the eastern region of india . following approval from our institutional ethics committee , clinical samples were collected over a period of 1-year ( february 2011 to january 2012 ) at a tertiary care hospital in kolkata , india . the study population included patients of all age groups and both sexes with suspected nosocomial infections ( infection developed after 48 h of hospital stay ) . patients with infections at the time of admission , within 48 h of hospital stay or 30 days after discharge were excluded . the clinical samples of urine , wound swab / pus , high vaginal swab and blood were inoculated on blood agar and macconkey agar . the causative bacteria were identified on the basis of colony characteristics , gram stain morphology , motility and biochemical tests ( catalase test , growth on bile aesculin agar , tolerance to 6.5% nacl , arginine dihydrolase test , and fermentation of arabinose , mannitol , raffinose , and sorbitol ) . the isolates were subjected to antimicrobial susceptibility testing by kirby - bauer disk diffusion method , as per clsi recommendations , using commercially available 6 mm disks ( himedia , mumbai , india ) on mueller - hinton agar . the disks used were vancomycin ( 30 g ) , ampicillin ( 10 g ) , erythromycin ( 15 g ) , ciprofloxacin ( 5 g ) , linezolid ( 30 g ) . for high - level gentamicin resistance ( hlgr ) detection , gentamicin ( 120 g ) disc was used . the diameter of the zone of inhibition of each antibiotic was measured and interpreted as sensitive , intermediate sensitive or resistant according to clsi criteria . for hlgr , resistance was indicated by no zone , and susceptibility , by a zone of diameter 10 mm . enterococcus faecalis atcc 29212 and e. faecalis atcc 51299 were used as the susceptible and resistant quality control strains . brain - heart infusion agar ( hi media , mumbai ) was supplemented with different concentrations of vancomycin . the test organism was grown in broth and the turbidity matched with mcfarland 0.5 standard ( approximately 1.5 10 cfu / ml ) . the isolates were subjected to antimicrobial susceptibility testing by kirby - bauer disk diffusion method , as per clsi recommendations , using commercially available 6 mm disks ( himedia , mumbai , india ) on mueller - hinton agar . the disks used were vancomycin ( 30 g ) , ampicillin ( 10 g ) , erythromycin ( 15 g ) , ciprofloxacin ( 5 g ) , linezolid ( 30 g ) . for high - level gentamicin resistance ( hlgr ) detection , the diameter of the zone of inhibition of each antibiotic was measured and interpreted as sensitive , intermediate sensitive or resistant according to clsi criteria . for hlgr , resistance was indicated by no zone , and susceptibility , by a zone of diameter 10 mm . enterococcus faecalis atcc 29212 and e. faecalis atcc 51299 were used as the susceptible and resistant quality control strains . brain - heart infusion agar ( hi media , mumbai ) was supplemented with different concentrations of vancomycin . the test organism was grown in broth and the turbidity matched with mcfarland 0.5 standard ( approximately 1.5 10 cfu / ml ) . 101 of 1194 urine isolates , 30 of 662 wound swab / pus isolates , 13 of 307 blood isolates and 09 of 203 high vaginal swab isolates were identified as enterococci [ table 1 ] . all isolates were further speciated as e. faecalis ( 90.85% ) , enterococcus faecium ( 8.50% ) and enterococcus gallinarum ( 0.65% ) . most urinary isolates were from maternity ward ( n = 25 ) , followed by gynecology ( n = 23 ) , and pediatric ward ( n = 10 ) respectively . enterococcal wound infections were also most commonly reported from gynecology ( n = 7 ) followed by surgical wards ( n = 5 ) . enterococci were the causative pathogen in high vaginal swab in patients from maternity , gynaecology and itu . mic of vancomycin observed in all enterococcal isolates was < 1 g / ml which corroborates the disc diffusion test result . the spectrum of disease produced by enterococci varied from uti , wound infection , soft tissue infection to bacteremia . urinary tract instrumentation or catheterization , genitourinary pathology , prior use of antibiotics , prolonged hospitalization were some of the predisposing factors for enterococcal infections . urinary tract was the most common site of enterococcal infection ( 66.01% ) in this study which often occurred in catheterized patients . the next common infection was wound infection ( 19.60% ) followed by bacteremia ( 8.50% ) which corroborates with the studies from different regions of india [ figure 1 ] . in india , e. faecalis is the predominant enterococcal species , which accounts for 80 - 90% of all clinical isolates , which is followed by e. faecium ( 5 - 15% ) . however , a progressive increase in e. faecium infections has been reported and is found to be more resistant to penicillin and aminoglycosides , which is attributed to production of the enzyme , 6-acetyl transferase and more penicillin - binding proteins . in our study , e. faecalis was the commonly isolated species followed by e. faecium and e.gallinarum , the incidence rates were comparable with previous reports . comparison between isolation rates of enterococcus in different clinical samples from different regions of india all enterococci isolates were found to be sensitive to vancomycin and linezolid which was consistent with other studies from india , however the prevalence of vancomycin resistant enterococci ( vre ) in india is reported to be between 0% and 30% . almost half of the isolates were resistant to ciprofloxacin ( 49.67% ) and gentamicin ( 45.75% ) [ figure 2 ] . in contrast , ampicillin showed a high level of sensitivity ( 77.12% ) among the nosocomial isolates [ table 2 ] . resistance to ciprofloxacin was also less prevalent among the wound isolates in comparison with the urine isolates . ampicillin and gentamicin showed a higher level of resistance among the wound isolates compared with urine isolates . isolates from nosocomial bacteremia showed a higher level of sensitivity to each of the drug tested [ table 3 ] . antibiotic resistance pattern of enterococcus as evident in different studies antimicrobial sensitivity pattern of the isolates in nosocomial infections comparison of percentage prevalence of antibiotic resistance among isolates from different sources the hlar to gentamicin was observed in 45.75% isolates with 43.88% in e. faecalis and 69.23% in e. faecium [ table 4 ] . hlar to gentamicin is universally reported to be in the range of 1 - 48% ( mean , 22.6 12.3 ) , although with an increasing trend recently . hlar to gentamicin nullifies the efficacy of combination therapy , which is used to treat serious enterococcal infections . nevertheless , empirically chosen combination therapy with ampicillin and gentamicin would be effective in 54.25% of nosocomial infections [ figure 3 ] . interestingly 7% of the isolates ( n = 11 ) in our study were mdr and vancomycin or linezolid were the only available option for treating these patients . the mdr strains were mostly reported from gynecology ( n = 3 ) and maternity wards ( n = 2 ) . comparison between -lactam ( ampicillin ) and aminoglycoside ( gentamicin ) susceptibility patterns of enterococci isolates from different sources ( n = 153 ) ( am : ampicillin , g : gentamicin , s : sensitive , r : resistant ) our study reveals the prevalence of high degree of resistance to macrolide and fluoroquinolone among the nosocomial enterococcal isolates , thereby limiting the use of these drugs for therapeutic purposes . the resistogram of the enterococcal isolates varied among specimens from different wards , but the pattern was constant among isolates within a particular ward . hence , the nosocomial outbreak in our hospital had not been disseminated from a single strain though isolates from a particular ward might be epidemiologically linked . the present study also revealed that despite recent trends of increasing resistance to aminoglycosides , a combination therapy of -lactam and aminoglycoside as first - line drugs would be currently the best choice . vancomycin or linezolid therapy should be restricted for use in patients infected with mdr strains only . judicious use of vancomycin and linezolid in serious infections and appropriate infection control measures would probably recede the possible emergence of vre outbreaks in our geographical area .

background : resistance to commonly used antibiotics by enterococci causing nosocomial infections is of concern , which necessitates judicious , responsible and evidence - based use of antibiotics . the present study was conducted to review the prevalence and identify therapeutic options for nosocomial enterococcal infections in our tertiary care hospital.materials and methods : isolates identified by morphological and biochemical characteristics were tested for antibiotic susceptibility using kirby - bauer method.result:153 of 2096 culture positive clinical samples comprised of 101 urine , 30 wound swab / pus , 13 blood and 09 high vaginal swab isolates were identified as enterococcus faecalis ( 90.85% ) , enterococcus faecium ( 8.50% ) and enterococcus gallinarum ( 0.65% ) . enterococci accounted for 8.45% , 4.53% , 4.23% , 4.43% of urinary , wound swab or pus , blood , high vaginal swab isolates respectively , causing 7.3% of all nosocomial infections . significant number of enterococci isolated from nosocomial urinary tract infection ( 66.01% ) and wound infections ( 19.6% ) were multidrug resistant ( mdr ) . although all isolates were sensitive to vancomycin and linezolid , resistance to erythromycin ( 71.24% ) and ciprofloxacin ( 49.67% ) was frequently observed . high - level gentamicin resistance was observed in 43.88% , and 61.53% of e. faecalis and e. faecium isolates respectively . minimal inhibitory concentration of vancomycin of all the isolates were 1 g / ml . 7% of the enterococcal isolates were mdr strains and vancomycin or linezolid were the only effective antibiotics.conclusion:a combination of vancomycin and/or linezolid were effective against enterococci causing nosocomial infections in our tertiary care facility , nevertheless continuous and frequent surveillance for resistance patterns are necessary for judicious and evidence based use of antibiotics .

INTRODUCTION MATERIALS AND METHODS Antibiotic susceptibility of Determination of minimum inhibitory concentrations RESULTS DISCUSSION CONCLUSION

the wideness of interest of a scientific discipline , and its impact on knowledge and the research progress may be estimated through the number of journals issued in its field , the quantity of articles published in a given timespan , and the number of citations these articles obtained . no doubt , this is a rough estimate but it may allow to quantitate and compare the data taken from qualified databases , and to foresee future developments . during the last fifteen years , the number of journals published in the biomedical field has significantly increased : for instance , based on the thomson reuters journal citation report ( https://jcr.incites.thomsonreuters.com ) the journals in the subject categories of biology increased from 51 to 85 , those in cell biology from 147 to 184 , in medicine ( experimental & research ) from 74 to 123 , and in oncology from 103 to 211 , with an impressive ( though hardly computable ) surge of published articles . research in biomedicine has taken advantage of the progress in several disciplines , first of all in molecular biology and related omics . microscopy and histochemistry also played a major role , especially in recent years , by providing new instrumentations and refined techniques . in the attempt to assess the impact of histochemistry in the biomedical field , it may be interesting to look at the articles recently published in qualified journals , reporting the results of investigations performed by histochemical methods . browsing the records of pubmed ( http://www.ncbi.nlm.nih.gov/pubmed/ ) , web of science ( http://thomsonreuters.com/en/products-services/scholarly-scientific-research/scholarly-search-and-discovery/web-of-science.html ) and scopus ( http://www.scopus.com/ ) , the articles published in qualified journals where histochemistry or immunohistochemistry are present in the title / abstract or as key - words were about 87,000 , 130,000 and 132,000 respectively , in the last five years ( the numerical differences are essentially due to the different journals which are indexed : more medicine - oriented in pubmed , and more widely biology & medicine - oriented in web of science and scopus ) . by grouping these publications in subject categories , according to their contents ( figure 1 ) , it is apparent that more than 40% deal with medical subjects ( pathology & experimental medicine ) ; articles on differentiation & stem cells or neurons & muscle ( more than 15% and 10% , respectively ) , as well as those on methods ( about 10% ) are also present at high percentages . about 5% are those on animal tissue biology and hard tissue & matrix , and only few on tissue engineering or on plant tissue biology . thus , in these five recent years , biomedical studies where histochemistry has been used as a major experimental approach included a wide variety of subjects , from basic to applied research on both humans and other animal or plant organisms . among the purely histochemical journals , the european journal of histochemistry - ejh has traditionally been devoted to publish papers on functional cell and tissue biology in animals and plants , cell differentiation and development , cell - to - cell interaction and molecular trafficking , nerve and muscle cell biology , with special attention to the cellular basis of diseases . it is worth observing ( figure 1 ) that some differences exist in the distribution of subjects of the papers published in the european journal of histochemistry during the same five - years period , compared to the overall histochemical articles in the literature . consistent with the latter ones , the majority ( about 35% ) of the published articles were on pathology & experimental medicine , and the percentages found for hard tissues & matrix , neurons & muscle and plant tissue biology were also similar as those for the whole histochemical production . on the contrary , the percentage of papers published on animal tissue biology , on methods and on tissue engineering was higher in the european journal of histochemistry , whereas the one on differentiation & stem cells was lower . looking at the distribution of articles on these subjects during the last five years ( figure 2 ) may help to understand how the interests of this journal s authors changed over time , particularly in the most recent time . the articles in pathology & experimental medicine ranged from more than 40% to about 20% , having been very numerous in 2013 , while progressively decreasing in 2014 and 2015 ; ; most of these papers were focused on the detection of tumor markers , but several were aimed at elucidating the mechanisms of carcinogenesis and tumor progression . there was steady interest for developmental investigations and stem cell biology with about 15% of the published articles in the last couple of years : molecular markers of cell differentiation were used to describe the process of placenta formation and organogenesis , and to phenotype stem cells . extracellular matrix and hard tissues have become increasingly important topics , demonstrating that histochemistry may be suitably applied in particular to investigate cartilage or dentin during the histogenetic process and in pathological conditions ; some papers were devoted to the temporomandibular joint in healthy and diseased subjects , while other proposed in vitro models for chondrogenesis or dentinogenesis . techniques for culture in vitro were also used for tissue engineering , using different supports for growing and differentiating various cell types : histochemistry thus proved to be an effective mean to characterize the structural and functional features of cells to be used for reconstructive medicine . the incidence of articles on neurons & muscle has progressively decreased from 2013 to present , although a series of interesting article was published especially for muscle cells . in particular , it was observed that both the ultrastructural organization and the nuclear function of myonuclei and satellite cells nuclei in the skeletal muscle from patients with myotonic dystrophy were similar to those in the muscles from sarcopenic individuals , suggesting that similar nuclear mechanisms may lead to skeletal muscle wasting . there was a steady and progressive rise in the percentage of papers presenting new methods or improved techniques : they were about 12% of the published articles in 2013 growing to more than 20% in 2014 and 2015 . actually , this is consistent with the scope of the european journal of histochemistry , which has always been an open forum for scientists to present new methods and techniques , and to discuss their experimental results , as it recently occurred for the molecules responsible for some characteristics of cell autofluorescence . in the attempt to localize specific molecules at high resolution , ultrastructural cytochemistry proved to be especially suitable , and demonstrated by the several papers published : in particular , diaminobenzidine photo - oxidation was effectively used to visualize the endocytotic pathways after staining the plasma membrane with a fluorescent dye , and to simultaneously detect immunogoldlabeled antigens at the high resolution of transmission electron microscopy . as a journal of functional cytology , the european journal of histochemistry has traditionally published many papers on cell and tissue biology in a variety of vertebrate and invertebrate species . after a relative decrease in 2013 and 2014 , in the present year more than 20% of the published papers were on these subjects , thus demonstrating that histochemical techniques are powerful tools for properly describing cell and tissue organization as well as functional microanatomy in different taxa of still poorly described organisms . in a comparative perspective , the histochemical evidence , in parallel with molecular data on protein and dna , may help to elucidate the origin and evolution of cell and tissue physiology . the overall influence of an academic journal on the scientific community is usually estimated by the impact factor , which is a measure reflecting the mean number of citations obtained by recent articles published in a given journal . this index does not take into account the subject category of the journals where the citing articles were published ; but especially for journals devoted to a variety of subjects and techniques , as the european journal of histochemistry is , it should be interesting to consider this aspect , too . the more than 200 articles published in the period 2011 - 2015 in this journal have been cited in 340 different periodicals . figure 3a shows the percentage of citations in these journal which have been grouped according to their main topic and their first subject category reported in the thomson reuters journal citation reports . as expected , the journals on histochemistry were those in which the articles published in the european journal of histochemistry were most frequently cited . in agreement with the evidence in figure 1 and 2 , a large fraction of the citations were in medical journals ( dealing with both general and experimental medicine , and with oncology ) , and in journals on cell and tissue biology or on biochemistry and molecular biology . in most of the articles published in the european journal of histochemistry , specific molecular complexes were detected , and their structural location in cells and tissues was related with function . in addition , several papers also described new methods or presented improvements of well - established techniques thus providing new opportunities for application to a wide variety of research subjects . it is therefore not surprising that citations are frequently found in journals of neurobiology , anatomy & morphology , pharmacology & toxicology , reproductive biology , veterinary sciences , physiology , endocrinology , tissue engineering & biomaterials , plant cell biology , as well as in multidisciplinary journals ; significantly , the citations in histochemical journals of the articles published in 2011 and 2012 progressively decreased from 2013 to 2015 , while increasing during the same timespan in the journal of medicine , pharmacology , physiology and tissue engineering ( figure 3b ) . in the last couple of decades , histochemistry has become a true molecular biology in situ , aimed at detecting single molecules in the very place where their functional role is exerted . the potential of histochemistry for diagnosis and prognosis has greatly increased by the use of refined techniques for investigating gene expression in situ . vital histochemistry ( chiefly , enzyme histochemistry ) provides evidence of the biochemical events driving and controlling cell physiology . the recent technological improvements in super - resolved light microscopy and in electron microscopy as well as the progress in correlative microscopy , and in imaging molecules in vivo allowed to shed light on the molecular processes responsible for cell differentiation or which account for the onset a pathological condition . it is easy to foresee that , in the future , the histochemical journals will ever more be an attended forum for basic and applied scientists in the biomedical field . but a journal of histochemistry should also be open to an audience as varied as possible within the biological area , publishing articles on organ , tissue and cell biology in very different experimental models . this open - minded attitude will keep histochemistry fresh and vital , and will also be beneficial for histochemical journals : offering hints for the applications of histochemical techniques also to non - histochemist scientists will expectedly increase the number of potential authors , thus expanding the research horizon to novel and possibly exclusive subjects .

especially in recent years , biomedical research has taken advantage of the progress in several disciplines , among which microscopy and histochemistry . to assess the influence of histochemistry in the biomedical field , the articles published during the period 2011 - 2015 have been selected from different databases and grouped by subject categories . as expected , biological and biomedical studies where histochemistry has been used as a major experimental approach include a wide range of basic and applied researches on both humans and other animal or plant organisms . to better understand the impact of histochemical publications onto the different biological and medical disciplines , it was useful to look at the journals where the articles published in a multidisciplinary journal of histochemistry have been cited : it was observed that , in the five - years period considered , 20% only of the citations were in histochemical periodicals , the remaining ones being in journals of cell & tissue biology , general and experimental medicine , oncology , biochemistry & molecular biology , neurobiology , anatomy & morphology , pharmacology & toxicology , reproductive biology , veterinary sciences , physiology , endocrinology , tissue engineering & biomaterials , as well as in multidisciplinary journals . it is easy to foresee that also in the future the histochemical journals will be an attended forum for basic and applied scientists in the biomedical field . it will be crucial that these journals be open to an audience as varied as possible , publishing articles on the application of refined techniques to very different experimental models : this will stimulate non - histochemist scientists to approach histochemistry whose application horizon could expand to novel and possibly exclusive subjects .

Introduction Main subjects of the histochemical articles published from 2011 to 2015 The last five-years record of a multidisciplinary journal of histochemistry Estimating the impact of the published articles through a survey of the citing journals Concluding remarks

candidemia and invasive candidiasis ( c / ic ) show an increasing incidence in the nosocomial setting . the crude mortality of those infections ranges between 36%63% depending on patient population.14 comorbidities , aging and age - associated physiological changes , higher rates of oropharyngeal colonization with candida species , and concomitant drug use make elderly patients ( > 65years old ) more vulnerable to infections . for this reason fungal infections have become a major problem in older adults , since age is a well - documented predisposing factor with increased impact on mortality.57 the cutoff age for the elderly population can not be clearly defined because aging is a continuous process . it is also clear that aging is a multifactorial process influenced by both genetic and environmental parameters . many studies dealing with candida infection in the elderly have used several different arbitrary cutoff points such as over 60 , 65 , or 70 years old . however , most prospective epidemiological studies define elderly population as the age group > 65 years.59 the elderly population is large and is growing in proportion to the general hospitalized population , but available data on epidemiology , clinical impact , and outcome of nosocomial fungal infections are limited.710 the indications for antifungal therapy are the same for older as well as younger individuals , and the initial antifungal therapy should be selected on the basis of the infecting organism and the local epidemiology.9,11 fluconazole is generally effective against c / ic but its use may be limited by the increasing prevalence of candida species ( spp . ) with acquired or intrinsic resistance . echinocandins are recommended as first - line treatment for c / ic in all patients , and more specifically in hemodynamically unstable patients or in those with prior azole exposure , or for invasive infections caused by c. krusei or c. glabrata because of their activity against azole - resistant strains , while amphotericin b remains the cornerstone of antifungal treatment.1,9,1115 in this review , we aim to discuss the management and treatment options of fungal infections in the elderly population , considering additionally the specific conditions and the impact of potential comorbidities and drug interactions . invasive candidiasis ( ic ) ( also called systemic ) , is the invasion of candida spp . in a human organ ( invasion via the bloodstream is called candidemia ) . if multiple organs such as the brain , heart , kidneys , lungs , and liver are affected , the condition is called disseminated candidiasis . are ubiquitous ( more than 200 species have been described ) ; most consist partly of the human microbiological flora , although only 10% of these species are known to be responsible for infections in humans.16 indeed , at least 17 different candida spp . cause ic in humans ; c. albicans is the most common worldwide , presenting a global average of 66% of all candida spp . , with large geographical discrepancies according to the artemis disk global antifungal surveillance study.17,18 candida spp . account for 8%10% of all nosocomial infections , next to coagulase - negative staphylococci ( 31% ) , staphylococcus aureus ( 20% ) , and enterococci ( 9% ) and have become the fourth most common cause of hospital - acquired bloodstream infections ( bsis ) in the usa.19 other studies in europe and canada , with the exception of denmark , reported a lower incidence of candidemia than that reported in the usa ( 1.9% in finland , 4.9% in iceland and spain ) , reflecting probable differences in patient demographics and comorbidities , as well as in medical practices and/or diagnostic methods.6,20 in all of these studies the highest incidence occurred at the extremities of the age spectrum ( infants of less than one year old and in adults > 65 years old).6,2124 candida albicans is the most common pathogen causing ic worldwide , though a shift towards non - albicans species has been noted over the years . c. glabrata represents an important fungal pathogen , ranking second to c. albicans as a cause of bloodstream infection.6 in the usa , c. glabrata accounts for 20%24% of all candida bsis but in other geographical regions , including europe , lower rates have been reported.6 the origins of these discrepancies are unclear but previous exposure to azoles , increasing patient age , presence of underlying diseases such as malignancies , and different geographic locations or technical methodologies regarding blood cultures older patients ( > 65 years ) have increased risk of candidemia due to c. glabrata and increased risk of dying ( 29% ) as well.25 systemic broad spectrum antibiotic use , central venous catheters , long stay in the intensive care unit ( icu ) , renal insufficiency , and total parenteral nutrition were identified as the most important risk factors . it is unclear whether the higher identified rate of oropharyngeal colonization with c. glabrata in older , compared to younger , adults , is related to candidemia.26,27 blot et al have studied the outcome of critically ill patients with candidemia and found that fungemia from c. glabrata was significantly associated with older age . older age , polymicrobial bloodstream infection and acute renal failure were independent predictors of mortality.11 a significant epidemiological shift towards c. glabrata as a cause of candidemia has been reported in oncology centers , compared to individual hospitals.6,25,2830 c. paraspilosis is an exogenous pathogen , found mostly on skin rather than mucosal surfaces , and is known for its ability to form biofilm on catheters and other implanted devices . it is spread through hand contamination in hospitals and nursing homes . about 38% of c. paraspilosis bsis are acquired outside the hospital , a finding consistent with the fact that older patients often receive home health care with indwelling catheter use due to various chronic diseases.26,3133 bsis due to this species are associated with a lower mortality rate than other candida spp.2,6 c. tropicalis is an important pathogen in neutropenic patients with hematologic malignancies and mucositis . this pathogen is very common in latin america , responsible for 22% of bsi isolates but , according to the artemis disk global antifungal surveillance study , its incidence worldwide has reached 4%7% with an increasing trend.18 c. krusei is another important pathogen among patients with hematologic malignancies , and among blood marrow transplant recipients , characterized by intrinsic resistance to fluconazole.1 c. krusei accounts for 2%5% of all candida infections worldwide , having emerged in oncology patients under prophylaxis with fluconazole.28,34 interestingly , it has been reported that exposure to piperacillin - tazobactam and vancomycin leads more often to c. krusei infections than does exposure to fluconazole , because the former drugs promote skin and gastrointestinal colonization , rendering the human host more vulnerable to c. krusei bsis.35 c. guillermondii and c. rugosa are common in latin america and responsible for clusters of hospital infection , exhibiting low susceptibility to fluconazole , while they are considered as rare causes of catheter - related candidemias in other countries.3639 c. incospicua and c. norvegensis are both phenotypically similar to c. krusei , exhibiting intrinsic resistance to fluconazole , causing candidemia in human immunodeficiency virus - infected patients and in patients with hematologic malignancies . c. norvegensis has been found mostly in respiratory specimens.4042 the aging process leads to variable changes in physiological and morphological functions , rendering older patients potentially more vulnerable to infections , particularly from fungal species ( table 1).7 aging leads to hyposalivation , which in turn alters the normal microflora of the oral cavity , so that it has fewer anaerobic bacteria , such as enterococci.43 less saliva production limits peptide and protein presence in the oral cavity , and the lack of substances with broad antimicrobial activity , such as lysozyme , contributes to oral candidiasis.43 in the supragingival plaque , which is responsible for caries formation , candida species are the predominant pathogens , especially in adults > 70 years old.44 moreover , in dental prostheses various candida species can be found , with c. albicans being the most prevalent , followed by c. glabrata and c. tropicalis.44 this colonization is further influenced by ( a ) poor oral hygiene , ( b ) drugs that irritate or damage the oral mucosa , such as cytostatics , ( c ) drugs that alter the oral flora synthesis , such as antibiotics , or ( d ) by concurrent diseases , such as iron deficiency anemia . once the oral cavity is colonized , it is easier for the yeasts to reach the respiratory system , and since candida is a commensal of the gut lumen and the cutaneous surfaces , the colonization index is increasing.43,45 candida spp . are common in the urine of the elderly , especially after treatment with broad spectrum antibiotics . differentiating asymptomatic candiduria , even in high concentrations ( > 10/ml urine ) , from a true infection which triggers a systematic inflammatory response is difficult , and treatment is influenced by the biofilm formation in the urinary catheter.43 the biofilm formation is an aggregate of microorganisms where cells adhere to each other on a surface . it may become a problem in patients with indwelling catheters , such as older people in hospitals or nursing homes . candida colonization is one of the main reasons why older people are so prone to bloodstream infections , but not the only reason . older age is always accompanied by normal physiological alterations and/or various metabolic disorders or neoplastic diseases , which disrupt the mucosal and cutaneous barrier and make the organism more vulnerable to candida infection ( table 2).6 thus , selection of a suitable drug is based not only on the specific microorganism , and on the clinical condition and its severity , but also on all the underlying pathophysiological characteristics of the patient s advanced age . aging is characterized by diminished immunological response to infection , especially due to functional insufficiency of monocytes and macrophages , which leads to inadequate phagocytosis.43 other antigen presenting cells , such as dendritic cells , are lacking , and so are naive t - cells due to thymus gland involution.46 mature t - cells lose their memory capacity and exhibit poor and/or altered cytokine production.46 moreover , the number of circulating b - cells is diminished and their response to antigenic challenges through immunoglobulin production is weaker.47 animal studies on the aging liver have shown modifications of the hepatic physiology which affect drug metabolism . possible mechanisms , occurring normally with aging , involve reduction in total liver mass , hepatic blood flow , and protein synthesis . these factors compromise drug metabolism , such as hydroxylation , dealkylation , and reduction ; reactions occurring in phase i drug metabolism , performed by microsomal cytochrome p450.48,49 phase i is necessary to prepare the drug or toxin to undergo phase ii metabolism ( conjugation , acetylation , and methylation ) , altering its form and promoting its effective excretion.49 renal function is also impaired with advanced age . this is not reflected in serum creatinine because a 25% rise in serum creatinine level actually represents a substantial fall in gfr , probably as much as 50% , due to the exponential rise in creatinine level with declining renal function.50 this decline of renal function is often underestimated , since serum creatinine is dependent on muscle mass , which also attenuates with age and remains almost normal . therefore , older and very sick patients , with a normal creatinine value , have a gfr of only 30% of that of a young , healthy adult . this is associated with serious clinical problems with drugs dependent on renal excretion.50 polypharmacy in the elderly is another important issue , relevant to both adverse effects and drugs interactions . a recent dutch study demonstrated that almost 75% of the elderly population was being treated with at least 4 drugs , suggesting that elderly patients are not only prescribed a greater number of medications than younger patients , but they also receive drugs in a more inappropriate manner.5153 analgesics , including nonsteroidal anti - inflammatory drugs ( nsaids ) ( eg , acetylsalicylic acid , ibuprofen , indomethacin , naproxen ) , narcotics ( eg , hydrocodone ) , non - narcotic pain medications ( eg , acetaminophen ) , or drugs with other mechanisms of action that act synergistically on pain relief ( benzodiazepines , tricyclic antidepressants ) , comprise the most popular drug categories.53 furthermore , polypharmacy involves , apart from pain relief , treatment of other diseases , such as hypertension , diabetes , chronic obstructive pulmonary disease , heart failure , or cancer . moreover , alterations in body composition such as decrease in total body water or increase in body fat may result in unexpected toxic effect or duration of action of various drugs . the prevalence of the effect of drug drug interactions on the liver is > 74% in older women of which 63% involve nsaid use.54 apart from multiple drug use , the mechanisms of drug - induced liver injury in the elderly include gender , dosage and treatment duration , drug formulations , nutritional status , genetic susceptibility , environmental factors ( eg , alcohol abuse ) , and underlying comorbidities.53 invasive candida infections include clinical syndromes of different severity where the diagnosis is a challenge , especially in critically ill , immunocompromised , or elderly patients . the signs and symptoms vary from silent or atypical , to that of a bacterial infection . newer culture methods have raised the sensitivity of candida detection to almost 70% , but it takes a minimum of 24 to 48 hours to become positive and this may come late in the course of the infection.55 moreover , patients are often under fluconazole prophylaxis which may render the cultures negative at time of testing.56 two antigen based tests are currently available for the early diagnosis of candidemia , relying on detecting components of the fungal cell wall . the first method detects mannan levels , which is a major component of the candida cell wall . in high risk patients , it is recommended to be performed two to three times per week , since its circulation in the bloodstream is intermittent . sensitivity and specificity of this test , when combined with anti - mannan antibodies detection in critically ill but not immunocompromised patients , are 83% and 86% respectively.5659 the second diagnostic tool is based on the detection of 1,3--d - glucan . this test has been evaluated mostly in critically ill patients and has demonstrated an overall sensitivity of 77% and specificity of 85% for subjects with proven or probable ic.5860 one single positive test is indicative of the infection , but it must be interpreted with caution due to false positive results.55 a negative 1,3--d - glucan test is associated with high negative predictive value ( > 90% ) and can be used to rule out ic , especially in patients with neutropenia.6062 finally , nucleic acid - based detection methods ( real - time polymerase chain reaction ) have been developed for five different candida spp . although these techniques have shown significant advances in the early and specific diagnosis of ic , further evaluation must be conducted in specific populations such as the elderly , considering the relatively weak immune systems and variable immunological responses among this age group , which render the diagnostic accuracy of the above methods less precise.55 the management of candida infection includes prophylactic , preemptive , empiric , and targeted treatment ( figure 1 ) . preemptive therapy is justified in the presence of positive inflammatory markers biomarkers in conjunction with certain predisposing risk factors . empiric therapy is warranted in patients with a currently unknown infection , for whom treatment is justified based on clinical judgment , while targeted treatment is administered when the diagnosis of a certain pathogen is documented . currently available drugs against ic include amphotericin b ( amb ) and its derived lipid formulations ( lfamb ) , azoles ( triazoles ) including fluconazole ( flu ) , voriconazole ( vor ) , posaconazole ( pos ) , and itraconazole , and echinocandins including . are susceptible to these agents , except for those with intrinsic or acquired resistance after exposure to other drugs . amb is a polyene macrolide antifungal regimen with fungicidal action , which was considered in the past to be the gold standard for the treatment of invasive fungal infections.62 derived from streptomyces spp . , it has a high affinity for the sterols of fungal and bacterial membranes , forming small transmembrane channels which lead to monovalent ion leakage and cause fungal cell death . derivatives of amb were developed in order to limit toxicity , especially renal failure , which has been rated between 49%65%.50,6365 three lipid formulations of amb ( lfamb ) are commercially available , all with a good fungicidal activity and no differences in efficacy . the formulations include a true liposome structure lfamb ( ambisome , gilead , cambridge , uk ) ; a ribbon - like structure amb lipid complex ( abelcet , sigma - tau pharmaceuticals , inc , gaithersburg , md , usa ) ; and a colloid dispersion , amphotericin b colloidal dispersion ( abcd ) ( amphocil / amphotec , evaluatepharma , london , uk ) with a disc - like structure . all lipid formulations have less nephrotoxicity than conventional amb - deoxycholate.6466 among these , the liposome structure of amb seems to have lower nephrotoxicity rates ( 15% ) than the ribbon - like structure ( > 40%).67 lfamb is used in a dose of 3 to 5 mg / kg daily in life - threatening mycoses , as well as for empirical treatment of suspected ic , or in situations where an antifungal agent with rapid time - kill rate and high post - antifungal effect is needed.11,68 however , in older patients it should be used with caution , especially if parameters such as dehydration , large cumulative dosage , abnormal baseline renal function , and concomitant nephrotoxic drug use exist . the azoles flu , itraconazole , vor , and pos inhibit the fungal cytochrome p450 enzyme 14-demethylase and prevent the conversion of lanosterol to ergosterol , which is essential for the fungal cell membrane integrity . all the above azoles demonstrate activity against candida spp . but reduce activity against c. glabrata and c. krusei . flu , with primarily fungistatic effect ( 800 mg or 12 mg / kg loading dose , followed by 400 mg or 6 mg / kg daily ) , has comparable efficacy to amb for the treatment of candidemia , while it is indicated as empirical and curative treatment in non - neutropenic patients.6970 flu is recommended for the treatment of c. parapsilosis bsi , while among all the triazoles , it possesses the greatest penetration to the cerebrospinal fluid and it is therefore indicated for the treatment of central nervous system and intraocular infections . prophylactic use of flu led to a shift to resistant candida species , and its use alters ( and may even increase ) the frequency of infection due to molds . prophylaxis ( 6 mg / kg daily ) is recommended in solid organ transplant recipients ( liver , pancreas , and small bowel ) , during induction chemotherapy and in stem cell transplant recipients during the period of neutropenia.11 flu is an inhibitor of the human cytochrome p450 system and therefore it decreases the metabolism or increases the concentration of any drug metabolized by these enzymes . this should be kept in mind , considering the high number of concomitant drugs that older people take , which also undergo hepatic metabolism , in order to avoid serious and life - threatening drug drug interactions . serum levels of warfarin , phenyntoin , or oral hypoglycemic agents are increased by the azoles , whereas serum digoxin levels may increase . another rare adverse event is the potential effect on electrocardiographic qt interval , whose elongation increases the risk of ventricular arrhythmias , especially if drugs are used concurrently , which also prolong its duration ( eg , macrolides , fluoroquinolones , anticholinergic , antihistamines , diuretics , and the gastroprokinetic agent cisapride ) . itraconazole has a broader spectrum of activity than flu ( in vitro activity against candida spp . , aspergillus spp . , and dimorphic fungi , but not as broad as vor or pos ) , but is not able to penetrate cerebrospinal fluid . , while it is active against c. krusei , c. guillermondii , and c. lusitaniae.71 intravenous vor is complexed to a cyclodextrin molecule and after two loading doses of 6 mg / kg every 12 hours , a lower maintenance dosage of 34 mg / kg twice daily is recommended . due to cyclodextrin accumulation , vor is not indicated in patients with renal dysfunction and creatinine clearance of < 50 ml / min.72 vor is effective in candidemia , but it offers little advantage over flu and is therefore recommended as a step - down oral therapy for c. krusei infection and for flu - resistant , vor - sensitive c. glabrata infection . oral vor does not require dosage adjustment in renal insufficiency , but is the only triazole that requires dosage reduction in patients with mild - to - moderate hepatic insufficiency . in a randomized , international , multicenter trial comparing vor with lfamb as empirical antifungal treatment , the authors suggested that vor could be a suitable alternative to lfamb in patients with neutropenia and persistent fever.73 pos is available only as an oral suspension with high oral availability and seems to be more active than the other triazoles . pos exhibits a broad spectrum activity against yeasts , molds , or rare fungal strains . echinocandins ( ecs ) are a new class of antifungal agents that target the fungal cell wall by inhibiting 1,3--d - glucan synthetase , leading to osmotic instability and cell death . the three members of the group , cfg ( loading dose of 70 mg , then 50 mg daily ) , mic ( 100 mg daily ) , and anidulafungin ( loading dose of 200 mg , then 100 mg daily ) , are all available only for parenteral use . each of these agents has been studied for the treatment of ic in comparative and noncomparative clinical trials.7477 the mics of the echinocandins are low for a broad spectrum of candida spp . , including c. krusei and c. glabrata . c. parapsilosis demonstrates less in vitro susceptibility ( higher mics ) than most other candida spp . , and that has raised the concern of its being less responsive.71,78,79 similarly , there have been reports of increased clinical failure and persistence of infection with this species , claiming that c. parapsilosis infection may indeed require higher echinocandin dosage.1,80 therefore , in the recent clinical practice guidelines for the management of candidiasis , flu is the treatment recommendation for c. parapsilosis infection , unless the patient has already received an echinocandin , is clinically improved , and has negative follow - up cultures.11 until now , this matter was still under debate , since a recent meta - analysis showed that ecs are effective for the treatment of candidemia or invasive candidiasis due to c. parapsilosis.81 another important issue , unique for the ecs , is the eagle effect , a term used to describe the paradoxical in vitro and in vivo growth of candida and aspergillus isolates when the dose of the drug gets over the mic level.82 this phenomenon has similarities to the eagle effect observed in other cell wall active antimicrobial agents , such as penicillins . although the clinical impact of this phenomenon has not been elucidated , it might be of some importance in biofilm tretment.62 none of the ecs require dosage adjustment for renal insufficiency or dialysis . both cfg and mic undergo minimal hepatic metabolism , but neither is a major substrate for cytochrome p450 and therefore they have minimal drug - drug interactions . anidulafungin has not hepatic metabolism ; it undergoes slow chemical degradation to a ring opened peptide with no antifungal activity . though concerns have been raised about the potential hepatotoxicity of mic due to tumor formation in rodents , cfg is the only ec for which dosage adjustment is recommended for patients with moderate to severe hepatic dysfunction . the clinical practice guidelines favor the use of an ec as initial therapy for candidemia in non - neutropenic as well as neutropenic adult patients , with moderate to severe illness.11 alternatively , flu and lfamb may be used , but for infection due to c. glabrata an ec is preferred , since the triazoles have diminished activity against this species . moreover , very little is known about the pharmacological / pharmacokinetic properties of antifungal drugs in the elderly . the diminished drug clearance that occurs naturally with aging , along with the presence of other comorbidities and drug use , make the pharmacodynamic and pharmacokinetic issues very intriguing . drug interactions and comorbidities are the main reasons why we would not recommend azoles or amphotericin b as our first therapeutic choice in this specific population . dose modification is not warranted unless indicated for other reasons ( eg , hepatic or renal dysfunction).83 monitoring of plasma levels could be an option , but it is time and resource consuming , even in health care facilities where the method is available . monitoring drug levels in plasma can not be indicated for routine use unless future studies provide us with more data . flucytocine ( or 5-fluorocytosine , 5fc ) is an antimetabolite that acts as an antifungal against candida spp . 5fc enters the fungal cell via cytosine permease , and is metabolized to 5-fluorouracil , which is incorporated extremely closely into the fungal rna , inhibiting both dna and rna synthesis . most of the drug is excreted unchanged in the urine , so that dose adjustment is necessary for patients with renal dysfunction . considering the fact that 5fc is rarely administered as a single agent but in combination with other antifungal drugs ( mainly lfamb ) for patients with ic , it is not suggested as a combination in elderly patients due to the accumulative nephrotoxicity risk . nosocomial candidemia is associated with increased mortality and this seems to be further aggravated in case of delay in antifungal drug initiation.8486 morrell et al have shown that initiating empiric antifungal treatment more than 12 hours after the first blood culture sample is associated with a greater risk of hospital mortality than when patients are started on antifungal therapy within the first 12 hours.84 in a 5-year study , parkins et al studied 207 patients with ic ; 64 patients ( 32% ) received empirical therapy , in 51 ( 26% ) of which was deemed appropriate.86 similarly , kumar et al demonstrated a 12% decreased survival probability for every hour s delay in patients with fungal septic shock.87 therefore , prompt initiation of early empiric therapy is warranted in high risk patients . knowledge of risk factors for ic may help to identify those patients who could benefit from early antifungal therapy ( table 3 ) . the candida score was first introduced by len et al and the epcan study group , from data available from the surveillance study of fungal infection and colonization in critically ill patients.88 clinical sepsis ( 2 points ) , multifocal colonization ( 1 point ) , surgery ( 1 point ) , and total parenteral nutrition ( 1 point ) are the risk factors that must be evaluated by the physician in order to identify patients who are candidates for empirical treatment . a score of > 2.5 showed 81% sensitivity and 74% specificity for the early administration of empirical treatment in icu patients.88,89 in summary , the increase in invasive candidiasis in older adults has become an important clinical problem , since the older population is growing and is nowadays more likely to take aggressive chemotherapeutic regimens for cancer , or immunosuppressive drugs for nonmalignant diseases . moreover , aging leads to variable physiological changes , rendering older patients potentially more vulnerable to fungal infections . elderly patients are more easily colonized by pathogenic fungi and have an increased incidence of c. glabrata fungemia , which has higher mortality rates as well as higher rates of resistance to fluconazole , especially after exposure to the drug . therefore , although clinical manifestations in older and younger adults may be similar , for the treatment of the former the use of an echinocandin is safer , since treatment with amphotericin b is associated with increased nephrotoxicity risk . azoles are less toxic but they must be used with caution , since older adults are usually under a number of medications and the risk of serious drug

fungi are major causes of infections among immunocompromised or hospitalized patients with serious underlying diseases and comorbidities . candida species remain the most important cause of opportunistic infections worldwide , affecting predominantly patients over 65 years old , while they are considered to be the fourth most common cause of nosocomial bloodstream infections . the rapidly growing elderly population has specific physiological characteristics , which makes it susceptible to colonization and subsequent infection due to candida species . comorbidities and multidrug use should be taken into account any time the therapeutic regimen is under consideration . different classes of antifungal drugs are available for the treatment of invasive fungal infections but echinocandins , apart from their activity against resistant strains ( candida glabrata and candida krusei ) , seem to be safe , with limited adverse events and minimal drug drug interactions in comparison to the other regimens . therefore , these agents are strongly recommended when dealing with elderly patients suffering from an invasive form of candida infection .

Introduction Epidemiology Physiological alterations in the elderly Diagnosis Therapeutic targets and drug selection Conclusion

percutaneous transluminal coronary angioplasty was introduced by gruntzig in the late 1970s as an alternative to coronary artery bypass graft surgery for coronary revascularization.1 since then , percutaneous coronary intervention ( pci ) has been accepted as a safe , reliable , and effective treatment for coronary artery disease , and its use has spread worldwide . nevertheless , in - stent restenosis ( isr ) , a complex phenomenon resulting in renewed symptoms , need for re - intervention , and poor patient outcome remained for many years the achilles heel of pci.2 the introduction , a decade ago , of first - generation drug - eluting stents ( dess ) transformed the practice of pci by drastically reducing the rate of this complication.3 the efficacy of dess has largely been demonstrated in large randomized trials , leading to their current widespread use in clinical practice . even in high - risk populations , isr incidence does not currently go above 5%10%.46 however , major concerns regarding the long - term safety of these first - generation dess have progressively arisen , especially the increased risk of late / very late stent thrombosis ( st)714 and the need for prolonged dual antiplatelet therapy ( dapt ) with an inherent increase of bleeding complications . aside from patient- and lesion - related factors , delayed re - endothelialization and recovery of endothelial function after stenting as well as inhibition of vascular repair after des implantation , all of which promote inflammation and thrombotic pathways , have been implicated in the pathophysiology of late / very late st . of note , stent polymer has also been cited as one of the main causes of these late events . in fact , the persistence of a durable polymer after complete release of the anti - proliferative drug has been shown to be responsible for local hypersensitivity and inflammatory reactions.15 these safety concerns prompted additional research , new trial design , and development of new - generation dess to reduce the rate of this rare but critical event . apart from the progress in stent platforms ( thinner struts and stent designs ) , recent research in this field has subsequently been focused on the development of new more biocompatible durable polymers or completely biodegradable polymers . third - generation dess using biodegradable polymers , like the biolimus - eluting stent ( bes ) ( nobori ; terumo corporation , tokyo , japan ) , have been developed to overcome the long - term adverse vascular reactions related to the durable polymer . in this article we then discuss the results of recent publications investigating the safety and effectiveness of the use of the third - generation bes ( nobori ) for the treatment of coronary artery lesions . the two major complications of pci , isr and st , have always been the trigger for new stent development.16 indeed , despite the fact that these events are multifactorial , stent structure has been suggested to be one of the leading causes of isr and/or st.1721 isr physiopathology has not yet been fully understood . barotrauma induced by pci is responsible for endothelial denudation and sub - intimal hemorrhages , leading to a local inflammatory response . this inflammatory process induced by vascular damage is thought to be one of the main contributors to the development of restenosis , by promoting vascular smooth muscular cell proliferation and extracellular matrix formation , resulting in neointimal hyperplasia . beside these mechanical factors , other factors have been identified as predictors for isr , including patient - related ( eg , diabetes mellitus , smoking , and renal failure ) and lesion - related ( eg , minimal lumen diameter after pci , severe calcifications , chronic total occlusions , tortuous vessel , and long lesion length ) factors.22 as mentioned above , isr remained the achilles heel of pci until the large use of dess , which were specifically developed to overcome this complication . before the introduction of bare - metal stents ( bmss ) , up to 50% of the patients treated by pci experienced restenosis . even in the bms era , isr remained one of the major limitations of this technique , with an average incidence of 20% , but that could increase up to 35% in complex lesions and diabetic patients.4,5 the introduction , 10 years ago , of first- and then second - generation dess transformed the practice of pci by drastically reducing the incidence of this complication to less than 10%.3 dess prevent restenosis by inhibiting vascular smooth muscle proliferation.2331 unfortunately , they also delay re - endothelialization of stent struts , leading to the potential risk of late / very late st and thereby the need for prolonged dapt . since the appearance of dess , st has become the major safety concern in contemporary pci practice . st is a rare adverse event ( 1% at 1 year and then 0.5% per year ) but remains associated with high morbidity and mortality rates.32 the overall prognosis is poor : most patients in whom st occurs present with stemi ( st - segment elevation myocardial infarction ) or out - of - hospital death , and up to 30% of those who arrive alive at hospital die within the first month . numerous factors have been implicated in st physiopathology , but studies have also shown that these predictors vary over time . these data highlight the complex physiopathology of st , depending on the timing of event occurrence . acute ( within 24 hours ) and early st ( within 30 days ) are likely related to mechanical issues concerning the stent ( eg , minimum stent area and suboptimal stent expansion ) , inadequate platelet inhibition , or patient prothrombotic factors.33 late ( up to 1 year ) and very late st ( after 1 year ) have been attributed to incomplete vascular healing and/or inadequate neointimal coverage , which in turn , promote inflammation and activation of thrombotic pathways15 and late or acquired stent malapposition . dapt associating aspirin with an oral p2y12 inhibitor has been shown to be the standard therapy following coronary stenting in order to significantly reduce cardiac events , especially st after pci.34 current guidelines support the use of dapt for 612 months after des implantation.3539 in 2006 , the potential risk of late / very late st after des implantation raised the question of prolonging dapt even beyond the first year.4041 however , prolonged dapt has also clearly been associated with an increased risk of bleeding.4247 availability of new biodegradable polymers and/or stents may shorten the duration of necessary dapt and therefore minimize the risk of major bleeding to which it is associated . first- and second - generation dess have three major components : the stent platform , the antiproliferative drug , and the polymer . all of these factors have been subject to modification and have become a target for research and development . it provides the radial force to prevent vessel occlusion provoked by vessel injury following pci . cobalt - chromium and later platinum - chromium platforms used in second - generation dess permitted similar stents radial strength all the while enabling a thinner strut design and subsequently significantly improved deliverability and a reduced rate of isr.4849 unfortunately , the presence of a permanent scaffold in the vessel constitutes a stimulus for platelet aggregation and may lead to st in patients with nonoptimal antiplatelet therapy and/or incomplete stent endothelialization . recently , bioabsorbable platforms that biodegrade over a period of months have been developed , with the purpose of allowing the restoration of a normal vascular physiology and function over time . ultimately , no foreign material is left exposed in the bloodstream . these stents may also potentially preserve reactive vasomotion and permit expansive remodeling.50 there are several antiproliferative drugs with different modes of action . the goal of these drugs is to inhibit vascular smooth cell proliferation and migration , without affecting endothelial regeneration , and have anti - inflammatory / anti - thrombotic properties . inhibitors of the mammalian target of rapamycin ( mtor ) are the dominant class of anti - proliferative drugs used for dess . the mtor inhibitors are cytostatic drugs resulting in arrest of the cell cycle at the g1 phase . aside from mtor inhibitors , tacrolimus , which acts as a calcineurin inhibitor , has also been used in dess and is a cytostatic agent with both antiproliferative and anti - inflammatory activities . finally , paclitaxel is a taxan drug , which acts as a cytotoxic drug through the stabilization of microtubules . however , all these antiproliferative agents have shown detrimental local effects on the vascular wall and on endothelial function recovery after stenting.11 new drugs ( biolimus , novolimus , and myolimus ) have been developed and have shown promising results.5152 compared with other mtor inhibitors , biolimus shows better lipophilicity . agents other than drugs are under investigation to limit restenosis , such as antibody - coated stents ( cd34 antibody - coated stents ) and nucleotide- or peptide - coated stents . the ultimate goal remains the inhibition of maladaptive neointimal proliferation , all the while promoting vascular healing . once drug elution has been completed , most polymers exert limited functions and act as a potential trigger for local inflammation and hypersensitivity and subsequently late / very late st . they promote an inflammatory response and eosinophilic infiltration in the arterial wall , causing hypersensitivity reactions and endothelial dysfunction responsible for delayed healing and lead to st.11,12,22,53 the biocompatibility , composition , formulation , degradation delay of the polymer , pharmacokinetics of the antiproliferative agent released by the polymer , and the management of variation in polymer degradation delay have become new difficult challenges for the development of stent polymers . an optimal polymer should mimic the endothelial lining in order to prevent late thrombotic complications , thus improving stent safety . given the issue of polymer - induced inflammation and thrombosis , more biocompatible durable polymers have been developed . second - generation dess are composed of these polymers , thereby improving arterial healing and potentially reducing the rate of late ischemic events . the limited function of stent polymers once the drug is eluted has also fuelled research in biodegradable polymers . third - generation dess use polylactic acids ( poly - l - lactic acid and poly d , l - lactide - co - glycolide ) as bioabsorbable polymers . the bes with biodegradable polymer ( nobori ) is one of these third - generation dess . finally , another field of research is the development of stents that elute antiproliferative drugs without the need for polymers . these polymer - free stents could prevent the potential adverse physical effects of the polymer , leading to sustained intima inhibition , improved healing , and a lessened activation of the inflammatory / thrombotic pathways . safety , efficacy , and deliverability are the main sought - out properties for the development of new dess . an optimal combination of these components , which are in part interdependent , is necessary for enhancing stent performance . the bes stent with biodegradable polymer ( nobori ) is one of the third - generation dess ( figure 1 ) . the platform is composed of stainless steel , and the strut thickness is 112 m . it is coated with a polylactic acid polymer on its abluminal surface , which is metabolized within 69 months to lactic acid , water , and carbon dioxide through interaction with the krebs cycle . the stent elutes an antiproliferative drug , biolimus ( 15.6 g / mm ) , for up to 30 days . the coating design of the stent combined with the lipophilicity of the drug is thought to optimize local drug distribution and to reduce its release into the general circulation . at the end , the nobori stent will leave only a bms in place . the nobori stent has already been compared with first- and second - generation dess with promising results . the nobori core trial54 reported late - loss with the nobori stent at 9 months , similar to that found using the sirolimus - eluting stent ( ses , cypher ; cordis corporation , bridgewater , nj , usa ) : 0.10 and 0.12 mm , respectively ( p=0.66 ) . however , the use of the nobori stent results in better endothelial recovery , with normal coronary vasodilatation in the adjacent stent segments after implantation , contrasting with the paradoxical vasoconstriction seen with first - generation dess.55,56 the nobori i trial showed non - inferiority to and subsequent superiority of the nobori stent over the paclitaxel - eluting stent ( taxus , boston scientific , maple grove , mn , usa ) : late - loss 0.11 versus 0.32 mm , p<0.001 . moreover , this trial demonstrated a lower rate of st with the nobori stent after a 9-month follow - up.57 three recent large randomized trials have compared safety and efficacy of this third - generation des with that of first- and second - generation dess with durable polymer ( table 1).58 the sort out v trial59 enrolled 2,468 patients who underwent pci in denmark and randomized them 1:1 using the nobori and cypher stents . this trial was a multicenter , prospective , non - inferiority trial ( non - inferiority margin chosen at 2% ) comparing the bes using biodegradable polymer with the ses using permanent polymer . a total of 1,229 patients were assigned to the bes group ( 1,532 lesions ) and 1,239 to the ses group ( 1,555 lesions ) . the primary endpoint was a composite of safety ( cardiac death , myocardial infarction [ mi ] , and st ) and efficacy ( target lesion revascularization [ tlr ] ) within 9 months of stent implantation ( intention - to - treat analysis ) . there were 75% male , and only 15% of the patients had a history of diabetes . altogether , 17% had undergone a previous coronary intervention . the main target vessel location was the left anterior descending ( lad ) artery ( 40.8% ) . procedure characteristics were similar between the two groups , except for maximum stent pressure , which was significantly higher in the ses group ( 18 versus 16 atm , p<0.0001 ) . composite primary endpoint occurred in 4.1% of cases in the bes group and 3.1% of cases in the ses group ( one - sided pnon - inferiority=0.06 , and p=0.22 for superiority ) . cardiac death and tlr were not significantly different between the bes and ses groups ( 0.7% versus 1% , p=0.38 ; 3.3% versus 2.1% , p=0.07 ) . of note , st occurred more frequently in the bes group ( especially within the first month ) , with no clear explanation , and the rate of st was particularly low in the ses group : 0.7% versus 0.2% , p=0.03 . the authors concluded that the bes did not show non - inferiority when compared with the first - generation ses at 9 months . the compare ii trial60 was a multicenter , open - label , randomized , controlled , non - inferiority trial ( non - inferiority margin chosen at 4% ) and aimed to compare the safety and efficacy of the bes using biodegradable polymer with a second - generation everolimus - eluting stent ( ees ) using durable biocompatible polymer ( xience v [ abbott vascular , santa clara , ca , usa ] or prime [ boston scientific corporation , natick , ma , usa ] ) . a total of 1,795 patients ( 2,638 lesions ) received a bes , and 912 patients ( 1,387 lesions ) an ees . the primary endpoint was a composite of safety ( cardiac death and non - fatal mi ) and efficacy ( tlr ) at 12 months . there were 74% male , 21.7% diabetics , and 20% had a previous history of mi . procedure characteristics were not significantly different between the groups but the rate of non - allocated stent implantation was higher in the bes group . primary endpoint occurred in 5.2% of cases in the bes group and 4.8% in the ees group ( one - sided pnon - inferiority<0.0001 , and p=0.69 for superiority ) . there was no difference in the rates of cardiac death ( 0.8% in each group , p=0.97 ) and mi ( 2.8% in the bes group versus 2.5% in the ees group ; p=0.63 ) . the rate of definite st ( 0.7% versus 0.4% , p=0.38 ) and definite / probable st ( 0.8% versus 1% , p=0.58 ) were very low and comparable between the two groups . target vessel revascularization did not differ between the bes group ( 2.9% ) and the ees group ( 2.6% , p=0.69 ) . the authors concluded that the bes was shown to be non - inferior to the ees in terms of safety and efficacy at 1-year follow - up after pci . the 5-year follow - up of the study should answer the question of long - term safety and efficacy of the bes compared with second - generation dess . the next trial61 was a prospective , multicenter , randomized , open - label , non - inferiority trial ( non - inferiority margin chosen at 3.4% ) comparing the bes ( nobori ) with the ees ( xience v or prime ) in terms of tlr at 1 year . may to october , 2011 , in 98 japanese centers , 3,235 patients were randomized 1:1 without any exclusion criteria to undergo pci with either the bes or the ees . altogether , 30% of included patients were over 75 years old , and 46% of the population in each group were diabetic . efficacy endpoint was any tlr at 1 year , whereas the primary safety endpoint was a composite of death and mi . primary efficacy endpoint occurred in 4.2% in both the bes group and the ees group ( one - sided pnon - inferiority<0.0001 , and p=0.93 for superiority ) . the rate of any mi and stroke was similar between the two groups ( 3.3% versus 3.1 [ p=0.77 ] and 1.4% versus 1.5% [ p=0.89 ] ) . no difference was observed in terms of mortality , 2.6% and 2.5% in the bes and ees groups , respectively , p=0.9 . with regard to the rate of st , the incidence was extremely low , and there was no difference between the bes group and the ees group ( 0.25% and 0.06% , p=0.18 ) . the angiographic sub - study shows that the difference in in - segment late loss between the two groups was 0.03 mm , demonstrating non - inferiority of the bes , with a margin of 0.195 mm . of note , the rate of stent fracture was significantly higher in the bes group ( 3.1% versus 0% ; p=0.004 ) . the authors concluded in the non - inferiority of the bes over the ees in the setting of stable coronary artery disease . clinical outcomes were excellent , with a low rate of tlr and extremely low rate of st in each group . third - generation dess using biodegradable polymer such as the nobori stent have been developed to overcome long - term adverse effects observed with first - generation dess related to the use of durable polymers , and to thereby shorten the duration of dapt and the ensuing risk of hemorrhagic complications . biodegradable bess should nonetheless possess similar efficacy to actual dess in preventing isr ( lower rate of tlr as compared with bmss ) . first studies evaluating bes efficacy and safety have shown promising results , with significantly lower in - stent late loss than with paclitaxel - eluting stents , and similar in - stent late loss as sess.54,57 however , the recent compare - ii , sort out v , and next trials , all three of which were sufficiently powered to compare clinical outcomes , showed contrasting results . to date , only the compare - ii and the next trials have evaluated a new third - generation des versus a second - generation des ( xience v or prime ) . the compare - ii trial demonstrated non - inferiority of the bes relative to second - generation eess , but procedural failure was significantly higher in the bes group.60 by contrast , non - inferiority of the bes relative to the first - generation ses was not shown in the sort out v trial due to a higher risk of early st without a clear explanation for this phenomenon.59 in fact , the rate of st was particularly low in the ses group in this study . the next trial is the largest trial , to date , evaluating the efficacy and safety of the bes as compared with the ees.61 in this trial , clinical and angiographic outcomes of the bes group were non - inferior to the ees group , and the device implantation success rate was comparable in both groups . of note , a higher rate of stent fracture was seen in the bes group in the angiographic sub - study of the next trial , but its imputability in very late st or tlr is uncertain and needs further exploration . of major importance , all these trials have enrolled low risk patients , and notably a low incidence of diabetics , with relatively simple angiographic lesions ( eg , large vessels , low rate of bifurcations , and lesion length < 20 mm ) . these facts are critical regarding the interpretation of the results and the low rate of adverse events observed in these three trials : the primary efficacy endpoint was lower than 5% in all studies , and any conclusion must be interpreted with caution , especially in non - inferiority trials . in addition , a safety comparison of modern dess is challenging . indeed , st , especially very late st , is a rare complication , and although 12-month results of standard trials usually enable safety or efficacy to be assessed , longer follow - up ( 5 years or more ) and large trials are mandatory in the present case in regard of late - event exploration . the rate of st in the present studies is particularity low ( around 0.5% at 1 year ) , and these trials are not powered enough to evaluate such a low frequency event . finally , if present , the expected benefit of third - generation dess like the nobori stent over older dess would logically appear only after 1 year . to date , no very late st with the nobori stent has been reported in the literature , with the limitation that long - term follow - up is currently only available for very few patients . the leaders trial was the first randomized study to evaluate bess against durable polymer first - generation dess . this trial included higher risk patients , and the rate of events was on average twice as high as compared with the compare ii , sort out v , and next trials . the leaders study compared another third - generation bes ( biomatrix flex , biosensors , newport beach , ca , usa ) with sess . the only difference between the biomatrix flex and the nobori stent is the presence of an ultra - thin non - degradable parylene coating between the stent and the polymer on the nobori stent to assure polymer attachment to the stent struts , which do not exist on the biomatrix flex. the final 5-year report has just been published62 and demonstrates that the bes is non - inferior to the ses with regard to the primary endpoint ( cardiovascular death , mi , and clinically driven target vessel revascularization ) ( 22.3% in the bes group versus 26.1% in the ses group ; p=0.071 ) . in addition , even though no significant difference was observed between the bes and the ses in total definite st at 5 years , the bes was associated with a significantly lower rate of very late st and secondary composite endpoint ( all cause death , any mi , any revascularization ) ( 0.6% in the bes group versus 2.2% in the ses group for very late st [ p=0.003 ] , and 35.1% versus 40.4% for secondary composite endpoint [ p=0.02]).62 a recently pooled analysis based on patient individual data from the isar - test 3 , the isar - test 4 , and the leaders trial63 has demonstrated that biodegradable polymer dess ( including : biodegradable polymer bes [ biomatrix flex ] , n=857 ; and biodegradable polymer ses , n=1,501 ) improve safety and efficacy over first - generation sess during long - term follow - up . the three trials separately showed no differences between bess and dess in the past , highlighting the lack of statistical power of these studies ( taken alone ) to detect relevant differences in very low frequency events such as st . in this meta - analysis , the benefit of bess was seen in the rate of st ( 0.2% versus 1.3% , p=0.004 ) , mi ( 1.5% versus 3% , p=0.03 ) , and cardiac death ( 3.9% versus 4.9% , p=0.05 ) . of note , bess also showed a significant reduction in tlr in this meta - analysis ( 12% versus 13.7% , p=0.029 ) . these trials were performed with sirolimus first - generation dess as control , and results can not be extended to other second - generation dess . further studies using second - generation dess as control are required to make any conclusions . interestingly , such a meta - analysis of trials that have evaluated the nobori stent may carry interesting conclusions in the next future . bmss and dess have changed the landscape of current pci by significantly reducing the rate of isr . however , first - generation dess have been associated with a higher rate of late or very late st . the physiopathology of this rare but serious event is multifactorial , but inflammation and delayed arterial healing , of which the durable polymer may be a putative mechanism , has an important role . the concept of biodegradable polymer and totally bioresorbable scaffolds looks , in consequence , very interesting . long - term follow - up is mandatory to see if the implantation of these third - generation dess will improve clinical outcomes with a lower rate of st and mi . further studies and results of their long - term follow - up would definitely shed light on patient outcomes with the use of such devices .

first - generation drug - eluting stents have raised concerns regarding the risk of late and very late stent thrombosis compared with bare metal stents and require prolonged dual antiplatelet therapy . despite extensive investigations , the physiopathology of these late events remains incompletely understood . aside from patient- and lesion - related risk factors , stent polymer has been cited as one of the potential causes . in fact , the persistence of durable polymer after complete drug release has been shown to be responsible for local hypersensitivity and inflammatory reactions . third - generation drug - eluting stents with more biocompatible or biodegradable polymers have subsequently been developed to address this problem . in this article , we evaluate and discuss the concept and clinical results ( safety and efficacy ) of a third - generation drug - eluting stent with biodegradable polymer : the nobori stent .

Introduction Rationale for new stent development Stent structure and concept for new development The BES with biodegradable polymer (Nobori) stent: design and clinical results Discussion Conclusion

the modern concept of decompression for traumatic brain injury ( tbi ) was introduced by harvey cushing before world war i . the monro - kellie hypothesis dictates that the amount of space within the skull is constant ; therefore , when the pressure is raised death occurs by herniation when the capacity for adjustment by fluid shifts from the cerebrospinal fluid and vascular compartments are already maximized . increasing the skull size by removing bone and opening the dura delays or prevents these limits from being reached . however , in the 90 years since cushing made these observations , medical , radiographic , and surgical advances in the management of tbi have obviated the need for an aggressive surgical approach in all but a minority of cases . in spite of the ability to control intracranial pressure ( icp ) elevation in most cases with removal of mass lesions , osmotic diuretics , ventricular drainage , sedative / hypnotic agents , and prevention of hypercapnea , occasional cases occur in which icp elevation accelerates in spite of maximal conservative medical therapy , and then so - called heroic measures are employed . these are considered at the ' option ' level in the american association of neurological surgeons criteria for management of severe brain injury because no large randomized trial has proven their efficacy . in the pediatric population , ruf and colleagues as well as taylor and associates have addressed the issue of using decompressive craniectomy as a more formal part of a head injury protocol before going to other ' option ' therapies once icp elevation is affirmed . bifrontal decompressive craniectomy is an aggressive approach described by kjellberg and prieto before the era of modern neuroimaging with computed tomography . this approach is particularly useful in the pediatric population , in which diffuse injury without mass lesions and with icp elevation is relatively common . hemicraniectomy represents a large cranial and dural decompression , often associated with removal of mass lesions such as subdural hematoma or traumatic intracerebral hematoma . bitemporal decompressions are unilateral or bilateral bony decompressions designed to take the pressure off the temporal lobes to prevent uncal herniation , and have been used in other cranial conditions such as pseudotumor cerebri . gower and coworkers reported 40% mortality and 50% favorable outcome in a study of subtemporal decompression for icp control in 10 patients with closed head injury who had failed medical therapy , including barbiturate coma . cerebellar decompression for mass lesion is a standard neurosurgical response to any process in the posterior fossa ( hemorrhage , tumor , infection , or stroke ) that threatens cerebellar tonsillar herniation . the paper by ruf and colleagues purports to be a pilot study employing decompressive craniectomy in a standardized approach following development of medically refractory icp in the pediatric population . of the six cases presented , three involve bifrontal craniectomies : two unilateral and one cerebellar . the patients underwent surgery between hospital days 1 and 6 and had icp as low as 20 and as high as 70 mmhg . glasgow outcome scale findings are not provided ; however , at least four of the patients appeared to have no more than a mild disability . they randomly assigned 27 patients to craniectomy or medical management alone and found that the craniectomy patients obtained lower icp and better outcomes . in the control group only 14% of children had a favorable outcome , as indicated by 6-month glasgow outcome scale scores . that study suggested ( as statistical significance was not met because of the small numbers ) that aggressive early decompressive craniectomy may benefit this patient group . polin and colleagues explored the use of decompressive bifrontal craniectomy in victims of tbi . this group used a cohort control matching protocol employing the traumatic coma databank to match subjects based on age , icp , radiographic findings , and admission glasgow coma scale score . in a conditional logistic regression analysis comparing all 92 control patients with the craniectomy population , those investigators detected a significant influence of the operation on favorable outcome ( wald = 6.097 ; p = 0.014 ) . medical management alone carried a 3.86-fold greater risk for unfavorable outcome than did decompressive craniectomy . a pediatric subgroup was identified that appeared to benefit from the procedure ( p = 0.025 ) . the authors further identified a subgroup of patients who received surgery within 48 hours and who never had sustained icp elevation over 40 mmhg . the pediatric patients in this subset had favorable outcomes in 8 out of 10 cases , with statistically improved outcomes compared with control individuals . we think it unlikely that decompressive craniectomy will become a commonplace management scheme for tbi . standard medical management allows control of icp while preserving the ability to conduct a neurologic examination . the role of decompression as compared to that of other ' option ' therapies , such as hypothermia and barbiturate coma , is still evolving . the advantage of decompressive craniectomy over these other therapies is the rapid and generally permanent decline in icp , maintenance of neurologic status , and even the ability to obtain a neurologic examination after the procedure is performed . the disadvantages are the need for at least two surgeries ( one to replace the bone flap ) and the theoretical development of bifrontal contusions after decompression . decompressive hemicraniectomy and duraplasty for evacuation of mass lesions and management of unilateral hemispheric swelling is more widely accepted in head injury management . most surgeons have first hand experience with delayed cerebral swelling after removal of a subdural hematoma , presumably caused by venous infarction and/or cerebral contusion . some will remove the bone flap prophylactically after such operations and others the paper by ruf and colleagues provides more ammunition for the argument that decompressive craniectomy is a safe and effective method of icp reduction in severe tbi with associated icp elevation refractory to standard management . however , this approach remains a second tier strategy recommended only at the option level . for this scheme to become more prevalent , a large - scale multicenter trial such as that being planned by coplin and colleagues would be necessary .

more frequently than adults , pediatric victims of severe traumatic brain injury experience diffuse severe cerebral edema without mass lesions . these patients require methods to reduce intracranial pressure quickly and reliably . surgical decompression provides rapid relief of increased intracranial pressure and is an alternative to maximal medical therapy for these individuals . based on previous trials , most of which are anecdotal but now include attempts at case controlled and cohort matched investigations , ruf and colleagues describe a series of six pediatric patients treated with a prospectively implemented protocol of decompressive craniectomy for severe traumatic brain injury . the heterogeneous approaches presented ( which include hemicraniectomy , bifrontal craniectomy , and suboccipital craniectomy ) undermine the applicability of the results . however , this report , coupled with similar papers , does highlight the need for a true controlled trial of this modality to examine whether craniectomy can emerge as more than a second line option for the management of increased intracranial pressure .

Introduction Trials of decompressive craniectomy in pediatric patients Discussion Competing interests Abbreviations

a 32-year male patient presented to the ed with severe dyspnea and agitation at 2:00 a.m. the patient was placed in orthopnea position and oxygen was administered . blood pressure was 165/50 mmhg , heart rate was 113 bpm , body temperature was 36.5 c , 25 breaths of respiratory rate per minute and oxygen saturation with pulse oximetry was 78% with nasal cannula . physical examination revealed rales as far as upper zones of both of lungs and at base of heart , and a diastolic grade 2/6 murmur of aortic regurgitation was heard . medical history included hypertension treated with angiotensin receptor blocker ( arb ) and smoking habit . electrocardiography ( ecg ) showed sinus tachycardia with t - wave inversion in leads di and vl . the working diagnosis was pulmonary edema , and use of bronchodilator inhaler was initiated , as well as intravenous nitrates and intravenous diuretic therapy . while taking arterial blood gas sample , patient s condition deteriorated and abdominal respiration pattern became obvious . elective tracheal intubation of patient was performed , and patient was admitted to coronary intensive care unit ( cicu ) with pulmonary edema and possible acute valvular insufficiency . transthoracic echocardiogram ( tte ) showed mild to moderate aortic regurgitation with 3.3 cm of sinus of valsalva . systolic function of ventricle was normal and left ventricular ( lv ) cavity widened to 5.1 cm at end - diastolic volume . arterial blood gas before intubation showed hypoxia with 76% arterial oxygen saturation and hypocarbia with metabolic acidosis of 7.13 ph . contrast - enhanced computed tomography ( cect ) of thorax was used to refine the diagnosis . as a team , the cardiologist , the cardiovascular surgeon , and the radiologist checked the computed tomography ( ct ) images . sinus of valsalva was 3.5 cm and no flap was found in ascending or descending aortas . as lab test results showed coronary ischemia with positive troponin i levels ( 1.016 ng / dl ) , it was decided that a diagnostic coronary angiography ( cag ) would be performed . in the catheterization laboratory , intubation of right coronary artery ( rca ) and left main coronary artery ( lmca ) was difficult , and the test required 1 hour to complete . pulmonary embolism and coronary artery disease as cause of acute dyspnea were ruled out , but the cause of dissection of aorta was still unclear because of possibility of a false negative ct scan . transesophageal echocardiography ( tee ) was selected as the next diagnostic tool due to high pretest probability of aortic dissection . tee showed a stanford type a dissection flap closing lmca ostia from beat to beat and compromising the aortic valve with moderate aortic regurgitation . determining the appropriate diagnosis for acute heart failure was very challenging , but at 6.00 a.m. , surgery to correct a stanford type a dissection localized in the valve and coronary ostia was performed . upon further careful review of ct scan images , a very tiny flap was observed at the ostium of lmca ( figure 1 ) . transverse cut view of contrast enhenced enhanced computed tomography shows a tiny flap at the ostium of lmca that was initially misdiagnosed . acute aortic dissection is a life - threatening medical emergency that can quickly lead to death . incidence is estimated to be 3 in a 1000 cases according to international registry of aortic dissection ( irad ) . if left untreated , 33% of individuals will die within 24 hours of presentation , and 50% die in the initial 48 hours . although diagnostic tests have improved , the condition remains undiagnosed in about half of patients because of variable symptoms and negative laboratory tests . after first admission tests of physical examination , vital signs , and electrocardiogram ( ecg ) ; the most frequently performed tests to diagnose aortic dissection are ct , tee and magnetic resonance imaging ( mri ) . a recent meta - analysis by shiga et al . reviewed published studies of diagnosis of aortic dissection by tee , helical ct and mri showed that these tests have equal and reliable diagnostic value . tee had 99% sensitivity and 95% specificity , helical ct had 100% sensitivity and 98% specificity , and mri had 98% sensitivity and 98% specificity [ 4 , 5 ] . in the present case , first admission physical examination and laboratory tests were supportive of aortic dissection . after a misdiagnosis was made by the radiologist , the cardiologist , and the surgeon based on ct of chest , cag was performed to clarify coronary ischemia , which is contraindicated in aortic dissection . correct diagnosis of aortic dissection was made with tee , and the patient was taken to surgery . further examination of ct scan images then revealed a tiny flap at the ostium of lmca ( figure 1 ) . aortic dissection may occur in a small part of aorta and not be seen in ct scan or laboratory test results . if suspicion of aortic dissection is high , diagnosis can be made with tte and tee in intensive care units ( icus ) . dyspnea is the major symptom of acute heart failure and an etiologic assessment must be made for every patient who presents with acute dyspnea . in the present case , aortic dissection compromised aortic valve and coronary ostium , increasing left ventricle end - diastolic pressure , which led to pulmonary edema and acute dyspnea . in episodes of acute dyspnea though a diagnostic tool like ct scan may have 100% sensitivity and 98% specificity , it may also contribute to a misdiagnosed aortic dissection . if high pretest probability is present and ct scan does not support the diagnosis , tee and mri may be used next to determine the correct diagnosis .

acute dyspnea is a major complaint of patients admitted to cardiology and emergency departments ( ed ) . acute dyspnea can be life - threatening , and is seen in cases of asthma , pulmonary embolism , acute heart failure and myocardial infarction . the present case is that of a 32-year - old man admitted to the ed with orthopnea position and agitation . physical examination , electrocardiogram ( ecg ) , transthoracic echocardiogram ( tte ) , contrast - enhanced computed tomography ( cect ) of thorax and coronary angiography ( cag ) helped to rule out chest disease pathologies such as pneuomo - thorax , pulmonary embolism and coronary artery disease , but were not enough to make an appropriate diagnosis in this case . because of high pretest probability of aortic dissection , transesophageal echocardiography ( tee ) was performed and a diagnosis of stanford type a dissection closing left main coronary artery ( lmca ) ostia from beat to beat was made .

CASE REPORT DISCUSSION

debilitating conditions , such as disc herniation or lumbar spinal stenosis , are one of the most common causes of lumbago and lumbar radiculopathy . it may be easily recognized by their clinical symptoms and signs . magnetic resonance imaging ( mri ) nevertheless , there are uncommon causes of lumbago or radiculopathy , which include intraspinal extradural masses , such as synovial cyst , ganglion cyst , pseudocyst , hematoma and metastatic tumor , as well as arachnoid cysts . on the basis of clinical examination and history taking , these uncommon causes are generally difficult to distinguish from those that are more common . of these uncommon causes , intraspinal extradural cysts that communicate with the intervertebral discs are quite rare . this is a case report of a lumbar discal cyst in a patient with low back pain and pain that radiates to the legs . a 40-year - old man disclosed a 6-months history of aggravated lumbago and pain that radiated to the left buttock and the leg . , he showed paravertebral tenderness in the lower lumbar spine with lumbar motion limitation secondary to pain . a neurologic examination was remarkable for a grade 3/5 dorsiflexion weakness in his left ankle . the straight leg raising test on the left was positive at an angle of 45 degrees ( 45 ) . the vas score of lumbago was 4 ( pain was scored on a vas from 0=no pain to 10=strongest pain imaginable ) and that of radiating pain was 8 . an mri scan revealed a disc extrusion at l3/4 and a large spherical lesion behind the l4 vertebral body . a mass lesion revealed a low signal intensity on t1-weighted image and high signal intensity on t2-weighted images , which were located in the left ventromedial aspect of the extradural space at the l4 body level , compressing the thecal sac and left l4 nerve root dorsally ( fig . 1 ) . there was a central disc bulging with a ligamentum flavum thickening at the l4/5 intervertebral disc levels , while a narrowing was detected at the left l4/5 intervertebral foramen . based on further enhanced imaging studies , the cystic mass was well enhanced and seemed to be connected like a stalk between the l3/4 intervertebral disc spaces . this patient unsuccessfully underwent a conservative treatment with rest , nsaids ( non - steroridal anti - inflammatory drugs ) and an epidural steroid injection at a local clinic . owing to worsening of sensory and motor abnormalities in the lower extremities , a left partial laminectomy was performed at l4 ( due to the location of a mass lesion behind the l4 body ) . a darkish red cyst was seen underneath the left l4 nerve root , compressing the thecal sac and displacing the left l4 nerve root dorsally . a meticulous dissection was necessary for the severe adhesions between the cystic mass and nerve root as well as dura mater ( fig . 2 ) . grossly , the cystic mass was round and 1.21.1 cm in size . serous hemorrhagic fluid was aspirated with a 22-gauge syringe and sent for both pathologic study and culture ( fig . histologically , the cyst walls were constituted with dense fibrous connective tissues without cell linings or disc materials . the patient 's radicular pain resolved promptly after surgery and only slight numbness overlying his left anterior thigh was reported during the follow - up observation period of two ( 2 ) years . many articles have been reported about intraspinal extradural masses such as synovial cysts , ganglion cyst , perineural cyst , chordoma and metastatic tumor . among them less than one hundred ( 100 ) case reports and small case series for discal cysts have been published to date1,10 ) . the fact that discal cysts may be present in the absence of additional spinal pathology and most commonly affect young males may be distinctive characteristics that are different from other degenerative conditions . several pathogeneses may be suggested , such as degenerative changes due to previous herniated disc material , hematoma associated with a disc herniation , and mucoid degeneration . kono et al . mentioned that the pathogenesis of discal cysts is similar to that of meniscal cysts of the knee and synovial cysts of the facet joint9 ) . based on this hypothesis , focal degeneration of the intervertebral disc may lead to leakage of fluid into the spinal canal producing an inflammatory reaction . proposed that discal cysts would arise from hemorrhage of an epidural venous plexus due to mechanical forces transmitted by a herniated annular fissure2 ) . suggested that discal cysts were similar to ganglion cysts of the posterior longitudinal ligament or annulus fibrosus12 ) . the clinical symptoms and signs of discal cysts resemble those found in patients with a lumbar disc herniation . plain radiographs have a low diagnostic value except for identifying other conditions , such as degenerative spondylolisthesis and spondylosis . mr imaging demonstrates the nature of the cystic lesion and its relationship to the corresponding disc . like a normal intervertebral disc , a discal cyst reveals a low signal on t1-weighted imaging and a high signal on t2-wighted imaging . in addition , the surrounding rim and contents of a cyst may show enhancement in a contrast enhanced mri . these mr imaging characteristics are the keys to differentiating discal cysts from lumbar disc herniation11 ) . further diagnostic evaluation for a suspected discal cyst may include discography and ct discography13 ) . ct - discography was not incorporated owing to the possibility of aggravating the patient 's motor weakness , which could be provoked by contrast media . moreover , it was possible to confirm the connection between the corresponding disc and cyst intraoperatively . the natural history of the discal cyst is still unknown and therapeutic guidelines have not been established . spontaneous regression of a discal cyst has been reported4,14 ) , as well as regression after intradiscal and epidural steroid injections8 ) . however , the management of a discal cyst is similar to that of a lumbar herniation6 ) . ct - guided percutaneous aspiration can be considered as another initial treatment option for a discal cyst , because it has several potential advantages , including faster recovery , low complication rates and avoidance of general anesthesia3,7,8 ) . surgical resection of a discal cyst is reserved for patients with persistent symptoms . in this case study , this patient 's symptoms totally resolved after surgical excision and he remained nearly asymptomatic postoperatively for two years and thereafter . . they should remain in the differential diagnosis of any intraspinal extradural mass lesion compressing the thecal sac and nerve root . mr imaging helps to confirm the diagnosis , while surgical excision of a cyst should be considered for persistently symptomatic patients .

discal cysts are a rare cause of lumbar radiculopathy . there are only a few reports of this disease in medical literature . the authors describe the case of a 40-year - old man with a lumbar discal cyst that led to radiculopathy . an intraspinal extradural cystic mass was responsible for low and high signal intensities observed in lumbar lesions on t1 and t2 weighted magnetic resonance images . this cyst was a grossly spherical mass with clear serous fluid , which was connected to an adjacent intervertebral disc . histopathology of the cystic walls revealed fibrous connective tissues without specific cell linings . clinical symptoms were promptly relieved after surgical resection . further research on the pathophysiology and treatment of discal cysts are needed .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

salmonella was the most common cause of foodborne outbreaks ( fbos ) in the european union ( eu ) in 2013 , with 1168 fbos of human salmonellosis reported by 22 member states , accounting for 22.5% of all notified outbreaks of foodborne illness in the eu according to the european food safety authority ( efsa , 2015 ) . the genus salmonella , which is closely related to the genus escherichia , groups gram - negative , non - spore - forming , is rod - shaped bacteria belonging to the enterobacteriaceae family . two species are distinguished : s. enterica , classified into six subspecies ( grimont et al . , 2007 ) , and s. bongori . salmonella enterica subspecies enterica is an intracellular pathogen of warm - blooded mammals comprising over 2500 serovars ( baker and dougan , 2007 ) . some salmonella serovars , such as s. enterica serovar enteritidis and s. enterica serovar typhimurium , are non - specific host pathogens that can colonise a broad range of animals , rarely causing clinical manifestations . in humans , however , they can lead to gastroenteritis or occasionally septicemia ( galanis et al . , the serovars most frequently associated with human illness in the eu are s. enteritidis ( 39.5% ) , s. typhimurium ( 20.2% ) , and monophasic s. typhimurium ( 8.6% ) ( efsa , 2015 ) . salmonella is the most common causative agent , reason for hospitalisation , and cause of death tracked by the foodborne diseases active surveillance network ( foodnet ) ( cdc , 2011 ) . beyond their health effects salmonella alone is responsible for approximately 1 million foodborne infections ( scallan et al . , 2011 ) and it costs 365 million $ in direct medical expenditures annually ( us department of agriculture , 2011 ) . common salmonellosis symptoms include diarrhea , fever , and abdominal cramps beginning 12 to 72 hours after the consumption of contaminated food or beverages ( shariat et al . , 2013 ) . more often , human s. typhimurium cases are associated with the consumption of contaminated pork and beef , whereas s. enteritidis cases are linked to eggs and poultry meat . subclinical infection in animals leads to herd or flock contamination , with intermittent or persistent shedding of bacteria ( efsa , 2013 ) . in laboratory testing for salmonella enterica , isolates are identified and typed by several phenotypic methods , including biochemical profiling , serotyping , and phage typing . standard serotyping methods , based on the detection of somatic ( o ) and flagellar ( h ) antigens , are tedious and time - consuming . although they lack the capacity to fingerprint strains in a sensitive manner , they remain useful in surveillance programmes ( herikstad et al . , 2002 ) . more recently , different genotyping methods have been developed for the genetic discrimination of salmonella isolates in outbreaks , such as multilocus sequence typing , multilocus variable - number tandem - repeat analysis , next generation sequencing . pulsed - field gel electrophoresis ( pfge ) was adopted for salmonella surveillance and outbreak research in the 1990s . because of its remarkable discriminatory power and high reproducibility , it has been successfully used in typing salmonella strains isolated from human patients , foods and feed ( zou et al . , 2013 ) . although pfge is also labour - intensive , public health surveillance laboratories use it to determine strain relatedness and to confirm fbos . indeed , the us cdc recommends pfge as the gold standard method for molecular characterisation in outbreak investigations . in this paper it was reported the investigation of a household / domestic kitchen fbo of salmonellosis occurred in biella on the 15 of january 2010 linked to homemade lasagne . microbiological , serological , and molecular assays on salmonella isolates from human and food samples were performed to determine the relatedness of the implicated salmonella strains , identify the sources of infection , and trace the routes of salmonella contamination in this circumscribed fbo . the study was conducted in collaboration between the food control laboratory at the institute for experimental veterinary medicine of piedmont , liguria and aosta valley , turin , the biella department of public health prevention , and the hospital of biella ospedale degli infermi . four adults ( 1 man and 3 women ; age range , 45 - 61 years ) sought medical attention at the emergency room of the degli infermi hospital , biella because of gastrointestinal symptoms on the 16 of january 2010 . from initial accounts , all four persons were hospitalised , and the local public health and prevention department was notified . a standardised questionnaire was administered by the public health officials in order to obtain demographic and clinical data , history of recent travel , food and water consumption , exposure to animals , and other illness during the week before becoming ill . in the interviews , the patients stated that they had consumed a homemade baked lasagne dish consisting of boiled pasta in which cheese and tomato sauce are mixed and then cooked in the oven . then , a mixture of raw eggs was added to homemade lasagne and left in switched off oven until the evening . before eating in addition , the patients had also eaten salad , turkey meat , and cake . people who consumed common meal on the 15 of january 2010 in biella , who had gastro enteric symptoms from 15 to 16 of january , had not gastro enteric symptoms before the 15 of january 2010 . the food sample was analysed to identify salmonella spp . ; the stool samples were tested for salmonella , shighella and campylobacter . salmonella from the food sample was isolated according to iso 6579:2002/cor 1:2004 ( iso , 2004 ) . this method entails several steps : pre - enrichment in non - selective liquid medium ( buffered peptone water ; biokar diagnostic , beauvais cedex , france ) ; enrichment in two selective liquid media [ rappaport - vassiliadis medium with soya broth ( microbiol diagnostici , cagliari , italy ) and muller - kauffmann tetrathionate novobiocin mkttn broth ( liofilchem srl , roseto , te , italy ) ] ; and plating on xylose lysine deoxycholate agar ( xld ) ( microbiol diagnostici ) and brilliant green agar ( bga ) ( microbiol diagnostici ) . an internal method for detecting salmonella , shighella and campylobacter specifically , for the isolation of salmonella and shighella have been used hektoen enteric agar ( oxoid , rodano , mi , italy ) , mac conkey agar ( oxoid ) and salmonella shighella agar ( oxoid ) ; for the isolation of campylobacter the stool samples were analyzed using campylobacter species ag rapid test ( li starfish s.r.l . , cernusco , mi , italy ) and campylobacter selective agar ( biomrieux , marcy letoile , france ) . all isolated strains were identified by conventional biochemical methods ( api 20e ; biomriux ) and the salmonella spp . were serotyped according to the kauffman - white - le minor classification scheme ( kauffmann , 1966 ; le minor and popoff , 1987 , grimont and weill , 2007 ) . the isolates were processed according to the pfge protocol described by the us centers for disease control and prevention ( cdc , 2013 ) using xbai ( carlo erba reagents srl , cornaredo , mi , italy ) and blni ( roche diagnostics corporation , indianapolis , in , usa ) restriction enzymes . pfge using a single restriction enzyme ( xbai ) is a standard method for genotyping s. enteritidis ( dewaele et al . , 2012 ) . thus , the ability to deduce the serotype of a salmonella isolate based on its pfge profile provides an alternative method for screening and identifying salmonella serotypes ( zou et al . , 2010 ) . the pfge fingerprint patterns were analysed with bionumerics software ( version 7.1 ; applied maths , sint - martins - latem , belgium ) and the pfge patterns were normalised by interpolation to the nearest reference lane ( salmonella braenderup strain atcc h9812 ) . for comparing the pfge profiles , values of optimization of 1.0% and position tolerance of 1.0% were applied to both enzymes . dice similarity coefficients were calculated on the basis of pairwise comparisons of the pfge profiles . four adults ( 1 man and 3 women ; age range , 45 - 61 years ) sought medical attention at the emergency room of the degli infermi hospital , biella because of gastrointestinal symptoms on the 16 of january 2010 . from initial accounts , all four persons were hospitalised , and the local public health and prevention department was notified . a standardised questionnaire was administered by the public health officials in order to obtain demographic and clinical data , history of recent travel , food and water consumption , exposure to animals , and other illness during the week before becoming ill . in the interviews , the patients stated that they had consumed a homemade baked lasagne dish consisting of boiled pasta in which cheese and tomato sauce are mixed and then cooked in the oven . then , a mixture of raw eggs was added to homemade lasagne and left in switched off oven until the evening . before eating in addition , the patients had also eaten salad , turkey meat , and cake . people who consumed common meal on the 15 of january 2010 in biella , who had gastro enteric symptoms from 15 to 16 of january , had not gastro enteric symptoms before the 15 of january 2010 . stool samples were collected from all patients . only residues of the baked lasagne could be collected for sampling and subsequent analysis . the food sample was analysed to identify salmonella spp . ; the stool samples were tested for salmonella , shighella and campylobacter . salmonella from the food sample was isolated according to iso 6579:2002/cor 1:2004 ( iso , 2004 ) . this method entails several steps : pre - enrichment in non - selective liquid medium ( buffered peptone water ; biokar diagnostic , beauvais cedex , france ) ; enrichment in two selective liquid media [ rappaport - vassiliadis medium with soya broth ( microbiol diagnostici , cagliari , italy ) and muller - kauffmann tetrathionate novobiocin mkttn broth ( liofilchem srl , roseto , te , italy ) ] ; and plating on xylose lysine deoxycholate agar ( xld ) ( microbiol diagnostici ) and brilliant green agar ( bga ) ( microbiol diagnostici ) . an internal method for detecting salmonella , shighella and campylobacter was carried out on the stool samples . specifically , for the isolation of salmonella and shighella have been used hektoen enteric agar ( oxoid , rodano , mi , italy ) , mac conkey agar ( oxoid ) and salmonella shighella agar ( oxoid ) ; for the isolation of campylobacter the stool samples were analyzed using campylobacter species ag rapid test ( li starfish s.r.l . , cernusco , mi , italy ) and campylobacter selective agar ( biomrieux , marcy letoile , france ) . all isolated strains were identified by conventional biochemical methods ( api 20e ; biomriux ) and the salmonella spp . were serotyped according to the kauffman - white - le minor classification scheme ( kauffmann , 1966 ; le minor and popoff , 1987 , grimont and weill , 2007 ) . the isolates were processed according to the pfge protocol described by the us centers for disease control and prevention ( cdc , 2013 ) using xbai ( carlo erba reagents srl , cornaredo , mi , italy ) and blni ( roche diagnostics corporation , indianapolis , in , usa ) restriction enzymes . pfge using a single restriction enzyme ( xbai ) is a standard method for genotyping s. enteritidis ( dewaele et al . , 2012 ) . thus , the ability to deduce the serotype of a salmonella isolate based on its pfge profile provides an alternative method for screening and identifying salmonella serotypes ( zou et al . , 2010 ) . the pfge fingerprint patterns were analysed with bionumerics software ( version 7.1 ; applied maths , sint - martins - latem , belgium ) and the pfge patterns were normalised by interpolation to the nearest reference lane ( salmonella braenderup strain atcc h9812 ) . for comparing the pfge profiles , values of optimization of 1.0% and position tolerance of 1.0% dice similarity coefficients were calculated on the basis of pairwise comparisons of the pfge profiles . epidemiological investigation revealed the main symptoms of nausea ( 50% ) , abdominal cramps ( 75% ) , diarrhea ( 100% ) , vomiting ( 100% ) , fever ( 100% ) , and low back pain ( 25% ) . the symptoms began 5 hours after eating the meal in the first case and 9 hours later in the last case . during the interview no history of recent travel , food and water consumption , exposure to animals , and other illness during the week before becoming ill was reported . a participant cooked the baked lasagne at the dinner and all the people ate the lasagne . was isolated in three patients ( stool samples ) and in the baked lasagne ( residues stored in the refrigerator ) ; one patient was tested negative for all pathogens . four s. enteritidis isolates were genotyped by pfge using xbai and blni restriction enzymes , which yielded consistent reproducible fingerprints . single enzyme restriction analyses by xbai distributed the four isolates into one cluster ( figure 1 ) . s. enteritidis is the major serovar associated with human salmonellosis linked to the consumption of contaminated poultry products , including eggs ( braden , 2006 ; much et al . , 2009 ) . in 2013 207 strong evidence outbreaks were attributed to the consumption of eggs and egg products and 59.9% of fbos were caused by s. enteritidis ( efsa , 2015 ) . a 2002 - 2003 us case - control study reported that a significant risk factor of salmonella infections ( or 2.7 , 95% ci 1.1 - 3.9 ) is consuming undercooked eggs or egg - containing dishes inside the home ( middleton et al . , 2014 ) . the household / domestic kitchen remains the most commonly reported setting in european fbos ( 38.5% ) followed by the public / commercial kitchens ( restaurant , caf , pub , bar , hotel categories ) in 2013 ( 22.2% ) ( efsa , 2015 ) . the domestic fbo outbreak described here was linked to the homemade lasagne contaminated with salmonella spp . we identified a common source of infection by using pfge , one of the most important molecular characterization methods of diagnostic testing to compare bacteria strains . how and at which point during food preparation the lasagne became infected with salmonella can not be definitively determined , but the data suggest opportunities for person to food , food to food , and equipment to food cross - contamination . generating a hypothesis about the likely sources , the most probable is that the eggs used in preparing the lasagne were probably contaminated with salmonella enteritidis and their adding after cooking process is responsible of the contamination of rte product . indeed the reheating process before consumption normally applied at low temperature is not enough to kill the bacteria and might even to promote the bacterial growth . a plausible alternative hypothesis is that the lasagne was contaminated with salmonella enteritidis after cooking due to cross - contamination . indeed , one of the most common causes of fbos is cross - contamination during food handling due to the use of improperly washed utensils and handling by ill or healthy carriers . serotyping according to the kauffman - white - le minor classification scheme and pfge analysis allowed identifying the source of food poisoning in this domestic fbo . however , it was arduous to single out which ingredient of the meal was the source of salmonella contamination . collaborative efforts by the hospital physicians , public health department officials , and laboratories using different laboratory analysis methods during the outbreak investigation were essential for identifying the causative agent . good sanitation , safe food handling and preparation practices in the entire food chain are key to preventing foodborne illness . consumers should be made aware of the risks of fbo and learn how to minimise their chances of becoming ill by considering food safety at each step , from food purchase , to cooking , cleaning , and storing leftovers appropriately ( international food information council foundation , 2014 ) .

in the latest year , and also in 2013 , salmonella was the most frequently detected causative agent in foodborne outbreaks ( fbos ) reported in europe . as indicated in efsa report ( 2015 ) the serotypes mostly associated to fbos are s. typhimurium and enteritidis ; while salmonella typhimurium is generally associated with the consumption of contaminated pork and beef , fbos due to salmonella enteritidis are linked to eggs and poultry meat . in this study it is described the investigation of a domestic fbo involving four adults and linked to homemade lasagne . investigations were performed to determine the relatedness of salmonella strains , identify the sources of infection , and trace the routes of salmonella contamination in this fbo . salmonella strains were isolated in 3 out of 4 patient stool samples and from lasagne and all of them were serotyped as s. enteritidis . pulsed - field gel electrophoresis ( pfge ) analysis revealed the genotypical similarity of all the strains . although serotyping and pfge analysis identified the common food source of infection in this fbo , it was not possible to determine how or at what point during food preparation the lasagne became contaminated with salmonella .

Introduction Materials and Methods Epidemiological investigation Microbiological investigation Results Discussion Conclusions

peripheral nerve injuries are commonly caused by trauma to the upper limbs and are present in an estimated 23% of all patients admitted to a level 1 trauma center ( noble et al . , 1998 ) . the economic impact of nerve injuries can be large with operative costs , hospital charges , rehabilitation visits , and lost time at work . only subtle improvements to peripheral nerve repair have been made recently , and our current knowledge of nerve physiology and regeneration vastly exceeds our current repair capabilities . poor outcomes of peripheral nerve injury are largely due to the slow process of axonal outgrowth . after nerve injury , severed proximal axons with intact cell bodies can grow up to 1mm / day . regenerating axons preferentially target appropriate end organ receptors but do not always take a direct route to their target ( fox et al . , 2012 ) . thus , functional recovery is not always obtained due to slow growth and poor alignment of motor and sensory fibers . muscle atrophy initiates immediately after muscle denervation , and if motor axons do not reach their target muscle within a critical time window , muscle tissue is less receptive to re - innervation . the most common current strategies to augment recovery after nerve injury such as decellularized nerve allografts , tissue matrices and nerve growth guides involve techniques to enhance axonal regeneration and decrease surrounding inflammation . despite these advancements , the combination of slow axonal outgrowth , wallerian degeneration , and muscle atrophy are still barriers to significant progress . invertebrate axonal fusion after nerve injury is a natural mechanism that promotes rapid and highly specific recovery of neurons . given the mechanism of axonal fusion is possible , peg has been investigated as an agent to promote axonal fusion in vertebrates . bittner et al . pioneered initial studies using peg to fuse crayfish and giant earthworms axons with improvement in axonal fusion when axonal endings were exposed to a calcium free saline solution ( bittner et al . the addition of an anti - oxidant such as methylene blue ( mb ) was noted to have an additional positive impact in rat sciatic nerve repair ( spaeth et al . , the proposed mechanism is peg - induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur . in an axonal injury without peg , axonal endings seal after an influx of calcium , preventing axonal fusion ( yoo et al . , 2003 ) . figure 1 demonstrates the bioengineered process of peg fusion . when severed axonal endings are exposed to calcium - free hypotonic saline and an antioxidant ( i.e. , methylene blue or melatonin ) , vesicle - mediated sealing peg is then applied to artificially induce closely apposed membranes of severed axonal ends to flow into each other and fuse . this produces a partial repair of the plasmalemmal membranes that are then perfused with calcium containing saline , which causes vesicles to accumulate and seal remaining holes at the injury site . mechanism of polyethylene glycol ( peg ) axonal fusion . reprinted from rodriguez - feo et al . the success of the peg - fusion technique is based on multiple steps and factors . peg , a hydrophilic compound , enhances axonal fusion in either severed or crushed settings and restores the ability to generate compound action potentials across the site of injury . peg potentially facilitates lipid bilayer fusion by removing water molecules from the lipid bilayer at or near the damage site ( figure 1 ) . sealing of axolemmal damage occurs through a calcium - dependent accumulation of membranous structures that interact with nearby undamaged membrane to form a plug . in severed nerves , this calcium - dependent system plugs the cut ends of axons preventing them from fusing with an adjacent axonal stump . our studies have taken advantage of this calcium - dependent process by incubating in a calcium - free solution prior to nerve fusion , which prevents axolemmal sealing thereby enhancing peg based fusion ( sexton et al . oxidative damage has also been shown to play a role in ischemia reperfusion injury after peripheral nerve injury . methylene blue has been previously shown to slow axolemmal sealing and thereby enhance behavioral recovery after nerve injury ( spaeth et al . , methylene blue has shown a protective effect if applied in the correct sequence ( i.e. , after nerve severance and before peg based fusion ) . taken together , these studies have shown that peg , with carefully controlled modification of the periaxonal environment can fuse simple nerve transections . we have used the fusogenic properties of peg , the advantages of a calcium - free irrigation solution , and the antioxidant properties of methylene blue to demonstrate a rapid and decisive recovery of completely severed sciatic nerves in a commonly accepted mammalian model of peripheral nerve injury . most current research efforts have focused on investigating peg fusion in a rat sciatic nerve model . the rat sciatic nerve is easily accessible through a 23 cm incision posterior to the femur . after a small cutdown to the nerve , the perineural tissue can be dissected easily to free the nerve for transection and repair . our group has demonstrated the efficacy of peg fusion after neurotmesis with direct repair , nerve autografts , and nerve allografts . compound action potentials ( cap ) are measurements of nerve conductivity and can be readily measured across healthy nerves . after nerve transection , caps can no longer be obtained due to axonal discontinuity . caps are not restored after a direct suture repairdue to lack of axonal fusion secondary to axolemmal sealing after injury . however , after peg axonal fusion , caps are restored . bittner et al . published cap data after direct suture repair with and without peg ( bittner et al . , 2012 ) . baseline caps were obtained in all rats prior to nerve injury ( mean 4.1 0.16 mv ) . postoperatively , caps were not measurable immediately after repair in the control group ( direct suture repair ) . however , caps were restored within minutes after repair ( mean 3.3 0.23 mv ) in the peg fusion group . , baseline caps were obtained in all rats prior to nerve injury ( mean 4.131 2.25 mv , n = 20 ) . postoperatively , caps were not measurable immediately after repair or after 72 hours in the control group ( direct suture repair ) . however , caps were restored immediately after repair ( mean 3.533 1.61 mv , n = 10 ) and after 72 hours ( mean 2.051 0.857 mv , n = 10 ) in the peg fusion group . our experience with cap restoration in the peg fusion group was similar when using peg fused allografts ( riley et al . , 2015 ) . our rat behavioral testing includes both the sciatic function index ( sfi ) and foot fault asymmetry score ( ff ) . behavioral recovery was superior in the peg fused group in direct repair at 12 weeks ( bittner et al . , 2012 ) as well as the peg fused group in nerve allografts at 6 weeks ( riley et al . our nerve autograft study evaluated rats up to 3 days , and the peg fused group already had superior behavioral outcomes ( sexton et al . , 2012 ) . figure 2 demonstrates long - term behavioral outcomes after a standard microsurgical repair compared to a peg fusion repair after nerve transection . when considering the electrophysiology and behavioral data together , it suggests at least a partial inhibition of wallerian degeneration after peg fusion . behavioral assessments of sciatic nerve function according to the sciatic functional index ( a ) and foot - fault asymmetry test ( b ) . sciatic function index ( sfi ) and foot fault asymmetry score ( ff ) scores ( mean se ) were assessed from 3 days to 12 weeks post - operatively for two groups : control ( black dotted line ) and polyethylene glycol ( peg ) ( light - gray dotted line ) . individual animals for all 12 weeks animals were graphed according to the aforementioned group color scheme ( data not yet published , manuscript in preparation ) . after axonal injury , motor and sensory nerve counts are decreased distal to the injury site . immunohistochemical staining using commercial antibodies against carbonic anhydrase ii ( ca2 ) and choline acetyletransferase is a method to stain for sensory and motor neurons respectively . our research has shown increased sensory and motor axon counts in peg - treated nerve repairs ( figure 3 ) . toluidine blue staining of peg fused nerves shows higher proportions of healthy myelinated axons compared to negative control nerves . additionally , our lab has demonstrated the expression of npy1 , a marker of wallerian degeneration , in negative control nerves , whereas this is not expressed in peg - fused nerves . these findings imply that we have delayed or even inhibited wallerian degeneration by fusing axons and maintained both axonal viability and function . control ( a c ) and polyethylene glycol ( peg ) ( d f ) stained for choline acetyltransferase to label motor axons at 1 week ( a , d ) , 4 weeks ( b , e ) , and 12 weeks ( c , f ) post - operatively ( data not yet published , manuscript in preparation ) . diffusion tensor imaging ( dti ) is a magnetic resonance imaging ( mri ) technique , which is an emerging diagnostic tool in nerve injury . the institute of imaging science at our institution has developed a novel imaging protocol using dti for peripheral nerves . utilizing this protocol , we have been able to demonstrate the restoration of axonal continuity after peg fusion repair ( figure 4 ) . representative tractography images show that axonal tracts do not extend more than a couple millimeters distal to the repair site in control nerves ( cut + suture , n = 6 ) . in peg fused nerves ( n = 6 ) , the peg group had a statistically significant increase in the number of tracts that traveled through the repair site ( data not published , manuscript in progress ) . our mri data supports the interpretation that peg - fused nerves establish axonal fusion which has been previously evaluated through behavioral testing and axon counts . representative tractography of transected nerves harvested immediately following repair : control ( left ) and polyethylene glycol ( peg ) ( right ) . green arrows indicate zone of repair ( data supplied by riley dc and kelm nd , data not yet published , manuscript in preparation ) . despite the effectiveness we have observed with peg fusion , there can be inconsistencies in morphological and functional recovery of peg - fused animals . as demonstrated in figure 4 , peg fusion has not fully restored dti characteristics in these nerves , perhaps suggesting that not all axons were fused . we speculate that a variability in the number of fused axons may in turn be responsible for the variabilities in morphological and functional recovery . we also suspect factors such as the quality of repair , time of surgical intervention , and a surgeon 's level of experience with the technique play a critical role in achieving successful peg fusion . our studies have included applying the peg solution manually through a syringe with the needle held at the surface of the neurorrhaphy . this method , potentially , could result in small movements of the needle resulting in sub - optimal delivery of the peg solution or disturbance of the neurorrhaphy . our current efforts in developing a device are focused on achieving a consistent nerve repair with uniform peg application to the neurorrhaphy site . we recently described our experience with peg fusion using a nerve tube , which helps eliminate the need for sutures at the repair site ( sexton et al . , 2015 ) . our ultimate goal is to develop a simple coaptation system that will allow acute repair of severed nerves , with or without segmental injury , with minimal microsurgical training . our lab has focused its efforts on small animal models , and the study of peg fusion has occurred across multiple laboratories and animal models . we have demonstrated repeated success in working in a rat sciatic nerve model . bittner et al . has demonstrated the efficacy of peg fusion in invertebrate and small animal models in the aforementioned studies ( bittner et al . , 1986 ; in addition , peg fusion has been investigated in large animal sciatic nerve and ex - vivo central nerve models in other laboratories demonstrating reproducibility of the technique ( donaldson et al . , 2002 ; looking forward , we are currently conducting a phase i clinical trial of peg fusion in human nerve repair , and we were also hoping to move towards more large animal studies in the near future . the ability to fuse severed nerves and regain rapid functional recovery could result in a paradigm shift in all aspects of patient care following peripheral nerve damage . because this technique involves the use of current microsurgical techniques , with the simple addition of peg , it could rapidly be incorporated into a neurorrhaphy and autografting regimen . our current clinical trial will investigate efficacy and safety of peg fusion in humans , and our development of a peg delivery device will make peg fusion reproducible with minimal training required to achieve superior results .

the management of traumatic peripheral nerve injury remains a considerable concern for clinicians . with minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury , outcomes of surgical intervention have been unpredictable . the inability to manipulate the pathophysiology of nerve injury ( i.e. , wallerian degeneration ) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration ( ~1 mm / day ) . when axons are severed , the endings undergo calcium - mediated plasmalemmal sealing , which limits the ability of the axon to be primarily repaired . polythethylene glycol ( peg ) in combination with a bioengineered process overcomes the inability to fuse axons . the mechanism for peg axonal fusion is not clearly understood , but multiple studies have shown that a providing a calcium - free environment is essential to the process known as peg fusion . the proposed mechanism is peg - induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur . this review highlights peg fusion , its past and current studies , and future directions in peg fusion .

Introduction Polyethylene Glycol (PEG) Fusion Mechanism None Future Directions Conclusion

synaptic plasticity or long - lasting alterations in the efficacy of synaptic connections between two neurons has been proposed to be the cellular substrate of learning and memory ( malinow and malenka , 2002 ; bredt and nicoll , 2003 ; collingridge et al . , 2004 ; neves et al . , 2008 ) . persistent changes in synaptic efficacy are thought to involve at least two distinct phases an early phase ( on the order of minutes to hours ) that is independent of new protein synthesis and a more long - lasting late phase ( on the order of hours to days ) that is dependent upon new protein synthesis ( bramham and wells , 2007 ; bramham , 2008 ; richter and klann , 2009 ) . the first mechanism typically involves activity - dependent alterations in the phosphorylation and number of postsynaptic alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors ( ampa receptor ) and n - methyl d - aspartate receptors ( nmda receptor ) , which are delivered to the postsynaptic membrane via exocytosis and removed via endocytosis ( collingridge et al . , 2004 ; lau and zukin , 2007 ; neves et al . , 2008 ; holtmaat et al . , 2009 ; newpher and ehlers , 2009 ) . the second more long - lasting mechanism , postulated to underlie long - lasting memory storage , involves local or on - site protein synthesis in dendrites by translational machinery strategically localized in and around synaptic sites ( bramham and wells , 2007 ; bramham , 2008 ; richter and klann , 2009 ) . the identities of the newly synthesized proteins that are required to maintain altered synaptic efficacy are , however , as yet unclear . considerable evidence indicates that alterations in synaptic strength are locked - in by alterations in structural remodeling . these morphological changes include induction of new dendritic spines , enlargement of spines already extant , and the splitting of single spines into two or more functional synapses ( bourne and harris , 2008 ; holtmaat et al . more recently , studies involving spine long - term potentiation ( ltp ) , in which single spines are activated via photolysis of caged glutamate and imaged by means of two - photon microscopy , demonstrate that ltp is accompanied by enlargement of dendritic spines ( matsuzaki et al . , 2004 ; harvey and svoboda , 2007 ; harvey et al . , 2008 ; lee et al . , 2009 ) . mrna translation , a complex process involving the participation of many proteins and rnas , is generally divided into three phases : initiation , elongation , and termination . although each of these steps serves as a point of regulation to control the amount of protein that is produced , initiation is by far the most important and the salient features of this segment are important to keep in mind when considering how synapse stimulation induces changes in the translational apparatus . initiation begins with the formation of a ternary complex comprised of gtp , met - trna , and the initiation factor eif2 ( pestova et al . , 2007 ) . together with additional initiation factors ( eif3 , eif5 , eif1 , and eif1a ) , the ternary complex associates with the 40s ribosomal subunit to form a 43s pre - initiation complex ( chaudhuri et al . , 1999 ; majumdar et al . , 2003 ) . in mammals , this large bolus of factors is recruited to the 5 end of the mrna through interactions with the initiation factors eif4 g and eif3 ; at this point , it is referred to as the 48s pre - initiation complex ( lamphear et al . , 1995 ) . the dexh box protein dhx29 , a putative helicase , is also a required component of this complex for the translation of mrnas with highly structured 5 utrs ( pisareva et al . , 2008 ) . proper positioning of the complex on the 5 end requires the mg cap - binding complex consisting of eif4e , eif4 g and eif4a , which together are called eif4f ( gingras et al . , 1999 ) . with the aid of the initiation factors eif1 , eif1a , and dhx29 , the 48s complex then scans the mrna in the 3 direction until it encounters an aug initiation codon in the correct context , generally the first aug , where it is joined by the 60s subunit to begin polypeptide elongation ( pestova et al . , 1998 ; pestova and kolupaeva , 2002 ; pisareva et al . , 2008 the association between eif4e , the cap binding factor , and eif4 g , is often regulated upon synaptic activity . the protein 4e - bp can bind eif4e and prevent eif4 g from joining with eif4e , thus abrogating cap - dependent translation . phosphorylation of 4e - bp either directly or indirectly by the kinase mammalian target of rapamycin ( mtor ) causes it to dissociate from eif4e , thereby liberating the factor to bind both the cap and eif4 g and initiate translation ( gingras et al . , 1999 ) . such localized stimuli include those that trigger synapse formation , pruning , neurite outgrowth / collapse , activity - dependent synaptic plasticity and injury - induced axonal regeneration . a critical regulator of activity - dependent protein synthesis in dendrites is metabotropic glutamate receptors ( mglurs ) . mglurs are g protein - coupled receptors enriched at excitatory synapses throughout the brain where they act to regulate glutamatergic neurotransmission ( nakanishi , 1994 ; pin and duvoisin , 1995 ; conn and pin , 1997 ) . group i mglurs ( mglur1/5 ) activate phospholipase c , leading to mobilization , and the extracellular signal - regulated kinase ( erk)mitogen - activated protein kinase ( mapk ) pathway through which they modulate signal - to - nucleus communication . signaling by mglur1/5 is critical to synaptic circuitry formation during development and is implicated in forms of plasticity including ltp ( aiba et al . , 1994 ) , long - term depression ( ltd ) ( aiba et al . , 1994 ; oliet et al . , 1997 ; palmer et al . , 1997 ; huber et al . , 2001 ; bear et al . , 2004 ) associative learning ( aiba et al . , 1994 ) and cocaine addiction ( kenny and markou , 2004 ) . mglur1/5 elicit synapse - specific modifications in synaptic strength and spine morphology by stimulating rapid local translation of dendritic mrnas including fmr1 , encoding fragile x mental retardation protein ( fmrp ) ( greenough et al . , 2001 ) . expression of mglur - ltd at schaffer collateral to ca1 pyramidal cell synapses is mediated by persistent internalization of ampars and in adolescent wild - type ( wt ) mice requires de novo protein synthesis ( huber et al . , 2000 ; snyder et al . , 2001 ) that is dependent on the mtor ( hou and klann , 2004 ) . the protein synthesis dependent is both age and region - specific in that ca1 mglur - ltd in neonates ( nosyreva and huber , 2005 ) and mglur - ltd at cortical synapses ( desai et al . , 2006 ) mammalian target of rapamycin is a central regulator of cell growth and proliferation , autophagy , ribosome biogenesis , and translation ( hay and sonenberg , 2004 ; klann and dever , 2004 ; sarbassov et al . , 2005 ) and a critical signaling pathway downstream of group i mglurs . in neurons , components of the mtor signaling cascade are present at synapses and are thought to influence synaptic plasticity via regulation of local protein synthesis ( tang and schuman , 2002 ) . mtor is activated in dendrites by stimulation of group i mglurs ( klann and dever , 2004 ) and is required for mglur - ltd at schaffer - collateral to ca1 synapses ( hou and klann , 2004 ; banko et al . , 2006 ; antion et al . , growing evidence indicates that dysregulation of mtor is associated with human diseases , including cancer , diabetes , and autism ( jaworski and sheng , 2006 ; sabatini , 2006 ; dann et al . , 2007 ) . the fragile x syndrome ( fxs ) is the most common heritable form of mental retardation ( chiurazzi et al . , 2003 ; jacquemont et al . , 2007 ; bassell and warren , 2008 ) as well as the most common known genetic cause of autism ( fryns et al . , 1984 ) . fxs is caused by loss - of - function mutations in the fmrp , which is encoded by the fmr1 gene located on the x chromosome . in humans , the fxs typically results from expansion of a cgg repeat sequence in the 5-untranslated region and silencing of the fmr1 gene ( feng et al . , 1995 ; patients with the fxs exhibit a wide - range of neurological deficits including cognitive impairment , seizures , emotional instability , sleep disorders , attention deficits , autonomic dysfunction , and autism ( hagerman and hagerman , 2002 ; o'donnell and warren , 2002 ; chiurazzi et al . , 2003 ; jin and warren , 2003 ) . fmrp , the gene product of the fmr1 gene , is an rna binding protein that associates with many mrnas , some of which encode proteins important for neuronal development and plasticity . fmrp controls activity - dependent dendritic mrna localization ( dictenberg et al . , 2008 ) , stability ( brown et al . , 2001 ; miyashiro et al . , 2003 ; d'hulst et al . 2007 ) and translational efficiency of dendritic mrnas in response to stimulation of mglurs ( weiler et al . , 1997 ; todd et al . , 2003 ; muddashetty et al . , 2007 ; westmark and malter , 2007 ; napoli et al . , 2008 ) . fmrp is detected in cell bodies , in dendritic shafts and branch points ( ferrari et al . , 2007 ) , and at the base of synaptic spines and spine heads ( feng et al . , 1997 ) . fmrp mrna is translated near synapses in response to neurotransmitter activation ( weiler et al . , 1997 ) ; moreover , different models of experience - dependent plasticity such as whisker stimulation ( todd and mack , 2000 ) , visual experience ( gabel et al . , 2004 ) and exposure to enriched environment ( irwin et al . , 2005 ) also promote fmrp mrna translation . exhibit abnormalities in dendritic spine morphology ( bagni and greenough , 2005 ) , cognitive deficits ( o'donnell and warren , 2002 ) , exaggerated , protein synthesis - dependent ltd at schaffer collateral to ca1 pyramidal cell synapses in adolescent wt mice , but protein synthesis - independent in neonatal wt mice ( nosyreva and huber , 2005 ) and in fmr1 ko mice ( huber et al . , 2002 ; koekkoek et al . , 2005 ; hou et al . , 2006 ; nosyreva and huber , 2006 ; zhang et al . , 2009 ) , and decreased ltp in the cortex ( li et al . , 2002 recent findings indicate that dysregulation of mtor signaling in the hippocampus is an important functional link between overactivated mglur signaling , aberrant protein synthesis , and exaggerated mglur - ltd in fmr1 ko mice ( sharma et al . , 2009 ) . for example , the fmr1 ko mouse exhibits aberrant numbers and/or activity of mglur5 ( giuffrida et al . , 2005 ; dolen et al . , 2007 ) , nmda ( gabel et al . , 2004 ; desai et al . , 2006 ) and ampa ( pilpel et al . , 2009 ; schuett et al . , a hallmark feature of the fxs in humans , also observed in the fmr1 ko mouse , is that of synaptic spine dysmorphogenesis . spines on hippocampal and cortical neurons of fmr1 ko mice exhibit enhanced density and are thinner and longer than those of age - matched wt mice , resembling an immature morphology . these observations indicate that fmrp normally controls processes involved in synapse maturation , stabilization , and elimination ( bagni and greenough , 2005 ) . electrophysiology experiments using cultured neurons indicate a delay in establishing synaptic connections during a brief window in brain development ( braun and segal , 2000 ) . in the somatosensory cortex , although spine dysmorphogenesis persists into adulthood ( galvez and greenough , 2005 ; restivo et al . , 2005 ) , it can be rescued if the animals ( mice ) are exposed to enriched environmental conditions ( restivo et al . , 2005 ; meredith et al . , 2007 ) synaptic activity in the brain can trigger long - lasting changes in synaptic strength ; i.e. , ltp and ltd . early in development , ltp seems to be important for retaining nascent synapses , whereas ltd is more directly involved in activity - guided synapse elimination . the combination of these two phenomena contributes to the essential brain function of learning and memory . impaired synaptic activity almost certainly results in impaired mental development ( vaillend et al . , 2008 ) . new proteins that contribute to re - arrangements and re - assembling of synapse are made locally from mrnas that are trafficked to dendrites ( steward and levy , 1982 ) . local changes in synaptic efficacy are protein synthesis dependent ( steward and schuman , 2003 ; martin and zukin , 2006 ; lin and holt , 2008 ) ; synaptic activity also promotes an increase in the number of polysomes at spines , presumably reflecting increased on - site local synthesis of proteins critical to alterations in synaptic efficacy and structural remodeling of spines ( ostroff et al . , 2002 ) . in neurons , fmrp is packaged in ribonucleoprotein ( rnp ) granules , some components of which have been identified ( ohashi et al . , 2002 ; brendel et al . ; dictenberg et al . , 2008 ; napoli et al . , 2008 ) . fmrp granules contain several proteins involved in translation ( pur alpha and beta , hnrnpu , staufen , ef1alpha , papb1 , eif4e , ribosomal proteins ) , cytoskeleton dynamics ( tubulin , tau , actin , rac1 , cyfip ) , and the movement of molecules and organelles ( dynein , myosins and kinesins ) . importantly , camkii and arc , targets of fmrp that are critical to synaptic plasticity and known to be synthesized de novo in response to neuronal activity , are detected in these granules ( kanai et al . , 2004 ) . fmrp - containing rnp granules are transported along microtubules in dendrites ( antar et al . , 2004 ; ferrari et al . , 2007 these data strongly support a role for fmrp in dendritic delivery of rna and regulation of local translation in specific synaptic compartments . the molecular mechanism(s ) by which fmrp controls translation has been investigated by assessing the polysome / mrnp distribution of the fmrp - containing complex . initially , fmrp was shown to co - sediment entirely with polyribosomes or even larger granules ( ohashi et al . 2004 ; stefani et al . , 2004 ) or to co - sediment essentially equally with polysomes and mrnps ( feng et al . , 1997 ; brown et al . , 2001 ; ohashi et al . , 2002 ) . other laboratories found that fmrp co - sedimented mainly with the mrnp fraction ( siomi et al . , 1996 ; ishizuka et al . , 2002 ; zalfa et al . , 2003 ; while this apparent discrepancy could arise owing to differences in methodologies , brain region or cell type , a recent study by bagni and colleagues shows that fmrp regulates synaptic translation at the level of initiation ( napoli et al . , 2008 ) . these authors showed that fmrp inhibits translation initiation through an interacting factor , cyfip1/sra1 , which binds the cap binding factor eif4e . interestingly , cyfip1 binds eif4e via a novel domain with a characteristic reverse l shaped structure that is also assumed by the canonical eif4e - binding motif ( marcotrigiano et al . , 1999 ) . the importance of cyfip1 in the fmrp - regulatory circuit was demonstrated by an increased level of proteins encoded by known fmrp target mrnas upon reduction of cyfip1 in neurons . furthermore , the brain cytoplasmic rna 1 , another fmrp binding partner , increases the affinity of fmrp for the cyfip1-eif4e complex in the brain ( napoli et al . , 2008 ) . fmrp has been reported to sediment in both the rnp fractions ( i.e. , less the 80s monosome ) as well as with polysomes in sucrose gradients . in the rnp fractions , , we show that cyfip1 inhibits translational initiation by binding initiation factor eif4e and preventing assembly of eif4e with eif4 g . in the polysome fractions , fmrp may inhibit protein synthesis by inhibiting polypeptide elongation ( i.e. , by retarding the rate at which ribosomes move along the mrna ) . 40s and 60s are the ribosomal subunits , 80s is the monosome or complex of the two subunits , and m7 g is the cap , a modified nucleotide containing a methyl tag , present at the 5 end of the mrna . in cultured neurons , protein synthesis can be activated by several synaptic stimuli such as bdnf and dhpg . bdnf stimulates translation at synapses via the mtor and erk mapk pathways , and likely involves the modulation of translational initiation . moreover , bdnf activates the translation of arc and camkii mrnas ( yin et al . , 2002 ; schratt et al . , 2004 ; napoli et al . , 2008 ) , two fmrp targets ( zalfa et al . , 2003 ; park et al . , stimulation of neurons by bdnf or dhpg promotes dissociation of cyfip1 from eif4e at synapses , an event associated with initiation of protein synthesis . thus , fmrp - dependent translational repression in brain is mediated , at least in part , by cyfip1 ( napoli et al . , 2008 ) . activity dependent changes in fmrp phosphorylation is observed in dendrites and show a temporal correlation with the translational profile of select fmrp target transcripts ( narayanan et al . , 2007 ) . post - translational modifications of fmrp and or cyfip1 may account for such reversible control of local translation . finally , the temporal activity of fmrp seems also to be regulated by mglur signaling . mglur - ltd induces a transient , translation - dependent increase in fmrp that is rapidly reversed through degradation by the ubiquitin - proteasome pathway ( hou et al . , 2006 ) . nmda receptor - dependent synaptic stimulation promotes a redistribution of proteasomes from dendritic shafts to synaptic spines and thus provides a mechanism for local protein degradation ( bingol and schuman , 2006 ) . synaptic proteasomal activity seems to affect a large number of synaptic proteins including specific postsynaptic scaffolds whose turnover after ubiquitination is enhanced . interestingly , the long - term activity - dependent increases of camkii and psd-95 , encoded by two mrnas that are translationally repressed by fmrp ( zalfa et al . , 2003 ; hou et al . , 2006 ; muddashetty et al . , 2007 ; these findings suggest an interplay between mrna localization and fmrp degradation that is integrated with neuronal activity and directly regulated by the ubiquitin - proteasome system . fragile x mental retardation protein key target mrnas encode proteins involved in synaptic structure and function ( bagni and greenough , 2005 ; bassell and warren , 2008 ) . among them an interesting target candidate is arc / arg 3.1 ( zalfa et al . , 2003 ; park et al . , 2008 arc / arg 3.1 mrna translation is rapidly up - regulated following mglur stimulation , an effect that is not detected in the absence of fmrp . arc / arg 3.1 mrna synaptic translation has been proposed to be critical for synapse - specific ltd ( park et al . , 2008 ) . thus arc / arg 3.1 reduces the number of glur2/3 receptors leading to a decrease in ampar - mediated synaptic currents ( rial verde et al . , key ltd synaptic proteins whose upregulation would lead to the impaired hippocampal mouse ltd observed in fmr1 ko mice . ( 2004 ) proposed that a net loss of synaptic ampa and nmda receptors , possibly due to an increased internalization mediated by one or several ltd proteins , would contribute to a weakened spine and possibly the dysmorphogenesis observed in the fxs . fragile x mental retardation protein can specifically recognize its mrna targets in multiple ways : directly via rna structures such as g - quartets and/or g - rich and/or u - rich sequences , via bc1 rna , or micrornas ( bagni and greenough , 2005 ; bassell and warren , 2008 ) . it is unclear whether the alternative binding motifs or non - coding rnas are specific for different messengers or , rather , function as alternative modes to target the same mrnas during development or synaptic response . further work is needed to understand the functional consequences of impaired local protein synthesis in the developing brain and how this correlates with the autistic and fxs phenotypes . the cpeb is an rna binding protein that recognizes target rnas via specific sequences in their 3-untranslated regions . cpeb promotes dendritic delivery of mrnas along microtubules ( huang et al . , 2003 ) and polyadenylation - induced translation of mrnas in response to synaptic activity ( wu et al . , 1998 du and richter , 2005 ) ( figure 2 depicts key features of cpeb - controlled translation ) . in neurons , cpeb localizes to the cell body and postsynaptic sites and , within dendrites , is packaged in rnp granules that localize near synapses . in neurons cpeb targets a number of mrnas that encode proteins critical to synaptic plasticity ( du and richter , 2005 ; pique et al . , 2008 ) . disruption of cpeb stunts the development of dendritic arbors and prevents neurons from integrating into the functional visual system of xenopus laevis ( bestman and cline , 2008 ) . moreover , loss of cpeb activity in mice prevents the formation of some forms of synaptic plasticity and interferes with memory formation ( alarcon et al . ; mcevoy et al . , 2007 ; miniaci et al . , 2008 ) . cpeb knockout mouse exhibit reduced theta burst - induced ltp at schaffer collateral to ca1 pyramidal cell synapses of the hippocampus ( alarcon et al . , 2004a ) . in addition , these mice exhibit a deficit in extinction of memory , a phenomenon whereby behavioral responses diminish and eventually become extinct when there is no reinforcement of the memory ( berger - sweeney et al . although extinction requires the formation of new memories , the underlying mechanisms by which it occurs are probably distinct from those of memory acquisition and consolidation ( abel and lattal , 2001 ) . these data suggest an important role for cpeb in linking local protein synthesis and morphological plasticity to synaptic plasticity . some mrnas contain a cytoplasmic polyadenylation element ( cpe ) , which is bound by cpeb . cpeb also interacts with maskin ( or neuroguidin , a functionally related protein ) , which in turn interacts with the cap ( m7 g ) binding protein eif4e . such mrnas are translationally inactive because maskin inhibits the association of eif4e and eif4 g , another initiation factor that helps recruit the 40s ribosomal subunit to the 5 end of the mrna . cues such as nmda receptor activation stimulate the kinase aurora a , which phosphorylates cpeb , an event that causes poly(a ) tail elongation . poly(a ) binding protein ( pabp ) binds the newly elongated poly(a ) tail and recruits eif4 g . the pabp - eif4 g dimer helps to displace maskin from eif4e , allowing eif4 g to bind eif4e and initiate translation . identified growth hormone ( gh ) as one protein whose level is reduced 10-fold in the cpeb ko hippocampus ( zearfoss et al . , 2008 ) . gh mrna contains no binding sites for cpeb , and both gh mrna and pre - mrna are reduced in the ko versus wt hippocampus , suggesting that an mrna encoding a transcription factor that regulates gh gene expression is under the control of cpeb . indeed , cpeb controls the synthesis of c - jun , which regulates the expression of the gh gene . gh also induces ltp in hippocampal slices ; gh - induced ltp , like electrical stimulation - elicited ltp , is reduced at ca1 synapses of the cpeb ko mouse . moreover , ltp induced by gh or theta burst stimulation is reduced if polyadenylation is inhibited by the drug cordycepin ( 3-deoxyadenosine ) , which terminates adenylate polymers . these and other results suggest that gh acts in both autocrine and paracrine fashion to regulate plasticity through cpeb - mediated control of c - jun translation . the cpeb is also present in invertebrates , where it may act as a polyglutamine - containing prion - like factor to control plasticity ; in aplysia sensory neurons where cpeb rna expression has been disrupted by an antisense oligonucleotide , long - term facilitation is not maintained ( si et al . , 2003b ) . the isoform of aplysia neuronal cpeb differs from the vertebrate cpeb noted above in that it contains a high concentration of glutamine residues . polyglutamine can be found in proteins that resemble a prion , an infectious agent consisting of self - reproducing protein . the authors ( si et al . , 2003a , b ) suggested that this cpeb isoform could assume a prion - like structure following synaptic experience and form an indelible synaptic tag . if true , then cpeb , rather than the proteins derived from cpeb - regulated translation , would comprise a tag that distinguishes experienced from naive synapses . si et al . ( 2003a ) demonstrated that aplysia cpeb resembled a prion in that it was protease resistant and had a fast sedimentation rate in sucrose gradients . more compelling evidence came from experiments in transduced yeast , where aplysia cpeb assumed two forms : one that aggregated ( i.e. , prion - like ) and one that did not . very unexpectedly , the aggregated form of cpeb bound to rna and converted the nonaggregated form into an aggregated form . such epigenetic inheritance is a hallmark of prion formation and lead the authors to opin that synaptic activity could cause neuronal aplysia cpeb to become prion - like and stimulate the translation of rnas , cause it to alter its substrate specificity , or release some mrnas from a translationally dormant state . ( 2003a ) also suggested that once it becomes prion - like , cpeb would need no further stimulation to maintain activity . vertebrates contain three additional cpeb - like genes , all of which are expressed in the brain ( theis et al . , 2003 ) . two of these cpeb - like proteins contain some polyglutamine but they do not have a strong affinity for the cpeb binding site and do not regulate polyadenylation ( huang et al . , 2006 ) . although the relationship between vertebrate cpeb proteins and prions , if any , remains to be determined , a drosophila cpeb isoform , orb2 , contains polyglutamine and is important for long term memory ; when the polyglutamine is deleted , long term memory is disrupted ( keleman et al . , 2007 ) . while this study did not examine prion formation , or even mrna translation , it does point to the glutamine stretch of the cpeb isoform as being potentially important for memory formation several studies provide strong evidence linking mrna trafficking and dendritic protein synthesis to synaptic plasticity . relatively little is known about how these processes are interrelated , including coupling of synaptic signalling to the translational machinery , selective translation of specific mrna at synapses , and specific delivery of newly transcribed mrnas to activated synaptic sites . two molecules that connect receptor signalling to local protein synthesis in spines are cpeb and fmrp . cpeb localizes to postsynaptic sites where it regulates translation of proteins critical to synaptic plasticity ( du and richter , 2005 ; pique et al . , 2008 ) , remodeling of dendritic arborization and organization of the functional visual system ( bestman and cline , 2008 ) . cpeb ko mice exhibit deficits in synaptic plasticity and memory formation ( alarcon et al . , 2004b ; berger - sweeney et al . , 2006 ; mcevoy et al . , 2007 ; miniaci et al . , 2008 ) . these findings suggest an important role for cpeb in linking local protein synthesis and dendritic remodeling to synaptic plasticity . similarly , fmrp localizes to synapses where it regulates translation of proteins critical to synaptic plasticity and structural remodeling . fmr1 ko mice exhibit abnormal dendritic spines , cognitive deficits , aberrant synaptic plasticity in different areas of the brain , and a reduced number of mglur and ampa receptors . pharmacological or genetic manipulations that attenuate activity of group i mglurs ( dolen et al . , 2007 ) or exposure to an enriched environment increases ampar subunit abundance in the visual cortex ( restivo et al . , 2005 ; meredith et al . , 2007 ) and rescues , at least in part , the hallmark behavioral and synaptic abnormalities of the fragile x phenotype . dissection of the molecular mechanisms underlying these rescue experiments represent a starting point in our understanding of fmrp function and of the linkage between neuronal activity , receptor activation , and local protein synthesis . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

localization of mrnas to dendrites and local protein synthesis afford spatial and temporal regulation of gene expression and endow synapses with the capacity to autonomously alter their structure and function . emerging evidence indicates that rna binding proteins , ribosomes , translation factors and mrnas encoding proteins critical to synaptic structure and function localize to neuronal processes . rnas are transported into dendrites in a translationally quiescent state where they are activated by synaptic stimuli . two rna binding proteins that regulate dendritic rna delivery and translational repression are cytoplasmic polyadenylation element binding protein and fragile x mental retardation protein ( fmrp ) . the fragile x syndrome ( fxs ) is the most common known genetic cause of autism and is characterized by the loss of fmrp . hallmark features of the fxs include dysregulation of spine morphogenesis and exaggerated metabotropic glutamate receptor - dependent long term depression , a cellular substrate of learning and memory . current research focuses on mechanisms whereby mrnas are transported in a translationally repressed state from soma to distal process and are activated at synaptic sites in response to synaptic signals .

Synaptic Plasticity General Features of mRNA Translation Synaptic Signaling The Molecular, Cellular and Physiological Basis of the Fragile X Syndrome FMRP and Local Protein Synthesis Cytoplasmic Polyadenylation Element Binding Protein (CPEB)-Regulated Synaptic Translation Conclusions Conflict of Interest Statement:

. main cause of acs is intra abdominal hypertension ( iah ) due to various factors such as abdominal surgery , ileus , intestinal obstruction , intraabdominal malignancy , and organomegaly . the most important clinical presentations of acs are symptoms due to abdominal distension , acute oliguria , and respiratory problems 1 . we report a patient with external compression of distal duodenal segment due to the metastatic lymph nodes and acute kidney injury ( aki ) as a result of acs . it was diagnosed with computed tomography ( ct ) and after surgical intervention his clinical status improved significantly . we report this case because to date , a case of acs due to metastatic prostate cancer has not been reported . a 78-year - old man admitted to emergency department with abdominal pain , distension , nausea , vomiting , and dyspnea for nearly 1 week and oliguria for 2 days . bilateral renal parenchymal echo increased consistent with the acute kidney failure and the bladder was empty . also there was no electrolyte disturbances that could contribute to kidney injury such as hypercalcemia , hypokalemia , hyperphosphatemia , or hyperuricemia . he had a history of prostate adenocarcinoma diagnosed 6 months ago but he did not accept the antineoplastic therapy performed for him . on physical examination , blood pressure was 115/80 mmhg , oral mucosa was wet and there was severe abdominal distension and tenderness . at admission laboratory values were as follows ; urea : 116 mg / dl , creatinine : 4.7 mg / dl , c - reactive protein ( crp ) : 77 mg / dl , chloride : 90 mmol / l , lactate dehydrogenase ( ldh ) : 556 /l , ph : 7.49 , pco2 : 35.9 mmhg , hco3 : 28 mmol / l . on urinalysis ; urine ph was < 5 and density was 1017 . on ct there were multiple lymph nodes in the abdominal cavity , also gastric and duodenal distension due to lymph node compression on distal duodenal segment ( fig.1 ) . intravesical pressure of the patient was 36 mmhg ( normal levels of intra - abdominal pressure : 05 mmhg ) . after nasogastric ( ng ) tube insertion , 8 l of gastric fluid discharged suddenly . after medical decompression intervention , urine output increased gradually and creatinine levels started to regress . but after the removal of ng tube , clinical status deteriorated again . therefore , surgical decompression ( gastrojejunostomy ) was performed . after decompression operation beside the clinical and radiological status ( fig.2 ) , laboratory values improved , but intravesical pressure was not measured again . after operation laboratory values were as follows ; creatinine : 2.4 mg / dl , crp : 27 mg / dl , ldh : 399 /l . during follow- up organ dysfunction due to iah is called as acs and it is a mortal condition especially for critically ill patients . acs is not a disease , however , it can have many causes and can develop during many disease processes . one of the most affected abdominal organs due to acs is kidney and aki is not rare in this condition . our case had an uncontrolled prostate cancer history and presented with abdominal pain , dyspnea and oliguria . at admission we determined oliguric aki and on ct gastric and duodenal distension due to metastatic lymph nodes one of the most important factors that contribute organ failure caused by iah is probably increased levels of proinflammatory cytokines . in a human model of iah induced by laparoscopic surgery , markers of both systemic inflammation and aki were increased . thus , it is not impossible to suggest that acs caused by iah is at least partly due to increased inflammation 2 . also in our patient during the acs period crp level was high and decreased with successful treatment . intestinal obstruction is a predictor of iah just as the fact that iah is a predictor of mortality . in the majority of ileus and/or intestinal occlusion cases there is iah and this is an early and important messenger of mortality in these conditions . intra- abdominal pressure ( iap ) higher than 2025 mmhg may cause acs and in this way acute functional loss of various abdominal and extra - abdominal organs . inflammatory and hemodynamic factors caused by iah may affect pelvic , thoracic , cranial and muscular areas beside abdominal organs 3,4 . 5 reported a patient with acs presented with aki caused by air - trapping and excessive auto - positive end - expiratory pressure during intubation and recovered after extubation . after abdominal surgeries iah is not rare and measurement of iap is thought to be a predictor of aki development for patients followed in intensive care units 6 . 7 reported a 72-year - old woman with acs due to gastric dilatation after the surgery of retroperitoneal sarcoma and presented with cardiorespiratory collapse and anuria . reported a pediatric patient from japan with acs due to hepatomegaly and neuroblastoma and after decompression operation and continuous hemodiafiltration his health status ameliorated 9 . iap may be detected by measuring the pressure of urinary bladder ( intravesical method ) and it well correlates with iap . treatment options of world society of the abdominal compartment syndrome are : improvement of abdominal wall compliance , evacuation of intra - luminal contents , evacuation of intra - abdominal space occupying lesions , optimization of fluid management and optimization of systemic and regional perfusion 3,4 . in the present case we detected aki due to acs in a patient with duodenal compression because of multiple metastatic intra - abdominal lymph nodes . during the follow - up period , especially for patients with malignant disorders with acute oliguria and respiratory problems who have abdominal distension , acs should be in mind as causative factor . for patients with acute oliguria and respiratory problems who have abdominal distension , acs and/or iah should be in mind as differential diagnosis among other possible disorders . the author declares that there is no conflict of interests regarding the publication of this article .

key clinical messageorgan dysfunctions caused by intraabdominal hypertension is named as abdominal compartment syndrome ( acs ) . a patient with prostate cancer admitted with dyspnea and oliguria . after decompression his health status improved . for patients with malignant disorders presented with oliguria and respiratory problems who have abdominal distension , acs should be in mind .

Introduction Case Report Discussion Conflict of Interest

lyme disease is a worldwide - distributed multisystem animal - borne disease , caused by borrelia burgdorferi ( bb ) sensu lato ( gram - negative spirochaetes ) which is a group of at least 12 closely related species . recent evidence shows that human cases of lyme disease may also be caused by more than one species of b. burgdorferi sensu lato , with rodents and small mammals as its animal reservoir . traditionally lyme disease is divided into four stages , and clinical manifestations may be both cutaneous and systemic and can have cardiovascular , neurological , and musculoskeletal involvement [ 46 ] . when lyme disease is treated with appropriate antibiotic therapy in the early stages , long - term outcomes are good . lyme disease is the most common vector - borne disease in the usa and europe with more than 300,000 new cases in the usa and 100.000 cases annually in europe ( estimated from available national data ) . however , this number is largely underestimated as case reporting is highly inconsistent and many infections go undiagnosed [ 1013 ] . global climate change expanding the range of tick vectors and an increase in the incidence suggest that this disease will remain an important health issue in the forthcoming decades . chronic lyme disease ( cld ) is considered a constellation of persistent symptoms in patients with or without evidence of bb infection [ 15 , 16 ] . some authors use the term cld only for patients who had been clinically diagnosed with lyme disease and have persisting symptoms lasting more than 6 months following antibiotic treatment . for this reason the term posttreatment lyme disease [ 17 , 18 ] or post - lyme disease syndrome [ 10 , 18 ] is often used . the category of probable lyme disease was recently added to the cdc surveillance case definition to describe patients with serologic evidence of exposure and physician - diagnosed disease in the absence of objective signs . recently , the term chronic arthropod - borne neuropathy ( can ) has been proposed as an alternative to chronic lyme disease , as calling the chronic illness there is growing and well - documented evidence to the concept of persistent bb infection in both animals [ 2131 ] and humans [ 3241 ] . recent evidence shows bb is able to escape from destruction by the host immune reactions , persist in host tissues , and sustain chronic infection and inflammation , despite aggressive antibiotic challenge [ 32 , 35 , 36 , 4244 ] . an estimated 20% of patients display recurrent symptoms after antibiotic treatment . a recent study showed that , at six months following antibiotic treatment , 36% of patients reported new - onset fatigue , 20% widespread pain , and 45% neurocognitive difficulties without evidence of depressive symptomatology . some studies have provided evidence on pleocytosis and the production of bb antibodies in the cerebrospinal fluid of patients with neuroborreliosis , while this is contradicted by other studies showing greater variability in such patients . when looking at the clinical and basic science research , it is apparent that persistent bb infection and associated inflammation and molecular mimicry mechanisms are associated with gradually increasing encephalopathy and gradually increasing mental symptoms . despite available evidence , both treatment - refractory infection and persistent symptoms that are related to bb infection remain subject to controversy in which national medical societies , patient advocacy groups , insurance companies , lawyers , doctors , the private health medical sector , and scientific journals all have become embroiled [ 10 , 5053 ] . the controversy is exacerbated by the lack of a clinical or microbiologic definition and the frequent occurrence of chronic symptoms in the general population . the lyme disease controversy can also be largely linked to the misconception that neurobehavioral effects of illness constitute evidence of nervous system infection . appropriate differentiation between neuroborreliosis ( nervous system borrelia burgdorferi infection ) and lyme encephalopathy ( altered nervous system function in individuals with systemic but not nervous system infection)or encephalopathies of other etiologies would lessen the controversy considerably , as the attribution of nonspecific symptoms to supposed ongoing central nervous system infection is a major factor perpetuating the debate . because of the absence of solid evidence on prevalence , causes , diagnostic criteria and treatment options , the role of autoimmunity to residual or persisting antigens , and the role of a toxin or other bacterial - associated products that are responsible for the symptoms and signs , cld remains a relatively poorly understood chronic disease construct . the role and performance of family medicine in the detection , integrative treatment , and follow - up of cld are not well studied either . the purpose of this paper is to describe the complexity of cld as a multidimensional chronic disease construct and its relevance to family medicine by means of a systematic literature review . this paper is a second paper in a series on chronic diseases that focus on complex chronic conditions . a systematic review method was used to document the complexity and multidimensionality of cld . in addressing the objective of this review , we used a parallel search strategy via medline , the cochrane library of systematic reviews , cinahl ( cumulative index to nursing and allied health literature ) , and pre - cinahl , complemented with a reference review of key articles . articles were selected if they dealt with ( 1 ) cld care complexity ; ( 2 ) cld case ( patient ) complexity ; ( 3 ) the complexity of cld quality assessment ; and ( 4 ) complexity of cld at the health system level . in addition the studies had to produce insights into the complexity of cld with relevance to family medicine . the search strategy included a combination of medical subject headings ( mesh ) terms with regard to lyme disease . we also scrutinised extra sources for further identification of studies by hand - searching the reference lists of all articles . by focusing on cld care , case , quality assessment , and health systems complexity , we further build on four major and interrelated components of complexity in chronic care that have been described by borgermans et al . . each of these components represents a range of elements that contribute to the picture of complexity in chronic care . two authors carried out independent screening of titles and abstracts using the specific inclusion and exclusion criterion reported on previously . we ordered the full text of all citations that met the eligibility criteria or appeared relevant or where relevance / eligibility was not clear from the abstract . in the final screening , two authors independently scrutinised the full texts of studies and recorded the reasons when articles were excluded resolving disagreements by discussion and when necessary referred to a third author . a tool - based assessment of the methodological quality of the studies was not part of this review . the flow diagram ( figure 1 ) shows our study selection process and the number of studies included . 72 studies provided insights on cld care complexity , 9 studies provided insights on cld case complexity , 2 studies provided insights on cld quality complexity , and 6 studies provided insights on cld health system complexity with relevance to family medicine . it is current practice for physicians to base themselves primarily on clinical signs and symptoms when cld is suspected . patients often can not recall being bitten by a tick or if erythema migrans has occurred . erythema migrans is pathognomonic and does not require any further laboratory investigations , but a careful analysis is recommended as a variety of unconventional histopathologic patterns may occur . in the case of no erythema migrans clinical manifestations are complemented by laboratory tests , including enzyme - linked immunosorbent assay ( elisa ) and western blot testing . although this two - tier testing is standard practice in both the united states and europe , the test kits generally differ . there is consistent evidence that the two - tier testing lacks sensitivity , can not distinguish between current and past infection , can not be used as a marker for treatment , is often dependent on subjectively scored immunoblots , and is considered expensive [ 64 , 65 ] . moreover , the diagnosis of cld based on clinical manifestations , serological findings , and detection of infectious agents often contradict each other [ 56 , 66 , 67 ] . for this reason a growing number of studies support two - tier testing followed by a sensitive and reliable dna sequencing for confirmation to support the diagnosis of cld [ 68 , 69 ] . other authors suggest the use of a single tier hybrid antigen immuno - pcr and detection of igg antibodies only . for cases with a high clinical suspicion of disease , the c6 peptide enzyme immunoassay ( eia ) is put forward as a stand - alone test or in the second tier of a 2-tiered algorithm . this assay is based on a peptide ( c6 ) , whose amino acid sequence reproduces a conserved , immunodominant region of a single protein of borrelia burgdorferi . overall , the use of pcr - based methods appears to be of importance because of the high sensitivity and specificity of these assays [ 7173 ] . however , pcr positivity in the absence of culture positivity following antibiotic treatment should be interpreted with caution since b. burgdorferi dna and mrna can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters . another novel method including culturing spirochetes from the serum of patients uses a two - step preenrichment process , followed by immunostaining with or without polymerase chain reaction ( pcr ) analysis . in patients who complied with the strict cdc surveillance case definition for lyme disease the procedure resulted in positive cultures in 47% at 6 days and 94% at week 16 . the centers for disease control and prevention has however raised concerns about false - positive results caused by laboratory contamination and the potential for misdiagnosis . some authors put forward the measurement of activity of two lysosomal exoglycosidases , -fucosidase ( fuc ) and -galactosidase ( gal ) , in the serum as potential markers of ld . recent evidence has shown that the presence or absence of chronic lyme borreliosis may be objectively adjudicated by tissue examinations which demonstrate or which fail to show pathogenic microbes in patients who have received a full course of antibiotics . the use of spect scans of the brain using tc and standard nuclear imaging techniques is considered an objective measure of abnormalities present in patients with otherwise difficult to objectify clinical findings . brain spect scans are abnormal in most patients with chronic lyme disease , and these scans can be used to provide objective evidence in support of the clinical diagnosis . overall , studies highlight the need for standardization in diagnostic ( serological ) testing , as well as the need for studies that discriminate between active disease and past infection . a factor that complicates the diagnosis of cld is that it does not present with isolated subjective symptoms . in undiagnosed patients or posttreatment , bb may mimic the symptoms and signs of other diseases disorders [ 80 , 81 ] and for this reason is called the illness with a thousand faces . these symptoms may include fatigue , insomnia , widespread pain , arthritis , chronic eye lid edema , cataract , dermatitis , cognitive complaints , bilateral hearing loss , adult respiratory distress syndrome , palpitations , tachycardia , syncope , tremors , epileptic crises , depression , anxiety , panic attacks , catatonia , and psychosis , amongst others [ 93 , 94 ] . lyme disease might become misdiagnosed as fibromyalgia , chronic fatigue syndrome [ 96 , 97 ] , amyotrophic lateral sclerosis , autism spectrum disorder , parkinson 's disease , rheumatoid arthritis , multiple chemical sensitivity , guillain - barre syndrome ( gbs ) , and dementia , as well as other numerous neurological [ 101 , 102 ] and psychiatric disorders [ 88 , 103 ] . overall , studies show the need for a careful differential diagnosis in patients with suspected cld and persistent complaints . the diagnosis of cld is even more complex when tick - borne coinfections occur in association with lyme disease , which is the rule rather than the exception . routine laboratory tests exhibit varying reported sensitivity and are usually unhelpful in diagnosis , as serology fails in terms of specificity and/or sensitivity . clinically relevant coinfections include bartonella species , babesia , anaplasma , rickettsia , yersinia enterocolitica , chlamydophila pneumoniae , chlamydia trachomatis , mycoplasma pneumoniae , and tick - borne encephalitis virus , amongst others [ 108 , 111 ] . a particular condition that gets growing attention in patients with cld is morgellons disease . morgellons disease is an emerging skin disease characterized by formation of dermal filaments associated with multisystemic symptoms . recent studies show that bb spirochetes were detected in the dermatological specimens from study patients , providing evidence that morgellons disease is associated with an infectious process [ 112115 ] . overall , studies highlight the importance of coinfections since they can complicate the diagnostic process and their pathological synergism can exacerbate cld or induce similar disease manifestations . treatment options are complicated since the population of individuals reporting cld are heterogeneous with guidelines contradicting each other . recommendations about the type and duration of treatments in patients with cld have no factual basis , although prolonged courses of antibiotics are likely to be helpful . recent biostatistical review reveals that retreatment has meaningful clinical benefit in patients who had prior antibiotic treatment [ 116 , 117 ] , with the use of longer ( parenteral ) antibiotic therapy often to be justified . doxycycline is often the preferred agent for oral treatment because of its activity against other tick - borne illnesses , but recent evidence shows that doxycycline - treatment only does not always lead to clinical improvements in either the persistent symptoms or quality of life in patients with cld . this finding can potentially be explained since single antibiotic use often fails to address the different morphological forms of bb as well as biofilm formations in patients with cld . studies indicate the need to include cell wall and cystic and intracellular drugs in any treatment as the different morphological forms of bb display differences in sensitivity to antibiotic treatment . as coinfections are present in most patients , the use of specific antibiotics is required . tetracyclines and macrolides are often used , with quinolones for alternative treatment , particularly gemifloxacin . for bartonella henselae , chlamydia trachomatis , and chlamydophila pneumoniae the combination with rifampicin is recommended . recent evidence shows that novel therapeutic targets for the treatment of the disease should acknowledge a central role of the neutrophil - activating protein a ( napa ) of bb in promoting both regulatory t - cell response and immune suppression in the cerebrospinal fluid of patients with chronic lyme borreliosis . overall , studies show that various therapeutic regimens are used in patients with cld reflecting the need for individualized approaches . the vector model of complexity ( vmc ) is a useful model to describe case complexity in patients with cld . the vector model proposes that the complexity of an individual patient arises out of interactions between different domains : biology , genetics , socioeconomics , environment , behaviour , culture , and the health system . in a chronic condition such as cld , complexity in cld along the biology / genetics axis is important since gender distribution in patients with lyme borreliosis has recently been demonstrated . patients with cutaneous manifestations of lyme borreliosis tend to be predominantly female , whereas those with noncutaneous manifestations are predominantly male . complexity in cld is introduced along the environment axis with a growing number of studies to document the important relationship between the increase in outdoor activities in wooded areas and the incidence of lyme disease . special environmental risk factors include the presence of deer ticks in the home environment , ground cover containing moist humus , and leaf litter in the yard . distribution areas as well as host and vector ranges of lyme borreliosis agents turned out to be much wider than previously thought [ 2 , 124 ] . another element from the environment that contributes to the complexity of cld is that the variety of bb genospecies leads to distinction in clinical manifestations of lyme borreliosis ( lb ) [ 125 , 126 ] . at present , the risk of exposure to multiple pathogens from a single tick bite and the sources of coinfected ticks are not well understood . complexity in cld is also introduced along the behavioral axis as cld has considerable implications on daily life . patients report a significant and severe decline in health status associated with chronic lyme disease . studies have shown that a history of severe , long - term , premorbid , and psychological stress is associated with increased incidence of chronic physical symptoms in lyme disease patients . traumatic psychological experiences predating onset of lyme disease symptoms may play an important etiologic role in the chronicity of these symptoms . as a consequence , the burden of cld is likely to be underestimated as a consequence of the inadequate recognition of the connection between mental and physical health . the complexity of quality assessment is reflected by the lack of tools at the present time that can assess the quality of care delivered to patients with cld . a limited number of quality indicators at the structure , process , or outcome level of care for patients with cld exist , which is in part a consequence of the lack of research on ( the construct of ) cld . in addition , publications that describe approaches to patient involvement in quality indicator development are scarce and to our knowledge nonexisting for patients with cld . there is an abundance of studies on health seeking behaviour highlighting the importance of health system characteristics and their influence on an individual 's behaviour at a given time and place [ 129 , 130 ] . these influences include the financing of care , access to care , coordination mechanisms , and existing stigma on unexplained medical conditions and values and norms . a large survey conducted in the usa in patients with cld demonstrated extensive delays in obtaining an initial diagnosis and having poor access to healthcare [ 46 , 131 ] . negative experiences are associated with reports of dismissive , patronizing , and condescending attitudes in health care providers . studies show that consultations with complementary and alternative medicine ( cam ) practitioners and use of cam therapies are common , representing a cost to patients to be exorbitant and prohibitive . we have outlined the importance of case , care , quality assessment , and health system complexity in patients with cld . the majority of studies focus on cld care and case complexity with a minority of studies to report on cld quality assessment and health system complexity . while specialists are an essential element of the total health care continuum , the majority of patients with cld will continue to access the health care system through family physicians , with hospitalization due to lyme disease to be rare . a range of persistent misconceptions in family medicine exists , ranging from the reliability of available diagnostic tools , the signs and symptoms of nervous system involvement , the importance of coinfections , the appropriate choice and duration of antimicrobial therapy , the importance of jarisch - herxheimer reaction after the commencement of treatment with antibiotics , the curability of the infection , and the cause of symptoms that may persist in some patients after treatment [ 136138 ] . lyme literate family physicians seem to be rare which provides argument to the diagnosis and treatment of cld within the context of a broad - based , multidisciplinary approach to determining the best approach . comprehensiveness is the ability of the family physician to address a broad range of patient problems , whether or not the conditions are within the traditional domain of the specialty in which the physician is trained . referral to lyme literate specialists is essential both to meet the patient 's needs and to establish an integrated care network where responsibilities and tasks are shared . an effective response to the health needs of those with cld will especially require family physicians and specialists to expand their collaborative efforts and knowledge of each other 's practices and treatments . collaboration between family physicians , dieticians , and physiotherapists is considered important since healthy lifestyles are essential in the recovery of patients with cld . another component of the care responsibility of family physicians is ensuring that patients receive preventive interventions . family physicians can contribute to public health interventions in high - incidence age groups focusing on accurately communicating risk , improving knowledge both of lyme disease symptoms and of ticks that carry the disease , as well as teaching preventive behaviors to help reducing tick - borne illness rates . research has shown that patients ' knowledge of tick - borne illnesses is poor and the frequency of practicing preventive behaviors is low . it must be noted , however , that a growing number of patients consult medical sources on the internet with the aim of increasing their health literacy on the subject . family physicians must encourage and support their patients in search of reliable sources of information . the controversy on cld needs to be solved because of the heavy burden of illness associated with cld in patients with or without evidence of bb infection . there is a need for the development and establishment of new clinical diagnostic tools with increased accuracy , sensitivity , and specificity , as well as novel treatment approaches that may reduce the burden of illness and concomitant costs posed by cld . family physicians have a crucial role to play in the prevention and treatment of the disease fostering an integrative multidisciplinary approach to care .

lyme disease has become a global public health problem and a prototype of an emerging infection . both treatment - refractory infection and symptoms that are related to borrelia burgdorferi infection remain subject to controversy . because of the absence of solid evidence on prevalence , causes , diagnostic criteria , tools and treatment options , the role of autoimmunity to residual or persisting antigens , and the role of a toxin or other bacterial - associated products that are responsible for the symptoms and signs , chronic lyme disease ( cld ) remains a relatively poorly understood chronic disease construct . the role and performance of family medicine in the detection , integrative treatment , and follow - up of cld are not well studied either . the purpose of this paper is to describe insights into the complexity of cld as a multidimensional chronic disease construct and its relevance to family medicine by means of a systematic literature review .

1. Introduction 2. Methods 3. Results 4. Conclusion

poly(3-hydroxybutyrate ) , p(3hb ) is a biodegradable thermoplastic polyester accumulated intracellularly by many microorganisms under unfavorable growth conditions . the high production cost of p(3hb ) can be decreased by strain development , improving fermentation and separation processes [ 24 ] , and/or using a cheap carbon source . in p(3hb ) production , about 40% of the total production cost is contributed by the raw material , whereby the cost of carbon feedstock alone accounts for 70 to 80% of the total raw material cost [ 6 , 7 ] . therefore , the utilization of renewable and sustainable substrates for the production of p(3hb ) has become an important objective for the commercialization of bioplastics . a lot of research have been carried out to discuss and propose the utilization of renewable biomass to replace commercial sugars as carbon source in order to reduce the production cost of p(3hb ) [ 812 ] . recently , we reported on the use of oil palm frond ( opf ) juice as the novel and renewable feedstock for the production of p(3hb ) . we demonstrated that opf juice is a good substrate for the production of p(3hb ) from wild - type cupriavidus necator ( ccug52238 ) , with better yield of product formation in comparison to technical grade sugars . this can be explained by the presence of minerals and nutrients in the opf juice which are essential for bacterial growth during fermentation . apart from contributing to higher product formation and microbial growth , the use of opf juice is advantageous compared to the other lignocellulose - based sugars due to the ease in its processing wherein no harsh pretreatment steps and enzymatic treatment will be needed in order to obtain the sugars . in our report , c. necator is well known as polyhydroxyalkanoate ( pha ) producer and its ability to accumulate pha more than 50 wt.% has been previously reported [ 8 , 14 ] . in general , p(3hb ) accumulation is favored by an excess carbon source and inadequate supply of macrocomponents such as nitrogen , phosphate , and dissolved oxygen or microcomponents such as magnesium , sulphate , iron , potassium , manganese , copper , sodium , cobalt , tin , and calcium [ 7 , 15 ] . moreover , it was also reported that the accumulation of p(3hb ) in microorganisms were influenced by several physical parameters including ph and agitation speed [ 1619 ] . in order to make the production of p(3hb ) feasible for industrial application , it is crucial to have high p(3hb ) production yield . in this study , we investigated the effect of initial medium ph , agitation speed , and ammonium sulfate ( nh4)2so4 concentration on p(3hb ) production from c. necator ( ccug52238 ) utilizing opf juice in shake flasks fermentation with the aim to clarify the effect of each fermentation parameter on the microbial growth and p(3hb ) formation . the effect of dissolved oxygen tension ( dot ) level on cell growth and p(3hb ) production was investigated by conducting batch fermentation in 2-l - bioreactor . p(3hb ) produced from this study was then characterized for its thermal and mechanical properties . in this study , c. necator ( ccug52238 ) was obtained from the culture collection , university of goteborg , sweden and used for the production of p(3hb ) . the inoculum preparation , media , and cultivation conditions for c. necator ( ccug52238 ) are similar to those reported by zahari et al . p(3hb ) biosynthesis was carried out through one - stage cultivation fermentation in shake flasks . opf juice in this study was obtained by pressing fresh opf following the method described earlier . opf juice which comprises fructose , glucose and sucrose was diluted from stock ( 55 g / l of total initial sugars and used as carbon sources throughout the study period . in order to study the effect of culture medium initial ph on biosynthesis of p(3hb ) , the initial ph value of each msm and opf juice was adjusted to ph 6.08.0 using 2 m naoh prior to autoclaving . another set of experiment was conducted to study the effect of agitation on p(3hb ) production by testing several agitation speed at 180 , 200 , 220 , 240 , and 260 rpm . for the effect of ammonium sulfate concentration , various concentrations of ( nh4)2so4 in the range of 02.0 the cultures were incubated at 30c under aerobic condition , and all experiments were conducted in duplicates . in order to study the effect of dissolved oxygen tension ( dot ) on cell growth and p(3hb ) production profile under the optimized condition obtained from the shake flask study , batch experiment was conducted in 2-l - bioreactor ( 1 l working volume ) at different dot levels of 20 , 30 , 40 , and 50% . 100 ml of pregrown cells from growing stage were transferred into 900 ml msm in 2l bioreactors ( sartorius , germany ) supplemented with opf juice at 30% ( v / v ) dilution . g / l of initial total sugars concentration was autoclaved separately prior to addition with the msm medium . the msm compositions were prepared as previously reported by zahari et al . , except that 0.5 the temperature inside the bioreactor was set at 30c , while dot level was set at various concentrations of saturation throughout the fermentation using cascade mode and supplied with air at 1.0 vvm . the ph value during fermentation was controlled at ph 7.0 0.05 by 2 m naoh / h2so4 . samples were withdrawn every 5 h for the period of 50 h for the determination of cdw , p(3hb ) concentration , residual sugars , and ammoniacal nitrogen ( nh3-n ) content . residual sugars concentration , cell dry weight measurement , and p(3hb ) analysis were done as previously described by zahari et al . . the samples from the bacterial fermentations were taken at the end of the cultivation period to measure the total dry weight and p(3hb ) content . each sample was centrifuged at 11,000 g for 5 min at 4c ( thermo fisher scientific , nc , usa ) and the solids were washed with distilled water and centrifuged for two consecutive times . dry weight measurements were carried out by drying the solids at 50c and cooling in a desiccator to constant weight . the p(3hb ) content and composition in the lyophilized cell were determined using the gas chromatography ( shimadzu gc-2014 ) . approximately , 20 mg of lyophilized cells were subjected to methanolysis in the presence of methanol and sulfuric acid [ 85% : 15% ( v / v ) ] . the organic layer containing the reaction products was separated , dried over na2so4 , and analyzed by gc according to the standard method using an id - bp1 capillary column , 30 m 0.25 mm 0.25 m film thickness ( sge ) . residual ammoniacal nitrogen ( nh3-n ) content analysis was done using nessler method according to standard procedures ( hach , usa ) which was previously described by zakaria . samples with appropriate dilution factor were filled to 25 ml in the sampling bottles . three drops of mineral stabilizer were added into solution and the bottle was inverted for several times . three drops of polyvinyl alcohol also were added into the solution and mixed well with inversion several times . lastly , 1.0 ml of nessler reagent was added to the mixtures and mix thoroughly by inversion . the standard solution was prepared by replacing the samples with deionised water as blank sample . the sample solution was determined at the wavelength ( ) 425 nm using dr/4000 spectrophotometer by following the manufacturer , instructions ( hach , usa ) . residual sugars were determined by a high performance liquid chromatography ( hplc ) ( agilent series 1200 , usa ) using the supelcosil lc - nh2 column ( sigma aldrich ) ( 25 cm 4.6 mm i d , 5 m particles ) with a ri detector operated at 30c . the mobile phase was acetonitrile : water ( 75% : 25% ) at a flow rate of 1.0 ml / min . the components were identified by comparing their retention times with those of authentic standards under analytical conditions and quantified by external standard method . thermal properties of the polymer were determined by differential scanning colorimetry ( dsc ) ( ta instruments ) . for dsc analysis , 57 mg of homopolymer samples were weighed and heated from 20 to 200c at heating rates 10c / min and held for 1 min . the second scan was used in reheating the samples at the same heating rates and was used in evaluating the thermal properties of the biopolymer . the tensile strength , young 's modulus and elongation to break were determined by using instron universal testing machine ( model 4301 ) at 5 mm / min of crosshead speed . mechanical tensile data were calculated from the stress - strain curves on average of five specimens . the effect of initial medium ph on biosynthesis of p(3hb ) from opf juice was studied by varying the ph between ph 6.0 and 8.0 due to the fact that c. necator can tolerate and produce pha at the aforementioned ph range . suitable initial medium ph is crucial for the cell growth and p(3hb ) accumulation by c. necator ( ccug52238 ) . as shown in table 1 , increasing the initial medium ph value at intervals of 0.5 units affected both the cell growth and p(3hb ) production . both the cell growth and p(3hb ) content were increased when the initial medium ph was increased from ph 6.0 to ph 7.0 , that is , from 6.42 g / l to 8.57 g / l for cdw and 20 wt.% to 34 wt.% for p(3hb ) content , respectively . however , further increase of initial medium ph above ph 7.0 decreased both the cdw and p(3hb ) content . from the results , it can be concluded that ph 7.0 was the optimum initial medium ph for the growth and biosynthesis of p(3hb ) by c. necator ( ccug52238 ) in which , 8.57 g / l of cdw and 34 wt.% of p(3hb ) accumulation was recorded . the optimal ph for the cell growth and p(3hb ) accumulation in this study was similar to those reported in the literature . it was reported that the optimum ph for growth and p(3hb ) production by a. eutrophus was ph 6.9 and that a ph of 5.4 inhibited its growth . on the other hand , lowest cdw and p(3hb ) content , 4.02 g / l and 10 wt.% , respectively , were obtained at ph 8.0 . lowest cell growth and p(3hb ) accumulation at this initial ph value were obtained might due to alkaline condition which could affect the p(3hb ) production . , recommended a ph range of between 6.0 to 7.5 for microbial growth and p(3hb ) production . although p(3hb ) production can be controlled by precisely manipulating the medium ph , it has been reported that ph values other than 7.0 affected p(3hb ) production . these results suggested that p(3hb ) production is sensitive to the ph of cultivation . table 2 displays the effect of agitation speed on biosynthesis of p(3hb ) using opf juice as substrate in shake flasks experiment . it is interesting to note that both cell growth and p(3hb ) production showed an increasing trend with the agitation speed up to 220 rpm . for the agitation speed of more than 220 rpm agitation not only provides mixing and homogeneous cell and heat dispersion in the fermentation broth , but also better aeration for the cells by increasing the oxygen transfer rate throughout the fermentation medium . generally , slower agitation speed may cause the possibilities of cells aggregation , making the culture medium more heterogeneous . this may cause the cell growth to be decreased and thus affecting the production of p(3hb ) . on the other hand , increasing agitation speed higher than its optimal level may reduce the p(3hb ) formation , and hence , the cdw . this is due to the fact that pha is only produced and stored as granules in the cell cytoplasm by microorganisms when they are under stress conditions , for example when there is limitation of nutrient or electron acceptor such as oxygen . in our study , the best condition for the biosynthesis of p(3hb ) is at moderate agitation speed which is at 220 rpm with the highest cdw and p(3hb ) content reaching up to 9.42 ( nh4)2so4 has been widely used as the inorganic nitrogen source for the biosynthesis of p(3hb ) by c. necator . it is important to optimize nitrogen content in fermentation medium as p(3hb ) accumulation in the microorganisms can be triggered when one of the nutrients ( n , p , mg , and o2 ) in the mineral salt is limited in the presence of excess carbon source [ 14 , 15 ] . the effect of different ( nh4)2so4 concentrations on biosynthesis of p(3hb ) by c. necator ( ccug52338 ) from opf juice is summarized in table 3 . in overall , it was observed that cdw was increased when ( nh4)2so4 concentration increased from 0 to 2.0 on the other hand , p(3hb ) accumulation decreased with the increase of ( nh4)2so4 concentration . highest p(3hb ) accumulation at 44 wt.% was achieved when there was no addition of ( nh4)2so4 , in the culture medium . however , unsatisfactory cell growth that is , 5.25 g / l of cdw was observed in the experiment . based on the results , it was also found that ( nh4)2so4 concentration at 0.5 g / l was the optimal concentration for p(3hb ) accumulation and cdw formation , giving 42 wt.% and 8.31 these results corroborate to the literature , which reported that p(3hb ) formation predominantly occurs under - nitrogen and oxygen - limited conditions [ 14 , 15 , 28 , 29 ] . it was discussed that excess nitrogen source may restrict acetyl - coa from entering p(3hb ) production pathways and otherwise channelling into tca cycle for biomass production [ 15 , 21 , 28 ] . biosynthesis of p(3hb ) was then carried out in shake flask under the optimized conditions : initial ph medium , 7.0 ; agitation speed , 220 rpm and ( nh4)2so4 concentration , 0.5 g / l . under these conditions , the p(3hb ) produced from this study is 40% higher compared to the p(3hb ) produced under nonoptimized condition as shown in our previous study . the results presented herewith demonstrated that ph , agitation speed , and nitrogen concentration indeed plays an important role in the p(3hb ) production by c. necator ( ccug52338 ) utilizing opf juice . biosynthesis of p(3hb ) from opf juice by c. necator ( ccug52338 ) was carried out through batch cultivation process in 2-l bioreactor . the effect of dot level in the bioreactor was studied for do concentrations of 20 to 50% and the results are shown in table 4 . it was observed that cdw was increased when dot level increased from 20 to 50% . on the other hand , p(3hb ) g / l ) and p(3hb ) content ( 46 wt.% ) were achieved at 50 and 20% dot level , respectively . based on the result , it was found that dissolved oxygen concentration in the fermentation medium improved the cell growth ; however , p(3hb ) accumulation was found to be increased towards oxygen limitation . this result suggested that appropriate level of oxygen is needed for cell development , and oxygen depletion was favorable for p(3hb ) accumulation . as shown in table 4 , p(3hb ) accumulation was tripled at lower dissolved oxygen concentration ( 20% ) compared to the higher ones ( 50% ) . this might be due to the fact that insufficient supply of oxygen to the bacteria may decrease oxidation of nadh and lead to p(3hb ) biosynthesis [ 15 , 21 , 28 ] . a similar observation was obtained in our previous study on the effect of different ( nh4)2so4 concentration on p(3hb ) production using opf juice in shake flask experiment . these results indicate that both nitrogen and oxygen limitation do not improve cell biomass development , but markedly improve the p(3hb ) accumulation . therefore , it can be suggested that besides nitrogen depletion , oxygen limitation is also important in getting the optimal level of p(3hb ) accumulation . in order to study cell biomass and p(3hb ) production profile by c. necator ( ccug52238 ) , batch cultivation process was carried out using opf juice in 2-l bioreactor with aeration supplied at 30% dot level and the results were depicted in figures 1(a ) and 1(b ) . it was observed that the culture entered the exponential phase after a lag of 15 h , and nitrogen was completely consumed within 35 h. highest cdw ( 11.37 g / l ) and p(3hb ) content ( 44 wt.% ) were achieved at 45 hr cultivation period . the biomass yield ( yx / s ) and p(3hb ) yield ( yp / s ) were 0.81 g biomass / g sugars consumed and 0.36 g p(3hb)/g sugars consumed , respectively . the maximum p(3hb ) productivity was 0.11 g / l / h . almost similar p(3hb ) content with some improvement in cell growth g / l ) and biomass yield ( 0.81 g biomass / g sugars consumed ) obtained in fermentor compared to shake flask were due to different conditions which prevail in the shake flasks and fermentor ; some of these conditions include aeration , agitation , and temperature . in fermentor , aeration was supplied via air sparging , and agitation is provided by an impeller or by the motion imparted to the broth ( liquid phase ) by rising gas bubbles . temperature is maintained at a constant and uniform value by circulation of cooling water through coils in the vessel or in a jacket surrounding the vessel . compared to our previous studies in shake flasks using technical grade sugars , batch studies in 2-l bioreactor using renewable sugars from opf juice showed superior results in c. necator ccug52238 probably due to the additional components in the opf juice that improve the fermentation performance . an almost similar observation was reported by koutinas et al . when wh and fe were used as renewable feedstock for p(3hb ) production . it was reported that the consumption of various carbon sources ( carbohydrates , amino acids , peptides ) presented in the feedstock resulted in high growth yields ( up to 1.07 g cells / g glucose ) as related to glucose . as shown in figures 1(a ) and 1(b ) , the microbial growth is mainly associated with ammoniacal nitrogen consumption . for the first 35 h , lower sugars consumption by c. necator was observed . the sugars consumption within the time range was only 8.03 g / l which is half of the total sugars in the culture broth . on the other hand , the nh3-n was found to be decreased drastically from initial and completely exhausted after 35 h of cultivation period . this result indicates that at initial , the microbial growth was mainly attributed by the consumption of nitrogen sources from ( nh4)2so4 supplied earlier as one of the medium composition in bioreactor . in addition to that , other organic compounds such as amino acids , carbohydrates , and other minerals which were previously characterized in the opf juice could be used as supplementary growth substrates by the bacterium . after that , the cell growth was mainly contributed by the cell expansion due to p(3hb ) accumulation inside the cells . it can be seen that the p(3hb ) accumulation was doubled that is , 20 wt.% to 40 wt.% , from 35 h to 40 h of cultivation period . from sugars consumption and p(3hb ) profiles , it can be observed that the detectable depletion of sugars in the medium from 35 h onwards can be associated with p(3hb ) accumulation . these results are in agreement with the findings of other researchers that reported p(3hb ) accumulation is favored by an excess of carbon source and limited supply of macrocomponents such as nitrogen and dissolved oxygen [ 7 , 16 , 29 ] . it is interesting to note that c. necator ccug52238 completely utilized the glucose in the opf juice . regardless of fructose ( due to too low concentration of fructose in the medium ) , it seems like the bacterium preferred to consume glucose compared to sucrose . glucose consumption rate by c. necator ccug52238 was much higher at 0.33 g / l / h , compared to that of sucrose that is , 0.049 g / l / h . the mechanical and thermal properties of the homopolymer produced in 2-l bioreactor are shown in table 5 . the mechanical and thermal properties of p(3hb ) obtained in this study showed an almost similar properties to those reported in the literature . for instance , the tensile strength and elongation to break for p(3hb ) produced in this study were 40 mpa and 8% , respectively , and it was comparable to the p(3hb ) produced from pure fructose . the melting temperature , tm of p(3hb ) obtained from opf juice ( tm = 162.2c ) , was slightly lower compared to the melting point 177c reported for p(3hb ) produced from pure fructose and other renewable sugars such as maple sap . this could be influenced by other properties of the p(3hb ) such as molecular weight . it has been reported that the molecular weight of p(3hb ) produced is mainly influenced by the type of bacterial strain , substrate , growth rate , and production temperature [ 15 , 29 , 32 ] . this study demonstrated that higher cell growth and p(3hb ) accumulation can be obtained by culturing cupriavidus necator strain ccug52238 at optimized condition using opf juice as the sole renewable carbon source . under the optimal conditions , the highest cell weight was 9.31 0.13 g / l with 45 1.5 wt.% of p(3hb ) contained in the cells , accounts of 40% increment for p(3hb ) content compared to the nonoptimized condition . g / l and p(3hb ) content of 44 wt.% . in the meanwhile , thermal and mechanical characterization of the p(3hb ) obtained from opf juice showed almost similar properties to those reported in the literature . it is worth to mention that this study may contribute to the process development for p(3hb ) production from renewable opf juice in pilot and industrial scale . furthermore , since opf is an abundant solid waste at oil palm plantation and is currently underutilized , it has a great potential to be used as sustainable , renewable , and cheap fermentation feedstock for the production of p(3hb ) .

factors influencing poly(3-hydroxybutyrate ) p(3hb ) production by cupriavidus necator ccug52238 t utilizing oil palm frond ( opf ) juice were clarified in this study . effects of initial medium ph , agitation speed , and ammonium sulfate ( nh4)2so4 concentration on the production of p(3hb ) were investigated in shake flasks experiments using opf juice as the sole carbon source . the highest p(3hb ) content was recorded at ph 7.0 , agitation speed of 220 rpm , and ( nh4)2so4 concentration at 0.5 g / l . by culturing the wild - type strain of c. necator under the aforementioned conditions , the cell dry weight ( cdw ) and p(3hb ) content obtained were 9.31 0.13 g / l and 45 1.5 wt.% , respectively . this accounted for 40% increment of p(3hb ) content compared to the nonoptimized condition . in the meanwhile , the effect of dissolved oxygen tension ( dot ) on p(3hb ) production was investigated in a 2-l bioreactor . highest cdw ( 11.37 g / l ) and p(3hb ) content ( 44 wt.% ) were achieved when dot level was set at 30% . p(3hb ) produced from opf juice had a tensile strength of 40 mpa and elongation at break of 8% demonstrated that p(3hb ) produced from renewable and cheap carbon source is comparable to those produced from commercial substrate .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

a substantial body of evidence indicates , that low - grade inflammation in the vascular endothelium promotes atherogenesis [ 1 , 2 ] . this is indirectly evidenced by the fact that several inflammatory markers are linked to an increased risk of atherosclerosis . high - sensitive crp ( hscrp ) has been reported to associate with an increased risk of myocardial infarction , stroke , sudden death from cardiac causes , and peripheral arterial disease . in addition , hscrp has been shown to predict the first cardiovascular event better than low - density lipoprotein cholesterol ( ldl - c ) . recruitment of inflammatory cells from the circulation and their transendothelial migration is one of the earliest phases of atherogenesis . vascular endothelium expresses cellular adhesion molecules ( cams ) in response to several inflammatory stimuli . the evidence for the role of cams in the pathogenesis of atherosclerosis has emerged from experimental studies which have demonstrated the focal expression of adhesion molecules in atherosclerotic plaques [ 46 ] . selectins and their ligands are involved in the first steps in leukocyte adhesion at sites of endothelial injury as they mediate the rolling and tethering of leukocytes in the vascular wall [ 1 , 2 ] . intercellular adhesion molecule-1 ( icam-1 ) and vascular cell adhesion molecule-1 ( vcam-1 ) induce firm adhesion of inflammatory cells to the vascular surface . overall , both soluble icam-1 ( sicam-1 ) and soluble vcam-1 ( svcam-1 ) have been linked to increase risk of atherosclerosis [ 79 ] . apoe is transported in lipoproteins , and it regulates the hepatic uptake of remnant lipoproteins and facilitates cholesterol efflux from foam cells . in addition , apoe has a central role in inflammation and in mediating the central nervous system response to injury [ 1113 ] . there are three common apoe isoforms , designated e2 , e3 , and e4 , which are encoded for by distinct alleles on human chromosome 19 . apoe isoforms are distributed in six different phenotypes e2/2 , e2/3 , e3/3 , e4/2 , e4/3 , and e4/4 . the presence of e4 isoform is associated with coronary heart disease and with the early onset of alzheimer 's disease . so far , there are sparse data on apoe polymorphism in relation to the parameters of inflammation . mnttri et al showed that dyslipidemic men with e4/4 , e4/3 , and e4/2 phenotypes exhibit lower crp values than men carrying other apoe phenotypes . in a recent report by mrz et al , carriers of apoe2 allele and apoe3/3 homozygotes had significantly higher crp than individuals with the genotypes apoe3/4 and apoe4/4 . in dyslipidemic men and women , c - reactive protein has been reported to be lower in subjects with one or two copies of e4 allele than in those with zero e4 allele . paradoxically , e4 allele has been associated with an enhanced inflammatory reaction after cardiopulmonary bypass . in line , transgenic mice expressing the e4 allele had greater systemic and brain elevations of proinflammatory cytokines than e3 carriers . we hypothesize that the apoe phenotype , as a critical component in inflammatory processes , may influence the levels of inflammation markers . hdl has been demonstrated to exhibit antiinflammatory properties by inhibiting cytokine induced expression of adhesion molecules [ 2123 ] . in contrast , acute inflammation process may decrease hdl concentration and thus compromise its antiatherogenic function [ 24 , 25 ] . therefore we studied the effects of apoe phenotype on the levels of crp and scams on a sample of finnish low - hdl families and in normolipidemic subjects . the study subjects were recruited according to the study protocol as reported previously . to identify the low - hdl families , first degree relatives of the probands were examined . the family members were coded as affected if their hdl - c levels were below the 10th percentile cut - off levels . if at least two affected subjects were identified in the family , all available second - degree relatives and spouses were contacted and included in the study . the blood samples for the lipid and lipoprotein analysis were taken at the same visit as the inflammation parameters and therefore the lipid and lipoprotein values in this study represent the values of that visit , and not those of the screening visit . altogether 211 low - hdl family members and 157 normolipidemic subjects had measures of their apoe phenotype , hscrp , and svcam-1 concentrations . the normolipidemic group comprised healthy volunteers and normolipidemic spouses from the low - hdl families . they were required to have no signs of chd or diabetes , and no lipid lowering medication . the smoking status of the patients was categorized into current smokers and nonsmokers ( including exsmokers ) . hypertension was defined as self - reported use of antihypertensive drugs , systolic blood pressure 140 mmhg , or diastolic blood pressure 90 mmhg . estrogen use was defined as any means of estrogen administration ( oral contraceptives , or oral or transdermal postmenopausal hormone replacement therapy ) . serum levels of sicam-1 , svcam-1 , and se - selectin were measured by using commercially available monoclonal antibody - based elisas ( r & d systems , minneapolis , mn , usa ) . serum highly sensitive crp ( hscrp ) was determined by a commercial kit from medix biochemica , kauniainen , finland . statistical comparisons of clinical and biochemical parameters were performed with spss 11.0 for windows ( spss inc . , chigaco , il , usa ) . the frequency distribution between the groups was compared with the chi - square test . the correlation coefficients between the biochemical and clinical characteristics were examined by pearson 's correlation for continuous variables . multiple regression analysis was performed to determine the relative contribution of different variables to hscrp and svcam-1 values . in the low - hdl group , because the subjects in the present cohort were not independent , we used the family number ( which indicates belonging to a certain family ) as a random variable . family - number - adjusted residuals of log crp and log vcam were used in the multiple regression model for low - hdl family members . since control subjects were not related , the family - number adjustment was performed only in the low - hdl group . the effects of apoe phenotype was studied by dividing subjects into two subgroups ( apoe groups ) : into the e3 group , which included e3/2 and e3/3 phenotypes , and into the e4 group which included e4/2 , e4/3 , and e4/4 phenotypes . table 2 shows that serum hscrp values were significantly lower in the e4 than in the e3 subgroup both in the low - hdl group ( p < .01 ) and in the normolipidemic group ( p < .05 ) after adjustment for age , bmi , serum apoa - i , apoa - ii , apob , hdl - c , ldl - c , triglycerides , smoking status , hypertension , and gender in normolipidemic subjects , and after additional adjustment for family number in the low - hdl group . the concentration of serum svcam-1 was significantly lower in the e4 group than in the e3 group in low - hdl subjects ( p < .01 ) but the significance disappeared after adjustment for family number ( table 2 ) . multiple regression analysis revealed that apoe phenotype was a significant predictor of serum hscrp values both in low - hdl and normolipidemic subjects ( p = .023 and p = .011 , resp ) ( table 3 ) . estrogen used was also a strong predictor of serum hscrp concentration in low - hdl subjects ( p = .026 ) as well as in normolpidemic subjects ( p = .036 ) ( table 3 ) the major finding of the present study is that subjects presenting the apoe4 allele have decreased levels of serum hscrp . this is an interesting observation , since apoe plays a central role in inflammation and atherosclerosis . apoe knockout mice are especially prone to atherosclerosis and to die from lipopolysaccharide ( lps)-induced sepsis . apoe participates in cholesterol efflux , and has been observed to be recycled from triglyceride - rich lipoproteins into hdl - particles [ 10 , 26 ] . in mice , exogenous apoe injection has been demonstrated to decrease the lps - induced production of proinflammatory cytokines , probable by decreasing the release of cytokines from macrophages , which was accompanied by reduced mortality . in addition , human crp transgene expression has been shown to cause accelerated atherosclerosis in apolipoprotein e - deficient mice . thus , according to these studies , the role of apoe in inflammation and atherosclerosis may be protective . despite the importance of apoe in the inflammatory process , only few studies have linked apoe polymorphisms to inflammatory parameters . in the central nervous system , apoe4 has been reported to be less effective than apoe3 or apoe2 at suppressing the activation of microglia in cell culture models of brain inflammation . transgenic mice expressing the apoe4 allele have significantly greater systemic and brain elevations of proinflammatory cytokines tnf - alpha and il-6 as compared with their apoe3 counterparts . in the present study , the hscrp concentration was significantly lower in subjects exhibiting apoe4 allele than in those exhibiting other alleles . likewise , mnttri and associates demonstrated , in a population of 179 dyslipidemic finnish men , that subjects with apoe4/3 phenotype had lower mean crp concentration than those with apoe3/3 phenotype . in japanese americans , crp values have been reported to be significantly higher in apoe2 carriers than in apoe3 or apoe4 carriers , and similar between apoe3 and apoe4 carriers . in line with the japanese data , german patients with or without coronary heart disease also showed higher crp values in apoe2 carriers than in apoe3/4 heterozygotes or in apoe4/4 homozygotes . however , crp was also significantly higher in apoe3/3 homozygotes than in apoe3/4 heterozygotes or in apoe4/4 homozygotes . similarly , in the cardiovascular health study , crp values were lower in apoe4 positive than in apoe4 negative individuals independently of the amount of used alcohol . thus , the present data extend these previous observations to a cohort of low - hdl family members as well as to normolipidemic subjects . the mechanisms behind low serum crp values in apoe4 carriers in this study , and in previous works [ 1618 , 31 ] are unclear . since apoe phenotype remained as a predictor of hscrp in multiple regression model , the difference between apoe4 and apoe3 groups can not be explained by confounding factors , such as estrogen use . one explanation , originally suggested by mrz and associates , could be that the metabolism of crp is related to mevalonate pathway . in carriers of apoe4 , the mevalonate pathway may be down - regulated resulting in decreased crp . the observed low serum crp values in apoe4 carriers in the present study , and reportedly low crp values in apoe4 carriers in previous works [ 1618 , 31 ] , are perplexing findings since apoe4 allele is associated to an increased risk of atherosclerosis . however , in the present study , the low - hdl family members within the e4 group had an atherogenic lipid profile : they had higher concentrations of apob , a trend for higher serum triglycerides , and lower concentration of hdl - c as compared with the e3 group . thus , the changes in lipoproteins rather than changes in inflammatory parameters may explain the atherogenity of apoe4 allele . we conclude that apoe phenotype has a strong influence on c - reactive protein . apoe may be a double - faced protein having different actions in lipid metabolism and inflammation . the clinical characteristics and lipid values of the study subjects . data are expressed as means sems . * p < .05 , p < .06 after adjustment for smoking . the concentrations of hscrp , svcam-1 , sicam-1 , and se - selectin in low - hdl subjects and normolipidemic subjects divided by apoe subgroups . data are expressed as means sems . * * p < .01 ; * p < .05 for the comparison with the e3 group after adjustment for age , gender , bmi , hypertension , smoking status , and lipoprotein variables ; and family number in the low - hdl group . multiple regression analysis of predictors of crp values in low - hdl and normolipidemic subjects . 0.202 ( p < .001 ) for low - hdl subjects and 0.326 ( p < .001 ) for normolipidemic subjects when all variables are included . the dependent variable in low - hdl family members is the family number adjusted residual of log crp , and in normolipidemic subjects , the dependent variable is log crp .

the study was aimed to compare inflammatory parameters between carriers of apoe4 isoforms ( apoe4/3 , apoe4/2 , and apoe4/4 phenotypes ) and those of carrying apoe3 isoform without apoe4 isoform ( apoe3/3 phenotypes and apoe2/3 phenotypes ) . the concentrations of serum hscrp , svcam-1 , sicam-1 , and se - selectin were measured in 211 subjects from finnish low - hdl families and in 157 normolipidemic subjects . the subjects with apoe4 isoform had lower concentrations of serum hscrp both in low - hdl family members ( p < 0.05 ) and in normolipidemic subjects ( p < 0.01 ) . the differences in serum crp values remained significant after adjustment for age , bmi , smoking status , hypertension , gender , lipoprotein variables , and family number . we conclude that apoe phenotype has a strong influence on serum crp values .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION Figures and Tables

the aim of this study was to examine the relationship between cartilage , ligament , and meniscus degeneration and radiographic alignment in severe varus knee osteoarthritis in order to understand the development of varus knee osteoarthritis . fifty - three patients ( 71 knees ) with primary varus knee osteoarthritis and who underwent total knee arthroplasty were selected for this study . there were 6 men and 47 women , with 40 right knees and 31 left knees studied ; their mean age at operation was 73.5 years . the ligament , meniscus , degeneration of joint cartilage , and radiographic alignments were examined visually . the tibial plateau tibial shaft angle was larger if the condition of the cartilage in the lateral femoral condyle was worse . the femorotibial angle and tibial plateau tibial shaft angle were larger if the conditions of the lateral meniscus or the cartilage in the lateral tibial plateau were worse . based on the results of this study , progression of varus knee osteoarthritis may occur in the following manner : medial knee osteoarthritis starts in the central portion of the medial tibial plateau , and accompanied by medial meniscal extrusion and anterior cruciate ligament rupture , cartilage degeneration expands from the anterior to the posterior in the medial tibial plateau . bone attrition occurs in the medial tibial plateau , and the femoro - tibial angle and tibial plateau therefore , the lateral intercondylar eminence injures the cartilage of the lateral femoral condyle in the longitudinal fissure type . osteoarthritis is a degenerative condition that increases in incidence and prevalence with age and is a major cause of morbidity in people older than 65 years . this condition most commonly occurs at the knee , particularly in the medial tibiofemoral compartment , and varus knee osteoarthritis accounts for more than 95% of knee osteoarthritis in japan . despite the occurrence of significant morbidity , the etiology and factors involved in the pathogenesis of osteoarthritis and the development of varus knee osteoarthritis have not been fully elucidated . there have been few reports that compared cartilage , ligament , and meniscus degeneration and radiographic alignment in patients with severe varus knee osteoarthritis . the aim of this study was to examine the relationship between cartilage , ligament , and meniscus degeneration and radiographic alignment in patients with severe varus knee osteoarthritis in order to understand the development of varus knee osteoarthritis , observing in detail the degeneration of the knee compartments of the patients who had total knee arthroplasty for their severe varus knee osteoarthritis . over a 4-year period , 53 patients ( 71 knees ) were selected for this study . the inclusion criteria for the study were primary osteoarthritis in a varus knee and total knee arthroplasty in our hospital . no previous history of anterior cruciate ligament ( acl ) damage was found in these patients , and no previous operations had been done in their affected knee joints . there were 6 men and 47 women , with 40 right knees and 31 left knees studied . the mean age at operation was 73.5 years ( range 60 - 86 years ) . the acl and posterior cruciate ligament ( pcl ) were also inspected and divided into 3 groups : intact , ruptured , or disappeared ( table 2 ) . the lateral meniscus ( lm ) and medial meniscus ( mm ) were also inspected and divided into 3 groups : intact , degenerated , and ruptured or disappeared . assessments of the anterior cruciate ligament ( acl ) , posterior cruciate ligament ( pcl ) , and lateral meniscus ( lm ) in severe varus knee osteoarthritis . intact : visually as having an intact synovial sheath , no fiber disruption , and solid insertion points in the femur and tibia , and clinical assessment demonstrated a hard end point . ruptured : the synovial sheath was not present , and fibers were visually attenuated or stretched , and when clinical assessment demonstrated a soft end point . disappeared : the synovial sheath was not present and the fibers were visually absent . degeneration of joint cartilage in the lateral and medial femorotibial joints were also examined visually . using the international cartilage repair society ( icrs ) classification , cartilage degeneration was divided into 5 grades : grade 0 was normal ; grade 1 was superficial lesions that included soft indentation and/or superficial fissures and cracks ; grade 2 was lesions extending down to less than 50% of cartilage depth ; grade 3 was cartilage defects extending down more than 50% of cartilage depth , as well as down to calcified layer and down to but through the subchondral bone ; and grade 4 was exposure of subchondral bone . the femorotibial joint was divided into 4 zones : lateral femoral condyle ( lfc ) , lateral tibial plateau ( ltp ) , medial femoral condyle ( mfc ) , and medial tibial plateau ( mtp ) . cartilage degeneration of the lfc and ltp was divided into 4 types : type i was defined as all areas of the lfc or ltp were grade 0 of the icrs classification ; type ii was defined as most of the degenerated cartilage area in the lfc or ltp was grade1 ; type iii was defined as most of the degenerated cartilage area in the lfc or ltp was grade 2 ; and type iv was defined as most of the degenerated cartilage area in the lfc or lpt was grade 3 or 4 . moreover , the medial compartment of the tibial plateau was subdivided into 3 zones using the icrs cartilage injury evaluation package : anterior , central , and posterior . based on the wear pattern of the mtp , severe varus knee osteoarthritis was divided into 4 types . ( a ) type a is defined as subchondral bone that is exposed only in the central part of the medial tibial plateau ( mtp ) . ( b ) type b is defined as subchondral bone that is exposed in the anterior and central part of the mtp . ( c ) type c is defined as subchondral bone that is exposed in the central and posterior part of the mtp . ( d ) type d is defined as subchondral bone that is exposed from the anterior to the posterior part of the mtp . the femorotibial angle ( fta ) of the lower limb in the frontal plane four angles were then measured to assess the varus deformity of each knee in figure 2 . four angles to assess the varus deformity of each knee on standard preoperative weightbearing anteroposterior x - rays . ( a ) femorotibial angle ( fta ) between the anatomical axis of the femur and that of the tibia . femoral shaft angle ( fcfs ) between the anatomical axis of the femur and the tangent to the subchondral plate of the femoral condyles . ( c ) femoral condylar tibial plateau angle ( fctp ) between the tangents to the subchondral plates of the femoral and tibial condyles . tibial shaft angle ( tpts ) between the tangent to the subchondral plate of the tibia and the anatomical axis of the tibia . the assessments of the acl , pcl , and lm were compared with the radiographic alignment , and cartilage degeneration of the lateral compartment ( lfc and ltp ) was compared with the radiographic alignment . the type of mtp was also compared with the assessments of the acl , pcl and mm , as well as the radiographic alignment . all data are expressed as mean and standard deviation , and statistical software ( spss version 22 , ibm , armonk , ny , usa ) was used for data analysis . statistical analyses were performed using the mann - whitney u test ( 2 groups ) , tukey - kramer test ( more than 2 groups ) , and the test . if the p value was less than 0.05 , the difference was considered significant . the assessments of the acl , pcl , and lm in severe varus knee osteoarthritis are shown in table 2 . the apl normal group was defined as one in which the acl , pcl , and lm were all intact in severe varus knee osteoarthritis , and the apl abnormal group was defined to include all other cases . with regard to each of the parameters of radiographic alignment , only the fta of the apl normal group was significantly smaller than that of the apl abnormal group ( table 3 ) . therefore , the fta of the patients who had normal acl , pcl , and lm in varus knee osteoarthritis was significantly smaller than the others . the apl normal group was defined as the group in which anterior cruciate ligament ( acl ) , posterior cruciate ligament ( pcl ) , and lateral meniscus ( lm ) were all intact in severe varus knee osteoarthritis , and the apl abnormal group was defined to include all other cases . there is a significant difference ( p = 0.0400 , mann - whitney u test ) . the figures in the parentheses indicate the number of cases . the comparison between the cartilage degeneration of the lfc and the radiographic alignment is shown in table 4 . the tibial plateau therefore , if the bone attrition in the medial tibia increased , the cartilage degeneration in the lateral femoral condyle increased . abbreviations : lfc , lateral femoral condyle ; tpts , tibial plateau tibial shaft angle ; fta , femorotibial angle ; ltp , lateral tibial plateau . there is a significant difference between types i and iv ( p = 0.0443 , tukey - kramer test ) . the figures in parentheses indicate the number of cases . there is a significant difference between types i and iv ( p = 0.0331 , tukey - kramer test ) . there are significant differences between types i and iv and between types ii and iv ( p = 0.0101 , tukey - kramer test ) . the figures in the parentheses indicate the number of cases . the comparison between the cartilage degeneration of the ltp and the radiographic alignment is shown in table 4 . the fta was significantly smaller in type i than in type iv , and the tpts was significantly larger in type iv than in types i and ii . there were no significant differences in the other parameters of radiographic alignment in the cartilage degeneration of the ltp . therefore , if the bone attrition in the medial tibia increased , the cartilage degeneration in the lateral tibial plateau increased . the comparison between the assessment of the lateral meniscus and the radiographic alignment is shown in table 5 . the fta was significantly smaller in type i than in type iii , and the tpts was significantly smaller in type i than in types ii and iii . there were no significant differences in the other parameters of radiographic alignment in the assessment of the lm . therefore , if the bone attrition in the medial tibia increased , the degeneration of the lateral meniscus increased . abbreviations : fta , femorotibial angle ; tpts , tibial plateau tibial shaft angle . there is a significant difference between types i and iii ( p = 0.0118 , tukey - kramer test ) . there are significant differences between types i and ii , and between types i and iii ( p = 0.0009 , tukey - kramer test ) . the figures in parentheses indicate the number of cases . there were no significant differences in the age at operation by medial tibial plateau type . the ratio of intact acl or pcl by the type of medial tibial plateau is shown in figure 3 . there was a significant positive correlation between intact acl and type a medial tibial plateau ( test ; p = 0.0096 ) . the ratio of surviving mm , including intact or degenerated , by the type of medial tibial plateau is shown in figure 4 . independent of the type of medial tibial plateau , the middle and posterior segments of the mm disappeared more than the other portion of the mm . the ratio of the intact anterior cruciate ligament ( acl ) or posterior cruciate ligament ( pcl ) by the type of medial tibial plateau ( mtp ) is shown . there is a significant positive correlation between intact acl and type a mtp ( p = 0.0096 , test ) . the ratio of survived medial meniscus , including intact or degenerated , by the type of medial tibial plateau ( mtp ) is shown . independent of the type of mtp , the middle and posterior segments of the medial meniscus ( mm ) disappeared more than the other portion of the mm . ah , anterior horn ; as , anterior segment ; ms , middle segment ; ps , posterior segment ; ph , posterior horn . in the present study , it appeared that , in the medial compartment , cartilage degeneration in the mtp first occurred in the central portion , and there was a significant positive correlation between intact acl and type a mtp . reported that the wear pattern in the mtp was limited mainly to the central portion in intact acl knees and was observed to enlarge into the adjacent zone in acl - deficient knees . because the femur moves on the tibial plateau in a combination of rolling and sliding , and it has a wider contact area compared with the tibia ; tibias presumably sustain constant mechanical stresses and , therefore , reflect the effect of mechanical stresses more than do femurs . biomechanical descriptions of knee flexion and axial rotation during kneeling or squatting are consistent with the more pronounced anteromedial and posterolateral cartilage wear patterns observed in saudi arabian knees . it can be suggested that changes in histomorphometric parameters of subchondral bone are secondary to cartilage damage and proceed deeper into subchondral bone with increasing cartilage degeneration . bone mineral density of the subchondral tibial bone is significantly correlated with future joint space narrowing and could be used as a predictor of knee osteoarthritis progression . the ratio of intact acl in patients with varus osteoarthritis undergoing total knee arthroplasty was reported to be 35.7% or 58.6% ; it was 44% in the present study . the ratio of disappeared acl was reported to be 25% or 7.1% ; it was 15.5% in the present study . acl rupture is not the primary etiology of the cartilage degenerative changes of the knee in patients affected by late arthritis . it seems that acl rupture might be caused by late degenerative changes due to femoral intercondylar notch abrasion and by the increased joint shear force due to the absence of the physiological cartilage layer . forty - six percent of pcls from patients in the osteoarthritis group had moderate or marked degenerative change on light microscopy , but macroscopically , it was 15% in the present study . the enlargement and displacement of the wear pattern in the mtp seems to be worsened by damage or absence of the mm . found that severe cartilage wear in the central portion of the mtp was a median of 20% when the mm was intact , suggesting the occurrence of a protective function of the meniscus , even when the acl was deficient . in the present study , the ratio of the middle segment of the mm was also the smallest in type a mtp . there was a strong correlation between the degree of mm subluxation and the severity of medial joint space narrowing . in the present study , independent of the type of mtp , the middle and posterior segments of the mm disappeared more than the other portion of the mm . hunter et al . reported that cartilage loss was thought to be the major determinant of alignment , and that other factors , including meniscal degeneration and position , bone attrition , osteophytes , and ligament damage , contribute to the variance in malalignment . the knee bone mineral density distribution in the proximal tibia of the 113 patients was negatively correlated with the axial deformity . increasing varus knee alignment was associated with increasing risk of medial compartment joint space narrowing and osteophytes . in the present study , if varus knee osteoarthritis progressed , the fta , especially the tpts , increased significantly . we thought that one of the main causes of progression of varus knee osteoarthritis was bone attrition in the mtp . reported that the sclerotic changes associated with late - stage osteoarthritis were a late manifestation of the disease . sclerotic changes probably reflected a response of the subchondral bone to the increased load bearing and friction that occurred after loss of the articular cartilage . varus thrust was a potent risk factor , identifiable by simple gait observation , for osteoarthritis progression in the medial tibiofemoral compartment . ackroyd reported that the degenerative process usually started in the anterior half of the tibial plateau and in the corresponding part of the medial femoral condyle when the knee was in 20 to 30 of flexion . subregional analysis suggested that , compared with healthy knees , cartilage changes in radiographic osteoarthritis might occur in both directions . based on the results of the present study , the progression of varus knee osteoarthritis may occur in the following manner : medial knee osteoarthritis starts in the central portion of the mtp , and , accompanied by medial meniscal extrusion and acl rupture , cartilage degeneration expands from the anterior to the posterior in the mtp . therefore , the lateral intercondylar eminence results in an injury of the cartilage of the longitudinal fissure type in the lateral femoral condyle , as shown in figure 5 . only the longitudinal fissure is shown in the lateral femoral condylar cartilage . in the comparison of tibial plateau tibial shaft angle ( tpts ) by lateral femoral condyle ( lfc ) type , type i tpts is significantly smaller than type iv . in medial knee osteoarthritis , it appears that the lateral intercondylar eminence first injure the longitudinal fissure in the lateral femoral condylar cartilage .

objectivesthe aim of this study was to examine the relationship between cartilage , ligament , and meniscus degeneration and radiographic alignment in severe varus knee osteoarthritis in order to understand the development of varus knee osteoarthritis.designfifty-three patients ( 71 knees ) with primary varus knee osteoarthritis and who underwent total knee arthroplasty were selected for this study . there were 6 men and 47 women , with 40 right knees and 31 left knees studied ; their mean age at operation was 73.5 years . the ligament , meniscus , degeneration of joint cartilage , and radiographic alignments were examined visually.resultsthe tibial plateau tibial shaft angle was larger if the condition of the cartilage in the lateral femoral condyle was worse . the femorotibial angle and tibial plateau tibial shaft angle were larger if the conditions of the lateral meniscus or the cartilage in the lateral tibial plateau were worse.conclusionbased on the results of this study , progression of varus knee osteoarthritis may occur in the following manner : medial knee osteoarthritis starts in the central portion of the medial tibial plateau , and accompanied by medial meniscal extrusion and anterior cruciate ligament rupture , cartilage degeneration expands from the anterior to the posterior in the medial tibial plateau . bone attrition occurs in the medial tibial plateau , and the femoro - tibial angle and tibial plateau tibial shaft angle increase . therefore , the lateral intercondylar eminence injures the cartilage of the lateral femoral condyle in the longitudinal fissure type . thereafter , the cartilage degeneration expands in the whole of the knee joints .

Objectives Design Results Conclusion Introduction Materials and Methods Results Discussion

the alarming increase in overweight and obesity has played a pivotal role in the rise of type 2 diabetes prevalence . the obesity epidemic has also been associated with an increased prevalence of sleep disturbances , particularly obstructive sleep apnea ( osa ) ( 2,3 ) . over the past decade , both laboratory and epidemiologic studies have identified poor sleep quality and osa as putative novel risk factors for type 2 diabetes ( 46 ) . osa is a treatable chronic sleep disorder characterized by recurrent episodes of complete ( apnea ) or partial ( hypopnea ) obstruction of the upper airway . osa leads to intermittent hypoxemia and hypercapnia , increased oxidative stress , cortical microarousals , sleep fragmentation , and chronic sleep loss . hypopnea index ( ahi ) 5 events per hour , in nondiabetic obese adults have ranged from 50 to 68% ( 7,8 ) . in recent years , evidence has accumulated to indicate that osa is both a risk factor for type 2 diabetes and an exceptionally frequent comorbidity with an adverse impact on glycemic control . five independent studies , totaling nearly 1,400 patients with type 2 diabetes , have shown that the prevalence of osa ( assessed by polysomnography [ psg ] ) ranges between 58 and 86% ( 9,10 ) . independent of adiposity and other known confounders between the presence and severity of osa and insulin resistance and glucose intolerance in nondiabetic adults ( 1114 ) . two studies that used the gold standard of in - laboratory psg to accurately quantify the severity of osa reported a robust association between increasing osa severity and increasing levels of hemoglobin a1c ( hba1c ) in patients with type 2 diabetes , after controlling for multiple potential confounders ( 9,15 ) . while the findings of these two studies suggested that the effective treatment of osa may be a nonpharmacologic strategy to improve glucose control , the results of the only randomized , placebo - controlled clinical trial examining the impact of continuous positive airway pressure ( cpap ) treatment on hba1c in patients with type 2 diabetes were surprisingly disappointing ( 16 ) . one potential reason for the failure of osa treatment to improve chronic glycemic control in patients with type 2 diabetes is insufficient cpap use . notably , the mean nightly cpap use in this clinical trial was 3.6 h. as most of rapid eye movement ( rem ) sleep occurs in the early morning hours before habitual awakening , one possibility is that with suboptimal adherence to cpap therapy , obstructive apneas and hypopneas during rem sleep were disproportionately untreated compared with events in non - rem ( nrem ) sleep . this may be relevant to glycemic control because it is now well established that compared with nrem sleep , rem sleep is associated with greater sympathetic activity in healthy subjects as well as in patients with osa ( 1719 ) . further , compared with events in nrem sleep , obstructive apneas and hypopneas during rem sleep last nearly 30 s longer and are associated with significantly larger oxygen desaturation ( 2022 ) . therefore , obstructive events during rem sleep , as compared with nrem sleep , may have a larger adverse effect on insulin release and action . this issue has major clinical implications for the duration of cpap use that is needed to reverse the negative consequences of osa on glycemic control in type 2 diabetes . we have therefore performed a detailed analysis comparing the contributions of nrem versus rem osa to glycemic control as assessed by levels of hba1c in a large cohort of adults with type 2 diabetes . we prospectively recruited subjects with established type 2 diabetes using an advertisement posted in the primary care and endocrinology clinics at the university of chicago , inviting participation in a research study on sleep and diabetes . eligible individuals had to meet the criteria for type 2 diabetes based on physician diagnosis using standard criteria ( 23 ) . in order to include individuals with newly diagnosed type 2 diabetes , we also recruited in the community using an advertisement inviting subjects at risk for type 2 diabetes based on age and adiposity to participate in a research study on sleep and metabolism . all participants without an established diagnosis of type 2 diabetes had to undergo a standard 75-g oral glucose tolerance test and meet the american diabetes association guidelines for the diagnosis of type 2 diabetes ( 23 ) . all participants were in stable condition and , when on pharmacological treatment , on a stable antidiabetic medication regimen for the preceding 3 months . exclusion criteria were unstable cardiopulmonary conditions , neurological disorders , psychiatric disease , shift work , chronic insomnia , or any prior or current treatment for osa ( upper airway surgery , cpap therapy , oral appliances , or supplemental oxygen ) . we previously reported the association of osa severity categories ( no , mild , moderate , and severe osa ) with chronic glycemic control in type 2 diabetes using 60 of the participants included in this analysis ( 9 ) . the study was approved by the university of chicago institutional review board , and all participants gave written informed consent . subjects were admitted to the clinical resource center or the sleep research laboratory of the sleep , health , and metabolism center at the university of chicago to undergo an overnight in - laboratory psg . self - reported ethnicity - based diabetes risk was categorized as low - risk category ( non - hispanic whites ) and high - risk category ( african americans , hispanics , and asians ) . the duration of type 2 diabetes and the number of medications were verified by questionnaires as well as review of the patients medical records . hba1c values ( defined as the proportion of hemoglobin that is glycosylated ) were obtained from the patient s chart if assessed during the previous three months ( n = 17,15% of subjects ) or measured on a single blood sample drawn on the morning after the psg ( n = 98 , 85% of the subjects ) . hba1c was measured by bio - rad variant classic boronate affinity - automated high - performance liquid chromatography ( bio - rad , hercules , ca ) . the intra - assay coefficient of variation was 0.51.0% , and the interassay coefficient of variation was 2.22.4% . bedtimes were from 11:00 p.m.12:00 a.m. until 7:00 a.m.8:00 a.m. each subject was recorded for a minimum of 7 h to determine the presence and severity of obstructive respiratory events across the entire night . psg ( neurofax eeg 1100 system , nihon kohden , foothill ranch , ca ) included recordings of six electroencephalographic channels , bilateral electro - oculograms , chin and tibialis electromyogram , electrocardiogram , airflow by nasal pressure transducer and oronasal thermocouples , chest and abdominal wall motion by piezo electrode belts , and oxygen saturation by finger pulse oximeter . all psgs were staged and scored according to the 2007 american academy of sleep medicine manual for the scoring of sleep and related events ( 24 ) . apneas were defined as total cessation of airflow for at least 10 s ( obstructive if respiratory effort was present and central if respiratory effort was absent ) . hypopneas were scored if the magnitude of the ventilation signal decreased by at least 50% of the baseline amplitude of the nasal pressure transducer for at least 10 s and were associated with either a 3% or greater drop in oxygen saturation as measured by finger pulse oximetry or an electroencephalographic microarousal ( 24 ) . the total ahi was defined as the number of obstructive apneas and obstructive hypopneas per hour of sleep . given the minimal presence of central apneas , we did not include these events in the calculation of ahi . the median central apnea index was 0.001 ( interquartile range of 0.0010.41 ) , and the highest central apnea index was 5 . a subject was considered to have mild osa if the ahi was 514 , moderate osa if the ahi was 1529 , and severe osa if the ahi was 30 . rem ahi was calculated as the number of apneas and hypopneas during rem sleep divided by total time in rem sleep in hours . nrem ahi was calculated by dividing the number of apneas and hypopneas during nrem sleep by total time in nrem sleep in hours . the oxygen desaturation index ( odi ) was defined as the total number of oxygen desaturations of at least 3% per total sleep time ( tst ) in hours . the microarousal index ( mai ) was calculated as the total number of microarousals per hour of sleep . differences between subjects with and without osa were tested using the student t test or mann whitney nonparametric test for continuous variables . categorical variables were reported as proportions and were compared using the square test or fisher s exact test . five multivariate linear models were successively fitted to examine associations between hba1c and measures of osa severity after controlling for multiple covariates . model 1 included demographic variables traditionally associated with glycemic control , namely , age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . lastly , model 5 included all the covariates in model 1 plus nrem ahi and rem ahi . since there are individuals who have a significant number of apneas and hypopneas during rem sleep while having an overall ahi below 5 ( hence no osa based on current definitions ) , we included all 115 subjects ( with or without osa ) in the multivariate regression models in order to explore the entire spectrum of rem and nrem events ( ahi , odi , and mai ) . we formally ruled out any evidence of collinearity among the variables entered in the models using standard statistics , including tolerance and variance inflation factor ( spss statistics v20 , ibm , armonk , ny ) . values for hba1c , years of type 2 diabetes , and rem and nrem ahi were submitted to natural log ( ln ) transformation . in order to deal with zero values , the total ahi , rem ahi , and nrem ahi were log transformed using the formula ahi = log(ahi + 0.01 ) . only one outlier was identified ( low hba1c ) , and sensitivity analysis excluding this subject was performed confirming the association between hba1c and measures of rem osa severity . goodness of fit of the models was assessed using diagnostic plots . in order to estimate the effect size of increasing severity of osa on hba1c in a clinically useful manner , models were fitted to estimate the change in adjusted hba1c based on quartiles of rem and nrem ahi . the models included all the covariates in model 5 ( age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use ) and replaced lnrem ahi with rem ahi quartiles while keeping lnnrem ahi in the model . this process was repeated , and lnnrem ahi was replaced with nrem ahi quartiles while keeping lnrem ahi in the model . similar models were fitted for rem and nrem odi and mai quartiles . to simulate the impact of various durations of nocturnal cpap therapy on hba1c , we calculated the mean profiles of cumulative minutes of rem and nrem sleep over 8 h of total recording time from the 115 polysomnograms . we then estimated mean percentages of rem and nrem sleep left untreated after 4 , 6 , and 7 h of optimal cpap treatment eliminating all events . for each duration of cpap use , we entered the number of rem and nrem obstructive events left untreated in a regression model predicting hba1c after adjustment for age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . all statistical analyses were performed using spss statistics v20 and verified using stata ( v10.1 , college station , tx ) . we prospectively recruited subjects with established type 2 diabetes using an advertisement posted in the primary care and endocrinology clinics at the university of chicago , inviting participation in a research study on sleep and diabetes . eligible individuals had to meet the criteria for type 2 diabetes based on physician diagnosis using standard criteria ( 23 ) . in order to include individuals with newly diagnosed type 2 diabetes , we also recruited in the community using an advertisement inviting subjects at risk for type 2 diabetes based on age and adiposity to participate in a research study on sleep and metabolism . all participants without an established diagnosis of type 2 diabetes had to undergo a standard 75-g oral glucose tolerance test and meet the american diabetes association guidelines for the diagnosis of type 2 diabetes ( 23 ) . all participants were in stable condition and , when on pharmacological treatment , on a stable antidiabetic medication regimen for the preceding 3 months . exclusion criteria were unstable cardiopulmonary conditions , neurological disorders , psychiatric disease , shift work , chronic insomnia , or any prior or current treatment for osa ( upper airway surgery , cpap therapy , oral appliances , or supplemental oxygen ) . we previously reported the association of osa severity categories ( no , mild , moderate , and severe osa ) with chronic glycemic control in type 2 diabetes using 60 of the participants included in this analysis ( 9 ) . the study was approved by the university of chicago institutional review board , and all participants gave written informed consent . subjects were admitted to the clinical resource center or the sleep research laboratory of the sleep , health , and metabolism center at the university of chicago to undergo an overnight in - laboratory psg . self - reported ethnicity - based diabetes risk was categorized as low - risk category ( non - hispanic whites ) and high - risk category ( african americans , hispanics , and asians ) . the duration of type 2 diabetes and the number of medications were verified by questionnaires as well as review of the patients medical records . hba1c values ( defined as the proportion of hemoglobin that is glycosylated ) were obtained from the patient s chart if assessed during the previous three months ( n = 17,15% of subjects ) or measured on a single blood sample drawn on the morning after the psg ( n = 98 , 85% of the subjects ) . hba1c was measured by bio - rad variant classic boronate affinity - automated high - performance liquid chromatography ( bio - rad , hercules , ca ) . the intra - assay coefficient of variation was 0.51.0% , and the interassay coefficient of variation was 2.22.4% . bedtimes were from 11:00 p.m.12:00 a.m. until 7:00 a.m.8:00 a.m. each subject was recorded for a minimum of 7 h to determine the presence and severity of obstructive respiratory events across the entire night . psg ( neurofax eeg 1100 system , nihon kohden , foothill ranch , ca ) included recordings of six electroencephalographic channels , bilateral electro - oculograms , chin and tibialis electromyogram , electrocardiogram , airflow by nasal pressure transducer and oronasal thermocouples , chest and abdominal wall motion by piezo electrode belts , and oxygen saturation by finger pulse oximeter . all psgs were staged and scored according to the 2007 american academy of sleep medicine manual for the scoring of sleep and related events ( 24 ) . apneas were defined as total cessation of airflow for at least 10 s ( obstructive if respiratory effort was present and central if respiratory effort was absent ) . hypopneas were scored if the magnitude of the ventilation signal decreased by at least 50% of the baseline amplitude of the nasal pressure transducer for at least 10 s and were associated with either a 3% or greater drop in oxygen saturation as measured by finger pulse oximetry or an electroencephalographic microarousal ( 24 ) . the total ahi was defined as the number of obstructive apneas and obstructive hypopneas per hour of sleep . given the minimal presence of central apneas , we did not include these events in the calculation of ahi . the median central apnea index was 0.001 ( interquartile range of 0.0010.41 ) , and the highest central apnea index was 5 . a subject was considered to have mild osa if the ahi was 514 , moderate osa if the ahi was 1529 , and severe osa if the ahi was 30 . rem ahi was calculated as the number of apneas and hypopneas during rem sleep divided by total time in rem sleep in hours . nrem ahi was calculated by dividing the number of apneas and hypopneas during nrem sleep by total time in nrem sleep in hours . the oxygen desaturation index ( odi ) was defined as the total number of oxygen desaturations of at least 3% per total sleep time ( tst ) in hours . the microarousal index ( mai ) differences between subjects with and without osa were tested using the student t test or mann whitney nonparametric test for continuous variables . categorical variables were reported as proportions and were compared using the square test or fisher s exact test . five multivariate linear models were successively fitted to examine associations between hba1c and measures of osa severity after controlling for multiple covariates . model 1 included demographic variables traditionally associated with glycemic control , namely , age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . lastly , model 5 included all the covariates in model 1 plus nrem ahi and rem ahi . since there are individuals who have a significant number of apneas and hypopneas during rem sleep while having an overall ahi below 5 ( hence no osa based on current definitions ) , we included all 115 subjects ( with or without osa ) in the multivariate regression models in order to explore the entire spectrum of rem and nrem events ( ahi , odi , and mai ) . we formally ruled out any evidence of collinearity among the variables entered in the models using standard statistics , including tolerance and variance inflation factor . values for hba1c , years of type 2 diabetes , and rem and nrem ahi were submitted to natural log ( ln ) transformation . in order to deal with zero values , the total ahi , rem ahi , and nrem ahi were log transformed using the formula ahi = log(ahi + 0.01 ) . only one outlier was identified ( low hba1c ) , and sensitivity analysis excluding this subject was performed confirming the association between hba1c and measures of rem osa severity . goodness of fit of the models was assessed using diagnostic plots . in order to estimate the effect size of increasing severity of osa on hba1c in a clinically useful manner , models were fitted to estimate the change in adjusted hba1c based on quartiles of rem and nrem ahi . the models included all the covariates in model 5 ( age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use ) and replaced lnrem ahi with rem ahi quartiles while keeping lnnrem ahi in the model . this process was repeated , and lnnrem ahi was replaced with nrem ahi quartiles while keeping lnrem ahi in the model . similar models were fitted for rem and nrem odi and mai quartiles . to simulate the impact of various durations of nocturnal cpap therapy on hba1c , we calculated the mean profiles of cumulative minutes of rem and nrem sleep over 8 h of total recording time from the 115 polysomnograms . we then estimated mean percentages of rem and nrem sleep left untreated after 4 , 6 , and 7 h of optimal cpap treatment eliminating all events . for each duration of cpap use , we entered the number of rem and nrem obstructive events left untreated in a regression model predicting hba1c after adjustment for age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . all statistical analyses were performed using spss statistics v20 and verified using stata ( v10.1 , college station , tx ) . therefore , the study was completed by 131 participants . those who obtained less than 4 h of tst during the psg were not included in the analysis ( n = 7 ) . participants were also excluded if the psg data could not be interpreted due to multiple artifacts in the airflow signal ( n = 8) . one patient showed severe oxygen desaturation not explained by apneas or hypopneas consistent with significant hypoventilation . of the 115 subjects included in the study , 56.5% were women , 58.3% were african american , 68.7% were obese , and the mean bmi was 34.5 kg / m . the median duration of type 2 diabetes was 4 years , and a quarter of the subjects were not on any antidiabetic medication . i.e. , neuropathy , nephropathy , retinopathy , coronary artery disease , or peripheral arterial disease ) . mild osa was present in 27% , moderate osa in 28.7% , and severe osa in 29.6% of the subjects . there were no significant differences in sex , race , bmi , years of type 2 diabetes , number of antidiabetic medications , insulin use , and hba1c level between subjects with and without osa , but participants without osa were on average 9 years younger than those with osa . the lack of statistically significant differences in bmi and hba1c may have been related to the small number of subjects without osa . there were no significant differences in total recording time and percentage of slow wave sleep between subjects with and without osa . however , subjects with osa had significantly less tst and percentage of rem sleep and significantly higher wake after sleep onset . within the participants with osa ( n = 98 ) , rem ahi , rem odi , and rem mai were all significantly higher than nrem ahi , nrem odi , and nrem mai . the odi was more than fourfold higher during rem than nrem sleep , but differences in mai were more modest . demographic and clinical features of 115 patients with type 2 diabetes table 2 describes the results of the five multivariate linear regression models predicting hba1c . model 3 shows that nrem ahi was not associated with hba1c ( p = 0.070 ) . in contrast , in model 4 , rem ahi was independently associated with hba1c ( p = 0.001 ) . in model 5 , rem ahi ( p = 0.008 ) remained a significant predictor of hba1c even after adjusting for nrem ahi ( p = 0.762 ) . in the final fully adjusted model 5 , the independent predictors of increased hba1c were rem ahi ( p = 0.008 ) , race risk ( p = 0.001 ) , years of type 2 diabetes ( p = 0.001 ) , and insulin use ( p < 0.001 ) . similar results were obtained when nrem ahi and rem ahi were replaced by the total number of events in nrem and rem sleep , respectively ( p = 0.023 for rem events and p = 0.355 for nrem events ) . multivariate linear regression models predicting natural log of hba1c in patients with type 2 diabetes in order to estimate the effect size of increasing levels of rem ahi and nrem ahi on hba1c , we performed multivariate linear regression models using quartiles of rem ahi and nrem ahi . 1 , after adjustment for age , sex , bmi , race risk , years of type 2 diabetes , insulin use , and lnnrem ahi , increasing quartiles of rem ahi were significantly associated with increasing levels of hba1c ( p = 0.044 for linear trend ) . the mean adjusted hba1c increased from 6.3% in subjects with rem ahi < 12.3 events per hour ( lowest quartile ) to 7.3% in subjects with rem ahi > 47 events per hour ( highest quartile ) . similarly , quartiles of rem odi and rem mai were significantly associated with increasing levels of hba1c . the mean adjusted hba1c increased from 6.5% in the lowest quartile of rem odi to 7.5% in the highest quartile ( p = 0.039 for linear trend ) . similarly , the mean adjusted hba1c increased from 7.6% in the lowest quartile of rem mai to 8.9% in the highest quartile ( p = 0.003 for linear trend ) . in contrast , increasing levels of nrem ahi , nrem odi , and nrem mai quartiles were not associated with hba1c ( fig . 1 ) . adjusted mean hba1c values for rem and nrem ahi , odi , and mai quartiles . for all the panels , multivariate linear regression models were fitted to estimate the mean natural ln hba1c adjusted for demographic variables traditionally associated with glycemic control such as age , sex , ethnicity - based diabetes risk , bmi , ln years of type 2 diabetes , and insulin use . in addition , panels are adjusted for ( a ) lnnrem ahi , ( b ) lnrem ahi , ( c ) lnnrem odi , ( d ) lnrem odi , ( e ) lnnrem mai , and ( f ) lnrem mai . age and bmi are centered at their means : 55 years old and 35 kg / m , respectively . the corresponding -coefficients for each quartile were then exponentiated to convert from ln hba1c to the standard values of hba1c . figure 2a illustrates the predominance of rem sleep in the later part of sleep . in our cohort , 3 and 4 h after lights off , on average only 25 and 40% of rem sleep had occurred , respectively . optimally titrated cpap use for 3 or 4 h would treat only 25 or 40% of rem sleep , respectively , and would leave most obstructive events during rem sleep untreated . figure 2b and c illustrate the simulated impact of 4 , 6 , and 7 h of cpap use in men and women with low and high race / ethnicity - based diabetes risk . this simulation clearly shows that the metabolic benefit of 4 h of cpap use , often considered as adequate cpap compliance , is modest , while a much more clinically significant effect can be obtained when treatment is extended to 6 h and beyond . cumulative minutes of rem and nrem sleep over 8 h of bedtime ( a ) and simulation of various hours of cpap use in men and women with type 2 diabetes based on race / ethnicity - based diabetes risk ( b and c ) . a : data are summarized as mean sd of cumulative rem and nrem sleep minutes from lights off to lights on in 115 subjects with type 2 diabetes . the mean duration of rem and nrem sleep in our cohort was 82 and 298 min , respectively . using cpap for 3 or 4 h from the time lights are turned off will cover only 25 or 40% of rem sleep , respectively , and will leave most obstructive events during rem sleep untreated . b and c : simulation of the impact of 4 , 6 , and 7 h of cpap use in four groups of subjects based on sex and race / ethnicity - related diabetes risk . with this simulation , 4 h of cpap use would treat 40% of rem sleep and would lead to a drop in adjusted hba1c of 0.230.28% . in contrast , 7 h of cpap therapy would treat 87% of rem sleep and lead to a decrease in adjusted hba1c between 0.87 and 1.1% . this study reveals that hba1c , a measure of chronic glycemic control in patients with type 2 diabetes , is adversely associated with obstructive apneas and hypopneas that occur in rem sleep ( rem ahi ) but not in nrem sleep ( nrem ahi ) . the independent association between rem ahi , rem odi , and rem mai and hba1c is robust and of clinical significance , with a difference of 1.0% hba1c between the lowest and highest quartiles of rem ahi as well as rem odi and of 1.3% hba1c between the lowest and highest quartiles of rem mai after adjusting for all the covariates . these effect sizes are comparable to what would be expected from widely used antidiabetic medications . the severity of osa in our cohort was greater in rem sleep than in nrem sleep , as evidenced by a higher ahi and a nearly fourfold higher odi . thus , despite the shorter duration of rem sleep , exposure to the adverse consequences of osa , particularly intermittent hypoxemia , was greater during rem than nrem sleep . surprisingly , in our diabetic participants with osa , nrem ahi only predicted approximately 25% of the variance of rem ahi . whether hyperglycemia plays a role in this relative independence of the severity of rem osa relative to nrem osa remains to be determined . multiple mechanistic pathways are likely to be involved in the link between rem osa and poorer glycemic control in subjects with type 2 diabetes . when compared with nrem sleep or quiet wakefulness , rem sleep is associated with increased sympathetic activation and reduced vagal tone in normal subjects and even more so in patients with osa ( 1719 ) . most endocrine organs releasing hormones involved in glucose regulation are sensitive to changes in sympathovagal balance . well - documented examples relevant to metabolic risk are pancreatic insulin secretion , hepatic glucose production , and adipocyte regulation of energy balance ( 2527 ) . in addition , peptidergic factors originating from the intestine ( glucagon - like peptide-1 and glucose - dependent insulinotropic polypeptide ) augment the insulin response induced by nutrients . the secretion of these incretin hormones is intimately linked to autonomous nervous system ( 2830 ) . however , it is important to point out that the impact of osa on sympathetic activation in patients with type 2 diabetes of long duration remains unclear and that long - standing hyperglycemia may lead to reduction in sympathetic activity . lastly , obstructive apneas and hypopneas during rem sleep lead to greater degrees of hypoxemia than in nrem sleep ( 21,22 ) . in the present cohort , rem odi was indeed much greater than nrem odi . intermittent hypoxemia has been shown to be toxic to -cell function in murine models of sleep apnea ( 31,32 ) . the findings from our analyses strongly suggest that rem - related obstructive respiratory events are of clinical significance for the severity of type 2 diabetes . two recent studies that performed continuous interstitial glucose monitoring simultaneously with psg directly support our hypothesis that rem - related osa may have adverse metabolic consequences ( 33,34 ) . one of these studies included 13 obese patients with type 2 diabetes with severe osa and compared them with 13 obese patients with type 2 diabetes without osa with similar demographic characteristics . although there was no difference in the mean diurnal glycemic level between the two groups , the mean glycemic level was 38% higher during rem sleep in those with osa ( 33 ) . they found that in the absence of osa , rem sleep leads to a larger decline in interstitial glucose concentration than nrem sleep . in contrast , osa during nrem sleep had no impact on interstitial glucose concentrations ( 34 ) . taken together , the evidence from studies assessing interstitial glucose levels supports our finding that obstructive events during rem sleep are adversely associated with glucose metabolism . while our participants did not meet any proposed definition of rem - related or rem - predominant osa ( 35,36 ) , our findings suggest that failure to recognize and treat osa in rem sleep may be of critical clinical significance for glycemic control in diabetic patients . in clinical practice , 4 h of nightly cpap use is considered adequate adherence to therapy ( 37 ) . indeed , a randomized controlled trial of cpap therapy in patients with type 2 diabetes reported an average use of 3.6 h per night ( 16 ) . the severity of residual osa was not estimated . the disappointing results of cpap trial in type 2 diabetes may reflect the failure to treat rem osa due to insufficient cpap use , leaving most obstructive events during rem sleep untreated . alternatively , there may be other factors beyond poor cpap adherence that led to a lack of improvement in glycemic control such as a poor reserve in -cell function . our analyses show that based on the distribution of cumulative rem sleep in our cohort , cpap therapy for the first 4 h after lights off would leave 60% of obstructive events during rem sleep untreated and would be associated with a decrease in the adjusted hba1c by only 0.250.28% . in contrast , 7 h of optimal cpap therapy would be associated with a decrease in the adjusted hba1c by 0.871.1% . first , we used hba1c , the most commonly used measure in clinical practice , to assess glycemic control . therefore , we can not ascertain whether the mediating pathways linking rem ahi to hba1c involve increased insulin resistance or impaired -cell function . moreover , we only measured hba1c at a single time point , which was not consistently on the same day as psg . however , treatment was stable for the preceding 3 months in all participants , and hba1c was measured on the morning after the psg in 98 out of 115 participants ( 85% ) . although hba1c reflects glycemic control 10 to 12 weeks before the assay , it mostly reflects glucose fluctuations during the last 6 weeks of the measurement . despite our efforts to ensure treatment stability in the prior 3 months , we can not exclude the possibility that fluctuations in adherence to medications may have influenced hba1c levels . our study did not assess associations between rem osa and glucose control in subjects with prediabetes or normal glucose tolerance . we also had a large proportion of african americans and subjects requiring little or no antidiabetic medications . therefore , it would be important for our findings to be replicated in larger and more diverse cohorts , including participants with more diabetes complications and/or longer disease duration as well as individuals with prediabetes or with normal glucose tolerance but at high risk for type 2 diabetes . also , we did not have a measure of habitual sleep duration , which may be important in evaluating chronic exposure to rem osa , and our only measure of adiposity was the bmi . lastly , the cross - sectional nature of the study does not address the direction of causality . indeed , only rigorously designed intervention studies will provide causal evidence between disordered breathing during rem sleep and glucose metabolism dysregulation . in summary , our findings support the notion that osa in rem sleep has a strong and clinically significant association with glycemic control in subjects with type 2 diabetes . since rem sleep is dominant during the latter part of the sleep period , rem - related osa may often remain untreated with 4 h of cpap use . our analyses suggest that to achieve clinically significant improvement in glycemic control in patients with type 2 diabetes , cpap use may need to be extended beyond 6 h per night . further research is needed to elucidate the mechanistic pathways linking osa during rem sleep and adverse metabolic outcomes .

objectiveseverity of obstructive sleep apnea ( osa ) has been associated with poorer glycemic control in type 2 diabetes . it is not known whether obstructive events during rapid eye movement ( rem ) sleep have a different metabolic impact compared with those during non - rem ( nrem ) sleep . treatment of osa is often limited to the first half of the night , when nrem rather than rem sleep predominates . we aimed to quantify the impact of osa in rem versus nrem sleep on hemoglobin a1c ( hba1c ) in subjects with type 2 diabetes.research design and methodsall participants underwent polysomnography , and glycemic control was assessed by hba1c.resultsour analytic cohort included 115 subjects ( 65 women ; age 55.2 9.8 years ; bmi 34.5 7.5 kg / m2 ) . in a multivariate linear regression model , rem apnea hypopnea index ( ahi ) was independently associated with increasing levels of hba1c ( p = 0.008 ) . in contrast , nrem ahi was not associated with hba1c ( p = 0.762 ) . the mean adjusted hba1c increased from 6.3% in subjects in the lowest quartile of rem ahi to 7.3% in subjects in the highest quartile of rem ahi ( p = 0.044 for linear trend ) . our model predicts that 4 h of continuous positive airway pressure ( cpap ) use would leave 60% of rem sleep untreated and would be associated with a decrease in hba1c by approximately 0.25% . in contrast , 7 h of cpap use would cover more than 85% of rem sleep and would be associated with a decrease in hba1c by as much as 1%.conclusionsin type 2 diabetes , osa during rem sleep may influence long - term glycemic control . the metabolic benefits of cpap therapy may not be achieved with the typical adherence of 4 h per night .

Introduction Research Design and Methods Participants Experimental Protocol PSG Statistical Analysis Results Conclusions Supplementary Material

peripheral veno - arterial extracorporeal membrane oxygenation ( va - ecmo ) is increasingly used in acute , severe cardiogenic shock refractory to conventional therapy . although percutaneous femoral insertion of va - ecmo is straightforward , the procedure is usually performed after intubation under general anaesthesia , and invasive ventilation is continued for the whole period of extracorporeal support . here , we show that va - ecmo can be successfully performed with neither ( non- ) invasive ventilation nor deep analgosedation . a 51-year - old man with a history of hypertension and diabetes mellitus presented to the local hospital in cardiogenic shock ( blood pressure , 80/40 mmhg ) . electrocardiography ( ecg ) revealed a ( sub)acute anterior myocardial infarction complicated by complete atrioventricular block and a slow ventricular escape rhythm of 30 beats per minute ( fig . 1 ) . after initial medical therapy ( volume resuscitation , inotropes ) , the clinical condition allowed urgent transport to an interventional centre . on arrival , immediate transvenous ventricular pacing echocardiography showed preserved wall thickness , but extensive apical and anteroseptal akinesia and inferior hypokinesia , with a large left ventricular ( lv ) apical thrombus ( left ventricular ejection fraction ( lvef ) , 10 % ) . 1serial electrocardiograms ( upper panel ) and transthoracic echocardiograms ( lower panel ) on presentation ( left ) and on recovery 6 weeks later ( right ) showing a large dilated anterior myocardial infarction with partial recovery of contractile function . arrow indicates lv apical thrombus , which dissolved over time after therapeutic anticoagulation ( recovery ) . rv right ventricle , lv left ventricle serial electrocardiograms ( upper panel ) and transthoracic echocardiograms ( lower panel ) on presentation ( left ) and on recovery 6 weeks later ( right ) showing a large dilated anterior myocardial infarction with partial recovery of contractile function . arrow indicates lv apical thrombus , which dissolved over time after therapeutic anticoagulation ( recovery ) . rv right ventricle , lv left ventricle since cardiogenic shock persisted , va - ecmo was initiated under local anaesthesia via femoral access ( 25f venous cannula , 19f arterial cannula ) as a bridge to recovery and additional therapy . during the procedure , the patient was fully awake , non - intubated and breathing oxygen through a non - rebreathing facemask . va - ecmo yielded 4-l / min flow , and a mean arterial pressure of 6070 mmhg was achieved . an adequately supported circulation was reflected by normal blood gas analysis , lactate levels , venous oxygen saturation and renal function ; sinus rhythm ensued on day 2 . coronary angiography revealed severe three - vessel disease ( proximally occluded right , significantly stenosed circumflex and subtotally occluded left anterior descending coronary artery ) unattractive for immediate revascularisation . the clinical course was complicated by obvious signs of pulmonary oedema on serial chest radiographs ( fig . 2 ) in the absence of dyspnoea ( respiratory rate of 1520 per minute ) and with adequate oxygenation ( sao2 , 97100 % ; pao2 > 100 mmhg ) . therefore , heart failure therapy ( captopril , eplerenone , isosorbide mononitrate ) was tailored with a pulmonary artery catheter ( pac ) and intensified ( furosemide , hydrochlorothiazide ) to accomplish negative fluid balances . daily gradual tapering of ecmo flow ( l / min ) during weaning off ecmo support ( lower panel ) serial chest x - rays indicating decrease of pulmonary congestion ( upper panel ) . daily gradual tapering of ecmo flow ( l / min ) during weaning off ecmo support ( lower panel ) after 4 days of va - ecmo under low - dose continued intravenous analgesia ( 1 mg morphine / h ) , support flow was gradually reduced ( 1 l / min per day ) under therapeutic heparinisation and pac monitoring . on day 8 , echocardiography revealed partial lv recovery ( lvef , 25 % ) during normally conducted sinus rhythm ( fig . 1 ) and 1 l / min support flow , allowing weaning off the va - ecmo . in a stable condition , the patient was mobilised and discharged from the intensive care unit ( icu ) on day 11 . based on myocardial viability studies ( mid anteroseptal viability on cardiac magnetic resonance perfusion imaging ) , a drug - eluting stent was placed in the left anterior descending artery on day 21 , followed by clinical discharge on day 37 . there is accumulating evidence that severe , refractory cardiogenic shock can be supported by percutaneous va - ecmo as a bridge to recovery , long - term mechanical support and eventually heart transplantation . yet , va - ecmo is cumbersome , as patients are generally managed and instrumented in the icu with invasive ventilation , analgosedation and feeding via a nasogastric tube . this renders va - ecmo a complex and invasive therapeutic approach with numerous potential icu - related complications . in contrast to va - ecmo standard care , we managed this case without intubation , ventilation or significant analgosedation , which has , to the best of our knowledge , not been reported in this way before . we used pac monitoring for haemodynamic tailoring and strived for optimal filling pressures ( wedge pressures < 15 mmhg ) , while allowing adequate systemic va - ecmo flow . during va - ecmo weaning , we monitored cardiac recovery by continuously measuring intrinsic cardiac output on gradual daily reduction of support flow ( fig . 1 ) moreover , spontaneous respiration was carefully monitored by pulse oximetry and repeated blood gas analyses , while pulmonary oedema was aggressively treated by combined diuretic therapy ( fig . 1 ) . this strategy seems imperative as , for example , pneumonia may render va - ecmo without intubation impossible . others have reported on awake va - ecmo requiring additional percutaneous atrioseptostomy to treat pulmonary congestion , which was not necessary in our patient . moreover , neurological performance remained unremarkable and could easily be evaluated in this cooperative patient . neurological assessment is of utmost importance during peripheral va - ecmo , especially on recovering cardiac function and pending pulmonary oedema , which might cause hypoxaemia of the proximal aortic arch , that is , the harlequin syndrome . after roughly a week of va - ecmo support , cardiac recovery was accompanied by immediate physical mobilisation creating optimal conditions for the subsequent clinical course . va - ecmo for severe and refractory cardiogenic shock may successfully be performed without mechanical ventilation . careful monitoring of volume status and respiratory condition is imperative and allows keeping patients comfortable and cooperative also during prolonged periods of extracorporeal support . the limited invasiveness of this stand - alone va - ecmo approach may encourage a more widespread and accessible use in cardiogenic shock .

we report on the use of percutaneous femoral veno - arterial extracorporeal membrane oxygenation ( va - ecmo ) in a fully awake , non - intubated and spontaneously breathing patient suffering from acute , severe and refractory cardiogenic shock due to a ( sub)acute anterior myocardial infarction . intensified heart failure therapy was closely monitored with a pulmonary artery catheter and allowed gradual weaning off the ecmo support without additional invasive measures , notably without mechanical ventilation . neurological assessment was possible at all times and complete physical mobilisation was straightforward directly after weaning from ecmo . this limited invasive approach may encourage a more widespread use of percutaneous va - ecmo .

Introduction Discussion Conclusion

gas storage in porous materials is a desirable technology that has been significantly developed in recent years , owing to its potential to address numerous persisting challenges in a number of industrial applications related to energy , environment , and health care sectors . in the context of clean energy , there is an amplified willingness to reduce greenhouse gas emissions , caused by energy production processes , as evidenced by the considerable ongoing research in academia and industry alike aiming to develop practical solutions to mitigate this problem . correspondingly , appropriate studies have been conducted in order to practically deploy relatively cleaner alternative fuels such as methane ( ch4 ) , a primary component of natural gas ( ng ) and biogas . ch4 is of great interest as a fuel for stationary and mobile applications due to ( i ) its high h to c ratio in comparison to other fossil fuels , resulting in relatively lower co and co2 emissions , and ( ii ) its lower sulfur and nitrogen contents , leading to lessened sox and nox emissions . the aforementioned attributes position ch4 as a fuel appreciably cleaner than gasoline and diesel . nonetheless , the main drawback of ch4 , in comparison to liquid fossil fuels , is its low volumetric energy density . therefore , the development of suitable and sustainable on - board vehicle methane storage solutions , close to room temperature , is vital to the successful deployment of methane as a conventional fuel for transport applications . highly porous materials represent an interesting category of adsorbents that display distinct structural advantages for ch4 storage . the appropriate combination of a high surface area associated with a considerable pore volume with a suitable pore shape and functionality , in a given porous material , is crucial to achieve the desired enhanced ch4 storage uptake and a practical working capacity at a set pressure and temperature . it is notable that the volumetric working capacity is an essential parameter to assess the material s performance toward ch4 storage . the working capacity represents the usable amount of ch4 derived by subtracting the unused adsorbed ch4 , corresponding to the uptake at the delivery pressure ( 5 bar ) , from the uptake at the maximum adsorption pressure ( 35 bar or higher ) . prominently , one of the pathways to enhance the methane working capacity of a given porous material is to regulate its methane uptake at relatively low pressures and subsequently reduce the unused ch4 uptake up to the 5 bar threshold . relatedly , the storage of other gases such as nitric oxide ( no ) and carbon dioxide ( co2 ) has been previously studied and explored for various relevant applications . in contrast , studies pertaining to high - pressure oxygen ( o2 ) storage are still scarce . the availability of high amounts of o2 is of prime importance in the health care domain , particularly in the treatment of respiratory insufficiencies and in hyperbaric oxygen changes for the treatment of carbon monoxide poisoning . correspondingly , a large amount of oxygen is used to enrich air during catalyst regeneration in the catalytic cracking units . markedly , there is a significant need to develop efficient pathways to store o2 for various industrial needs . metal organic frameworks ( mofs ) , a special class of solid - state materials , have emerged as modular and functional porous materials that can offer potential to address many enduring challenges pertaining to energy and environmental sustainability . principally , advances in mof chemistry have permitted the successful implementation of reticular chemistry ; predesigned building blocks were directed to assemble into a preset network topology . namely , the molecular building block ( mbb ) approach has been deployed to construct targeted functional mofs , where desired structural and geometrical attributes are incorporated into the mbbs prior to the assembly process . in effect , isolating reaction conditions that consistently permit in situ formation of the requisite inorganic mbbs , in the presence of a suitable organic linker , is essential for the successful implementation of the mbb approach and the subsequent formation of the targeted mof with the desired network topology . advantageously , the mbb approach permits the rational assembly of targeted mofs and their subsequent structural fine tuning using isoreticular chemistry . the deliberate alteration of a mof dimensionality and functionality without changing its underlying topology , isoreticular chemistry , is regarded as a powerful pathway for the development of new functional materials with distinctive properties . evidently , the selection of an appropriate mof platform , with desired topological attributes for the logical practice of isoreticular chemistry , offers the potential to readily access porous materials suitable to address the ongoing gas storage challenges . practically , several key requisites have been considered and targeted in order to facilitate the attainment of a high - storage mof media : ( i ) inorganic mbbs based on light and abundant elements , ( ii ) organic mbbs amenable to size , shape , and functionality adjustments via expansion and decoration , ( iii ) an elect mof platform that permits access to isoreticular mofs with concomitant high surface area , large pore volume , and fine - tuned pores in the micropore domain , and ( iv ) an mof platform based on an underlying topology that prohibits interpenetration upon mof expansion . in this context , considering the aforementioned requisites , we identified the mof platform based on the soc topology ( square - octahedral ) as a promising platform to access isoreticular mof materials for potential use in gas storage and separation applications . the soc - mof platform offers interesting structural features where the pore system comprised of cavities and channels can be fine tuned in the micropore domain by the judiciously fine tuning the square building unit ( the tetracarboxylate ligand ) . it is notable that the first reported soc - mof materials ( in- , ga- , and fe - based soc - mof ) revealed an exceptionally high gas storage density for h2 and ch4 despite their associated moderate surface area and pore volume in comparison to the best storage mof materials . congruently , we found it compelling to target soc - mofs with relatively larger surface areas and pore volumes via isoreticular chemistry , where the expansion strategy is employed to construct isoreticular soc - mofs based on selected / compatible and expanded organic mbbs , and subsequently evaluate their performance for storage of valuable commodities such as ch4 , h2 , co2 , and o2 . markedly , the construction of a highly microporous mof with a soc topology requires the judicious selection of an expanded rectangular organic linker that facilitates the in situ formation of the targeted inorganic oxo - centered trinuclear m(iii ) cluster [ m3(3-o)(o2c)6 ] ( m = in , al , fe , etc . ) . specifically , targeting mofs based on the trinuclear aluminum(iii ) cluster will permit the development of a relatively low cost material with tailored properties for gas storage applications . it is important to note that mofs based on the trinuclear al(iii ) cluster [ al3(3-o)(o2c)6 ] , a 6-connected mbb , are scarce , with only a few examples reported in the literature due to challenges in isolating reaction conditions that allow the in situ formation of the aforementioned inorganic mbb . in this work , we report the synthesis and the structure , on the basis of single - crystal x - ray diffraction ( scxrd ) studies , of the first aluminum mof having the soc topology and possessing an exceptional porosity . importantly , this is the first report disclosing the assembly of the oxo - centered trinuclear aluminum(iii ) cluster ( oxo - centered aluminum(iii ) trimer ) [ al3(3-o)(o2c)6 ] , with a quadrangular ligand into a given mof , namely the highly microporous al - soc - mof-1 with more than 2 cm / g pore volume and 6000 m / g apparent langmuir surface area . furthermore , the use of similar reaction conditions , which afforded the synthesis of the parent al - soc - mof-1 , in the presence of functionalized tetracarboxylate linkers ( with naphthalene or anthracene replacing the phenyl core in the parent ligand ) resulted in two new isoreticular structures : namely the naphthalene species al - soc - mof-2 and the anthracene species al - soc - mof-3 . extensive gas adsorption studies were carried out on these isoreticular soc - mofs with different gases ( n2 , co2 , ch4 , o2 ) at low pressures ( cryogenic temperatures ) and at high pressures . in particular , ch4 and o2 adsorption isotherms were investigated experimentally at different temperatures and over a wide range of pressures up to 85 and 120 bar , respectively . it was found that al - soc - mof-1 has one of the highest ever total and working gravimetric ch4 uptakes at 35 bar and higher pressures at any given temperature . in contrast to all other best mofs reported to date in the open literature for ch4 storage , the parent al - soc - mof-1 sorption studies revealed an enhancement in the volumetric ch4 storage working capacity when the temperature was decreased . particularly , at 258 k and 80 bar , the al - soc - mof-1 fulfilled the department of energy ( doe ) target ( both gravimetric and volumetric ) and exhibited the highest working volumetric capacity of 264 cm ( stp)/cm . correspondingly , to the best of our knowledge , al - soc - mof-1 showed the highest gravimetric total capacity for co2 and o2 among microporous mofs . furthermore , molecular simulation studies supported and confirmed our experimental results for ch4 storage and thus encouraged us to explore various plausible theoretical isoreticular al - soc - mofs , based on expanded ( or contracted ) and/or functionalized tetracarboxylate organic building blocks , for ch4 storage . this study permitted us to ( i ) pinpoint various prospective al - soc - mofs with outstanding ch4 storage capabilities similar to those of the parent al - soc - mof-1 , confirming the superior volumetric and gravimetric storage capabilities of the parent al - soc - mof-1 , and ( ii ) gain a better understanding of the structure property relationship , deriving a better correlation between the soc - mof gas storage properties and the makeup of the soc - mof porous system ( pore shape and size , ligand dimensions and functionalities ) . in our effort to isolate the first aluminum - based soc - mof , numerous attempts have been carried out to isolate reaction conditions that consistently allow the in situ formation of the desired trinuclear aluminum(iii ) mbb [ al3(3-o)(o2c)6 ] . accordingly , we designed and synthesized the tetratopic ligand 3,3,5,5-tetrakis(4-carboxyphenyl)-p - terphenyl ( h4tcpt ) ( h4l1 ) that can act as a rectangular mbb . successfully , reactions between h4l1 and alcl36h2o in acidic solution containing a mixture of n , n-dimethylformamide ( dmf ) and acetonitrile ( ch3cn ) afforded colorless homogeneous crystals with a cube - shaped morphology , characterized and formulated by scxrd as [ al3o(tcpt)1.5(h2o)3]|cl| ( 1 ) . the crystal structure of 1 reveals a 3-periodic framework built up from 3-oxo - centered trinuclear al(iii ) inorganic mbbs [ al3(3-o)(h2o)3(o2c)6 ] . each aluminum cation ( al ) displays an octahedral coordination environment and coordinates to six oxygen atoms : namely , four bis - monodentate deprotonated carboxylate oxygen atoms from four independent tcpt ligands and one 3-oxo anion , and a terminal aqua ligand to complete the coordination sphere . the trinuclear al(iii ) mbbs are bridged by six independent tcpt ligands , resulting in the formation of a 3-periodic cationic framework , al - soc - mof-1 ( figure 1 ) . the charge balance is provided by the presence of chloride ions , which was confirmed by an x - ray photoelectron spectroscopy ( xps ) experiment ( figure s11 in the supporting information ) . crystallographic analysis affirms that the chloride ions are disordered over six positions around the trinuclear al(iii ) cluster with equal probability . this analysis was also supported by a al solid - state nmr spectroscopy experiment ( figure s12 in the supporting information ) . crystal structure of 1 showing the assembly of the trinuclear aluminum(iii ) mbb [ al3(3-o)(h2o)3(o2c)6 ] with the organic ligand h4tcpt ( h4l1 ) ( middle ) and topological analysis of 1 , where the 6-connected trinuclear al(iii ) mbb can be viewed as a trigonal - prismatic sbu , while the organic ligand can be rationalized as a 4-connected building unit to give ( 4,6)-c soc - net ( left ) or can be viewed as 3-c sbus resulting in a ( 3,6)-c derived net edq ( right ) . topological analysis reveals that 1 has the anticipated edge transitive ( 4,6)-connected net with the soc underlying topology . the trinuclear al(iii ) mbb [ al3(3-o)(o2c)6 ] can be regarded as a trigonal - prismatic secondary building unit ( sbu ) with the six points of extension corresponding to the carbon of the carboxylate moieties matching the vertex figure of the 6-c node in the soc net . the 6-c inorganic mbbs are joined by the rectangular organic ligand , 4-c node , into a primitive cubic system arrangement ( figure 1 ) . alternatively from a topological perspective , the 4-c rectangular ligand can be regarded as comprised of two interconnected 3-c triangular sbus that are further linked through the 6-c trigonal - prismatic sbus to afford a mof related to a ( 3,6)-c derived net edq , with transitivity 2 2 ( figure 1 and figures s25 and s26 in the supporting information ) . in this paper , the reported al - mofs will be referred to as al - soc - mofs . in order to isolate other isoreticular analogues of al - soc - mof-1 , the phenyl ring located at the core of the h4tcpt ligand was substituted by 1,4-naphthalenyl and 9,10-anthracenyl cores to give the naphthalene- and anthracene - functionalized ligands 3,3,5,5-tetrakis(4-carboxyphenyl)-1,4-diphenylnaphthalene ( h4tcdpn ) ( h4l2 ) and 3,3,5,5-tetrakis(4-carboxyphenyl)-9,10-diphenylanthracene ( h4tcdpa ) ( h4l3 ) , respectively ( figure 2a ) . as anticipated , under reaction conditions similar to those used to isolate the al - soc - mof-1 , cube - shaped crystals were obtained and characterized using scxrd and powder x - ray diffraction ( pxrd ) studies ( figure 2b ) , revealing the construction of two isoreticular al - soc - mof compounds , naphthalene al - soc - mof-2 ( 2 ) and anthracene al - soc - mof-3 ( 3 ) with the formula [ al3o(ligand)1.5(h2o)3]|cl| . ( a ) representation of the organic mbbs used to construct isoreticular al - soc - mofs . ( c ) nitrogen isotherms at 77 k for the isoreticular al - soc - mofs . the phase purity of each al - soc - mof compound ( 13 ) was confirmed by whole profile pattern matching using the le bail method ( figures s13s15 in the supporting information ) . the al - soc - mof-1 structure encloses cubic - shaped cages 14.3 in diameter delimited by six tcpt ligands , which occupy the faces of the cage , and eight inorganic trinuclear al(iii ) clusters located on the vertices of the cuboidal cage . the cage is accessible through apertures of approximately 5.6 8.4 , taking van der waals ( vdw ) radii into consideration . the structure also encloses two well - defined 1d infinite channels with estimated dimensions of 14 ( vdw ) , which is approximately at the border of microporous materials ( figures s23 and s24 in the supporting information ) . the corresponding solvent - accessible free volumes for 13 were estimated to be 80.5% , 79% , and 75% , respectively , by summing voxels more than 1.2 away from the framework using platon software . in light of the extraordinarily pure microporous architecture exhibited by 13 , optimization of the conventional activation conditions ( drying under vacuum and heating ) showed that the guest solvent in the pores could be easily removed using a traditional approach ( vacuum and heating ) without altering their microporosity . nitrogen ( n2 ) adsorption measurements at 77 k were carried out on the acetonitrile - exchanged samples , showing a fully reversible type i isotherm representative of porous materials with permanent microporosity ( figure 2c ) . g for 2 , and 5212 and 4849 cm / g for 3 ( table 1 ) . it is notable that the resultant high microporosity ( surface area and pore volume ) is exceptional and has not been observed , prior to this work , using a traditional activation method that often causes pore collapse in the case of highly porous mofs . such a unique feature is of prime importance for the implementation and deployment of 13 as gas storage media for onboard or stationary gas storage applications . abet and alang are the experimental bet and langmuir specific surface areas . pvtheo and pvexp are the calculated pore volume from crystal structures and the experimentally measured pore volume , respectively . the successful use of the conventional activation method was confirmed by the excellent agreement between the experimental and the optimal theoretical pore volumes ( pvexp = 2.3 , 2.1 , and 1.8 cm / g and pvtheo = 2.3 , 2.2 , and 1.9 cm / g for 13 , respectively ) . furthermore , al - soc - mof structures preserved their optimal porosity after heating up to 340 c under vacuum ( figures s27b , s34b , and s36b in the supporting information ) , another essential feature that is rarely observed for highly porous mofs . the high thermal stability was also confirmed using variable - temperature pxrd studies and thermal gravimetric analysis ( figures s17s22 in the supporting information ) . interestingly , the extremely open structure , exclusively concerted in the microporous range , combined with the distinctive structural features ( presence of cages and channels ) place al - soc - mofs as ideal adsorbent candidates for gas storage studies . accordingly , ch4 adsorption on 13 was extensively studied experimentally at variable temperatures and up to 80 bar as well as at low pressure and 112 k ( boiling point of ch4 ) . the methane adsorption isotherms at 112 k for 13 revealed remarkable ch4 uptakes near saturation pressures e.g. , 1336 , 1205 , and 1055 cm ( stp)/g at p / p0 = 0.95 , respectively ( figures s28a , s35a , and s37a in the supporting information ) . importantly , high - pressure ch4 adsorption isotherms at variable temperatures , depicted in figure 3 , showed that 1 has one of the highest ch4 gravimetric uptakes ever reported ( ca . 361 cm ( stp)/g ) for any microporous mof materials under the disclosed doe operational storage conditions ( 298 k and 35 bar ) . mesoporous mof-210 and dut-49 displayed uptakes of around 210 and 364 cm(stp)/g under the same conditions . interestingly , the doe ch4 gravimetric uptake target of 700 cm ( stp)/g ( 0.5 g / g ) was addressed and reached for relatively high pressures at temperatures below 288 k : e.g. 50 bar at 258 k and 85 bar at 288 k ( figure 3 ) . single - component gas adsorption isotherms for ch4 at different temperatures for al - soc - mof-1 , showing total ch4 gravimetric uptakes surpassing the doe target at particular pressures and temperatures . additionally , analysis of the volumetric ch4 adsorption isotherms , using the al - soc - mof-1 crystal density , revealed an enhancement in the volumetric ch4 storage working capacity when the temperature was decreased ( figure 4 ) . specifically , the volumetric ch4 storage working capacity for 1 increased from 201 cm ( stp)/cm to 264 cm ( stp)/cm when the temperature was decreased from 298 to 258 k at working pressures between 80 bar ( adsorption ) and 5 bar ( desorption ) . this attribute , unique to al - soc - mof-1 , is unprecedented , as all available ch4 storage data for mofs have shown a conventional decrease in the volumetric ch4 storage working capacity with a decrease in temperature , as illustrated in figure 4 and table s3 in the supporting information for utsa-76 , hkust-1 , ni - mof-74 , nu-111 , and pcn-14 . it is notable that mof-519 was not included in this comparative assessment , as the associated data at low temperatures were not available for this highly ch4 adsorbing mof . comparison of the ch4 volumetric working capacities ( 580 and 565 bar ) at different temperatures ( 258 , 273 , and 298 k ) for al - soc - mof-1 with the best microporous mof materials reported to date . a comprehensive comparison of absolute ch4 uptakes and working capacities for al - soc - mof-1 ( 1 ) with the various best mof materials reported so far under different temperature and pressure conditions is presented in figure 5 and figure s38 in the supporting information . interestingly , although the total volumetric ch4 uptake for 1 is relatively lower than those of some of the highly adsorbing mofs , 1 displayed mutually high volumetric and gravimetric working capacities at different working temperatures and pressures . this notable and rare compromise between the gravimetric and the volumetric capacities for 1 is a result of the reduced unused ch4 uptake below 5 bar and the linear trend of the ch4 isotherms at relatively high pressures , a desirable attribute for an appropriate gas storage medium . in contrast to the best mofs reported so far for ch4 storage , 1 exhibits a relatively low ch4 heat of adsorption ( 11 kj / mol at low loading ) in the relatively low ch4 loading region , only slightly higher than the ch4 latent heat of evaporation ( figure s30 in the supporting information ) . markedly , the comparatively favorable methane adsorption at relatively high pressures can be attributed to enhanced ch4ch4 interactions regulated by the appropriate pore size of al - soc - mof-1 . therefore , the combination of both aforementioned effects , governing the ch4 adsorption at low and high pressures , in a single material afforded the exceptional ch4 working capacities observed for the al - soc - mof-1 , especially at relatively low temperatures . it is notable that 1 exhibits the second highest ch4 volumetric working capacity at 298 k and 580 bar working pressure range : namely , 201 cm ( stp)/cm vs 230 cm ( stp)/cm for the recently reported mof-519 . noticeably , the two synthesized isoreticular al - soc - mofs ( 2 and 3 ) also exhibited high gravimetric and volumetric total and working ch4 uptakes that were only slightly lower than the uptake values derived for 1 . the experimental results for 2 and 3 are summarized in figure s41 and table s4 in the supporting information . total ( a d ) at 65 and 80 bar and 565 and 580 bar working ( e h ) ch4 gravimetric and volumetric uptakes for al - soc - mof-1 ( 1 ) in comparison to the best mof materials reported to date at 298 , 270 , and 240 k. the data for al - soc - mof-1 ( 1 ) were collected at 298 , 273 , and 258 k. with the aim of gaining a better understanding of the structure property relationship governing the resultant high methane storage capacities in the al - soc - mofs , we assessed theoretically the plausible ch4 storage capacity of various plausible theoretical isoreticular al - soc - mofs based on expanded ( or contracted ) and/or functionalized tetracarboxylate organic building blocks . in order to derive a better correlation between the soc - mof gas storage properties and the makeup of the soc - mof porous system ( pore shape and size , ligand dimensions and functionalities ) , a comprehensive molecular simulation study was carried out on various hypothetical / isoreticular al - soc - mofs constructed using hypothetical organic building blocks . first , we envisioned validating our molecular simulation methodology on the parent 1 ( al - soc - mof-1 ) and corroborating the mechanism of ch4 adsorption at 5 bar and at higher pressures , resulting in the outstanding ch4 working capacity . second , we simulated and evaluated the absolute and working ch4 uptakes for various hypothetical isoreticular soc - mofs , constructed using different optimized ( i ) elongated , ( ii ) functionalized , and ( iii ) contracted organic mbbs . in effect , the simulated isoreticular al - soc - mofs were assembled using both experimentally synthesized and hypothetically designed linkers obtained by modifying the arms and/or the core of the al - soc - mof-1 ligand as shown in figure 6 and figure s47 and scheme s2 in the supporting information . figure 6 depicts the naming scheme employed to label the hypothetical al - soc - mof linkers . for example , the original linker is shown in figure 6 ( left ) , where p - p denotes phenyl phenyl : both the arm and the core have one phenyl group . phenyl for the arm and acetylene phenyl acetylene for the core , as shown in figure 6 ( right ) . a total of 18 theoretical analogues were hypothetically assembled , and their associated ch4 adsorption isotherms were simulated . for clarity the new simulated al - soc - mof structures will be named and referred to using the associated linker name . scheme illustrating the adapted naming for the employed hypothetical organic ligands and associated hypothetical al - soc - mofs . initially , grand canonical monte carlo ( gcmc ) simulations of methane adsorption were performed for the parent compound 1 in order to compare first the resulting simulated results with available experimental adsorption data , and subsequently validate the simulation approach adopted in this study ( figure 7a and figure s44 in the supporting information ) . more details about the employed simulation method are described in the supporting information . as shown , the theoretical ch4 adsorption isotherm for p - p is in a good agreement with the experimental data ( 1 ) . the corresponding screenshots of methane adsorption at different relevant pressures , i.e. 5 bar ( limiting desorption pressure ) and 35 , 65 , and 80 bar ( storage pressures ) , are shown in figure 7b . the relatively very low simulated unused methane uptake at 5 bar ( both gravimetric and volumetric ) for p - p equivalent to 1 , at 5 bar , was confirmed by performing adsorption isotherms at different temperatures : namely , at 298 , 295 , 273 , and 258 k ( figure s45 in the supporting information ) . this is reflected in the observed nonpreferential positions of ch4 molecules in the framework of p - p ( figure 7b ) : i.e. , no specific preferential adsorption sites at 5 bar . ( a ) methane sorption in al - soc - mof-1 at 298 k : simulation ( red filled circles ) vs experiment ( black filled squares ) . ( b ) screenshots of methane adsorption in p - p ( equivalent to al - soc - mof-1 ) at 298 k at different pressures : 5 , 35 , 65 , and 80 bar . the purple spheres surrounding the framework represent the methane molecules . delightfully , the simulated ch4 adsorption isotherms on the optimized structure , on the basis of a molecular mechanics simulation approach ( details in the supporting information ) , of the parent 1 ( p - p ) are in good agreement with the corresponding ch4 adsorption isotherms simulated on the experimental structure ( figure s46 in the supporting information ) . accordingly , the same molecular mechanics optimization procedure was employed to construct 18 hypothetical isoreticular al - soc - mofs and subsequently simulate their associated total and working ch4 uptakes using the gcmc approach . the simulated absolute volumetric and gravimetric ch4 adsorption isotherms for p - p ( 1 ) and the other isoreticular al - soc - mofs were simulated at various temperatures and up to 80 bar total pressure ( figures s76s84 in the supporting information ) . interestingly , these results showed that the use of ( i ) elongated , ( ii ) functionalized , and ( iii ) contracted linkers resulted in three distinguished al - soc - mof groups in terms of gravimetric volumetric uptake tradeoff . in fact , the use of ( i ) elongated arms and/or cores , such as pp - pp , led to an increase in the gravimetric uptake at the expense of the volumetric uptake under any pressure and temperature conditions evaluated in this study . ( ii ) functionalizing the phenyl core of the linker led generally to lower gravimetric uptake but still with good gravimetric volumetric uptake tradeoff under any pressure and temperature conditions explored here . in a particular case , strategies for functionalization of the phenyl cores with different functional groups , such as cf3 and br , led to relatively good working volumetric uptakes ( due to the relatively higher framework density ) , albeit with much lower working gravimetric uptakes . interestingly , ( iii ) contraction of the arms and/or the core , for instance the a - p structure , offer potential for a collective improvement ( ideal compromise ) of the gravimetric and volumetric uptakes in comparison to 1 at any temperature , particularly at intermediate pressures of 35 bar ( figure 8) . principally , in terms of ch4 working ( 535 bar ) volumetric and gravimetric capacity tradeoff , contraction of the arms showed a notable improvement in the absolute ch4 uptake at 298 k. this unique compromise can be attributed to the enhanced ch4 adsorption uptake at relatively high pressures below 35 bar , due to the reduced channel dimensions in the a - p structure in comparison to the parent al - soc - mof-1 ( 9.3 10.0 vs 14.0 14.2 ) . it is important to note that the al - soc - mof-1 ( p - p ) still exhibits both enhanced volumetric and gravimetric tradeoff for 565 and 580 bar working pressures at any temperature ( figure 8) . total ( a ) at 35 bar and ( b ) 535 bar working ch4 gravimetric and volumetric uptakes for p - p and a - p simulated structures in comparison to the best mof materials reported to date at 298 k. theoretical total ( left , c and e ) and working ( right , d and f ) gravimetric vs volumetric capacities for selected hypothetical isoreticular al - soc - mofs under a wide range of pressures ( 35 , 65 , and 80 bar ) at different temperatures ( 298 and 258 k ) in comparison to al - soc - mof ( 1 ) . the purple area represents the desired range of the best compromise between gravimetric and volumetric total and working uptakes . a summary of the theoretical results in terms of ch4 absolute and working capacities , volumetric and gravimetric , at 298 , 273 , and 258 k under a wide range of pressures are presented in figures s48s66 in the supporting information . the exceptional methane storage capabilities of al - soc - mof-1 have inspired us to extend this study to other important commodities , namely o2 and co2 . accordingly , we recorded various o2 and co2 adsorption isotherms for al - soc - mof-1 . notably , the amounts of o2 and co2 adsorbed in al - soc - mof-1 near saturation pressures ( 0.95 p / p0 ) , derived from adsorption isotherms at 90.2 and 195.15 k for o2 and co2 , respectively , were found to be remarkably high ( 1757 and 1236 cm ( stp)/g , respectively ) ( figure s29 in the supporting information ) . markedly , the combination of experimentally accessible low pressure ( at 90.2 k ) and high - pressure o2 adsorption data up to 115 bar ( at 298 k ) ( combined with the toth model ) revealed that al - soc - mof-1 exhibits a record of 29 mmol / g absolute gravimetric o2 uptake at 140 bar , which is much higher than those of hkust-1 ( 13.2 mmol / g ) and nu-125 ( 17.4 mmol / g ) , reference materials for this application ( figure 9 ) . additionally , al - soc - mof-1 displayed a record deliverable capacity between 5 and 140 bar of 27.5 mmol / g , vs 11.8 and 15.4 mmol / g for hkust-1 and nu-125 , respectively ( figure 9a ) . consequently , by neglecting the effect of packing density and the void space occupied by the material , a 1 l cylinder filled with al - soc - mof-1 will potentially enhance the volumetric o2 storage capacity ( 172 cm / cm ) by 70% at 100 bar , in comparison to a conventional empty cylinder ( figure 9b ) . it is notable that , if we assume a prospective 25% loss associated with packing density , the al - soc - mof-1 volumetric o2 storage capacity still offers a 25% enhancement over an empty cylinder . analysis of the o2 adsorption recorded at variable temperatures indicated that 1 exhibits a relatively low o2 heat of adsorption ( 10 kj / mol at low loading ) over the whole o2 loading range , slightly higher than the o2 latent heat of evaporation ( figure s33 in the supporting information ) . ( a ) single - component gravimetric gas adsorption isotherm for o2 at 298 k showing that 1 exhibits the highest deliverable uptake reported so far . ( b ) volumetric o2 adsorption isotherm compared to the storage capacity in a pressurized container . additionally , the co2 adsorption studies revealed that al - soc - mof-1 exhibits an exceptional absolute gravimetric co2 uptake at 40 bar of 2 g / g ( 1020 cm ( stp)/g ) vs 1.5 g / g for mof-177 , setting a new record among microporous mofs ( figure s39 in the supporting information ) . consequently , al - soc - mof-1 displays the uppermost working capacity between 1 and 40 bar of 1.90 g / g ( 967 cm ( stp)/g ) vs 1.46 g / g ( 742 cm ( stp)/g ) for mof-177 . an analysis of variable - temperature co2 adsorption data showed that al - soc - mof-1 exhibits a relatively low co2 heat of adsorption ( 17 kj / mol at low loading ) over the whole co2 loading range ( figure s31b in the supporting information ) . it is important to note that the mesoporous mof-210 exhibits the highest absolute gravimetric co2 uptake at 50 bar ( 2.8 g / g ) . finally , it is worth noting that al - soc - mof-1 also exhibits an excellent h2 storage capacity at 77 k ( ca . 11 wt % ) ( figure s40 in the supporting information ) at high pressure ( 30 bar ) in comparison to other highly porous materials . similarly , o2 and co2 adsorption studies were performed for the two isoreticular al - soc - mofs ( 2 and 3 ) , which revealed that the naphthalene and anthracene analogues exhibit only slightly lower o2 and co2 adsorption uptakes under the same conditions in comparison to 1 ( figures s42 and s43 and table s4 in the supporting information ) . in summary , we successfully employed the molecular building block approach to synthesize the first aluminum soc - mof isoreticular materials . specifically , reaction conditions that consistently permit the in situ generation of the [ al3(3-o)(h2o)3(o2c)6 ] mbb were isolated and used for the construction of a highly porous ( 4,6)-connected aluminum based soc - mof , al - soc - mof-1 , with more than 6000 m / g langmuir specific surface area . importantly , tedious activation using dry supercritical co2 is not required in order to activate the al - soc - mof-1 and its naphthalene and anthracene analogues . in particular , the conventional activation technique , i.e. a simple combination of heating and vacuum ( or n2 flush ) , is sufficient for full activation prior to gas loading unloading cycles . extensive gas adsorption studies were carried out on the al - soc - mof platform with different gases ( n2 , co2 , ch4 , o2 , etc . ) . methane adsorption isotherms were completed at different temperatures and over a wide range of pressures , up to 85 bar . interestingly , it was found that al - soc - mof-1 exhibits one of the highest total and working gravimetric ch4 uptakes at 35 bar . in contrast to the other reported best mofs for ch4 storage , al - soc - mof-1 showed enhanced ch4 storage working capacity as the temperature was decreased . particularly , at 258 k and 80 bar , al - soc - mof-1 fulfils the doe target and exhibits the highest working volumetric capacity of 264 cm ( stp)/cm . to the best of our knowledge , this is the first time that a porous material has fulfilled both the challenging gravimetric and volumetric targets for the ch4 working capacity . the collective experimental and gcmc simulation studies indicated that the parent al - soc - mof-1 , in contrast to various hypothetical isoreticular al - soc - mofs based on contracted , elongated , and functionalized ligands , exhibits the best compromise between the volumetric and gravimetric total and working uptakes over a wide range of pressure and temperature conditions . details on the synthesis of the organic ligands used in this study , 3,3,5,5-tetrakis(4-carboxyphenyl)-p - terphenyl ( h4tcpt ) , 3,3,5,5-tetrakis(4-carboxyphenyl)-1,4-diphenylnaphthalene ( h4tcdpn ) , and 3,3,5,5-tetrakis(4-carboxyphenyl)-9,10-diphenylanthracene ( h4tcdpa ) , are provided in the supporting information . single - crystal diffraction data were collected at beamline i19 , diamond light source , didcot , u.k . , using the wavelength 1.0402 at 250 k. fourier - transform infrared ( ft - ir ) spectra ( 4000600 cm ) were collected in the solid state on a nicolet 700 ft - ir spectrometer . the peak intensities are described in each of the spectra as very strong ( vs ) , strong ( s ) , medium ( m ) , weak ( w ) , broad ( br ) , and shoulder ( sh ) . powder x - ray diffraction ( pxrd ) measurements were performed on a panalytical mpd xpert pro x - ray diffractometer at 45 kv and 40 ma for cu k ( 1.5418 ) equipped with a variable - temperature stage , with a scan speed of 20/min . the sample was held at the designated temperature for at least 10 min between each scan . high - resolution dynamic thermal gravimetric analysis ( tga ) was performed under a continuous n2 flow and recorded on a ta instruments high - resolution tgaq500 thermogravimetric analyzer . low - pressure gas sorption measurements were performed on a fully automated autosorb-1c gas sorption analyzer ( quantachrome instruments ) . high - pressure gas sorption studies were performed with a magnetic suspension balance marketed by rubotherm ( bochum , germany ) . a solution of alcl36h2o ( 29 mg , 0.015 mmol ) and h4l1 ( 7.1 mg , 0.01 mmol ) in n , n - dimethylformamide ( dmf ) ( 1 ml ) , acetonitrile ( ch3cn ) ( 1 ml ) , and nitric acid ( 3.5 m , 0.5 ml ) was prepared in a 20 ml scintillation vial and subsequently placed into a preheated oven at 130 c for 12 h to give pure small colorless cube - shaped crystals . suitable single crystals were obtained using the same synthetic procedure , but with an increase in the amount of hno3 to l ml . crystals of al - soc - mof-1 were harvested , washed with ch3cn , and air - dried . ft - ir ( 4000650 cm ) : 3349 ( br ) , 1605 ( s ) , 1592 ( s ) , 1423 ( s ) , 1387 ( vs ) , 1312 ( w ) , 1243 ( w ) , 1100 ( w ) , 1018 ( w ) , 854 ( w ) , 830 ( w ) , 783 ( s ) , 771 ( s ) , 701 ( s ) . a solution of alcl36h2o ( 29 mg , 0.015 mmol ) and h4l2 ( 7.6 mg , 0.01 mmol ) in n , n - dimethylformamide ( dmf ) ( 1 ml ) , acetonitrile ( ch3cn ) ( 1 ml ) , and nitric acid ( 3.5 m , 0.5 ml ) was prepared in a 20 ml scintillation vial and subsequently placed into a preheated oven at 130 c for 12 h to give pure small colorless cube - shaped crystals . suitable single crystals were obtained using the same synthetic procedure , but with an increas in the amount of hno3 to l ml . crystals of al - soc - mof-2 were harvested , washed with ch3cn , and air - dried . ft - ir ( 4000650 cm ) : 3349 ( br ) , 1606 ( s ) , 1545 ( m ) , 1422 ( s ) , 1384 ( s ) , 1241 ( w ) , 1100 ( w ) , 1015 ( w ) , 851 ( w ) , 851 ( w ) , 771 ( s ) , 705 ( m ) . a solution of alcl36h2o ( 29 mg , 0.015 mmol ) and h4l3 ( 8.1 mg , 0.01 mmol ) in n , n - dimethylformamide ( dmf ) ( 1 ml ) , acetonitrile ( ch3cn ) ( 1 ml ) , and nitric acid ( 3.5 m , 0.3 ml ) was prepared in a 20 ml scintillation vial and subsequently placed into a preheated oven at 130 c for 12 h to give a pure microcrystalline yellow powder . suitable single crystals were obtained using the same synthetic procedure , but with an increase in the amount of hno3 to 1 ml crystals of al - soc - mof-3 were harvested , washed with ch3cn , and air - dried . ft - ir ( 4000650 cm ) : 3349 ( br ) , 1606 ( s ) , 1547 ( s ) , 1442 ( s ) , 1387 ( s ) , 1312 ( w ) , 1241 ( w ) , 1181 ( w ) , 1016 ( w ) , 852 ( m ) , 771 ( s ) , 706 ( s ) .

the molecular building block approach was employed effectively to construct a series of novel isoreticular , highly porous and stable , aluminum - based metal organic frameworks with soc topology . from this platform , three compounds were experimentally isolated and fully characterized : namely , the parent al - soc - mof-1 and its naphthalene and anthracene analogues . al - soc - mof-1 exhibits outstanding gravimetric methane uptake ( total and working capacity ) . it is shown experimentally , for the first time , that the al - soc - mof platform can address the challenging department of energy dual target of 0.5 g / g ( gravimetric ) and 264 cm3 ( stp)/cm3 ( volumetric ) methane storage . furthermore , al - soc - mof exhibited the highest total gravimetric and volumetric uptake for carbon dioxide and the utmost total and deliverable uptake for oxygen at relatively high pressures among all microporous mofs . in order to correlate the mof pore structure and functionality to the gas storage properties , to better understand the structure property relationship , we performed a molecular simulation study and evaluated the methane storage performance of the al - soc - mof platform using diverse organic linkers . it was found that shortening the parent al - soc - mof-1 linker resulted in a noticeable enhancement in the working volumetric capacity at specific temperatures and pressures with amply conserved gravimetric uptake / working capacity . in contrast , further expansion of the organic linker ( branches and/or core ) led to isostructural al - soc - mofs with enhanced gravimetric uptake but noticeably lower volumetric capacity . the collective experimental and simulation studies indicated that the parent al - soc - mof-1 exhibits the best compromise between the volumetric and gravimetric total and working uptakes under a wide range of pressure and temperature conditions .

Introduction Results and Discussion Conclusions Experimental Section

each year , an estimated 10 million health care office visits to gynecologists are due to vulvovaginitis . vulvovaginitis refers to a variety of inflammatory lower genital tract disorders that may be secondary to infection , irritation , allergy , or systemic disease . infectious causes of vulvovaginitis include bacterial vaginosis , candidiasis , and trichimoniasis ; while noninfectious causes include exposure to chemicals , allergens , genital atrophy , and trauma . of the infectious vaginitides , only vulvovaginitis due to candida offers over the counter medications for women to self diagnose and treat their condition . these nonprescription antifungals , introduced to the market in 1990 , are among the top ten best selling over - the - counter drugs in the us with annual sales of approximately $ 250 million . one survey reports that 73% of women with recurrent vulvovaginitis have resorted to over - the - counter medications to reduce health care cost and avoid an expensive office visit . further , homeopathic drug sales were estimated at $ 201 million in 1995 and have steadily risen to an estimated 300450 million in 2003 [ 3 , 4 ] . this article attempts to clarify the efficacy of the available over - the - counter options for women seeking self treatment for vaginitis symptoms and further discern which products are appropriate for treating self - diagnosed yeast vaginitis . we visited four common venues frequented by women in their search for vaginitis treatment ; one grocery store , one drug store , one health food store , and the ubiquitous wal - mart . we went to the feminine hygiene section of each store and purchased one of each available product that could be considered a treatment option by the average consumer . cures most vaginal yeast infections were purchased as well as any products claiming to relieve vaginal itching . we next conducted a review of the literature , searching for rcts evaluating the efficacy of each product . our search for published literature included conventional search engines ( pub med , ovid , cochcrane , cinahl , apc journal club , google ) , and alternative medicine databases ( longwood herbal taskforce , nccam , natural medicines com - prehensive database ) . the products purchased could be grouped into three categories : intravaginal imidazoles , vaginal anti - itch creams , and homeopathic treatments . the intravaginal imidazoles were as a whole the more expensive products ranging from $ 6$19 , with an average cost of $ 12 . the homeopathic remedies ranged in price from $ 4$13 , with an average cost of $ 8.50 . finally , the vaginal anti - itch creams range in price from $ 2$6 , with an average price of $ 4 . further , a review of five rcts found no significant difference in treatment durations of one to fourteen days of imidazole therapy for uncomplicated vulvovaginitis . the cdc recommends that pregnant women use the seven - day treatment course due to the lower average concentration of medication . finally , the one , three , and seven - day regimens all deliver the same total dosage of active ingredient ; they differ solely in the number of doses and therefore strength of each dose . our review of the literature failed to reveal studies confirming the efficacy of vaginal anti - itch creams for treatment of infectious vaginitis . these products may confuse women seeking self treatment for vaginitis due to their proximity on the shelves , as well as their claims to relieve vaginal itching , the primary symptom of yeast vaginitis . we could not locate any rcts proving the effectiveness of any of the homeopathic treatments . systemic reviews have not found homeopathy to be a definitively proven treatment for any medical condition . despite the introduction of otc drugs for treatment of vulvovaginitis , the costs of health care office visits to treat this disorder are still rising , to an estimated of 3.1 billion dollars by 2014 . this may be due to the fact that women have proven to be inadequate in self diagnosis . in fact , in one study only one third of the women correctly diagnosed themselves with a yeast vaginitis . further , women with a previous clinical diagnosis of candida infection were not more accurate at identifying their current condition . the most common cause of infectious vulvovaginitis is bacterial vaginosis which has been found to be twice as prevalent as yeast vaginitis . while vaginitis was once thought not to be clinically perilous , there is a mounting body of evidence linking infectious vaginitis with more serious adverse reproductive outcomes . infection with bacterial vaginosis and trichomoniasis have been shown to increase one 's risk of acquiring hiv and other sti 's including pid , which can lead to infertility [ 2 , 9 ] . further , there has been a positive link between bacterial vaginosis and increased risk of preterm labor . bv also increases a women 's risk of postabortion uterine infection and posthysterectomy cuff infection . empowering women to correctly identify and treat their symptoms with the proper medications will alleviate their discomfort and prevent adverse outcomes from lack of recognition of symptoms necessitating a physi- cian visit . since our findings did not prove any significant differences between brand or formulation of any of the intravaginal imidazoles , women should chose their treatment based on personal preference . knowing that these products are equally effective allows the consumer to base her decision on price , route of administration , and ease of use . it should be noted that women with chronic or persistent yeast infections are less likely to respond to short courses of therapy and should consult with their doctor about a specific treatment regimen . vaginal anti - itch creams are grouped on the shelves next to the vaginal yeast infection treatments . these products such as vagisil , vagi - gard , summers eve , and equate vaginal cream are marketed to the consumer as anti - itch creams , making them a seemingly enticing treatment option for symptomatic relief of pruritis associated with candida . these creams utilize ingredients including anesthetics ( benzocaine ) , external analgesics ( resorcinol ) , and anti - pruitics ( hydrocortisone ) . women should be aware that if they purchase these creams they may experience only temporary alleviation of symptoms and this may delay appropriate medical treatment . further , these creams may act as an irritant in some women and in fact exacerbate their symptoms . a wide variety of nontraditional therapies have been touted for their potential to combat yeast infections . the most commonly cited alternative therapies in a literature review were yogurt containing live acidophilus , boric acid tablets , garlic , and tea tree oil . however , our search did not uncover any otc products utilizing these ingredients that bore a label claiming to cure most vaginal yeast infections . of the alternative therapies widely available over - the - counter claiming to treat yeast infections , homeopathic remedies predominate . a variety of homeopathic treatments were not only available at the health food stores , but at least one homeopathic product was on the shelves at each venue we visited , including eckerd drug and wal - mart . homeopathic remedies as a genre were significantly less expensive with a mean price of $ 3.50 less than the intravaginal imidazoles . simplistically , the theory of homeopathy is to administer small doses of toxic substances to stimulate the body 's own immune response . homeopathic products are often so diluted that they no longer contain even a single molecule of the active substance . this accounts for the fact that homeopathic substances have little proven beneficial or harmful effects . studies have found that women with recurrent vaginal yeast infections are more likely to experiment with alternative therapies due to the lower relative cost and the difficulty of obtaining a last minute doctors appointment . an estimated 42% of patients with recurrent vaginal yeast infections have resorted to alternative therapies . the use of products not proven efficacious or approved by the cdc may delay treatment of more serious medical conditions or promote more adverse outcomes . self - treating allows women to minimize out - of - pocket costs , and avoid a costly and time consuming visit to their health care provider . however , choosing a treatment for vaginitis can be cumbersome due to the number of available products , causing the consumer to feel overwhelmed and confused . most topical agents for treating vulvovaginal candidiasis are available otc , packaged in the one , three , or seven - day treatments . these products are of equal efficacy , allowing the consumer to purchase a treatment based on the ease of use and price . consumers should be aware that vaginal anti - itch creams and homeopathic remedies have not - proven efficacy in any rtcs and homeopathic treatments likely do not contain enough active substance to merit an effect . the price discrepancy among treatments may lure the consumer towards treatments that have little or no proven efficacy . it should be noted that these products cost an average of two to three times more than the vaginal anti - itch creams and homeopathic remedies . the authors recommend that physicians be directive in their counseling of patients about which otc products women should purchase once diagnosed with a yeast infection .

background . the fda approved over - the - counter ( otc ) use of vaginal antifungals in 1990 . subsequently , a plethora of otc products have become available to women on drugstore shelves . objectives . the purpose of this study was to determine the availability of otc products marketed for the treatment of vaginitis and to determine if their efficacy had been confirmed by published prospective randomized control trials ( rcts ) . materials and methods . the authors chose four retail locations frequented by women seeking vaginitis treatment . all products deemed a viable treatment option were purchased . results . all intravaginal imidazoles purchased , regardless of treatment duration or active ingredient , were found to be of proven efficacy . we were unable to find an rct confirming the effectiveness of vaginal anti - itch creams and homeopathic treatments for vaginitis . conclusion . 45% of products available to women in the feminine hygiene section of the stores surveyed could not be confirmed to be effective for treating infectious vaginitis .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION 5. CONCLUSION

we retrospectively reviewed the cholangiographic findings and hospital records of 67 patients who between january 1997 and may 1999 had undergone metallic biliary stent placement for the palliation of malignant extrahepatic strictures including klatskin tumors and peripheral cholangiocarcinoma invading the confluence of the intrahepatic ducts . patients with recurrence of biliary obstruction after bilioenteric anastomosis ( n = 4 ) or inadequate follow - up for the evaluation of stent patency ( n = 4 ) were excluded , and a total of 59 patients were thus enrolled in this study . thirty - six were male and 23 were female , and their mean age was 63.6 ( range , 25 - 82 ) years . the causes of extrahepatic strictures were as follows : common duct cancer ( n = 5 ) , klatskin tumor ( n = 5 ) , peripheral cholangiocarcinoma invading the hepatic confluence ( n = 5 ) , gall bladder cancer ( n = 5 ) , cancer of the pancreas ( n = 16 ) , and metastasis ( n = 13 ) ( 12 gastric cancer and one adenocarcinoma of unknown origin ) . malignancy was confirmed by surgery in 18 cases , by percutaneous needle biopsy in 19 , by bile cytology with and without brushing in eight , and on the basis of the imaging findings of cholangiography and ct in 14 . in all 59 patients , when reviewing the cholangiograms obtained during percutaneous transhepatic biliary drainage ( ptbd ) and before stent placement , we focused on the angulation formed by the distal and proximal portion of the ehd ( fig . 1 ) and the location of strictures within it ( fig . all cases except one showed a varying degree of angulation ( 80 - 170 ) between the proximal and distal portions of the ehd ; in the exceptional case , the course of the ehd from the biliary confluence to the ampulla of vater was straight . the area of angulation within the ehd was termed ' bending portion ' . to facilitate analysis , the classical method of describing ehd strictures ( 16 ) according to the location of the distal margin of the stricture relative to the ampulla of vater and the bending portion of the ehd , lesions were categorized as one of four types . type a represented a lesion whose distal margin was located within 3 cm of the ampulla of vater , regardless of the bending portion ( fig . 2a ) ; a type - b lesion was one whose distal margin was located more than 3 cm above the ampulla of vater and at or below the bending portion ( fig . 2b ) ; type c represented a lesion whose distal margin was located not more than 3 cm above the bending portion ( fig . 2c ) , while a lesion whose distal margin was located over 3 cm from the bending portion was categorized as type d ( fig . the number of type a , b , c and d cases was 27 , 7 , 9 , and 16 , respectively ( table 1 ) . a case in which a straight extrahepatic duct without angulation was noted was categorized as type a because the stricture was located within 3 cm of the ampulla . fifty - seven patients had undergone ptbd before stent placement , and the remaining two underwent placement as a single step . the mean time lag between ptbd and stent placement was 9.5 ( range , 0 - 25 ) days . informed consent was obtained from all patients . using self - expandable stainless stents ( hanaro stent ; mi tech , seoul , korea ) in 48 patients and nitinol stents ( stentech , seoul , korea ) in 11 , the stainless stent was made of 0.25-mm stainless steel wire bent in a zigzag pattern , with alternating different leg lengths forming a spiral cylindric configuration , and for the nitinol stent , 0.18 or 0.20-mm nitinol wire bent in a mesh was used . the length of stent was determined by the length of the stricture . in order to leave sufficient safety margin at each end of the stent within the bile duct , we generally selected a stent at least 4 cm longer than the stricture . if a stent was placed across the ampulla of vater ( transpapillary placement ) , a longer one , which would cover the distal duct , was chosen . mean stent length was 6.2 ( range , 3 - 8 ) cm for the suprapapillary method , and 7.3 ( range , 5 - 9 ) cm for the transpapillary . the routes of stent insertion were right transhepatic in 25 patients , left transhepatic in 24 , and bilateral trans - hepatic in ten . to simplify analysis , methods were classified only from the viewpoint of location of the distal end of the stent : whether or not it crossed the ampulla of vater . the term ' suprapapillary method ' was used to denote a stent placement technique which did not cross the ampulla of vater regardless of the location of the distal end of the stent within the ehd . the term ' transpapillary method ' denoted a placement technique that crossed the ampulla , the distal end of the stent thus being located in the duodenal lumen . in this way the stent was placed using the suprapapillary method in 35 cases and the transpapillary method in 24 ( table 1 ) . to permit bile drainage until the following morning , an 8.5-fr drainage catheter ( simp - loc ; cook , bloomington , ind . ) was positioned after stent placement , and the tube was then clamped for one day . when no problem was evident after clamping the drainage tube and the cholangiogram indicated good passage of contrast media into the duodenum , the drainage tube was removed . when the remaining waist of the stent hindered the passage of contrast media , as occurred in a limited number of cases , a balloon catheter ( olbert ; boston scientific , watertown , mass , u.s.a . ) with a diameter of 8 or 10 mm was used to relieve the waist . if it was found that a previously inserted stent inadequately decompressed the biliary system , a further stent was placed . many type - a lesions were managed using the transpapillary method , whereas for all type - d lesions , the suprapapillary method was used ( table 1 ) . in the 35 cases in which the superapapillary method was employed initially , another stent was used by way of secondary intervention in five lesions because the initial stent failed to maintain patency . there were four such type - a lesions and one type c. their numbers are shown in parentheses in the table . if passage of contrast media through the stent was poor , the procedure was regarded as inadequate . using the fisher exact test , initial patency rates for both the suprapapillary and transpapillary method were compared for each of the four types . the causes of inadequate procedures were analyzed using the cholangiograms obtained before , during , and after stent placement . regardless of the cause of reobstruction , recurrence of symptoms within two months of initial stent placement was defined as ' early recurrence ' . using fisher 's exact test , early recurrence rates were also compared for each of the four types . the duration of stent patency was defined as the interval between initial placement and recurrence of symptoms because of obstruction . patient survival and duration of stent patency were calculated with regard to type of lesion and the method of stent placement . using the kaplan - meier method , differences in long - term patency were compared . we retrospectively reviewed the cholangiographic findings and hospital records of 67 patients who between january 1997 and may 1999 had undergone metallic biliary stent placement for the palliation of malignant extrahepatic strictures including klatskin tumors and peripheral cholangiocarcinoma invading the confluence of the intrahepatic ducts . patients with recurrence of biliary obstruction after bilioenteric anastomosis ( n = 4 ) or inadequate follow - up for the evaluation of stent patency ( n = 4 ) were excluded , and a total of 59 patients were thus enrolled in this study . thirty - six were male and 23 were female , and their mean age was 63.6 ( range , 25 - 82 ) years . the causes of extrahepatic strictures were as follows : common duct cancer ( n = 5 ) , klatskin tumor ( n = 5 ) , peripheral cholangiocarcinoma invading the hepatic confluence ( n = 5 ) , gall bladder cancer ( n = 5 ) , cancer of the pancreas ( n = 16 ) , and metastasis ( n = 13 ) ( 12 gastric cancer and one adenocarcinoma of unknown origin ) . malignancy was confirmed by surgery in 18 cases , by percutaneous needle biopsy in 19 , by bile cytology with and without brushing in eight , and on the basis of the imaging findings of cholangiography and ct in 14 . in all 59 patients , when reviewing the cholangiograms obtained during percutaneous transhepatic biliary drainage ( ptbd ) and before stent placement , we focused on the angulation formed by the distal and proximal portion of the ehd ( fig . 1 ) and the location of strictures within it ( fig . all cases except one showed a varying degree of angulation ( 80 - 170 ) between the proximal and distal portions of the ehd ; in the exceptional case , the course of the ehd from the biliary confluence to the ampulla of vater was straight . the area of angulation within the ehd was termed ' bending portion ' . to facilitate analysis , the classical method of describing ehd strictures ( 16 ) according to the location of the distal margin of the stricture relative to the ampulla of vater and the bending portion of the ehd , lesions were categorized as one of four types . type a represented a lesion whose distal margin was located within 3 cm of the ampulla of vater , regardless of the bending portion ( fig . 2a ) ; a type - b lesion was one whose distal margin was located more than 3 cm above the ampulla of vater and at or below the bending portion ( fig . 2b ) ; type c represented a lesion whose distal margin was located not more than 3 cm above the bending portion ( fig . 2c ) , while a lesion whose distal margin was located over 3 cm from the bending portion was categorized as type d ( fig . the number of type a , b , c and d cases was 27 , 7 , 9 , and 16 , respectively ( table 1 ) . a case in which a straight extrahepatic duct without angulation was noted was categorized as type a because the stricture was located within 3 cm of the ampulla . fifty - seven patients had undergone ptbd before stent placement , and the remaining two underwent placement as a single step . the mean time lag between ptbd and stent placement was 9.5 ( range , 0 - 25 ) days . informed consent was obtained from all patients . using self - expandable stainless stents ( hanaro stent ; mi tech , seoul , korea ) in 48 patients and nitinol stents ( stentech , seoul , korea ) in 11 , the stainless stent was made of 0.25-mm stainless steel wire bent in a zigzag pattern , with alternating different leg lengths forming a spiral cylindric configuration , and for the nitinol stent , 0.18 or 0.20-mm nitinol wire bent in a mesh was used . the length of stent was determined by the length of the stricture . in order to leave sufficient safety margin at each end of the stent within the bile duct , we generally selected a stent at least 4 cm longer than the stricture . if a stent was placed across the ampulla of vater ( transpapillary placement ) , a longer one , which would cover the distal duct , was chosen . mean stent length was 6.2 ( range , 3 - 8 ) cm for the suprapapillary method , and 7.3 ( range , 5 - 9 ) cm for the transpapillary . the routes of stent insertion were right transhepatic in 25 patients , left transhepatic in 24 , and bilateral trans - hepatic in ten . to simplify analysis , methods were classified only from the viewpoint of location of the distal end of the stent : whether or not it crossed the ampulla of vater . the term ' suprapapillary method ' was used to denote a stent placement technique which did not cross the ampulla of vater regardless of the location of the distal end of the stent within the ehd . the term ' transpapillary method ' denoted a placement technique that crossed the ampulla , the distal end of the stent thus being located in the duodenal lumen . in this way the stent was placed using the suprapapillary method in 35 cases and the transpapillary method in 24 ( table 1 ) . to permit bile drainage until the following morning , an 8.5-fr drainage catheter ( simp - loc ; cook , bloomington , ind . ) was positioned after stent placement , and the tube was then clamped for one day . when no problem was evident after clamping the drainage tube and the cholangiogram indicated good passage of contrast media into the duodenum , the drainage tube was removed . when the remaining waist of the stent hindered the passage of contrast media , as occurred in a limited number of cases , a balloon catheter ( olbert ; boston scientific , watertown , mass , u.s.a . ) with a diameter of 8 or 10 mm was used to relieve the waist . if it was found that a previously inserted stent inadequately decompressed the biliary system , a further stent was placed . many type - a lesions were managed using the transpapillary method , whereas for all type - d lesions , the suprapapillary method was used ( table 1 ) . in the 35 cases in which the superapapillary method was employed initially , another stent was used by way of secondary intervention in five lesions because the initial stent failed to maintain patency . there were four such type - a lesions and one type c. their numbers are shown in parentheses in the table . if passage of contrast media through the stent was poor , the procedure was regarded as inadequate . using the fisher exact test , initial patency rates for both the suprapapillary and transpapillary method were compared for each of the four types . the causes of inadequate procedures were analyzed using the cholangiograms obtained before , during , and after stent placement . regardless of the cause of reobstruction , recurrence of symptoms within two months of initial stent placement was defined as ' early recurrence ' . using fisher 's exact test , early recurrence rates were also compared for each of the four types . the duration of stent patency was defined as the interval between initial placement and recurrence of symptoms because of obstruction . patient survival and duration of stent patency were calculated with regard to type of lesion and the method of stent placement . using the kaplan - meier method , all 24 cases involving the transpapillary method and 23 of 35 ( 65.7% ) in which the suprapapillary method was used showed good initial patency . the difference in initial patency rate between the two methods was statistically significant ( p < .001 ) . in type a , only one ( 14.3% ) involving the seven cases of the suprapapillary method showed good patency , whereas all 20 in which the transpapillary method was employed showed good passage through the stent ( fig . the causes of failure of initial patency were unpredicted stricture of the distal extrahepatic duct near the ampulla of vater in three cases ( fig . 4 ) , aggravation of distal angulation after stenting in two , and misplacement of the stent resulting in inadequate coverage of the stricture at the distal portion of the stent in one . with regard to type - b strictures , four of the five cases involving the suprapapillary method ( 80.0% ) showed good patency ( fig . 5 ) ; the cause of failure of initial patency in one case was aggravation of angulation after stent placement . in two cases the transpapillary method was used , and both were also patent . for type c , three of the seven cases employing the suprapapillary method showed good patency . in the other four , the cause of failure of initial patency was aggravation of the preexisting angulation of the duct due to longitudinal rigidity of the distal portion of the stent ( fig . two cases involved the transpapillary method , and both were also patent . for type d , stricture management relied in all cases on the suprapapillary method , and 15 of 16 ( 93.8% ) showed good patency . the cause of failure of initial patency in one case was aggravation of angulation , which can be prevented if the stent length chosen is appropriate . because of the small sample size , statistical significance for types b , c , and d of the six type - a cases which showed poor patency after initial stent placement , four underwent additional placement across the ampulla of vater , and in two balloon dilatation of the unpredicted stricture of the distal ehd near the ampulla of vater was performed ( fig . 4b ) . in one type - b case of poor patency due to aggravation of angulation at the distal portion of the stent , further intervention was not attempted . among the four type - c cases with poor patency , one underwent transpapillary placement of an additional stent immediately after initial placement , but in the other three there was no further intervention . in one type - d case of poor patency , after reintervention there were changes in the numbers of cases in which the suprapapillary / transpapillary method was employed . the new totals are shown in parentheses ( see table 1 ) . among the 30 cases in which the suprapapillary method was used , there were seven in which early recurrence occurred , including five of poor patency at initial stent placement but without additional placement . there were just two cases of early recurrence in the remaining patients , all of whom showed good patency of the biliary tree after stent placement . in four of 29 cases involving the transpapillary method ( 13.8% ) , early recurrence the six - month and one - year patency rates were 58.9% and 39.4% , respectively , while the median patency intervals for type a , b , and c were 201 , 317 , and 50 days , respectively . we were unable to calculate the median patency interval for type d. all differences were statistically insignificant ( p = .39 ) . the median patency intervals for cases in which the suprapapillary and transpapillary methods were used were 317 and 253 days , respectively , with no significant statistical difference ( p = .57 ) . all 24 cases involving the transpapillary method and 23 of 35 ( 65.7% ) in which the suprapapillary method was used showed good initial patency . the difference in initial patency rate between the two methods was statistically significant ( p < .001 ) . in type a , only one ( 14.3% ) involving the seven cases of the suprapapillary method showed good patency , whereas all 20 in which the transpapillary method was employed showed good passage through the stent ( fig . the causes of failure of initial patency were unpredicted stricture of the distal extrahepatic duct near the ampulla of vater in three cases ( fig . 4 ) , aggravation of distal angulation after stenting in two , and misplacement of the stent resulting in inadequate coverage of the stricture at the distal portion of the stent in one . with regard to type - b strictures , four of the five cases involving the suprapapillary method ( 80.0% ) showed good patency ( fig . 5 ) ; the cause of failure of initial patency in one case was aggravation of angulation after stent placement . in two cases the transpapillary method was used , and both were also patent . for type c , three of the seven cases employing the suprapapillary method showed good patency . in the other four , the cause of failure of initial patency was aggravation of the preexisting angulation of the duct due to longitudinal rigidity of the distal portion of the stent ( fig . two cases involved the transpapillary method , and both were also patent . for type d , stricture management relied in all cases on the suprapapillary method , and 15 of 16 ( 93.8% ) showed good patency . the cause of failure of initial patency in one case was aggravation of angulation , which can be prevented if the stent length chosen is appropriate . because of the small sample size , statistical significance for types b , c , and d of the six type - a cases which showed poor patency after initial stent placement , four underwent additional placement across the ampulla of vater , and in two balloon dilatation of the unpredicted stricture of the distal ehd near the ampulla of vater was performed ( fig . 4b ) . in one type - b case of poor patency due to aggravation of angulation at the distal portion of the stent , further intervention was not attempted . among the four type - c cases with poor patency , one underwent transpapillary placement of an additional stent immediately after initial placement , but in the other three there was no further intervention . in one type - d case of poor patency , after reintervention there were changes in the numbers of cases in which the suprapapillary / transpapillary method was employed . among the 30 cases in which the suprapapillary method was used , there were seven in which early recurrence occurred , including five of poor patency at initial stent placement but without additional placement . there were just two cases of early recurrence in the remaining patients , all of whom showed good patency of the biliary tree after stent placement . in four of 29 cases involving the transpapillary method ( 13.8% ) , early recurrence the six - month and one - year patency rates were 58.9% and 39.4% , respectively , while the median patency intervals for type a , b , and c were 201 , 317 , and 50 days , respectively . we were unable to calculate the median patency interval for type d. all differences were statistically insignificant ( p = .39 ) . the median patency intervals for cases in which the suprapapillary and transpapillary methods were used were 317 and 253 days , respectively , with no significant statistical difference ( p = .57 ) . for the effective palliation of malignant ehd strictures , it is essential to consider the anatomic characteristics of the ehd . these include the length of the bile duct and the location of cystic duct insertion , as well as the size and anatomic level of the obstructing tumor itself within the ehd ( 4 , 8) . for the prevention of tumor overgrowth it is very important to cover the bile duct for as great a disttance as possible ( over - stenting ) , leaving safety margins below and above the tumorous stricture ( 3 , 8) . when stenting the strictures located adjacent to the ampulla of vater , however , it is sometimes impossible to leave adequate distal safety margins . in our series , when the stent was placed by means of the suprapapillary method in such lesions ( type a ) , we were often faced with poor patency just after stent placement ; this was due to unexpected narrowing distal to the stent or angulation of the bile duct below the stent . though we can not adequately explain the presence of an unexpected stricture , it may have been caused by edematous change or spasm of the ampulla of vater associated with the procedure , by extrinsic compression of metastatic lymphadenopathy , or by infiltration of the tumor . in addition , we emphasize the importance of natural bending ( angulation ) of the ehd for successful palliation of the obstruction . the course of the extrahepatic duct from the confluence of the hepatic duct to the ampulla of vater is , in most cases , not straight . anatomically , the supraduodenal and retroduodenal segments of the extrahepatic duct slant obliquely from the right to the left , and below them the pancreatic segment curves rather sharply to the right to enter the descending duodenum via its posteromedial surface ( 18 ) . the overall course of the extrahepatic duct results in convexity or angulation to the left , the degree of angulation varying according to the patient . by reviewing the cholangiographic findings of 270 patients , liu et al . ( 9 ) measured the degree of angulation , the mean value of which was 140 - 150. a tumor such as pancreatic cancer around the duct sometimes aggravates the degree of angulation ( 9 ) . we often encountered cases showing aggravation by the distal portion of the stent itself of preexisting angulation of the ehd after stent placement . sometimes the angulation , which hindered effective bile flow , progressed on follow - up due to the longitudinal stretching force ( straightening ) of the metallic stent . in such cases another stent was required for distal angulation , and the function of ampulla of vater had to be sacrificed . ( 4 ) reported an interesting case of stent malfunction caused by exaggerated angulation at the proximal portion of a stent placed for the palliation of distal ehd stricture . they pointed out certain disadvantages of angulation , such as ineffective drainage and increased likelihood of damage to the bile duct epithelium by sharp wires ( 4 ) . stoker and lameris ( 10 ) encountered two cases of proximal occlusion caused by straightening of the stent , a phenomenon also observed by huibergtse ( 19 ) , using a wallstent . they found that the straightened proximal tip of a stent embedded in the bile duct wall hindered bile flow , and this facilitated sludge formation . stoker and lameris ( 10 ) suggested that in selected cases , a longer stent or one with a smaller diameter and less expansive force should be used . self - expanding metallic stents will eventually straighten with time , to a certain extent , although the longitudinal flexibility of a wallstent is slightly less than that of the stents we used ( 20 , 21 ) . this implies that if the proximal or distal ends of the stent are located near the bending portion of the bile duct when the rule of simple over - stenting is applied , it is important to overcome the problem of angulation . it is therefore wise to choose a stent which is long enough to successfully traverse the bending portion of the bile duct ( 4 ) . we recommend the transpapillary method for the palliation of type - a or c lesions in which the stricture is close to the ampulla of vater or the bile duct is angulated below the stricture . for type - b or d lesions in which the stricture is far from the ampulla of vater or bile duct angulation occurs apart from the stricture so that the stent can be placed without aggravation of preexisting angulation , a conventional suprapapillary method can easily be applied . if the suggested indication is applied , the number of cases amenable to the transpapillary method increases very considerably . ( 22 ) placed transpapillary stents in 22 of their 65 patients , even though the method was applied in restricted cases . when they were sure that the papilla was not involved , suprapapillary placement was attempted . in our series , where our indication was applied , the transpapillary method was indicated in 38 of 59 patients ( 64% ) . although some authors have suggested that duodenal ulceration and subsequent bleeding or perforation of the duodenum , as well as both reflux of duodenal content into the bile duct ( once believed to be a cause of early stent dysfunction ) and pancreatitis are complications of the transpapillary method of stent placement , the cases they encountered were limited ( 12 - 15 ) . in spite of concerns over these complications , most reports describing the long - term results of metallic stent insertion have demonstrated that their incidence is very low ( 2 , 12 ) . interestingly , most reports of bleeding involved cases in which the prototype wallstent was used ( 15 ) . nowadays , wallstents and other commercially available stents have relatively smooth edges and this problem does not , therefore , exist . ( 2 ) reported that they did not encounter such complications , though stoker and lameris ( 10 ) described two cases of duodenal pressure ulcer among 135 patients who had undergone transpapillary wallstent placement . though we were unable to analyze the complications arising from the methods employed in this retrospective study , the transpapillary method gave rise to no serious complication . there were , however , relatively frequent incidences of epigastric discomfort after placement , and these might be caused by inadvertent dilatation of the ampulla of vater by the stent . within two or three days , however , they subsided spontaneously . ( 22 ) , no radiologic report has compared the efficiency with which the suprapapillary and transpapillary methods of stent placement palliate malignant biliary obstruction . ( 22 ) found that the occlusion rate of a stent was not influenced by its position . a further report on the same subject ( 23 ) dealt with endoscopically - placed plastic stents , concluding that there was no significant difference in patency and complication rates . we observed similar results : there were no significant differences in early recurrence and long - term patency rates between the two methods . in conclusion , a careful review of cholangiograms with regard to stricture location and ehd angulation , as well as proper application of stent placement methods , are mandatory procedures if malignant biliary strictures are to be successfully palliated . for strictures involving distal ehd and associated with angulation of the bile duct below the stricture ,

objectiveto compare the efficacy of suprapapillary and transpapillary methods of transhepatic biliary metallic stent placement in malignant biliary strictures and to specify the indications of each method applied.materials and methodsstents were placed in 59 patients . strictures were categorized as type a ( within 3 cm of the ampulla , n = 27 ) , type b ( over 3 cm from ampulla , n = 7 ) , type c ( within 3 cm of the bending portion , n = 9 ) , or type d ( over 3 cm above the bending portion , n=16 ) . the stenting method was suprapapillary in 34 cases and transpapillary in 25 . the rates of initial and long - term patency and of early recurrence were compared.resultsinitial patency rates for the suprapapillary and transpapillary methods were 1/7 ( 14.3% ) and 20/20 ( 100% ) respectively for type a ( p < 0.0001 ) , 4/5 ( 80.0% ) and 2/2 for type b , 3/7 ( 42.9% ) and 2/2 for type c , and 15/16 ( 93.8% ) and 0/0 for type d. early recurrence rates were 7/30 ( 23.3% ) using the suprapapillary method and 4/29 ( 13.8% ) using the transpapillary method ( p = 0.51 ) . the long - term patency rate did not differ significantly according to either type ( p = 0.37 ) or method ( p = 0.62).conclusionfor good initial patency , the transpapillary method is recommended for strictures of the distal extrahepatic duct near the ampulla and just above the bending portion . long - term patency is not influenced by the stenting method employed .

MATERIALS AND METHODS Patients Lesion characteristics Procedure Analysis RESULTS Initial patency Secondary intervention Early recurrence Long-term patency DISCUSSION

they may have similar presentations but possibly different treatment modalities , ie , hysterectomy for adenomyosis versus conservative management or hysterectomy for leiomyomas , or both . however , only limited studies have been performed on the efficacy of conservative treatment of adenomyosis , and these typically do not improve or preserve fertility . . mri provides better tissue contrast and may be superior to other imaging modalities in certain pelvic pathologies , such as congenital uterine anomalies , leiomyoma , adenomyosis , and endometriomas . some reports suggest mri is a reliable preoperative tool available to physicians to assist in differentiating between fibroids and adenomyoma / adenomyosis . in this study , we evaluated the predictive value of the pre - operative mri in differentiating between leiomyoma and adenomyosis compared with pathologic findings . a 5-year retrospective chart review was performed from january 1999 to december 2004 in a university - based hospital among patients who were admitted to undergo hysterectomy , myomectomy , or both . although 1517 charts were reviewed , only 153 patients who underwent a preoperative mri were selected for this study . patients ranged in age from 21 to 69 with a mean age of 41 and a median age of 39 . the presenting symptoms were as follows : 73(48% ) had menorrhagia , 34(22% ) had dysmenorrhea , 31(20% ) had pelvic pain , 40(26% ) had a pelvic mass , 16(10% ) had irregular bleeding , 14(9% ) had infertility , and 2(1.3% ) had postmenopausal bleeding . of these patients , 50 had a hysterectomy and the other 103 had a myomectomy . mri studies were performed in axial , sagittal , and coronal planes with various t1-weighted spin - echo and t2-weighted turbo spin - echo or spin - echo on a 1.5 tesla system ( gyro scan , phillips medical systems , bothell , washington , usa or ge open magnet mri scanners , general electric company , fairfield , connecticut , usa ) in different centers . contrast enhanced gadolinium was used as an additive technique in 50 cases to achieve better tissue contrast in the patients suspicious for leiomyoma . leiomyoma appeared as low - signal intensity in t2-weighted images and intermediate- to high - signal intensity in t1-weighted images . adenomyosis was described as a focal or diffuse widening of junctional zone above 12 mm , uterine enlargement , or both , with focal or diffuse low - signal intensity myometrial area in t2-weighted images . also on contrast enhanced t1-weighted images , ( gadolinium scan ) small hypointense myometrial spots mri interpretation was performed by different observers in different centers including private radiology centers , community and university hospitals . mri studies were performed in axial , sagittal , and coronal planes with various t1-weighted spin - echo and t2-weighted turbo spin - echo or spin - echo on a 1.5 tesla system ( gyro scan , phillips medical systems , bothell , washington , usa or ge open magnet mri scanners , general electric company , fairfield , connecticut , usa ) in different centers . contrast enhanced gadolinium was used as an additive technique in 50 cases to achieve better tissue contrast in the patients suspicious for leiomyoma . leiomyoma appeared as low - signal intensity in t2-weighted images and intermediate- to high - signal intensity in t1-weighted images . adenomyosis was described as a focal or diffuse widening of junctional zone above 12 mm , uterine enlargement , or both , with focal or diffuse low - signal intensity myometrial area in t2-weighted images . also on contrast enhanced t1-weighted images , ( gadolinium scan ) small hypointense myometrial spots were indicative of adenomyosis . mri interpretation was performed by different observers in different centers including private radiology centers , community and university hospitals . the results were used to confirm the mri findings for the presence of leiomyoma and adenomyosis . according to the pathology reports , 120 had only leiomyoma , 23 had leiomyoma with coexisting adenomyosis , and 9 had adenomyosis . one patient in the hysterectomy group had a normal mri and pathology report that revealed no myoma or adenomyosis . fifteen patients had other pelvic pathologies coexisting with leiomyoma or adenomyosis , or coexisting with both . mri studies correctly diagnosed 136 of 144 patients with leiomyoma . in the remaining 8 patients with leiomyoma data analyses were as follows : sensitivity 94% , specificity 33% , positive predictive value 95% , and negative predictive value 27% . for adenomyosis , in 31 patients , mri revealed true positive , false - positive , and false - negative results in 12 , 11 , and 19 patients , respectively . the positive and negative predictive values of mri for adenomyosis were 52% , 85% , respectively , with 38% sensitivity and 91% specificity . as mentioned previously mri revealed correct diagnoses in only 9/23 patients , in 12/23 it revealed only myoma , and in 2/23 it diagnosed only adenomyosis . adenomyosis and leiomyoma are 2 benign uterine conditions with similar clinical presentations and different pathogeneses . adenomyosis or internal endometriosis is a disease of parous women with the peak incidence between 40 to 50 years of age . it is characterized by the presence of endometrial glands and stroma inside the uterine myometrium . according to different histological interpretations of the hysterectomy specimens , frequency of adenomyosis uterine involvement may be focal or diffuse , and the size of the lesion ranges from a gross macroscopic well - defined lesion ( adenomyoma that mimics myoma ) to a microscopic lesion . uterine leiomyoma is a benign smooth muscle tumor with 25% prevalence in women of reproductive age with a predominance among african american women . furthermore , pathology examination of surgical specimens revealed an incidence of leiomyoma as high as 77% . a definitive diagnosis for both categories is possible based on histological examination of surgical specimens , but imaging techniques are increasingly used for preoperative evaluation and diagnosis . transvaginal ultrasound and pelvic mri are increasingly used as a preoperative diagnostic tool for adenomyosis . although both techniques have been reported to have almost the same sensitivity ( 68% versus 70% ) , the specificity is greater for mri ( 86% versus 65% ) . pelvic mri is also used in the diagnosis of leiomyoma when a patient 's obesity or extreme uterine retroversion , or both , hinder pelvic ultrasound performance . pelvic mri is highly specific in the diagnosis of adenomyosis ( 91% ) and highly sensitive for leiomyoma ( 94% ) according to our findings . currently , pelvic ultrasound , a relatively affordable and effective method , is widely used as an office procedure in the diagnosis of leiomyoma . it is more efficacious to limit the use of mri for specific conditions when uterine preservation is the goal of therapy , such as localization of leiomyoma in an infertile patient or when the patient is a candidate for uterine artery embolization . our results are different from the results in previously published reports that indicated a higher sensitivity ( 70% vs. 38% ) of mri for adenomyosis and higher specificity ( 100% vs. 33% ) for leiomyoma . this can possibly be explained because our study was influenced by selection bias . because the study was performed on a diseased population with a previous diagnosis of myoma or adenomyosis , or both , where ultrasound alone was thought by the physician to be inadequate . therefore , there were very few patients in our study with negative findings , leading to a low specificity . however , it was not possible to select another population , because of the retrospective nature of the study . also , mri studies were done in different centers and interpreted by different radiologists , which may produce interobserver bias . like other radiological studies , mri is highly operator dependent , and the results may be influenced by technical and observer interpretation . however , this may be a strength of the study in the sense that in reality there will not always be the same specialist reviewing all the studies . of 153 patients who had mri as a preoperative diagnostic tool , 103 underwent myomectomies , so there may be an inherent bias against identifying false - negative adenomyosis . at the present time , pelvic ultrasound is used as a first line for the diagnosis of uterine pathology . however , it may be helpful for diagnosis of pelvic pathologies that are not well defined with ultrasound studies . we suggest mri be used as a complementary diagnostic tool in patients whose ultrasound examination does not clarify the diagnosis , such as in extreme uterine retroversion or obesity , or both , and in patients whose ultrasound examination is indicative of adenomyosis and there is a desire for conservative management . if the preoperative mri shows no adenomyosis , patients can be counseled of the low likelihood of having adenomyosis ( 15% ) and therefore conservative management can be offered .

objective : we evaluated the role of mri as a preoperative diagnostic tool for leiomyoma and adenomyosis.method:this is a retrospective chart review at a university - based hospital . the study included 1517 women who underwent hysterectomy or myomectomy over a 5-year period , and 153 women with a preoperative pelvic mri were included . comparisons were made between the results of the mri and postoperative pathology reports.results:the mri and pathology report were the same for 136 of 144 women with leiomyoma and 12 of 31 women with adenomyosis . the mri had 94% sensitivity and 33% specificity for leiomyoma and 38% sensitivity and 91% specificity for adenomyosis . positive and negative predictive values of mri for leiomyoma were 95% and 27% with 90% accuracy . positive and negative predictive values of mri for adenomyosis were 52% and 85% , respectively , with 80% accuracy.conclusion:mri has a high sensitivity and a low specificity for diagnosing leiomyoma and a high specificity and a low sensitivity for diagnosing adenomyosis . due to the high cost and technical variations , we suggest using mri only as an adjunctive diagnostic tool when ultrasound is not conclusive and differentiation between the 2 pathologies ultimately affects patient management .

INTRODUCTION METHODS MRI Studies RESULTS DISCUSSION CONCLUSION

the utility of this model stems from the fact that few assumptions are needed to determine hazard ratios based on the coefficients . the stratified cox ( sc ) model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption . parametric models are used only occasionally in analyzing clinical studies of survival despite offering some advantages over semiparametric models . parametric regression analysis is an attractive alternative to the widely used cox model when hazard functions themselves are of primary interest , or when relative survival times are the primary measure of association . when empirical information is available , parametric models can provide insight into the shape of the baseline hazard and baseline survival . furthermore , difficulty choosing the appropriate family of distributions leads many researchers to prefer the cox model . some parametric models are accelerated failure time ( aft ) models which assume that the relationship between the logarithm of survival time and covariates is linear one approach to address these difficulties is fitting the generalized gamma ( gg ) distribution . this extensive family contains nearly all commonly used distributions including the exponential , weibull , and log normal , making it particularly useful for estimating individual hazard functions as well as both relative hazards and relative survival times . in this analysis , we applied both semiparametric and parametric models under different conditions of the ph and aft assumptions and compared their results . the data in this study describe 12,531 women diagnosed with breast cancer in british columbia during 19901999 , and followed till 2010 . these were coded using binary variables equal to one if the subject received the treatment and zero otherwise . we defined survival time as a period between the diagnosis of disease and death or the end of patient 's follow - up . a binary censoring variable was used to indicate whether a patient died of breast cancer . for choosing the appropriate parametric model this distribution is a three - parameter family with location ( ) , scale ( > 0 ) , and shape ( ) parameters , in which all the three parameters depend on covariates . it should be noted that the conventional aft model holds only when covariate effects are modeled through the beta parameter . if we extend the analysis to covariates having effects through the sigma and/or lambda parameters , these are no longer conventional aft models . the gg family contains nearly all of the most commonly used distributions in survival analysis , including the exponential ( = = 1 ) , weibull ( = 1 ) , and log normal ( = 0 ) . if a general parametric distribution includes other distributions as special cases , the general distribution is called a nesting ( larger ) family of the specific distributions . the gg distribution includes three specific distributions , and thus represents a nesting family of them , allowing us to evaluate the appropriateness of the specific distributions relative to each other . testing the appropriateness of a family of distributions is equivalent to testing whether a subset of parameters in its nesting distribution are equal to specific values , and can be performed using a likelihood ratio test . the log - logistic distribution is a commonly used distribution in survival analysis , which is not nested in the gg family . to compare the selected parameter distribution with the log - logistic distribution , we used simple procedures based on baysian information criterion ( bic schwarz , 1978 and akaike information criterion ( aic ; akaike , 1969 ) , in which r is defined as . in our comparisons , the candidate distribution with the largest r value was considered the best fit . for aft models , we can estimate relative survival time by exponentiating the coefficient of a variable . for other models , we can calculate the relative time , rt ( p ) , using appropriate formulae . the relative times are defined for 0 < p < 1 as the ratio of the corresponding quantile functions , rt ( p ) = t1(p)/t0(p ) . the interpretation of rt ( p ) is that the time required for proportion p of individuals in the exposed or treated population to experience the event of interest is rt(p)-fold the time for the same proportion of events to occur in the reference population . links between quantiles of the gamma and gg facilitats use of software to obtain the percentiles of the gg . cox was the first to propose the model , specifying the hazard function as a function of time and the covariates : h ( t , x , b ) = h0(t ) exp(xb ) . with this parameterization , the hazard ratio is : hr ( t , x1 , x0 ) = exp(b(x1 - x0 ) ) . the cox ph model assumes that the hazard ratio for any two specifications of predictors is constant over time , and schoenfeld residuals can be used to assess the ph assumption . the sc model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption.the predictor that does not satisfy the ph assumption is being adjusted by stratification , whereas the predictor that satisfies the ph assumption is being adjusted by its inclusion in the model . the hazard ratio value for the effect of variables in each stratum can be estimated . nevertheless , the hazard ratio value for the effect of a stratified variable can not be estimated . furthermore , we applied the likelihood ratio ( lr ) test to check the interaction between stratified variable and variables in each stratum . a standard treatment protocol for breast cancer is determined mainly by the patient 's age , stage of cancer , and tumor sensitivity to certain hormones . breast cancer stage has an important role in choosing the treatment , and a patient 's response to treatment depends on her age , so we divided the dataset according to the age of patient at diagnosis of disease ( age < 50 , age 50 ) and the stage of cancer ( i , ii , iii , iv ) . this produced eight combinations of age and stage with different conditions of ph and aft assumptions , and we could compare the treatment effect on patient survival with parametric and semi - parametric models . the presence of hormone receptors has been proven to have an effect on survival time of patient , and was included in all models . for each combination of age and stage , we only included variables for which more than 10 patients received and did not receive the treatment . the data in this study describe 12,531 women diagnosed with breast cancer in british columbia during 19901999 , and followed till 2010 . these were coded using binary variables equal to one if the subject received the treatment and zero otherwise . we defined survival time as a period between the diagnosis of disease and death or the end of patient 's follow - up . a binary censoring for choosing the appropriate parametric model , we started by fitting the saturated gg distribution . this distribution is a three - parameter family with location ( ) , scale ( > 0 ) , and shape ( ) parameters , in which all the three parameters depend on covariates . it should be noted that the conventional aft model holds only when covariate effects are modeled through the beta parameter . if we extend the analysis to covariates having effects through the sigma and/or lambda parameters , these are no longer conventional aft models . the gg family contains nearly all of the most commonly used distributions in survival analysis , including the exponential ( = = 1 ) , weibull ( = 1 ) , and log normal ( = 0 ) . if a general parametric distribution includes other distributions as special cases , the general distribution is called a nesting ( larger ) family of the specific distributions . the gg distribution includes three specific distributions , and thus represents a nesting family of them , allowing us to evaluate the appropriateness of the specific distributions relative to each other . testing the appropriateness of a family of distributions is equivalent to testing whether a subset of parameters in its nesting distribution are equal to specific values , and can be performed using a likelihood ratio test . the log - logistic distribution is a commonly used distribution in survival analysis , which is not nested in the gg family . to compare the selected parameter distribution with the log - logistic distribution , we used simple procedures based on baysian information criterion ( bic schwarz , 1978 and akaike information criterion ( aic ; akaike , 1969 ) , in which r is defined as . in our comparisons , the candidate distribution with the largest r value was considered the best fit . for aft models , we can estimate relative survival time by exponentiating the coefficient of a variable . for other models , we can calculate the relative time , rt ( p ) , using appropriate formulae . the relative times are defined for 0 < p < 1 as the ratio of the corresponding quantile functions , rt ( p ) = t1(p)/t0(p ) . the interpretation of rt ( p ) is that the time required for proportion p of individuals in the exposed or treated population to experience the event of interest is rt(p)-fold the time for the same proportion of events to occur in the reference population . links between quantiles of the gamma and gg facilitats use of software to obtain the percentiles of the gg . cox was the first to propose the model , specifying the hazard function as a function of time and the covariates : h ( t , x , b ) = h0(t ) exp(xb ) . with this parameterization , the hazard ratio is : hr ( t , x1 , x0 ) = exp(b(x1 - x0 ) ) . the cox ph model assumes that the hazard ratio for any two specifications of predictors is constant over time , and schoenfeld residuals can be used to assess the ph assumption . the sc model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption.the predictor that does not satisfy the ph assumption is being adjusted by stratification , whereas the predictor that satisfies the ph assumption is being adjusted by its inclusion in the model . the hazard ratio value for the effect of variables in each stratum can be estimated . nevertheless , the hazard ratio value for the effect of a stratified variable can not be estimated . furthermore , we applied the likelihood ratio ( lr ) test to check the interaction between stratified variable and variables in each stratum . a standard treatment protocol for breast cancer is determined mainly by the patient 's age , stage of cancer , and tumor sensitivity to certain hormones . breast cancer stage has an important role in choosing the treatment , and a patient 's response to treatment depends on her age , so we divided the dataset according to the age of patient at diagnosis of disease ( age < 50 , age 50 ) and the stage of cancer ( i , ii , iii , iv ) . this produced eight combinations of age and stage with different conditions of ph and aft assumptions , and we could compare the treatment effect on patient survival with parametric and semi - parametric models . the presence of hormone receptors has been proven to have an effect on survival time of patient , and was included in all models . for each combination of age and stage , we only included variables for which more than 10 patients received and did not receive the treatment . for choosing the appropriate parametric model , we started by fitting the saturated gg distribution . this distribution is a three - parameter family with location ( ) , scale ( > 0 ) , and shape ( ) parameters , in which all the three parameters depend on covariates . it should be noted that the conventional aft model holds only when covariate effects are modeled through the beta parameter . if we extend the analysis to covariates having effects through the sigma and/or lambda parameters , these are no longer conventional aft models . the gg family contains nearly all of the most commonly used distributions in survival analysis , including the exponential ( = = 1 ) , weibull ( = 1 ) , and log normal ( = 0 ) . if a general parametric distribution includes other distributions as special cases , the general distribution is called a nesting ( larger ) family of the specific distributions . the gg distribution includes three specific distributions , and thus represents a nesting family of them , allowing us to evaluate the appropriateness of the specific distributions relative to each other . testing the appropriateness of a family of distributions is equivalent to testing whether a subset of parameters in its nesting distribution are equal to specific values , and can be performed using a likelihood ratio test . the log - logistic distribution is a commonly used distribution in survival analysis , which is not nested in the gg family . to compare the selected parameter distribution with the log - logistic distribution , we used simple procedures based on baysian information criterion ( bic schwarz , 1978 and akaike information criterion ( aic ; akaike , 1969 ) , in which r is defined as . in our comparisons , the candidate distribution with the largest r value was considered the best fit . for aft models , we can estimate relative survival time by exponentiating the coefficient of a variable . for other models , we can calculate the relative time , rt ( p ) , using appropriate formulae . the relative times are defined for 0 < p < 1 as the ratio of the corresponding quantile functions , rt ( p ) = t1(p)/t0(p ) . the interpretation of rt ( p ) is that the time required for proportion p of individuals in the exposed or treated population to experience the event of interest is rt(p)-fold the time for the same proportion of events to occur in the reference population . links between quantiles of the gamma and gg facilitats use of software to obtain the percentiles of the gg . cox was the first to propose the model , specifying the hazard function as a function of time and the covariates : h ( t , x , b ) = h0(t ) exp(xb ) . with this parameterization , the hazard ratio is : hr ( t , x1 , x0 ) = exp(b(x1 - x0 ) ) . the cox ph model assumes that the hazard ratio for any two specifications of predictors is constant over time , and schoenfeld residuals can be used to assess the ph assumption . the sc model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption.the predictor that does not satisfy the ph assumption is being adjusted by stratification , whereas the predictor that satisfies the ph assumption is being adjusted by its inclusion in the model . the hazard ratio value for the effect of variables in each stratum can be estimated . nevertheless , the hazard ratio value for the effect of a stratified variable can not be estimated . furthermore , we applied the likelihood ratio ( lr ) test to check the interaction between stratified variable and variables in each stratum . a standard treatment protocol for breast cancer is determined mainly by the patient 's age , stage of cancer , and tumor sensitivity to certain hormones . breast cancer stage has an important role in choosing the treatment , and a patient 's response to treatment depends on her age , so we divided the dataset according to the age of patient at diagnosis of disease ( age < 50 , age 50 ) and the stage of cancer ( i , ii , iii , iv ) . this produced eight combinations of age and stage with different conditions of ph and aft assumptions , and we could compare the treatment effect on patient survival with parametric and semi - parametric models . the presence of hormone receptors has been proven to have an effect on survival time of patient , and was included in all models . for each combination of age and stage , we only included variables for which more than 10 patients received and did not receive the treatment . results of comparison between parametric regression models have been summarized in table 2 . also , the results of fitting parametric model and cox model are shown in tables 3 and 4 respectively . number of patients receiving treatment by stage at diagnosis comparison between parametric regression models in each category of age and stage relative time estimated in parametric models relative hazard estimated in cox model for patients under age 50 years in disease stages i and iv , the ph and aft assumptions were satisfied . in patients under age 50 years with stage i cancer , the best - fitting parametric model used a conventional gg distribution in which chemotherapy ( p < 0.001 ) and erposneg ( p = 0.002 ) were significant . for the cox model , radiotherapy ( p = 0.001 ) , chemotherapy ( p < 0.001 ) , and hormone therapy ( p = 0.01 ) were significant . 50 years with stage iv cancer , a conventional lognormal model was the best candidate in gg family . the aic and bic criteria were the same for conventional lognormal and conventional log - logistic models , but the cox - snell residual plot indicated better fit for the lognormal . radiotherapy ( p = 0.01 ) , hormone therapy ( p = 0.01 ) , and erposneg(p = 0.01 ) were meaningful in lognormal model . for the cox model , radiotherapy ( p = 0.02 ) and erposneg ( p = 0.001 ) were significant and no interactions were significant in any of the models . snell residual plots for the lognormal , log - logistic , and cox ph model are given in figures 13 . cox - snell residual plot for fitted conventional lognormal model for patients under age 50 years with stage iv cancer cox - snell residual plot for fitted conventional log - logistic model for patients under age 50 years with stage iv cancer cox - snell residual plot for proportional cox model for patients under age 50 years with stage iv cancer for patients aged 50 years or more with stage i cancer , the ph assumption was not satisfied , but the aft assumptions held . in parametric models , the conventional gg had the best fit and radiotherapy ( p = 0.014 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p 0.001 ) were significant . in semiparametric model , the covariates radiotherapy ( p = 0.003 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p < 0.001 ) were meaningful ; and since the variable erposneg did not hold the ph assumption , it was lost due to stratification and the effects of other parameters were estimated by stratifying the model by this variable . no interactions were significant in each model . for patients aged 50 years or more with stage iv cancer , none of the ph and aft assumptions held . in parametric model , the gg family fitted better than the log - logistic according to aic and bic criteria . in models based on the gg family , surgery ( p = 0.001 ) , radiotherapy ( p = 0.004 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p < 0.001 ) were significant and the interaction of hormone therapy and erposneg ( p = 0.021 ) was meaningful . since the saturated lognormal is not an aft model , the relative survival time was calculated for the 25 , 50 , and 75 quantiles . surgery ( p = 0.001 ) , radiotherapy ( p = 0.03 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p = 0.001 ) were significant . since the ph assumption only held for surgery , the effect of this variable was estimated by stratifying on erposneg . age50 for all patients with stage ii cancer , and patients aged 50 and older with stage iii cancer , neither the ph nor the aft assumptions were satisfied . in semiparametric models , most variables did not meet the ph assumption and their effect could not be estimated ; in parametric models , the saturated gg fitted better than other distributions . for patients under age 50 years in disease stages i and iv , the ph and aft assumptions were satisfied . in patients under age 50 years with stage i cancer , the best - fitting parametric model used a conventional gg distribution in which chemotherapy ( p < 0.001 ) and erposneg ( p = 0.002 ) were significant . for the cox model , radiotherapy ( p = 0.001 ) , chemotherapy ( p < 0.001 ) , and hormone therapy ( p = 0.01 ) were significant . in both models , in patients under age 50 years with stage iv cancer , a conventional lognormal model was the best candidate in gg family . the aic and bic criteria were the same for conventional lognormal and conventional log - logistic models , but the cox - snell residual plot indicated better fit for the lognormal . radiotherapy ( p = 0.01 ) , hormone therapy ( p = 0.01 ) , and erposneg(p = 0.01 ) were meaningful in lognormal model . for the cox model , radiotherapy ( p = 0.02 ) and erposneg ( p = 0.001 ) were significant and no interactions were significant in any of the models . snell residual plots for the lognormal , log - logistic , and cox ph model are given in figures 13 . cox - snell residual plot for fitted conventional lognormal model for patients under age 50 years with stage iv cancer cox - snell residual plot for fitted conventional log - logistic model for patients under age 50 years with stage iv cancer cox - snell residual plot for proportional cox model for patients under age 50 years with stage iv cancer for patients aged 50 years or more with stage i cancer , the ph assumption was not satisfied , but the aft assumptions held . in parametric models , the conventional gg had the best fit and radiotherapy ( p = 0.014 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p the covariates radiotherapy ( p = 0.003 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p < 0.001 ) were meaningful ; and since the variable erposneg did not hold the ph assumption , it was lost due to stratification and the effects of other parameters were estimated by stratifying the model by this variable . for patients aged 50 years or more with stage iv cancer , none of the ph and aft assumptions held . in parametric model , the gg family fitted better than the log - logistic according to aic and bic criteria . in models based on the gg family , surgery ( p = 0.001 ) , radiotherapy ( p = 0.004 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p < 0.001 ) were significant and the interaction of hormone therapy and erposneg ( p = 0.021 ) was meaningful . since the saturated lognormal is not an aft model , the relative survival time was calculated for the 25 , 50 , and 75 quantiles . surgery ( p = 0.001 ) , radiotherapy ( p = 0.03 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p = 0.001 ) were significant . since the ph assumption only held for surgery , the effect of this variable was estimated by stratifying on erposneg . age50 for all patients with stage ii cancer , and patients aged 50 and older with stage iii cancer , neither the ph nor the aft assumptions were satisfied . in semiparametric models , most variables did not meet the ph assumption and their effect could not be estimated ; in parametric models , the saturated gg fitted better than other distributions . when ph and aft assumptions were satisfied , both the parametric and semiparametric models were appropriate . the models indicate different significant variables , but parametric models have some advantages over semiparametric models in general . with small sample sizes , the ph assumption is satisfied , some studies indicate that parametric ph models are a better approach than the cox model . further , some studies have shown the robustness of parametric aft models to misspecification because of their log - linear form . finally , one advantage of a parametric model compared to a cox model is that the parametric likelihood accommodates right- , left- , or interval - censored data . the cox likelihood , by contrast , handles right - censored data but does not directly accommodate left- or interval - censored data . when the ph assumption is not satisfied but aft assumptions hold , the parametric model can be used as a substitute for the cox model . when neither the ph nor the aft assumptions hold , a member of the gg distribution , the saturated lognormal , can be used to calculate relative survival times in different quantiles . many settings , including breast cancer analysis , where the proportionality assumption does not hold , the lognormal model has been shown to be appropriate.[1214 ] . a meta - analysis has shown that saturated parametric models provide better results than conventional models for comparing treatments . when the ph and aft assumptions do not hold and the saturated gg distribution fits better than other distributions , the gg family contains many different distributions such as the inverse weibull and inverse lognormal . our analysis applied the most commonly used parametric distributions in survival analysis , but we could not determine the best fit . for one category of age and stage in our study , according to aic and bic criteria , the log - logistic distribution gave the same fit as the lognormal model within the gg family . the log - logistic distribution belongs to the generalized f distribution , which includes the gg distribution . when a member of the gg family does not fit satisfactorily , the best distribution can be found through the generalized f distribution family . if the scales of the parameters in cox 's model and the parametric models differ , neither parameter estimates nor their estimated variances are suitable for comparisons . if the cox model is compared to parametric ph models , the efficiency of parameter estimates can be compared by wald - type statistics . for patients with stage iv cancer , semiparametric and parametric models showed lower hazards and longer survival times for patients receiving treatments than those not receiving treatments . for patients with stage i cancer in both the under 50-year and over 50-year age groups , semiparametric and parametric models showed higher hazards and smaller survival times for patients receiving treatments , which might reflect other covariates that caused the patients to receive a certain treatment . for example , studies have shown the ethnicity of patients affect their use of treatments that are more common in lower stages of cancer . in both parametric and semiparametric models applied to different combinations of age and cancer stage , the expression of hormone receptors was associated with a longer survival time and lower hazard , which has been confirmed in many other studies . a major strength of this study is that we fitted models and performed comparisons using a large set of real - life data from a population - based cancer registry . the major limitation is that our findings describe associations with survival , and not causes of survival . a patient 's survival does not necessarily depend on the treatment they receive ; rather , the treatment that a patient receives often depends on disease characteristics that predict survival . in particular , it would be of interest to determine whether the findings are sustained in more - recent patient cohorts where newer treatments have been used . however , there are some recent studies that describe the factors associated with survival time of patients in developing countries . when ph and aft assumptions were satisfied , semiparametric and parametric models provided two different valid approaches for exploring breast cancer patients survival , and the models can be seen as complementary . when ph assumptions were not satisfied but aft conditions held , the parametric model should be used instead of the cox model . when neither the ph nor the aft assumptions were met , the log normal distribution , a member of the gg family , provided an alternative approach to semiparametric model . more generally , when ph assumptions are not satisfied , parametric models should be considered , whether or not aft assumptions are met .

background : the goal of this study is to extend the applications of parametric survival models so that they include cases in which accelerated failure time ( aft ) assumption is not satisfied , and examine parametric and semiparametric models under different proportional hazards ( ph ) and aft assumptions.methods:the data for 12,531 women diagnosed with breast cancer in british columbia , canada , during 19901999 were divided into eight groups according to patients ages and stage of disease , and each group was assumed to have different aft and ph assumptions . for parametric models , we fitted the saturated generalized gamma ( gg ) distribution , and compared this with the conventional aft model . using a likelihood ratio statistic , both models were compared to the simpler forms including the weibull and lognormal . for semiparametric models , either cox 's ph model or stratified cox model was fitted according to the ph assumption and tested using schoenfeld residuals . the gg family was compared to the log - logistic model using akaike information criterion ( aic ) and baysian information criterion ( bic).results : when ph and aft assumptions were satisfied , semiparametric and parametric models both provided valid descriptions of breast cancer patient survival . when ph assumption was not satisfied but aft condition held , the parametric models performed better than the stratified cox model . when neither the ph nor the aft assumptions were met , the log normal distribution provided a reasonable fit.conclusions:when both the ph and aft assumptions are satisfied , the parametric and semiparametric models provide complementary information . when ph assumption is not satisfied , the parametric models should be considered , whether the aft assumption is met or not .

INTRODUCTION MATERIALS AND METHODS Study design Statistical analysis Parametric model Semiparametric model RESULTS Situations when both PH and AFT assumptions are satisfied Situations when only AFT assumptions hold Situations where none of the PH and AFT assumptions are satisfied DISCUSSION CONCLUSION

despite many promising lupus treatments reaching early - phase human studies over the past several decades , none except belimumab has yet demonstrated efficacy in phase iii trials . attempts to explain the disappointing results of most clinical trials in lupus have pointed to pitfalls in the application and interpretation of clinical endpoints.1 according to recommendations by the food and drug administration , european league against rheumatism and outcome measures in rheumatology , the ideal endpoint should be able to detect both improvement and worsening in different manifestations and discern acute lupus disease activity from chronic damage and changes related to other causes.2 detection of both improvement and worsening is not possible using a global measure of disease activity such as the systemic lupus erythematosus disease activity index ( sledai ) , and it is not a trivial undertaking even with an organ - based assessment such as the british isles lupus assessment group ( bilag ) since different events within one organ may not be differentiated even with that complex instrument . this led to proposals of composite endpoints to detect improvement without worsening , two of which have already been widely incorporated in clinical trials.35 the sle responder index ( sri ) was derived in part by posthoc analysis of data from a failed phase ii study of belimumab.2 this was subsequently used as the primary outcome measure in two successful phase iii trials ( bliss-52 and bliss-76 ) , leading to belimumab approval by regulatory agencies.3 4 the sri consists of scores derived from the sledai and the bilag index : ( 1 ) 4-point reduction in sledai global score , ( 2 ) no new severe disease activity ( bilag a organ score ) or more than one new moderate organ score ( bilag b ) and ( 3 ) no deterioration from baseline in the physician 's global assessment ( 10% of scale ) . although a requirement for no change in treatment was not formally included in the sri definition , initiation of off - protocol medications defined non - response in the bliss studies . based on the success of the bliss programme , sri is currently being used in a number of clinical trials for sle . nonetheless , the treatment effect achieved by adding belimumab to standard of care using the original sri ( four - point drop in sledai ) was at best modest , and it remains unclear whether this is the optimal discriminatory endpoint . the bilag - based composite lupus assessment ( bicla ) was derived by expert consensus6 and employed as the primary endpoint in the emblem trial of epratuzumab in lupus , where it appeared to discriminate well between standard of care and epratuzumab added to standard of care , at least in some doses tested.5 bicla response is defined as ( 1 ) at least one gradation of improvement in baseline bilag scores in all body systems with moderate or severe disease activity at entry ( eg , all a ( severe disease ) scores falling to b ( moderate ) , c ( mild ) , or d ( no activity ) and all b scores falling to c or d ) ; ( 2 ) no new bilag a or more than one new bilag b scores ; ( 3 ) no worsening of total sledai score from baseline ; ( 4 ) 10% deterioration in physicians global assessment and ( 5 ) no initiation of non - protocol treatment . it should be noted that the emblem protocol was more restrictive than the bliss protocols in the increases allowed in background medications at baseline . the clinical components could be studied in isolation of the medication restrictions in order to simply determine presence or absence of clinical improvement . adding medication restrictions ( as has been done in clinical trials ) response to an intervention given at baseline without clouding that assessment by the impact of other medications that might have been added when patients were not responding to that treatment . direct comparison of bicla and sri has been addressed by only few studies to date,7 8 and the impact of the requirement for no medication changes on the simple determination of improvement has not yet been examined for either instrument . in this real - world exercise , the bicla and sri definitions were compared with physician 's clinical assessment of change in disease state with and without medication restriction rules to distinguish between improvement and response . all patients underwent informed consent procedures and health insurance portability and accountability act disclosures in compliance with good clinical practice . individuals were identified who met the 1997 modified american college of rheumatology classification criteria for sle,9 had two cohort study encounters at which the bilag , sledai and physician 's global assessment ( pga ) had been scored and had a sledai6 at the earlier ( baseline ) visit . in a minor deviation from either the selena - sledai ( used in the sri ) or the sledai-2k ( used in the bicla ) , a hybrid sledai is used for this cohort ( identical to the selena - sledai except for the scoring of proteinuria , which uses the sledai-2k definition ) . during a quality check of the data , where discrepancies were found between sledai and bilag scores and the original clinic note , these were corrected based on the original source documentation . the following data were determined retrospectively based on the medical record : some pga values that had not been scored at the time of the clinic encounters , and an overall physician impression of clinical change at the later encounter ( follow - up visit ) as compared with the first visit , determined by review of clinic notes and laboratory values but without consideration of what treatments had been given in the interim . this was categorised as clinically significant improvement ( physician - rated improvement ( pri ) ) , deterioration or no change . medication changes between baseline and follow - up and at the follow - up visit were recorded , and the sri-4 ( sri ) and bicla composite responses were calculated , both with and without consideration of the medication restrictions that characterised the major clinical trials . sri-3 and sri-5 were computed similarly to sri-4 , except for a minimal three - point or five - point improvement in sledai being required , respectively.2 when medication criteria were applied , improvement criteria were not considered to be met at any follow - up visit where lupus medication changes were made after the initial changes that were instituted at baseline with the exception of nsaids or topical agents . the performance of bicla and sri was compared against clinician - rated improvement . potential causes of discrepancies in each instrument were explored , including the role of each component endpoint . descriptive statistics ( mean sd ) were used to describe measures of disease activity ( pga , sledai , global bilag and number of bilag domains involved ) at baseline and follow - up . comparison of disease activity measures between baseline and follow - up was performed by paired t test . disease activity measures between pri responders that did or did not meet the bicla or sri endpoints were compared by mann spearman 's rank test was used to correlate pri with sri and bicla responses . the number of bilag domains with persistent and/or b scores at follow - up among pri responders stratified by the number of bilag a and/or b scores at baseline was compared by fisher 's exact test . descriptive statistics ( mean sd ) were used to describe measures of disease activity ( pga , sledai , global bilag and number of bilag domains involved ) at baseline and follow - up . comparison of disease activity measures between baseline and follow - up was performed by paired t test . disease activity measures between pri responders that did or did not meet the bicla or sri endpoints were compared by mann spearman 's rank test was used to correlate pri with sri and bicla responses . the number of bilag domains with persistent and/or b scores at follow - up among pri responders stratified by the number of bilag a and/or b scores at baseline was compared by fisher 's exact test . in total , 91 patients eligible for the analysis were identified , including 86 women and 5 men with sle . the mean age at the time of the baseline visit was 41 years ( sd 11.8 ) , ranging from 21 to 68 . also , 46 subjects were caucasian , 22 african - american , 14 native american , 5 caucasian / hispanic and 4 asian . the patients in this analysis were all assessed between june 2009 and april 2012 with a mean interval of 6.24 months between baseline and follow - up visits ( range 125 months ) . in total , 68 patients improved by pri , 17 remained the same and 6 deteriorated . disease activity in patients who did or did not improve by pri was compared using the pga , sledai , global bilag scores and the number of bilag organ domains involved ( table 1 ) . a statistically significant improvement between baseline and follow - up in all disease activity instruments was found in patients improving by pri , but no differences in those indices were observed in group not deemed by the physician to be improved . this supports previous literature that outcome measures discriminate clinically relevant differences.10 11 disease activity at baseline and follow - up among patients clinically improving ( n=68 ) or not ( n=23 ) bilag , british isles lupus assessment group ; pga , physician 's global assessment ; pri , physician - rated improvement ; sledai , systemic lupus erythematosus disease activity index . of 68 patients determined to be improving by the physician ( pri ) , 58 met the clinical criteria for sri and 52 met the bicla endpoint , with sensitivity of agreement with pri 85% and 76% , respectively ( table 2 ) . this evaluation was for improvement not response and did not include the restriction that change of medications over - rules response to the baseline treatment . of the 23 patients not improving by pri , 17 did not meet the sri clinical criteria and 18 did not improve by the bicla ; therefore , specificity of sri and bicla for pri was 74% and 78% . spearman 's rank correlations to pri were sri-3 0.605 , sri-4 0.563 , sri-5 0.541 and bicla 0.492 ( all p values<0.000001 ) . sensitivity and specificity of sri and bicla compared with pri bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . of the 10 out of 68 patients improving by pri but not by sri , 7 did meet the bicla improving criteria . these seven sri - negative / bicla - positive discordant cases failed the sri because of less than four - point improvement on sledai , which scores mild , moderate or severe arthritis four points and mild , moderate or severe rash two points ( six individuals had a two - point improvement and one had a three - point improvement ; therefore , all did have complete resolution of at least one feature , but the scores given to these particular features were not high enough to define improvement by sri ) . among 16 patients who improved by pri but these 13 sri - positive / bicla - negative discordant cases failed bicla because of one or more organ systems without even partial improvement , despite complete resolution of at least one other feature counting four points on the sledai . persistent b ( moderate ) scores in the musculoskeletal and mucocutaneous domains were present in six patients each , and there was one persistent b in each of the following domains : gastrointestinal ( lupoid hepatitis ) , cardiorespiratory ( pleurisy ) and renal ( proteinuria ) . one patient had a persistent a ( severe ) score in the cardiorespiratory domain ( interstitial lung disease ) . six of the twenty - three patients who did not improve by pri met the sri criteria , three of whom did not improve by bicla . these three sri / bicla discordant cases achieved four - point reduction in sledai due to resolving mild manifestations ( rash , alopecia , mucosal ulcers ) despite other ongoing ( but not worsening ) more severe organ involvement . five pri failures met the bicla , two of which did not meet the sri . one of these two patients developed new bilag b arthritis , and one new b score is allowed in the bicla definition of improvement . another had only moderate improvement in arthritis ( musculoskeletal bilag a score ( severe arthritis ) decreased to a high b ( significant moderate arthritis ) ) , but the patients were globally judged as overall clinically unchanged . since patients improving by pri often failed bicla ( false negative ) because improvement did not occur in all domains , we hypothesised this may be more common if there is a greater number of organs active at baseline . therefore , the total number of bilag domains involved in bicla improvement versus non - improvement was assessed in those patients meeting pri ( table 3 ) . comparison of disease activity indices between pri responders that met ( n=52 ) or not ( n=16 ) the bicla endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index . bicla responders tended to have fewer domains involved at baseline compared with non - responders , an observation that was more pronounced when only organs with baseline a ( severe disease ) and b ( moderate disease ) were counted . this supports the hypothesis that the more bilag organs are significantly involved at baseline , the more likely it is to have persistent disease in one or more organs at follow - up despite significant improvement in some aspects of the disease and an overall clinical impression of improvement . among patients improving by pri , 33.3% of those with two or more bilag a or b scores at baseline had persistent a or b scores at follow - up ( bicla failure ) compared with 10.3% in those with one or less bilag a or b scores at baseline ( fisher 's exact test , p=0.0924 ) . this suggests that the bicla might be less sensitive at detecting improvement when more organs are involved . pri responders meeting the bicla criteria also had less disease activity at follow - up compared with those not meeting the bicla , evident by the pga , sledai and global bilag scores , as well as the number of bilag organs involved ( p<0.02 for all comparisons ) , indicating the possibility that the bicla may provide some additional meaningful discrimination beyond what is captured by pri or be considered a higher bar for determining improvement . disease activity indices at baseline and follow - up were not as informative when comparing those improved by pri who did or did not meet the sri criteria ( table 4 ) . with the exception of pga , there was no difference in baseline disease activity measures in those improving by sri versus failures . comparison of disease activity indices between pri responders that met ( n=58 ) or not ( n=10 ) the sri endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index ; sri , systemic lupus responder index . clinical trial endpoints using the sri and bicla constructs to determine response to a treatment initiated at baseline have used restrictions in rescue medications in the definition of response ( underscoring a distinction between the concepts of improvement and response ) . a requirement for no off - protocol medication changes was included in the bicla response definition , used in the embem trial.5 in the bliss trials of belimumab in sle where the sri was used,3 4 medication increases were restricted during the latter months , and protocol deviations defined non - response accomplishing a similar end , although medication restrictions were less restrictive in those protocols than in the emblem study . to examine the impact of medication restrictions on how bicla and sri compare to physician 's determination of improvement , we performed the sri and bicla determination in this real - world sample of patients ( no protocolised restriction on treatment ) . all pharmacological changes in treatment after baseline interventions and prior to and/or at follow - up visit , except for nsaids or topical agents , excluded the designation of improvement by bicla or sri in this analysis . as expected , medication restrictions dramatically decreased the number of patients meeting bicla and sri response definitions . surprisingly , when medications were factored in as response criteria , this increased specificity for pri ( decreased the likelihood that bicla and sri will detect improvement when the physician did not agree ) , even though the physician was not considering the treatment changes in the pri assessment ( table 5 ) . sensitivity and specificity of sri and bicla with medication criteria bilca , british isles lupus assessment group - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . in total , 91 patients eligible for the analysis were identified , including 86 women and 5 men with sle . the mean age at the time of the baseline visit was 41 years ( sd 11.8 ) , ranging from 21 to 68 . also , 46 subjects were caucasian , 22 african - american , 14 native american , 5 caucasian / hispanic and 4 asian . the patients in this analysis were all assessed between june 2009 and april 2012 with a mean interval of 6.24 months between baseline and follow - up visits ( range 125 months ) . in total , 68 patients improved by pri , 17 remained the same and 6 deteriorated . disease activity in patients who did or did not improve by pri was compared using the pga , sledai , global bilag scores and the number of bilag organ domains involved ( table 1 ) . a statistically significant improvement between baseline and follow - up in all disease activity instruments was found in patients improving by pri , but no differences in those indices were observed in group not deemed by the physician to be improved . this supports previous literature that outcome measures discriminate clinically relevant differences.10 11 disease activity at baseline and follow - up among patients clinically improving ( n=68 ) or not ( n=23 ) bilag , british isles lupus assessment group ; pga , physician 's global assessment ; pri , physician - rated improvement ; sledai , systemic lupus erythematosus disease activity index . of 68 patients determined to be improving by the physician ( pri ) , 58 met the clinical criteria for sri and 52 met the bicla endpoint , with sensitivity of agreement with pri 85% and 76% , respectively ( table 2 ) . this evaluation was for improvement not response and did not include the restriction that change of medications over - rules response to the baseline treatment . of the 23 patients not improving by pri , 17 did not meet the sri clinical criteria and 18 did not improve by the bicla ; therefore , specificity of sri and bicla for pri was 74% and 78% . spearman 's rank correlations to pri were sri-3 0.605 , sri-4 0.563 , sri-5 0.541 and bicla 0.492 ( all p values<0.000001 ) . sensitivity and specificity of sri and bicla compared with pri bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . of the 10 out of 68 patients improving by pri but not by sri , 7 did meet the bicla improving criteria . these seven sri - negative / bicla - positive discordant cases failed the sri because of less than four - point improvement on sledai , which scores mild , moderate or severe arthritis four points and mild , moderate or severe rash two points ( six individuals had a two - point improvement and one had a three - point improvement ; therefore , all did have complete resolution of at least one feature , but the scores given to these particular features were not high enough to define improvement by sri ) . among 16 patients who improved by pri but not bicla , these 13 sri - positive / bicla - negative discordant cases failed bicla because of one or more organ systems without even partial improvement , despite complete resolution of at least one other feature counting four points on the sledai . persistent b ( moderate ) scores in the musculoskeletal and mucocutaneous domains were present in six patients each , and there was one persistent b in each of the following domains : gastrointestinal ( lupoid hepatitis ) , cardiorespiratory ( pleurisy ) and renal ( proteinuria ) . one patient had a persistent a ( severe ) score in the cardiorespiratory domain ( interstitial lung disease ) . six of the twenty - three patients who did not improve by pri met the sri criteria , three of whom did not improve by bicla . these three sri / bicla discordant cases achieved four - point reduction in sledai due to resolving mild manifestations ( rash , alopecia , mucosal ulcers ) despite other ongoing ( but not worsening ) more severe organ involvement . five pri failures met the bicla , two of which did not meet the sri . one of these two patients developed new bilag b arthritis , and one new b score is allowed in the bicla definition of improvement . another had only moderate improvement in arthritis ( musculoskeletal bilag a score ( severe arthritis ) decreased to a high b ( significant moderate arthritis ) ) , but the patients were globally judged as overall clinically unchanged . since patients improving by pri often failed bicla ( false negative ) because improvement did not occur in all domains , we hypothesised this may be more common if there is a greater number of organs active at baseline . therefore , the total number of bilag domains involved in bicla improvement versus non - improvement was assessed in those patients meeting pri ( table 3 ) . comparison of disease activity indices between pri responders that met ( n=52 ) or not ( n=16 ) the bicla endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index . bicla responders tended to have fewer domains involved at baseline compared with non - responders , an observation that was more pronounced when only organs with baseline a ( severe disease ) and b ( moderate disease ) were counted . this supports the hypothesis that the more bilag organs are significantly involved at baseline , the more likely it is to have persistent disease in one or more organs at follow - up despite significant improvement in some aspects of the disease and an overall clinical impression of improvement . among patients improving by pri , 33.3% of those with two or more bilag a or b scores at baseline had persistent a or b scores at follow - up ( bicla failure ) compared with 10.3% in those with one or less bilag a or b scores at baseline ( fisher 's exact test , p=0.0924 ) . this suggests that the bicla might be less sensitive at detecting improvement when more organs are involved . pri responders meeting the bicla criteria also had less disease activity at follow - up compared with those not meeting the bicla , evident by the pga , sledai and global bilag scores , as well as the number of bilag organs involved ( p<0.02 for all comparisons ) , indicating the possibility that the bicla may provide some additional meaningful discrimination beyond what is captured by pri or be considered a higher bar for determining improvement . disease activity indices at baseline and follow - up were not as informative when comparing those improved by pri who did or did not meet the sri criteria ( table 4 ) . with the exception of pga , there was no difference in baseline disease activity measures in those improving by sri versus failures . comparison of disease activity indices between pri responders that met ( n=58 ) or not ( n=10 ) the sri endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index ; sri , systemic lupus responder index . clinical trial endpoints using the sri and bicla constructs to determine response to a treatment initiated at baseline have used restrictions in rescue medications in the definition of response ( underscoring a distinction between the concepts of improvement and response ) . a requirement for no off - protocol medication changes was included in the bicla response definition , used in the embem trial.5 in the bliss trials of belimumab in sle where the sri was used,3 4 medication increases were restricted during the latter months , and protocol deviations defined non - response accomplishing a similar end , although medication restrictions were less restrictive in those protocols than in the emblem study . to examine the impact of medication restrictions on how bicla and sri compare to physician 's determination of improvement , we performed the sri and bicla determination in this real - world sample of patients ( no protocolised restriction on treatment ) . all pharmacological changes in treatment after baseline interventions and prior to and/or at follow - up visit , except for nsaids or topical agents , excluded the designation of improvement by bicla or sri in this analysis . as expected , medication restrictions dramatically decreased the number of patients meeting bicla and sri response definitions . surprisingly , when medications were factored in as response criteria , this increased specificity for pri ( decreased the likelihood that bicla and sri will detect improvement when the physician did not agree ) , even though the physician was not considering the treatment changes in the pri assessment ( table 5 ) . sensitivity and specificity of sri and bicla with medication criteria bilca , british isles lupus assessment group - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . the performance of two composite indices of lupus disease improvement and/or response was compared using as reference a clinician 's global rating of improvement . although the pga , when performed in a paper patient exercise , has been demonstrated to have poor agreement between different physicians,12 a retrospective overall determination of whether or not a patient has a clinically meaningful improvement might be a valuable tool when determined in a consistent manner by one qualified assessor . this was supported in the current exercise , where changes in disease activity ( including sledai and bilag performed prospectively at the time of the visits ) were consistent with the global clinician ratings . retrospectively assessing clinical change between visits is possibly limiting the validity of our observation and warrants conformation prospectively . although the visits used to determine improvement or response were separated by various timepoints in the current study , this did not affect the global clinical assessment of change between visits and is consistent with the manner in which sri and bicla are used in clinical trials as landmark assessments performed at different timepoints compared with a baseline visit . the assessment of response to an intervention is not the same as a measurement of clinical improvement ( which may occur without response to a given intervention if rescue treatments have been given ) . a comparison of bicla and sri without using the medication criteria for response to the baseline treatment was first performed to determine their utility in simply defining clinical improvement . in this assessment , when only those patients ranked as improved by the physician were evaluated , bicla but not sri seemed to define patients with greater improvement in disease activity , suggesting the possibility that it could be more discriminatory in some settings . since more organs involved at baseline decreased the sensitivity of the bicla , it can be hypothesised that this instrument might be particularly useful in patients with less widespread organ involvement , including those patients considered suitable for clinical trials in which less background medication is allowed . in fact , in the emblem trial of epratuzumab in lupus , which limited background medication changes more restrictively than the belimumab trials , the bicla response ( including the medication criteria ) at 12 weeks effectively discriminated the 2400 mg / month cumulative epratuzumab dosage groups from placebo with placebo responses lower than in the bliss trials.5 a comparison of two outcome measures used in different trials does not account for potential differences in patient populations , background treatments or efficacy of the test articles . however , it is worth observing that the apparent improved discriminatory capacity in the emblem trial ( using the bicla ) did not appear to be due to increased efficacy rates in the treatment group but to lower rates in the placebo ( standard of care ) group,7 which does not suggest increased sensitivity in detection of improvement , but might either reflect a more generally ill population , less background treatments or , indeed , the possibility of increased specificity of the improvement measurement . in posthoc analysis of this same trial dataset using the sri,7 sri rates were higher than bicla rates in all arms , including the placebo group , losing discriminatory capacity with a loss of significant differences between treatment and placebo . disagreement between bicla and sri in the emblem trial may , however , have been driven by the baseline distribution of items with high sledai weights that tended to improve at follow - up , with the greatest difference in groups with activity scored as eight - point sledai items ( vasculitis , lupus headache ) at baseline . scoring sledai descriptors for mild / moderate cutaneous vasculitis and headache risks sledai scores that are high in relation to the degree of illness of the patients . sri discriminatory performance might improve in a protocol , restricting the scoring of these features . in the current analysis , no lupus headaches were scored ( consistent with the evaluation here that they are very rare ) , and although four individuals had cutaneous vasculitis at baseline , this resolved in only one at follow - up , thereby not accounting for discrepancies in sri and bicla performance found here . some pri responders failed the sri , providing potential insight into limitations of the sri . patients rated as improving by the physician usually fail to meet the sri improvement criteria due to less than four - point improvement in sledai , which requires not only improvement but complete resolution of at least one manifestation . as expected , this limitation was less evident for sri-3 at the expense of lower specificity for the detection of pri , whereas the opposite was the case for the sri-5 . our results are consistent with the posthoc sensitivity analysis of data from the bliss-76 trial , where modifications of the sri using higher thresholds for selena - sledai improvement ( 5-point to 10-point reductions ) increased differentiation of belimumab from placebo at 52 and 76 weeks but occurred less frequently.4 in an assessment using a physician 's global rating of clinically significant improvement , the bicla may be less sensitive than the sri , particularly in determining improvement in patients with sle with multiple organs involved at baseline . on the other hand , the bicla may be more discriminatory than the sri in selecting those patients with a greater change in disease activity . when sri and bicla were discrepant , this was usually due to the bicla requiring only partial improvement but in all organs versus the sri requiring full improvement and not necessarily in all organs . each instrument could likely be an optimal primary endpoint depending on the population under study and the design of a given clinical trial .

objectivesto compare two measures of systemic lupus erythematosus ( sle ) response : the british isles lupus assessment group ( bilag)-based composite lupus assessment ( bicla ) and the systemic lupus responder index ( sri ) against a clinician 's assessment of improvement.methodsninety-one lupus patients were identified with two visits at which systemic lupus erythematosus disease activity index ( sledai ) and bilag had been scored and with active disease ( sledai6 ) at the first visit . a physician rated the disease activity at the second visit as clinically significant improvement , no change or worsening . sri and bicla were scored both with and without the medication criteria often used in trials to restrict response definitions.results68 patients were considered improved , 17 same and 6 worse at follow - up . sri versus bicla , performed without considering medication changes , captured physician - rated improvement with 85% vs 76% sensitivity and 74% vs 78% specificity . with medication limits both instruments had 37% sensitivity and 96% specificity for physician - assessed improvement . seven patients considered improved by the clinician met the bicla but not the sri definition of improvement by failing to achieve a four - point improvement in sledai . 13 clinician - rated responders met sri but not bicla by improving in less than all organs.conclusionsshortfalls of sri and bicla may be due to bicla only requiring partial improvement but in all organs versus sri requiring full improvement in some manifestation(s ) and not all organs . sri and bicla with medication restrictions are less likely to denote response when the physician disagrees and could provide stringent proof of efficacy in appropriately powered clinical trials .

Introduction Materials and methods Statistical analysis Results Demographic characteristics The PRI was associated with improvements in disease activity scores between baseline and follow-up Sensitivity of SRI compared with BICLA in detecting PRI Those considered improved by the physician but failed the BICLA had more active organs at baseline Response differs from improvement: the addition of medication criteria dramatically decreased the number of patients meeting the BICLA and SRI response definitions Discussion Conclusions

treatment of elbow dislocation with irreparable radial head fracture needs replacement of radial head to achieve stability of elbow . an alternate method in cases of elbow dislocation with radial head fracture can be resection of radial head with repair of medial collateral ligament . we report a retrospective analysis of cases of elbow dislocation with irreparable radial head treated by excision head of radius and repair of mcl . nine patients of elbow dislocation with associated irreparable fractures of the head of the radius were included in this analysis ( 6 f:3 m , age : 35 - 47 years ) . radial head excision was done through the lateral approach and mcl was sutured using no 3 ethibond using medial approach . above elbow plaster all patients were assessed at final followup using mayo elbow performance score ( meps ) . there was no extension deficit when compared to opposite side with mean range of flexion of 138.8 6.97 ( range 130 -145 ) . mean pronation was 87.7 4.4 ( range 80 - 90 ) and mean supination was 87.7 4.62 ( range 80 - 90 ) . radial head excision with mcl repair is an acceptable option for treatment of patients with elbow dislocation and irreparable radial head fracture . elbow dislocations without any associated fracture need no special care except closed reduction under sedation or short anesthesia , splintage for a few days followed by active mobilization for elbow rehabilitation.1 however , those with associated fracture head of radius need repair of head radius or replacement of the radial head along with the reduction of the dislocation to achieve a stable reduction . results of repair or replacement of radial head are not always satisfactory for supination pronation movements , although , the elbow remains stable.2 excision of isolated fracture of the head radius without elbow dislocation gives very satisfactory results,3 as mcl is always intact , though proximal migration of radius after many years has been reported.456 while , excision of head of radius alone , in elbow dislocation gives unsatisfactory results as elbow remain unstable due to loss of both stabilizers of the elbow i.e. head of radius , and mcl.7 hence if the radial head is repairable , it must be repaired in such situation.8 elbow dislocation , with irreparable fracture of the head of radius , needs replacement of the radial head to achieve stable elbow . if , decision is done to excise , isolated head of the radius fracture without dislocation , it must be confirmed under anesthesia , that elbow is stable , and it is not a case of spontaneous , reduction of elbow before patient has reported to the health care services . irreparable fracture head of the radius is said to be present when there are more than three fragments.2 it also becomes irreparable in the absence of adequate implants on the operating table as well as with a surgical team lacking experience.9 instead of doing inadequate fixation which gives restriction of rotation , it is safer to excise the head and repair the mcl . in elbow dislocation , the mcl is always injured.1 if excision of radial head is done for irreparable fracture , there is an increased chance of redislocation of elbow . results of replacement of radial head are not consistent and many long term and short - term failures are reported.8 repair of the comminuted fracture is technically difficult.10 the internal fixation should be reserved for minimally comminuted fractures with three or fewer articular fragments , in associated fracture - dislocations of the elbow.2 when the mcl is not injured in an isolated fracture of the head of the radius , head excision gives consistent results.3 similarly in an elbow dislocation , when the head of the radius is irreparable and needs replacement , if the radial head is excised along with repair of the mcl , it mimics the isolated radial head fracture situation and gave consistently good results.2 we report a retrospective analysis of cases of elbow dislocation with irreparable radial head treated by excision head of radius and repair of mcl . nine patients of elbow dislocation with associated irreparable fractures of the head of the radius were included in this analysis ( 6 f;3 m , age : range 35 - 47 years ) . two cases had an associated type grade 1 coronoid fracture which did not need repair . all elbow injuries with simple dislocations without fracture head of the radius , cases where the head was repairable , cases of terrible triad elbow which needs complex treatment and open fractures were excluded from this analysis . all patients presented immediately after injury . the elbow dislocation was confirmed on plain x - rays . elbow was reduced under sedation in all cases except one where short general anesthesia was used . the exact fracture pattern was assessed on post reduction x - rays [ figure 1 ] . x - ray elbow joint lateral ( a , b ) and anteroposterior ( c ) views of a 47 year old male ( case 6 ) presented with acute injury showing right elbow dislocation and displaced fractures radial head . closed reduction was done to relocate the joint and medial laxity can be well appreciated after reduction the joint was exposed using the lateral approach under general anesthesia and tourniquet . mcl was exposed , by a medial approach , remaining anterior to the medial condyle , avoiding the ulnar nerve . it was observed that the mcl was completely torn in all cases [ figures 25 ] . medial flexor muscles were also injured in all cases ; in six of them , it was injured in the muscle bulk , and in three cases , it had avulsed from the medial condyle . x - ray elbow joint anteroposterior and lateral views of a 36 year old female with elbow dislocation showing relocation of elbow with comminuted fracture radial head intraoperative demonstration of gross medial laxity ( arrow is on medial side ) intraoperative photographs excision of the radial head ( a ) showing lateral approach to the lebow with damaged soft tissues ( b ) irreparable excised radial head intraoperative photographs showing ( a ) the two ends of medial collateral ligament that are approximated ( b ) appearance after suturing the medial collateral ligament mcl with thick nonabsorbable no 3 ethibond sutures and medial stability was established ( c ) appearance after suturing the medial colareral ligament the mcl was sutured with thick nonabsorbable 03 number ethibond ( johnson and johnson , new jersey , usa ) sutures and medial stability was established . muscles , which were torn , were sutured . an above - elbow plaster slab was given for 6 weeks , for soft tissue healing . all patients were followed up till 4 months after removal of plaster , and were assessed for range of motion and elbow stability . all the patients were available for followup with mean followup of 19.55 7.12 months ( range 14 - 36 months ) . no patients reported pain at elbow or any subjective instability . there was no extension deficit when compared to opposite side with mean flexion of 138.8 6.97 ( range 130 - 145 ) . mean pronation was 87.7 4.4 ( range 80 - 90 ) and mean supination was 87.7 4.62 ( range 80 - 90 ) [ table 1 ] . the mean meps was 98.8 3.33 ( range 90 - 100 ) [ table 2 ] . no patient had any sensory complaints , or subluxation , or redislocation till final followup . none of the patients had to change their occupation and all were able to carry out their daily activities without disability . the primary stabilizers of the elbow are the intact bones , namely , olecranon and coronoid , head of radius , and lower end of the humerus . the radial head provides 30% of the valgus stability . with an intact mcl complex , removal of head of radius results in no instability . irreparable fracture head radius is said to be present when there are more than three fragments.2 the repair of multi fragment head of radius is demanding procedure as it requires quality implants and surgical skills of high order . this injury used to be treated by keeping the shattered head intact and elbow cast immobilization for 4 weeks when radial head prosthesis was not available . the delayed excision of radial head was after about 2 to 3 months was advocated to regain supination / pronation . unfortunately , range of motion did not return fully in these cases and poor results were more frequent.12 later , time , head of the radius was excised at time of primary treatment , and elbow was stabilized by steinmann pin till soft tissue healing ( 3 weeks ) . however , it was observed that the elbow could not be maintained in a stable position and had restriction of movements . results improved with the excision of the head of the radius and repaire of the mcl . fracture of the head of the radius can not be excised with associated elbow dislocation , where the mcl is always torn . if the head is excised , both primary and secondary stabilizers are lost and the elbow usually re - dislocates even in plaster . so if any one of the stabilizer is intact , the elbow remains stable like in the case of an excised head without dislocation where the mcl is intact . likewise , if the irreparable head of radius is replaced without repairing the torn mcl , the elbow remains stable . however , controversy still exists regarding which fractures are optimally treated with reduction and internal fixation as well as whether a fracture may be too comminuted to fix . tashjian and katarincic7 concluded that the radial head is a constraint secondary to the mcl for both valgus displacement and internal rotation . isolated repair of the ligament was superior to isolated prosthetic replacement and may be sufficient to restore valgus and internal rotatory stability after excision of the radial head in mcl deficient elbows . moro et al.8 opined that the radial head should be preserved , if possible , to lower the risk and prevent severe instability ; and if the radial head must be resected , suturing of torn ligaments and muscles at the epicondyles was essential . ring et al.9 reported that at the 12-year followup , following open reduction and internal fixation of radial head fractures , 80% had good or excellent results ; however , only 30% were completely pain - free , and 45% had evidence of arthritis . to conclude , repairing the mcl is a better , easier , and more predictable approach , when irreparable head radius fractures needs excision , in complex elbow dislocation .

background : treatment of elbow dislocation with irreparable radial head fracture needs replacement of radial head to achieve stability of elbow . an alternate method in cases of elbow dislocation with radial head fracture can be resection of radial head with repair of medial collateral ligament . we report a retrospective analysis of cases of elbow dislocation with irreparable radial head treated by excision head of radius and repair of mcl.materials and methods : nine patients of elbow dislocation with associated irreparable fractures of the head of the radius were included in this analysis ( 6 f:3 m , age : 35 - 47 years ) . radial head excision was done through the lateral approach and mcl was sutured using no 3 ethibond using medial approach . above elbow plaster was given for 6 weeks and gradual mobilization was done thereafter . all patients were assessed at final followup using mayo elbow performance score ( meps).results : mean followup was 19.55 7.12 months ( range 14 - 36 months ) . there was no extension deficit when compared to opposite side with mean range of flexion of 138.8 6.97 ( range 130 -145 ) . mean pronation was 87.7 4.4 ( range 80 - 90 ) and mean supination was 87.7 4.62 ( range 80 - 90 ) . the mean meps was 98.8 3.33 ( range 90 - 100 ) . no patient had pain , sensory complaints , subluxation or redislocation . all were able to carry out their daily activities without disability.conclusion:radial head excision with mcl repair is an acceptable option for treatment of patients with elbow dislocation and irreparable radial head fracture .

Background: Materials and Methods: Results: Conclusion: I M R D

ld is a nationally notifiable condition , and cases are reported by state and local health departments to the centers for disease control and prevention ( cdc ) through the national notifiable diseases surveillance system in accordance with previously established protocols ( 7 ) . , a revised case definition was implemented that altered the laboratory criteria and distinguished between confirmed and probable cases ; cases reported during 20082013 included both categories ( 8) . weighting was applied to state- and county - specific numbers of cases to account for variations in completeness of ethnicity data . descriptive statistics and comparisons were calculated by using sas version 9.3 ( sas institute , cary , nc , usa ) . we compared median age of hispanics and non - hispanics with ld using the kolmogorov - smirnov 2-sample test . cdc human subjects review of the protocol determined it was not research involving human subjects . a total of 374,338 ld cases were reported to cdc , of which 148,444 ( 39.7% ) reports contained information about ethnicity and were included in this analysis . among these , most ( 54.8% ) hispanics with ld were male ; median age was 32 years ( interquartile range 1546 years ) . annual incidence of reported ld among hispanics was 0.8 cases/100,000 persons , compared with 4.0/100,000 among non - hispanics . during 20002001 , hispanics comprised 2.8% of all persons with ld , whereas during 20092013 , hispanics comprised 3.7%4.9% of persons with reported ld . in comparison , the proportion of hispanics in the us population increased slightly during this period , from 13% in 2000 to 16% in 2010 ( 5 ) . although a bimodal age distribution was evident among both hispanics and non - hispanics with ld , the peak in children was less pronounced among hispanics ( figure ) . highest incidence among hispanic children was in boys 1014 years of age , whereas among non - hispanic children , incidence was highest in boys 59 years of age . in adults , highest incidence among both hispanics and non - hispanics was in men 6574 years of age . age- and sex - specific incidence of lyme disease among hispanics ( a ) and non - hispanics ( b ) , united states , 20002013 . for persons hispanics were significantly less likely than non - hispanics to have disease onset during the summer months ( rr 0.85 , 95% ci 0.830.88 ) and more likely to have disease onset during the fall months ( rr 1.15 , 95% ci 1.071.24 ) ( table 1 ) . although erythema migrans was the most commonly reported clinical feature for both groups , it was less commonly reported among hispanics than non - hispanics ( rr 0.83 , 95% ci 0.800.86 ) . manifestations of disseminated disease , such as arthritis and facial palsy , were more commonly reported among hispanics than non - hispanics ( table 1 ) . iqr , interquartile range ; ld , lyme disease ; rr , risk ratio . percentage of persons with ld for whom sex is known ( n = 5,442 hispanics , n = 142,625 non - hispanics ) . the substantial difference in median age between the us hispanic population ( 27 y ) and the us non - hispanic population ( 42 y ) most likely accounts for the difference seen here . defined as 1 of the 13 highest - incidence states that accounted for 95% of all reported confirmed cases of ld in 2010 : connecticut , delaware , maine , maryland , massachusetts , minnesota , new hampshire , new jersey , new york , pennsylvania , vermont , virginia , and wisconsin . overall were reported from high - incidence states , although hispanics with ld were slightly less likely to report residence in a high - incidence state ( rr 0.90 , 95% ci 0.820.98 ) . all of the statistical associations were similar when analysis was restricted to confirmed cases only , with the exception of residence in a high - incidence state , which became nonsignificant ( rr 0.99 , 95% ci 0.891.10 ) . after weighting , nearly half of all estimated cases of ld among hispanics were from new york or new jersey ( table 2 ) . among counties with at least 75 estimated ld cases among hispanics during the study period , highest incidence among hispanics occurred in columbia county , new york ( 170.4 cases/100,000 persons ) ; sussex county , new jersey ( 111.4/100,000 ) ; and hunterdon county , new jersey ( 106.3/100,000 ) . after correcting for missing ethnicity data . calculated as follows : ( no . reported cases)/x = ( % with ethnicity information)/100 , where x is the weighted number of cases . incidence calculated as number of annual estimated cases in county/100,000 hispanic residents in county . only counties with a substantial number of cases were included in this comparison . overall , the epidemiology of ld among hispanics was similar to that among non - hispanics : bimodal age distribution , slight predilection in males , and clustering in states to which ld is highly endemic were apparent ( 10 ) . most notably , hispanics with ld were significantly more likely than non - hispanics with ld to have signs of disseminated infection and symptom onset during fall months . although the overall incidence of ld in hispanics was lower than that in non - hispanics , additional research is needed to determine the reasons underlying these differences and the extent of any ld underdiagnosis in the hispanic population . inadequate healthcare access , language barriers , and lack of ld awareness could cause both underdiagnosis and delays in diagnosis in the hispanic population . during 20092013 , a total of 41.5% of hispanics lacked health insurance , compared with 15.1% of non - hispanic whites ; 15.5% of hispanics described delay in or nonreceipt of medical care because of cost ( 11 ) . furthermore , whether the predilection toward symptom onset in the fall months for hispanics results from delays in medical care or other factors , such as seasonal outdoor labor patterns , is unclear . lastly , because a larger proportion of hispanics than the overall us population live in urban areas ( 12 ) , the risk for ld might be differentially diluted in hispanics . first , we had to exclude more than half of reported ld cases because of missing ethnicity data . although we have no reason to believe that case reports with missing ethnicity data differed otherwise from those included in this study , we can not exclude this possibility . finally , surveillance data are limited by underreporting and reporting bias , which might differ by state and between hispanic and non - hispanic populations . although some educational materials about prevention of tickborne diseases have been translated to spanish ( 13,14 ) , additional translations and modifications to address cultural differences would be helpful . furthermore , targeted educational campaigns could enhance use of these materials and improve the reach , retention , and overall impact of prevention education . direct and more in - depth assessments regarding prevention practices , knowledge , and ld epidemiology on local and national scales will further the understanding of ld risk in this population and guide future targeted prevention and education efforts .

hispanics comprise a growing portion of the us population and might have distinct risk factors for tickborne diseases . during 20002013 , a total of 5,473 lyme disease cases were reported among hispanics through national surveillance . hispanics were more likely than non - hispanics to have signs of disseminated infection and onset during fall months .

The Study Conclusions

chronic cardiovascular diseases ( cvds ) are considered the leading cause of mortality worldwide ( 1,2 ) . mortalities by cvds occur mainly due to coronary heart disease ( chd ) , stroke , rheumatic heart , and myocardial infarction ( mi ) . approximately 80% of these deaths occur in low and middle income families and in poor countries ( 3 ) . it is estimated by who that approximately 20 million individuals are likely to succumb to cvds in the year 2015 ( 4 ) . in addition , non - communicable diseases are to account for over 75% of mortalities worldwide ( 1 ) . infectious diseases , tuberculosis , and malaria followed by nutritional disorders are the primary cause of such mortalities ( 5 ) . cvds are also the largest contributor to global mortality of heart patients ( 3,6 ) . the epidemiology of heart diseases has been described by many researchers in the literature . for the epidemiological studies of heart diseases , a significant percentage of cvd mortalities occur in younger individuals and children in developing nations due to malnutrition , low income , poverty , unemployment , excessive alcohol consumption , tobacco smoking , car accidents , and other factors ( 5 ) . approximately 41% individuals die between the age of 35 and 64 years in south africa , 35% in india , 28% in brazil , 12% in the usa , and 9% in portugal ( 7 ) . the median age of heart attack and first stroke and death from ischemic heart disease ( ihd ) as well as acute myocardial infarction ( ami ) are very important ( 3 ) . hypertension , high blood pressure ( hbp ) , high blood cholesterol , obesity , diabetes , reduced physical activity , psychological stress and other factors are also responsible for cvds . the historical trends in cv mortality and tobacco use in north america increased between 1900 and 1990 ( 8) . in the uk , a 38-year follow - up study of men showed that baseline differences in tobacco use , hbp pressure , and cholesterol were associated with a 10- to 15-year shorter life expectancy ( 9 ) . the rise in cigarette smoking in young individuals indicates increased tobacco - related mortalities in many countries ( 10 ) . by the year 2030 , who ( 2008 ) projects that over 80% of tobacco - related deaths are to occur in young people in developing countries . currently , malnutrition and obesity occur within the same population in many low and middle income families ( 11,12 ) . malnutrition is common in many low and middle income countries of africa , latin america , and south asia ( 7 ) . the global prevalence of overweight in children aged 517 years is 10% , varying from under 2% in sub - saharan africa to over 30% in the usa ( 14 ) . the epidemiological research of cvds suggests that dietary changes are associated with nutritional transition , specifically the increasing consumption of energy - dense diets high in fats , oils , sodium , and sugars , which contribute to an increase in cvd incidence in low and middle income families ( 15 ) . the kilo caloric intake from selected food groups in china , india , mexico , egypt , and south africa from 1980 to 1982 were compared to the kilo caloric intake in 2001 to 2003 using data from the food and agriculture organization of the united nations statistical database ( faostat ) ( 16 ) . in china , total lcd ( low - calorie diet ) increased from 2,327 to 2,940 kcal , and meat consumption increased by over 246% from 1980 to 2003 . there was also a significant increase in the intake of oils in china , with three types of oils increasing over 100% : palm oil ( + 640% ) , soybean oil ( + 635% ) , and vegetable oils ( + 259% ) . on the positive side , the intake of fruits and vegetables also increased by 600 and 367% , respectively ( 16 ) . between 1980 and 2003 , there has been an increase in meat as well as greasy food consumption in china , as well as increased use of tropical oils ( which is associated with high cholesterol and not healthy for heart ) in preparing food . similarly , in india , mexico , south africa and other nations the elevated kcal intake of palm oil in the diets was reported ( 16 ) . the causes of the decline in cvds in developed countries offer potential lessons for achieving similar results in developing countries . the major risk factors for cvds have been reported for childhood and adolescence ( 1720 ) . these include use of tobacco , diets and physical activities , overweight and obesity , and adverse childhood experiences . poor social life circumstances in childhood have also been linked to cvds later in life in different cohort studies conducted in the usa and europe ( 21,22 ) . a cohort study of 20,040 individuals four measures of health were examined : smoking , low physical activity , low plasma vitamin c levels ( used as a proxy for fruit and vegetable intake ) , and not drinking alcohol in moderation ( abstaining from alcohol or consuming more than 14 drinks per week ) and the results indicated that one or more of the abovementioned risk factors alone or in combination with other risk factors constituted a > 2-fold increase in stroke incidence ( 23 ) . this is consistent with prior findings in large cohort studies on men and women in the usa whereby a healthy diet and lifestyle of not smoking , regular exercise , moderate alcohol consumption , and not being overweight was associated with an almost 80% lower risk of ihd compared to having none of the abovementioned healthy lifestyle components ( 24 ) . the major risk factors for cvd in young individuals or adults include fats , abnormal blood lipids , tobacco smoking , alcohol , abdominal obesity , psychosocial factors , hypertension , and diabetes . there is strong evidence that , excessive salt and sodium intake significantly increases cvd risk and that reduction in sodium intake decreases incidence of cvds ( 25 ) . three major dietary patterns were identified : i ) oriental ( high intake of tofu and soya ; ii ) western ( high in fried foods , salty snacks , eggs , and meat ) ; and iii ) prudent ( high in fruits and vegetables ) . the results showed the western dietary pattern was associated with an increased risk of chd in animals and humans globally , whereas the prudent pattern with fruits and vegetables was associated with a lower risk of heart diseases in children , young and adult individuals ( 26 ) . increased daily consumption of alcohol has been reported as a great risk factor of chd in individuals of all ages in low and middle income groups ( 27 ) . the who estimates that the harmful use of alcohol was responsible for 3.8% deaths and 4.5% of the global burden of heart diseases in 2004 ( 3 ) . in the past few decades , consumption of alcohol has increased in countries undergoing nutritional transition , such as india and china , and russia , where it contributes significantly to overall mortality among men ( who expert committee , 2007 ) ( 2 ) . excessive alcohol intake is associated with increased risk for hypertension , stroke , coronary artery disease , and other forms of cvds ; however , evidence conducted in various populations suggests that light to moderate intake of alcohol may reduce the risk of chd ( 2 ) . the relationship between one drink per day for women and no more than two drinks per day for men constitute moderate drinking for health of heart ( 28 ) . physical exercise is also important for the health of heart as who and the food and agriculture organization ( fao ) suggest the importance of physical activity as a key determinant of obesity , cvd , and diabetes ( 29 ) . obesity or overweight issues , reported from developed and developing countries are considered risks of cvds ( 30 ) . malnutrition remains highly prevalent compared to overweight and obesity , especially among women and children ( 31 ) . the intake of cereals is also important in china , where 58% of total calorie intake is consumed compared to meat , 13% , and cooking oils , 17% ( 32 ) . the national data from the office of national statistics indicates that cereals and related products account for 31% of calories with other major categories including meat , 15% ; milk and related products , 19% ; and beverages , 10% ( 33 ) . in a study on hbp it was found that approximately 54% of individuals experience stroke , 47% ihd , 75% hypertensive disease and 25% other cvds that were attributable to hypertension . approximately 7.6 million individuals succumb to hypertension , with 13.5% of the total number of annual global deaths being attributable to hbp ( 34 ) . it is estimated that the current age - standardized prevalence rate of hypertension is 17.7% , which amounts to approximately 177 million individuals , while 20% of individuals in china may succumb to hbp due to a high intake of salt ( 35,36 ) . the framingham study showed a connection between hypercholesterolemia and increased risk of chd in 1960 with the finding that lower levels of high - density lipoprotein ( hdl ) cholesterol as well as elevated levels of low - density lipoprotein ( ldl ) cholesterol were associated with increased risk of chd ( 37,38 ) . the results of several randomized controlled trial studies have shown that the reduction of ldl cholesterol , both in primary and secondary prevention , is associated with a lower incidence of chds ( 4143 ) . the interheart study found that abnormal blood lipids were the most important risk factor for mi , by odds ratio in all global regions ( 44 ) . elevated cholesterol levels were the third leading risk factor for the worldwide mortality of young and elderly individuals , after hypertension and smoking ( 45 ) . the risk of cvd associated with diabetes is even greater in women and younger individuals . having diabetes may be equivalent to aging by at least 15 years with regard to the clinical manifestations of cvd ( 46,47 ) . diabetes is associated with serious health consequences and is a major risk factor for chd and stroke . depression and depressive symptoms are also associated with behaviors that increase cvd risk ( 48 ) . anxiety , anger , hostility , chronic and acute stress , and lack of social support are factors associated with increased cvd morbidity and mortality . the acute stress from traumatic injury , death of a relative , earthquakes , terrorist attacks or other factors have been associated with significant temporal increases in the incidence of mi in young or adults ( 49 ) . research has also shown that air pollution and respiratory problems have adverse health outcomes associated with lung - heart or cardiovascular effects ( 50 ) . the burning of coal and wood used for cooking is also a significant contributor of air pollution in developing countries . the epidemiological studies of heart diseases have also shown that long - term exposure to smoke can increase the risk of cvd ( 51 ) . molecular genetics and advanced statistical methods , used for the study of heart diseases and proteomics methods can lead to potential biomarkers that can identify risk of cvds , which in turn , can improve the prediction of acute cardiovascular events ( 52 ) . pharmacogenetics can be useful in the development of more effective medications , and the management of cvd in children , young and adult population . rheumatic heart disease ( rhd ) , chagas disease , and infectious pericarditis and cardiomyopathies continue to cause a substantial burden of disease and death in some low and middle income countries ( 53 ) . a better epidemiological understanding of infectious forms of cvd in developing countries , and efforts to improve health care are needed to prevent and treat these diseases . infectious diseases such as hiv / aids , and tuberculosis ( tb ) , and other diseases such as malaria , typhoid and diabetes are also important for their association with cvd ( 53 ) . in order to study cvds in the developing and developed nations , efficient epidemiological programs should be designed . chd accounts for the maximum proportion of cvds for which risk factors are hypertension , smoking , diabetes , high cholesterol , higher level of ldl , obesity and overweight causing death of heart patients . assessment of short- or long - term risk prediction algorithms is useful to identify patients that can benefit most from risk factor interventions . the evaluation of new risk factors and screening for subclinical atherosclerosis are imperative in the identification of individuals that are at the highest risk of cvd . the prevention of chds must focus on identifying and managing risk factors in individual cases as well as the population through primordial , primary , and secondary prevention . epidemiological studies have provided a hypothesis based on clinical trials for chd and the efficacy of risk - factor interventions , which are the basis of prevention of heart diseases ( 55 ) . further investigations on screening and intervention strategies is important to determine the effect on the prevention of chds and cvds . recent advances in the epidemiology , pathogenesis and prognosis of acute heart failure and cardiomyopathy have been described from africa ( 56 ) . a large prospective registry showed that acute decompensated heart failure is caused by hypertension , cardiomyopathy and rhd in 90% of cases . this is a pattern that is in contrast with the dominance of coronary artery disease in north america and europe ( 56 ) . acute heart failure is a disease of young individuals with a mean age of 52 years and occurs equally in men and women . this observation was associated with high mortality at 6 months ( approximately 18% ) , but was similar to that observed in non - african heart failure registries , suggesting that heart failure was a dire prognosis globally , regardless of the aetiology . investigations into the molecular genetics of dilated cardiomyopathy , hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy in populations in africa are consistent with observations elsewhere ( 56 ) . in addition , the study of genotype - phenotype correlations in a large number of cardiomyopathy individuals is due to the same mutation ( 56 ) . thus the use of proven treatments , and the control of hypertension in various populations should be increased . the american heart association ( aha ) has recommended following a mediterranean diet for heart patients ( 57 ) , in which more fruits and vegetables are included . at least 16 countries border the mediterranean sea and their diets vary among these countries . there are numerous differences in culture , ethnic background , religion , economy and agricultural production that result in a difference in mediterranean diets . the common mediterranean dietary pattern has these characteristics : high consumption of fruits , vegetables , bread and other cereals , potatoes , beans , nuts and seeds , olive oil which is an important source of monounsaturated fats , dairy products , fish and poultry consumed in low to moderate amounts , and a modest amount of red meat ( 57 ) . in such a diet , eggs are consumed 04 times a week and wine in low - to - moderate amounts . individuals who adhere to an average mediterranean diet usually eat less saturated fats than those who consume an average american diet . it is known that over 50% of fat calories in a mediterranean diet are derived from monounsaturated fats , mainly from olive oil . monounsaturated fats do not raise the level of blood cholesterol in the same way as saturated fat . the incidence of heart disease in mediterranean countries is lower than that in the united states , as are mortality rates . diet as well as lifestyle factors , such as more physical activity and extended social support systems may also be important in heart diseases . in recent advances in preventive cardiology and lifestyle medicine , public policy approaches to the prevention of heart disease and stroke have been described ( 58 ) . the global epidemiology of heart disease and stroke in the 20th century showed marked variations in cause - specific mortality rates between developed and developing countries due to trends of social and political origins of cardiovascular risk . thus , governments and the private sectors are attempting to educate the public and coordinate public efforts to retard the global epidemic of heart disease and stroke ( 59 ) . the osaka declaration has emphasized factors for the health sector that serve as impediments to heart health ( 60 ) . success in the treatment of heart diseases and conditions of mi , valvular heart disease , and arrhythmias has improved patient survival . in the elderly , congestive heart failure is the leading cause for admission to hospitals in the united states , which is extremely expensive for health care ( 61 ) . african - americans , aged 4565 years , have a 70% higher rate of heart failure as shown in the census bureau and the centers for disease control and prevention , with a mortality rate 2.5-fold higher than that of the caucasian population . the hispanic / latino population has a higher incidence of heart failure than the caucasian non - hispanic population . however , more population - based studies are needed to estimate the prevalence of heart failure in the hispanic population ( 62 ) . useful sites for heart disease research include : the american college of cardiology ( http://www.acc.org/ ) , for up to date heart failure patient 's information ( http://www.uptodate.com/home ) ; national library of medicine , ( https://www.nlm.nih.gov/ ) ; national heart , lung , and blood institute ( http://www.nhlbi.nih.gov/ ) ; european society of cardiology ( http://www.escardio.org/ ) ; and heart failure online , http://www.heartfailure.org/. recent investigations on cvds in europe for 2014 have shown that , epidemiological aspects have been updated ( 63 ) . cvds and in particular chd and stroke have been previously demonstrated in european countries ( 63 ) . cvds cause more deaths among europeans than other countries , and cause more than twice as many deaths as cancer . it has also been reported that for some eastern european countries , including russia and the ukraine , the mortality rate for chd in 55- to 60-year - old individuals is greater than the equivalent rate in france . the epidemiology of cvd in the uk in 2014 was described with regard to mortality , prevalence and treatment costs ( 64 ) . in the year 2012 , cvd was the most common cause of mortality in the uk for women at 28% of all female deaths , but not for men , whereas cancer is currently the most common cause of mortality at 32% of all male deaths . mortality from cvd varies widely throughout the uk , with the highest age - standardized cvd death rates in scotland ( 347/100,000 ) and the north of england ( 320/100,000 in the north west ) . the prevalence of chds is also highest in the north of england ( 4.5% in the north east ) and scotland ( 4.3% ) . thus , primary and secondary prevention measures are important to reduce the burden of cvd and inequalities in cvd mortality rate and prevalence . the epidemiology of heart failure has been reported and it has been suggested that chd is prevalent ( 65 ) . the excessive use of tobacco smoking , alcohol consumption and other factors are also responsible for heart diseases . the present review focused on recent epidemiological aspects of cvd and chd as well as the benefits of a mediterranean diet for heart patients and its usefulness in preventing cardiovascular disease . in general , there are several risk factors such as high blood cholesterol , high blood pressure , obesity and family history that are associated with cvd and chd results obtained from several studies support the positive effects of the mediterranean diet for the primary prevention of cardiovascular disease . the mediterranean diet is characterized by a high intake of olive oil , fruit , vegetables , and cereals ; a moderate intake of fish and poultry , and a low intake of dairy products , red meat , processed meats , and sweets , as well as wine in moderation , consumed with meals . future studies may shed more light on the notable effects of the mediterranean diet on the prevention of cardiovascular disease .

cardiovascular diseases ( cvds ) are the leading cause of mortality worldwide . coronary heart disease ( chd ) is the main cause of mortality in heart patients following stroke , rheumatic heart disease and myocardial infarctions . approximately 80% of individuals succumb to cvds , due to poor living conditions in low and middle income families and malnutrition . infectious diseases , human immunodeficiency , tuberculosis , malaria , high blood pressure or hypertension , obesity and overweight , and nutritional disorders including smoking , excessive alcohol consumption , high salt and sugar intake , as well as other factors are responsible for cvds and chds in young as well as elderly individuals . the focus of the present review are recent epidemiological aspects of cvd and chd as well as the usefulness of a mediterranean diet for heart patients and the prevention of heart diseases .

Introduction Mediterranean diet for heart patients Epidemiological studies Mediterranean diet for heart patients Conclusion

previously documented sites of primary disease include the ovaries , melanoma , breast , lung and the colon and are typically associated with high disease burden . splenic metastases from the colon involving low levels of disease burden are rare in the literature and to date , there have been only two documented cases of metastases to the spleen originating from a caecal tumour . a 60 year old male with no significant past medical history presented to the emergency department with a three week history of lower abdominal pain , nausea and a palpable mass in the right iliac fossa . computerised tomography ( ct ) of the abdomen and pelvis demonstrated a large circumferential obstructing tumour of the caecum without evidence of distant metastases ( fig . the patient was prepared for surgery , consisting of a right hemicolectomy with an uncomplicated postoperative course . histology from the excised specimen revealed a poorly differentiated adenocarcinoma with signet ring cell permeation through the muscularis propria . lymph node analysis comprised twenty one lymph nodes , all noted to contain metastatic spread . the presenting tumour was therefore classified pathologically as stage iii caecal adenocarcinoma ( pt3n2m0 ) . carcinoembryonic antigen measured prior to surgery was recorded within normal limits ( 3 ng / l ) . two months post surgical resection , the patient underwent adjuvant chemotherapy utilising the folfox protocol as discussed at the multidisciplinary conference . he received 12 cycles of chemotherapy however the dose of oxaliplatin was reduced after cycle six due to development of thrombocytopaenia and a slight rise in cea level , both of which were attributed to the chemotherapeutic agent ( table 1 ) . the patient was then entered into a standard programme of routine surveillance consisting of regular clinical reviews , interval measurement of cea levels , colonoscopy and yearly ct of thorax , abdomen and pelvis . although surveillance studies proved negative initially for disease recurrence , a routine ct thorax , abdomen and pelvis at twenty three months post initial presentation revealed left sided para aortic lymphadenopathy and two suspicious splenic lesions measuring 2.7 2.5 cm and 2.4 2.2 cm respectively which were not present on imaging , performed twelve months previously ( fig . 2 a and b ) . the splenic lesions were biopsied under ultrasound guidance using an 18 gauge tru cut needle without complication ( fig . subsequent histology of the biopsied lesions revealed normal splenic parenchyma infiltrated by poorly differentiated adenocarcinoma with abundant signet ring cells , morphologically identical to the initial presentation with an obstructing caecal tumour ( fig . the disease although of low burden and primarily localised to the spleen was associated with the presence of suspicious para aortic lymphadenopathy prompting chemotherapy rather than operative intervention . the oncology team advised further systemic chemotherapy comprising 5 fluorouracil , oxaliplatin and avastin . the patient remains well on chemotherapy and further imaging studies are planned in 4 months time to assess response to treatment . metastases to the spleen from any primary tumour are rare despite the highly vascular nature of the organ and it 's proximity to many potential primary sites including the pancreas , stomach , lung , breast and the colon . a number of theories have been put forward to explain the hostile nature of the spleen preventing tumour implantation and proliferation of neoplastic cells . anatomical features thought to play a role include the sharp angle at the origin of the splenic artery with the coeliac axis , the rhythmic contraction of splenic sinusoids and the scarcity of lymphatic vessels extending to the intrasplenic parenchyma . immunological factors considered to negatively impact on the rate of malignant cell implantation include the presence of abundant kupffer cells , immunoglobin synthesis and opsonin production . in most of the reported cases of colorectal metastases to the spleen , the primary tumour is located on the left side of the colon or in the rectum . this distribution of colorectal metastases favours the theory that neoplastic cells reach the spleen via retrograde dissemination through the inferior mesenteric vein . to the best of our knowledge , there have been only two previous cases of caecal adenocarcinoma metastasising to the spleen recorded in the literature without infiltration of other solid organs . furthermore , there have only been 4 additional cases of right sided colonic cancer ( all ascending colon ) with metastases to the spleen recorded . given the additional presence of left sided para aortic lymphadenopathy in our patient , this could possibly indicate tumour spread via the lymphatic system . it is not routinely used as a screening tool as it lacks sensitivity and specificity . however it is widely used as part of surveillance programmes to monitor for disease recurrence in conjunction with colonoscopy , imaging and follow up clinic appointments . it has been shown that in 3040% of cases of disease recurrence there is no corresponding rise in cea levels . furthermore in cases where cea levels were recorded as normal at initial presentation , it was reported that in cases of recurrence , a corresponding elevation in cea is only observed in 27% of cases . in one large study , it was noted that cea monitoring did not result in improved survival . this brings into question the value of interval cea monitoring as part of surveillance programmes post resection for colorectal cancers . historically the technique of percutaneous splenic biopsy was not widely performed due to the perceived high complication rates mostly involving haemorrhage . with the advent of improved imaging modalities , splenic biopsies can now be performed relatively safely using either ultrasound or ct guided techniques . the differential of a solid splenic lesion includes metastases , an infarct or a splenic abscess . adequate collection of targeted tissue suitable for histological analysis has been recorded at 88% in one series . complication rates were found to be directly proportional to the gauge of needle used in the procedure . given the limited number of cases recorded in the literature , the optimal treatment for splenic metastases from a colorectal primary is not known . in cases of isolated splenic metastases from a colonic source , splenectomy is preferred to chemo / radio therapy . in the literature , this has been performed by both the traditional open technique and more recently laparoscopically . to date disease free intervals and survival are reported to be similar for both techniques . chemotherapy is currently favoured in circumstances where disease is recorded in other sites including the spleen . the current data available is too small to draw any definitive conclusions regarding the optimal treatment for splenic metastases in the context of low disease burden . firstly , multimodal imaging is important in the context of asymptomatic patients and especially given the limited sensitivity of cea meaurements . second , modern image guided biopsy techniques facilitate accurate and safe percutaneous biopsy of suspicious splenic lesions . third , further research is necessary to determine the optimal treatment of splenic metastases from a colonic source with a low disease burden . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . kevin barry helped in the final review of article prior to submission including editing and layout .

introductionthe spleen is a highly vascular organ and is in close proximity to many potential primary sites such as the stomach , breast , pancreas and colon . it is however an unusual site for metastatic disease . the reasons for this are not fully understood at the present time . a number of hypotheses have been postulated . definitive diagnosis and subsequent treatment of metastatic disease to the spleen presents a number of challenges for the surgeon and the wider multi disciplinary team.presentation of casea 60 year old male presented with a three week history of lower abdominal pain , distension , nausea and a palpable mass in the right iliac fossa . imaging revealed a large circumferential caecal mass consistent with malignancy with secondary small bowel obstruction . the patient underwent an emergency right hemicolectomy and was subsequently treated with systemic chemotherapy for lymph node positive caecal adenocarcinoma . two years following initial presentation , two suspicious lesions were noted within the spleen during routine surveillance imaging with computerised tomography of the thorax , abdomen and pelvis . of note , one month prior to this the patient had a normal surveillance colonoscopy performed with multiple interval carcinoembryonic antigen ( cea ) levels recorded within the normal range . image guided splenic biopsy and subsequent histology confirmed metastatic caecal adenocarcinoma.discussionsplenic metastases from any source including the colon are a rare phenomenon.conclusionthis case questions the value of routine post operative cea monitoring , underlines the importance of multimodal pathways of surveillance and highlights recent advances in image guided splenic biopsy techniques .

Introduction Case report Discussion Conclusion Conflict of interest statement Funding Ethical approval Author contributions

recent advances in repair of thoracoabdominal aortic aneurysms ( taaa ) allow one to choose between three therapeutic options : conventional open surgery , a hybrid open - endovascular procedure or endovascular repair . despite significant progress in operative technique and procedural anesthesia , a conventional open surgical repair of taaa is still accompanied by significant morbidity and mortality . a number of large studies dealing with outcomes following open taaa surgical treatment indicate that the 30-day mortality of elective cases ranges between 5% and 19% , while one - year mortality increases to even 30% [ 1 , 2 ] . however , only experienced high - volume vascular centers can treat taaa surgically with a mortality rate below 10% . in high - risk patients , hybrid procedures combining extra - anatomic bypasses and endovascular techniques with branched and fenestrated aortic stent grafts have been used to reduce the risk of conventional taaa with the repair of visceral arteries . in patients with crawford type iv taaa , employment of fenestrated aortic stent grafts has been proved to provide better early results than open surgical procedures . endovascular repairs in patients with taaa are limited so far to anatomical factors , such as ostial stenosis , severe vessel angulations and tortuosity as well as narrow iliac , renal and visceral arteries . other limitations to the common use of fenestrated stent grafts include high costs and the time delay between qualification and manufacture of the tailored grafts . today , there are only a few studies in the literature reporting application of fenestrated stent grafts in the treatment of complex abdominal aortic aneurysms involving four visceral vessels . we describe a case of a high - risk patient with taaa involving the visceral vessels in which the endovascular technique was chosen as the most appropriate therapeutic option . a 56-year - old man was diagnosed with asymptomatic type iv taaa with a maximal diameter of 55 mm ( fig . a number of risk factors that could increase predicted mortality and morbidity rate such as hypertension , type ii diabetes mellitus treated with insulin , symptomatic coronary artery disease ( canadian cardiovascular society , ccs class ii ) and severe chronic obstructive pulmonary disease ( fev1 below 1l ) were diagnosed . the pre - operative computed tomography angiography ( cta ) that confined the whole descending aorta from the arch to bifurcation and further to the common femoral arteries demonstrated a type iv taaa and patent normal celiac trunk as well as the superior mesenteric artery originating from the aneurysm . ( a ) ct scan with thoracic aorta ( 24 mm in diameter ) above an aneurysm . ( b ) ct scan at the level of the aneurysm with maximal diameter of 55 mm . ( c ) ct scan with abdominal aorta below the aneurysm the stent graft was customized after meticulous calculations such as length and diameter at the proximal and distal implantation sites within normal aortic segments ( fig . 1 ) , diameters of both iliac and femoral arteries , all angulations , the ostial diameter of the renal arteries , the superior mesenteric artery and the celiac trunk as well as distances between these vessels were done . the stent graft diameter at both ends was 28 mm , length was 178 mm , and it had four holes ( two 8 mm and the other two 6 mm in diameter ) . they were exposed surgically through longitudinal skin incisions approximately 5 cm in length in both groins . after the arteries were dissected free intravenous heparin then a purse - string stitch with 5 - 0 monofilament polypropylene non - absorbable suture was placed on the right femoral artery and an endovascular graft was delivered using the seldinger technique ( the sheath size was 20 fr ) . after an appropriate position was achieved in the region of the aortic aneurysm , a stent graft was deployed . then the superior mesenteric and both renal arteries were successfully cannulated from the left common femoral artery . the covered advanta v12 stents ( atrium medical corporation , nh , usa ) were placed to secure the interface between the stent graft fenestrations and superior mesenteric and both renal arteries orifices . the sizes of the renal and superior mesenteric stents were 6 mm 22 mm and 8 mm 38 mm , respectively . afterwards , the celiac trunk was cannulated from the left brachial artery open surgical approach ( as described above ) and was stented with a covered 8 mm 60 mm fluency stent ( bard peripheral vascular , tempe , ar , usa ) ( fig . consecutive steps of endovascular procedure under guidance of digital subtraction angiography ( dsa ) complete exclusion of the aneurysm with no endoleak and with preservation of flow in the celiac trunk , the superior mesenteric artery and both renal arteries was confirmed . the sites of cannulation of surgically exposed peripheral arteries ( both femoral and left brachial ) were fixed with previously placed purse - string stitches and the wounds were closed with layers with absorbable sutures . the total stent graft implantation time was 250 minutes and the fluoroscopy time was 54 minutes . follow - up cta at 6 months after endovascular repair showed successful sealing with complete exclusion of the aneurysm and proper perfusion in the four target vessels ( fig . properly deployed stent graft in the abdominal aorta and stents in the proximal segments of the visceral vessels ( celiac trunk , upper mesenteric and both renal arteries ) pararenal and taaa repair by fenestrated or branched stent grafts as a minimally invasive technique seems to offer good short- and medium - term results in comparison to conventional open surgery . a conventional operation for taaa requires extensive exposure that requires opening not only the abdominal but also not uncommonly the pleural cavity . this surgical excess itself is associated with at least moderate risk for the development of serious postoperative complications . moreover , rehabilitation after open surgery is long , and mobilization of the patient is only possible a few days after the operation . our patient was mobilized and rehabilitation was initiated on the second day after surgery . additionally , we are convinced it enabled us to avoid many possible complications related to mechanical ventilation of an individual with advanced chronic obstructive pulmonary disease . thus , the indications for endovascular repair of type iv taaa are high - risk patients ( e.g. elderly subjects , individuals with several comorbidities ) with favorable anatomy that makes it possible to manufacture the customized fenestrated stent grafts . adequate visualization of all visceral branches and proper sizing of the fenestrated stent grafts are of crucial importance to ensure success in the endovascular treatment of taaa . these devices were manufactured on the basis of cta data with bespoke fenestrations at the level of the orifices of four target vessels . considerable experience in endovascular grafting is necessary , especially during deployment of the fenestrated graft in an accurate position and fixation of the graft fenestrations with renal and visceral orifices by implantation covered stents . stenting of the fenestrated graft to the renal and visceral orifices as well as the branched graft have a favorable impact on preservation of blood flow through the graft - ostial junctions and target arteries . additionally , these covered stents placed in the graft - ostial tracts may also prevent graft migration and endoleaks as well as renal and visceral artery occlusions . the significant injury during open surgery especially in high - risk patients leads to the development of serious perioperative complications . meanwhile , during endovascular repair there are real chances to avoid or at least significantly decrease the rate of organ complications . despite the good outcome of endovascular repair of taaa postoperative complications occurred in 11% to 14% of patients and included heart failure or myocardial infarction ( 5.5% to 6.5% ) , stroke ( 1.4% to 2.5% ) , respiratory insufficiency ( 3.8% to 6.8% ) , neurological events such as paraplegia or paraparesis ( 1.1% to 2.7% ) and renal dysfunction ( 1.4% ) . in the perioperative period prevalence of endoleaks the early occurrence of renal dysfunction may be provoked by the use of a large volume of contrast agents predisposing to contrast - induced nephropathy or embolization of the renal arteries during deployment of the stent graft . moreover , the use of fenestrated and branched stent grafts was proved to be beneficial , particularly in patients with preexisting severe cardiovascular , pulmonary , neurological or renal dysfunction . for these reasons , even complex endovascular therapy with fenestrated stent grafts may offer shorter hospitalization and faster post - procedural rehabilitation . our uncomplicated case demonstrated at 6 months after the procedure complete aneurysm exclusion with 18 mm reductions in aneurysm sac diameter and preserved perfusion in the stent graft and four visceral arteries . thus , complex and diffuse aortic pathologies such as thoracoabdominal aneurysms may be treated successfully with fenestrated stent grafts . however , appropriate patient selection , proper graft design and a multidisciplinary team experienced in endovascular procedures are of paramount importance in successful treatment of this particular group of patients .

conventional open surgical repair of thoracoabdominal aortic aneurysm ( taaa ) is associated with high perioperative mortality and morbidity risk . our report of successful treatment of a 56-year - old patient with taaa involving all visceral arteries and with many comorbidities with a fenestrated stent graft supports its application in high - risk taaa patients .

Introduction Case study Discussion Disclosure

renal cell carcinoma ( rcc ) may metastasize to any site of the body , but clinically evident metastatic intestinal involvement by rcc is extremely rare . to our knowledge , simultaneous duodenal and colonic metastases have not been reported in the english literature . we report a case of pathologically proven simultaneous duodenal and ascending colonic metastases about four years after a left nephrectomy for rcc . a 76-year - old female patient who had undergone a left radical nephrectomy 4 years previously for rcc ( mixed clear and granular cell type , tnm stage iii ) presented with a 1-month history of dyspepsia , lethargy and pain in the right upper abdomen . her blood pressure was 110/70 mmhg , pulse rate 80/min , respiration rate 22/min and body temperature 36.8c . abdominal examination revealed slight tenderness but normal peristalsis ; however , a movable mass was palpated in the right upper abdomen . the hematocrit was 28.1% and the white cell count was 9700/mm with 77% polymorphonuclear cells and 14% lymphocytes . serum sodium was 134 meq / l , potassium 3.5 meq / l , chloride 101 meq / l and calcium 9.5 mg / l . the result of liver function tests were as follows : total protein 6.5 g / dl , albumin 3.5 g / dl , cholesterol 165 mg / l , bilirubin 0.6 mg / dl , alkaline phosphatase 105 iu / l , ast 28 iu / l and alt 21 iu / l . tumor marker levels , such as carcinoembryonic antigen ( cea ) , carbohydrate antigen 19 - 9 ( ca19 - 9 ) and -fetoprotein ( afp ) , were within normal limits . an abdominopelvic ct scan showed circumferential wall thickening with high enhancement at the second portion of the duodenum and additional enhancement of an irregular protruding mass into the lumen of the ascending colon with multiple lymphadenopathy in the aortocaval area ( figure 1 ) . a gastroscopy showed a large , irregular multi - lobed , partially necrotic and ulcerative protruding mass nearly obstructing the second portion of the duodenum . a colonoscopy revealed a polypoid , nodular mass in the ascending colon , which was purplish in color , had sharp margins and was pliable ( figure 2 ) . microscopic findings of biopsy specimens showed features identical to those of the renal cell carcinoma which was resected 4 years earlier in this patient and immunohistochemical stainings for vimentin was positive ( figure 3 ) . interferon was given at a dose of 2 million iu per square meter three times a week . however , interferon therapy was discontinued after three months due to anemia , anorexia and general weakness . the clinical course of the disease ranges from months to several decades and even spontaneous regression has been documented . approximately 25% of patients with rcc have metastatic disease at the time of diagnosis . the delayed occurrence ( as late as 31 years after a nephrectomy ) of metastatic rcc is well known . solitary metachronous metastases from rcc are rare ; however , they can occur very late in the course of the disease . renal cell carcinoma may metastasize to almost every organ of the body , but 95% of the metastatic lesions involve the lung , lymph nodes , liver , bone , adrenal glands and the opposite kidney . however , there are scattered reports in the literature of clinically evident intestinal metastasis from rcc . to the best of our knowledge , a secondary tumor involving the intestinal tract may be caused by direct extension , lymphatic spread , peritoneal or hematogenous dissemination . in the present case , duodenal and colonic metastases are not generalized carcinomatosis because any other metastasis is not occurred in common metastatic sites and duodenal and ascending colonic involvement are remote from a primary cancer of the left kidney . hypervascularity of the present tumor , as suggested by a ct scan , gastroscopy and colonoscopy , was compatible with the characteristics of metastatic renal cell carcinoma . also , positive immuno - histochemical stain for vimentin elucidated that the duodenal and colonic masses are compatible with carcinoma rather than adenocarcinoma . surgical excision of the local recurrence is the best procedure for therapy , but this can be radical only when the recurrence can be completely excised . chemotherapies , including hormonal and interferon therapies , are effective in some patients with metastatic renal cell carcinoma . in our case although interferon treatment was used to treat metastatic rcc , it is not easy to evaluate its effectiveness because of premature discontinuance of the treatment . in our opinion , endoscopists should consider the possibility of intestinal metastasis of rcc when endoscopic and ct studies show a hypervascular mass in a patient with a previous history of rcc .

we report a case of pathologically proven simultaneous duodenal and colonic metastases about four years after nephrectomy for mixed clear and granular cell type renal cell carcinoma ( rcc ) . a 76-year - old female patient who had undergone a left radical nephrectomy 4 years previously for rcc presented with a 1-month history of dyspepsia and pain in the right upper abdomen . an abdominopelvic ct scan showed circumferential wall thickening with high enhancement at the second portion of the duodenum and additional enhancement of an irregular protruding mass into the lumen of the ascending colon . a gastroscopy showed a large and ulcerative protruding mass nearly obstructing the second portion of the duodenum . a colonoscopy revealed a polypoid , nodular and purplish mass in the ascending colon . microscopy of the biopsy specimen showed the features identical to those of the rcc which was resected 4 years earlier in this patient . we believe this to be the first case illustrating a metastatic renal cell carcinoma as simultaneous duodenal and colon masses .

INTRODUCTION CASE REPORT DISCUSSION

sarcoidosis is a multisystem disease of undetermined cause which is characterized by the widespread presence of non - caseating granuloma . autopsy - based series report prevalence rates of renal involvement between 7 and 27% , and the disease is well known for its protean manifestations [ 24 ] . two non - mutually exclusive clinical and pathological patterns account for the great bulk of renal sarcoidosis , namely ( i ) nephrolithiasis and/or nephrocalcinosis - related to hypercalcaemia and hypercalciuria and ( ii ) interstitial nephritis with or without granulomatous formation [ 5 , 6 ] . in contrast , renal vascular involvement is believed to be an exceptional complication of sarcoidosis and extensive retrospective series have failed to document any such complication [ 5 , 6 ] . likewise , renal imaging findings are scanty and , if present , usually restricted to nephrocalcinosis , striated nephrograms or urinary tract obstruction [ 79 ] . here we report the case of a 22-year - old patient in whom the manifestation of renal sarcoidosis consisted of severe hypertension associated with bilateral vascular lesions and granulomatous interstitial nephritis . a 22-year - old white woman with a medical history of idiopathic epilepsy and sarcoidosis was referred to our institution for severe hypertension . six months before her referral to our institution she had been diagnosed with sarcoidosis during the follow - up of clinical features consistent with lfgren 's syndrome . during diagnostic work - up , blood pressure ( bp ) was shown to be within normal ranges ( 128/66 mmhg ) . corticosteroid therapy was considered unnecessary at that point and symptoms resolved within a couple of weeks . upon admission in our department , the patient 's bp was 169/123 mmhg under therapy of diltiazem 200 mg lp once daily , without clinical or echocardiographic repercussions . ( 68 mol / l , i.e. egfr = 98 ml / min/1.73 m , using the 4-variable mdrd [ modication of diet in renal disease ] ) formula . haematuria and urine infection were excluded by dipstick examination and microalbuminuria was within physiological ranges ( 8 mg / l ) . hormonal investigations , carried out under euvolaemic conditions , substantiated secondary hyperaldosteronism with elevated levels of plasma renin and aldosterone levels in the supine ( 75 mu / l and 31 ng / dl ; reference ranges 1025 and 2.814.4 , respectively ) and upright positions ( 298 mu / l ; and 2919 ng / dl ; reference ranges 1550 and 2001000 ) . the 24-h urine aldosterone levels were high ( 2487 ng/24 h ; reference range 5051694 ) . additional testing evidenced elevated levels of 25(oh ) vitamin d3 ( 85 nmol / l ; reference range 1157 ) but normal values of calcaemia ( 2.27 mmol / l ) , calcium urinary excretion ( 2.42 mmol / l ) and angiotensinogen - converting enzyme ( 11 mu / ml ) . an abdominal computed tomography scan ( ct ) revealed multiple bilateral wedge- and nodular - shaped hypodense foci affecting the renal cortex and medulla ( figure 1 ) , with no evidence of urinary tract obstruction . renal angiography was prompted by persistent suspicion of underlying renal ischaemia : multiple cortical and sub - cortical vascular defects were evidenced in both kidneys ( figure 2 ) . in view of the equivocal nature of the lesions , there were no signs of vessel destruction or of small - vessel angeitis in this specimen , and the rest of the examination was unremarkable ( figure 3 ) . 1.transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . 2.selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . 3.first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . the patient was subsequently started on tapering doses of prednisone ( 1 mg / kg / day ) and ramipril ( 5 mg once daily ) . bp control was achieved and scr remained stable ( scr , 68 mol / l ; egfr , 98 ml / min/1.73 m ) . an abdominal mri performed 9 months after the initiation of corticosteroid therapy exhibited only minimal cortical defects in both kidneys whereas the lesions located in the medulla had receded . two years following the patient 's initial referral and progressive corticosteroid tapering , the patient presented with a recurrent bout of uveitis . concurrently , renal impairment was evidenced ( scr , 126 mol / l ; egfr , 45 ml / min/1.73 m ) . corticosteroids were reinitiated but proved to be unsuccessful as the patient 's kidney function failed to improve during the following year . three years after initial referral , ct imaging ( figure 4 ) substantiated major bilateral cortical atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted . there were also multiple left lateral aortic and caval lymph nodes , of increased size compared with the reference ct . 4.transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . a 22-year - old white woman with a medical history of idiopathic epilepsy and sarcoidosis was referred to our institution for severe hypertension . six months before her referral to our institution she had been diagnosed with sarcoidosis during the follow - up of clinical features consistent with lfgren 's syndrome . during diagnostic work - up , blood pressure ( bp ) was shown to be within normal ranges ( 128/66 mmhg ) . corticosteroid therapy was considered unnecessary at that point and symptoms resolved within a couple of weeks . upon admission in our department , the patient 's bp was 169/123 mmhg under therapy of diltiazem 200 mg lp once daily , without clinical or echocardiographic repercussions . ( 68 mol / l , i.e. egfr = 98 ml / min/1.73 m , using the 4-variable mdrd [ modication of diet in renal disease ] ) formula . haematuria and urine infection were excluded by dipstick examination and microalbuminuria was within physiological ranges ( 8 mg / l ) . hormonal investigations , carried out under euvolaemic conditions , substantiated secondary hyperaldosteronism with elevated levels of plasma renin and aldosterone levels in the supine ( 75 mu / l and 31 ng / dl ; reference ranges 1025 and 2.814.4 , respectively ) and upright positions ( 298 mu / l ; and 2919 ng / dl ; reference ranges 1550 and 2001000 ) . the 24-h urine aldosterone levels were high ( 2487 ng/24 h ; reference range 5051694 ) . additional testing evidenced elevated levels of 25(oh ) vitamin d3 ( 85 nmol / l ; reference range 1157 ) but normal values of calcaemia ( 2.27 mmol / l ) , calcium urinary excretion ( 2.42 mmol / l ) and angiotensinogen - converting enzyme ( 11 mu / ml ) . an abdominal computed tomography scan ( ct ) revealed multiple bilateral wedge- and nodular - shaped hypodense foci affecting the renal cortex and medulla ( figure 1 ) , with no evidence of urinary tract obstruction . renal angiography was prompted by persistent suspicion of underlying renal ischaemia : multiple cortical and sub - cortical vascular defects were evidenced in both kidneys ( figure 2 ) . in view of the equivocal nature of the lesions , there were no signs of vessel destruction or of small - vessel angeitis in this specimen , and the rest of the examination was unremarkable ( figure 3 ) . 1.transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . 2.selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . 3.first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . the patient was subsequently started on tapering doses of prednisone ( 1 mg / kg / day ) and ramipril ( 5 mg once daily ) . bp control was achieved and scr remained stable ( scr , 68 mol / l ; egfr , 98 ml / min/1.73 m ) . an abdominal mri performed 9 months after the initiation of corticosteroid therapy exhibited only minimal cortical defects in both kidneys whereas the lesions located in the medulla had receded . two years following the patient 's initial referral and progressive corticosteroid tapering , the patient presented with a recurrent bout of uveitis . concurrently , renal impairment was evidenced ( scr , 126 mol / l ; egfr , 45 ml / min/1.73 m ) . corticosteroids were reinitiated but proved to be unsuccessful as the patient 's kidney function failed to improve during the following year . three years after initial referral , ct imaging ( figure 4 ) substantiated major bilateral cortical atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted . there were also multiple left lateral aortic and caval lymph nodes , of increased size compared with the reference ct . 4.transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . this case reports an unusual presentation of renal sarcoidosis with prominent clinical features consisting of severe hypertension with bilateral vascular injury . despite a history strongly suggestive of sarcoidosis , renal biopsy indeed , the presence of nodular corticomedullar - located hypodense foci entertained the possibility of a coexisting tumoural process . only sparse case reports have documented this rare pseudonodular presentation [ 913 ] . in the even fewer instances where a renal biopsy was conducted , this lesional pattern was almost invariably related to extensive sarcoid granulomatous inflammation as in our case [ 1113 ] . renal sarcoidosis is usually associated with marked renal impairment on presentation . nevertheless , in three cases of bilateral , pseudotumoral , renal sarcoidosis ( out of a total of four reports ) the initial kidney function was shown to be normal as in the case presented here [ 1315 ] . renal pathology examination was also motivated by the triangular - shaped lesions located in the cortex which could be interpreted as post - ischaemic or evidence of infectious insults . renal angiography ultimately confirmed the suspicion of renal ischaemia by demonstrating multiple vascular voids in the renal cortex and sub - cortex . although no pathological process involving the renal arterioles could be demonstrated on pathology , this may be ascribed to a sampling bias given the focal distribution of the renal perfusion defects . regardless of the underlying mechanism , this process caused severe hypertension associated with secondary hyperaldosteronism and , in fine , widespread glomerular ischaemia and tubulointerstitial fibrosis . although sarcoidosis is known to embrace a wide variety of clinical and pathological manifestations ( table 1 ) , this case is different from the other rarely reported clinical patterns consisting of severe hypertension associated with small and medium - vessel injury . aside from its rarity , sarcoidosis - induced vascular injury has been shown to involve all types of renal vessels . in most cases , renal vascular damage is but one of the multiple manifestations of multisystem vasculitis , a rare life - threatening complication associated with sarcoidosis . in the case of small and medium - sized vascular involvement , the prominent clinical features also include pulmonary angiitis , cerebral vasculitis and skin sarcoidosis consistent with leukocytoclastic vasculitis and granulomatous vasculitis [ 17 , 18 ] . large vessel damage may either present as renal artery stenosis with concurring severe hypertension or aortic and renal aneurisms placing patients at high risk of subsequent rupture [ 18 , 20 ] . in stark contrast , at no point of the disease process did we record any extra - renal vascular involvement . furthermore , vascular wall involvement with elastic lamina dissolution as exemplified by previous case reports was not observed on pathological examination in our case [ 17 , 21 ] . table 1.summary of clinical and pathological patterns of renal sarcoidosispattern and/or cause of renal injuryfrequencyrenal function on presentationprominent laboratory featurespathological findings on kidney biopsyabdominal imaging findings ( on ct scan)granulomatous or non - granumolatous tubulointersititial nephritis+++altered , severe impairment is frequentmild proteinuria , microhematuria , sterile leukocyturia,+/ renal tubular acidosis+/ fanconi syndrometubulo - interstial nephritis+/ non necrotizing granuloma with epithelioid - like giant cells+/ schaumann bodiesnormal + + striated nephrogram + /pseudotumoral renal masses ( rare)nephrocalcinosis / nephrolithiasis++altered , severe impairment is frequenthypercalcaemia , hypercalciuria , elevated levels of 1 - 25(oh)vitd3+/renal tubular acidosis+/ fanconi syndromeintratubular and peritubular aggregation of calciumintrarenal calcifications+/ urinary obstructionglomerular diseaseraregenerally normal , mild to moderate impairment is possiblenephrotic range proteinuriamembranous nephritis + + + iga nephritis + + nrrenal vascular involvementvery rare??coexisting gin granuloma - associated vascular injuryrenal artery stenosis or aneurysmsmall- and medium - vessel occlusionmultisystem vascular involvementretroperitoneal fibrosisvery rare ? ? , no consistent data given the small number of cases ; a , most frequently described glomerular patterns ; gin , granulomatous interstitial nephritis ; nr , not relevant . , no consistent data given the small number of cases ; a , most frequently described glomerular patterns ; gin , granulomatous interstitial nephritis ; nr , not relevant . corticosteroids are regarded as the mainstay of renal sarcoidosis and retrospective studies have highlighted their effectiveness . however , several pitfalls should be kept in mind : ( i ) reversal of renal impairment is not universal and some patients may require other immunosuppressive therapy such as azathioprine and mycophenolate mofetil , ( ii ) residual renal failure is common , and not all patients regain their baseline renal function , ( iii ) relapses are frequent on cortiscosteroid weaning although patients may respond on reinitiation of therapy and ( iv ) response to therapy is negatively correlated to the extent of renal fibrosis [ 6 , 22 ] . however , by the time repeat imaging and biopsy were carried out the bilateral hypodense lesions thought to indicate the presence of granulomatous inflammation had resolved . instead , these lesions had given way to extensive renal fibrosis and the reintroduction of corticosteroids was of no avail . our case conveys further evidence that hypodense renal masses upon ct imaging may be regarded as a possible , albeit rare , manifestation of sarcoidosis . it also suggests that renal ischaemia and hypertension may arise via extrinsic compression from granulomatous inflammation in the absence of renal or extra - renal vasculitis . the occurrence of severe hypertension in a patient with a history of sarcoidosis should urge physicians to consider this complication . a.h . and d.n . provided the expertise in the imaging and histopathological examinations , respectively . c.r . undertook the data collection and analysis as well as the initial drafting of the report .

renal sarcoidosis embraces a wide variety of clinical patterns . renal vascular involvement has seldom been reported and usually in the setting of systemic vasculitis . we report the case of a 22-year - old patient in whom inaugural manifestation of renal sarcoidosis consisted of severe hypertension associated with bilateral perfusion defects and tumour - like nodules . in the setting of renal sarcoidosis , our case suggests that renin - dependant hypertension may arise from renal ischaemia as a result of extrinsic compression of kidney blood vessels due to severe granulomatous inflammation .

Background Case presentation Clinical history Follow-up Discussion Conclusion Author's contributions

although the basic principles of resuscitation were described by versalius more than 500 years ago , the practice of cardiopulmonary resuscitation in its modern form only starts 50 years ago . despite advances in the understanding and practices of airway management , ventilatory support , external cardiac compression and drug therapy the outcome of patients undergoing cardiopulmonary resuscitation remained poor . patients may have spontaneous circulation restored and admitted to the intensive care unit , but then developed complications related to ischaemic insult to the brain as well as to the rest of the body . the term post - resuscitation disease was coined by the russian resuscitologist vladimir a. negovsky in 1972 to describe the constellation of pathological processes caused by ischaemia and reperfusion associated with cardiac arrest and the subsequent resuscitation . this is more recently renamed post - cardiac arrest syndrome because " the term resuscitation is now used more broadly to include treatment of various shock states in which circulation has not ceased ... (and ) the term postresuscitation implies that the act of resuscitation has ended ... " 4 key components contribute to the development of this syndrome : 1 ) post - cardiac arrest brain injury ; 2 ) post - cardiac arrest myocardial dysfunction ; 3 ) systemic ischaemia / reperfusion response ; and 4 ) persistent precipitating pathology . there is evidence to support that proper management in the post - resuscitation phase can improve outcome of these patients and therapeutic hypothermia is one important component of such management . to avoid confusion , there is a need to define terminology used in relation to manipulation of body temperature : hypothermia is defined as core body temperature of less then 36 regardless of the cause.induced hypothermia is defined as an intentional reduction of a patient 's core temperature below 36.therapeutic hypothermia is defined as controlled induced hypothermia ; i.e. induced hypothermia with the potentially deleterious effects such a shivering , being controlled or suppressed.controlled or therapeutic normothermia is defined as bringing down core temperature in a patient with fever , and maintaining temperature within a range of 36 - 37.5 , with the potentially deleterious effects such a shivering , being controlled or suppressed . hypothermia is defined as core body temperature of less then 36 regardless of the cause . induced hypothermia is defined as an intentional reduction of a patient 's core temperature below 36. therapeutic hypothermia is defined as controlled induced hypothermia ; i.e. induced hypothermia with the potentially deleterious effects such a shivering , being controlled or suppressed . controlled or therapeutic normothermia is defined as bringing down core temperature in a patient with fever , and maintaining temperature within a range of 36 - 37.5 , with the potentially deleterious effects such a shivering , being controlled or suppressed . the degree of therapeutic hypothermia can mild ( 34.0 - 35.9 ) , moderate ( 32.0 - 33.9 ) , moderately deep ( 30.0 - 31.9 ) or deep ( early studies showed that induced hypothermia improved outcome in cardiac arrest patients . although both studies were small ( 12 patients had induced hypothermia in each study ) case series . however , further progress was hindered by 1 ) lack of understanding of the underlying pathophysiology , 2 ) lack of reliable methods to control body temperature , and 3 ) lack of intensive care facilities to manage possible complications . the early hypothesis that beneficial effect of hypothermia was due to reduction of metabolism leads to the adoption of more profound cooling ( around 30 ) . the result was serious complications which can not be handled effectively without the support of intensive care , which was then only at its infancy . further studies in the recent decades showed that the mechanism of cerebral protection by hypothermia is more complicated and neurological outcome can be improved by mild to moderate degree of hypothermia . two studies published in 2002 provided the evidence basis that hypothermia can improve neurological outcome . coupled with advances in technology in hypothermia and intensive care in general , the study population of both the study by bernard et al . and the haca study group are patients suffering from out - of - hospital ventricular fibrillations . there is evidence that cooling patients with presenting rhythms other than vf or vt following out - of - hospital arrests does no harm : 2 retrospective cohort studies found that the outcomes for patients presenting with pea / systole did not differ from those observed among the group presenting with vf or vt . there is little published evidence to support the use of therapeutic hypothermia following in - hospital cardiac arrest . an analysis of patients entered into arrich and erchacars group found that 13% of procedures were performed post in - hospital cardiac arrest . 43% of these were cooled and although the initiation of hypothermia was faster than that for the out - of- hospital arrests , there was no difference in outcome between the patients treated with hypothermia and those with normothermia . the haca in - hospital multi - center trial is currently investigating whether therapeutic hypothermia is beneficial for patients following in - hospital cardiac arrest . in both the haca and bernard studies , patients who are hypotensive ( map < 60 mmhg or sbp < 90 mmhg despite adrenaline infusion ) were excluded . the underlying reason being the possible deleterious effect of hypothermia on cardiovascular function and coagulation . however , there are several publications suggesting that post - cardiac arrest patients in cardiogenic shock could actually also benefit from moderate therapeutic hypothermia . reduction in platelet count and platelet dysfunction is noted when body temperature is dropped to < 35 ; and when temperature is below 33 , synthesis and kinetics of clotting enzymes and plasminogen activator inhibitors are also affected . however , it was found in a recent study which assessed the risk and severity of bleeding during simultaneous use of mild hypothermia and thrombolysis that bleeding risks were similar to historical controls treated with thrombolytics alone , although there was a trend toward more red blood cell units being required to reach target hematocrit in hypothermic patients who developed bleeding complications and needed transfusions . . the procedures of therapeutic hypothermia can be divided into 4 phases : induction , maintenance , de - cooling ( or rewarming ) and normothermia . this is the initial phase during which the patient 's body temperature is lowered to the target of 32 - 34. continuous core temperature measurement should be started before induction of therapeutic hypothermia to ensure that the patient 's body temperature is within the target range and to avoid overshoot . this is preferably achieved by bladder , rectal , central venous , or oesophageal measurement . however , it should be noticed that bladder temperature may poorly reflect core temperature if the patient is oliguric , and other monitoring sites are preferred . this phase is associated with many possible complications most of which can be minimized by rapid cooling . rapid cooling can be achieved by rapid bolus administration of 30 to 40 ml / kg cold ( 4 ) isotonic resuscitation fluid over 1 hour . despite theoretical problems this method when core temperature drops to 32 , metabolic rate decreases to 50 - 65% of normal . the issue of how to interpret arterial blood gases in hypothermic patients ( alpha - stat versus ph - stat ) is a topic of some controversy and readers are referred to review articles for detailed discussion . hypothermia can lead to hypokalaemia , hypophosphataemia and hypomagnesaemia as a result of intracellular shift as well as urinary loss because of tubular dysfunction . it is particularly important to monitor and replace potassium in order to avoid arrhythmia . on the other hand , it is the result of increased fat metabolism , leading to an increase in the levels of glycerol , free fatty acids , ketonic acids , and lactate . hyperglycaemia due to reduction of insulin secretion and insulin resistance should be controlled by close monitoring and insulin therapy . the main sources of relevant data come from patients with hypothermia induced during cardiac surgery . cardiac output decreased along with the heart rate ; however , the hypothermia - induced decrease in metabolic rate usually equaled or exceeded the decrease in cardiac output , so that the balance between supply and demand remained constant or improved . however , the actual cardiac output is also affected by the volume status and effect of sedation . systolic function of myocardium may actually be improved during mild to moderate hypothermia but diastolic function is usually impaired . it is important to note that the effect of hypothermia on myocardial contractility is strongly dependent on heart rate . if the heart rate is allowed to decrease along with the temperature , myocardial contractility as measured by systolic function usually increases , although there may be a mild degree of diastolic dysfunction . however , if the heart rate is artificially increased through administration of chronotropic drugs or a pacing wire , myocardial contractility decreases significantly . this phenomenon has been demonstrated in animal studies and also in patients undergoing cardiothoracic surgery . thus , the effect of hypothermia on myocardial function strongly depends on whether the heart rate is allowed to decrease [ 20 - 22 ] . maintaining euvolemia is one important aspect of management during therapeutic hypothermia and one challenge is cold induced diuresis . the underlying mechanisms leading to cold - induced diuresis include increased venous return caused by constriction of peripheral vessels ( particularly in the skin ) due to hypothermia - induced increases in plasma noradrenaline levels and activation of the sympathetic nerve system , activation of atrial natriuretic peptide , decreased levels of antidiuretic hormone and renal antidiuretic hormone receptor levels , and tubular dysfunction . if uncorrected , diuresis can cause hypotension as well as electrolyte depletion and increase in blood viscosity . the risk for hypovolemia increases significantly if the patient is simultaneously treated with diuretic agents such as mannitol . however , hypotension can be quite easily prevented by avoiding or promptly correcting hypovolemia , and by avoiding excessive stimulation of heart rate . while profound hypothermia of < 28 is associated with severe arrhythmia , mild to moderate hypothermia could stabilize membranes . study in normal human showed that the threshold for vasoconstriction is about 36.5 and that for shivering is 35.5. the threshold is slightly higher in female but the difference is < 0.5 . shivering of postoperative patients may lead to increased risk of morbid cardiac events especially in older patients with heart disease : the result of increased rate of metabolism and oxygen consumption , leading to excess work of breathing and tachycardia . however , the situation may be different in sedated patients . while hypothermia in an awake patient causes tachycardia , inducing hypothermia intentionally in sedated patients shivering will increase oxygen consumption , but as the patient is on mechanical ventilation , there will be no increase in the work of breathing . nevertheless , it is important to prevent or aggressively treat shivering because it significantly complicates hypothermia induction , and leads to an undesirable increase in metabolic rate and oxygen consumption . shivering may be controlled by one and more of the following methods : 1 ) drugs lowering shivering threshold , e.g. acetaminophen ( paracetamol ) , aspirin , and nonsteroidal antiinflammatory drugs ; 2 ) drugs suppressing shivering response paralyzing agents , sedatives , opiates , and others ; and/or 3 ) skin counterwarming . drugs lowering shivering threshold are not quite effective in general . of the drugs which suppress shivering , it was found that there is significant variation in sedation protocols amongst the 68 icus included in the selected studies . midazolam ( 5 mg / h to 0.3 mg / kg / h ) being most commonly used , followed by propofol ( up to 6 mg / kg / h ) . a quarter of icus do not use any analgesic , the rest use either fentanyl ( 0.5 - 10 mcg / kg / h ) or morphine . use of neuromuscular blocking agents , however , is associated with several disadvantages some of which can be reduced by adequate sedation : 1 ) attempts of the brain to generate a shivering response will not cease ; 2 ) they can mask seizure ; 3 ) they lack of vasodilating effect of sedatives ; and 4 ) their use is associated with development of critical illness polyneuromyopathy . the importance of adequate sedation and suppression of hypothermia - induced stress responses is underscored by observations from animal experiments suggesting that some or all of hypothermia 's neuroprotective effects can be lost if cooling is used in non - sedated animals . it was found that 4 increase in skin temperature could " compensate " for 1 drop in core temperature to prevent a shivering response . some areas of the body ( hands , feet and face ) have a higher concentration of " temperature sensors " and warming of those areas could have a greater effect on suppressing shivering . by reducing shivering , counterwarming can reduce metabolic rate and oxygen consumption of hypothermic patients . during this phase , the goal is to maintain core temperature at 32 - 34 for 12 - 24 hours . the patient is usually more stable during this phase with fewer disturbances in their haemodynamic , volume or electrolyte status , as well as less shivering . however , the patient is subjected to other hazards including changes in pharmacokinetics , nosocomial infections and bedsores . conventionally , patients are maintained cool by surface cooling through exposure , placing ice packs to the neck , groins and axillae , and/or spraying with water . rubber cooling blankets can also be used but should be placed over the patient . placing those blankets under the patient can lead to skin damage due to vasoconstriction in already pressurized areas . although widely available and inexpensive , conventional surface cooling is difficult to control as there is no internal feedback mechanism . these may be classified as non - invasive surface cooling devices such as hydrogel - coated water circulating pads or water - circulating wrapping garments , and invasive core cooling devices using intravascular catheters . a problem with surface cooling is that 40 - 90% the patient 's surface area needs to be covered and carries a risk of skin lesions . but they can be started immediately using nurse - driven protocols without direct physician intervention . core - cooling methods , on the other hand , are highly reliable once the catheter is in place , with a cooling rate of 2.0 - 4.5/hour . however , insertion of catheter is required and it can lead to complications such as thrombosis and infection . even short duration of hypothermia during perioperative period for intracranial aneurysm increase the risk of postoperative bacteraemia . a study has shown that 3 days of prophylaxis with ampicillin - sulbactam can reduce early onset nosocomial pneumonia but not the incidence of late - onset nosocomial pneumonia . after 12 - 24 hours of cooling , patient can be allowed to warm up slowly . the goal should be 0.2 - 0.33 per hour until the patient is 36.5 - 37.0. more rapid warming must be prevented to avoid electrolyte disturbance due to transcellular shift , hypoglycaemia due to increased insulin sensitivity , as well as aggravated brain destruction . this can be prevented by slow rewarming , allowing the kidneys to excrete the excess potassium . however , this should not be a reason to accept hypokalaemia in the induction or maintenance phase . for patients with renal dysfunction , renal replacement therapy this may be the result of nosocomial infection or part of the post - cardiac arrest syndrome . furthermore , cerebrovascular reactivity may be impaired following hypothermia treatment and thus increase the potential harmful effects of fever even more . there was a case report in which a post - cardiac arrest patient who initially improved after rewarming deteriorated after development of fever and subsequently died . it is recommended that patients should be maintained normothermic after decooling until 72 hours have elapsed since restoration of spontaneous circulation . current evidence supports that induction of therapeutic hypothermia in selected patients after cardiac arrest can improve neurological outcome . lack of familiarity and lack of a concrete protocol are the most important amongst the list of perceived barriers . it is hoped that by summarizing the current state of knowledge on the subject and highlighting issues on clinical management will enable more patients to benefit from the therapy .

pioneer works on therapeutic hypothermia ( th ) half a century ago already showed promising results but clinical application was limited by a lack of understanding of the underlying pathophysiology , lack of reliable method for temperature control and lack of intensive care facilities to deal with possible complications . more recently , 2 studies in 2002 supported the application of moderate th ( 32.0 - 34.0 ) in post - cardiac arrest patients . although the studies included only patients suffering from out - of - hospital vf , many icus world - wide are applying the therapy to all post - cardiac arrest patients irrespective of site or presenting rhythm . while primary coagulopathy and cardiogenic shock are usually stated as relative contraindications , evidences are accumulating to support the application of th in patients with cardiogenic shock . th can be divided into 4 phases : induction , maintenance , de - cooling and normothermia . induction is usually achieved by infusion of cold isotonic fluid . the precautions included avoidance of over - cooling , hypokalaemia , hyperglycaemia , and shivering . th can be maintained by many different methods , varying in their level of invasiveness , cost and effectiveness . issues including changes in pharmacokinetics and haemodynamics , and susceptibility to infection need to the addressed . the optimal duration of maintenance is unknown but the usual practice is 12 - 24 hours . de - cooling and rewarming is especially challenging because complications can be serious if temperature rise by more than 1 every 3 - 5 hours . life - theatening hyperkalaemia can occur especially if patient suffers from renal insufficiency . fever is a frequent complication either due to infection or post - cardiac arrest syndrome but patient must be kept normothermic for 72 hours .

Introduction Indications and Contraindication Induction Maintenance De-cooling/Rewarming Normothermia Conclusion

myctophids ( myctophidae ) are mesopelagic fishes distributed in all the world s oceans ( dalpadado and gjster 1988 ; cherel et al . 2010 ) , playing an important role in the marine ecosystems ( gjster and kawaguchi 1980 ; cherel et al . 2010 ) as trophic links between zooplankton ( kinzer and schulz 1985 ; shreeve et al . 2009 ) and piscivorous predators ( giske et al . 1990 ; markaida and sosa - nishizaki 2003 ; dokster et al . they are also likely to play a major role in the vertical flux of organic matter from the upper productive layer to deeper layers ( radchenko 2007 ; hernandez - leon et al . their vertical distribution and diel vertical migration behavior affect such interactions and processes , making it important to reveal these patterns . the myctophid glacier lanternfish ( benthosema glaciale ) and the sternoptychidae pearlside ( maurolicus muelleri ) are the dominant mesopelagic fish in norwegian fjords ( aksnes et al . 2004 ; kristoffersen and salvanes 2009 ) . in masfjorden , pearlside dominates acoustic scattering layers ( sl s ) shallower than 200 m , while glacier lanternfish dominates the sl s below 200250 m ( giske et al . 1990 ; bagien et al . 2001 ; kaartvedt et al . 2009 ; dypvik et al . diel vertical migration ( dvm ) is commonly referred to as a trade - off between feeding opportunities and predation risk induced by changes in light intensity ( clark and levy 1988 ; pearre 2003 ; cohen and forward 2009 ; ringelberg 2010 ) . thus , light is the proximate cause of dvm influencing the time of migration and vertical extent of migration ( ringelberg and van gool 2003 ; staby and aksnes 2011 ) , while the distribution of food and predation risk are regarded as major drivers for fish vertical distribution and migration ( clark and levy 1988 ; bailey 1989 ; neilson and perry 1990 ; sutton and hopkins 1996 ) . however , size ( busch and mehner 2012 ) , internal factors such as energy reserves ( hays et al . 2001 ) and hunger ( pearre 2003 ) , and external factors such as currents ( bennett et al . 2002 ) and temperature ( wurtsbaugh and neverman 1988 ; sogard and olla 1996 ; mehner et al . 2010 ) may also influence vertical distribution and migration . in normal dvm ( ndvm ) , individuals ascend toward the surface at night before descending to deeper waters during the day . in the less common inverse dvm ( idvm ) , individuals ascend in the water column at daytime and descend during the night ( pearre 2003 ) . glacier lanternfish is known to carry out ndvm ( halliday 1970 ; roe and badcock 1984 ; sameoto 1988 ) , idvm ( kaartvedt et al . 2012 ) or display no diel vertical migration ( nodvm ) ( roe and badcock 1984 ; albikovskaya 1988 ; kaartvedt et al . the depth distribution of myctophids is size dependent with larger fish distributed deeper than smaller individuals ( willis and pearcy 1980 ; gartner et al . 1994 ) , vision ( capabilities ) for detecting prey ( warrant and locket 2004 ) , internal state ( satiation and hunger ) ( cailliet and ebeling 1990 ; staby et al . 2011 ) , and motivation ( rosland and giske 1997 ; busch and mehner 2012 ) are expected to result in a mixture of migration patterns , which may occur simultaneously . glacier lanternfish feeds on a variety of zooplankton ( gjster 1973 ; roe and badcock 1984 ; sameoto 1988 ) , but seems to prefer calanoid copepods , especially calanus ( sameoto 1988 , 1989 ; balio and aksnes 1993 ; dypvik et al . 2012 ) . the main pattern in seasonal zooplankton abundance and vertical distribution at the site for this study is established from previous studies at the same or adjacent locations . in spring and summer , most zooplankton are distributed in the upper part ( < 30 m ) of the water column ( rasmussen and giske 1994 ) . however , during autumn and winter , the highest biomass of zooplankton is below 150 m ( giske et al . this is because zooplankton vanishes from upper layers as primary production declines , but also because of seasonal vertical migration among the main calanoid copepod species , calanus spp . , which leaves upper waters , descending for overwintering in mid - waters ( bagien et al . myctophids are capable of feeding at mesopelagic depths ( roe and badcock 1984 ; sameoto 1988 ; pusch et al . 2004 ) , so that the seasonal migration of calanus to deep waters may represent an important food source in the daytime habitat of the fish ( dypvik et al . in norwegian fjords , glacier lanternfish exercise a strong predation pressure on overwintering calanus ( bagien et al . mesopelagic fish can be studied by use of echo sounders as they tend to aggregate into sl s ( holton 1969 ; god et al . normally , acoustic studies of mesopelagic fish are carried out in periods restricted by time or seasonality ( collins et al . 2009 ) , and to our knowledge , there is no systematic study addressing how migration patterns in a population of glacier lanternfish , or other myctophids , may vary throughout a year . however , the use of moored echo sounders can give long - time acoustic data series ( brierley et al . here we take advantage of the unique opportunity for a long - term study offered by a deep fjord where populations of mesopelagic fish are easily accessible . we present data from 16 months of continuous acoustic registrations ( july 2007october 2008 ) , enabling us to address the seasonal patterns of diel vertical behavior , unveiling the relative occurrence and consistency of ndvm , idvm , and nodvm by glacier lanternfish . at the outset of this study , we hypothesized that the patterns of glacier lanternfish dvm would vary seasonally , as recently documented for the pearlside ( staby et al . given the general seasonal zooplankton dynamics of norwegian fjords , we hypothesize that during spring and summer , when the abundance of potential prey is high in near - surface waters , the glacier lanternfish migrate to the upper part of the water column at night ( ndvm ) to feed , avoiding visual predators in the bright surface waters during daytime . conversely , we hypothesize that during autumn and winter , when food is sparse in upper waters , and at seasonal high in mid - waters , as seasonally migrating copepods have descended to their overwintering habitat , glacier lanternfish restrain from migrations to the surface at night , rather exploiting the prey in mid - waters by performing idvm toward favored light conditions in shallower waters during daytime ( dypvik et al . furthermore , for the nodvm layer , we investigate two alternative hypotheses : ( 1 ) the population really does split into migrating and non - migrating components ( pearcy et al . 1979 ) or ( 2 ) migrations are undertaken also among the nodvm component , but not detected by standard methods because they are asynchronous , so that a proportion of the population always is present in deep waters ( sutton and hopkins 1996 ; pearre 2003 ) . since our approach facilitates studies of individuals , we have the possibility of addressing these competing hypotheses . the study was undertaken in masfjorden ( ~6052n , ~524e ) , norway ( fig . 1 ) . the deeper parts of the water column ( ~200390 m ) are particularly in focus as glacier lanternfish dominates the acoustic backscattering , and idvm and nodvm occur in this interval ( dypvik et al . depicting location of the bottom - mounted echo sounder ( gray circle ) at 392 m depth in masfjorden , norway map depicting location of the bottom - mounted echo sounder ( gray circle ) at 392 m depth in masfjorden , norway a mooring with an upward - facing 38 khz simrad ek60 split - beam echo sounder ( 7.1 beam width ) , cabled to shore for power and transmission of data , was deployed at 392 m in july 2007 and retrieved in october 2008 . detailed descriptions of the echo sounder system and parameter settings are given in kaartvedt et al . data were recorded continuously , apart for intermittent periods following power failures , giving a total of 218 full days of records spanning all seasons . a minimum number of 4 days per month were included in this study ( i.e. sept . 2007 and we classify september november as autumn , december february as winter , march may as spring , and june august as summer . the results are presented as average echograms illustrating the average sv ( db ) values for the entire water column ( ~0390 m ) for each month , and in greater detail from selected months representing each season for the section ~200390 m , which is of particular interest for this study ( called monthly echograms ) . sv values are logarithmic measurements of the volume acoustic backscattering or accumulated ts ( target strength ) , ts representing the echo of a single individual ( maclennan and simmonds 1992 ) . the seasonal patterns of the vertical distribution and migration of glacier lanternfish is assessed by studying the scattering layers in monthly echograms . these monthly echograms were made in matlab by dividing each day into 30 s intervals , and for each time interval average all the pings for the given month . in this way , all the acoustic records from the entire registration period are included in the presentations . in addition , to better illustrate any activity in the nodvm layer , which by definition was expected to have little vertical migration , we randomly picked 1 day for each season ( 18 october 2007 ; 2 february ; 12 march 2008 ; 8 august 2008 ) , focusing on the 300390 m depth interval . for these days , we furthermore selectively chose examples where vertical swimming was seen in the compressed , daily echograms , and portrayed these at higher resolution for better depicting individual behavior . abundance estimates to identify the variation in concentration of glacier lanternfish in different depth intervals between day and night were made by echo integration . this was done with day and depth divided into blocks of 1 h and 10 m respectively between ~250 and 390 m for days where 24 h of echo data were successfully retrieved ( from min . time for sunrise and sunset was set to the 15th of each corresponding month , as an approximate for each month , determining time allocated to day and night . glacier lanternfish makes up the main part of the volume backscattering ( sv ) at 90 db , while larger fish results in sv values greater than 64 db ( bagien et al . 2001 ) . thus , values retrieved by echo integration at 64 db were subtracted from the echo integration at 90 db to exclude fish larger than glacier lanternfish ( bagien et al . the concentrations ( individuals m ) were derived from measurements of sv and target strength ( ts , see description below ) by dividing the linear sv values with the linear ts ( maclennan and simmonds 1992 ) . ts depends on the size , anatomy , and behavior of the organism , as well as the frequency of the echo sounder ( maclennan and simmonds 1992 ) . for precise estimation of concentration , the ts of glacier lanternfish was obtained monthly by means of automatic target tracking ( tt ) , performing tt for 3 days of every month within a range of 1050 m from the transducer ( ~340380 m depth ) . this limited range was chosen as the resolution of acoustic data decreases with distance from the transducer , and so does the precision of the ts estimates . minimum track length was set at 10 ping , maximum ping gap to 1 ping and gating range to 0.3 m during tracking . ts values between 65 and 50 db were regarded as glacier lanternfish , as ts values stronger than 50 db indicate fish of greater size ( foote 1980 ; maclennan and simmonds 1992 ) . a minimum value of 65 db was chosen in accordance with a previous acoustic study of glacier lanternfish ( torgersen and kaartvedt 2001 ) . tracks of individual fish obtained by tt were also used for analysis of swimming behavior . vertical swimming speeds of individuals were analyzed together with visual inspection of daily 24 h echograms in order to investigate any asynchronicity in the dvm pattern of the deeper living part of the population . both tt and echo integration were performed in the post processing program sonar_5 pro version 5.9.9 ( balk and lindem 2007 ) . the migration patterns below 250 m as assessed from the monthly echograms were compared with changes in concentration as assessed based on combining the measurements of sv and ts . the maximum concentration of glacier lanternfish estimated below 250 m each day was assumed to reflect the total population below 250 m. increase and/or decrease in concentration , before and after the time of migration of a specific layer of glacier lanternfish , was used as a proxy for the relative proportion of glacier lanternfish with nodvm , idvm , and ndvm . the speed of migration by individuals in sl s was calculated by analyzing the distance of descent / ascent over time in sl s . the continuous acoustic measurements were complemented by sampling during research cruises 14 november 2007 with r / v haakon mosby ( university of bergen , imr ) and 37 october 2008 with r / v trygve braarud ( university of oslo ) . results from the physical oceanography , zooplankton , and trawling studies from these field campaigns are used in the interpretations , but are presented elsewhere ( staby et al . the behavioral patterns described in this study can with confidence be ascribed to glacier lanternfish . previous studies covering spring and summer ( rasmussen and giske 1994 ; kaartvedt et al . unpublished ) , autumn ( bagien et al . 2001 ; kaartvedt et al . 2009 ) , winter ( giske et al . 1990 ; balio and aksnes 1993 ; bagien et al . 2001 ) , and trawl data obtained during the present study ( staby et al . 2011 ; dypvik et al . 2012 ) , as well as subsequent studies ( kaartvedt et al . unpublished ) have shown that glacier lanternfish are the main cause of backscattering deeper than 250 m in masfjorden . catches at these depths consist of specimens > 4.5 cm , with a prevalence of individuals > 6 cm below 300 m ( kaartvedt et al . 2009 ; dypvik et al . 2012 ) , that is adult individuals ( gjster 1981 ) . pelagic shrimps are also common in the deep waters of masfjorden ( kaartvedt et al . 1988 , 2009 ) and may contribute somewhat to the backscatter . however , the sl s below 200 m in masfjorden are pronounced also at 18 khz ( kaartvedt et al . 2008 ) , signifying that the backscatter mainly consists of mesopelagic fish ( torgersen and kaartvedt 2001 ; love et al . 2004 ; god et al . 2009 ; kloser et al . 2009 ) . furthermore , the average ts of the targets studied here ( see results ; target strength and concentration estimates in deep water ) is in accordance with previous studies of lanternfishes ( torgersen and kaartvedt 2001 ; yasuma et al . 2009 ) , while shrimps have a ts about one magnitude weaker than that of glacier lanternfish ( benoit - bird and au 2001 ; torgersen and kaartvedt 2001 ) . in summer , there were four sl s performing ndvm to the surface at night , and one deep layer with no apparent diel behavior ( fig . 2 ) . the upper of these layers ( ~70200 m ) were ascribed to pearlside as they are found to dominate at these depths ( the seasonal dvm patterns of pearlside are discussed in staby et al . the two deeper layers ( > 250 m during daytime ) were ascribed to glacier lanternfish with ndvm and nodvm , respectively . zooming in on the deeper parts of the water column illustrates more clearly how organisms carrying out ndvm in summer descended to waters below ~250 m at daytime , and ascended at night ( fig . 2monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . 3monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) in autumn , ndvm was still present above ~200 m , but was less noticeable below ~200 m ( fig . 2 ) . idvm of acoustic targets ascribed to glacier lanternfish was now evident below the pearlside sl ( figs . 2 , 3 ) . an apparent non - migrating layer of glacier lanternfish occurred from 300 m to the bottom ( figs . 2 , 3 ) . in winter , there were strong echoes of vertical migrating juvenile pearlside , and largely non - migrating adult pearlside in the upper 200 m ( fig . 2 ) . the sl of individuals with idvm ( glacier lanternfish ) was still apparent , although less pronounced than in autumn ( figs . 2 , 3 ) . the deepest non - migrating layer was now at its seasonal high in level of backscatter ( fig . 2 ) . in spring , several sl s with ndvm appeared between ~100210 m in daytime ( march and april ) and in the upper ~100 m at night ( fig . 2 ) . , individuals performing ndvm descended to ~300 m in daytime , indicating a large portion of the glacier lanternfish population with this behavior ( figs . 2 , 3 ) . m ( glacier lanternfish ) , yet with a seasonal low in backscatter ( fig . 2 ) . estimated proportions with different dvm modes were calculated for each month based on the echograms and the echo integration from 250 to 390 m ( fig . ndvm was performed by ~2555 % of these fish in summer , while this behavior ceased in autumn and winter ( fig . however , individuals performing ndvm re - appeared in early spring , and the proportion performing ndvm increased toward late spring ( from ~13 % in march to ~51 % in may ) ( fig . 4 ) . the proportion performing idvm increased from late summer ( ~15 % in august ) to the beginning of autumn ( 70 % in september 2007 ) , then decreased throughout the winter ( 24 % in february 2008 ) . in spring and the first part of summer , idvm was not recorded ( fig . nodvm was suggested for ~4575 % of the population at these depths during spring and summer , but this proportion decreased from summer to autumn ( ~3040 % in september 2007 and 2008 ) , before it increased toward the winter months ( fig . 4the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively visual inspection of 24 h echograms suggested little systematic vertical swimming among the nodvm component in relation to time of day , as exemplified by 1 day each season in fig . fish at these depths commonly appeared to drift up and down in a consistent pattern suggesting internal wave motions ( fig . the limited vertical movement was reflected in the results from the year - round tt . of more than 200,000 tracks retrieved by tt however , intermittent vertical swimming was recorded in which individuals ascended or descended in a step - wise pattern ( fig . the maximum vertical swimming speed detected was ~18.4 cm / s . in comparison , the approximately ascending and descending speeds of the migrating layers were 34 cm / s for the ndvm layer and 0.50.8 cm / s for the idvm layer . in addition to such marked vertical relocations , seemingly passively drifting fishes once in a while slightly adjusted their vertical distribution , subsequently taking up their apparent torpid behavior.fig . 5 a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . time is given as utc ( local standard time 1 h ) a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . time is given as utc ( local standard time 1 h ) the average ts ( db ) of glacier lanternfish as measured between ~340 and 380 m were always in the range of approximately 57.5 to 59.5 db . within this limited range , the ts distribution of glacier lanternfish showed two peaks ( in both summers ) and decreased from the autumn to spring ( fig . 6average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) the ts values were used in assessing the numerical densities of glacier lanternfish . the concentration of fish was usually in the range of 0.0050.015 individuals m ( fig . however , the maximum density , just below 0.02 individuals m , was recorded in july 2008 in the deepest interval ( 300390 m ) , and in october 2008 in the shallower interval ( 250300 m ) ( fig . 7a , b).fig . 7 a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration in spring and summer , when ndvm is the dominating mode of migration , the daytime concentration of the interval ~250300 m was higher than during night ( fig . below 300 m , where the nodvm mode were dominating , relatively little concentration fluctuations between day and night occurred , except for june august 2008 ( fig . 7b ) when the ndvm layers entered those depths at daytime ( fig . 2 ) . in the months where idvm were the dominating mode of migration ( i.e. september february ) , the concentration of glacier lanternfish decreased during daytime in the interval ~250300 m ( fig . 7a ) , as the inverse vertical migrators that were distributed from ~220 to 300 m during nighttime ascended to mid - waters ( in the lower part of the strong sl between ~150220 m ) during the day ( figs . 2 , 3 ) . in summer , there were four sl s performing ndvm to the surface at night , and one deep layer with no apparent diel behavior ( fig . 2 ) . the upper of these layers ( ~70200 m ) were ascribed to pearlside as they are found to dominate at these depths ( the seasonal dvm patterns of pearlside are discussed in staby et al . the two deeper layers ( > 250 m during daytime ) were ascribed to glacier lanternfish with ndvm and nodvm , respectively . zooming in on the deeper parts of the water column illustrates more clearly how organisms carrying out ndvm in summer descended to waters below ~250 m at daytime , and ascended at night ( fig . 2monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . 3monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) in autumn , ndvm was still present above ~200 m , but was less noticeable below ~200 m ( fig . 2 ) . idvm of acoustic targets ascribed to glacier lanternfish was now evident below the pearlside sl ( figs . 2 , 3 ) . an apparent non - migrating layer of glacier lanternfish occurred from 300 m to the bottom ( figs . 2 , 3 ) . in winter , there were strong echoes of vertical migrating juvenile pearlside , and largely non - migrating adult pearlside in the upper 200 m ( fig . 2 ) . the sl of individuals with idvm ( glacier lanternfish ) was still apparent , although less pronounced than in autumn ( figs . 2 , 3 ) . the deepest non - migrating layer was now at its seasonal high in level of backscatter ( fig . 2 ) . in spring , several sl s with ndvm appeared between ~100210 m in daytime ( march and april ) and in the upper ~100 m at night ( fig . 2 ) . , individuals performing ndvm descended to ~300 m in daytime , indicating a large portion of the glacier lanternfish population with this behavior ( figs . 2 , 3 ) . m ( glacier lanternfish ) , yet with a seasonal low in backscatter ( fig . 2 ) . estimated proportions with different dvm modes were calculated for each month based on the echograms and the echo integration from 250 to 390 m ( fig . 4 ) . ndvm was performed by ~2555 % of these fish in summer , while this behavior ceased in autumn and winter ( fig . however , individuals performing ndvm re - appeared in early spring , and the proportion performing ndvm increased toward late spring ( from ~13 % in march to ~51 % in may ) ( fig . the proportion performing idvm increased from late summer ( ~15 % in august ) to the beginning of autumn ( 70 % in september 2007 ) , then decreased throughout the winter ( 24 % in february 2008 ) . in spring and the first part of summer , nodvm was suggested for ~4575 % of the population at these depths during spring and summer , but this proportion decreased from summer to autumn ( ~3040 % in september 2007 and 2008 ) , before it increased toward the winter months ( fig . . 4the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively visual inspection of 24 h echograms suggested little systematic vertical swimming among the nodvm component in relation to time of day , as exemplified by 1 day each season in fig . fish at these depths commonly appeared to drift up and down in a consistent pattern suggesting internal wave motions ( fig . the limited vertical movement was reflected in the results from the year - round tt . of more than 200,000 tracks retrieved by tt , ~85 % revealed vertical relocation speed < 0.4 cm / s . however , intermittent vertical swimming was recorded in which individuals ascended or descended in a step - wise pattern ( fig . 5e h ) . the maximum vertical swimming speed detected was ~18.4 cm / s . in comparison , the approximately ascending and descending speeds of the migrating layers were 34 cm / s for the ndvm layer and 0.50.8 cm / s for the idvm layer . in addition to such marked vertical relocations , seemingly passively drifting fishes once in a while slightly adjusted their vertical distribution , subsequently taking up their apparent torpid behavior.fig . 5 a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . time is given as utc ( local standard time 1 h ) a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . the average ts ( db ) of glacier lanternfish as measured between ~340 and 380 m were always in the range of approximately 57.5 to 59.5 db . within this limited range , the ts distribution of glacier lanternfish showed two peaks ( in both summers ) and decreased from the autumn to spring ( fig . 6average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) the ts values were used in assessing the numerical densities of glacier lanternfish . the concentration of fish was usually in the range of 0.0050.015 individuals m ( fig . however , the maximum density , just below 0.02 individuals m , was recorded in july 2008 in the deepest interval ( 300390 m ) , and in october 2008 in the shallower interval ( 250300 m ) ( fig . 7 a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration in spring and summer , when ndvm is the dominating mode of migration , the daytime concentration of the interval ~250300 m was higher than during night ( fig . below 300 m , where the nodvm mode were dominating , relatively little concentration fluctuations between day and night occurred , except for june august 2008 ( fig . 7b ) when the ndvm layers entered those depths at daytime ( fig . 2 ) . in the months where idvm were the dominating mode of migration ( i.e. september february ) , the concentration of glacier lanternfish decreased during daytime in the interval ~250300 m ( fig . 7a ) , as the inverse vertical migrators that were distributed from ~220 to 300 m during nighttime ascended to mid - waters ( in the lower part of the strong sl between ~150220 m ) during the day ( figs . 2 , 3 ) the 16-month acoustic data series revealed three different diel behavioral modes of glacier lanternfish . in ndvm , individuals ascend toward the surface at night and descend back to deeper waters in the morning . in idvm , individuals ascend toward mid - waters at daytime , before descending at night . in nodvm a clear seasonal pattern , with ndvm prevailing during spring and summer , and idvm prevailing during autumn and winter , was also apparent . all through the year , nodvm occurred in the water column deeper than ~300 m. this diversity in diel behavior of glacier lanternfish has previously been observed during autumn in masfjorden by kaartvedt et al . note , however , that while the acoustic results unveil the main patterns in diel behavior for the part of the population below ~200 m , they do not provide the entire picture for the whole population . only idvm was recorded acoustically in october and november , yet trawl catches documented some ndvm ( kaartvedt et al . also previous studies have documented ndvm of glacier lanternfish in fall and winter ( kaartvedt et al . 1988 ; giske et al . 1990 ; bagien et al . 2001 ) . some shallower - living glacier lanternfish intermingle with , and thus become inseparable in the acoustic signatures of the sl dominated by adult pearlside ( torgersen and kaartvedt 2001 ) . shallower - living glacier lanternfish are mostly smaller ( dypvik et al . 2012 ) and might be too small to be properly detected at 38 khz . the largely consistency in ts values in deep water underlines that we likely have addressed the adult population throughout this study period . the slight apparent seasonal variation in ts does not seem to correlate with behavioral changes . during spring and summer , , the concentration of zooplankton peaks in the surface layers , and significantly lowers in the deeper waters ( atkinson and peck 1988 ; rasmussen and giske 1994 ; richter 1994 ) . ndvm among lanternfishes is usually related to avoidance of visual predators in bright surface waters at daytime and subsequent feeding on zooplankton in the surface layers at night ( holton 1969 ; dalpadado and gjster 1987 ; kinzer et al . 1993 ) . the pay - off from ndvm will decrease as surface plankton becomes less abundant throughout fall and winter , while at the same time , seasonally migrating copepods return to depth ( speirs et al . 2006 ; broms and melle 2007 ) . . however , small individuals with less energy reserves , larger motivation , or less capability for feeding in deep water may continue to migrate toward the surface at night ( giske and aksnes 1992 ) , while larger fish may opt for other strategies such as staying in deeper waters feeding on overwintering calanoid copepods . in accordance with this , mainly 2-year or older glacier lanternfish are distributed deeper than ~250 m , while smaller individuals are distributed in shallower waters , during autumn ( dypvik et al . similar size - dependent depth distribution has also been observed in other studies of lanternfishes ( willis and pearcy 1980 ; gartner et al . idvm became apparent in august and was recorded until march . in winter , the bulk of zooplankton biomass is distributed in mid - waters ( giske et al . ( 2012 ) showed that inversely migrating glacier lanternfish were foraging on overwintering calanus in mid - waters during the day . other studies have shown that calanus is an important part of the glacier lanternfish diet during fall and winter ( gjster 1973 ; balio and aksnes 1993 ) . the seasonal occurrence of idvm seems to harmonize well with the overwintering period for calanus finmarchicus ( bagien et al . 2001 ; speirs et al . 2006 ) , in both the timing of initiation of this behavior , and its termination in spring ( heath 1999 ; astthorsson and gislason 2003 ; speirs et al . 2006 ; broms and melle 2007 ) . by staying in deeper waters performing idvm , glacier lanternfish may optimize their energy budget and reduce the predation risk ( dypvik et al . in addition , light decreases rapidly with depth in seawater ( srnes and aksnes 2006 ; aksnes et al . 2009 ) . a lanternfish swimming toward shallower waters , while still staying below ~200 m , will thus experience an increase of light in the surroundings , which would favor visual feeding by the low - light adapted lanternfish ( warrant and locket 2004 ) . thus , it seems likely that individuals with idvm ascend toward the overwintering layer of calanus at daytime to feed at favored light levels , sinking back to deeper layers at night . however , the proportion of the population with idvm decreases through the winter , which may reflect the reduced concentration of calanus from autumn through winter ( bagien et al . individuals of glacier lanternfish with nodvm occurred all year at depths greater than ~300 m. dypvik et al . ( 2012 ) found that the individuals with nodvm during autumn were the largest individuals of the glacier lanternfish population . individuals of greatest size tend to have larger eyes and might be better adapted than smaller individuals to detect prey in low light ( warrant and locket 2004 ) . they are also better adapted for handling large prey such as krill and shrimps , which occur deep in the water column ( kaartvedt et al . 2012 ) , and which represent a much higher dividend than copepods due to their much larger size ( falk - petersen 1981 ; tande 1982 ) . we therefore suggest that individuals with nodvm are able to feed in the restricted light levels of the deep waters . the behavior of individuals in deep water ( > 300 m ) suggested that the nodvm group indeed largely consisted of non - migrating individuals , and that consistent backscatter in deep water is not a result of asynchronous migrations , but that the population rather splits into migrating and non - migrating individuals , as initially suggested by pearcy et al . ( 1979 ) . nevertheless , single individuals were observed swimming upwards or downwards in a step - wise fashion , as also observed by kaartvedt et al . intermittent vertical relocations , ascent / descent in a step - wise fashion , would help the fish scan different parts of the water column for prey . fish may also use their lateral lines for tactile prey detection in darkness ( janssen et al . 1999 ) , and such behavior would be in accordance with both visual ( obrien et al . 5b ) suggests a prominence of ascent at day , so we can not reject the possibility that some of these deep - living individuals took part in idvm at that time . yet , most step - wise movements seemed to represent infrequent re - locations within the layer . a wide variety of lanternfishes are capable of feeding at depths greater than several hundreds of meters ( pearcy et al . 1979 ; roe and badcock 1984 ; sameoto 1988 ) , and nodvm has previously been observed among several species of lanternfish ( pearcy et al . however , different species of calanus occur in different geographic regions and , as well as other genera , perform seasonal vertical migration , overwintering in deeper water ( atkinson and peck 1988 ; richter 1995 ; peterson 1998 ; broms and melle 2007 ) . we therefore hypothesize that idvm and nodvm are widespread behaviors in areas where concentrations of potential prey are distributed in deeper waters , and that similar plasticity in dvm behavior as observed for glacier lanternfish in masfjorden might occur in other populations of lanternfishes in areas with seasonally migrating zooplankton , such as overwintering calanoid copepods . long - term acoustic measurements might be used to reveal such dvm plasticity in other areas , information that could be used to gain knowledge about interactions between lanternfishes , their prey and predators , as well as lanternfishes contribution to the vertical flux of organic matter . the vertical distribution and diel vertical migration of lanternfishes can affect such interactions and processes , making it imperative to further reveal these patterns .

the seasonal variations in glacier lanternfish ( benthosema glaciale ) vertical distribution and diel vertical migration ( dvm ) were studied by use of a bottom - mounted upward - facing 38 khz echo sounder deployed at 392 m depth and cabled to shore in masfjorden ( ~6052n , ~524e ) , norway . acoustic data from july 2007october 2008 were analyzed , and scattering layers below ~220 m during daytime were attributed to glacier lanternfish based on net sampling in this , and previous studies , as well as from analysis of the acoustic data . at these depths , three different diel behavioral strategies were apparent : normal diel vertical migration ( ndvm ) , inverse dvm ( idvm ) , and no dvm ( nodvm ) . nodvm was present all year , while idvm was present in autumn and winter , and ndvm was present during spring and summer . the seasonal differences in dvm behavior seem to correlate with previously established seasonal distribution of prey . we hypothesize that in regions with seasonally migrating zooplankton , such as where calanoid copepods overwinter at depth , similar plasticity in dvm behavior might occur in other populations of lanternfishes .

Introduction Materials and methods Results Main patterns in acoustic backscatter Relative proportion of migration patterns Individual behavior of glacier lanternfish Target strength and concentration estimates in deep water Discussion

acute liver failure ( alf ) can occur as a result of various etiologies including hepatic injury by drugs and poison , viral hepatitis , ischemia , or other causes . the mechanisms by which liver cells are destroyed as well as the processes mediating liver regeneration , remain largely unknown . it has become evident that liver cell death can occur via distinct biochemical pathways and morphological alterations , including apoptosis , autophagic cell death , and necrosis . apoptosis is defined by chromatin condensation , nuclear fragmentation , cell shrinkage , blebbing of the plasma membrane , and formation of apoptotic bodies that contain nuclear or cytoplasmic material ( kerr et al . , 1972 ; autophagic cell death , on the other hand , is characterized by a massive accumulation of double - membrane containing vacuoles , called autophagosomes , that subsequently fuse with lysosomes . necrotic cell death is often negatively defined as a form of cell death that lacks signs of apoptosis or autophagy . typically , necrotic cells show cytoplasmic swelling , dilation of organelles , and mechanical rupture of the plasma membrane . although necrosis has been deemed to be a mainly passive process , the initiation , and modulation of necrotic cell death are currently under intense investigation at the molecular level . the relative contribution of apoptosis or necrosis to organ dysfunction in alf remains controversial ( schulze - osthoff and bantel , 2011 ) . necrosis is typically the consequence of acute metabolic perturbation with atp depletion , whereas apoptosis represents an atp - dependent cell death program . furthermore , in several cases , the nature and duration of cellular injury determine if cells die by apoptosis , necrosis , or other mechanisms . at low doses , a variety of injurious stimuli often induce apoptosis , but the same stimuli can result in necrosis at higher doses . therefore , in many situations cell death might be not executed as a clear - cut form of cell death , but as a continuum with intermediate features of both apoptosis and necrosis . a distinction of different cell death forms is therefore not only relevant for semantical reasons , but has important clinical implications when considering the therapeutic targeting of cell death processes . thus , an understanding of the cell death processes is most important for development of effective interventions to prevent hepatocellular death in acute liver damage ( fischer and schulze - osthoff , 2005 ) . apoptosis represents a programmed form of cell death that is required for the maintenance of tissue homeostasis by counterbalancing cell proliferation and eliminating damaged , infected , or transformed cells . this process is particularly important in the liver as an organ that is naturally exposed to toxins , drugs , and viruses . apoptosis results from a collapse of cellular infrastructure through internal proteolytic digestion , which leads to cytoskeletal disintegration , metabolic derangement , and genomic fragmentation . members of the caspase family of proteases form the core engine of apoptosis and are involved in initiation , execution , and regulatory phases of the pathway . caspases are cysteine proteases that cleave substrates after aspartate residues within specific peptide recognition sequences . to preclude unwarranted cell death , caspases are expressed as inactive zymogens consisting of a prodomain followed by two subunits with the catalytic domain . caspases operate in hierarchical cascades that serve to amplify the apoptotic signal ( los et al . , 1999 ) . based on their structure and order in cell death pathways effector caspases such as caspase-3 , -6 , and -7 cleave diverse cellular substrates including structural proteins such as cytokeratin-18 and many others ( fischer et al . , in contrast , initiator caspases , such as caspase-8 , -9 , and -10 , exert regulatory roles by activating downstream effector caspases . caspases are activated by two major signaling routes , namely the extrinsic death receptor and the intrinsic mitochondrial pathway , that both depend on the formation of large multi - protein complexes ( schulze - osthoff et al . , 1998 ; initiator caspase-8 is the key mediator of the extrinsic pathway . in a simplified model , binding of death ligands such as trail or cd95l or this event then results in the recruitment of the adapter protein fadd and the initiator caspase-8 into a death - inducing signaling complex ( disc ) , wherein caspase-8 becomes activated by dimerization and autoproteolytic cleavage ( figure 1 ) . subsequently , caspase-8 cleaves and activates the effector caspase-3 , culminating in the demise of so - called type i cells . in most cells including hepatocytes , however , only low amounts of initiator caspases are activated at the disc , which is insufficient for cell death . in those type ii cells , the extrinsic receptor pathway must be amplified by the intrinsic mitochondrial apoptotic pathway through the caspase-8-mediated cleavage of bid , a pro - apoptotic bcl-2 family protein , which subsequently initiates together with the bcl-2 family members bak and bax the release of mitochondrial pro - apoptotic mediators ( schwerk and schulze - osthoff , 2005 ) . interestingly , cd95-induced hepatocyte apoptosis is delayed but not inhibited in bak / bax - deficient mice indicating that hepatocytes can act as type i cells in the absence of pro - apoptotic bcl-2 proteins ( hikita et al . , activation of nf-b is mediated by traf-2 , rip-1 , and other signaling molecules that lead to activation of ib kinase and subsequent activation of nf-b target genes . fadd and caspase-8 are the essential adapter proteins involved in apoptosis , which in hepatocytes requires a mitochondrial amplification loop through caspase-8-mediated cleavage of bid . the subsequent translocation of bax and bak results in mitochondrial outer membrane permeabilization , cytochrome c release , and effector caspase-3 activation . under conditions of impaired apoptosis , tnf receptor-1 can induce necroptosis , which involves rip-1 and rip-3 kinases . among other effects , rip-3 can increase the production of reactive oxygen species ( ros ) due to increased oxidative phosphorylation , resulting in intracellular calcium overload , mitochondrial membrane permeability transition ( mpt ) , depletion of atp , and necrosis . apap - induced necrosis is essentially mediated by a toxic metabolite , which depletes glutathione and forms apap protein adducts , triggering oxidative stress , compromised respiratory function , and atp depletion . although apap treatment can instigate the mitochondrial pathway of apoptosis , high doses of apap will ultimately mediate liver cell death by necrosis . this has been impressively demonstrated in mice that died rapidly of liver failure with massive hepatocyte apoptosis when agonistic anti - cd95 antibody was injected ( ogasawara et al . , 1993 ) . similarly , treatment of mice with tnf- in combination with a transcription - blocking agent , such as d - galactosamine ( d - galn ) or actinomycin d , induces lethal hepatitis ( leist et al . , 1994 , 1995 ; another well established mouse model of alf consists of a combined treatment with d - galn and lipopolysaccharide ( lps ) , which induces tnf- expression and an inflammatory response that is predominantly directed toward the liver ( galanos et al . , 1979 ) . it has been suggested that the toxicity in the murine tnf- and anti - cd95 models resembles viral forms of acute hepatic failure in patients ( keppler et al . , 1968 ; el - mofty et al . , 1975 ) . cd95/cd95l expression has been shown to be upregulated in viral hepatitis and to correlate with disease activity and hepatocyte apoptosis ( hiramatsu et al . , 1994 ; mita et al . , 1994 ; pianko et al . , 2001 ; lee et al . , we have recently demonstrated that diseased , e.g. , hcv - infected , livers show an upregulation of trail receptors and increased susceptibility toward trail - induced apoptosis ( volkmann et al . , 2007 ) . these data implicate that viral forms of alf are associated with death receptor - induced cell death . recent data show that the cd95 system is involved in human alf caused not only by viral hepatitis but also by wilson s disease ( strand et al . , 1998 ; increased levels of death ligands or receptors such as cd95l , tnf- , or tnf receptors ( tnf - r ) were found in blood of patients with alf ( ryo et al . particularly , high serum levels of soluble death receptor cd95 have also been found in drug - induced alf ( tagami et al . , 2003 ; rutherford et al . , 2007 ) . silencing of cd95 or caspase-8 protected mice from alf or fulminant hepatitis induced by agonistic cd95 antibody or concanavalin a , respectively ( song et al . however , cd95 and caspase-8 also promotes liver regeneration by inducing differentiation of stellate cells and possibly of other non - parenchymal liver cells ( desbarats and newell , 2000 ; canbay et al . , 2003 ; ben moshe et al . , 2007 ) . additionally , tnf- plays a pivotal role in liver regeneration by activation of transcription factors such as nf-b , which induces the transcription of a huge number of cytokines and growth - promoting target genes ( wullaert et al . , 2007 ) . the activation of nf-b by tnf- is mediated by distinct adapter proteins that are recruited to tnf - r-1 upon ligand binding ( figure 1 ) . whether increased levels of circulating death receptors or ligands in human alf mirror apoptotic cell death or liver regeneration and whether death receptor - induced cell death depends on special alf etiologies remains unknown . in contrast to viral infection , drug - induced liver injury is mainly associated with signaling pathways triggered by mitochondrial damage ( chan et al . , 2005 ) . in the intrinsic pathway , apoptosis is mediated by translocation of pro - apoptotic bcl-2 molecules , such as bax and bak , from the cytosol to mitochondria to form pores in the outer mitochondrial membrane ( los et al . , 1999 ) . this process is followed by the mitochondrial release of cytochrome c and other pro - apoptotic factors . cytochrome c normally functions in electron transport processes of the respiratory chain to generate atp . in the cytosol of apoptotic cells , however , it serves as a cofactor for the adapter protein apaf-1 . upon binding of cytochrome c and datp , apaf-1 oligomerizes , and recruits the initiator caspase-9 to trigger the formation of the apoptosome thus , similar to the disc , the apoptosome is a high - molecular weight complex that serves as a caspase activation platform . once assembled in the apoptosome , caspase-9 becomes activated and subsequently triggers the caspase cascade ( schulze - osthoff et al . as mentioned above , there is also considerable crosstalk between the extrinsic and intrinsic pathways . for example , during death receptor - mediated apoptosis caspase-8 can proteolytically activate the bcl-2 protein bid , which facilitates cytochrome c release and amplifies the apoptotic signal following death receptor activation . in contrast to apoptosis , necrosis is mediated by opening of the mitochondrial membrane permeability transition ( mpt ) pore , which triggers the collapse of the membrane potential and cessation of atp formation . the resulting mitochondrial swelling leads to the rupture of the outer mitochondrial membrane with the release of intermembrane proteins and subsequent nuclear dna fragmentation . other prominent features include massive energy depletion , formation of reactive oxygen species ( ros ) , and activation of non - apoptotic proteases . the elevated calcium levels in the cytosol trigger mitochondrial calcium overload , leading to depolarization of the inner mitochondrial membrane and a shut - down of atp production . while depletion of atp impedes the function of membrane channels , increased calcium activates calcium - dependent proteases , such as calpains . calcium fluxes , atp depletion , and oxidative stress involve complex and interactive feedback loops , which self - amplify and potentiate each other leading to exaggerated cell death . the relative amount of atp might be an important factor that determines whether hepatocytes die by apoptosis or necrosis ( ferrari et al . when the necrotic cell ruptures , an inflammatory response follows due to the release of intracellular contents . in contrast , inflammation is not typical of apoptosis , because phagocytic cells rapidly engulf apoptotic cells and thereby prevent the release of noxious intracellular compounds . acetaminophen ( paracetamol , n - acetyl - p - aminophenol ; apap ) overdose represents one of the most common causes of alf in developed countries ( larson et al . , 2005 ) . apap - induced hepatotoxicity is due to the formation of the toxic metabolite n - acetyl - p - benzoquinone imine by the cytochrome p450 system , which causes glutathione depletion , oxidative stress , alterations of calcium homeostasis , resulting in mpt , loss of mitochondrial membrane potential , and atp depletion ( hinson et al . , 2010 ) . although necrosis has been thought to be the predominant mode of cell death in apap - induced liver injury , conflicting in vitro and animal data have emerged suggesting a potential role of apoptosis in acetaminophen - induced hepatotoxicity ( el - hassan et al . mice treated with a toxic dose of acetaminophen showed 40% apoptotic and 60% necrotic hepatocytes ( ray et al . , 1996 ) . it was demonstrated that mice with defective cd95 receptor ( lpr mice ) or cd95 ligand ( gld mice ) were partially protected from apap - induced liver injury ( liu et al . , 2004 ) . moreover , increased circulating levels of cd95 have been found in humans with apap intoxication ( tagami et al . , 2003 ) . it has been recently suggested that apap hepatotoxicity is caused by the mitochondrial apoptosis pathway and facilitated by chemokine ( c - x - c motif ) receptor 2 ( cxcr2 ) signaling . in this study caspase inhibition prevented dna fragmentation , although the authors did not investigate whether caspase inhibition was also associated with cell survival ( hu and colletti , 2010 ; schulze - osthoff and bantel , 2011 ) . in contrast , in another study no caspase activation was observed and , accordingly , caspase inhibition did not protect from liver injury in apap - treated mice ( jaeschke et al . , 2006 ) . further data from animal models revealed also no evidence that apoptotic cell death contributes to apap - induced liver injury . for instance , following the application of an apap overdose in mice , less than 1% of the parenchymal cells revealed an apoptotic morphology ( gujral et al . , 2002 ) . another study in mice showed that knockdown of the cd95 receptor protected against 300 mg / kg apap overdose but not against 700 mg / kg overdose ( zhang et al . , 2000 ) , indicating that the mode of cell death might at least partially depend on the apap dose . indeed , several studies demonstrated that apap induces mitochondrial dysfunction with atp depletion which even interrupts initial cd95-induced mitochondrial signaling pathways ( lawson et al . on the other hand , subliminal cd95 activation can also increase apap - induced liver injury ( tinel et al . , 2004 ) , indicating that death receptor signaling might influence the extent of apap - induced necrotic liver injury . conflicting data exist also about the role of tnf- in apap - induced liver injury . increased tnf- expression in liver and circulating tnf- levels have been observed after apap poisoning ( blazka et al . . however , the role of tnf- in apap - induced necrosis remains controversial , as tnf- inhibitors exerted either protection or no effect ( blazka et al . , 1995 ; simpson et al . in addition , tnf- knockout mice showed similar sensitivity to acetaminophen compared to wildtype mice ( boess et al . , 1998 ) . it has been recently shown that inhibition of c - jun n - terminal kinase ( jnk ) , a member of the mitogen - activated protein kinase family , reduced paracetamol - induced toxicity in mice by inhibiting hepatic tnf- production ( henderson et al . , 2007 ) . furthermore , apap - induced jnk activation has been linked to activation of the pro - apoptotic bcl-2 protein bim . in line , apap - induced necrotic liver injury was shown to be reduced in bim knockout mice ( badmann et al . , however , neither jnk1 nor jnk2 knockdown did protect mice from apap - induced liver toxicity , raising concerns about a major role of jnk in apap - induced liver injury ( gunawan et al . , 2006 ; henderson et al . , 2007 ; bourdi et al . , 2008 ) . altogether , although apoptotic alterations can occur , profound energy depletion , and mitochondrial failure presumably divert cell death to necrosis as the principal mode of apap - induced liver toxicity . autophagy represents another process that might influence the outcome of apap - induced liver toxicity . autophagy is a catabolic mechanism by which long - lived proteins and organelles are recycled in order to maintain energy and protein synthesis . it is characterized by the appearance of numerous cytosolic vacuole - like structures , called autophagosomes , which encapsulate cytosolic materials and fuse with lysosomes . although the role of the autophagy in protection during nutrient starvation is accepted , its function in programmed cell death remains controversial . under normal physiological conditions autophagy occurs at low basal levels , contributing to the turnover of cytoplasmic components and promoting cell survival during stress conditions . interestingly , it has been recently demonstrated that apap induces autophagy in mouse liver and primary human hepatocytes and that activation of autophagy protects against apap - induced hepatotoxicity ( ni et al . , it was suggested that the induction of oxidative stress might play an important role in apap - induced autophagy . moreover , it was shown that pharmacological inhibition of autophagy increased apap - induced liver injury . clearly , more work is needed to elucidate the role of autophagy in apap - induced hepatotoxicity and the complex crosstalk with other cell death pathways . in addition to measuring death ligands , several other cell death biomarkers have been proposed for monitoring the clinical outcome of alf and other liver diseases ( volkmann et al . , 2006 ; rutherford et al . caspases cleave the intermediate filament protein cytokeratin ( ck)-18 into specific fragments that are released into circulating blood and can be detected by the m30 elisa ( bantel et al . , 2001b , 2004 ; seidel et al . , 2005 ) . moreover , when this assay is combined with a second elisa that detects the total release of caspase - cleaved and uncleaved ck-18 ( m65 elisa ) , even different forms of cell death , such as necrosis and apoptosis , can be discriminated . using these serological assays , it has been recently demonstrated that the predictive sensitivity of total ck-18 for lethal outcome was comparable to the model for end - stage liver disease ( meld ) score at time of admission of alf patients ( bechmann et al . , 2010 ) . moreover , modification of the meld score by substitution of bilirubin for total ck-18 significantly increased the prediction of alf outcome . interestingly , we have demonstrated that alf patients display considerable caspase activity and high levels of caspase - cleaved ck-18 in the serum , which was unexpectedly higher in spontaneous survivors than in patients that required transplantation or died ( volkmann et al . , 2008 ) . nevertheless , despite a weaker activation of caspases , liver biopsies of patients without spontaneous recovery revealed extensive tunel reactivity , which detects both apoptotic and necrotic cell death . moreover , sera from those patients contained increased levels of total ck-18 , but reduced levels of its caspase - generated fragments as compared to patients with spontaneous recovery . these findings therefore indicate that necrosis but not apoptosis is the predominant cell death in those critically ill alf patients , whereas in alf patients with spontaneous recovery apoptotic cell death predominates . in contrast , in another study , detection of caspase - cleaved ck-18 could not adequately predict alf outcome ( rutherford et al . however , unlike in our study in which apap - induced alf only played a minor role , in this study a relevant number of patients with apap - induced alf was included . as mentioned above , in those patients necrotic cell death might predominate irrespective of the outcome . in line with this observation , patients with apap - induced alf showed higher levels of total ck-18 compared to caspase - cleaved ck-18 levels ( bechmann et al . 2008 ; craig et al . , 2011 ; bantel and schulze - osthoff , 2012 ) . caspase activation might also play a role in liver regeneration and this might explain our observation of higher caspase activity in spontaneous survivors . caspases can cleave different cytokine precursors to generate active cytokines and thus create an environment that could be essential for liver regeneration . experiments in mice showed that caspase activation is associated with chemokine production and inflammation in the liver , whereas caspase-3 inhibition strongly reduced activity of pro - inflammatory transcription factors and chemokines ( faouzi et al . , 2001 ) . a role of caspases in liver regeneration is best exemplified by a recent report showing that a hepatocyte - specific knockout of caspase-8 attenuates hepatocyte proliferation after partial hepatectomy ( ben moshe et al . , 2007 ) . in addition to cell death biomarkers , circulating levels of nucleosomes and high - mobility group box 1 ( hmbg1 ) protein , both of which are released during hepatocyte death , have been shown to be elevated in patients with alf 2011 ) . however , those biomarkers were not able to predict alf outcome , and no significant differences in hmbg1 or nucleosome levels between paracetamol- and non - paracetamol - induced liver injury were found ( craig et al . , 2011 ) . one explanation for the lacking correlation of those biomarkers with alf outcome might be that the release of hmbg1 and nucleosomes are not strictly related to necrotic cell death , but also occur during apoptosis ( bell et al . certainly , further larger cohort studies are required to evaluate the predictive value of cell death markers in patients with paracetamol- or non - paracetamol - induced alf . an admixture of necrosis and apoptosis occurs in alf and might therefore open up novel strategies for therapeutic intervention ( fischer and schulze - osthoff , 2005 ) . more extreme injury leads to necrotic killing , whereas milder injury may result in apoptosis . whether inhibition of apoptosis using available pharmacological caspase inhibitors can indeed prevent liver cell death or will just simply shift the mode of cell death to necrosis , remains to be shown ( los et al . , 2002 ) . as caspases not only play a role in apoptosis but also in processes of liver regeneration , the possibility of adverse effects should not be ignored when considering the therapeutic use of caspase inhibitors in alf treatment . death receptor - blocking agents , such as cd95-fc or trail - r - fc proteins , might be promising candidates in virus - induced alf ( bantel and schulze - osthoff , 2003 ) . micrornas ( mirnas ) are non - coding rnas which have been implicated in the posttranscriptional regulation of various cellular pathways . it was recently demonstrated that overexpression of mirna-221 delays cd95-induced fulminant liver failure in mice ( sharma et al . , 2011 ) . thus , mirnas involved in cell death signaling pathways might serve as potential therapeutic targets in alf . due to the pleiotropic effects of tnf- , inhibitors of this cytokine might not only influence liver cell death but also immune response and liver regeneration . treatment of apap intoxicated mice with anti - tnf- partially prevented hepatotoxicity ( blazka et al . , other studies showed no alterations of apap toxicity in mice treated either with anti - tnf- antibody or soluble tnf receptor ( simpson et al . whether the observed discrepancy is due to the variant experimental conditions , such as different mouse strains , remains to be elucidated ( jaeschke et al . , there is increasing evidence suggesting that tnf- triggers cell death not only by apoptosis , but also by a necrosis - like process , which has been recently called necroptosis ( schulze - osthoff et al . whereas tnf--induced apoptosis involves caspase-8 , tnf--induced necroptosis is essentially mediated by the kinases rip-1 and rip-3 that are recruited to tnf - r1 ( figure 1 ) . interestingly , specific inhibitors of rip-1 , called necrostatins , have been recently designed as novel cytoprotective agents that might be a promising tool to positively influence alf outcome , at least in conditions in which death receptor - mediated necrosis predominates . therapeutic targets to improve apap - induced alf outcome might be jnk or other members of the mitogen - activated protein kinase family , such as p38 . jnk inhibition by sp600125 , a small - molecule reversible atp - competitive inhibitor , or by d - jnkii , a peptide inhibitor that inhibits the interaction of jnk with substrates , markedly reduced mortality in murine paracetamol - induced hepatotoxicity , with a significant reduction of hepatic apoptosis and necrosis ( bennett et al . , 2001 ; borsello et al . , 2003 ; henderson et al . , 2007 ) . since glutathione depletion and subsequent oxidative stress formation are key pathogenic mechanisms of apap - induced hepatocyte death , application of n - acetylcysteine , a pro - drug for glutathione synthesis , which has been shown to reduce apap - induced liver necrosis , is the current standard therapy in apap - induced alf ( corcoran et al . , 1985 ; saito et al . , 2010 ) in addition , other drugs with anti - oxidant effects might improve alf outcome . in this respect , application of cyclooxygenase inhibitors exerted protective effects in an experimental alf mouse model with decreased oxidative stress formation and marked reduction of hepatic necrosis ( chang et al . a novel therapeutic target in alf that connects the immune system with cell death might be cyclophilin a. this intracellular protein exerts pro - inflammatory and hepatotoxic activity when released from necrotic liver cells in apap - induced liver injury . conversely , inhibition of cyclophilin reduced inflammatory response to necrotic liver ( dear et al . , 2011 ) . thus , targeting of cyclophilin might represent an opportunity for a novel therapeutic approach in acetaminophen poisoning . as mentioned above , pharmacological inhibition of autophagy exacerbated apap - induced liver toxicity . vice versa , induction of autophagy by rapamycin was shown to inhibit apap - induced hepatotoxicity ( ni et al . , 2012 ) . thus , autophagy induction might be a further strategy to improve alf outcome in apap intoxicated patients . taken together , there are many promising therapeutic approaches for inhibition of apap- and non - apap - induced alf . however , it should be kept in mind that most of the described targets exert a pleiotropic role and might interfere not only with cell death but also with survival pathways . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

acute liver failure ( alf ) can be the consequence of various etiologies , that might vary between different geographic regions . most frequent are intoxications with acetaminophen , viral hepatitis , or liver damage of unknown origin . alf occurs when the extent of hepatocyte death exceeds the regenerative capacity of the liver . the mode of liver cell death that is predominantly induced in alf , i.e. , apoptosis or necrosis , is still controversial and presumably determined by the etiology , duration , and magnitude of liver injury . severe liver damage involves oxidative stress and depletion of atp resulting in necrosis . in contrast , maintenance of atp stores is required for the execution of apoptosis . recent data suggest that necrosis resulting from severe liver damage is associated with poor outcome of alf patients . discrimination between apoptosis and necrosis might be therefore useful for the identification of alf patients requiring liver transplantation . identification of the molecular cell death mechanisms remains an important issue not only for early prediction of alf outcome , but also for therapeutic interventions . in view of the pleiotropic functions of critical mediators of cell death and tissue regeneration , a particular challenge will be to reduce hepatocellular death without inhibiting the regenerative capacity of the liver . here , we review the molecular mechanisms of hepatocyte injury and the pathways leading to apoptosis and necrosis , which might represent potential diagnostic and therapeutic targets in alf .

Introduction Death Receptor Signaling in Acute Liver Failure Role of Mitochondrial Damage in Acute Liver Failure Cell Death Mechanisms Involved in Acetaminophen-Induced Acute Liver Failure Cell Death Biomarkers for Monitoring Acute Liver Failure Possible Therapeutic Strategies for Cell Death Inhibition in Acute Liver Failure Conflict of Interest Statement

age - related macular degeneration ( amd ) is subdivided into the dry form and the wet form . according to bressler et al . the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular age - related macular degeneration ( marina ) evaluated the benefit of ranibizumab in a dose of 0.5 mg per month . at 12 months , 95% of patients had lost < 15 early treatment diabetic retinopathy study ( etdrs ) letters from baseline and 34% of patients had a best corrected visual acuity ( bcva ) gain of 15 etdrs letters . the study ranibizumab in patients with subfoveal choroidal neovascularization ( cnv ) secondary to age - related macular degeneration ( sustain study ) was designed to further evaluate the safety , tolerability , and efficacy of optical coherence tomography ( oct)/bcva - guided , individualized , and flexible pro re nata ( prn , as needed ) dosing regimen for ranibizumab . at 12 months , 92.5% of patients had lost < 15 etdrs letters from baseline and 19.3% of patients had a bcva gain of 15 etdrs letters . whether the fixed or prn regimen was used in the treatment of wet amd , the therapeutic response of individual patients was not always the same . current observations suggest that the factors which affect the response to the initial treatment with ranibizumab are individual . the aim of this study was to assess the importance of various factors ( age , gender , baseline bcva , baseline macula thickness , and type and size of cnv ) for early morphological therapeutic response to ranibizumab in clinical practice . the treatment of patients with wet amd is centralized into 9 tertiary referral centres in the czech republic ( see appendix ) . anonymised data on treatment efficacy and safety have been consecutively entered into the czech national database amadeus ( age - related macular degeneration in patients in the czech republic ) since september 2008 . the main aim of this registry is to collect basic epidemiologic data on patients diagnosed with wet amd in the czech republic , document standard diagnostic and therapeutic patterns , and assess treatment efficacy in standard clinical practice . the data collection is independent of all treatment decisions ; it does not affect a patient 's access to treatment and fully complies with all ethical as well as legal requirements for noninterventional data collection in the czech republic . all patients have given written informed consent to the treatment , as well as data collection . the reported investigations were in accordance with the principles of the current version of the declaration of helsinki . the data are recorded from the moment of diagnosis and start of treatment at regular 3-month intervals for half a year . in the following period , the record is completed every 6 months . each record presumes biomicroscopic examination of the retina , determination of bcva using the etdrs chart , and an oct examination ( oct 3 stratus ) . the first visit involves fluorescein angiography ( fa ) ; indocyanine green angiography ( icga ) is used only if it is necessary for determining a diagnosis . the compulsory data always include bcva expressed by the number of etdrs letters , the central thickness of macula in 1 mm of macula in m , and volume in 6 mm of macula in mm . the first visit also involves recording age and gender and measuring type and size of cnv using fa or icga . patients with diagnosed wet form of amd who meet the czech society of ophthalmology criteria for initiation of treatment with the ranibizumab are entered into the registry . ranibizumab therapy in the czech republic is indicated in patients with amd who are older than 50 years , with predominantly classic , minimally classic , or occult cnv in subfoveal localization , a bcva score between 70 and 35 letters ( 20/4020/200 snellen equivalent ) , total macular lesion area 8 disc area ( da ) , and submacular haemorrhage 25% of the total macular lesion area . minimally classic and occult cnv must show signs of activity in the form of the presence of hard exudates , subretinal haemorrhages , or decrease in bcva within the last 3 months by 10 letters of the etdrs chart . in patients treated with ranibizumab in a dose of 0.5 mg , there are two separate phases : the loading phase , followed by a prn phase . in the loading phase , patients receive 3 consecutive monthly injections of ranibizumab ( months 02 ) , followed by a prn phase when further treatment is given between and including months 3 and 11 according to the retreatment criteria . retreatment with ranibizumab is performed if the patient 's bcva worsened against bcva recorded in the previous visit and if there is a demonstrable macular edema on oct examination . the prn method of application is also followed in the second year and all succeeding years of patient treatment . our study assessed the influence of age , gender , baseline bcva , baseline macula thickness , and type and size of cnv on early morphological therapeutic response to ranibizumab in clinical practice . we studied these factors in terms of anatomical changes in the macula after 3 consecutive monthly injections of ranibizumab in the loading phase ( months 02 ) . the monitored factors : age , gender , baseline best corrected visual acuity , baseline macula thickness , and type and size of choroidal neovascularization nor any others parameters ( visual acuity , oct ) had no influence on the treatment scheme in the loading phase . from 01/09/2008 to 24/6/2013 , 1153 newly diagnosed , treatment - nave patients treated with ranibizumab were entered into the registry . following the 3 initial injections of ranibizumab , the patients were divided into two groups based on the morphological findings in the macula : a group with active cnv and a group with inactive cnv . in the group with active cnv , oct screening revealed intraretinal macular edema , subretinal fluid accumulation , retinal pigment epithelium ( rpe ) detachment , fibrovascular rpe detachment , or the combination of all these findings . oct screening revealed restored foveal depression and a scar at the site of cnv with no signs of exudation was apparent . no intraretinal macular edema , subretinal fluid accumulation , rpe detachment , or fibrovascular rpe detachment was found . in both groups , we assessed the following parameters : gender and age of patients , type and size of cnv , baseline bcva on the etdrs chart , and baseline macular thickness . standard descriptive statistics were applied in the analysis : absolute and relative frequencies for categorical variables and median supplemented with 5th95th percentiles and mean supplemented by 95% confidence interval for continuous variables . the statistical significance of differences between groups was analyzed using pearson 's chi - square test for categorical variables and mann - whitney u test for continuous variables. = 0.05 was adopted as the level of statistical significance in all analyses . ( spss , inc . 2010 ) and performed by the institute of biostatistics and analyses at masaryk university , brno , operating independently of any amd treating centre in the czech republic . the sample included 1092 patients , 38.6% men , average age 73.3 years ( sd : 8.4 ) , and 61.4% women , average age 74.2 years ( sd : 8.6 ) . the analysis included 1153 treated eyes ; the right eye was treated 561 times , the left eye 592 times ( both eyes were treated 61 times ) . after the initial 3 injections ( day 0 , month 1 , and month 2 ) of ranibizumab , in month 3 we examined the sample of 1153 eyes . of these there were 841 eyes with active cnv ( the active group ) and 312 eyes with inactive cnv ( the inactive group ) . the sample in the inactive group included 41% of men and 59% of women ( p = 0.338 , pearson 's chi - square test ) . the active and the inactive group included 29.3% and 27.9% of patients at the age < 70 years , 43.4% and 42.9% of patients aged 7080 years , and 27.3% and 29.2% of patients at the age > 80 years ( p = 0.237 , mann - whitney u tests ) , respectively . the active and the inactive group included 31% and 20.2% of patients with predominantly classic cnv , 21% and 19.9% with minimally classic cnv , and 47.9% and 59.9% with occult cnv , respectively . the inactive group showed statistically significantly higher presence of occult membranes and statistically significant lower presence of predominantly classic cnvs compared with the active group ( p < 0.001 , pearson 's chi - square test ) . the active and the inactive group included 23.8% and 26.9% of patients with cnv < 2 disc areas ( da ) , 66.8% and 70.2% of patients with cnv 25 da , 9.4% and 2.9% of patients with cnv > 5 da , respectively . the inactive group showed statistically significantly lower presence of cnv > 5 da compared with the active group ( p < 0.001 , pearson 's chi - square test ) . the baseline bcva in the range of 1530 etdrs letters was shown in 10.2% of patients in the active group and 10.9% of patients in the inactive group . the bcva in the range of 3160 etdrs letters was shown in 62.5% of patients in the active group and 55.1% of patients in the inactive group . the baseline bcva of > 60 etdrs letters was shown in 27.2% of patients in the active group and 34% of patients in the inactive group . the median of the baseline bcva was 54 ( 595 percentiles : 2273 ) and 55 ( 595 percentiles : 2375 ) in the active and inactive group , respectively . we found no statistically significant difference in the value of the baseline bcva between the groups ( p = 0.066 , mann - whitney u test ) . 19.3% of patients in the active group and 15.6% of patients in the inactive group had a baseline macular thickness of < 250 m . the baseline macular thickness in the range of 250400 m was shown in 51.7% of patients and 58.5% of patients , respectively , and baseline macular thickness of > 400 m was shown in 29% and 25.9% of patients , respectively . the median of the baseline macular thickness was 330 m ( 595 percentiles : 190600 ) and 337 m ( 595 percentiles : 201535 ) in the active and inactive group , respectively . there was no statistically significant difference in the value of the baseline macular thickness between groups ( p = 0.663 , mann - whitney u test ) . in the literature , we found no publications describing prognostic factors for early morphological therapeutic response to treatment with ranibizumab in patients with wet amd . sarks , killingsworth , and gonzales noted that occult cnv may have a good functional treatment response . and killingsworth demonstrated histologically that the onset of cnv is characterized by intrachoroidal neovascularization followed by sub - rpe fibrovascular proliferation . occult cnv which is fibrovascular tissue in the sub - rpe space may in part represent an earlier stage of cnv because it is in the same tissue plane . up to 50% of occult cnv occult cnv as an earlier stage of the disease is assumed to be associated with less damage to photoreceptors in macula and successful treatment has a better prognosis . in our study , we evaluated the impact of gender , age , baseline bcva , baseline macular thickness , and type and size of cnv on early morphological therapeutic response following the 3 initial injections of ranibizumab . in the inactive group of 312 patients with complete regression of cnv activity after the initial 3 injections of ranibizumab , we found a statistically significantly higher proportion of occult membranes , statistically significant lower presence of predominantly classic cnv ( p < 0.001 ) , and statistically significantly lower incidence of cnv > 5 da ( p < 0.001 ) compared with the active group . we observed that smaller and occult cnv lesions have potentially better morphological therapeutic response with the disappearance of the cnv activity and resorption of the macular edema . we found no significant impact of gender , age , value of baseline bcva , or baseline macular thickness on early morphological therapeutic response after the initial 3 injections of ranibizumab . additional studies are needed to further clarify the relationship of morphological and functional results in ranibizumab treated patients with wet age - related macular degeneration . the results showed positive early morphological therapeutic response with restored foveal depression and no signs of exudation on oct in patients with higher incidence of occult cnv , lower incidence of predominantly classic cnv , and lower incidence of cnv > 5 da . there was no evidence of any effect of age , gender , baseline best corrected visual acuity , or baseline macula thickness on the early anatomical restoration of the macula . we believe that occult cnv and a cnv smaller than 5 da are optimistic for better morphological therapeutic response at the beginning of ranibizumab therapy . to determine if other factors influence the morphological response to ranibizumab treatment in patients with wet amd , further clinical studies are needed .

aim . to assess the significance of age , gender , baseline best corrected visual acuity , baseline macula thickness , and type and size of choroidal neovascularization in early morphological therapeutic response to ranibizumab treatment in patients with the wet form of age - related macular degeneration . methods . from 09/2008 to 06/2013 we evaluated 1153 newly diagnosed , treatment - nave patients treated with ranibizumab . based on the morphological findings in the macula following the initial 3 injections of ranibizumab , the patients were divided into two groups based on active and inactive choroidal neovascularization . results . after the initial 3 injections of ranibizumab , we examined the sample of 841 eyes with active cnv and 312 eyes with inactive cnv . in the inactive group , we found a statistically higher proportion of occult cnv ( p < 0.001 ) and lower incidence of cnv greater than 5da ( p < 0.001 ) compared with the active group . we found no statistically significant difference in age , gender , baseline best corrected visual acuity , or baseline macula thickness between the inactive and active groups . conclusion . occult cnv and cnv smaller than 5da are optimistic factors for a better morphological therapeutic response at the beginning of ranibizumab treatment .

1. Introduction 2. The Population and Methodology 3. Results 4. Discussion 5. Conclusion

few similar cases are reported in literature but none of them is associated with mirizzi syndrome . radiological images could help the surgeon to choose the surgical strategy and to evaluate the presence of associated diseases . in our case laparoscopic surgery showed to be an excellent approach for both the cholecystectomy and the repair of the defect of the abdominal wall . the patient was an 85 year - old man with history of hypertension , previous surgery for perforated diverticular disease ( hartmann procedure followed by reversal of colostomy ) . the physical examination showed a 5 cm mass in the right upper quadrant tender to palpation . ultrasound and ct confirmed the presence of a lithiasic gallbladder herniated through the abdominal wall ( fig.1 ) , with a dilatation of the common bile duct that measured 11 mm , due to a 17 mm obstructive infundibular stone as it happens in mirizzi syndrome type i ( fig.2 ) . lithiasic gallbladder herniated through the abdominal wall the routine complete blood test , including bilirubin level , was unremarkable . therefore the patient underwent a laparoscopic cholecystectomy and a repair of the hernia . during surgery , multiple adhesions were found and released carefully to identify the gallbladder that was fully included in the right upper quadrant abdominal wall covered by peritoneum . the gallbladder was completely released and the dissection of the pedicle showed a short cystic duct and a normal common bile duct . most of the cases reported in literature are internal hernias through the winslow foramen ( 1 ) . the herniation of the gallbladder through acquired defects of the abdominal wall ( incisional hernia ) has been much less reported . ( 2 ) presented the case of a gallbladder herniated through a fascial defect of a subcostal incision . garcia reported a patient who presented a gallbladder hernia through a parastomal defect ( 3 ) . more recently , the case of a gallbladder strangulation through an abdominal wall defect on the site of a previous colostomy was described ( 4 ) . in this case , the most remarkable thing is that herniation does not occur through a natural orifice or an acquired defect , such as respectively for the winslow foramen or for an incisional hernia , but directly into the abdominal wall . to our knowledge , there are only 3 published cases of spontaneous herniation of the gallbladder through the abdominal wall ( 5,6,7 ) . another interesting aspect of this case is the presence of a chronically distended gallbladder ( gallbladder hydrops ) , associated with extrinsic compression of the common hepatic duct by an impacted stone in the infundibulum ( mirizzi syndrome type i ) ( 8) . it is probably the gallbladder dilatation that plays an important role in the development of the hernia pressing constantly the gallbladder against the abdominal wall . usually the clinical picture is represented by a right upper quadrant pain , associated with variable degrees of a compromised general condition . in our experience the preoperative study based on computed tomography is essential for the diagnosis of the gallbladder hernia , and it provides also additional information on the abdominal wall defect . the management of this type of hernia consists in reducing the content and repairing the abdominal wall defect . in this case we suggest the laparoscopy as surgical approach of first choice because it allows to solve three problems at the same time : first , the reduction of the gallbladder s incarcerated hernia ; second , it enables to perform a cholecystectomy that was indicated for the presence of gallstones and hydrops ; third , it allows to repair the hernia defect preferably with a mesh , if the local conditions are favorable . in our experience , preoperative imaging has been proved to detect the gallbladder disease and the morphology of the fascial defect . mesh repair is the gold standard for hernias ; however , acute cholecystitis still remains a contraindication for mesh repair due to the high risk of infection .

a gallbladder incarcerated hernia associated with mirizzi syndrome is a very rare entity and to our knowledge this is the first case ever described in literature . an 85-year - old man presented at the emergency department with a tender right upper quadrant mass . computed tomography ( ct ) revealed the presence of a gallbladder lithiasis with signs of acute cholecystitis , herniated through the abdominal wall with an associated mirizzi syndrome . laparoscopic cholecystectomy and repair of the abdominal wall defect were performed . the patient recovered very well and the postoperative period was uneventful .

INTRODUCTION CASE PRESENTATION DISCUSSION

neglected tropical diseases ( ntds ) is an umbrella term used to describe infectious diseases that primarily affect low - income populations in tropical regions and are often widespread , chronic , and debilitating . although the number of diseases that are officially recognized as ntds by various public health authorities ranges from less than 10 to over 40 , the list of 17 diseases currently classified as ntds by the world health organization ( who ) is widely accepted as accurate and comprehensive ( 20 ) . of these 17 diseases , four are caused by bacterial pathogens ( buruli ulcer , leprosy , trachoma , and yaws ) , three are caused by protozoan parasites ( chagas disease , human african trypanosomiasis and leishmaniasis ) , two are viral ( dengue and rabies ) , and the other eight are caused by various species of parasitic worms ( 20 ) . the most recent data available indicating the estimated number of cases of each ntd are listed in table 1 . estimated number of cases of neglected tropical diseases ( ntds ) worldwide . prevalence ( total number of existing and new cases ) is reported for chronic ntds and incidence ( total number of new cases ) is reported for acute ntds or ntds with a high rate of recurrence . cdc , centers for disease control ; cfsph , center for food security and public health , iowa state university ; n / a , data not available ; who , world health organization . ntds exert an immensely deleterious effect on the public health and economies of much of the developing world . yet , by definition , ntds are issues for which the levels of awareness and resources necessary to develop effective solutions have not been sufficiently garnered . because ntds are largely confined to poor areas and have relatively low mortality rates , the policy makers and financiers of global health campaigns may underestimate or simply not be aware of the magnitude of the problem . it is for precisely these reasons ( that ntds are chronically debilitating , affect a large number of indigent people , and are often overlooked ) that it is crucial to increase the amount of education , research , and public outreach devoted to their study and treatment . even though ntds are largely confined to developing countries , there are a number of reasons why addressing ntds is important to developed countries such as the united states , and to the educational systems therein . the argument for increasing the focus on teaching about ntds can be distilled into three main points : ( i ) the sheer magnitude of the impact that ntds have on global health and interconnected world economies warrants a proportionately significant amount of attention in our classrooms ( ii ) increased awareness of ntds in western countries is crucial because successful ntd research programs and public health campaigns can significantly benefit from leadership , expertise , and funding from those with the greatest resources , and ( iii ) teaching and learning about ntds provides a unique opportunity to bridge myriad connections between basic science and other disciplines and to promote civic engagement and critical thinking . this is nearly 40 times the number of people living with hiv ( 37 million ) and more than six times the annual number of cases of malaria reported in recent years ( approximately 216 million ) ( 12 , 19 ) . although schistosomiasis alone is responsible for approximately 200,000 deaths per year , most ntds have relatively low mortality rates ( 20 ) . however , ntds can be permanently debilitating or disfiguring , and significantly limit the educational opportunities , productivity , and wage earning potential of affected individuals ( 16 ) . for example , trachoma , the leading cause of preventable blindness in the world , currently impairs the sight of over 8 million people and costs approximately $ 2.9 billion in lost productivity each year ( 7 , 16 ) . even more striking is the centers for disease control ( cdc ) estimate that the economic benefit of a successful campaign to eliminate lymphatic filariasis could exceed $ 55 billion ( 7 ) . as shown in table 1 , these two diseases are far from being the most widespread ntds , which underscores the immensity of this issue . the total impact of ntds is difficult to estimate due to a variety of factors including the chronic nature of these diseases , the confounding effects of multiple comorbidities , and difficulties in accurately tracking and treating diseases in large , diverse , poor populations . the effect of ntds is perhaps most accurately expressed as the amount of healthy productive years of life lost due to a combination of chronic disability and premature death of affected individuals . worldwide , ntds are responsible for the loss of approximately 4657 million years of healthy life per annum ( 16 ) . the only disease that currently causes a greater burden of this type is aids ( 16 ) . in addition to causing loss of human life and productivity , the pathogens that cause ntds further devastate poor communities by harming the livestock on which subsistence farmers depend for their livelihood . african trypanosomes , closely related to those that cause human african trypanosomiasis , are responsible for a widespread and devastating disease in livestock called nagana , which kills three million cattle per year , amounting to a loss of $ 4 billion from struggling african economies . similarly , cysticercosis causes a substantial economic toll by reducing the market value of cattle and pigs , which are rendered unsafe to eat when infected ( 20 ) . because we live in an increasingly interconnected world , the economic and social ramifications of ntds are not confined to endemic nations ; when the health and productivity of one - fifth of the world s population is adversely affected by disease , other nations are indirectly impacted by the moral obligation to provide assistance and the cost of providing that aid . more than 300,000 individuals currently residing in the united states are infected with trypanosoma cruzi , the causative agent of chagas disease , for which there is no vaccine available ( 7 ) . although most of these cases are attributed to infections that occurred outside of the country , at least 31 people have been infected inside the continental united states via vectoral transmission or transfusion of infected blood products ( 5 , 13 ) . and , as recently as 2008 , soil - transmitted helminth infections in the united states were estimated at nearly 4 million , occurring mostly in poor regions of appalachia and the south ( 10 ) . even ntds of which there are few , if any , domestic human cases reported can still exert a great impact on a country . for example , over $ 300 million is spent each year in the united states alone to vaccinate animals against rabies in order to prevent human infection ( 7 ) . the incongruous amount of attention devoted to ntds compared to other less prevalent diseases , such as aids , malaria , and tuberculosis ( often referred to as the big three diseases ) , extends beyond the allocation of funding for scientific research and public health campaigns and into the arena of public education . peter hotez , dean of the national school of tropical medicine at baylor college of medicine , has described ntds as the most common diseases you have never heard of , a characterization that should be actively changed through modifications to our nation s education system ( 9 ) . the typical american student enrolled in a general microbiology course is likely to know and learn significantly less about ntds than hiv / aids , even though the incidence of ntds is much higher . for example , the textbook chosen by this author s department for use in all microbiology courses , which is a highly regarded text described by the publisher , john wiley & sons , inc . , as a bestseller for multiple editions , discusses ntds on less than 10% of approximately 200 pages devoted to describing infectious diseases . although the prevalence of a disease is only one of many factors to consider when allocating class time to discussing various pathogens , the striking epidemiology of ntds , along with the other factors discussed herein , make a strong case for devoting a significant amount of time to the study of these diseases . the educational merits of teaching about ntds and the effects of these diseases on american citizens are not the only reasons to teach our students about them . the united states plays a crucial role in the global fight against ntds , yet our efforts as a nation have been historically inadequate ; as of 2012 , only 1.3% of the money spent by the united states on global health programs was devoted to ntds ( 16 ) . in contrast , malaria and hiv / aids ( which are much less prevalent ) account for 60% of the nation s global health budget ( 16 ) . non - governmental foundations based in the united states , such as the gates foundation , also provide a significant portion of the global budget for combating ntds ; however , there is widespread pressure to tighten budgets and decrease funding of global health programs in both the public and private sector . equally important to the funding of public health initiatives is the expertise of american scientists and physicians in developing chemotherapeutics that target the causative agents of ntds , and funding for basic science research has been drastically cut in recent years . our efforts must be expanded , not stymied , in order to make progress in eradicating ntds . increasing awareness of ntds through education , and promoting the interest of our nation s students in becoming ntd researchers and health policy makers , is likely the most promising way to ensure the continuation and redoubling of our nation s efforts to combat these diseases . due to the tremendously far - reaching and long - lasting impact that ntds exert on the human race , teaching about these diseases provides a prime opportunity to incorporate interdisciplinary learning and civic engagement into science education . the benefits of interdisciplinary learning , which involves the integration of knowledge from multiple fields of study around a central theme , include helping students access prior knowledge in order to form connections with new knowledge , the promotion of critical thinking and higher - order cognitive skills , improvement of content retention , enhancement of creativity , increased motivation to learn , and increased sensitivity to ethical issues ( 11 , 14 ) . microbiology lessons about ntds could easily be paired with an array of courses to facilitate interdisciplinary learning by emphasizing the link between the biology of ntds and socioeconomic issues . pairing a microbiology course with a public health course could facilitate interdisciplinary learning by focusing on the development of improved health policies and practices geared towards ameliorating the effects of ntds in developing nations . collaboration with epidemiology or mathematics courses could focus on quantifying the impact of ntds , and utilize real - world ntd - related problems as examples for teaching mathematical concepts . an interdisciplinary approach involving chemistry or pharmacology courses could focus on the development of improved chemotherapeutics to treat ntds , which is an area of global medicine significantly lacking in funding and progress . strategies for mobilizing and managing resources to treat and eliminate ntds and to sever the connection between ntds and the propagation of poverty could be explored in business , economics , or public policy courses . because the influence of ntds on the health and economies of endemic countries has been so forceful and enduring as to shape the histories of those countries , african studies , asian studies , and latin american studies programs the broad implications of ntds could be further explored in sociology courses by examining the way these diseases have shaped societies over time , and the ethical implications of how those with the resources to make a difference respond to the plight of those in need . all of the aforementioned issues could be further explored by integrating a writing - intensive course in which students can thoroughly develop and communicate their understanding and reactions . civic engagement , or the application of knowledge and skills into actions that promote and improve the well being of communities , is becoming increasingly emphasized as an important part of high school and university curricula . as with interdisciplinary learning , the extensive impact ntds have on communities around the world makes teaching about ntds well suited to the incorporation of civic engagement . the thought of civic engagement related to ntds may conjure images of students conducting fieldwork in remote tropical locations , but that is only one way to incorporate civic engagement in the study of ntds . because ntds leave almost no one in the world completely unaffected due to the potential for infection in non - endemic regions , the indirect economic impact that ntds exert on essentially every nation , and the moral obligation of those with the resources to help , any project that increases awareness of these issues in any community could be a valid civic engagement endeavor . interdisciplinary learning and civic engagement promote active learning by engaging students in critical thinking , reflective reasoning , data analysis , assessing the validity of data sources , synthesizing ideas and solutions , and interpreting knowledge with cultural literacy . active learning , in turn , helps propel students through the levels of bloom s taxonomy of learning ( 2 ) , from knowledge acquisition to applying , analyzing , and evaluating scientific information to synthesize solutions to problems . there are a number of pedagogical tools available to facilitate teaching and learning about ntds . in addition to the material about ntds presented in textbooks , an array of materials that can be used to supplement instruction is freely available . the global health division of the cdc maintains an easily navigable encyclopedic resource about ntds on its website ( http://www.cdc.gov/globalhealth/ntd/ ) , providing information about the biology , disease signs and symptoms , diagnostic and treatment options , epidemiology and risk factors , and prevention and control measures for each disease . the scope and presentation of this material makes it a felicitous resource for engaging students in research and active learning . the who provides a similar collection of information about ntds on its website ( http://www.who.int/neglected_diseases/diseases/en/ ) , including a cache of interesting facts about each ntd that are not all easily found on the cdc website ; however , the organization and presentation of information by the who is less accessible than on the cdc website , making it better suited as a secondary , complementary resource . additional resources include : alliance for case studies for global health ( http://www.casestudiesforglobalhealth.org ) , global network for neglected tropical diseases ( http://www.globalnetwork.org ) , pan american health organization ( http://new.paho.org ) , end neglected tropical diseases in africa ( end in africa ) ( http://endinafrica.org/ ) , the neglected tropical disease nongovernmental development organizations network ( http://ntd-ngdonetwork.org/ ) , and the united states agency for international development ntd program ( http://www.neglecteddiseases.gov/ ) . these websites may be useful for promoting interdisciplinary discussion and critical thinking because they provide a wealth of information about current and planned public health initiatives , as well as facts , anecdotes , and photographs that illustrate the human toll and vast socioeconomic implications of these diseases . some courses may benefit from the analysis and discussion of primary literature , a plethora of which is available via national center for biotechnology information ( 15 ) . the scholarly journal plos neglected tropical diseases is devoted exclusively to ntds , and many other scientific journals regularly publish primary research , review articles , and commentaries about ntds . the national center for case study teaching in science ( nccsts ) at the university of buffalo maintains a database of peer - viewed case studies that are freely available for use in the classroom . the nccsts database is continually being expanded , and currently includes at least five case studies about ntds , one on african trypanosomiasis ( 3 ) , two about chagas disease ( 4 , 18 ) and two on dengue fever ( 1 , 17 ) . these cases are presented as multipart narratives interrupted by questions and active learning exercises . for instructor use only , a password - protected answer key is provided , along with a set of teaching notes describing how the authors implemented the case in their own classroom and tips they have for other instructors who wish to use the case . many pedagogical tools are utilized in the case studies , including small and large group discussions , brainstorming , problem - based learning , and group presentations that incorporate a research component and peer feedback . to complete the case studies , students analyze scientific data and contextual information to identify a particular ntd - causing pathogen , and evaluate existing public health campaigns in order to synthesize their own plan for effectively treating and preventing the spread of that disease . these case studies provide a framework for examining the interdisciplinary connection of microbiology with fields such as economics , epidemiology , public health , and sociology , and promote civic engagement by emphasizing the connection between biology and the complex real - world problems facing communities affected by ntds . to promote hands - on civic engagement , there are a number of established programs available for educators who have the opportunity to incorporate a study - abroad component into their curriculum . two programs specifically designed to engage undergraduate students in laboratory and field - based research and community outreach projects in areas that are endemic for ntds are the public health brigades , overseen by the global brigades association headquartered in seattle , washington , and the tropical disease research program directed by dr . both of these programs are open to qualified students at colleges around the country and are primarily associated with public health projects related to chagas disease . the public health brigades program involves a week - long excursion in rural honduras , where students directly engage with locals in home infrastructural development , community leader training , and health education programs ( 8) . the tropical disease research program engages students in hands - on modules involving field studies , laboratory research , and healthy living development projects in rural ecuador over a period of two to six weeks ( 19 ) . many other opportunities for qualified students to conduct research and fieldwork exist , but due to variability in the availability and specifications of these programs they will not be described here . to reiterate , relevant civic engagement projects need not involve a travel - abroad component as long as they increase the awareness of the risks , social issues , or need for funding associated with ntds . this is nearly 40 times the number of people living with hiv ( 37 million ) and more than six times the annual number of cases of malaria reported in recent years ( approximately 216 million ) ( 12 , 19 ) . although schistosomiasis alone is responsible for approximately 200,000 deaths per year , most ntds have relatively low mortality rates ( 20 ) . however , ntds can be permanently debilitating or disfiguring , and significantly limit the educational opportunities , productivity , and wage earning potential of affected individuals ( 16 ) . for example , trachoma , the leading cause of preventable blindness in the world , currently impairs the sight of over 8 million people and costs approximately $ 2.9 billion in lost productivity each year ( 7 , 16 ) . even more striking is the centers for disease control ( cdc ) estimate that the economic benefit of a successful campaign to eliminate lymphatic filariasis could exceed $ 55 billion ( 7 ) . as shown in table 1 , these two diseases are far from being the most widespread ntds , which underscores the immensity of this issue . the total impact of ntds is difficult to estimate due to a variety of factors including the chronic nature of these diseases , the confounding effects of multiple comorbidities , and difficulties in accurately tracking and treating diseases in large , diverse , poor populations . the effect of ntds is perhaps most accurately expressed as the amount of healthy productive years of life lost due to a combination of chronic disability and premature death of affected individuals . worldwide , ntds are responsible for the loss of approximately 4657 million years of healthy life per annum ( 16 ) . the only disease that currently causes a greater burden of this type is aids ( 16 ) . in addition to causing loss of human life and productivity , the pathogens that cause ntds further devastate poor communities by harming the livestock on which subsistence farmers depend for their livelihood . african trypanosomes , closely related to those that cause human african trypanosomiasis , are responsible for a widespread and devastating disease in livestock called nagana , which kills three million cattle per year , amounting to a loss of $ 4 billion from struggling african economies . similarly , cysticercosis causes a substantial economic toll by reducing the market value of cattle and pigs , which are rendered unsafe to eat when infected ( 20 ) . because we live in an increasingly interconnected world , the economic and social ramifications of ntds are not confined to endemic nations ; when the health and productivity of one - fifth of the world s population is adversely affected by disease , other nations are indirectly impacted by the moral obligation to provide assistance and the cost of providing that aid . more than 300,000 individuals currently residing in the united states are infected with trypanosoma cruzi , the causative agent of chagas disease , for which there is no vaccine available ( 7 ) . although most of these cases are attributed to infections that occurred outside of the country , at least 31 people have been infected inside the continental united states via vectoral transmission or transfusion of infected blood products ( 5 , 13 ) . and , as recently as 2008 , soil - transmitted helminth infections in the united states were estimated at nearly 4 million , occurring mostly in poor regions of appalachia and the south ( 10 ) . even ntds of which there are few , if any , domestic human cases reported can still exert a great impact on a country . for example , over $ 300 million is spent each year in the united states alone to vaccinate animals against rabies in order to prevent human infection ( 7 ) . the incongruous amount of attention devoted to ntds compared to other less prevalent diseases , such as aids , malaria , and tuberculosis ( often referred to as the big three diseases ) , extends beyond the allocation of funding for scientific research and public health campaigns and into the arena of public education . peter hotez , dean of the national school of tropical medicine at baylor college of medicine , has described ntds as the most common diseases you have never heard of , a characterization that should be actively changed through modifications to our nation s education system ( 9 ) . the typical american student enrolled in a general microbiology course is likely to know and learn significantly less about ntds than hiv / aids , even though the incidence of ntds is much higher . for example , the textbook chosen by this author s department for use in all microbiology courses , which is a highly regarded text described by the publisher , john wiley & sons , inc . , as a bestseller for multiple editions , discusses ntds on less than 10% of approximately 200 pages devoted to describing infectious diseases . although the prevalence of a disease is only one of many factors to consider when allocating class time to discussing various pathogens , the striking epidemiology of ntds , along with the other factors discussed herein , make a strong case for devoting a significant amount of time to the study of these diseases . the educational merits of teaching about ntds and the effects of these diseases on american citizens are not the only reasons to teach our students about them . the united states plays a crucial role in the global fight against ntds , yet our efforts as a nation have been historically inadequate ; as of 2012 , only 1.3% of the money spent by the united states on global health programs was devoted to ntds ( 16 ) . in contrast , malaria and hiv / aids ( which are much less prevalent ) account for 60% of the nation s global health budget ( 16 ) . non - governmental foundations based in the united states , such as the gates foundation , also provide a significant portion of the global budget for combating ntds ; however , there is widespread pressure to tighten budgets and decrease funding of global health programs in both the public and private sector . equally important to the funding of public health initiatives is the expertise of american scientists and physicians in developing chemotherapeutics that target the causative agents of ntds , and funding for basic science research has been drastically cut in recent years . our efforts must be expanded , not stymied , in order to make progress in eradicating ntds . increasing awareness of ntds through education , and promoting the interest of our nation s students in becoming ntd researchers and health policy makers , is likely the most promising way to ensure the continuation and redoubling of our nation s efforts to combat these diseases . due to the tremendously far - reaching and long - lasting impact that ntds exert on the human race , teaching about these diseases provides a prime opportunity to incorporate interdisciplinary learning and civic engagement into science education . the benefits of interdisciplinary learning , which involves the integration of knowledge from multiple fields of study around a central theme , include helping students access prior knowledge in order to form connections with new knowledge , the promotion of critical thinking and higher - order cognitive skills , improvement of content retention , enhancement of creativity , increased motivation to learn , and increased sensitivity to ethical issues ( 11 , 14 ) . microbiology lessons about ntds could easily be paired with an array of courses to facilitate interdisciplinary learning by emphasizing the link between the biology of ntds and socioeconomic issues . pairing a microbiology course with a public health course could facilitate interdisciplinary learning by focusing on the development of improved health policies and practices geared towards ameliorating the effects of ntds in developing nations . collaboration with epidemiology or mathematics courses could focus on quantifying the impact of ntds , and utilize real - world ntd - related problems as examples for teaching mathematical concepts . an interdisciplinary approach involving chemistry or pharmacology courses could focus on the development of improved chemotherapeutics to treat ntds , which is an area of global medicine significantly lacking in funding and progress . strategies for mobilizing and managing resources to treat and eliminate ntds and to sever the connection between ntds and the propagation of poverty could be explored in business , economics , or public policy courses . because the influence of ntds on the health and economies of endemic countries has been so forceful and enduring as to shape the histories of those countries , african studies , asian studies , and latin american studies programs the broad implications of ntds could be further explored in sociology courses by examining the way these diseases have shaped societies over time , and the ethical implications of how those with the resources to make a difference respond to the plight of those in need . all of the aforementioned issues could be further explored by integrating a writing - intensive course in which students can thoroughly develop and communicate their understanding and reactions . civic engagement , or the application of knowledge and skills into actions that promote and improve the well being of communities , is becoming increasingly emphasized as an important part of high school and university curricula . as with interdisciplinary learning , the extensive impact ntds have on communities around the world makes teaching about ntds well suited to the incorporation of civic engagement . the thought of civic engagement related to ntds may conjure images of students conducting fieldwork in remote tropical locations , but that is only one way to incorporate civic engagement in the study of ntds . because ntds leave almost no one in the world completely unaffected due to the potential for infection in non - endemic regions , the indirect economic impact that ntds exert on essentially every nation , and the moral obligation of those with the resources to help , any project that increases awareness of these issues in any community could be a valid civic engagement endeavor . interdisciplinary learning and civic engagement promote active learning by engaging students in critical thinking , reflective reasoning , data analysis , assessing the validity of data sources , synthesizing ideas and solutions , and interpreting knowledge with cultural literacy . active learning , in turn , helps propel students through the levels of bloom s taxonomy of learning ( 2 ) , from knowledge acquisition to applying , analyzing , and evaluating scientific information to synthesize solutions to problems . there are a number of pedagogical tools available to facilitate teaching and learning about ntds . in addition to the material about ntds presented in textbooks , an array of materials that can be used to supplement instruction is freely available . the global health division of the cdc maintains an easily navigable encyclopedic resource about ntds on its website ( http://www.cdc.gov/globalhealth/ntd/ ) , providing information about the biology , disease signs and symptoms , diagnostic and treatment options , epidemiology and risk factors , and prevention and control measures for each disease . the scope and presentation of this material makes it a felicitous resource for engaging students in research and active learning . the who provides a similar collection of information about ntds on its website ( http://www.who.int/neglected_diseases/diseases/en/ ) , including a cache of interesting facts about each ntd that are not all easily found on the cdc website ; however , the organization and presentation of information by the who is less accessible than on the cdc website , making it better suited as a secondary , complementary resource . additional resources include : alliance for case studies for global health ( http://www.casestudiesforglobalhealth.org ) , global network for neglected tropical diseases ( http://www.globalnetwork.org ) , pan american health organization ( http://new.paho.org ) , end neglected tropical diseases in africa ( end in africa ) ( http://endinafrica.org/ ) , the neglected tropical disease nongovernmental development organizations network ( http://ntd-ngdonetwork.org/ ) , and the united states agency for international development ntd program ( http://www.neglecteddiseases.gov/ ) . these websites may be useful for promoting interdisciplinary discussion and critical thinking because they provide a wealth of information about current and planned public health initiatives , as well as facts , anecdotes , and photographs that illustrate the human toll and vast socioeconomic implications of these diseases . some courses may benefit from the analysis and discussion of primary literature , a plethora of which is available via national center for biotechnology information ( 15 ) . the scholarly journal plos neglected tropical diseases is devoted exclusively to ntds , and many other scientific journals regularly publish primary research , review articles , and commentaries about ntds . the national center for case study teaching in science ( nccsts ) at the university of buffalo maintains a database of peer - viewed case studies that are freely available for use in the classroom . the nccsts database is continually being expanded , and currently includes at least five case studies about ntds , one on african trypanosomiasis ( 3 ) , two about chagas disease ( 4 , 18 ) and two on dengue fever ( 1 , 17 ) . these cases are presented as multipart narratives interrupted by questions and active learning exercises . for instructor use only , a password - protected answer key is provided , along with a set of teaching notes describing how the authors implemented the case in their own classroom and tips they have for other instructors who wish to use the case . many pedagogical tools are utilized in the case studies , including small and large group discussions , brainstorming , problem - based learning , and group presentations that incorporate a research component and peer feedback . to complete the case studies , students analyze scientific data and contextual information to identify a particular ntd - causing pathogen , and evaluate existing public health campaigns in order to synthesize their own plan for effectively treating and preventing the spread of that disease . these case studies provide a framework for examining the interdisciplinary connection of microbiology with fields such as economics , epidemiology , public health , and sociology , and promote civic engagement by emphasizing the connection between biology and the complex real - world problems facing communities affected by ntds . to promote hands - on civic engagement , there are a number of established programs available for educators who have the opportunity to incorporate a study - abroad component into their curriculum . two programs specifically designed to engage undergraduate students in laboratory and field - based research and community outreach projects in areas that are endemic for ntds are the public health brigades , overseen by the global brigades association headquartered in seattle , washington , and the tropical disease research program directed by dr . both of these programs are open to qualified students at colleges around the country and are primarily associated with public health projects related to chagas disease . the public health brigades program involves a week - long excursion in rural honduras , where students directly engage with locals in home infrastructural development , community leader training , and health education programs ( 8) . the tropical disease research program engages students in hands - on modules involving field studies , laboratory research , and healthy living development projects in rural ecuador over a period of two to six weeks ( 19 ) . many other opportunities for qualified students to conduct research and fieldwork exist , but due to variability in the availability and specifications of these programs they will not be described here . to reiterate , relevant civic engagement projects need not involve a travel - abroad component as long as they increase the awareness of the risks , social issues , or need for funding associated with ntds . ntds are often marginalized in the spheres of global health policy and scientific research , as well as in the classroom . there is a strong argument for restructuring microbiology curriculum to increase the emphasis on teaching and learning about ntds due to the fact that these diseases affect more people worldwide than almost any other group of diseases and because educating students about the seriousness of ntds is integral to ensuring that the future leaders of the world will focus sufficient resources on ameliorating the global plight caused by this group of diseases . ntds also provide an opportunity to teach and learn about a diverse and interesting group of organisms , while promoting interdisciplinary learning , civic engagement , and critical thinking . to promote the teaching of ntds , educators have an array of educational tools to choose from , including informational websites , a case study database , and inter - institutional study - abroad programs .

neglected tropical diseases constitute a significant public health burden , affecting over one billion people globally , yet this group of diseases is underrepresented in the appropriation of both monetary and intellectual capital for developing improved therapies and public health campaigns . the topic of neglected tropical diseases has been similarly marginalized in the biology classrooms of our nation s high schools and colleges , despite offering an opportunity to teach and learn about a diverse area of microbiology with far - reaching public health , social , and economic implications . discussed herein is an argument for increasing the representation of neglected tropical diseases in microbiology education as a means to generate increased interest in these diseases among the generation of future researchers and policy - makers , and to promote interdisciplinary learning , civic engagement , and critical thinking .

INTRODUCTION DISCUSSION The worldwide impact of NTDs The role of the American education system in the fight against NTDs NTD education: an opportunity for integrating interdisciplinary learning, civic engagement, and critical thinking into basic science courses Resources for teaching and learning about NTDs CONCLUSION

severely obese adults seeking lifestyle interventions report impaired physical , mental , and obesity - specific quality of life ( qol ) [ 13 ] . thus , several studies have included qol as a primary outcome in evaluation of multicomponent lifestyle interventions for these individuals [ 47 ] . these studies have proposed physical activity to be a contributor to unexplained improvements in qol [ 47 ] . . demonstrated improvements in the physical component summary ( pcs ) score and mental component summary ( mcs ) score of the medical outcomes study short - form 36 health survey ( sf-36 ) at the end of one - year , partly residential interventions . reported similar findings after a six - month , outpatient intervention for overweight to obese adults . repeated measures in two of these studies revealed improvements in both pcs and mcs after an initial , intensive intervention phase whereas longer - term maintenance varied [ 4 , 7 ] . the results reported by blissmer et al . . revealed positive associations between weight loss and improvements in pcs but not mcs . with regard to obesity - specific qol , the swedish obese subjects ( sos ) study developed and used the instrument obesity - related problems ( op ) scale . after four and ten years , the authors found significant improvements in severely obese adults who had received conventional treatment . however , the treatment was not standardised but was provided in accordance with local routines by many primary health care centres . in contrast , despite weight regain , kaukua et al . demonstrated improvements in op at the end of a two - year follow - up of severely obese individuals who completed a four - month , outpatient intervention . compared to physical , mental , and obesity - specific qol , life satisfaction is a broader qol construct representing a subjective and global assessment of all major dimensions of life . using single item measures on life satisfaction , obesity was associated with impaired life satisfaction in two u.s . population studies [ 10 , 11 ] , whereas a danish epidemiological study , controlling for a cluster of lifestyle - related factors including body mass index ( bmi ) , found independent positive associations between self - reported physical activity and life satisfaction . multicomponent lifestyle interventions for severe obesity aim for a sustainable change of behaviour related to diet and physical activity . physical activity is beneficial for body composition and fitness in obese individuals undergoing dietary energy restriction and reduces adverse cardiovascular outcomes of obesity [ 15 , 16 ] . in the eight - year follow - up of the look ahead study , self - reported physical activity was associated with initial and maintained weight loss in overweight to obese subjects with type 2 diabetes . cross - sectional , unadjusted analyses in studies on treatment - seeking severely obese adults have demonstrated positive correlations between self - reported physical activity and pcs [ 18 , 19 ] and mcs . we found positive independent associations between objectively assessed physical activity and life satisfaction prior to a lifestyle intervention for severely obese adults and did a series of studies on patterns of change in the participants during the intervention . we used accelerometers to objectively measure physical activity , collected data at multiple time points , and found positive associations in the patterns of change between physical activity and aerobic fitness , fat mass , and lipoproteins , confirming the importance of physical activity for clinical and anthropometric outcomes in lifestyle interventions . to our knowledge , a similar design has not been used to examine associations between change in physical activity and qol outcomes over time . therefore , the present study examines associations between change in objectively assessed physical activity as the independent variable and change in physical , mental , and obesity - specific qol and life satisfaction as the dependent variables during a two - year , multicomponent lifestyle intervention . this study is part of the haugland obesity study , a prospective cohort study on severely obese adults who participated in a publicly funded two - year lifestyle intervention at red cross haugland rehabilitation centre ( rchrc ) in western norway . data were collected between february 2010 and october 2012 and the present study used data from four time points : baseline prior to the intervention ( t0 ) , six weeks later , at the end of the first residential stay , ( t1 ) , and prior to the residential stays one ( t2 ) and two ( t3 ) years from baseline . referral of patients was done by general practitioners in accordance with the right to admission to the norwegian specialist health services ( i.e. , bmi 40 kg / m or 35 kg / m with comorbidities ) . in total , 53 eligible patients from the age of 18 to 60 years , divided into four groups , had their first residential stay between february 2010 and october 2011 . exclusion criteria included previous obesity surgery or referral to obesity surgery ; severe cardiovascular disease ; pregnancy ; substance or alcohol abuse ; and impaired physical functioning or mental problems which could interfere with adherence to the intervention . briefly , the patients spent a total of 15 weeks at rchrc divided into four stays of six , three ( after three months ) , three ( at year one ) , and three ( at year two ) weeks . the overall goal was to improve the qol of the patients , while weight loss , improved mental health and physical fitness , and reduction of obesity - related medical problems served as secondary goals . the group - based cognitive behavioural therapy , consisting of eleven sessions over two years , targeted qol and self - management of physical activity and eating . scheduled physical activity in the residential periods consisted of brisk walking , swimming , strength training , ball games , and aerobics and amounted to nine to eleven hours weekly divided into bouts of 2060 minutes . each patient developed a plan for physical activity , modified to his or her preferences , limitations , and home situation . the diet followed the nordic nutrition recommendations and consisted of three high - fibre , low - fat , and energy - reduced meals and two to three snacks . the patients were advised to follow a similar diet at home . in the home periods , pcs ranges from 15.4 to 62.1 and mcs from 10.1 to 64.0 ( with higher scores representing better qol ) [ 28 , 29 ] . the pcs and mcs have been standardised to a population normal distribution , with a mean of 50 and a standard deviation ( sd ) of 10 . sf-36 has been widely applied in obesity research [ 1 , 30 ] , discriminates between subgroups of severely obese adults , and is sensitive to change during lifestyle interventions [ 4 , 5 ] . this is an eight - item measure of obesity - specific qol including questions about restaurant visits , holidaying , participation in community activities , swimming in public places , trying on and buying clothes , and intimate / sexual situations . the calibrated score ranges from 0 to 100 ( < 40 mild , 40 to < 60 moderate , 60 to < 80 severe , and 80 extreme problems ) . op is reliable and valid in severely obese adults [ 2 , 31 ] . in the present study , life satisfaction was assessed using a global question on current satisfaction with life with seven response alternatives from very satisfied to very dissatisfied . one - item measures on life satisfaction have demonstrated reliability and validity in health research [ 33 , 34 ] . to assess physical activity , we used the actigraph gti m accelerometer ( actigraph , pensacola , fl , usa ) , which is an electronic movement sensor . the accelerometer registers vertical acceleration and converts it into the unit counts which increase with the magnitude of the work rate for walking . the participants were instructed to wear the accelerometer over the right hip for seven consecutive days while awake , except during water activities . the t0 , t2 , and t3 assessments took place during home periods , while the t1 assessment was carried out at the end of the first residential period . the criterion for a valid measure was wear - time of ten hours per day for four days . non - wear - time was defined as periods of 60 consecutive minutes without counts , allowing for up to two minutes of counts within these 60 minutes [ 35 , 36 ] . the overall physical activity , given as counts per minute , was calculated as total counts divided by total valid wear - time . the accelerometer has shown validity in severely obese individuals and accelerometer assessed physical activity offers more accuracy than self - reported data . height was measured in the standing position without shoes using a stadiometer and reported to the nearest 0.5 cm . fat mass and weight were measured on a bioelectrical impedance analysis device ( bc 420s ma , tanita corp . , tokyo , japan ) in the morning , in a fasting state , in light clothes , and after voiding . waist circumference was measured twice at exhalation at the level of the umbilicus and reported as the mean of the two measurements . we obtained written , informed consent from all participants prior to the study in accordance with the helsinki declaration . ethical approval was given by the regional committee for medical and health research ethics for south - east norway ( registration number 2010/159 ) . the scores on life satisfaction were reversed before analyses so that higher scores indicated better satisfaction with life . subject characteristics are presented as means and sd for continuous variables and percentages for categorical data . observed values for qol measures are presented as means and sd . the effect size ( es ) for differences between pcs and msc population norms and the study population scores at the four time points were calculated by subtracting the norms from the mean score of the participants divided by the sd of the latter . we performed an attrition analysis using the chi - squared test for difference in gender and the independent samples t - test for differences in other variables . a linear mixed model based on restricted maximum likelihood estimation with random intercept for subjects was used in all analyses for change ( ) over time , using least significant difference from baseline . the associations between the independent and the dependent variables were analysed using linear regression , applying delta scores between each time point ( y1 = y1 y0 ; x1 = x1 x0 ; y2 = y2 y1 , etc . ) , giving a total of n = 73 ( pcs and mcs ) , 72 ( op ) , and 71 ( life satisfaction ) observations . for physical activity , pcs , mcs , op , life satisfaction , and bmi , the differences between t0 and t1 ( 1 ) , t1 and t2 ( 2 ) , and t2 and t3 ( 3 ) were used . gender , age , and change in bmi served as covariates in the adjusted regression analyses . a 1000-repetition bootstrap analysis was used to calculate 95% confidence intervals ( ci ) of the regression coefficients . the changes from baseline to each of the time points in qol measures , physical activity , and bmi , obtained from the linear mixed model , are presented as means with 95% ci . the es for changes in the dependent variables were calculated by subtracting the mean t1 , t2 , and t3 estimates from the mean t0 estimate , divided by the sd of t0 . a secondary analysis was performed using a baseline - observation - carried - forward approach for missing values . effect sizes were judged against the standard criteria proposed by cohen : trivial ( < 0.2 ) , small ( 0.2 to < 0.5 ) , moderate ( 0.5 to < 0.8 ) , and large ( 0.8 ) . statistical analyses were conducted using spss for windows , version 20.0 ( spss inc . , chicago , usa ) . a two - sided p value of 0.05 indicated statistical significance . forty - nine patients ( 37 women ; 43.6 9.4 years ; bmi 42.1 6.0 kg / m ) consented to participate in the study . baseline sociodemographic and anthropometric characteristics are presented in table 1 . at year two , 16 women and six men ( 44.9% ) were lost to follow - up ( figure 1 ) . the rest dropped out of the intervention itself , due to referral to obesity surgery , pregnancy , reaching personal weight goals , health problems , inability to attend the residential stays , or unknown reasons . the noncompleters did not differ from the completers with regard to gender , age , bmi , physical activity , qol measures , or changes from t0 to t1 in bmi , physical activity , or qol measures . table 2 documents changes in the qol measures and the related es . over the first six weeks , , mcs and life satisfaction had returned to baseline levels , whereas the improvement in pcs was partly maintained . the es for within - group change was small for op , moderate for pcs and mcs , and large for life satisfaction after six weeks . at year one , the es was small for op and moderate for pcs . finally , at year two , the es were small for pcs and moderate for op . physical activity increased significantly during the first residential stay and was partly maintained at year one . at year two scores on pcs , mcs , op , and life satisfaction are presented in table 4 including es for differences between the study population and the norwegian sf-36 population norm . for pcs , figure 2 illustrates that correlations between change in physical activity and change in qol measures were strongest for mcs and weakest for op . this is also demonstrated in table 5 which presents the results of the regression analyses . in the adjusted analyses , changes in pcs , mcs , op , and life satisfaction the explained variance was moderate for pcs and mcs and small for op and life satisfaction . change in bmi was correlated with change in pcs , mcs , and life satisfaction in the unadjusted analyses ; however , this association was not statistically significant in the full models . replacement of change in bmi with change in waist circumference or fat mass did not alter any results ( data not shown ) . we tested for the interaction between physical activity and gender in all four models and found that the women had a stronger association between change in physical activity and change in pcs than the men ( p = 0.012 ) . this study has 71 to 73 observations for the main outcomes ( table 5 ) . given 71 observations , a power of 0.80 , and significance level of 0.05 , the study should have power to detect a standardized coefficient of 0.32 ( medium effect size ) . results from this study suggest that change in physical activity is independently associated with change in physical , mental , and obesity - specific qol and life satisfaction in severely obese adults participating in a lifestyle intervention . in correspondence with the present study , an independent dose - response relationship between exercise and physical and mental sf-36 subscales was reported from a randomized , controlled pre - post study on a six - month intervention for overweight to obese , menopausal , and hypertensive women . a cross - sectional study on overweight to obese subjects with type 2 diabetes also reported associations between self - reported physical activity and mcs , but not pcs , independent of bmi . by contrast , ross et al . reported that physical fitness , which may be a proxy for physical activity , did not mediate the association between weight reduction and improvements in sf-36 subscales in obese women enrolled in a six - month intervention . however , the six - minute walk test , which was utilized to assess fitness in the study by ross et al . used self - reported data on physical activity from two time points and found associations with pcs , but not mcs , in obesity surgery - seekers , although these results did not control for bmi . described improvements in op alongside fluctuations in self - reported physical activity during a lifestyle intervention but did not examine correlations . regarding life satisfaction , our cross - sectional baseline study from the haugland obesity study found an independent association with physical activity . population data revealed a positive relationship with self - reported physical activity , though this relationship did not control for bmi . a comparison of lifestyle interventions found no difference in life satisfaction between intervention and control groups during a one - year follow - up . so some studies do not support our finding of associations between change in physical activity and change in all qol outcomes . one explanation may be that the intervention of the present study clearly differed from other lifestyle interventions in that the overall goal was improvement of qol . other interventions have weight management [ 2 , 7 ] or behaviour change related to physical activity and diet ( which should lead to weight loss ) [ 46 ] as primary goals . the inconsistencies across studies may also relate to the cross - sectional design of several of them [ 10 , 20 , 43 ] , problems with the reliability of self - reported physical activity , the variety of weight classes included in the studies , and other heterogeneities of participants , context , or research designs . interestingly , although the unadjusted analyses revealed correlations between changes in bmi and pcs , mcs , and life satisfaction , weight loss did not moderate the associations between the independent and the dependent variables in the adjusted analyses . associations between weight loss and improvements in pcs have been found in several studies on patients undergoing lifestyle interventions [ 4 , 5 , 37 ] but not in the study by blissmer et al . . neither did kolotkin et al . after obesity surgery , whereas karlsen et al . our finding on op , as the only variable which had a nonsignificant correlation with change in bmi in the unadjusted analyses , is contradictive to the sos study which demonstrated short- and long - term decreases in obesity - related problems associated with weight loss [ 2 , 8 ] . noticeably , with regard to the purpose of the present study , none of the above - mentioned studies included physical activity as a variable . after an initial , in - patient period and kaukua et al . at the end of a four - month outpatient programme using the rand-36 questionnaire , equivalent to the sf-36 , and the op however , despite the statistically significant effect of physical activity on qol measures in our analyses , many of the improvements in the dependent variables were left unexplained . thus , several other aspects may have played a role , such as experience of peer support [ 46 , 47 ] , reduction of anxiety , improved eating pattern , and improved self - regulation and self - efficacy which have been demonstrated by others after intensive intervention phases , as well as our explicit intervention focus on improvement of qol . regarding the patterns of longer - term changes , there are variations across studies . our finding that mcs had returned to baseline at year two while moderate es for the change in pcs was maintained is opposite to two - year changes reported by blissmer el al . on overweight to obese subjects . the es for one - year changes reported for lifestyle intervention completers by karlsen et al . is similar to the es of the present study for pcs ( = 0.47 ) , but higher ( = 0.32 ) for mcs . yet the baseline scores on pcs ( mean = 39 , sd = 10 ) and mcs ( mean = 42 , sd = 11 ) were lower compared to our study which may indicate a greater potential for long - term improvements . so , although lifestyle treatment - seekers generally report better qol than obesity surgery - seekers and worse than non - treatment - seekers [ 1 , 3 ] , variations across study populations in lifestyle interventions may contribute to disparities in research outcomes . moreover , it may be unrealistic to expect the initial peaks in pcs , mcs , and life satisfaction to last in the long run . in fact , that would imply better pcs and mcs scores than in the general population . for future studies , examining how favourable outcomes of intensive phases of lifestyle interventions can be maintained , at least partly , over time will be worthwhile . and , in that regard , our finding of continued improvement in the op is of interest . an intervention that can help participants experience less obesity - related problems , despite modest weight loss , may be seen as positive . change of health - related behaviours is challenging and the interaction of time is an aspect that deserves consideration . often , lifestyle interventions are reported to be shorter than the present , for example , four months , six months , or one year [ 4 , 5 ] . the sos study has published data on long - term follow - up , but the length of the included conventional treatment is not standardised or described [ 2 , 8 ] . the look ahead study , though including patients of all weight classes from overweight to severely obese , does provide detailed information about lifestyle intervention for adults with type 2 diabetes and is unique with regard to the large number of participants ( n = 5,145 ) , the randomisation , and the length of the intervention , that is , eight years . clearly , more studies are needed to develop a better understanding of long - term effects of lifestyle interventions on qol outcomes . first , the use of accelerometers to collect data on physical activity increased reliability over self - reporting . second , as recommended , both general and condition - specific qol instruments were used . third , data was collected at four time points and used to reveal associations of patterns of change . since change of health - related behaviour is complex , increase and maintenance of physical activity are challenging , and subjective constructs like qol are not straightforward , these findings on patterns of change contribute uniquely to the body of knowledge about lifestyle interventions for severely obese adults . a limitation of this study was that the number of participants lost to follow - up challenged the statistical power . high attrition is not unusual in research on lifestyle interventions [ 6 , 7 ] but differences between completers and noncompleters vary across studies from none [ 5 , 6 ] to one [ 4 , 7 ] or some . in the present study , the attrition analyses revealed no statistical difference in key variables between the drop - outs and the completers . the secondary analysis confirmed the statistical level of change in the qol measures , counts per minute , and bmi ( data not shown ) . to deal with the challenge of statistical power , all valid data were included in the linear mixed model . regarding the sample size , the study was powered to detect medium sized effect sizes as found in the regression analysis of this study . inclusion of a control group amongst the referred patients was not possible due to their right to treatment . this study examined associations and , therefore , causal relationships could not be inferred . and the study did not control for change of diet , a possible confounder in the associations we examined [ 51 , 52 ] . in addition , we controlled for change in bmi which may be a proxy for diet , because generally diet modifications produce more weight loss than physical activity [ 54 , 55 ] . lastly , the patients were a self - selected , treatment - seeking group although public funding of the intervention gave equal access to all and , therefore , diminished the risk of socioeconomic bias . it has been proposed that lifestyle interventions for obese individuals should focus less on weight loss as the primary outcome and pay more attention to independent benefits of physical activity such as reduction of obesity - related health hazards and improvements of qol . the present study contributes uniquely to the literature on severe obesity , physical activity , and self - reported outcomes and indicates that improved qol may be a valid result of increased physical activity in multicomponent lifestyle interventions . these findings should be further tested in various settings , in larger samples , and with control groups .

it is unknown how changes in physical activity may affect changes in quality of life ( qol ) outcomes during lifestyle interventions for severely obese adults . the purpose of this study was to examine associations in the patterns of change between objectively assessed physical activity as the independent variable and physical , mental , and obesity - specific qol and life satisfaction as the dependent variables during a two - year lifestyle intervention . forty - nine severely obese adults ( 37 women ; 43.6 9.4 years ; body mass index 42.1 6.0 kg / m2 ) participated in the study . assessments were conducted four times using medical outcomes study short - form 36 health survey ( sf-36 ) , obesity - related problems ( op ) scale , a single item on life satisfaction , and accelerometers . the physical component summary ( pcs ) score and the mental component summary ( mcs ) score were used as sf-36 outcomes . associations were determined using linear regression analyses and reported as standardized coefficients ( stand . coeff . ) . change in physical activity was independently associated with change in pcs ( stand . coeff . = 0.35 , p = .033 ) , mcs ( stand . coeff . = 0.51 , p = .001 ) , op ( stand . coeff . = 0.31 , p = .018 ) , and life satisfaction ( stand . coeff . = 0.39 , p = .004 ) after adjustment for gender , age , and change in body mass index .

1. Introduction 2. Design and Methods 3. Results 4. Discussion 5. Conclusions