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, studies included in the detect-2 database , in which retinopathy data had been collected , were invited to provide these data for this analysis . additional studies with retinopathy data identified by coinvestigators through personal contact or literature search also were invited to contribute datasets . this analysis focuses on nine studies from five countries that had retinopathy data by grading . participants aged 2079 years , including those with known diabetes and with gradable retinal photographs and at least one measure of glycemia , were included . all studies were approved by respective institutional review boards and were conducted according to the declaration of helsinki . retinopathy was classified as present or absent for initial analysis . where data were available , those with retinopathy were further classified as those having minimal nonproliferative diabetic retinopathy ( npdr ) , mild npdr , moderate npdr , severe npdr , or proliferative diabetic retinopathy ( pdr ) based on the information provided by individual studies using the modified airlie house classification levels ( 19 ) , modified early treatment diabetic retinopathy study levels ( 20 ) , or the fukuda standard ( 21 ) . levels 1420 indicate minimal npdr , levels 3035 or the fukuda standard a1 indicate mild npdr , levels 4047 or the fukuda standard a2 indicate moderate npdr , levels 5053 or the fukuda standard a3 indicate severe npdr , and levels 6090 or the fukuda standards a4 and b1b4 indicate pdr . the final retinopathy grading for each participant was based on the diagnosis in the more severely affected eye . the primary outcome used in this study was diabetes - specific retinopathy , which we defined as moderate or more severe levels of retinopathy . all nine studies measured plasma glucose , and six studies that measured a1c used high - performance liquid chromatography , of which five used a diabetes control and complications trial ( dcct)-aligned assay ( 5,12,14,15,18 ) . prevalence of diabetes - specific retinopathy was examined by 1 ) 0.5-unit intervals of glycemic measures and 2 ) vigintiles ( dividing participants into 20 equally sized groups ) of the distribution for each measure of glycemia . logistic regression models were applied to test the relationships between diabetes - specific retinopathy and glycemia by 0.5-unit intervals and by vigintiles of each glycemic measure , with the lowest range as the reference . the discriminatory power of each measure of glycemia for retinopathy was assessed as the area under the receiver - operating characteristic ( roc ) curve ( auc ) . an auc of 1 indicates perfect discriminatory power and an auc of 0.5 indicates that the discrimination is no better than chance . the impact of various thresholds on the prevalence of diabetes was examined by applying these values to 16,381 participants without known diabetes who had all three measures of glycemia . sensitivity analysis was performed on 1 ) studies in which a dcct - aligned assay for a1c was used ( ausdiab , chennai urban rural epidemiological study [ cures ] , multi - ethnic study of atherosclerosis and air pollution [ mesa ] , nhanes iii , and singapore malay eye study [ simes ] ) ; 2 ) studies in which one of the authors ( t.y.w ) was personally involved in the grading of retinopathy using the modified early treatment diabetic retinopathy study ( atherosclerosis risk in communities [ aric ] , ausdiab , blue mountains eye study [ bmes ] , mesa , and simes ) ; 3 ) studies in which participants were predominantly caucasian ( aric , ausdiab , bmes , mesa , and nhanes iii ) ; 4 ) studies in which participants were asian ( cures , hiroshima study , and simes ) ; and 5 ) studies in which participants had all three measures of glycemia . all statistical analyses were performed using sas 9.1 for windows ( sas institute , cary , nc ) and spss 16.0 for windows ( spss , chicago , il ) . retinopathy was classified as present or absent for initial analysis . where data were available , those with retinopathy were further classified as those having minimal nonproliferative diabetic retinopathy ( npdr ) , mild npdr , moderate npdr , severe npdr , or proliferative diabetic retinopathy ( pdr ) based on the information provided by individual studies using the modified airlie house classification levels ( 19 ) , modified early treatment diabetic retinopathy study levels ( 20 ) , or the fukuda standard ( 21 ) . levels 1420 indicate minimal npdr , levels 3035 or the fukuda standard a1 indicate mild npdr , levels 4047 or the fukuda standard a2 indicate moderate npdr , levels 5053 or the fukuda standard a3 indicate severe npdr , and levels 6090 or the fukuda standards a4 and b1b4 indicate pdr . the final retinopathy grading for each participant was based on the diagnosis in the more severely affected eye . the primary outcome used in this study was diabetes - specific retinopathy , which we defined as moderate or more severe levels of retinopathy . all nine studies measured plasma glucose , and six studies that measured a1c used high - performance liquid chromatography , of which five used a diabetes control and complications trial ( dcct)-aligned assay ( 5,12,14,15,18 ) . prevalence of diabetes - specific retinopathy was examined by 1 ) 0.5-unit intervals of glycemic measures and 2 ) vigintiles ( dividing participants into 20 equally sized groups ) of the distribution for each measure of glycemia . logistic regression models were applied to test the relationships between diabetes - specific retinopathy and glycemia by 0.5-unit intervals and by vigintiles of each glycemic measure , with the lowest range as the reference . the discriminatory power of each measure of glycemia for retinopathy was assessed as the area under the receiver - operating characteristic ( roc ) curve ( auc ) . an auc of 1 indicates perfect discriminatory power and an auc of 0.5 indicates that the discrimination is no better than chance . the impact of various thresholds on the prevalence of diabetes was examined by applying these values to 16,381 participants without known diabetes who had all three measures of glycemia . sensitivity analysis was performed on 1 ) studies in which a dcct - aligned assay for a1c was used ( ausdiab , chennai urban rural epidemiological study [ cures ] , multi - ethnic study of atherosclerosis and air pollution [ mesa ] , nhanes iii , and singapore malay eye study [ simes ] ) ; 2 ) studies in which one of the authors ( t.y.w ) was personally involved in the grading of retinopathy using the modified early treatment diabetic retinopathy study ( atherosclerosis risk in communities [ aric ] , ausdiab , blue mountains eye study [ bmes ] , mesa , and simes ) ; 3 ) studies in which participants were predominantly caucasian ( aric , ausdiab , bmes , mesa , and nhanes iii ) ; 4 ) studies in which participants were asian ( cures , hiroshima study , and simes ) ; and 5 ) studies in which participants had all three measures of glycemia . all statistical analyses were performed using sas 9.1 for windows ( sas institute , cary , nc ) and spss 16.0 for windows ( spss , chicago , il ) . in total , 44,623 participants had information on both the presence and severity of retinopathy ( table 1 ) . a total of 1,589 participants had minimal npdr , 762 had mild npdr , 430 had moderate npdr , 50 had severe npdr , and 171 had pdr . the number of participants available for each measure of glycemia was 41,334 for fpg , 21,334 for 2-h pg , and 27,933 for a1c . of these , 27,445 participants had at least two measures and 18,533 participants had all three measures . the characteristics of participants by study are shown in supplementary table 1 in the online appendix ( available at http://care.diabetesjournals.org/cgi/content/full/dc10-1206/dc1 ) . summary of studies included in these analyses * number of participants aged 2079 years included in the analysis . the overall prevalence of any retinopathy was 6.7% and 1.5% for diabetes - specific retinopathy . in people with known diabetes , the prevalence of diabetes - specific retinopathy was 9.4% , in newly diagnosed diabetes 1.0% , in impaired glucose tolerance ( 1 ) 0.1% , in impaired fasting glucose ( 1 ) 0.1% , and with normal glucose tolerance 0.1% . figure 1 shows the prevalence of retinopathy by 0.5-unit intervals for each measure of glycemia for diabetes - specific retinopathy . diabetes - specific retinopathy was virtually absent ( prevalence < 0.4% ) at low levels for each glycemic measure but began to increase from the fpg category of 6.06.4 the curve for 2-h pg was flatter than for fpg and a1c , and no definite interval of increase for 2-h pg was obvious . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) with 95% confidence intervals , number of retinopathy cases , and participants within each interval by 0.5 unit intervals for fpg and 2-h pg , and a1c . logistic regression adjusted for study center showed that the first interval where the odds ratio ( or ) for diabetes - specific retinopathy was significantly different from the reference fpg level of 4.04.4 mmol / l was 6.56.9 mmol / l ( or 6.0 [ 95% ci 2.117.1 ] ; p < 0.01 ) . the corresponding result for a1c was 6.56.9% ( 16.8 [ 2.3123.7 ] ; p = 0.01 ) compared with an a1c of 4.04.4% . figure 2 shows the prevalence of diabetes - specific retinopathy by vigintiles of the glycemic distributions . the prevalence of diabetes - specific retinopathy was very low until the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ) and until the 17th vigintile for fpg ( 6.46.8 mmol / l ) and for a1c ( 6.16.2% ) . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) by vigintiles of the distribution of fpg , 2-h pg , and a1c . logistic regression models adjusted for study center confirmed a statistically significant difference in the or for diabetes - specific retinopathy compared with the first vigintile that occurred from the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ; or 10.1 [ 95% ci 1.379.4 ] ; p = 0.03 ) , from the 17th vigintile for fpg ( 6.46.8 mmol / l ; 2.5 [ 1.25.2 ] ; p = 0.01 ) , and from the 18th vigintile for a1c ( 6.36.7% ; 4.5 [ 1.415.2 ] ; p = 0.01 ) . the overall discriminatory power determined by aucs was uniformly high for diabetes - specific retinopathy for each measure of glycemia ( 0.87 [ 95% ci 0.850.89 ] for fpg , 0.89 [ 0.870.91 ] for 2-h pg , and 0.90 [ 0.880.92 ] for a1c ) . the overlapping cis suggests that there is no statistical difference between the three measures of glycemia . the performance of a wide range of thresholds was examined , with particular attention to those that overlapped from the continuous and vigintile distribution plots . mmol / l for fpg , 13.0 mmol / l for 2-h pg , and 6.4% for a1c ( table 2 ) . if these thresholds were used for diagnosing diabetes , the prevalence of newly diagnosed diabetes would be 11.9 , 8.0 , and 6.3% according to fpg , 2-h pg , and a1c , respectively . the differences in performance based on roc curve statistics for the three measures of glycemia were minor for threshold values around the above values ( supplementary table 2 ) . threshold ranges for diabetes - specific retinopathy ( moderate npdr or more severe retinopathy ) derived from logistic regression models ( adjusted for center ) of the glycemic measures by continuous distribution and vigintile distribution and roc curve analysis sensitivity analyses showed that the five studies in which t.y.w used the same retinopathy grading system or the five studies that used dcct - aligned assays for a1c measurements provided similar results to the overall study . in total , 44,623 participants had information on both the presence and severity of retinopathy ( table 1 ) . a total of 1,589 participants had minimal npdr , 762 had mild npdr , 430 had moderate npdr , 50 had severe npdr , and 171 had pdr . the number of participants available for each measure of glycemia was 41,334 for fpg , 21,334 for 2-h pg , and 27,933 for a1c . of these , 27,445 participants had at least two measures and 18,533 participants had all three measures . the characteristics of participants by study are shown in supplementary table 1 in the online appendix ( available at http://care.diabetesjournals.org/cgi/content/full/dc10-1206/dc1 ) . summary of studies included in these analyses * number of participants aged 2079 years included in the analysis . the overall prevalence of any retinopathy was 6.7% and 1.5% for diabetes - specific retinopathy . in people with known diabetes , the prevalence of diabetes - specific retinopathy was 9.4% , in newly diagnosed diabetes 1.0% , in impaired glucose tolerance ( 1 ) 0.1% , in impaired fasting glucose ( 1 ) 0.1% , and with normal glucose tolerance 0.1% . figure 1 shows the prevalence of retinopathy by 0.5-unit intervals for each measure of glycemia for diabetes - specific retinopathy . diabetes - specific retinopathy was virtually absent ( prevalence < 0.4% ) at low levels for each glycemic measure but began to increase from the fpg category of 6.06.4 mmol / l and from the a1c category of 6.06.4% . the curve for 2-h pg was flatter than for fpg and a1c , and no definite interval of increase for 2-h pg was obvious . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) with 95% confidence intervals , number of retinopathy cases , and participants within each interval by 0.5 unit intervals for fpg and 2-h pg , and a1c . logistic regression adjusted for study center showed that the first interval where the odds ratio ( or ) for diabetes - specific retinopathy was significantly different from the reference fpg level of 4.04.4 mmol / l was 6.56.9 mmol / l ( or 6.0 [ 95% ci 2.117.1 ] ; p < 0.01 ) . the corresponding result for a1c was 6.56.9% ( 16.8 [ 2.3123.7 ] ; p = 0.01 ) compared with an a1c of 4.04.4% . figure 2 shows the prevalence of diabetes - specific retinopathy by vigintiles of the glycemic distributions . the prevalence of diabetes - specific retinopathy was very low until the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ) and until the 17th vigintile for fpg ( 6.46.8 mmol / l ) and for a1c ( 6.16.2% ) . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) by vigintiles of the distribution of fpg , 2-h pg , and a1c . logistic regression models adjusted for study center confirmed a statistically significant difference in the or for diabetes - specific retinopathy compared with the first vigintile that occurred from the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ; or 10.1 [ 95% ci 1.379.4 ] ; p = 0.03 ) , from the 17th vigintile for fpg ( 6.46.8 mmol / l ; 2.5 [ 1.25.2 ] ; p = 0.01 ) , and from the 18th vigintile for a1c ( 6.36.7% ; 4.5 [ 1.415.2 ] ; p = 0.01 ) . the overall discriminatory power determined by aucs was uniformly high for diabetes - specific retinopathy for each measure of glycemia ( 0.87 [ 95% ci 0.850.89 ] for fpg , 0.89 [ 0.870.91 ] for 2-h pg , and 0.90 [ 0.880.92 ] for a1c ) . the overlapping cis suggests that there is no statistical difference between the three measures of glycemia . the performance of a wide range of thresholds was examined , with particular attention to those that overlapped from the continuous and vigintile distribution plots . the thresholds that optimized sensitivity and specificity were 6.6 mmol / l for fpg , 13.0 mmol / l for 2-h pg , and 6.4% for a1c ( table 2 ) . if these thresholds were used for diagnosing diabetes , the prevalence of newly diagnosed diabetes would be 11.9 , 8.0 , and 6.3% according to fpg , 2-h pg , and a1c , respectively . the differences in performance based on roc curve statistics for the three measures of glycemia were minor for threshold values around the above values ( supplementary table 2 ) . threshold ranges for diabetes - specific retinopathy ( moderate npdr or more severe retinopathy ) derived from logistic regression models ( adjusted for center ) of the glycemic measures by continuous distribution and vigintile distribution and roc curve analysis sensitivity analyses showed that the five studies in which t.y.w used the same retinopathy grading system or the five studies that used dcct - aligned assays for a1c measurements provided similar results to the overall study . mmol / l , for a1c 6.46.5% , and for 2-h pg 10.111.2 mmol / l . the current diagnostic criteria for diabetes were derived from analyses of the relationship between retinopathy and measures of glycemia ( 1 ) . our study is the largest to examine this association , using data from 45,000 participants from five countries , and provides the statistical power for a more detailed and precise examination of glycemic thresholds for diabetes - specific retinopathy ( moderate nonproliferative and more severe retinopathy ) . previous studies ( 7,16 ) have only reported the association of glycemic measures with any retinopathy , which is less specific for hyperglycemia and is very frequently detected in people without diabetes . the association between glycemic measures and retinopathy has traditionally been investigated by plotting the prevalence of retinopathy against the decile distribution ( the population divided into 10 equal groups ) of each glycemic measure ( 1,2 ) . our large dataset allows analysis using vigintile distributions ( the population divided into 20 equal groups ) , which narrows the glycemic range of each group . based on logistic regression analysis of these vigintile distributions , glycemic thresholds for diabetes - specific retinopathy were observed in the range of 6.46.8 mmol / l for fpg , 9.810.6 . the large size of this dataset enables diabetes - specific retinopathy to be plotted against measures of glycemia as a continuous variable . a curvilinear relationship was observed , especially for fpg and a1c , as opposed to the linear association observed between blood pressure and cardiovascular disease . diabetes - specific retinopathy was rare at low levels of glycemia but increased from a range of 6.06.4 mmol / l for fpg and 6.06.4% for a1c . a threshold for increasing retinopathy was less obvious for 2-h pg , probably related to the smaller number of study participants with this measure and diabetes - specific retinopathy . change point analyses , which were used previously in two population - based studies ( 22 ) , were applied to these curves in an attempt to identify statistically significant thresholds , but we were unable to demonstrate a clear threshold for any glycemic measure by this method . this could suggest that within the ranges of visually detected thresholds for the three measures , changes in the prevalence of diabetes - specific retinopathy remain somewhat linear . the continuous and vigintile plots provided a similar range of threshold values for fpg and a1c . roc curve analyses were then used to compare performance in relation to optimizing sensitivity and specificity of glycemic values in the range around these thresholds . these analyses suggest thresholds of 6.6 mmol / l for fpg and 6.4% for a1c . the results derived from the vigintile distribution , continuous plots , and roc curve analyses suggest cut point values of 6.5 mmol / l for fpg and 6.5% for a1c , which could be considered in deliberations on modifying the current diagnostic criteria for diabetes . the results for 2-h pg were too inconsistent to consider modifying the current diagnostic cut point of 11.1 mmol / l . it should be noted that these values do not result in equivalent estimates for prevalent diabetes . this has been an ongoing issue with the current diagnostic criteria , whereby using fpg alone or an oral glucose tolerance test to diagnose diabetes gives different diabetes prevalence ( 23 ) . from our data ( supplementary table 2 ) , lowering the fpg to 6.5 mmol / l would result in a diabetes prevalence of 13.0% based on fpg alone and 18.6% based on an oral glucose tolerance test using an fpg of 6.5 mmol / l or a 2-h pg of 11.1 the prevalence of diabetes defined by an a1c of 6.5% is considerably lower ( 5.7% ) . this discrepancy in prevalence may be problematic for epidemiological studies but is not necessarily a disadvantage for individual patient care . an a1c of 6.5% was associated with a higher sensitivity and specificity than an fpg of 6.5 mmol / l and a higher specificity than a 2-h pg of 11.1 mmol / l . in other words , fewer people would be identified as having diabetes , but this would not compromise the identification of people with diabetes - specific retinopathy . whether this would have any deleterious ramifications in relation to identifying individuals at increased risk of other microvascular or macrovascular disease remains to be determined . there have been reports of differences in a1c levels independent of glucose between black , white , and south asian populations ( 24,25 ) . in our study , subgroup analysis by asian and predominantly caucasian populations showed no difference in the optimal a1c threshold ( 6.4% for both ) . however , our study was not designed to have and did not have sufficient numbers to examine a potential black / white difference . strengths of this study include its large sample size , which was drawn from populations across different countries and racial / ethnic groups ; the ability to focus on diabetes - specific retinopathy ; and availability of data to examine three glycemic measures . first , this study was based on cross - sectional data , whereas diagnostic thresholds would ideally be informed by incidence data of diabetes complications . second , the methods used to assess and classify retinopathy differed between studies , and it was not possible to independently review the grading of all photographs . nevertheless , inter- and intraobserver consistency for retinopathy in the different studies was of the order of 8098% ( 3,10,15 ) and misclassification , especially for moderate or more severe forms of retinopathy , is likely to be minimal but can not be entirely eliminated . furthermore , analysis of the studies in which t.y.w . was involved in the standardized grading of retinal photographs showed cut points for fpg and a1c similar to our entire study cohort . third , no quality assurance of measures of glycemia could be applied across the studies . nevertheless , all studies measured a1c using high - performance liquid chromatography , and analysis of the five studies that used a dcct - aligned assay showed an a1c cut point of 6.46.5% . fourth , the hiroshima study , with its large sample size , and the pima indian study , with its high prevalence of diabetes - specific retinopathy , may have influenced the results . however , sensitivity analyses that excluded these two studies did not alter the overall results . finally , not all included studies were randomly sampled populations ( e.g. , mesa ) and some ( e.g. , ausdiab ) oversampled people with diabetes and/or prediabetes . common to all such analyses is the issue of whether to include people with previously diagnosed diabetes . if people with known diabetes currently receiving blood glucose lowering treatment are included , the population - based characteristics of the study sample are maintained , but a bias associated with treatment - induced effects on glycemia is introduced and the level of glycemia assessed in each study may be lower than that which led to retinopathy . excluding people with treated diabetes from the analyses eliminates this bias but changes the characteristics of the population by eliminating many individuals with retinopathy , making it much more difficult to identify a threshold ( 2,7 ) . large incidence studies are needed to resolve these issues and determine the optimal levels of glycemia that predict the development of diabetes - specific retinopathy . in summary , this pooled analysis of glycemia and diabetes - specific retinopathy among close to 45,000 participants substantially broadens the evidence based on glucose - specific and a1c diabetes diagnostic thresholds . our results demonstrate narrow glycemic threshold ranges for the presence of diabetes - specific retinopathy and suggest that the current diabetes diagnostic level for fpg should be lowered to 6.5 mmol / l and that an a1c of 6.5% is a suitable alternative diagnostic criterion .

objectiveto re - evaluate the relationship between glycemia and diabetic retinopathy.research design and methodswe conducted a data - pooling analysis of nine studies from five countries with 44,623 participants aged 2079 years with gradable retinal photographs . the relationship between diabetes - specific retinopathy ( defined as moderate or more severe retinopathy ) and three glycemic measures ( fasting plasma glucose [ fpg ; n = 41,411 ] , 2-h post oral glucose load plasma glucose [ 2-h pg ; n = 21,334 ] , and a1c [ n = 28,010 ] ) was examined.resultswhen diabetes - specific retinopathy was plotted against continuous glycemic measures , a curvilinear relationship was observed for fpg and a1c . diabetes - specific retinopathy prevalence was low for fpg < 6.0 mmol / l and a1c < 6.0% but increased above these levels . based on vigintile ( 20 groups with equal numbers ) distributions , glycemic thresholds for diabetes - specific retinopathy were observed over the range of 6.46.8 mmol / l for fpg , 9.810.6 mmol / l for 2-h pg , and 6.36.7% for a1c . thresholds for diabetes - specific retinopathy from receiver - operating characteristic curve analyses were 6.6 mmol / l for fpg , 13.0 mmol / l for 2-h pg , and 6.4% for a1c.conclusionsthis study broadens the evidence based on diabetes diagnostic criteria . a narrow threshold range for diabetes - specific retinopathy was identified for fpg and a1c but not for 2-h pg . the combined analyses suggest that the current diabetes diagnostic level for fpg could be lowered to 6.5 mmol / l and that an a1c of 6.5% is a suitable alternative diagnostic criterion .

RESEARCH DESIGNS AND METHODS Classification of retinopathy Statistical analysis RESULTS Study participants Prevalence of retinopathy CONCLUSIONS

the medical records of 227 patients diagnosed with retinoblastoma at seoul national university children 's hospital between march 1997 and september 2007 were retrospectively studied . the diagnosis of retinoblastoma was based on indirect ophthalmoscopy , computed tomography , and ultrasonography findings . the stage was assessed with r - e classification and the international classification of retinoblastoma ( icr ) . upon diagnosis of retinoblastoma , a brain magnetic resonance imaging and bone scan were performed to evaluate systemic metastasis . of the patients diagnosed with retinoblastoma , patients who had been treated with primary chemotherapy with or without the combination of various local therapeutic methods were selected and included in this study . patients whose tumors were small and able to be treated with local therapeutic methods alone were not included . patients with documented evidence of systemic metastasis or with evidence of extraocular extension at diagnosis were also excluded . patients who had chosen to undergo primary enucleation after thorough discussion of the available treatment options and their prognoses were also not included in this study . the chemotherapeutic regimen consisted of a 13-cycle combination of cisplatin ( carboplatin in some patients ) , etoposide , and vincristine , which is used in the treatment of central nervous system tumors [ 8,18 - 20 ] . an ophthalmoscopic examination was done either on an outpatient basis or under general anesthesia at the beginning of each cycle according to the patient 's condition . additionally , every patient was evaluated by a pediatric oncologist regularly for possible adverse effects . the chemotherapy regimen was changed to regimen 2 if the tumor showed a minimal response to the basic regimen or if adverse effects , such as nephrotoxicity , were detected . the chemotherapy regimen was changed to regimen 3 if the treatment response continued to be minimal and if growth of the tumor or new lesions were detected despite treatment either with the basic regimen or regimen 2 . the regimen was also modified according to the patients ' general condition and if any adverse effect of a specific chemotherapeutic agent was suspected . local therapy such as thermotherapy , cryotherapy , and laser photocoagulation was added depending on the status of each tumor after each cycle of chemotherapy . thermotherapy was done in patients with tumors with a base less than 3 mm and a thickness less than 3 mm without vitreous seeds located posterior to the equator . laser photocoagulation was done in similar cases , but also in slightly larger tumors by surrounding the tumor and closing off feeding vessels . the patients were grouped according to tumor classification ( r - e classification , icr ) , therapeutic methods used , and the status of the contralateral eye . enucleation was performed in eyes with tumors that showed persistent viable appearance with no signs of regression or in eyes with newly appearing lesions after two or more cycles of treatment . enucleation was also performed in eyes with media opacities rendering their tumors impossible to follow . the patients in which tumors had totally regressed and therefore avoided enucleation were considered to have had a favorable outcome . ebrt was not performed as a salvage treatment due to the superior efficacy of enucleation in tumor control and the possible occurrence of secondary malignancy and disfigurement of the orbit . informed consent was obtained from every patient at the time of diagnosis and initiation of treatment and also before the beginning of each cycle of chemotherapy . the chemotherapeutic regimen consisted of a 13-cycle combination of cisplatin ( carboplatin in some patients ) , etoposide , and vincristine , which is used in the treatment of central nervous system tumors [ 8,18 - 20 ] . an ophthalmoscopic examination was done either on an outpatient basis or under general anesthesia at the beginning of each cycle according to the patient 's condition . additionally , every patient was evaluated by a pediatric oncologist regularly for possible adverse effects . the chemotherapy regimen was changed to regimen 2 if the tumor showed a minimal response to the basic regimen or if adverse effects , such as nephrotoxicity , were detected . the chemotherapy regimen was changed to regimen 3 if the treatment response continued to be minimal and if growth of the tumor or new lesions were detected despite treatment either with the basic regimen or regimen 2 . the regimen was also modified according to the patients ' general condition and if any adverse effect of a specific chemotherapeutic agent was suspected . local therapy such as thermotherapy , cryotherapy , and laser photocoagulation was added depending on the status of each tumor after each cycle of chemotherapy . thermotherapy was done in patients with tumors with a base less than 3 mm and a thickness less than 3 mm without vitreous seeds located posterior to the equator . laser photocoagulation was done in similar cases , but also in slightly larger tumors by surrounding the tumor and closing off feeding vessels . the patients were grouped according to tumor classification ( r - e classification , icr ) , therapeutic methods used , and the status of the contralateral eye . enucleation was performed in eyes with tumors that showed persistent viable appearance with no signs of regression or in eyes with newly appearing lesions after two or more cycles of treatment . enucleation was also performed in eyes with media opacities rendering their tumors impossible to follow . the patients in which tumors had totally regressed and therefore avoided enucleation were considered to have had a favorable outcome . ebrt was not performed as a salvage treatment due to the superior efficacy of enucleation in tumor control and the possible occurrence of secondary malignancy and disfigurement of the orbit . informed consent was obtained from every patient at the time of diagnosis and initiation of treatment and also before the beginning of each cycle of chemotherapy . of the 227 patients diagnosed with retinoblastoma in our institution , a total of 52 patients , 65 eyes were included in this study . overall , 34 eyes achieved total regression of the tumor and were able to avoid enucleation . the probability of ocular survival was 46.566.70% using the kaplan - meier method ( fig . treatment outcomes of patients according to the r - e classification and icr are shown in table 3 . a relatively similar number of patients were in each group of r - e classification , but the majority of the eyes included in this study were in group d according to the icr . the majority of eyes with tumors of group c or lower were excluded from the study because they were eligible for local therapy alone . the eyes with group a and b tumors that were included in this study had undergone primary chemotherapy because of a group d tumor in the contralateral eye . there was a marked difference in the treatment outcome among patients classified as r - e group i to ii and r - e group iii to v. in patients with r - e classification i and ii tumors , complete remission of the tumors was observed in 100% and 86.7% of patients , respectively . in contrast , only 33.3% , 40% , and 20% of the r - e classification iii , iv , and v group patients avoided enucleation , respectively . in icr group the local modalities of treatment used in this study included cryotherapy , thermotherapy , and laser photocoagulation . twenty eyes in 18 patients received chemotherapy only , while 28 eyes in 21 patients were treated with chemotherapy and one modality of local therapy . seventeen eyes in 13 patients were treated with chemotherapy and two modalities of local therapy . the treatment outcome of patients with unilateral and bilateral involvement is shown in table 5 . three out of 23 eyes with unilateral involvement were saved while 13 out of the 16 eyes of patients with bilateral involvement who had undergone primary enucleation of one eye were saved . eighteen of the 26 eyes in patients with bilateral involvement and no enucleation were saved . tumors in the eyes with bilateral involvement and enucleation of the more severe eye had a significantly lower r - e classification ( p=0.001 ) . complications of the therapy included mild and manageable complications such as myelosupression , neutropenic fever , alopecia , mild nephrotoxicity , and ileus . the probability of overall survival was 96.773.17% using the kaplan - meier method ( fig . there was one case of hyphema and vitreous hemorrhage and one case of cataract , which was thought to be due to local therapy , but the direct relationship remains uncertain . in both cases enucleation primary chemotherapy , when combined with local therapy , showed a probability of ocular survival of 46.56% in retinoblastomas not treatable with local therapy alone . forty - three cases ( 66.2% ) in this study were group d or e tumors , and 16 of these 43 eyes ( 37.2% ) avoided enucleation . systemic chemoreduction followed by local therapy has shown various tumor control rates up to 78 to 85% . however , when comparing the treatment outcomes of these studies , the severity of the tumors in the subjects included in the studies should be taken into consideration . these studies usually have a heterogeneous severity of tumors included , often including a fair proportion of tumors of low severity . our study included only tumors that were considered otherwise untreatable with conventional treatment methods , which renders direct comparison of these previous survival rates with our results irrational . recently , successful tumor control has even been reported with chemotherapy alone in more advanced tumors . reported results of chemotherapy ( with six cycles of vincristine , etoposide , and carboblatin ) in group d heritable retinoblastoma . successful tumor control with chemotherapy was obtained in only 2 eyes ( 11% ) , although 9 more eyes ( 50% ) underwent successful salvage treatment with ebrt . shields et al . reported results of chemoreduction with 6 cycles of vincristine , etoposide , and carboplatin with or without prophylactic low - dose ebrt for group e retinoblastoma , with a globe salvage rate of 20/42 ( 48% ) in the chemoreduction only group and 4/5 ( 80% ) in the chemoreduction and prophylactic low - dose ebrt group . our results of a globe salvage rate of 37.2% in group d or e tumors without salvage treatment with ebrt is comparable to the results of previous studies . this treatment method can be considered an effective method with minimal complications ; it is especially valuable in bilateral cases when preservation of the eye and vision is more critical , or in cases when a patient strongly refuses enucleation . there was a tendency towards a better outcome in patients who were treated with more local treatment modalities . the salvage rate was 3/23 ( 13.0% ) in unilateral cases , compared to a salvage rate of 18/26 ( 69.3% ) in bilateral cases , indicating that there was a tendency towards enucleation in unilateral cases . this may have been due to a predilection to completely eradicate the tumor when even the slightest amount of evidence of progression was present . these findings suggest that with more vigorous treatment of the tumor with all possible additional local therapeutic methods , the overall eyeball salvage rate could be even higher . also , the local treatment modalities were selected according to the individual patient 's conditions and were not standardized . however , many of these shortcomings also apply to other studies regarding the treatment of retinoblastoma . in conclusion , chemoreduction and local therapy can be considered as a first - line treatment in low grade tumors . in higher grade tumors , although enucleation is still the treatment of choice in the most severe cases with vitreous seeding or massive tumors involving over half the retina , chemotherapy combined with local therapy can also be given as a choice of treatment to parents in intermediate cases , especially in bilateral cases with one eye already requiring enucleation . further study is necessary to determine the most effective chemotherapy regimen and local therapy protocol . the tendency of a better outcome in patients treated with more modalities of local therapy requires further study .

purposeto evaluate the efficacy of primary chemotherapy combined with local therapy in the treatment of retinoblastomas not treatable with a single therapeutic method.methodswe performed a retrospective chart review of 227 patients diagnosed with retinoblastoma . sixty - five eyes in 52 patients had tumors not treatable with a single therapeutic method and received primary chemotherapy combined with local therapy as needed.resultstumor control and eye salvage was achieved in 34 of the 65 eyes ; the probability of ocular survival was 46.56% using the kaplan - meier method . forty - three of the 65 eyes were group d or e tumors , in which tumor control and eye salvage was achieved in 16 eyes . twenty eyes were treated with chemotherapy only , while 28 eyes received one additional modality of local therapy , and 17 eyes received two modalities of local therapy . of the eyes treated with chemotherapy only , tumor control was achieved in 5 eyes.conclusionsprimary chemotherapy combined with local therapy can be effective and safe in the treatment of retinoblastomas otherwise untreatable with other therapeutic methods , such as group d and e retinoblastomas . more vigorous treatment with more local therapeutic methods combined may yield even better results .

Materials and Methods Chemotherapy Local therapy Results Discussion

coal - tar pitch is an important feedstock of carbochemical origin used for the production of i.a . carbon anodes , graphite electrodes , fireproof materials , carbon - carbon composites and carbon fibres , as coking additive and as binder in many insulating and sealing materials used in construction and road building [ 1 , 2 ] . bitumens originating from coal , similarly as petroleum asphalts can be treated as colloidal - dispersive systems . micelles composed of 2 components and partly of 1 components form dispersed phase while oils ( components ) and partly components are the dispersing phase . because of the presence of carcinogenic aromatic hydrocarbons in coal - tar pitch , there is a need for its modification to obtain material safe for natural environment . research on the preparation of modified bituminous substances originating from coal , carried out for many years in the institute of chemistry of warsaw university of technology in plock has led to the elaboration of method reducing the level of carcinogenicity of coal - tar pitch . modification of coal - tar pitch with polymers influences also significantly its group composition and properties . the direction and scale of changes depend on chemical structure of the modifier and its amount . chemical structure is a factor directly determining properties of each of the components of bitumen - polymer compositions and simultaneously has an impact on the possibility to form a specific structure of a given composition , on which in turn its properties depend [ 59 ] . because of profitable properties of pitch - polymer mixtures , it would be advisable to check the possibilities to use waste polymers for the modification of the pitch . it leads to the search for new techniques and methods of evaluation of their homogenicity and application properties . one of them are microscopic studies , which play significant role in the determination of the structure and colloidal stability of bitumens and bitumen - polymer systems , including pitch - polymer compositions . in the studies of bitumen - polymer systems , fluorescence microscope is used , particularly for the evaluation of polymer - asphalt materials [ 1113 ] . however , there is no significant development in the evaluation of pitch - polymer compositions with the use of such microscope . the aim of this work was to determine the utility of fluorescence microscope for the evaluation of the structure of coal - tar pitch compositions with selected waste polymers and to study their influence on pitch properties . the raw materials used in this study were coal - tar pitch ( ctp ) and selected waste polymers : poly(ethylene terephthalate ) ( pet ) , poly(methylene methacrylate ) ( pmma ) and phenol - formaldehyde resin ( pf ) . pitch - polymer compositions containing from 10 to 50 wt% waste polymer were prepared in the conditions allowing to obtain homogeneous and stable mixtures . depending on the applied waste polymer , the components were homogenized in the temperatures from 150 to 270 c , during 0.52.5 h. the homogenization of the composition components was carried out at as low as possible temperatures ( from which it was possible to mix the components ) . idea was to eliminate of the destruction and/or degradation processes of waste polymers in coal - tar pitch during the preparation of compositions . the composition of mixtures and preparation conditions are presented in table 1.table 1preparation conditions and properties of pitch - polymer compositionscompositions(wt%)preparation of compositionssp(c)cv(wt%)ti(wt%)qi(wt%)ctp107.053.0334.217.1490 ctp + 10 pet260 c0.5 h127.053.6245.8013.3175 ctp + 25 pet166.050.1043.1029.4350 ctp + 50 pet236.038.0068.5062.7990 ctp + 10 pmma270 c1 h137.060.0835.628.7875 ctp + 25 pmma145.048.3329.369.2250 ctp + 50 pmma200.028.7923.0310.4990 ctp + 10 pf150 c2.5 h128.054.1655.1810.1475 ctp + 25 pf141.053.6763.044.4850 ctp + 50 pf*54.0788.544.21*infusible composition preparation conditions and properties of pitch - polymer compositions * infusible composition for coal - tar pitch and pitch - polymer compositions , the following measurements were carried out : softening point by ring and ball method ( sp ) according to the pn - en 1427:2009 standard , coking value ( cv ) according to the pn - c-97093:1993 standard , content of components insoluble in quinoline ( qi ) according to the pn - c-97058:1999 standard andcontent of components insoluble in toluene ( ti ) according to the method elaborated in the institute of chemistry , warsaw university of technology in plock . softening point by ring and ball method ( sp ) according to the pn - en 1427:2009 standard , coking value ( cv ) according to the pn - c-97093:1993 standard , content of components insoluble in quinoline ( qi ) according to the pn - c-97058:1999 standard and content of components insoluble in toluene ( ti ) according to the method elaborated in the institute of chemistry , warsaw university of technology in plock . the colloidal structure of pitch - polymer compositions by olympus bx41 microscope was carried out . in the study of colloidal structure of pitch - polymer compositions , in ultraviolet light , press moulding of compositions on the microscopic slide by a hydraulic press was on adopted technique of preparing of samples for study . parameters of press moulding of samples : pressure from 2.5 to 3.5 mpa , time of press moulding 5 min , temperature of press moulding from 130 to 190 c . selected results of measurements of physicochemical properties of coal - tar pitch and pitch - poly(ethylene terephthalate ) compositions are presented in table 1 . the addition of waste pet significantly influenced the softening point of ctp . with the increase of pet in the compositions , the softening point increased . for the composition containing 50 wt% of pet , the softening point compared to the unmodified pitch increased by 129 c . with the increase of waste pet in the compositions , the cooking value decreased and so the yield of residue after high - temperature carbonization process . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 15 wt% compared to coal - tar pitch . the content of ti component in pitch - pet compositions was 4569 wt% and qi components was 1363 wt% . with the increase of waste in the compositions , the content of ti and qi components increased . in fig . 1 , microscopic images of pitch - pet compositions obtained using fluorescence microscope are presented . on obtained microscopic images , similarly as in the case of petroleum bitumens , carbon bitumen phase is observed in black colour.fig . 1microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet structures of compositions containing 25 wt% pet , had uniform level of dispersion , and dispersed polymer particles had round and regular shapes . the increase of waste pet amount in compositions caused the increase of the amount and size of dispersed particles . for the composition containing 50 wt% of waste poly(ethylene terephthalate ) agglomerates of irregular shape could be observed , which were formed from aggregates containing polymer macromolecules and probably components of coal - tar pitch . it has to be presumed that with the increase of waste pet amount in compositions , the development of micellar areas occurred due to strong physical interactions between the components , which is indicated by i.a . , high softening temperatures . selected results of measurements of physicochemical properties of pitch - poly(methylene methacrylate ) compositions are presented in table 1 . addition of waste pmma influenced the change of physicochemical properties of coal - tar pitch . for the compositions containing 10 or 25 wt% of waste pmma , the softening point compared to the unmodified pitch increased by 30 and 38 c , respectively . the highest softening point was observed for the compositions containing 50 wt% of waste pmma . addition of waste pmma into ctp caused the decrease of coking value and so the yield of residue after high - temperature carbonization process . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher coking value than ctp ( by 7.05 wt% ) . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 24.20 wt% compared to coal - tar pitch . increase of waste pmma addition into ctp caused the decrease of content of ti components and increase of content of qi components . the pitch - pmma compositions had lower content of ti components and had higher content of qi components compared to coal - tar pitch . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher content of ti components by 1.41 wt% compared to ctp . in fig . 2 , microscopic images of pitch - pmma compositions obtained using fluorescence microscope are presented.fig . 2microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma structures of pitch - waste pmma compositions containing up to 25 wt% of additive assumed the shapes of round particles of different shapes , chaotically dispersed in continuous phase of the bitumen . with the increase of waste pmma in compositions increased the amount and size of dispersed aggregates of irregular shapes , composed of macromolecules of a polymer and probably of components of coal - tar pitch . for the composition containing 50 wt% of waste pmma , phase inversion was observed in the dispersion . selected results of measurements of physicochemical properties of pitch - phenol - formaldehyde resin compositions softening point increased with increasing content of waste pf . in the case of compositions containing 50 wt% of this component , the measurements of softening point was not possible because of the impossibility to melt them . for the compositions containing pf , independently from the amount of waste , coking values were similar to the value of coal - tar pitch . addition of waste pf into ctp caused changes of group composition of the bitumen . with the increase of waste pf in the compositions , the content of ti components increased . for the composition containing 50 wt% of waste pf , the content of ti components increased by 54.33 wt% compared to the unmodified pitch . the content of qi components in pitch - pf compositions was 4.24.5 wt% and was lower than ctp . an exemption from this rule was the composition containing 10 wt% of waste pf , which had higher content of qi components compared to coal - tar pitch ( by 3 wt% ) . in fig . 3 , structures of compositions containing 25 wt% of pf , similarly as in the case of compositions containing pet in the amount of 25 wt% , exhibited uniform level of dispersion , and dispersed particles had round shape . further increase of the amount of phenol - formaldehyde resin in compositions up to 50 wt% caused formation of agglomerates of irregular shapes , being assemblies of aggregates composed of polymer macromolecules and probably of components of coal - tar pitch ( fig . 3microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf selected results of measurements of physicochemical properties of coal - tar pitch and pitch - poly(ethylene terephthalate ) compositions are presented in table 1 . the addition of waste pet significantly influenced the softening point of ctp . with the increase of pet in the compositions , the softening point increased . for the composition containing 50 wt% of pet , the softening point compared to the unmodified pitch increased by 129 c . with the increase of waste pet in the compositions , the cooking value decreased and so the yield of residue after high - temperature carbonization process . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 15 wt% compared to coal - tar pitch . the content of ti component in pitch - pet compositions was 4569 wt% and qi components was 1363 wt% . with the increase of waste in the compositions , the content of ti and qi components increased . in fig . 1 , microscopic images of pitch - pet compositions obtained using fluorescence microscope are presented . on obtained microscopic images , similarly as in the case of petroleum bitumens , carbon bitumen phase is observed in black colour.fig . 1microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet structures of compositions containing 25 wt% pet , had uniform level of dispersion , and dispersed polymer particles had round and regular shapes . the increase of waste pet amount in compositions caused the increase of the amount and size of dispersed particles . for the composition containing 50 wt% of waste poly(ethylene terephthalate ) agglomerates of irregular shape could be observed , which were formed from aggregates containing polymer macromolecules and probably components of coal - tar pitch . it has to be presumed that with the increase of waste pet amount in compositions , the development of micellar areas occurred due to strong physical interactions between the components , which is indicated by i.a . selected results of measurements of physicochemical properties of pitch - poly(methylene methacrylate ) compositions are presented in table 1 . addition of waste pmma influenced the change of physicochemical properties of coal - tar pitch . for the compositions containing 10 or 25 wt% of waste pmma , the softening point compared to the unmodified pitch increased by 30 and 38 c , respectively . the highest softening point was observed for the compositions containing 50 wt% of waste pmma . addition of waste pmma into ctp caused the decrease of coking value and so the yield of residue after high - temperature carbonization process . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher coking value than ctp ( by 7.05 wt% ) . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 24.20 wt% compared to coal - tar pitch . increase of waste pmma addition into ctp caused the decrease of content of ti components and increase of content of qi components . the pitch - pmma compositions had lower content of ti components and had higher content of qi components compared to coal - tar pitch . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher content of ti components by 1.41 wt% compared to ctp . in fig . 2 , microscopic images of pitch - pmma compositions obtained using fluorescence microscope are presented.fig . 2microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma structures of pitch - waste pmma compositions containing up to 25 wt% of additive assumed the shapes of round particles of different shapes , chaotically dispersed in continuous phase of the bitumen . with the increase of waste pmma in compositions increased the amount and size of dispersed aggregates of irregular shapes , composed of macromolecules of a polymer and probably of components of coal - tar pitch . for the composition containing 50 wt% of waste pmma , phase inversion was observed in the dispersion . selected results of measurements of physicochemical properties of pitch - phenol - formaldehyde resin compositions are presented in table 1 . softening point increased with increasing content of waste pf . in the case of compositions containing 50 wt% of this component , the measurements of softening point was not possible because of the impossibility to melt them . for the compositions containing pf , independently from the amount of waste , coking values were similar to the value of coal - tar pitch . addition of waste pf into ctp caused changes of group composition of the bitumen . with the increase of waste pf in the compositions , the content of ti components increased . for the composition containing 50 wt% of waste pf , the content of ti components increased by 54.33 wt% compared to the unmodified pitch . the content of qi components in pitch - pf compositions was 4.24.5 wt% and was lower than ctp . an exemption from this rule was the composition containing 10 wt% of waste pf , which had higher content of qi components compared to coal - tar pitch ( by 3 wt% ) . in fig . 3 , structures of compositions containing 25 wt% of pf , similarly as in the case of compositions containing pet in the amount of 25 wt% , exhibited uniform level of dispersion , and dispersed particles had round shape . further increase of the amount of phenol - formaldehyde resin in compositions up to 50 wt% caused formation of agglomerates of irregular shapes , being assemblies of aggregates composed of polymer macromolecules and probably of components of coal - tar pitch ( fig . 3microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf the application of fluorescence microscope for the evaluation of colloidal structure of pitch - polymer compositions can be a valuable supplement to other methods used to evaluate the homogenicity and level of dispersion of a polymer in bitumen - polymer compositions , and also it can allow to explain the phenomena occurring during processes of modification of coal - originated bitumens with macromolecular compounds . the changes of physicochemical properties of coal - tar pitch depended on the type and amount of applied waste polymer . addition of waste pet into ctp caused increase of softening point and the amount of components insoluble in toluene and quinoline , while coking value in pitch - pet compositions decreased . addition of waste pmma into ctp caused increase softening point and the amount of components insoluble in quinoline , decrease coking value and the amount of components insoluble in toluene . addition of waste pf into ctp caused increase softening point and the amount of components insoluble in toluene , decrease the amount of components insoluble in quinoline and caused slightly changes of coking value . pitch - polymer compositions can be treated as microheterogeneous systems , colloidal and biphase , generally exhibiting uniform dispersion of particles composed of polymer macromolecules and probably of components of coal - tar pitch . the size of dispersed particles depends on the type and amount of waste polymer added to the coal - tar pitch . observed microscopic images allowed to point out the following dependences between the properties of pitch - polymer compositions and their structure : for pitch - pet compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti and qi components and reduction of coking value , for compositions containing waste pmma , the increase of the size of dispersed aggregates caused the increase of softening point and content of qi components , reduction of coking value and content of ti components andfor pitch - pf compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti components and decrease content of qi components , while coking value changed insignificantly . for pitch - pet compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti and qi components and reduction of coking value , for compositions containing waste pmma , the increase of the size of dispersed aggregates caused the increase of softening point and content of qi components , reduction of coking value and content of ti components and for pitch - pf compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti components and decrease content of qi components , while coking value changed insignificantly .

in this work , the results of studies on the evaluation of colloidal structure of coal - tar pitch compositions with selected waste polymers by fluorescence microscope . for pitch - polymer compositions containing 1050 wt% waste polymer , softening point , coking value and content of components insoluble in toluene and quinoline were carried out . the results indicate that pitch - polymer compositions can be treated as microheterogeneous systems , colloidal and biphase , generally exhibiting uniform dispersion of particles composed of polymer macromolecules and probably of components of coal - tar pitch .

Introduction Experimental Results and discussion Pitch-poly(ethylene terephthalate) compositions Pitch-poly(methylene methacrylate) compositions Pitch-phenol-formaldehyde resin compositions Conclusions

current techniques in acl surgery have been associated with satisfactory long - term results in the majority of patients . however , there remains a considerable subset , up to 30% of patients , with unsatisfactory outcomes [ 2 , 7 ] . specifically , patients report problems relating to rotational instability and return to previous level of activity [ 3 , 10 ] . it has been suggested that a more anatomical approach to restore the original acl anatomy may benefit these patients [ 4 , 17 ] . some authors have advocated placing a single graft in a position closer to the oblique femoral attachment of the acl [ 14 , 15 ] . however , it is not possible to fully restore normal knee kinematics with a single graft , regardless of the position [ 13 , 16 ] . the double bundle reconstruction technique ( dbt ) for acl reconstruction aims at restoring the acl anatomy with its two bundles and is gaining popularity [ 11 , 17 ] . anatomic studies have demonstrated the presence of two functional bundles within the acl , the anteromedial ( am ) and the posterolateral ( pl ) bundle [ 1 , 8 ] . although it is somewhat of a simplification , the double bundle description of the acl is generally accepted as an anatomic model for understanding the complex structure and function of the ligament [ 6 , 9 ] . the am bundle often obscures the pl bundle , and it may appear that only one bundle is present without careful inspection . the goal of this study was to describe the presence of the double bundle structure from an arthroscopic point of view , and to evaluate the value of different portals in knee arthroscopy . we prospectively examined 60 knees during standard arthroscopy . in each knee , the double bundle acl structure was evaluated , along with the usefulnes of different portals for visualization . all knees that were included in the study had a previous x - ray and mri in order to rule out any significant changes to the bone , and to ensure that the acl was intact . all patients were less than 60 years and had no history of acl injury . surgical indications for the 60 total subjects examined included 31 cases treated for meniscal findings alone , 21 treated for articular cartilage findings alone , and 8 cases of a combined repair of meniscus and cartilage . there were 25 female and 35 male knees , 29 right knees , and 31 left knees included in the study . age distribution ranged between 16 and 60 , with an average age of 40.2 years . arthroscopy started with an anterolateral portal ( alp ) located just lateral to the patellar tendon using the inferior pole of the patella as a vertical landmark , and an anteromedial portal ( amp ) approximately 0.5 cm medial to the edge of the patella tendon , 1 cm superior to the joint line , and 1 cm inferior to the tip of the patella . for each knee , the acl anatomy and the visibility of the pl bundle through the alp and amp were evaluated with and without retraction of the am bundle according to the description of the two bundles by girgis et al . and arnoczky . gross biomechanics of the two bundles using a probe were also assessed . for statistical analysis with the arthroscope in the alp , we were able to distinguish the am and pl bundle in 17 cases ( 28.3% ) ( fig . 1 ) . in the remaining cases ( n = 43 , 71.7% ) , the pl bundle was obscured by the am bundle , and visualization was only possible with retraction of the am bundle with a probe ( fig . 2 ) . switching the arthroscope to the amp , differentiation of the am and pl bundle without using a probe to retract am was possible in 40 cases ( 66.7% ) ( fig . 3 ) . in the remaining cases ( n = 20 , 33.3% ) , visualization of the pl bundle was possible only after retraction of the am bundle ( table 1 ) . there was a statistically significant better visibility of the pl bundle using the amp ( p < 0.05).fig . 2anterolateral portal : pl bundle with retraction of am bundle , pl bundle loose with knee flexionfig . bundlepl bundleanterolateral portaln = 17 ( 28.3%)anteromedial portaln = 40 ( 66.7% ) * * p < 0.05 anterolateral portal : pl bundle without retraction of am bundle anterolateral portal : pl bundle with retraction of am bundle , pl bundle loose with knee flexion anteromedial portal : pl bundle without retraction of am bundle visualization of the pl bundle the femoral insertion site of the acl was semilunar at the inner surface of the lateral condyle . the centre of the pl bundle visualized more shallow than the centre of the am bundle with the knee held in 90 flexion , while both insertion sites were oriented horizontally ( fig . 4 ) . the pl bundle insertion was located at the posterolateral aspect of the tibial attachment with a close approximation of the pl bundle to the posterior root of the lateral meniscus . gross assessment using a probe while applying a flexion - extension motion pattern to the knee demonstrated a taut am bundle throughout the range of motion and a tightening of the pl bundle with knee - extension ( figs . 2 , 5 ) . pl bundle tight with knee extension anteromedial portal : pl bundle without retraction of am bundle anteromedial portal : pl bundle tight with knee extension the principal finding of the present study was that it is possible to distinguish the double bundle structure in acl anatomy as described by girgis et al . and arnoczky by applying a two portal knee arthroscopy technique . the visualization of the acl anatomy seems to be improved through the use of a medial portal . the anteromedial portal not only helps in visualization of the double bundle structure but also in distinguishing the am and pl portions of the femoral attachment sites . consistent with the literature , we found the pl bundle tightening when the knee is extended [ 6 , 12 ] . this study is limited by the fact that we observed only acl intact knees and did not consider cases with a torn acl , where the anatomic position of the attachment sites is sometimes obscured by the knee injury and bony changes that occur between injury and reconstruction . therefore , we stress the need for further arthroscopic evaluation of different portals in acl reconstruction . the clinical relevance of this study is that it can be beneficial to establish an additional medial working portal . the amp can be used for the arthroscope and a better visualization of the femoral insertion site of the acl at the inner surface of the lateral condyle and a precise femoral tunnel placement can be achieved . reported the use of an additional medial portal without any increase in morbidity to patients for acl surgery .

in order to describe the arthroscopic presence of the double bundle structure and to evaluate the value of different portals in knee arthroscopy , we assessed the am and pl bundle anatomy . we prospectively examined the knees of 60 patients undergoing arthroscopic surgery for pathology unrelated to the acl . arthroscopy was performed in a two portal technique using an anterolateral ( alp ) and an anteromedial ( amp ) portal . with the arthroscope in the alp , we could distinguish an am and pl bundle in 28% . switching the arthroscope to the amp , differentiation of the bundles was possible in 67% . in all remaining cases visualization of the pl bundle was possible after retraction of the am bundle . use of amp increased visualization of the pl bundle . it seems reasonable to perform arthroscopy for acl reconstruction with the arthroscope in the amp and to establish an additional medial working portal to increase the visualization of the femoral acl insertion sites for optimal femoral tunnel placement .

Introduction Materials and methods Results Discussion Conclusion

many stroke patients with stroke have a balance problems while standing because they tend to put their weight on their non - paralyzed lower limb to increase swing of their upper body1 . falling accidents due to such decline in balance ability is on the increase annually . even when there is no injury from falling , frequent falling can sufficiently disrupt physical and social activities2 . because deterioration of muscular strength and balance ability increases the risk of falling , it is significantly important to perform exercises to promote upper limb muscular strength and training for balance3 . regular exercise of walking and for muscular strength and balance may increase muscular strength and balance to prevent falling of patients4 . walking , in particular , is known to be a safe type of aerobic exercise for patients , having the merit that it can be performed gradually by considering appropriate intensity , frequency , period , and phases of exercise for abilities of each patient5 . recently efficiency of walking training using a treadmill has attracted more attention as when such training has been reported to have better effects than walking on even ground . some researchers reported that walking on a treadmill is effective in improving balance and walking ability6 , 7 . with recent developments of relevant computer programs , new methods of rehabilitation exercise such exercises can increase interest and participation of patients in treatment and improve their significant functions by enabling them to perform various forms of tasks appropriate for goals of individual patients in virtual reality . virtual reality indicates a type of interactive simulation using computer hardware and software , in which users can have close - to - reality experiences8 . virtual reality training using visual feedback enables the participants to undergo the training while seeing their movements with their own eyes , a feature that helps them adjust their inaccurate body center caused by body image damage suffered by most stroke patients . in addition , training serves as a catalyst in active participation and performance of tasks by inducing interest and pleasure , providing immediately visual feedback on the result to enhance motor education ability9 . many recent researches of virtual reality games with higher accessibility reported that such games showed significant effects on voluntary control and coordination of children with cerebral palsy and increases in the motor ability of patients with parkinson disease10 . those types of games were also effective on recovery of upper limb functions and induced significant improvement in balance and walking of stroke patients , showing that the games can be used as an effective method of intervention11 , 12 . as a means of overcoming limitations of the existing intervention methods , virtual reality shows an availability it has to being applied in the rehabilitation of stroke patients . however , devices of virtual reality in previous studies are so large that they are difficult to be used in general social institutes or homes in terms of scale and cost . furthermore , they are targeted to a small number of patients . in this context , the purpose of this study was to perform treadmill exercise and virtual reality training exercise using nintendo wii , a device that can be easily used without regard to place or facility , in order to investigate the effects on balance and walking of stroke patients . all of the subjects were sufficiently explained of this study and voluntarily consented to their participation in the experiment . this study was approved by the research agency , and all participants provided written informed consent . those who could perform communication , comply with instructions in this study , perform balancing and walking independently , had no pain limiting execution of exercise , and had no disability in sight , hearing , and the vestibular organs were selected to be the subjects of this study . the forty stroke patients were randomly divided into two exercise program groups with one group using virtual reality training ( n=20 ) and another group using a walking exercise program using a treadmill ( n=20 ) . age for the virtual reality group , the age was 62.2 7.2 years old , the height was 162.8 6.7 cm , the weight was 58.1 5.4 kg , and the onset period was 30.4 5.4 months . age for the treadmill group , the age was 63.2 5.4 years old , the height was 164.7 7.5 cm , the weight was 60.2 4.5 kg , and the onset period was 31.6 7.4 months . the virtual reality group underwent training using the wii - fit software by nintendo , japan . when playing the nintento wii , the participants used wireless controllers to interact with the avatars on the screen via the virtual reality motion detection system . the controllers were attached by acceleration detecting sensors responding to changes in direction and speed . due to the detector being installed on the television , the screen showed the movements of the controller just as the player performed the movements13 . the virtual reality system used in this study was the wii board balance system , in which users played games while the balancing board sensed weight shift and distribution . the subjects of the virtual reality group performed yoga , muscular strength exercise , aerobic exercise , and balancing exercise , all for 10 minutes each for a total of 40 minutes . the treadmill group used low - speed treadmills for patients to directly control their speed based on their walking ability while walking for 40 minutes . the treadmills were equipped with handles on the front and on both sides , which the users could grip when they lost balance walking . we used a pedoscan ( rsscan 1 m , germany ) to measure balance of the subjects . the device consisted of 4,096 sensors for measuring pressures on both feet in static standing , computerizing left / right weight - bearing and anterior / posterior weight - bearing by separating each area . the patients were asked to stand up straight on the device without any movement for 30 seconds . the device was attached to the dominant foot of a subject , measuring the anterior and posterior pressures of the foot by the pressure detecting sensors on its insole . the insole between the foot and the board sensed the vertical reaction force to measure the stance phase , swing phase , cadence of the dominant foot . the measured signals were temporarily saved by the portable compact controller attached to the ankle by receiving the walking data of the subject and then were computerized and calculated . before the walking exercise , the smart step insoles were inserted in the shoes and were injected with air to maintain appropriate pressure . after putting on the shoes and being attached to the controller on their ankles , the subjects walked along a straight footpath for 10 meters , looking straight ahead . a paired t - test was used for before and after comparisons , and an independent t - test was used for a between group comparison with a significance level of =0.05 . for balance , there were significant differences in both the virtual reality group and the treadmill group after completing of the exercise program . for walking , the virtual reality group showed significant differences after completing of the exercise program . significant differences between the groups were shown in balance after completing of the exercise program ( table 1table 1.the comparison of balance and gait in virtual reality and treadmillperiodvirtual reality group ( n=20 ) treadmill group ( n=20)m sdm sdleft / right weight - bearing ( % ) pre17.1 5.516.9 6.5post10.6 4.8**13.1 5.8*anterior / posterior weight - bearing ( % ) pre15.4 6.715.7 4.6post10.3 4.7**12.9 5.1*affected side stance phase ( % ) pre53.7 6.454.2 7.6post57.5 5.7 * 55.3 6.4affected side swing phase ( % ) pre46.2 6.445.7 7.6post42.4 5.7 * 44.6 6.4cadence ( steps / min)pre44.2 5.443.7 6.4post48.1 6.1 * 45.2 6.1*paired t - test , independent t - test , * p<0.05 , * * p<0.01 ) . * paired t - test , independent t - test , * p<0.05 , * * p<0.01 falling can occur in all age groups , but older people and patients show higher frequency of falling . planned and consistent exercise is needed to prevent falling and strengthen muscles , and exercises promoting weight bearing , resistance , and aerobic process are known to be effective for such purposes14 . exercises for lower limb muscular strength and balancing exercises are effective for the prevention of falling and improvement of stamina of patients , but such exercises have some demerits because the content is simple and thus boring . to supplement the demerits , new methods of exercise owing to development of scientific techniques have been recently introduced where patients can perform various tasks in virtual reality15 , 16 . therapeutic intervention via virtual reality is more advantageous than other intervention methods in that it can provide relatively easy environmental control , availability in selecting environments , training based on patients abilities via phase - in task difficulty , rapid and precise feedback on performing tasks , and self - learning in safe environment17,18,19 . we applied an eight - week exercise program to stroke patients who were divided into a virtual reality exercise group and a treadmill walking group , in order to investigate effects of each exercise on balance and walking . the results of measuring balance and walking effects as the purpose of the virtual reality exercise program showed that significant differences were shown in all the domains of the left / right and anterior / posterior weight - bearing in balancing and the stance phase , the swing phase , and the cadence in walking . for the treadmill group , significant differences were displayed in the left / right and anterior / posterior weight - bearing in balancing . although the walking exercise program using a treadmill showed significant differences in balancing ability , the exercise program using virtual reality training displayed significant differences in balancing and walking because the patients could directly adjust their weight center and shift due to visual feedback . in addition , various similar programs such as jogging may further influence their balancing and walking . the results of this study suggest that virtual reality training may be appropriate to provide planned and consistent exercises for stroke patients who need improved balance and walking ability by giving visual feedback to induce their interest . meanwhile , the results of this study are consistent with those reporting increases in left / right , anterior / posterior stability of stroke patients who performed of virtual reality training , or reporting significant improvement in the berg balance test for the elderly who underwent a training using virtual reality games20 . in addition , there were researches in which significant differences were observed in balancing ability of the elderly with their eyes open after they underwent a virtual reality exercise program , or in which significant differences were found in balancing ability of the elderly after an eight - week virtual reality exercise program . meanwhile , the increase in walking ability shown in this study was consistent with the results of studies in which electrotherapy based on virtual reality significantly increased walking speed of stroke patients , or in which exercise using visual feedback was effective on the walking ability of stroke patients8 , 14 , 21 . in this study also , virtual reality training was effective in producing positive improvements in balance and walking of stroke patients . however , there remains insufficient research related to virtual reality because the technology has not been fully commercialized yet . further studies are needed to deal with virtual reality for many patients to use it in performing various indoor exercise programs and developing their abilities . the results of this study may indicate that virtual reality training providing visual feedback enables stroke patients to directly adjust their wrong weight center and shift visually , and that it is appropriate for the patients who need improved balance and walking ability by inducing their interest for them to perform planned exercise on a consistent basis .

[ purpose ] the purpose of this study is to investigate the effects of virtual reality training using nintendo wii on balance and walking for stroke patients . [ subjects and methods ] forty stroke patients with stroke were randomly divided into two exercise program groups : virtual reality training ( n=20 ) and treadmill ( n=20 ) . the subjects underwent their 40-minute exercise program three times a week for eight weeks . their balance and walking were measured before and after the complete program . we measured the left / right weight - bearing and the anterior / posterior weight - bearing for balance , as well as stance phase , swing phase , and cadence for walking . [ results ] for balance , both groups showed significant differences in the left / right and anterior / posterior weight - bearing , with significant post - program differences between the groups . for walking , there were significant differences in the stance phase , swing phase , and cadence of the virtual reality training group . [ conclusion ] the results of this study suggest that virtual reality training providing visual feedback may enable stroke patients to directly adjust their incorrect weight center and shift visually . virtual reality training may be appropriate for patients who need improved balance and walking ability by inducing their interest for them to perform planned exercises on a consistent basis .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

since the introduction of antiretroviral therapy in 1996 , the clinical management of hiv infection revolves around a complex assemblage of potent pharmacological molecules that inhibit viral replication . the goal of this lifelong treatment regimen is to achieve ( and maintain ) viral suppression and to reach a threshold where copies of the virus are no longer detectable in the blood the lowest level of detection generally considered 4075 copies / ml ( hhs 2011 ) . an undetectable plasma viral load is widely interpreted as the marker of individual therapeutic success and is also used extensively to conceptualize health in the context of hiv ( persson et al . the person living with hiv who is able to achieve this kind of viral suppression through strict adherence can be understood as a good patient and deserving of the ongoing investment in their health , irrespective of the complexity of their lives and the numerous barriers to adherence many individuals face ( remien and mellins 2007 ) . however , in recent years , plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively ( terzian et al . in particular need of critical appraisal is the development of techniques to measure , map and monitor community viral load. this new epidemiological technology blurs the traditional boundaries of clinical care and public health work , thus raising questions about its implications . for the purpose of this article , we argue that community viral load should be examined in relation to the theoretical framework of foucault ( 1984 ) coined this concept to account for the ways through which capillary forms of power insert themselves into actions , attitudes , and discourses to produce a particular kind of person and population - productive , but docile and easily governed . miller and rose ( 2008 , p. 63 ) describe governmentality as a domain of strategies , techniques and procedures through which different forces seek to render programmes operable , and by means of which a multitude of connections are established between the aspirations of authorities and the activities of individuals and groups. these tactics , in their programmatic form , act directly and indirectly on the population ( and individuals who compose it ) to improve its conditions , welfare , wealth , longevity , health and so on ( foucault 1991 ) . these tactics become inseparable from a knowledge and understanding of processes related to population ( foucault 1991 ) . this form of governance is particularly concerned with processes that modulate health collectively rather than individually ; and this is why these processes have to be understood and managed using a broad range of tactics ( foucault 1991 ) . to govern , therefore , implies an ideological shift from the isolated person to the existing relations that are known to interfere with the production of a healthy population : relations between people and people , people and things , people and events , [ people and spaces] ( rose et al . these relations can then be administered directly or indirectly through tactics that make possible the optimization of health and so forth ( foucault 1991 ) . based on the work of foucault ( 1991 , 2003 ) , this article critically examines the use of community viral load as a new strategy of governmentality . also drawing on the work of miller and rose ( 2008 ) , this article further reconceptualizes specifically , it explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific micro - locales , and the government ( in the foucauldian sense ) of specific bodies which are seen as it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america . 2 ) describe community viral load as an aggregate biological measure of viral load for a particular geographic location for example the city of san francisco or a particular neighborhood and for a particular group of people who share socio - demographic characteristics. in order to determine the community viral load of any particular group or area of interest , one must take into account two distinct measures ; mean community viral load and total community viral load ( das et al . mean community viral load is the average of the most recent viral load of all reported hiv - positive persons in a particular target(ed ) population or geographical area ( das et al . total community viral load is the sum of the most recent viral loads of all reported hiv - positive persons in a particular target(ed ) population or geographical area ( das et al . these measurements make it possible to determine the mean average viral burden and the absolute level of the virus in any given population or geographical area . community viral load is a relatively new concept but has gained significant momentum in the field of hiv in recent years . this is due in part to the implementation of seek , test and treat ( now seek , test , treat and retain ) initiatives which rely on the premise that scaling up treatment collectively will result in a dramatic reduction of hiv transmission ( montaner et al . community viral load is considered by many researchers and clinicians to be a valuable marker of the direct relationship between hiv concentration ( in specific populations and geographical areas ) and overall hiv incidence ( wood et al . , community viral load has become an important concept under which to advocate for the expansion of testing and surveillance in the community and , most importantly , the reconceptualization of treatment as a prevention tool ( montaner et al . 2010 ) . recent studies in canada and the united states have demonstrated the value of measuring and mapping hiv viral load at the community level . in vancouver and across the province of british columbia ( canada ) , community viral load is increasingly being used as an epidemiological surveillance tool and an evaluation tool to monitor the outcomes of initiatives for the expansion of testing and treatment coverage ( montaner et al . 2010 ) . building on research findings , which suggest that community viral load is correlated with hiv incidence ( wood et al . 2009 ) , these initiatives were implemented across the province ( and more aggressively in the downtown eastside of vancouver ) and have led to a progressive decline in community viral load ( montaner et al . in some postal code areas of the province , however , the proportions of potentially infectious individuals ( as stated by the authors ) remain high regardless of the expansion of testing and treatment coverage ( lourenco et al . 2012 ) . similar findings have been reported in san francisco ( california , united states ) following the intensification of surveillance , the expansion of testing and earlier initiation of treatment ( das et al . 2011 ) . in addition to a documented decline in community viral load and new reported cases of hiv , the department of public health was also able to map areas with the highest concentration of viral load and further target them to produce improved outcomes ( das et al . areas such as tenderloin , south market , mission and castro were subsequently identified as hotspots ( as stated by the authors ) or areas with particularly high risk of hiv transmission due to the distribution of community viral load ( das et al . it was , therefore , recommended that more community - level interventions be implemented in these areas and that more aggressive prevention efforts ( including the use of treatment as a prevention tool ) be deployed to reduce community viral load . a similar mapping exercise was conducted in washington ( district of columbia , united states ) over a period of 4 years ( castel et al . ( 2012 ) included the most recent viral load data in the city , two indicators of socioeconomic status ( income and education ) , and race . their data were statistically analyzed and presented on various maps of the city of washington . these maps showed that the neighbourhoods with the worst socioeconomic indicators also had the highest hiv / aids prevalence rates ( castel et al . ( 2012 ) , can inform surveillance and the implementation of interventions that target populations with the highest viral load burden . it was determined that community viral load and its geospatial distribution are particularly useful to inform targeted public health interventions ( castel et al . similar arguments are presented by researchers who conducted studies on community viral load in new york city ( new york , united states ) from 2005 to 2009 ( laraque et al . based on their findings , they suggest that community viral load is valuable to public health agencies because it has the potential to identify high risk groups and target interventions to the groups whose viral control if achieved will most likely result in a rapid lowering of community viral load ( terzian et al . furthermore , it has the potential to identify groups at risk for sustained high viral load such as bronx residents who , according to terzian et al . ( 2012 ) , are more likely to have a detectable viral load and suffer from hiv - related disparities . identifying groups who are at risk or known to have sustained high viral load can thus provide an opportunity for the implementation of outreach initiatives in the community and the scale - up of efforts to educate , test , treat and engage members of the community in hiv care ( terzian et al . the inner workings of community viral load have not been critically examined in the hiv literature . the aim of this article is to interrogate the use of community viral load through a deductive process using theory to analyse empirics . theory makes it possible to engage in a critical analysis of a relatively new tool in the field of hiv and a limited body of empirical literature . it also allows us to disrupt normalizing discourses in the field of hiv and challenge the ways in which community viral load has been introduced as seemingly benign . this is particularly important considering that community viral load is now becoming normalized , standardized , and deployed in various public health agencies , without its assumptions and claims having been interrogated . in our view , it is imperative that researchers engage in discussions and debates around the use of community viral load and how it is intrinsically linked to the logics of governmentality . for this reason , we argue that a theory - based analysis of community viral load is of great value to these discussions and debates . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . these spaces are calculated , reconfigured , and imagined , with new levels of sophistication . first , community viral load signals an important shift in the way viral load is seen , imagined and represented both visually and spatially . specifically , we draw upon philo 's ( 2000 ) reading of foucault to expose three distinctive but interconnected representations of viral load . the first representation has to do with the tabulation of laboratory results that can attest to the progression of hiv and the response to treatment at the individual level . it draws a picture of how viral replication actually appears at every stage of hiv infection and what response is to be expected once treatment is initiated . here , tables and graphics of viral load patterns provide a reference point to assess individuals in the clinical setting and systematically position each of them accordingly in relation to an ideal patient with optimal viral suppression . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . finally , the third representation allows for hiv to be located in space through mapping techniques . the location of the virus in space is compatible with foucault 's analysis of the shift from leper colony to plague city , wherein the risk of transmission is already present and requires the segmentation of space ( foucault 1990 ) . foucault explicitly linked mapping and government , and the need to have a rational plan to manage space ( crampton 2007 , p. 224 ) . in fact , he identified the ordering of space as central to the strategy for containment of biological risks and the government of processes that modulate health collectively rather than individually ( foucault 1990 , legg 2005 ) . these particular kinds of spaces take the form of specific neighbourhoods and locations where hiv is highly concentrated often located in geographical areas marked by high levels of marginalization , poverty , oppression and social exclusion . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . huxley ( 2006 ) explains that in order to better appreciate the articulation of spatial rationalities in the fabrication of governable spaces , we must pay close attention to the way authorities imagine these spaces and make them amenable to regulation . expanding on foucault 's work , huxley ( 2006 ) identifies three spatial rationalities : dispositional , generative and vitalist . for the purpose of this article , we will only refer to the dispositional and generative spatial rationalities . a dispositional rationality has to do with the production of boundaries and the spatial disposition of bodies ( huxley 2006 ) . it operates through the logics of quadrillage ( a term coined by foucault ) to prevent the spread of diseases and ensure a more effective regulation of processes that are responsible for epidemics ( huxley 2006 ) . in other words , it allows for the segmentation of space and the management of bodies to achieve a given end . entails systems or surveillance , as these are seen as essential to enforcing the ordering of spaces and bodies. generative rationality , on the other hand , concerns the effects of space on contagion , health , disease and death ( huxley 2006 ) . here , the problem is not so much the disposition of spaces but the concentration of diseased bodies in geographical areas ( huxley 2006 ) . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . with this in mind , biomarkers become an important tool to circumscribe infectiousness and transmissibility ( or the risk thereof ) geographically . the emphasis is no longer on the disposition of diseased bodies in space but rather on areas where disease is highly concentrated and where the risk of transmission is intensified ( huxley 2006 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . we make a point here of referring to the virus because it is the real focus of this geographical ( and political ) undertaking even though we acknowledge that it reduces the bodies of people living with hiv to vectors of disease ( brown 1995 ) . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . what it does , however , is provide the necessary arguments to intensify surveillance , testing and prevention efforts in areas where we can find a number of people living with hiv whose viral load is unsuppressed. while our objective is not to portray people living with hiv as vectors of disease , we do have to stay true to the imaginations and rationales of those who see in community viral load a promising way to circumscribe infectiousness and transmissibility geographically . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as risky , dangerous , and in need of attention ( lupton 1995 ) . not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. routine testing in institutions located in targeted areas , point of care testing in certain neighbourhoods , treatment as prevention initiatives in specific locations , and so forth . we shall now examine how community viral load cultivates specific ties between persons and communities through these new programmes of interventions . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . this new territory has a number of significant features ( miller and rose 2008 ) . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. lifestyle communities , moral communities , risk communities and so on ; miller and rose 2008 ) . second , it operates through the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 , p. 90 ) . in other words , it makes use of allegiances and specific ties between individuals and communities to regulate , reform and mobilize ( miller and rose 2008 ) . from this perspective , members of particular communities are encouraged to practice active personal responsibility and conduct themselves accordingly ( miller and rose 2008 ) . as a sign of social citizenship , they must take responsibility , they must show themselves capable of calculated action and choice , they must shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . third , it concerns the identification of individuals as members of particular communities and the work required to make these individuals aware of their allegiances , for example , with the disability community , the gay community or the aids community . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . that is to say that these particular communities are created and marketed despite the fact that they may appear to be natural and may resonate with our own personal identity ( miller and rose 2008 ) . drawing on a number of examples , miller and rose ( 2008 ) explain that government through community works , even when it works upon pre - existing bonds of allegiance , transforms them , invests them with new values , affiliates them to expertise and reconfigures relations ( p. 93 ) in a productive way . miller and rose ( 2008 ) explain that the construction of community involves various strategies for making individuals aware of their personal allegiances . one of these strategies is to raise awareness ( i.e. awareness campaigns ) , educate ( i.e. community training sessions ) , communicate ( i.e. media and other communication tools ) , and make sure that individuals identify themselves as members of that community ( miller and rose 2008 ) . drawing on health promotion programmes in hiv and aids , miller and rose ( 2008 ) explain that government through community produces new personal allegiances and works upon pre - existing ones to make individuals aware of their collective affiliations . these affiliations , explain miller and rose ( 2008 ) , are to be celebrated , encouraged , nurtured , shaped and instrumentalized in the hope of producing consequences that are desirable for all and for each ( p. 93 ) . affiliations to particular communities , then , create new relations of identification and , incidentally , new relations of mutual obligation ( miller and rose 2008 , p. 88 ) . as such , they open up questions of personal responsibilities and obligations . these questions are particularly relevant to our analysis because community viral load is not just a means of identification and community affiliation . it is also used to govern individuals who are located here and not there , that can be expected to behave and engage in certain ways and not others , and whose distribution is spatially correlated with other characteristics that makes them part of risk communities ( viral load , serological status , gender , ethnicity , socio - economic status , sexuality , etc . ) ( brown and knopp 2010 ) . from this perspective , members of risk communities must practice active personal responsibility and are expected to shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . professional gaze , where expertise now focuses on conduct itself and the cognitive and moral organization of perception , intention , action and evaluation ( miller and rose 2008 , p. 106 ) . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . this form of governance relies on the deployment of expert knowledge from various fields ( i.e. nursing , social work , medicine , etc . ) and practices of empowerment . empowerment , then , is a matter of experts teaching , coaxing , requiring their clients to conduct themselves within their respective communities , according to certain prescribed codes of active personal responsibility and moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . the use of the term community is a powerful rhetorical tool and appeals to a common sense of belonging indeed , do we not all belong to some community ? yet , public health is not interested in all communities equally . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is inclusive of virtual communities of individuals who share particular risk factors but are geographically dispersed to communities of individuals who share geographical coordinates and whose virus is unsuppressed. what is important to understand here is not just that community viral load allows for these communities to be imagined , but that it is part of a large - scale operation to seek , test and treat more aggressively in target areas with the highest community viral load . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . seek , test and treat initiatives are arguably the best examples of high impact prevention efforts that specifically target such neighbourhoods and use on - site outreach to implement more aggressive forms of testing , surveillance and treatment -including mass testing fares with incentives meant to draw in the most marginalized . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the one hand , it concerns the identification of individuals who reside in areas where hiv is most heavily concentrated as members of a community. on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . they may also become increasingly politicized . while this phenomenon may be beneficial for particular communities , it also tends to extend and reinforce the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 ) . seek , test and treat initiatives rely on the assumption that individuals who are made aware of their personal allegiances with a community will be more inclined to enrol , mobilize and act more responsibly ( miller and rose 2008 ) . one way to practice active personal responsibility is to take part in testing and link with prevention services . another way to practice active personal responsibility is to initiate antiretroviral treatment as soon as possible after diagnosis and demonstrate optimal treatment adherence . with this is mind , it is important to understand that seek , test and treat initiatives make room for new relations of expertise and new opportunities for experts to intervene directly in the community. in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . they also rely on the ongoing presence and visibility of experts in the community , working in neighbourhoods and locations that are most affected by the hiv / aids epidemic . again , this may be beneficial for some communities but it is important to acknowledge that the expansion of testing and treatment coverage are not substitute to the removal of vulnerabilities that place people at risk of infection in the first place ( which incidentally , overlap with vulnerabilities preventing access to [ hiv care and ] treatment ( nguyen et al . 2011 , p. 292 ) . while our goal is not refute the scientific evidence in support of high impact prevention , which is the guiding framework for seek , test and treat initiatives , we consider that these programmes require more reflection . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . in light of our analysis , we consider that this new framework has very little to do with the care of individuals who live with hiv/ aids and much more to do with the production of a healthy population and the control of certain bodies . it is concerned with matters of life and death , with health and illness , with infectiousness and transmissibility , and with the collective processes that contribute to the spread of hiv . it acts upon the health of the population as a whole by targeting geographical areas where hiv is highly concentrated , seeking and testing populations conceived as inherently risk - prone , vulnerable or unstable , and treating individuals who are found to be hiv positive in order to achieve individual viral suppression . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . it was introduced to the field of hiv as a way to act upon communities who are located in specific geographical areas rather than individuals themselves . however , in order for it to be effective , it has to create specific ties between individuals who reside in areas where hiv is most heavily concentration and their respective risk communities. this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . as such , we are concerned that community viral load has become a proxy for naming risk communities , but with new levels of sophistication . in particular , we are concerned with the potential to increase stigma directed at populations who occupy areas that are identified as highly virulent. this phenomenon has yet to be addressed in the literature and in research conducted on community viral load . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . these spaces are calculated , reconfigured , and imagined , with new levels of sophistication . first , community viral load signals an important shift in the way viral load is seen , imagined and represented both visually and spatially . specifically , we draw upon philo 's ( 2000 ) reading of foucault to expose three distinctive but interconnected representations of viral load . the first representation has to do with the tabulation of laboratory results that can attest to the progression of hiv and the response to treatment at the individual level . it draws a picture of how viral replication actually appears at every stage of hiv infection and what response is to be expected once treatment is initiated . here , tables and graphics of viral load patterns provide a reference point to assess individuals in the clinical setting and systematically position each of them accordingly in relation to an ideal patient with optimal viral suppression . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . finally , the third representation allows for hiv to be located in space through mapping techniques . the location of the virus in space is compatible with foucault 's analysis of the shift from leper colony to plague city , wherein the risk of transmission is already present and requires the segmentation of space ( foucault 1990 ) . foucault explicitly linked mapping and government , and the need to have a rational plan to manage space ( crampton 2007 , p. 224 ) . in fact , he identified the ordering of space as central to the strategy for containment of biological risks and the government of processes that modulate health collectively rather than individually ( foucault 1990 , legg 2005 ) . these particular kinds of spaces take the form of specific neighbourhoods and locations where hiv is highly concentrated often located in geographical areas marked by high levels of marginalization , poverty , oppression and social exclusion . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . huxley ( 2006 ) explains that in order to better appreciate the articulation of spatial rationalities in the fabrication of governable spaces , we must pay close attention to the way authorities imagine these spaces and make them amenable to regulation . expanding on foucault 's work , huxley ( 2006 ) identifies three spatial rationalities : dispositional , generative and vitalist . for the purpose of this article a dispositional rationality has to do with the production of boundaries and the spatial disposition of bodies ( huxley 2006 ) . it operates through the logics of quadrillage ( a term coined by foucault ) to prevent the spread of diseases and ensure a more effective regulation of processes that are responsible for epidemics ( huxley 2006 ) . in other words , it allows for the segmentation of space and the management of bodies to achieve a given end . entails systems or surveillance , as these are seen as essential to enforcing the ordering of spaces and bodies. generative rationality , on the other hand , concerns the effects of space on contagion , health , disease and death ( huxley 2006 ) . here , the problem is not so much the disposition of spaces but the concentration of diseased bodies in geographical areas ( huxley 2006 ) . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . with this in mind , biomarkers become an important tool to circumscribe infectiousness and transmissibility ( or the risk thereof ) geographically . the emphasis is no longer on the disposition of diseased bodies in space but rather on areas where disease is highly concentrated and where the risk of transmission is intensified ( huxley 2006 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . we make a point here of referring to the virus because it is the real focus of this geographical ( and political ) undertaking even though we acknowledge that it reduces the bodies of people living with hiv to vectors of disease ( brown 1995 ) . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . what it does , however , is provide the necessary arguments to intensify surveillance , testing and prevention efforts in areas where we can find a number of people living with hiv whose viral load is unsuppressed. while our objective is not to portray people living with hiv as vectors of disease , we do have to stay true to the imaginations and rationales of those who see in community viral load a promising way to circumscribe infectiousness and transmissibility geographically . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. routine testing in institutions located in targeted areas , point of care testing in certain neighbourhoods , treatment as prevention initiatives in specific locations , and so forth . we shall now examine how community viral load cultivates specific ties between persons and communities through these new programmes of interventions . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . this new territory has a number of significant features ( miller and rose 2008 ) . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. lifestyle communities , moral communities , risk communities and so on ; miller and rose 2008 ) . second , it operates through the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 , p. 90 ) . in other words , it makes use of allegiances and specific ties between individuals and communities to regulate , reform and mobilize ( miller and rose 2008 ) . from this perspective , members of particular communities are encouraged to practice active personal responsibility and conduct themselves accordingly ( miller and rose 2008 ) . as a sign of social citizenship , they must take responsibility , they must show themselves capable of calculated action and choice , they must shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . third , it concerns the identification of individuals as members of particular communities and the work required to make these individuals aware of their allegiances , for example , with the disability community , the gay community or the aids community . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . that is to say that these particular communities are created and marketed despite the fact that they may appear to be natural and may resonate with our own personal identity ( miller and rose 2008 ) . drawing on a number of examples , miller and rose ( 2008 ) explain that government through community works , even when it works upon pre - existing bonds of allegiance , transforms them , invests them with new values , affiliates them to expertise and reconfigures relations ( p. 93 ) in a productive way . miller and rose ( 2008 ) explain that the construction of community involves various strategies for making individuals aware of their personal allegiances . one of these strategies is to raise awareness ( i.e. awareness campaigns ) , educate ( i.e. community training sessions ) , communicate ( i.e. media and other communication tools ) , and make sure that individuals identify themselves as members of that community ( miller and rose 2008 ) . drawing on health promotion programmes in hiv and aids , miller and rose ( 2008 ) explain that government through community produces new personal allegiances and works upon pre - existing ones to make individuals aware of their collective affiliations . these affiliations , explain miller and rose ( 2008 ) , are to be celebrated , encouraged , nurtured , shaped and instrumentalized in the hope of producing consequences that are desirable for all and for each ( p. 93 ) . affiliations to particular communities , then , create new relations of identification and , incidentally , new relations of mutual obligation ( miller and rose 2008 , p. 88 ) . these questions are particularly relevant to our analysis because community viral load is not just a means of identification and community affiliation . it is also used to govern individuals who are located here and not there , that can be expected to behave and engage in certain ways and not others , and whose distribution is spatially correlated with other characteristics that makes them part of risk communities ( viral load , serological status , gender , ethnicity , socio - economic status , sexuality , etc . ) ( brown and knopp 2010 ) . from this perspective , members of risk communities must practice active personal responsibility and are expected to shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . professional gaze , where expertise now focuses on conduct itself and the cognitive and moral organization of perception , intention , action and evaluation ( miller and rose 2008 , p. 106 ) . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . this form of governance relies on the deployment of expert knowledge from various fields ( i.e. nursing , social work , medicine , etc . ) and practices of empowerment . empowerment , then , is a matter of experts teaching , coaxing , requiring their clients to conduct themselves within their respective communities , according to certain prescribed codes of active personal responsibility and moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . the use of the term community is a powerful rhetorical tool and appeals to a common sense of belonging indeed , do we not all belong to some community ? yet , public health is not interested in all communities equally . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is inclusive of virtual communities of individuals who share particular risk factors but are geographically dispersed to communities of individuals who share geographical coordinates and whose virus is unsuppressed. what is important to understand here is not just that community viral load allows for these communities to be imagined , but that it is part of a large - scale operation to seek , test and treat more aggressively in target areas with the highest community viral load . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . seek , test and treat initiatives are arguably the best examples of high impact prevention efforts that specifically target such neighbourhoods and use on - site outreach to implement more aggressive forms of testing , surveillance and treatment -including mass testing fares with incentives meant to draw in the most marginalized . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the one hand , it concerns the identification of individuals who reside in areas where hiv is most heavily concentrated as members of a community. on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . they may also become increasingly politicized . while this phenomenon may be beneficial for particular communities , it also tends to extend and reinforce the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 ) . seek , test and treat initiatives rely on the assumption that individuals who are made aware of their personal allegiances with a community will be more inclined to enrol , mobilize and act more responsibly ( miller and rose 2008 ) . one way to practice active personal responsibility is to take part in testing and link with prevention services . another way to practice active personal responsibility is to initiate antiretroviral treatment as soon as possible after diagnosis and demonstrate optimal treatment adherence . with this seek , test and treat initiatives make room for new relations of expertise and new opportunities for experts to intervene directly in the community. in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . they also rely on the ongoing presence and visibility of experts in the community , working in neighbourhoods and locations that are most affected by the hiv / aids epidemic . again , this may be beneficial for some communities but it is important to acknowledge that the expansion of testing and treatment coverage are not substitute to the removal of vulnerabilities that place people at risk of infection in the first place ( which incidentally , overlap with vulnerabilities preventing access to [ hiv care and ] treatment ( nguyen et al . 2011 , p. 292 ) . while our goal is not refute the scientific evidence in support of high impact prevention , which is the guiding framework for seek , test and treat initiatives , we consider that these programmes require more reflection . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . in light of our analysis , we consider that this new framework has very little to do with the care of individuals who live with hiv/ aids and much more to do with the production of a healthy population and the control of certain bodies . it is concerned with matters of life and death , with health and illness , with infectiousness and transmissibility , and with the collective processes that contribute to the spread of hiv . it acts upon the health of the population as a whole by targeting geographical areas where hiv is highly concentrated , seeking and testing populations conceived as inherently risk - prone , vulnerable or unstable , and treating individuals who are found to be hiv positive in order to achieve individual viral suppression . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . it was introduced to the field of hiv as a way to act upon communities who are located in specific geographical areas rather than individuals themselves . however , in order for it to be effective , it has to create specific ties between individuals who reside in areas where hiv is most heavily concentration and their respective risk communities. this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . as such , we are concerned that community viral load has become a proxy for naming risk communities , but with new levels of sophistication . in particular , we are concerned with the potential to increase stigma directed at populations who occupy areas that are identified as highly virulent. this phenomenon has yet to be addressed in the literature and in research conducted on community viral load . the objective of this article has been to formulate a critique and propose a novel way of theorizing community viral load through a foucauldian analytics of governmentality with an attention to the role of space and community . we have shown that particular kinds of spaces are being reconfigured through a combination of clinical measures and epidemiological techniques to map the distribution of infected and risky bodies . these spaces then become key targets of public health intervention in the form of high impact treatment and prevention technologies that necessitate an unprecedented investment in identifying individuals in need of treatment ( nguyen et al . once on treatment , individuals become part of the community viral load loop their individual viral load becomes used to assess their we have also shown that the term community and its uses represents a discursive terrain imbued with power and ideology and requires unpacking to surface how it is being used and to what ends ( lynn 2006 ) . in light of our analysis , we are struck by what appears to be a shift back to the beginnings of the aids epidemic when various groups were singled out as infectious ( novitsky and essex 2012 ) and characterized as dangerous [ for historical overview and critique of this , see epstein 1996 , patton 1996 ] . the concern became that identifying people in this way perpetuated stigma and could result in the potential for an even greater number of infections ( parker and aggleton 2003 , peretti - watel et al . , epidemiology moved to monitoring risk behaviours as a way of de - stigmatizing these groups . with the invention and deployment of community viral load , we see a return to this previous logic , but resting on the seeming neutrality of space as if all people freely inhabit spaces of their own choosing . the implications of this , some of which we have explored , are only speculative at this time as calculating community viral load in all jurisdictions has various logistical and policy challenges . there are however concerted efforts underway to reduce these remaining barriers as suggested by the most recent national hiv / aids strategy in the united states ( office of national aids policy 2010 ) and guidelines published by the centers for disease control and prevention ( 2012 ) on community viral load measures , definitions and methods for calculation . this return to previous ways of thinking about , and managing hiv , albeit through more sophisticated techniques than were available at the beginnings of the epidemic , raises important questions about how hiv is being seen by governments , policy makers and public health agencies . require testing and mandatory treatment on a scale seen only in dictatorships ( p. 263 ) . however , this bold statement fits within larger debates about hiv as a threat to inter / intra state security ( elbe 2005 , 2009 ) . how do the changing politics of hiv and the growing interest in the securitization of health and illness help inform our thinking about mapping community viral load what are the implications of these clinical advancements for other health issues , and the potential outcomes to those who reside in spaces characterized by high rates of hiv and other forms of chronic illness . we hope that by closing on such a note that others will be encouraged to revisit hiv , and these new treatment and new prevention technologies , as a site for critical debate .

hiv plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual hiv plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively using techniques to measure community viral load. this article seeks to formulate a critique and propose a novel way of theorizing community viral load . based on the salient work of michel foucault , especially the governmentality literature , this article critically examines the use of community viral load as a new strategy of government . drawing also on the work of miller and rose , this article explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific microlocales , and the government ( in the foucauldian sense ) of specific bodies which are seen as risky , dangerous and therefore , in need of attention . it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america .

Introduction Defining community viral load Community viral load research in Vancouver, San Francisco, New York and Washington Mapping community viral load Governing through communities Beyond the rhetoric of community Final remarks

endovascular aortic aneurysm repair ( evar ) has developed into a feasible and successful alternative to open surgery for the treatment of abdominal aortic aneurysms . evar can be offered to many patients with a suitable anatomy of the aorta and iliac arteries , regardless of comorbid conditions [ 13 ] . despite the known excellent early results of evar in terms of the reduction in perioperative mortality , rate of complications and length of hospitalization , many patients require re - intervention during the middle and long - term follow - up because of procedure - related complications . for this reason , surveillance of these patients is crucial to determine the long - term performance of these devices [ 4 , 5 ] . because of the rapid diffusion of evar and the increased number of patients who undergo multidetector ct ( mdct ) follow - up , the radiologist should be familiar with the full spectrum of possible procedure - related complications in order to allow their early diagnosis and treatment . the purpose of this article is to present a spectrum of post - evar complications as seen with mdct . regardless of the scanner available ( 4 , 16 or 64 row ) , a thick - slice unenhanced acquisition can be performed to visualize the position of the stent - graft to reveal calcifications and to plan the following contrast - enhanced examination by selecting the acquisition volume and placing a region of interest in the aorta at the level of the celiac trunk ( if bolus - tracking software is used ) . contrast - enhanced images in the arterial phase are obtained during bolus intravenous injection of 90130 ml of iodinated high - concentration non - ionic contrast medium , administrated with an automated injector at a flow rate of 34 ml / s through an antecubital vein . ct acquisition protocol parameters and scanning coverage ranges are illustrated table 1 and fig . 1 . scanning should start when the examined structures have reached an ideal level of opacification ; therefore , the scan delay must be individualized to the patient by using bolus - tracking software to capture 100 hu on the abdominal aorta . at the end of the arterial acquisition , delayed images focused on the graft must be acquired , performed at least 60 s and up to 120 s after contrast material injection . the cta examination can be complemented by postprocessing reconstructions , including maximum- intensity projection ( mip ) , curvilinear reformation ( cvr ) and volume rendering ( vr ) .table 1ct acquisition protocol parametersscannerrotation time ( s)collimationtable feed ( mm / s)slice thickness ( mm)slice interval ( mm)duration ( s)4 slice0.54 1 mm251.25125 - 3016 slice0.516 0.625 mm27.50.6250.62525 - 3064 slice0.564 0.625 mm800.6250.625<15fig . 1scanning coverage of unenhanced and enhanced ct - acquisition ct acquisition protocol parameters scanning coverage of unenhanced and enhanced ct - acquisition an aortic stent - graft is a device composed of a metallic portion ( nitinol , elgiloy , and stainless steel ) and graft material ( polyester , ptfe ) . on ct images only the metallic portion is visualized . on the basis of the general morphology , there are three types of stent - grafts available for treating abdominal aortic aneurysms : straight , aorto - uni - iliac grafts and bifurcated . straight aortic tube grafts have the proximal and distal attachment sites in the aorta , above the aortic bifurcation ( aorto - aortic ) . the aorto - uni - iliac device is a stent - graft that is deployed from the supra - aneurysmal aorta to one iliac artery only ; the opposite iliac artery is then occluded with an endovascular occlusion device in order to prevent retrograde blood flow into the aneurysm sac , and a femoro - femoral crossover graft maintains blood flow into the opposite limb ( fig . 2 ) . bifurcated stent - grafts are extended to the iliac arteries . on the basis of proximal fixation , the suprarenal fixation device has an uncovered metallic portion placed above the ostia of the renal arteries and a covered portion placed below the renal arteries : the radiological markers placed between the uncovered and covered portion are represented by two metallic points located on the stent - graft metallic structure at the opposite position [ fig . the fenestrated device has a covered metallic portion of the graft that incorporates any visceral vessels such as the renal arteries , superior mesenteric artery or celiac trunk ; the patency of the incorporated vessels is maintained by the associated placement of covered stents ( fig . 2vrt ( a ) and mip ( b ) images : aorto - uni - iliac device associated with a surgical femoro - femoral cross - over graft . 3scout view and axial images showing the two metallic radiological markers placed in the opposite site between the uncovered and covered portion in a suprarenal fixation devicefig . 4mip ( a ) and vrt ( b ) images show a fenestrated stent - graft with a covered metallic portion that incorporates the renal arteries , which are made patent by the associated placement of covered stents ( c ) vrt ( a ) and mip ( b ) images : aorto - uni - iliac device associated with a surgical femoro - femoral cross - over graft . the left iliac limb is occluded by an occluder ( arrows ) scout view and axial images showing the two metallic radiological markers placed in the opposite site between the uncovered and covered portion in a suprarenal fixation device mip ( a ) and vrt ( b ) images show a fenestrated stent - graft with a covered metallic portion that incorporates the renal arteries , which are made patent by the associated placement of covered stents ( c ) stent - graft and distal branches are considered patent if a uniform and homogeneous contrast enhancement is detected within them . on the other hand , graft thrombosis is recognized as an intraluminal , concentric or eccentric hypodense area within the stent - graft on ct images obtained after contrast - media injection . angulation of the prosthesis could lead to a stenosis or thrombosis with consequent reduction of either the inflow or outflow , with increased possibility of in - stent thrombosis and stenosis , which can be manifest in about 319 % of cases [ 8 , 9 ] . graft occlusion appears as a complete non - enhancing intraluminal area within the stent - graft . graft limb occlusion may be directly due to compression of the limb in the proximal neck or in a narrow distal aneurysm neck , or to kinking or compression of the limb in a narrow or tortuous iliac artery . in case of iliac limb thrombosis , a femoro - femoral cross - over graft can be made , whereas a main body occlusion needs a complete surgical conversion . 5axial ( a ) and cpr ( b c ) images show a stent graft thrombosis causing significant intraluminal stenosis axial ( a ) and cpr ( b c ) images show a stent graft thrombosis causing significant intraluminal stenosis fractures or distortions of the metallic stent - graft structure are rare but important complications : these complications are better visualized on 3d rather than axial images ( fig . another complication that must be promptly recognized is stent - graft migration that occurs because of poor attachment of the stent to the aortic wall , which can cause sac reperfusion and subsequent aneurysm rupture.fig . 6mpr ( a ) and mip ( b ) images show a distortion of the metallic stent - graft structure . the integrity of the graft material as demonstrated by the linear and defined margin of the contrast - medium inside the stent - graft needs to be observed ( arrows ) . a type - ii endoleak is also detected ( * in b ) mpr ( a ) and mip ( b ) images show a distortion of the metallic stent - graft structure . the integrity of the graft material as demonstrated by the linear and defined margin of the contrast - medium inside the stent - graft needs to be observed ( arrows ) . unless the graft can be extended proximally , treatment is usually carried out by conversion to open repair . only migration of more than 4 mm this is related to a number of factors , including inadequate proximal fixation ( incorrect sizing , conical - shaped neck , short neck and angulation of the neck ) , progressive dilatation of the proximal neck and aneurysm size , as well as iliacal fixation [ 1012 ] . mdct angiography clearly detects minimal stent - graft migration : the diagnosis is based on the comparison of 1-month follow - up ct images evaluating the relationship between the proximal end of the stent - graft and lumbar vertebral body or renal artery origin on 3d and axial images , respectively ( fig . 7).fig . 7one month ( a ) and 1 year ( b ) post - evar follow - up sagittal mip images . at 1 year follow - up the stent graft appeared to have migrated distally with a significant angulation of the main body one month ( a ) and 1 year ( b ) post - evar follow - up sagittal mip images . at 1 year follow - up the stent graft appeared to have migrated distally with a significant angulation of the main body since aneurysm exclusion is expected to be correlated with shrinkage , surveillance of aneurysm dimensions is mandatory for asserting the adequacy of aneurysm exclusion from the blood flow and for guiding the need for reintervention in selected cases . the aneurysm volume may increase slightly immediately after evar ( 34 mm ) , given the additional volume and the external force exerted by the stent - graft or , alternatively , the swelling of the aneurysm sac induced by perigraft thrombosis . on the other hand , the volume tends to increase if sac perfusion is present or decreases during the follow - up in the absence of endoleaks . diameter measurements are most accurate if performed on axial images perpendicular to the aortic long axis , but volume assessment has been proven to be more accurate than diameter in the early detection of aneurysm growth ; however , volume assessment is time - consuming and requires advanced processing , dedicated equipment and skilled operators . endoleak is defined as a persistent blood flow within the sac excluded from the stent - graft , and it occurs in 2.445.5 % of patients after evar . on cta images , the endoleak appears as a high attenuation area outside the graft but within the aneurysm sac , detected on arterial and/or delayed phase images , but generally absent on unenhanced images . unenhanced images can be helpful to avoid false - positive diagnoses , allowing the differentiation of calcifications in the aneurysm sac from an endoleak ( fig . because endoleaks have variable flow rates , they can be detected at variable times after contrast material injection . for this reason , a delayed phase has been recommended : in detail , this phase could detect endoleaks not visualized during the arterial phase , the so - called low - flow endoleaks . early endoleaks occur in the first 30 days following evar , while endoleaks that fail to seal within 30 days are called persistent endoleaks . proper classification of an endoleak is important for its subsequent management [ 16 , 17].type i is caused by separation of the device from the arterial wall , resulting in leaks originating at the proximal and/or distal attachment sites of the graft because of a technical ( e.g. , suboptimal stent - graft diameter ) or anatomical ( e.g. , a short , irregular , ulcerated or angulated landing zone without an optimal conformation of the stent - graft to the curved aortic contour ) problems , or to its caudal migration . on cta images , it often appears as a huge and circumferential leak , adjacent to the proximal or distal end of the prosthesis ( figs . 9 and 10).type ii endoleaks are caused by back - filling of the aneurysm sac via branch vessels , such as the lumbar arteries and inferior mesenteric artery excluded by the stent - graft . on cta images , the type ii endoleak is most pronounced at the periphery of the aneurysmal sac , with little or no contact with the prosthesis , is commonly located in a posterior or lateral position , and is associated with opacification of the lumbar arteries . if an endoleak is located in the anterior position , a retrograde flow into the sac by the inferior mesenteric artery must be suspected ( fig . 11).type iii endoleaks arise from a fabric tear , modular or graft disconnection , and are more likely when multiple prostheses with short overlapping areas are used . on cta images , the leak is strictly adjacent to the prosthesis , with little or no contact with margins of the aneurysmal sac , without opacification of the lumbar arteries or inferior mesenteric artery ( fig . 12).type iv involves vascular flow caused by the high porosity of the graft , most likely created by the numerous suture holes holding the graft material to the stent . they are usually only detected on conventional angiograms performed at the end of the procedure.type v or endotension refers to a growth of the aneurysm sac but without demonstrable reperfusion defects ( fig . the combined analysis of unenhanced ( a ) , arterial ( b ) and late - phase enhanced images ( c ) allows the correct characterization of the thin hyperdensity within the sac ( arrows ) as a linear calcificationfig . 9axial ( a ) and mip- ( b ) images show the stent - graft caudal migration with subsequent proximal type - i leak ( * ) fig . 10axial ( a ) and cpr ( b ) images show an incomplete distal attachment of the right iliac limb causing a type i leak , as confirmed by dsa ( c)fig . axial ( a ) and thin mip ( b , c ) images show back - filling of the aneurysm sac through the inferior mesenteric arteryfig . 12vrt ( a , b ) , mip ( c ) and cpr ( d ) images show a type iii endoleak due to a right iliac limb disconnection and distal migrationfig . follow - up mdct examinations performed at 6 months ( a ) and 12 months ( b ) show an increase in sac diameter without evidence of endoleaks type i is caused by separation of the device from the arterial wall , resulting in leaks originating at the proximal and/or distal attachment sites of the graft because of a technical ( e.g. , suboptimal stent - graft diameter ) or anatomical ( e.g. , a short , irregular , ulcerated or angulated landing zone without an optimal conformation of the stent - graft to the curved aortic contour ) problems , or to its caudal migration . on cta images , it often appears as a huge and circumferential leak , adjacent to the proximal or distal end of the prosthesis ( figs . 9 and 10 ) . type ii endoleaks are caused by back - filling of the aneurysm sac via branch vessels , such as the lumbar arteries and inferior mesenteric artery excluded by the stent - graft . on cta images , the type ii endoleak is most pronounced at the periphery of the aneurysmal sac , with little or no contact with the prosthesis , is commonly located in a posterior or lateral position , and is associated with opacification of the lumbar arteries . if an endoleak is located in the anterior position , a retrograde flow into the sac by the inferior mesenteric artery must be suspected ( fig . type iii endoleaks arise from a fabric tear , modular or graft disconnection , and are more likely when multiple prostheses with short overlapping areas are used . on cta images , the leak is strictly adjacent to the prosthesis , with little or no contact with margins of the aneurysmal sac , without opacification of the lumbar arteries or inferior mesenteric artery ( fig . type iv involves vascular flow caused by the high porosity of the graft , most likely created by the numerous suture holes holding the graft material to the stent . they are usually only detected on conventional angiograms performed at the end of the procedure . type v or endotension refers to a growth of the aneurysm sac but without demonstrable reperfusion defects ( fig . the combined analysis of unenhanced ( a ) , arterial ( b ) and late - phase enhanced images ( c ) allows the correct characterization of the thin hyperdensity within the sac ( arrows ) as a linear calcification axial ( a ) and mip- ( b ) images show the stent - graft caudal migration with subsequent proximal type - i leak ( * ) axial ( a ) and cpr ( b ) images show an incomplete distal attachment of the right iliac limb causing a type i leak , as confirmed by dsa ( c ) type ii endoleak . axial ( a ) and thin mip ( b , c ) images show back - filling of the aneurysm sac through the inferior mesenteric artery vrt ( a , b ) , mip ( c ) and cpr ( d ) images show a type iii endoleak due to a right iliac limb disconnection and distal migration type v endoleak ; axial images . follow - up mdct examinations performed at 6 months ( a ) and 12 months ( b ) show an increase in sac diameter without evidence of endoleaks type i and type iii endoleaks require prompt treatment as they are associated with a high risk of sac rupture . however , the most common endoleak found in endovascular stent - grafting is the type ii endoleak . renal artery stenosis , occlusion and dysfunction represent the main concern with evar , especially in patients with inadequate anatomy of the proximal aneurysm neck , pre - existing renal disease or in whom suprarenal fixation was used . axial ct images are commonly used to monitor the patency of these arteries following stent - graft placement and to detect renal infarction following suprarenal stent grafting . however , both mip and cpr are useful and complementary to axial images for the detection of stenosis and occlusions because of the tortuous course of the renal arteries . the additional views provided by ct angiography allow displaying the renal arteries in multiple planes and projections , which is often necessary for depiction of stenosis . in renal infarction , contrast - enhanced ct scans demonstrate a sharply demarcated , wedge - shaped area of decreased attenuation in the kidney ( fig . however , to date no significant correlation has been found between evar and renal impairment [ 1920].fig . the right kidney appeared decreased in size and showed an area of parenchymal hypoperfusion due to infarction mip images obtained before ( a ) and after ( b ) evar . the right kidney appeared decreased in size and showed an area of parenchymal hypoperfusion due to infarction usually a transfemoral approach is used to perform evar , exposing the femoral artery by a vertical groin incision . the large - caliber delivery sheaths used for evar , up to 26 fr , increase the morbidity rate compared to the usual catheter size used for routine diagnostic angiography . the minimum vessel diameter required to allow passage of the evar device is 78 mm . access site complications include dissection , demonstrated by a linear endoluminal hypodense area ( intimal flap ) ( fig . 15a b ) or arterial rupture , with contrast material extravasation outside the artery . other complications are pseudoaneurysms , especially close to the entrance site , hematomas near the femoral incision , demonstrated by a high - attenuating area ( fig . 15c ) , infections and lymphoceles .fig . 15axial ( a ) and mip ( b ) images show an endoluminal hypodense line within the right external iliac artery representing a dissection ; c axial image shows bilateral small hematomas at the femoral incision site axial ( a ) and mip ( b ) images show an endoluminal hypodense line within the right external iliac artery representing a dissection ; c axial image shows bilateral small hematomas at the femoral incision site mdct is the method of choice both in the pre- and postoperative setting of evar procedures . a better understanding of the procedure followed , the devices used , the normal postoperative imaging features and the possible procedure - related complications ensures an optimal planning and follow - up of patients who undergo an evar procedure . finally , in order to better determine the long - term performance of these devices , strict surveillance of all patients is mandatory this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

backgroundmultidetector computed tomography ( mdct ) angiography represents the standard of reference in the follow - up of patients after endovascular abdominal aortic aneurysm repair ( evar ) , being effective in the detection of the full spectrum of possible complications on both axial and 3d images.methodsthe purpose of this article is to review the normal ct angiography findings of the different types of stent - grafts and to describe the radiological findings of early and late complications after evar on axial and reconstructed images . a selection of cases of post - evar mdct angiography is presented to learn the techniques most commonly used for endovascular treatment , the correct ct scanning technique to acquire the data , the full gamut of possible procedure - related complications and how these complications usually appear on ct images.conclusionmdct angiography is an effective and specific technique in both the pre- and postoperative settings of evar procedures . a better understanding of the procedure , the devices , the normal postoperative imaging features and the possible procedure - related complications ensures optimal planning and follow - up of patients undergoing an evar procedure .

Introduction Multidetector CT technique Stent-grafts Patency Integrity Position of the stent-graft Sac aneurysm changes Endoleak Renal artery patency Access site complications Conclusion Open Access

multiple sclerosis ( ms ) is considered a chronic inflammatory disease of the central nervous system of autoimmune origin . onset of disease is most frequently in young adulthood , between 20 to 40 years of age.1 although many aspects of ms pathogenesis have been elucidated , the exact causal mechanisms are still not fully understood . an interplay between environmental factors in genetically susceptible individuals is assumed.2 this concept is supported by a wealth of research findings.35 unambiguously , once the disease has developed , it continues lifelong , and there is still no cure . the course of the disease can be relapsing remitting , which means that episodes with exacerbation of neurological symptoms alternate with periods of remission.6 over time , these relapses often do not fully resolve , leading to a stepwise accumulation of disability . moreover , after 1015 years , one - half of patients with initial relapsing remitting disease course will develop a secondary progressive disease7 that is characterized by a progressive increase of disability , independent of relapses and predominantly caused by deterioration of walking ability . only about 10%15% of patients exhibit a primary progressive disease course , which is defined by at least one year disease progression from onset on.8 a clinically isolated syndrome ( cis ) is diagnosed in patients with a first clinical event suggestive of ms and evidence of disease dissemination in space , but not yet evidence of dissemination in time , thus chronicity of disease . one of the hallmarks of ms is a high variability of the natural disease course . while some patients , apart from relapses , never experience any handicap , the majority of patients will ultimately develop disability to a variable degree and after a variable time interval after onset of disease . as first shown by confavreux et al9 and based on a concept of a two - stage disease with focal inflammation in the early stage and diffuse inflammation and neurodegeneration in a second - disease stage , leray et al recently confirmed highly interesting data on time to disability accumulation in ms:10 while duration of the first stage from onset of disease until reaching a milestone of kurtzke disability status scale ( dss ) 311 ( corresponding to moderate disability ) ranged from less than 3 years to more than 15 years , duration of phase ii ( kurtzke dss 3 to dss 6 , the latter is defined by requiring unilateral assistance to walk 100 meters ) remained nearly identical in all patients with 69 years , irrespective of the duration of phase i. this observation suggests that , once a clinical threshold of irreversible disability is reached , further progression of disability becomes inevitable . support of this hypothesis can be derived from two typical findings of the chronic progressive phase of ms . even in the absence of new or contrast - enhancing magnetic resonance imaging ( mri ) lesions , disability usually increases in the chronic progressive phase of the disease , and once the chronic progressive phase is reached , efficacy of disease - modifying therapies often declines.12,13 although relapse rate and progression of the expanded disability status score ( edss)11 are widespread parameters of disease activity , many patients with ms do not only suffer from physical disability ; they also suffer from neurocognitive decline , fatigue , or depression.14,15 hence , the diagnosis of ms is a life - changing event with a significant impact on family , society , and the social welfare system . fortunately , during the last decades , several disease - modifying therapies ( dmt ) have been licensed that ameliorate the course of the disease.16 the aim of these dmt is to reduce inflammation , disease activity as measured by mri , and relapse rate . thereby , a delay of transition into the secondary progressive phase and prevention of permanent disability ought to be achieved . due to the chronic nature of ms , dmts are usually applied continuously over many years , or even decades . to keep in mind , dmts are applied in a preventive manner , since neuronal damage can not be reversed . ms is the most frequent nontraumatic cause for disability among young adults in north america and europe.17 worldwide , about 2.5 million people are affected by ms ; although traditionally , the incidence is supposed to vary by geographical latitude.18 the highest prevalence with more than 30 ms patients per 100,000 inhabitants can be found in northern europe and north america . a medium prevalence of 530 per 100,000 is observed in southern europe and the southern united states ; whereas , a low prevalence of fewer than 5 per 100,000 has been reported for asia and south america.19 population genetics , the geographically determined physical environment , and socioeconomic structures have been proposed as possible reasons for continental differences in ms prevalence.20 in the united states alone , about 400,000 individuals suffer from ms ; each week , more than 200 persons are newly diagnosed with the disease.18 according to estimates for 2010 , ms has been diagnosed in 540,000 subjects of the european population.21 however , recent epidemiological studies found an increasing incidence and prevalence of ms , especially in women.20 this seems to be the case also in areas of the world that were formerly classified as low - prevalence regions . for example , ms prevalence was recently reported as 3155 per 100,000 inhabitants in the arabian gulf region and 0.8321.5 per 100,000 individuals in latin america and the caribbean.22,23 factors contributing to an increasing incidence of ms have not been fully elucidated . earlier ms diagnosis , due to revised diagnostic criteria ; better ascertainment of diagnosis ; less sunlight exposure ; higher standards in hygiene ; and cigarette smoking , have all been proposed as likely candidates.20,24 the economic impact of ms can be divided into direct costs and indirect costs . direct costs account for health care costs , such as pharmaceuticals , inpatient care costs , outpatient care costs , additional treatments like physiotherapy , and medical aids . in contrast , indirect costs are caused by productivity loss of patients and caregivers.25 this might be due to sick leave , reduced daily working time , unemployment , or premature retirement . in europe , mean annual cost per person with ms is estimated to be around 27,000 , ranging from 7,227 in bulgaria to 44,565 in luxembourg . on average , about two - thirds of the totals are caused by direct costs , while approximately one - third are derived from indirect costs.21 in 1998 , all - over costs of ms in the united states were us$6.8$11.9 billion annually , which averages about us$34,000 per patient , per year.26 in a survey among patients receiving dmt in the united states in 2004 , total average costs per patient and year were estimated as high as us$47,000.27 of these , 63% accounted for direct costs , including 34% of total costs ( us$16,000 ) for dmt . of note , patients having suffered a relapse were found to have higher costs than patients without relapse.27,28 furthermore , several studies have shown that total costs rise with increasing disability.2832 in line with this result , a secondary and primary progressive disease course was found to be more costly than a relapsing remitting one , due to a rise of indirect costs . besides degree of physical disability , age , depression , and fatigue have been identified as independent factors of an increase of costs.32 quality of life remarkably and constantly declines with advancing stages of disability , and the occurrence of relapses negatively affects quality of life measures.28,31 it was in 1993 that interferon beta-1b was introduced as the first dmt of ms . still , it represents a very common first - line treatment that is widely used . remitting ms , and secondary progressive ms with superimposed relapses.33 there are three different recombinant interferon beta preparations on the market . interferon beta-1b ( betaferon , bayer healthcare pharmaceuticals , berlin , germany ; betaseron , bayer healthcare pharmaceuticals , berlin , germany ; extavia , novartis , basel , switzerland ) is recombinantly expressed in escherichia coli and is subcutaneously injected every other day . interferon beta-1a is recombinantly expressed in chinese hamster ovary cells and is identical to the human interferon beta.34 there are two preparations of interferon beta-1a . rebif ( merck serono , geneva , switzerland ) is subcutaneously administered three times a week . avonex ( biogen idec , cambridge , ma , usa ) is applied once weekly as an intramuscular injection . placebo - controlled trials have demonstrated a significant reduction of disease activity for all three interferon beta preparations.3537 the pivotal trial on efficacy of interferon beta-1b in ms , which led to the initial approval of the drug for treatment of ms , demonstrated an annual relapse rate of 1.27 for placebo and 0.84 for 250 g interferon beta-1b.35 similar results were observed for interferon beta-1a . in the prevention of relapses and disability by interferon beta-1a subcutaneously in multiple sclerosis ( prisms ) trial on subcutaneous interferon beta-1a , the annual relapse rate was 1.28 for placebo , 0.91 for 22 g , and 0.87 for 44 g.37 intramuscular interferon beta-1a reduced the annual relapse rate to 0.61 , compared with 0.90 in the placebo group.36 these findings are supported by significant reduction of mri disease activity in all three trials , while only in the latter two trials , a beneficial effect on disability progression was recorded during this 2-year study period . while these therapies have been introduced , a better understanding of ms pathophysiology especially the knowledge of unrecoverable axonal loss already in early stages of the disease has prompted a fundamental shift in the ms treatment approach and provided the rational for early ms treatment.33,34 based on this consideration , dmts are initiated early in the course of the disease and before irreversible neuronal damage and disability have occurred . following the concept of early ms treatment , interferon beta treatment has been investigated in patients with cis and has shown a beneficial effect in terms of conversion to definite ms , edss progression , and mri disease activity.3841 in the betaferon/betaseron in newly emerging multiple sclerosis for initial treatment ( benefit ) trial,40 patients with cis were randomized to receive interferon beta1-b or placebo for 2 years , or until clinically definite multiple sclerosis ( cdms ) was diagnosed , according to the poser et al criteria.42 within the 2-year study period , 45% of patients in the placebo group versus 28% of the interferon beta group progressed to cdms , according to poser et al ; and 85% of the placebo versus 69% of the interferon beta group were diagnosed with definite ms , according to the mcdonald criteria of 2001.43 both results reached statistically significant difference . these data revealed a significant lower number of newly active lesions , cumulative number of new t2-weighted mri lesions and gadolinium - enhancing lesions , as well as cumulative volume of gadolinium - enhancing lesions in the interferon beta - treated patients group . of note , there was no change of health - related quality of life observed during the 2 years . after having finished the placebo - controlled core study , patients were offered to enroll in an open - label interferon beta-1b 250 g follow - up trial to investigate effects of early treatment in cis , versus delayed treatment after diagnosis of cdms or after 2 years on study . three years after initial randomization , 37% of the early treatment group versus 51% of the delayed - treatment group have developed cdms ( risk reduction of 41% ) ; 16% of the early treatment group versus 24% of the delayed - treatment group experienced confirmed edss progression ( risk reduction 40%).44 a subgroup analysis revealed lower treatment effects in patients with less clinical or mri activity at the time of the first event ; patients with initial multifocal presentation and high lesion load would benefit more from early treatment . in the 5-year active treatment extension follow - up study , risk reduction of cdms remained 37% in the early treatment group ; whereas , no statistically significant difference in confirmed disability progression was observed any more between early and delayed interferon beta treatment.45 in both treatment groups , median edss remained 1.5 during the 5-year study period . finally , in the benefit extension trial , a long - term international observational study of patients having completed the initial placebo - controlled core study , data on follow - up for more than 8 years are available.46 at the discretion of the physician and according to local standards , patients were treated with any of the disease - modifying drugs or received no treatment . during the study periods , 5.1% of patients received no treatment ; 77.6% received no other drug than interferon beta-1b ; 6% , interferon beta-1a ; 5.8% , glatiramer acetate ; and 6.6% of patients received any escalation therapy . in the overall study population , disability as quantified by the edss changed only slightly over 8 years , with the median edss remaining 1.5 at year 8 . however , 55.5% of patients , who had received early interferon beta treatment in the core study , developed clinically definite ms , while clinically definite ms was diagnosed in 65.8% of patients with delayed interferon beta treatment . moreover , a difference of the overall annualized relapse rate was still observed over the 8-year period 0.20 early treatment versus 0.26 delayed treatment.47 placebo - controlled clinical trials on interferon beta-1a in cis have yielded very similar results,38,41 suggesting a class effect of interferon beta treatment in early ms and cis . by numerous investigational trials and the clinical experience in interferon beta for two decades , safety and tolerability profile of this drug is well - known and has proven favorable , also , in long - term application . side effects mainly include injection site reactions , flu - like symptoms , leukopenia , and liver - enzyme elevation.48 moreover , up to 40% of patients develop antibodies against interferon beta , which antagonize bioactivity in a significant proportion of patients.4951 if neutralizing antibodies against interferon beta persistently occur , therapeutic efficacy of the drug is reduced or abolished.5256 besides interferon beta glatiramer acetate ( [ ga ] , copaxone , teva , petah tikva , israel ) represents a common first - line therapy with daily subcutaneous injections , which is approved for treatment of patients with relapsing remitting ms , as well as for patients with cis and typical mri features of ms . the precise ( early glatiramer acetate treatment in delaying conversion to clinically definite multiple sclerosis in subjects presenting with a clinically isolated syndrome ) study demonstrated that ga reduced the risk of developing clinically definite multiple sclerosis by 45% , compared with placebo during a 3-year study period.57 interferon beta and ga are the only agents that are licensed for treatment of cis . however , for the sake of completeness , it needs to be mentioned that there are several other therapeutics licensed for baseline and escalation therapy in definite ms , and more agents are likely to enter the market.16 approval of all these agents is usually based on efficacy and safety data of a 2-year clinical trial . in ms phase ii and phase iii interventional clinical trials , a duration of about 6 months to a few years has become common , since demonstration of efficacy usually needs several months of treatment . on the other hand , duration of clinical trials longer than 2 years will make a well - controlled trial less feasible to accomplish and might hold back an effective treatment from ms patient care . given an increasing number of available dmt in ms , trials investigating the long - term efficacy are needed . this is to briefly summarize the terms and types of pharmacoeconomic evaluations used in the studies reviewed here ( adapted from clifford goodman unless cited otherwise).25 cost - effectiveness analyses ( cea ) measure costs in monetary units and consequences in natural units , such as life - years gained , or relapses avoided.58 in cost - utility analyses ( cua ) , a variant of cea , consequences are measured in terms of preference - based measures of health,58 most commonly based on quality - adjusted life - years ( qaly).59 a qaly is a unit that adjusts gained or lost life - years subsequent to an intervention by the quality of life during those years . gained or lost life - years are multiplied by a weighting factor ( utilities ) ranging from 0.00 ( representing death ) to 1.00 ( representing a perfect health state ) . cea and cua always involve comparison of alternative interventions , expressed as incremental cost - effectiveness ratio ( icer ) and incremental cost - utility ratio ( icur ) , respectively . therefore , incremental costs are divided by the change of effectiveness or utility ( eg , qualys gained ) . depending on the perspective of pharmacoeconomic evaluations ( eg , society overall , third - party payers , patients ) , different types of costs are of interest . costs need to be corrected for effects of inflation ; both costs and outcomes should be discounted . discounting reflects the fact that costs and benefits tend to have less value in the future than in present . the former approach involves collection of original data ( eg , randomized controlled studies ) . quantitative modeling - based pharmacoeconomic analyses simulate costs and consequences of pharmacological interventions , based on existing data and estimates of key variables where there are no data available . in this type of analysis it is based on predefined sets of health states ( eg , edss levels ) . its time horizon is divided into equal increments of time , referred to as markov cycles . for each cycle , costs , outcomes , and transition probabilities of moving from one state to another or of remaining in the same state are estimated.60 finally , sensitivity analyses determine robustness of results of model - based analyses by varying the estimates of key variables within plausible ranges ( eg , by applying a multivariate approach ) . we conducted a systematic literature search in medline ( pubmed ) on december 14 , 2012 , applying the medical subject headings ( mesh ) terms multiple sclerosis and costs and cost analysis . we deliberately chose the comprehensive mesh term costs and cost analysis to cover all potentially relevant studies . four hundred fourteen publications met our search criteria , including 40 non - english publications , which were excluded ; titles and abstracts of the resulting 374 publications were reviewed by fc and db . inclusion criteria were as follows : ( 1 ) original research on the pharmacoeconomics of dmt in ms ; ( 2 ) comparative study ; and ( 3 ) comparators ( initiation of dmt at the time of the first demyelinating event versus treatment after cdms ) . because our search strategy resulted in no more than four relevant studies,6164 we did not exclude the study by curkendall et al,62 although the authors only report on costs and not on health - outcome measures of dmt in cis . the remaining three studies were rated for quality of health - economic analyses , based on the quantitative scoring system that has been proposed by chiou et al ( range 0100 ; 0 representing the lowest ; 100 representing the highest quality).65 the 16-item weighted scoring system was independently applied by fc and db . our scoring results were in accordance with the results reported by yamamoto and campbell , who chose the same approach ( table 1).66 as outlined before , early initiation of dmt with interferon beta or ga has been proven to reduce time to cdms , relapse rate , and disease progression . this observation led to the approval of interferon beta and ga for treatment of relapsing remitting ms , as well as cis . therefore , initiation of dmt may be started even before definite diagnosis of ms is established . to date , four studies have been published that address the economic impact of starting dmt at the time of the incident cis versus after conversion to cdms ( table 1).6164 the swedish study by caloyeras et al estimates cost - effectiveness of interferon beta-1b if initiated in cis compared with delayed treatment after the diagnosis of cdms has been made.61 the authors applied a markov model analysis , based on twelve health states defined by edss scores , the assigned diagnosis ( ie , cis versus cdms ) , and the course of disease ( ie , relapsing or nonrelapsing forms of ms , and death ) . transition probabilities after 6-month cycles were estimated for a lifetime time horizon of 50 years . the authors assumed that treatment discontinuation rates would not differ between both treatment arms ; all hypothetical patients would discontinue dmt after roughly 25 years . switching to another dmt and the core input data for the statistical model were derived from the benefit trial.40,44,45 moreover , extrapolations of benefit data and best available data from the published literature were used as model parameters . results of the cua demonstrate that , during a 50-year time horizon , patients assigned to the early treatment group gained 12.9 qalys , as compared with 12.4 qualys in the delayed - treatment group . on average , patients in the delayed - treatment group progressed 2 years earlier to cdms ( estimated conversion rate 99.54% ) , compared with patients in the early treatment group ( estimated conversion rate 99.31% ) . direct medical costs ( ie , interferon beta-1b drug costs , costs for inpatient and outpatient care , and costs related to testing , other drugs , and relapse events ) were higher in the early treatment arm , whereas indirect costs ( ie , costs related to early retirement and short - term absence ) and direct nonmedical costs ( ie , informal care , services , investments ) in the delayed - treatment group exceeded those in the early treatment group . when all costs were summed up over the 50-year time horizon , total costs in the delayed - treatment arm were higher than those in the early treatment arm ( difference of approximately 344,000 sek per patient ) . in summary , the markov model - based estimation revealed that early interferon beta-1b treatment may decrease total costs and increase qalys . however , sensitivity analyses revealed that costs per qaly gained were very high for short time horizons . in fact , only for time horizons longer than 10 years , early treatment was less costly and more effective with respect to qalys gained . this study reached the highest quality of health economic studies ( qhes ) score of the articles reviewed here ( 99 points ) . curkendall et al applied a different approach to assess the pharmacoeconomics of early dmt.62 their analysis was based on insurance claims data in the united states for the years 20002008 , extracted from the thomson reuters marketscan commercial and medicare supplemental databases ; therefore , only health care expenditures and utilizations were analyzed . the core inclusion and exclusion criteria were as follows : ( 1 ) no diagnosis of ms during 12 months before and at the time of a cerebral mri ; this mri scan was defined as start date of the 12-months observation period ; ( 2 ) at least one symptom that was documented 6 months before or 1 month after the index date and that was likely to be attributable to ms ; ( 3 ) initiation of treatment with interferon beta-1a , interferon beta-1b , or ga sometime during the follow - up period . thus , cis patients who may have received dmt after the follow - up period were not included . patients were categorized into two groups an early dmt and a delayed dmt group depending on whether they received dmt before or after the diagnosis of ms . ms diagnosis was defined as one inpatient claim or two outpatients claims coded with icd-9-cm diagnosis code 340 . three thousand nine hundred fifty one patients were enrolled ; 227 and 3,724 were assigned to the early and delayed - treatment cohort , respectively . the mean follow - up was approximately 3 years in both groups , yet the pharmacoeconomic analysis was restricted to the first year after the index mri . the mean time interval between index mri and initiation of dmt was , as expected , significantly shorter in the early treatment group than in the delayed - treatment group ( 122 days versus 184 days ) . however , this difference was small , and more than 90% of the patients assigned to the delayed - treatment group received dmt within the first year after index mri . within the observation period , the early dmt cohort had significantly fewer hospitalizations compared with the delayed dmt cohort ( 0.20 versus 0.33 ) . total drug expenditures were higher in the early dmt cohort ; whereas , total ms - related expenditures were significantly lower in the early dmt group , compared with the delayed dmt group after taking out costs for dmt . both all - cause and ms - related total financial expenditures were not significantly different between the two groups ( all - cause total expenditures us$31,184 in the early dmt group , us$30,051 in the delayed dmt group ) . the authors conclude that the high drug costs of early dmt might be compensated by savings in other medical expenditures . however , the observation period was short , and the analysis was conducted from a third - party payer s perspective alone . the qhes quality score could not be applied to this study , as it does not report on outcome measures , thus not fulfilling the criteria of pharmacoeconomic analyses . in contrast to the other articles reviewed here , the strength of this study lies in the analysis of real costs , albeit restricted to health care expenditures . lazzaro et al applied an open cohort epidemiological model from an italian perspective.63 two treatment arms were analyzed . one group of patients received interferon beta-1b after diagnosis of cis ; the other group was treated with interferon beta-1b after diagnosis of cdms . observation period lasted 25 years with 2,000 cis patients being added to each treatment arm every year , resulting in two cohorts of 50,000 patients each . the conversion rates for years 12 were based on the benefit data,40 while the conversion rates for the remaining 22 years were assumed to decline asymptotically . nineteen years after cdms diagnosis , approximately 50% of the patients would progress to secondary progressive ms . moreover , the observation period was arbitrarily divided into four periods ( year 0 ; years 110 ; years 1120 ; and years 2124 ) . based on the report by kobelt et al,67 edss scores were converted into utility parameters , which were assumed to stay constant within each period . costs were adopted from both the italian national health service ( inhs ) and the societal viewpoints . most of the cost estimates were based on the italian cost - of - illness study by amato et al;68 all costs were reported in 2006 euro . according to this analysis , patients gained 7.84 and 7.49 qalys in the early and delayed treatment arm , respectively ; the difference of 0.35 qalys reached the level of significance . the cua showed that , from the inhs perspective ( ie , only health care resources were considered ) , the icur for early versus delayed interferon treatment was 2,575 per qualy gained and lay considerably below a recently proposed italian willingness to pay of 12,00060,000 per qaly.69 when patient and family resources ( ie , the societal perspective ) were additionally taken into account , early treatment would be even less costly than delayed treatment ( 220,416 versus 226,022 per patient for early versus delayed treatment , respectively ) . the authors conclude that , from the health service and societal perspective , early treatment with interferon beta-1b may be cost - effective , compared with treatment initiation after cdms . however , this interpretation did not hold true for short time horizons as there was a sharp decline in icur estimates over the first years ; eg , from the societal perspective , the icur started from approximately 135,000 per qualy gained at year 1 , decreased below 60,000 after year 3 , and , finally , became negative after year 6 onward . the qhes score of this study reached 75 points and lay considerably below the quality score of the swedish study reviewed here . iskedjian et al performed a pharmacoeconomic analysis of treatment with interferon beta-1a administered intramuscularly once weekly in cis patients ; the study was conducted from a canadian perspective.64 the aim of this study was to investigate : ( 1 ) cost - effectiveness of interferon beta-1a treatment based on the gain of additional monosymptomatic life - years ( mlys ) ; and ( 2 ) cost - utility of long - term interferon beta treatment based on the gain of quality - adjusted monosymptomatic life - years ( qamlys ) . monosymptomatic life - years were defined as years after diagnosis of cis and before diagnosis of cdms . for cis patients , high - dose intravenous steroid pulse therapy ( plus tapering ) for the index event ( current treatment ) and high dose intravenous steroid pulse therapy ( plus tapering ) for the index event plus interferon beta-1a . once the diagnosis of cdms was established , all patients received the same treatment regime with interferon and high dose steroids for treatment of relapse . the time horizon of the cea and cua was 12 and 15 years , respectively , starting from diagnosis of cis and extending to various edss stages of cdms . for the cea and cua , two markov models were developed : the state of cis and various edss states defined the markov cycles ( cycle length , 1 year ) . the conversion rate to cdms was derived from the efficacy results of the controlled high risk avonex multiple sclerosis study ( champs ) study.38 transition probabilities through the various stages of edss were derived from a canadian ms study by weinshenker et al.7 cost analyses , including unemployment rates and the average hospital length of stay within each edss level , were based on data from the literature7072 and canadian health authorities . costs were reported in 2002 canadian dollars ( ca$ ) ; total costs were estimated from the perspective of the canadian ministry of health ( moh ) and from the canadian societal perspective . the former perspective was limited to direct medical costs ; the latter additionally covered direct nonmedical and indirect costs . the input parameters tested in the sensitivity analyses comprised the progression rate to cdms , the indirect costs associated with the duration from cis diagnosis to cdms , the time horizon , the discount rate , and the utilities . the cea demonstrates that the incremental cost - effectiveness ratio ( icer ) of interferon beta per mly gained was ca$53,110 and ca$44,789 from the moh and societal perspectives , respectively . as opposed to the moh perspective , interferon beta treatment was considered cost - effective from the societal perspective , as costs of current treatment per mly gained were higher ( ca$75,444 ) than the icer of interferon beta treatment . the cua was conducted , based on the utility estimates derived from both a canadian and a swedish study.30,70 from the moh perspective , incremental cost of interferon beta per qamly gained ( icur ) was ca$227,586 ( ca$116,071 for the swedish utility estimates ) ; whereas , from the societal perspective , incremental cost of interferon beta reached ca$189,286 ( ca$91,228 , swedish utility estimates ) . the sensitivity analyses showed that both the cea and cua were sensitive to variations of the time horizon with shorter horizons producing higher icer / ucer and vice versa . the multivariate sensitivity analysis of the cea showed that in 87% and 6% of the resulting scenarios the icer of interferon beta therapy would be lower than the costs of current treatment per mly gained from the societal and moh perspective , respectively . therefore , the authors conclude that interferon beta treatment in cis may be cost - effective . however , this conclusion could be drawn from the cea of the societal perspective , only . moreover , concerning the results of the cost - utility analysis , the authors omit to address the issue of decision makers willingness to pay . of the three pharmacoeconomic studies reviewed here , ms is a chronic disease with high economic burden on patients , families , the health system , and society . worldwide incidence and prevalence seems to be increasing with a total of about 2.5 million individuals currently being affected by ms . as reviewed here , several studies have demonstrated that progression of disease severity is accompanied by a rise of total costs as well as a change in the distribution of costs . during early stages of the disease , direct costs , which are mainly caused by dmt , dominate the total costs ; whereas , with accumulation of disability during later stages of the disease especially indirect costs significantly increase.73 moreover , occurrence of relapses has been associated with a peak of costs . noteworthy , quality of life is substantially reduced with increase of disability and during relapse . due to a better understanding of ms pathogenesis and the experience that efficacy of available dmt is high in early stages of ms but declines in the progressive phase of the disease , the concept of early ms treatment has been established . therefore , dmts are usually initiated after diagnosis of relapsing remitting ms or even before definite diagnosis of ms is made at the stage of cis . since dmts are , in general , considered as rather expensive treatments , there has been much debate whether it is economically justifiable to widely prescribe these drugs . a major concern is that clinical trials for approval of dmt even though they have clearly demonstrated efficacy on parameters such as relapses , mri activity , and sometimes also on edss progression during the trial have hardly addressed long - term disability . numerous studies have investigated the pharmacoeconomics of dmt in ms , though only few have explicitly analyzed the pharmacoeconomic effect of early initiation of dmt in cis.6164 these studies indicate that early versus delayed treatment with interferon beta may be overall cost - effective in the long term . reduction of relapses , hospitalization , and indirect costs and a gain of qalys seem to outweigh the costs of dmts . however , to keep in mind , all these analyses and models highly depend on estimates of the applied input parameters . for instance , becker et al74 recently demonstrated the impact of cohort selection by replicating a previously published model - based analysis75 on the cost - effectiveness of interferon beta in ms . based on a different patient cohort with longer follow - up , costs per relapse avoided turned out to be approximately 45% lower than in the original model . in their study reviewed here , iskedjian et al showed that , from the canadian societal perspective , estimated incremental costs of early interferon beta treatment would be 189,286 cad per qamly gained . when applying different utility estimates , the same analysis resulted in 91,228 cad , which is less than 50% of the former result.64 however , it seems to be ambiguous which of the applied utility estimates may be more valid than the other . thus , results of pharmacoeconomic studies and inferences that may be drawn by decision makers largely depend on the analysis approach , the applied simulations and estimations , the perspective of the analysis , and particularly on the quality of the pharmacoeconomic analysis . as shown in our review and for instance in the recent work by yamamoto et al , the latter has turned out to be considerably variable.66 we believe that this is especially critical in a chronic , heterogeneous , and complex disease , such as ms . in this context , one should note , that several pharmacoeconomic studies in ms , including the four studies reviewed here , have been funded by manufacturers of dmt ( table 1 ) . obviously , the results of all four reviewed studies can not be readily generalized to other national settings as several key input data ( eg , costs ) were country - specific.76 besides interferon beta and ga , new dmts have been introduced , which are used as baseline and/or escalation therapy . the wider range of dmts , as well as escalation strategies , needs to be taken into account in future pharmacoeconomic studies , which will be even more challenging . therefore , we need more data on long - term efficacy of dmt and costs in ms in the real - life setting . biomarkers , which will help to stratify patients at early stages with respect to severity of the disease course and response to therapy , are of great need from an economic point of view and even more so for patient care .

multiple sclerosis ( ms ) is a common neurological disease with increasing incidence and prevalence . onset of disease is most frequently in young adulthood when productivity is usually highest ; it is of chronic nature and , in the majority of patients , it will result in accumulation of disability . due to loss of productivity in patients and caregivers as well as high expenses for medical treatment , ms is considered a disease with high economic burden for patients and society . several drugs have been approved for treatment of ms . while treatment ameliorates the course of the disease , it is very costly ; therefore , pharmacoeconomics , evaluating costs and effects of disease - modifying treatment in ms , has become an important issue . here , we review the economic impact and treatment strategies of ms and discuss recent studies on pharmacoeconomics of early treatment with interferon beta .

Introduction Epidemiology and economic impact of MS Efficacy of early initiation of DMT in MS using interferon beta Methodological aspects of pharmacoeconomic analyses Literature search and quality assessment Early treatment with interferon beta from a pharmacoeconomic perspective Conclusion

the head and neck regions are common sites where intra - dermal or subcutaneous firm nodules of 0.5 - 3 cm are found . although its histological findings are well - described , the radiological features of chondroid syringoma have been reported in only few cases . a 59-year - old man presented with a painless mass on the anterior aspect of the upper thigh with a slow enlargement over the past 5 years [ figure 1 ] . dermatological examination showed a relatively firm , mobile 5 3 cm nodule covered by normal skin . laboratory studies including complete blood count , erythrocyte sedimentation rate ( esr ) , urine analysis , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , blood urea , creatinine were normal . sonographic examination revealed mixed , echogenic solid mass measuring approximately 4.5 3 2 cm . on doppler ultrasound , magnetic resonance images ( mri ) revealed a 4.7 2.9 2.2 cm , well - circumscribed , lobular mass that showed heterogeneous enhancement predominantly in the peripheral portion of the mass after intravenous contrast administration [ figure 2 ] . diffusion - weighted imaging ( dwi ) showed areas of increased signal intensity that were indicative of a highly cellular tumor [ figure 3 ] . according to the mri findings , we considered benign subcutaneous lipomatous tumors with cystic and solid components such as angiolipoma or fibrolipoma or well - circumscribed low - grade soft tissue liposarcoma without surrounding tissue extension in our differential diagnosis . because the vascularity of the lesion and heterogeneous pattern with contrast enhancement on mri examination , we excluded lipoma . relatively firm , 5 3 cm , lipoma - like mass covered by normal skin post - contrast axial fat - saturated t1-weighted images show well - circumscribed , lobular mass with heterogeneous enhancement diffusion - weighted imaging ( b : 800 ) signs of increased restriction in diffusion , a finding indicative of a highly cellular tumor we decided to perform trucut biopsy . histopathological examination revealed a group of cells , which have eosinophilic cytoplasm and regular small nuclei , exhibited epithelial - like pattern in the myxoid stroma . however , as adipocytes were variable in size and focally showed a lipoblast - like appearance , some tumor areas were reminiscent of a well - differentiated lipoma - like liposarcoma [ figure 4 ] . microscopic examination shows a focal area of lipoblast - like appearance in the myxoid background ( h and e , 10 ) the patient underwent surgical excision of the mass . there were foreign body reactions against the epidermoid cysts within the tumor and surrounding tissue . epidermoid cysts were seen ( h and e , 5 ) epithelial nests and tubuloalveolar structures in a chondromyxoid stroma . the head and neck regions are common sites ; less commonly , axillary region , hand , foot , abdomen , penis , vulva , scrotum can be involved . chondroid syringoma usually presents as a solitary , asymptomatic , intracutaneous or subcutaneous nodule , mainly seen in middle - aged or older males . the size is usually between 0.5 and 3 cm , but it may be very large . most chondroid syringomas are benign with a slow progression over years . in our case , malignant lesions are usually larger than 3 cm , but benign one measuring 10 cm is also reported . histologically , a wide variety of structures may be found in chondroid syringoma of skin . eccrine types have multiple small syringoid elements and are rich in mucin . in the apocrine type , in our case , there was abundant lipomatous stroma that made diagnostic difficulty in radiological imaging and histopathological examination . previous case reports have described the mr imaging findings of benign and malignant chondroid syringomas.[79 ] although the mr imaging appearance of chondroid syringoma is relatively non - specific , mr imaging generally may allow characterization of the origin of tissue components in the tumor and depiction of the extent and relationships of the lesion to adjacent structures . in our case , the signal intensity characteristics of the lesion ( heterogeneous lesion of mixed fat and fluid - like intensity ) , while not specific for chondroid syringoma , allowed for some analysis of the consistency of the tumor . the benign neoplasms of the subcutaneous tissue that may have similar appearances with our case include superficial lipoma and variants of lipoma ( angiolipoma , myolipoma , spindle - cell and pleomorphic lipoma , chondroid lipoma , and hibernoma ) . although subcutaneous in location , superficial lipoma consists entirely of fat , unlike our lesion . variants of lipoma are commonly heterogeneous and include more than one type of tissue , which may be fat , muscle , chondroid matrix , vascular elements , or myxoid tissue with calcification and hemorrhage . due to the presence of vascular channels on doppler examination , the malignant tumors that may bear similar characteristics with mixed tumor of the skin include well - differentiated liposarcoma , myxoid liposarcoma , extraskeletal myxoid chondrosarcoma , myxofibrosarcoma , and malignant peripheral nerve sheath tumors . after tru - cut biopsy , we considered many of the above entities in the differential diagnosis . there were no cytologic atypia , increased mitotic figures , tumor necrosis , or infiltrative margins , thus excluding malignant chondroid syringoma . the mixed fat and fluid - like signal intensity lesion of skin is somewhat of a diagnostic enigma due to its non - specific imaging and histologic presentation . clearly a soft tissue lesion of mixed fat and fluid - like signal intensity , as in this case , while not pathognomonic for chondroid syringoma , allows for a limited differential diagnosis . as this case shows , chondroid syringoma should be included in the differential diagnosis of a slowly growing , nodular heterogeneous mass in the skin or subcutaneous tissue . 1 . diffusion - weighted imaging , which showed signal intensity changes indicative of a highly cellular tumor in this case , is not routinely done in chondroid syringoma . to our knowledge , this is the first case of chondroid syringoma mimicking low - grade liposarcoma reported in the literature .

chondroid syringoma , or mixed tumor of skin , is a relatively rare , usually benign sweat gland tumor , most often seen in the head - and - neck region . rare malignant examples have been reported , commonly involving the extremities . we report here a case radiologically mimicking a malignant neoplasm , but histologically - proven benign subcutaneous chondroid syringoma , arising in the anterior aspect of the upper thigh of a 59-year - old male .

Introduction Case Report Discussion

women have relatively weak neck muscles , which can be a primary cause of chronic fatigue syndrome related to the muscular system , resulting in higher risk of chronic neck pain1 . in relation to pain a study by fisher found that 62% of women wear heels that are over 5 cm , and the ones who enjoy wearing high heels are reported to suffer from foot , knee , and back pain , as well as an alteration of the normal gait patterns2 . according to previous studies , wearing high - heeled footwear can alter the static posture and dynamic movements of the body , and is regarded as a cause of musculoskeletal problems related to the spine3,4,5 . walking in high - heeled shoes changes the kinetic characteristics of the lower extremity joints , causing a reduction in ankle plantar flexor muscle moment , and power during stance phase and increases in forefoot peak pressure . walking in high heels produces an upward displacement of the center of mass of the body and possibly a more unstable posture compared to that producted by low - heeled walking6,7,8,9 . additionally , shock and ground reaction forces can lead to increased axial pressure onto the intervertebral discs , resulting in increased erector spinal muscle activation10 . walking in high - heeled shoes produce an increase in the ground reaction force compared to flat heeled shoes . in theory , the increase in ground reaction force with increased height may result from decreased subtalar joint pronation at heel strike and a lengthened tibiofemoral lever arm . as a result , trunk muscle activity increases with increasing heel height , which changes the posture and ground reaction force , leading to discomfort of the back muscle in women who wear high heels11 . in addition , the effects are magnified with increased activation of the erector spinae muscles , which contribute to compression of the spine . therefore , wearing high heels for an extended period can lead to an increase in paraspinal musculature activation of the lumbar and cervical spine , causing prolonged overload and fatigue in the trunk and neck muscles1 . a recent study reported that walking in heels induced a significant increase in the activation of cervical muscles in healthy subjects . this study was conducted in order to examine the effects of wearing high heels during gait on the cervical and lumbar musculature in patients with chronic neck pain and in healthy women . thirteen healthy women were recruited for this study . an information leaflet describing the study method and procedure was used for the recruitment of subjects . the inclusion criteria for patients were as follows : ( 1 ) history of neck pain for the past three months or less , ( 2 ) between the age of 2045 years , ( 3 ) not experiencing orthopedic or neurological problems other than neck pain , and ( 4 ) able to wear high heels without pain . this study excluded subjects who felt acute worsening of pain symptoms during their maximal effort or who were in an unstable condition with an acute flare up during the time of testing . table 1table 1.demographic data of the collective intention to treat ( n=13)variablemeanstandard deviationage ( yrs)31.56.6height ( cm)161.14.2body mass ( kg)55.17.5 shows a list of common characteristic of the participants in this study . a test on the general characteristics of the subjects was filled out prior to conduct of this study . to determine the effects of the different heights of heels on the muscle activation , the paraspinalis cervicis ( cervical spine ) and erector spinae ( lumbar spine ) were measured at the time of heel strike and toe off during gait . after preparing the skin by shaving , cleaning with alcohol , and sanding , two differential surface electrodes were placed at a distance of 2 cm from each other . surface electromyography ( emg ) electrodes were placed on the paraspinalis cervicis and erector spinae of the subjects dominant side . all electrodes were placed over the muscle mass , which was visible during maximally resisted voluntary contraction . two foot switches were placed and attached on the shoe exterior , underneath the first metatarsal head area and the heel of the shoe on the dominant side . to familiarize the subjects to the testing condition with surface electromyography ( emg ) attached to their body , they were instructed to walk down the walkway three times prior to the testing condition . each subject was instructed to walk naturally at a self - selected velocity on a flat and even surface under three different testing conditions ( barefoot , 4 cm stiletto heels , and 10 cm stiletto heels with a base of 1 cm ) . prior to each gait trial , subjects were instructed to stand still with their feet together in a comfortable position , and then were asked to look straight up and walk down the 10 m walkway in front of them . to prevent muscular fatigue , emg data collected during the 10 m walk , the data during the middle 6 m block was used for analysis . data from the three trials were averaged for analysis using the heel strike and toe off events for each gait trial . all subjects with neck pain were in a stable condition without an acute flare up during the time of testing . differences in the emg activity of the paraspinalis cervicis and erector spinae in patients with neck pain during heel strike and toe off during gait were assessed using the spss 12.0 program ( spss inc . , a one - way repeated anova was used for the identification of significant differences among the barefoot , 4 cm high heel , and 10 cm high heel conditions ; and a post - hoc test using the least - significant - difference ( lsd ) method was employed for comparing the conditions . muscle activation in the paraspinalis cervicis and erector spinae at heel strike and toe off ( except that of paraspinalis cervicis at toe off event ) differed significantly with the 10 cm high heels , compared to the barefoot condition . at heel strike , muscle activation of the paraspinalis cervicis was significantly greater with the 10 cm high heels as compared to that with the 4 cm high heels ( table 2table 2.muscle activation ( % mvic ) in normal subjects under the three conditions ( n = 13)musclesgait cyclebarefoot4 cm high - heeled10 cm high - heeledcervical paraspinaeheel strike7.62.17.72.49.43.2toe off7.51.87.11.68.22.5erector spinaeheel strike24.78.229.510.531.212.3toe off28.310.530.311.136.218.3meansd . % mvic : % maximum voluntary isometric contraction . * significant differences compared to barefoot condition . % mvic : % maximum voluntary isometric contraction . * significant differences compared to barefoot condition . this study demonstrated the effects of walking in high heels on the muscular activity of the cervical and lumbar muscles in healthy women . previous studies have reported the muscle activation or biomechanical effects of walking in high heels in healthy women . mika and coworkers studied the effects of walking on barefoot , 4 cm high heels , and 10 cm high heels on the activation of the cervical paraspinalis in young women ( 2025 years ) and older women ( 4555 years ) . the results showed a significant increase in cervical muscle activation while walking in 10 cm high heels . subjects in the older group showed a significant increase in the cervical muscle activation at heel strike while walking in 4 cm high heels and 10 cm high heels . a higher magnitude of vertical ground reaction force was demonstrated with high heels , and the effects were amplified with the presence of more active erector spinae muscles , which could act partially to compress the spine12 , 13 . results of previous studies have demonstrated an increase in the compensated muscular activities with an increasing heel height . wearing high heels changes the overall body posture and loading on the spine and the joints in the lower extremities , resulting in a change in the body s center of mass ( com ) . snow and williams reported that wearing excessively high heels induces a shift in the body s com anteriorly and superiorly . ankle is shifted superiorly and anteriorly , and the base of support ( bos ) shifts anteriorly . as the body tries to maintain this posture without falling , compensatory activity of both the erector spinae and the cervical paraspinalis increases14 . cervical spine movement assists in maintaining the head stability in space , assists in dynamic postural control , and compensates for trunk motion to maintain head stability during gait . with neck extension , the center of mass of the head neck extension may also assist in balancing the head over the trunk due to the center of mass of the head lying anterior to the cervical spine . previous studies also attempted to determine the lumbar curve angle , center of mass , and activation of the erector spinae during the gait of healthy women wearing high - heeled shoes6 , 15 . the results indicated a significant decrease in the lumbar curve angle and , in the vertical movement of the body s center of mass , and a significant increase in activation of the erector spinae . our results showed a significant increase in muscle activation of the erector spinae at heel strike and toe - off events while wearing 10 cm high heel , as compared to that in barefoot condition in all subjects . the results of our study demonstrated that wearing high - heeled shoes increases the activation of cervical and lumbar musculature in healthy women16 . according to mika and colleagues , even healthy people wearing high - heeled shoes experience increased back muscle activity during gait , which could promote local muscle fatigue , in turn leading to tissue deformation , such as swelling or decreased movement13 . in addition , these symptoms may be more remarkable while wearing stiletto type high heels with unstable balance . further studies are needed to evaluate the long - term effects between high - heeled shoes and the mechanism of muscle activation of the cervical and lumbar spine . another limitation of this study was the small number of recruited subjects . for a more accurate observation of the relationship between neck pain and high - heeled shoes , a follow - up study with a longer period of time and future studies will be necessary to evaluate the effect of high - heels on cervical spine kinematics and muscle fatigue over an extended period of time . in addition , consideration of changes in body alignment and muscle activation caused by extrinsic factors , such as high - heeled shoes , may be a more effective approach for planning treatment strategies .

[ purpose ] the purpose of this study was to investigate the effects of different height of high heels on muscle activation of the paraspinalis cervicis and erector spinae in healthy young women . [ subjects and methods ] thirteen healthy women were recruited in this study . to examine the effects of different heights of heels on muscle activation , the paraspinalis cervicis ( cervical spine ) and erector spinae ( lumbar spine ) were measured at the time of heel strike and toe off during gait on three different conditions ( barefoot , 4 cm high heels , and 10 cm high heels ) . there are no previous trials or reports that have evaluated this approach in patients with chronic neck pain . [ results ] a significant increase in muscle activation of the paraspinalis cervicis and erector spinae at heel strike and toe off ( except that of the paraspinalis cervicis at toe off in healthy subjects ) was observed in the under 10 cm high heel condition as , compared to that with barefoot condition , in all the subjects . [ conclusion ] the height of the high heels affects to the activation demand of the paraspinalis cervicis and erector spinae in patients with neck pain .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

life expectancy is among the objective indexes for measuring health for a person , society , or population . in 1880 - 1882 , life expectancy at birth for females in jamaica was 39.8 years which was 2.79 years more than that for males . one hundred and twenty - two year ( 2002 - 2004 ) , this health disparity increased to 5.81 years : life expectancy at birth for female was 77.07 years . for the world , the difference in life expectancy for the sexes was 4.2 years more for females than males : for 2000 - 2005 , life expectancy at birth for females was 68.1 years . within the expanded conceptual framework offered by the world health organization ( who ) in the late 1940s , health is more than the absence of morbidity as it includes social , psychological and physiological wellbeing . some scholars opined that using the opposite of ill - health to measure health is a negative approach is health is more than this biomedical approach . brannon and feist forwarded a positive approach which is in keeping with the biopsychosocial framework developed by engel . engel coined the term biopsychosocial when he forwarded the perspective that patient care must integrate the mind , body and social environment[58 ] . he believed that mentally patient care is not merely about the illness , as other factors are equally influence the health of the patient . although this was not new because the who had already stated this , it was the application which was different from the traditional biomedical approach to the study and treatment of ill patients . embedded in engel 's works were wellbeing , wellness and quality of life and not merely the removal of the illness , which psychologists like brannon and feist called the positive approach to the study and treatment of health . recognizing the limitation of life expectancy , who therefore developed dale disability adjusted life expectancy which discounted life expectancy by number of years spent in illness . the emphasis in the 21 century therefore was healthy life and not length of life ( i.e. life expectancy ) . dale is the years in ill health which is weighted according to severity , which is then subtracted from the expected overall life expectancy to give the equivalent healthy years of life . using healthy years , statistics revealed that the health disparity between the sexes in jamaica was 5 years in 2007 , indicating that health status of females on average in jamaica is better than that for males . this is not atypical to jamaica as females in many nations had a greater healthy life expectancy than males . the discipline of public health is concerned with more than accepting the health disparity as indicated by life expectancy or healthy life expectancy , as it seeks to improve the quality of life of the populace and the various subgroups that are within a particular geographical border . in order for this mandate to be attained , we can not exclude the study of females health merely because they are living longer than males and accept this as a given ; and that there is not need therefore to examine their health status . many empirical studies that have examined health of caribbean nationals were on the population[1115 ] ; elderly[1625 ] ; children ; adolescents[2830 ] and females have been omitted from the discourse . the values for the healthy life expectancy can not be enough to indicator the health status of females neither can use health status of population , children , elderly and adolescents to measure that of females . who forwarded a position that there is a disparity between contracting many diseases and the gender constitution of an individual , suggesting that population health can not be used to measure female health . females have a high propensity than males to contract particular conditions such as depression , osteoporosis and osteoarthritis . a study conducted by mcdonough and walters revealed that women had a 23 percent higher distress score than men and were more likely to report chronic diseases compared to males ( 30% ) . it was found that men believed their health was better ( 2% higher ) than that self - reported by females . mcdonough et al used data from a longitudinal study named canadian national population health survey ( nphs ) . the current study fills the gap in the health literature by investigating health of females in jamaica . the objectives of the current study are 1 ) to examine the health conditions ; 2 ) provide an epidemiological profile of changing health conditions in the last one half decade ( 2002 - 2007 ) ; 3 ) evaluate whether self - reported illness is a good measure of health status ; 4 ) compute the mean age of females having particular health conditions ; 5 ) calculate the mean age of being ill compared with those who are not ill ; and 6 ) assess the correlation between health status and income quintile . the current study extracted subsample of females from two secondary cross - sectional data collected by the planning institute of jamaica and the statistical institute of jamaica . in 2002 , a subsample of 12,675 females was extracted from the sample of 25,018 respondents and for 2007 ; a subsample of 3,479 females was extracted from 6,783 respondents . the survey is called the jamaica survey of living conditions ( jslc ) which began in 1989 . the jslc is modification of the world bank 's living standards measurement study ( lsms ) household survey . trained data collectors are used to gather the data ; and these individuals are trained by the statistical institute of jamaica the survey was drawn using stratified random sampling . this design was a two - stage stratified random sampling design where there was a primary sampling unit ( psu ) and a selection of dwellings from the primary units . the psu is an enumeration district ( ed ) , which constitutes a minimum of 100 residences in rural areas and 150 in urban areas . this means that the country was grouped into strata of equal size based on dwellings ( eds ) . based on the psus , a listing of all the dwellings was made , and this became the sampling frame from which a master sample of dwelling was compiled , which in turn provided the sampling frame for the labor force . one third of the labor force survey ( i.e. lfs ) was selected for the jslc . self - reported illness ( or health conditions ) : the question was asked : is this a diagnosed recurring illness ? the answering options are : yes , cold ; yes , diarrhea ; yes , asthma ; yes , diabetes ; yes , hypertension ; yes , arthritis ; yes , other ; and no . self - rated health status ( health status ) : how is your health in general ? and the options were very good ; good ; fair ; poor and very poor . the first time this was collected for jamaicans , using the jslc , was in 2007 . social class : this variable was measured based on the income quintiles : the upper classes were those in the wealthy quintiles ( quintiles 4 and 5 ) ; middle class was quintile 3 and poor those in lower quintiles ( quintiles 1 and 2 ) . health care - seeking behavior : this is a dichotomous variable which came from the question has a doctor , nurse , pharmacist , midwife , healer or any other health practitioner been visited ? with the option ( yes or no ) . the data were collected , stored and retrieved in spss for windows 16.0 ( spss inc ; chicago , il , usa ) . descriptive statistics were used to provide information on the socio - demographic variables of the sample . cross tabulations were employed to examine correlations between non - metric variables , and analysis of variance ( anova ) was utilized to examine statistical associations between a metric and non - metric variable . the level of significance used in this research was 5% ( i.e. 95% confidence interval ) . bryman and cramer correlation coefficient values were used to determine , the strength of a relation between ( or among ) variables : 0.19 and below , very low ; 0.20 to 0.39 , low ; 0.40 to 0.69 , moderate ; 0.70 to 0.89 , high ( strong ) ; and 0.90 to 1 is very high ( very strong ) . the data were collected , stored and retrieved in spss for windows 16.0 ( spss inc ; chicago , il , usa ) . descriptive statistics were used to provide information on the socio - demographic variables of the sample . cross tabulations were employed to examine correlations between non - metric variables , and analysis of variance ( anova ) was utilized to examine statistical associations between a metric and non - metric variable . the level of significance used in this research was 5% ( i.e. 95% confidence interval ) . bryman and cramer correlation coefficient values were used to determine , the strength of a relation between ( or among ) variables : 0.19 and below , very low ; 0.20 to 0.39 , low ; 0.40 to 0.69 , moderate ; 0.70 to 0.89 , high ( strong ) ; and 0.90 to 1 is very high ( very strong ) . the data were collected , stored and retrieved in spss for windows 16.0 ( spss inc ; chicago , il , usa ) . descriptive statistics were used to provide information on the socio - demographic variables of the sample . cross tabulations were employed to examine correlations between non - metric variables , and analysis of variance ( anova ) was utilized to examine statistical associations between a metric and non - metric variable . the level of significance used in this research was 5% ( i.e. 95% confidence interval ) . bryman and cramer correlation coefficient values were used to determine , the strength of a relation between ( or among ) variables : 0.19 and below , very low ; 0.20 to 0.39 , low ; 0.40 to 0.69 , moderate ; 0.70 to 0.89 , high ( strong ) ; and 0.90 to 1 is very high ( very strong ) . in 2002 , 14.7% of sample reported an illness and this increased by 19.1% in 2007 . over the same period , health insurance coverage increased by 81.0% ( to 21.0% in 2007 ) ; those seeking medical care increased to 67.6% ( from 66.0% ) ; the mean age in 2007 was 30.621.9 years which marginal increased from 29.4 22.3 years ; diabetic cases exponentially increased by 227.7% ( in 2007 , 15.4% ) ; hypertension decline by 45.5% ( to 24.8% in 2007 ) and arthritic cases felt by 66.1% ( to 9.4% in 2007 ) . urbanization was evident between 2007 and 2002 as the number of females who resided in urban areas increased by 114.7% ( to 30.4% in 2007 ) , with a corresponding decline of 19.4% in females zones . table 1 revealed that the increase in self - reported illness was substantially accounted for by increased cases in the rural sample ( from 12.9% in 2002 to 20.0% in 2007 ) . the drastic increase in health insurance coverage in 2007 was due to public establishment of public health insurance coverage . the greatest increase was observed in semi - urban areas 17.8% ) followed by urban ( 9.6% ) and rural ( 7.8% ) ( table 1 ) . the increases in self - reported illness can be accounted for by diabetes mellitus , asthma and other dysfunctions . concurrently , most of the increased cases were diabetic in semi - urban zones ( 17.1% ) ; other health conditions in semi - urban areas ( 12.4% ) and asthma in urban zones ( 12.0% ) ( table 1 ) . sociodemographic characteristics of sample by area of residence , 2002 and 2007 there was a significant statistical correlation between health status and self - reported illness - ( df = 4 ) = 700.633 , p < 0.001 ; with there being a negative moderate relation between the variables correlation coefficient = - 0.412 ( table 2 ) . based on table 2 , 10.7% of those who reported an illness had had very good health status compared to 40.2% of those who did not indicate an illness . on the other hand , 2.5% of those who did not report a dysfunction had at least poor health status compared to 19.8% of those who indicated having an illness . even after controlling health status and self - reported illness by age , marital status and per capita annual expenditure , a moderate negative correlation was found - correlation coefficient = - 0.362 . health status by self - reported illness , 2007 on further examination of the self - reported illness by age , it was found that in 2002 the mean age of individual who reported an illness was 43.97 26.81 years compared to 27.05 20.41 years for who without an illness t - test = 30.818 , p < 0.001 . in 2007 , the mean age of reporting an illness was 42.83 26.53 years compared to 28.16 19.95 years for those who did not report an ailment t - test = 15.263 , p < 0.001 . based on figure 1 , there is an increase in the mean age of females being diagnosed with diarrhea ( 32.00 36.2 years ) and asthma ( 21.73 20.51 years ) . however , there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007 ) . a cross tabulation between health status and income quintile revealed a significant statistical correlation - ( df = 16 ) = 54.044 , p < 0.001 ; with the relationship being a very weak one - correlation coefficient = 0.126 ( table 3 ) . based on table 3 , the wealthy reported the greatest health status ( i.e. very good ) compared to the wealthiest 20% ( 36.7% ) ; with the poorest 20% recorded the least very good health status . health status by income quintile , 2007 no significant statistical correlation was found between diagnosed self - reported illness and income quintile - ( df = 28 ) = 36.161 , p > 0.001 ( table 4 ) . in 2002 , 14.7% of sample reported an illness and this increased by 19.1% in 2007 . over the same period , health insurance coverage increased by 81.0% ( to 21.0% in 2007 ) ; those seeking medical care increased to 67.6% ( from 66.0% ) ; the mean age in 2007 was 30.621.9 years which marginal increased from 29.4 22.3 years ; diabetic cases exponentially increased by 227.7% ( in 2007 , 15.4% ) ; hypertension decline by 45.5% ( to 24.8% in 2007 ) and arthritic cases felt by 66.1% ( to 9.4% in 2007 ) . urbanization was evident between 2007 and 2002 as the number of females who resided in urban areas increased by 114.7% ( to 30.4% in 2007 ) , with a corresponding decline of 19.4% in females zones . table 1 revealed that the increase in self - reported illness was substantially accounted for by increased cases in the rural sample ( from 12.9% in 2002 to 20.0% in 2007 ) . the drastic increase in health insurance coverage in 2007 was due to public establishment of public health insurance coverage . the greatest increase was observed in semi - urban areas 17.8% ) followed by urban ( 9.6% ) and rural ( 7.8% ) ( table 1 ) . the increases in self - reported illness can be accounted for by diabetes mellitus , asthma and other dysfunctions . concurrently , most of the increased cases were diabetic in semi - urban zones ( 17.1% ) ; other health conditions in semi - urban areas ( 12.4% ) and asthma in urban zones ( 12.0% ) ( table 1 ) . sociodemographic characteristics of sample by area of residence , 2002 and 2007 there was a significant statistical correlation between health status and self - reported illness - ( df = 4 ) = 700.633 , p < 0.001 ; with there being a negative moderate relation between the variables correlation coefficient = - 0.412 ( table 2 ) . based on table 2 , 10.7% of those who reported an illness had had very good health status compared to 40.2% of those who did not indicate an illness . on the other hand , 2.5% of those who did not report a dysfunction had at least poor health status compared to 19.8% of those who indicated having an illness . even after controlling health status and self - reported illness by age , marital status and per capita annual expenditure , a moderate negative correlation was found - correlation coefficient = - 0.362 . health status by self - reported illness , 2007 on further examination of the self - reported illness by age , it was found that in 2002 the mean age of individual who reported an illness was 43.97 26.81 years compared to 27.05 20.41 years for who without an illness t - test = 30.818 , p < 0.001 . in 2007 , the mean age of reporting an illness was 42.83 26.53 years compared to 28.16 19.95 years for those who did not report an ailment t - test = 15.263 , p < 0.001 . based on figure 1 , there is an increase in the mean age of females being diagnosed with diarrhea ( 32.00 36.2 years ) and asthma ( 21.73 20.51 years ) . however , there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007 ) . a cross tabulation between health status and income quintile revealed a significant statistical correlation - ( df = 16 ) = 54.044 , p < 0.001 ; with the relationship being a very weak one - correlation coefficient = 0.126 ( table 3 ) . based on table 3 , the wealthy reported the greatest health status ( i.e. very good ) compared to the wealthiest 20% ( 36.7% ) ; with the poorest 20% recorded the least very good health status . health status by income quintile , 2007 no significant statistical correlation was found between diagnosed self - reported illness and income quintile - ( df = 28 ) = 36.161 , p > 0.001 ( table 4 ) . in 2002 , 14.7% of sample reported an illness and this increased by 19.1% in 2007 . over the same period , health insurance coverage increased by 81.0% ( to 21.0% in 2007 ) ; those seeking medical care increased to 67.6% ( from 66.0% ) ; the mean age in 2007 was 30.621.9 years which marginal increased from 29.4 22.3 years ; diabetic cases exponentially increased by 227.7% ( in 2007 , 15.4% ) ; hypertension decline by 45.5% ( to 24.8% in 2007 ) and arthritic cases felt by 66.1% ( to 9.4% in 2007 ) . urbanization was evident between 2007 and 2002 as the number of females who resided in urban areas increased by 114.7% ( to 30.4% in 2007 ) , with a corresponding decline of 19.4% in females zones . table 1 revealed that the increase in self - reported illness was substantially accounted for by increased cases in the rural sample ( from 12.9% in 2002 to 20.0% in 2007 ) . the drastic increase in health insurance coverage in 2007 was due to public establishment of public health insurance coverage . the greatest increase was observed in semi - urban areas 17.8% ) followed by urban ( 9.6% ) and rural ( 7.8% ) ( table 1 ) . the increases in self - reported illness can be accounted for by diabetes mellitus , asthma and other dysfunctions . concurrently , most of the increased cases were diabetic in semi - urban zones ( 17.1% ) ; other health conditions in semi - urban areas ( 12.4% ) and asthma in urban zones ( 12.0% ) ( table 1 ) . there was a significant statistical correlation between health status and self - reported illness - ( df = 4 ) = 700.633 , p < 0.001 ; with there being a negative moderate relation between the variables correlation coefficient = - 0.412 ( table 2 ) . based on table 2 , 10.7% of those who reported an illness had had very good health status compared to 40.2% of those who did not indicate an illness . on the other hand , 2.5% of those who did not report a dysfunction had at least poor health status compared to 19.8% of those who indicated having an illness . even after controlling health status and self - reported illness by age , marital status and per capita annual expenditure , a moderate negative correlation was found - correlation coefficient = - 0.362 . health status by self - reported illness , 2007 on further examination of the self - reported illness by age , it was found that in 2002 the mean age of individual who reported an illness was 43.97 26.81 years compared to 27.05 20.41 years for who without an illness t - test = 30.818 , p < 0.001 . in 2007 , the mean age of reporting an illness was 42.83 26.53 years compared to 28.16 19.95 years for those who did not report an ailment t - test = 15.263 , p < 0.001 . based on figure 1 , there is an increase in the mean age of females being diagnosed with diarrhea ( 32.00 36.2 years ) and asthma ( 21.73 20.51 years ) . however , there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007 ) . a cross tabulation between health status and income quintile revealed a significant statistical correlation - ( df = 16 ) = 54.044 , p < 0.001 ; with the relationship being a very weak one - correlation coefficient = 0.126 ( table 3 ) . based on table 3 , the wealthy reported the greatest health status ( i.e. very good ) compared to the wealthiest 20% ( 36.7% ) ; with the poorest 20% recorded the least very good health status . health status by income quintile , 2007 no significant statistical correlation was found between diagnosed self - reported illness and income quintile - ( df = 28 ) = 36.161 , p > 0.001 ( table 4 ) . the current study found a moderate significant correlation between the two aforementioned variables , suggesting that self - reported illness is a relatively good measure of female 's health . in this study it was revealed that 60 out of every 100 who reported an illness had at most fair health status , with 20 out every 100 indicated a least poor health . it is evident from the findings that health status is wider than illness , which concurs with the literature , which is keeping with the propositions of the who that health must be more than the absence of illness . health status is people 's self - rated perspective on their general health status , which includes a percentage of poor health ( or ill - health ) . the other components of this status include life satisfaction , happiness , and psychosocial wellbeing . using a sample of elderly barbadians , hambleton et there is a disparity between the current study and that of hambleton 's work as more of health status of the elderly is explained by current illness with this being less for females in jamaica . concomitantly , there is an epidemiological shift in the typology of illnesses affecting females as the change is towards diabetes mellitus . in 2007 over 2002 , the 15 out of every 100 females reported being diagnosed with diabetes mellitus compared to 5 in 100 in 2002 indicating the negative effects of life behavior of female 's health status . another important finding of the current study is that diagnosed illnesses are not significant different based on income quintile in which a female is categorized . however , the health status of females in different social standing ( measured using income quintile ) is different . embedded in this finding is the role of income plays in improving health status . like marmot , this study found that income is able to buy some improvement in health status ; but this work goes further as it found that income it does not reduce the typology is health conditions affecting the female . before this discussion can proceed , the discourse must address the biases in subjective indexes which are found in studies like this one . any study on subjective indexes in the measurement of health ( for example , happiness , life satisfaction ; health status , self - reported illness ) needs to address the challenges of biases that are found in self - reported data in particular self - reported health data . the discourse of subjective wellbeing using survey data can not deny that it is based on the person 's judgment , and must be prone to systematic and non - systematic biases . diener argued that the subjective measure seemed to contain substantial amounts of valid variance , suggesting that there is validity to the use of this approach in the measurement of health ( or wellbeing ) like the objective indexes such as life expectancy , mortality or diagnosed morbidity . a study by finnas et al opined that there are some methodological issues surround the use of self - reported ( or self - rated ) health and that these may result in incorrect inference ; but that this measure is useful in understanding health , morbidity and mortality . using life expectancy and self - reported illness data for jamaicans , bourne found a strong significant correlation between the two variables ( correlation coefficient , r = - 0.731 ) , and that self - reported illness accounted for 54% of the variance in life expectancy . when bourne disaggregated the life expectancy and self - reported illness data by sexes , he found a strong correlation between males ( correlation coefficient , r = 0.796 ) than for females ( correlation coefficient , r = 0.684 ) . self - reported data therefore do have some biases ; but that it is good measure for health in jamaica and more so for males . in spite of this fact , the current research recognized some of the problems in using self - reported health data ( read finnas et al . for more information ) , while providing empirical findings using people 's perception on their health . now that the discourse on objective and subjective indexes of is some out of the way , the next issue of concern is the reduced aged of reported illness and age of being diagnosed with particular chronic illness . in 2002 , the mean age recorded for those who self - reported an illness was 44 years and this fell by 1 year in 2007 , indicating that on average females are becoming diagnosed with an illness 2 months earlier . when self - reported illness was disaggregated into acute and chronic health conditions , it was revealed that on average females were being diagnosed 7.41 years earlier with arthritis in 2007 over 2002 ; 4.95 years earlier with hypertension and 1.13 years earlier with diabetes mellitus . the current study has revealed that , although healthy life expectancy for females at birth in jamaica is 66 years , improvements in their health status can not be neglected as there are shits in health conditions ( to diabetes mellitus ) as well as the decline in ages at which females are being diagnosed with particular chronic illnesses .

background : the 21st century can not see the examination of health status of elderly , population , children and adolescents ; but not for females.aims:current study are 1 ) to examine the health conditions ; 2 ) provide an epidemiological profile of changing health conditions in the last one half decade ( 2002 - 2007 ) ; 3 ) evaluate whether self - reported illness is a good measure of health status ; 4 ) compute the mean age of females having particular health conditions ; 5 ) calculate the mean age of being ill compared with those who are not ill ; and 6 ) assess the correlation between health status and income quintile.methods and results : in 2002 , a subsample of 12,675 females was extracted from the sample of 25,018 respondents and for 2007 ; a subsample of 3,479 females was extracted from 6,783 respondents.results:there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007).conclusion : although healthy life expectancy for females at birth in jamaica was 66 years , improvements in their health status can not be neglected as there are shits in health conditions ( to diabetes mellitus ) as well as the decline in ages at which females are being diagnosed with particular chronic illnesses .

Introduction Materials and Methods None Statistical analysis Results None Demographic characteristic of sample Health Discussion Conclusions

frontometaphyseal dysplasia ( fmd ) , also called gorlin cohen syndrome , is a hereditary x - linked dominant syndrome described in 1969 with less than 30 cases described in the literature . this case report of a child with fmd is presented owing to the rarity of the syndrome and the anticipated difficult airway , which was successfully managed by using a combination of dexmedetomidine and ketamine while preserving spontaneous ventilation . a 2-year - old female child , a known case of fmd , presented for open reduction of the left hip with osteotomy of femur . physical examination revealed a slender undernourished girl of 8 kg with prominent supraorbital ridges , ocular hypertelorism , low set ears and a wide bridge nose with prominent eyes . airway examination revealed a mallampatti score of iii with significant retrognathia , high arched palate with malocclusion of teeth . in addition , she had dorsolumbar scoliosis , pectus carinatum , bowing of long bones with distal phalangeal hypoplasia and multiple joint dislocations [ figure 1 ] . pre - operative blood investigations , echocardiography and chest x - ray were within physiological limits . on arrival to the operation theatre , monitors were connected and child pre - oxygenated for 5 minutes . injection dexmedetomidine 1 g / kg was administered for 10 min and then a continuous infusion at 1 g / kg / h was set for the duration of the remaining procedure . ketamine was administered in increments of 5 mg up to 12 mg until there was no response to jaw thrust while ensuring spontaneous respiration . just before direct laryngoscopy intravenous lignocaine rigid laryngoscopy with miller 1 straight blade offered a grade iv cormack and lehane view . after optimal external laryngeal manipulation , the visible glottic chink was sprayed with topical lignocaine and tracheal intubation was successfully performed using an uncuffed 4 sized endotracheal tube . anesthesia was continued with n2o in 40% o2 along with a continuous dexmedetomidine and atracurium infusion . adequate padding was provided at pressure points and extreme caution was exercised during positioning . at the end of fmd belongs to the otopalatodigital spectrum syndromes that includes four phenotypically related conditions , otopalatodigital syndrome types 1 and 2 , fmd and melnick - needles syndrome . the most common manifestations include supraorbital hyperostosis , hypertelorism , down - slanting palpebral fissures , broad nasal bridge and micrognathia with anomalies of teeth and generalized skeletal dysplasia . congenital heart disease , subglottic tracheal narrowing and genitourinary anomalies , muscular hypotonia . micrognathia , microstomia and malocclusion of teeth may make direct laryngoscopy impossible ; therefore , a well - planned airway strategy is mandatory . ketamine was preferred in our case of anticipated difficult airway due to it 's inherent sympathomimetic actions devoid of respiratory depression alongwith provision of excellent analgesia and amnesia . dexmedetomidine a specific and selective 2-adrenoceptor agonist known for its sedative , anxiolytic , analgesic properties was used to complement ketamine . at the same time dexmedetomidine offsets the sympathomimetic effects of ketamine , this unique pharmacological combination in the present case preserved the respiratory drive , allowed maintenance of a patent airway and provided sufficient sedation , analgesia and anesthesia to allow successful airway control . in addition , topical lignocaine was used as per recommendation of aroni et al . which states that ketamine does not depress coughing or swallowing reflexes . available literature describes the use of combination of both these drugs in children during procedural anesthesia and not as a complete anesthesia protocol in a challenging case . the present experience of using this combination successfully paves the way to emerging new solutions for management of a difficult pediatric airway . hence safety profile , rapid onset of action with adequate sedation and analgesia provided by the ketamine and dexmedetomidine make them a distinctive drug combination in the pediatric difficult airway situation in a child with fmd .

frontometaphyseal dysplasia ( fmd ) , also called gorlin - cohen syndrome , is a rare hereditary x - linked dominant craniotubular bone disorder . the presentation describes the airway management of a 2-year - old child suffering from fmd with significant retrognathia , posted for major long bone corrective osteotomy . induction with a combination of dexmedetomidine and ketamine preceded a successful endotracheal intubation under spontaneous ventilation .

Introduction Case Report Discussion

for a prospective study of lead exposure and early development , we recruited pregnant women from a lead smelter town and from an unexposed town in yugoslavia and followed their children through 7 years of age . in this paper we consider associations between lifetime lead exposure , estimated by the area under the blood lead ( bpb ) versus time curve ( auc7 ) , and intelligence , with particular concern for identifying lead 's behavioral signature . the wechsler intelligence scales for children - version iii ( wisc - iii ) was administered to 309 7-year - old children , 261 of whom had complete data on intelligence , blood lead , and relevant sociodemographic covariates ( i.e. , home observation for the measurement of the environment ( home ) , birth weight , gender , sibship size , and maternal age , ethnicity , intelligence , and education ) . these showed anticipated associations with 7-year intelligence , explaining 41 - 4% of the variance in full scale , performance , and verbal iq . before covariate adjustment , auc7 was unrelated to intelligence ; after adjustment , auc7 explained a significant 2.8%-4.2% of the variance in iq . after adjustment , a change in lifetime bpb from 10 to 30 micro / dl related to an estimated decrease of 4.3 full scale iq points ; estimated decreases for verbal and performance iq were 3.4 and 4.5 points , respectively . auc7 was significantly and negatively related to three wisc - iii factor scores : freedom from distractibility , perceptual organization , and verbal comprehension ; the association with perceptual organization was the strongest . consistent with previous studies , the iq / lead association is small relative to more powerful social factors . findings offer support for lead 's behavioral signature ; perceptual - motor skills are significantly more sensitive to lead exposure than are the language - related aspects of intelligence.imagesfigure 1 . afigure 1 . bfigure 1 . cfigure 1 . dfigure 2 .

Images

multiple sclerosis ( ms ) is a chronic , autoimmune , demyelinating disease of the central nervous system ( cns ) . presenting symptoms can vary greatly , but most commonly involve weakness , paresthesia , gait difficulty , or visual deficits . virtually any area of the cns white matter can be involved , though this most classically involves the periventricular white matter . brain stem involvement is common , though isolated cranial nerve palsies are rare signs in ms . previous studies have suggested that isolated cranial nerve palsies in ms are more commonly found as presenting symptoms than as relapsing symptoms . among isolated cranial nerve palsies in ms , the fifth nerve is most commonly involved ( 4.8% ) , followed by the seventh nerve ( 3.7% ) , and the sixth nerve ( 1.0% ) . thus , abducens palsy is a rare isolated ms finding , either as a presenting sign or during disease exacerbation . patients with abducens palsy typically present with diplopia upon horizontal gaze , and examination can reveal a slow ipsilateral lateral rectus movement [ 4 , 5 ] . ms has been implicated as the cause of unilateral abducens palsy in 49% of cases , though mri may not detect brain stem lesions in all cases . one 2002 study investigating nontraumatic causes of sixth nerve palsies in patients 2050 years of age found ms to be the cause in 24% of cases . in addition to ms , the differential diagnosis for an abducens nerve palsy includes mass lesions , lyme disease , viral infection , syphilis , sarcoidosis , and vascular disease . here , we report a patient who presented with a unilateral isolated abducens palsy as the initial sign of ms . a 28-year - old man with a past medical history of hypertension and obesity presented to the emergency department with a 1-day history of double vision , most prominent upon left lateral gaze . the double vision resolved with covering either eye . in addition , he had a 12-week history of paresthesia of the distal right hand and forearm , and the fifth digit of the right foot . initial laboratory findings revealed only a mild leukocytosis ( 11.3 10/l ) and no other hematologic or electrolyte abnormalities . several focal areas of increased t2 signal intensity were noted within the periventricular white matter of the frontal and parietal lobes , subcortical white matter of the left temporal lobe , and the left pons . these findings likely represented multiple lesions distributed in both time and space , and , along with the patient 's clinical history and examination , were suggestive of a diagnosis of ms . the patient was admitted and started on intravenous methylprednisolone . further workup to rule out other etiological causes of illness revealed an elevated esr of 25 mm / h , negative viral and lyme serologies , nmo igg negative and a negative ana . he was treated with methylprednisolone 500 mg intravenously for 3 days and then discharged home . he was re - evaluated in the office 2 days after hospital discharge and had complete resolution of the cn vi palsy . isolated nerve palsies occur in only 10.4% of patients with ms , with abducens palsy as the third most common isolated nerve palsy , occurring in 1.0% of patients with ms , behind trigeminal ( 4.8% ) and facial ( 3.7% ) . these palsies occur most commonly at disease onset , though they can also occur during the course of the disease . the lesion in the medial pons was enhancing and thus consistent with an active lesion , and the location was consistent with his cn vi palsy . the sixth nerve nucleus is located in the pontine tegmentum , and a compact fiber tract bundle containing motor neurons from this nucleus runs medially towards the ventral region , where the cranial nerve exits the pons [ 3 , 8 ] . our patient 's pontine lesion appeared to be located along the fiber tract emerging from the sixth nerve nucleus , and correlates with his diplopia . while an mri lesion was detected clearly in this case , it should be noted that previous authors have found that mri does not always detect brain stem lesions accounting for these palsies in ms patients [ 4 , 6 ] . in any patient presenting with isolated cranial nerve palsies , ms must be considered within the differential diagnosis . in patients younger than 50 years of age ( as with our patient ) , infectious causes ( lyme disease , viral infections , syphilis ) , sarcoidosis , and autoimmune vasculitis should also be considered on the differential diagnosis , whereas for older patients small vessel vascular disease should be considered as well [ 7 , 8 ] . the presence of multiple presenting neurological deficits including cranial nerve palsies is suspicious for ms and should be investigated with mri . though mri may not always detect brain stem lesions responsible for cranial nerve palsies , it can reveal other white matter lesions of the cns that can aid in the diagnosis of ms , as was the case with our patient .

while brain stem involvement in multiple sclerosis ( ms ) is relatively common , isolated cranial nerve palsies are rare , especially when they represent the initial presenting sign of a new diagnosis of ms . this report describes a patient with no prior history of ms whose sole presenting sign was an isolated abducens palsy . an enhancing pontine lesion was found on mri which correlated with his abducens palsy , and additional nonactive lesions on mri led to a diagnosis of ms . this case demonstrates the importance of considering ms as part of the differential diagnosis of patients with isolated cranial nerve palsies .

Introduction Case Description Discussion Statement of Ethics Disclosure Statement

according to the world health organization ( who ) , a maternal death is defined as death of a woman while pregnant or within 42 days of termination of pregnancy , irrespective of the duration and site of pregnancy , from any cause related to or aggravated by pregnancy or its management ( icd-10 ) . about 99% of these women are from developing world with over 90% concentrated in africa and asia . the risk of a woman dying as a result of pregnancy and childbirth during her lifetime is about 1 in 6 in afghanistan compared with 1 in 30,000 in northern europe . united nation ( un ) report card on millennium development goal-5 concluded that little progress had been made in sub - saharan africa where half of all maternal deaths take place . the progress shown by the south asian countries including india which accounts for 25% of all maternal deaths is also not impressive . most of the evidence for maternal mortality is obtained through hospital data and community based reports , which are situated mostly in urban areas , whereas most of the maternal deaths are from rural areas . this study was done to assess maternal mortality in a tertiary medical college hospital situated in semi urban part of western maharashtra where large numbers of patients are referred from rural parts of western maharashtra and north karnataka . this study was done to assess the local causes of maternal mortality and suggest remedial measures to reduce the same . to calculate the maternal mortality rate in our hospital.to assess the epidemiological aspects of maternal mortality.to assess the causes of maternal mortality.to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital . to assess the epidemiological aspects of maternal mortality . to assess the causes of maternal mortality . to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital.to assess the epidemiological aspects of maternal mortality.to assess the causes of maternal mortality.to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital . to assess the epidemiological aspects of maternal mortality . to assess the causes of maternal mortality . to suggest ways to reduce the mmr our hospital is a semi - urban , tertiary care center ; situated in the southernmost part of western maharashtra . it gets a large number of referrals from maternity homes , primary health centers from rural parts of western maharashtra , and also from north karnataka . the present study was a retrospective study , conducted in the department of obstetrics and gynecology of this hospital . data regarding maternal mortality was collected from maternal mortality register after obtaining permission from the medical superintendent of the hospital . the details of maternal deaths from january 2001 to december 2010 were collected and analyzed with respect to following epidemiological parameters . locality wise distribution of maternal deaths.literacy wise distribution of maternal deaths.gravidity wise distribution of maternal deaths.maternal deaths according to receipt of antenatal care.distribution of maternal deaths according to socioeconomic status.causes of maternal deaths . the details of number of live births from january 2001 to december 2010 were collected from labor ward register . maternal mortality rate for the study period was calculated by using the formula- mean maternal mortality ratio for the study period was calculated by calculating the mean of yearly mmr of the entire study period . during the study period , january 2001 to december 2010 , there were a total of 39704 live births and 120 maternal deaths . maximum maternal deaths ( 49.16% ) were reported in the age group of 20 to 24 years . ( 43.33% ) , more maternal deaths were reported in women from rural areas ( 69.16% ) as compared to women from urban areas ( 30.83%).maximum maternal deaths were reported in unbooked patients ( 83.33% ) as compared to booked patients ( 16.66% ) . epidemiological characteristics of maternal deaths ( n = 120 ) in the study period , 72.5% of maternal deaths were due to direct causes . year - wise distribution of direct causes of maternal deaths is shown in table 2 . the classical triad of hemorrhage ( 26.66% ) , eclampsia ( 26.66% ) , and sepsis ( 18.33% ) was the major direct causes of maternal deaths , whereas only one maternal death ( 0.83% ) was due to obstructed labor . in the study period , anemia , jaundice , and heart disease accounted for 10% , 9.16% , and 3.33% of maternal deaths respectively and miscellaneous cause like acute gastroenteritis accounted for 5% of maternal deaths as shown in table 3 . high incidence of maternal deaths reflects poor quality of maternal services , late referral and low socioeconomic status of the community . various studies done in india in the last 15 years have shown wide variation in mmr ranging from 47/100000 to 625/100000 births.[49 ] madhu jain has reported a very high mmr of 2270/100000 . this study has comparatively high mmr , which could be due to the fact , that our hospital is a tertiary care hospital and receives a lot of complicated referrals from rural areas of southern maharashtra and also from north karnataka at a very late stage . in our study , 70% of maternal deaths were in the age group of 20 to 29 years , as highest numbers of births are reported in this age group . more maternal deaths were reported in women from rural areas ( 69.16% ) , unbooked patients ( 83.33% ) , illiterate women ( 65% ) , and women belonging to low socioeconomic status . ( 83.33% ) all our findings were similar to studies by jain , jadhav , pal , onakewhor . in our study , hemorrhage ( 26.66% ) , eclampsia ( 26.66% ) , and sepsis ( 18.33% ) were the major direct causes of maternal deaths . our findings were consistent with studies by jain , jadhav , pal , onakewhor , and shah . even today large number of maternal deaths is due to the classical triad of hemorrhage , sepsis , and eclampsia . all these are preventable causes of maternal mortality provided the treatment is instituted in time . unfortunately , in many cases , patients were referred very late , in critical condition , unaccompanied by health care worker . many patients had to travel a distance of 70 to 80 kilometers in a private vehicle to reach our tertiary center . most of these deaths are preventable if patients are given appropriate treatment at periphery and timely referred to higher centers . training of medical officers and staff nurses working in rural areas by programs like basic emergency obstetrics care ( bemoc ) and skilled attendant at birth ( sab ) training gives a ray of hope of reducing maternal mortality . anemia , jaundice , and heart disease were responsible for 10% , 9.16% , and 3.33% of maternal deaths , respectively . these findings were consistent with studies by jain , jadhav , pal , and onakewhor . maternal deaths can be prevented by improving the health care facilities in rural areas by ensuring round the clock availability of certain basic drugs like injection magnesium sulfate , tablet misoprostol as most maternal deaths in rural areas are still due to eclampsia and post partum hemorrhage . early detection of high risk pregnancies and referring them to a tertiary center at the earliest can reduce the complications of high risk pregnancies . national rural health mission ( nrhm ) can play a major role in reducing maternal mortality by advocating institutional deliveries and timely referral of high risk cases . although we have not actually evaluated the impact of aforementioned educational programs on maternal mortality , it would be interesting to direct future studies in this regard . even today most maternal deaths are seen in patients from rural areas , unbooked , illiterate patients and patients from low socioeconomic status . hemorrhage , eclampsia and sepsis are the major causes of maternal deaths . improvement in primary health care in rural areas and proper implementation of nrhm programs and up gradation of hospitals in rural areas can definitely bring down the number of maternal deaths .

background : epidemiological data pertaining to maternal mortality is valuable in each set up to design interventional programs to favourably reduce the ratio . this study was done to evaluate the maternal mortality rate in our hospital , to assess the epidemiological aspects and causes of maternal mortality , and to suggest recommendations for improvement.methods:this was a 10 year retrospective study . epidemiological data was collected from the hospital register and maternal mortality ratio , epidemiological factors and causes affecting maternal mortality were assessed.results:a total of 120 maternal deaths occurred . most maternal deaths occurred in the age group of 2024 years , multiparous women ( 56.66% ) , women from rural areas ( 69.16% ) , illiterate women ( 65% ) , unbooked patients ( 83.33% ) , and patients of low socioeconomic status ( 83.33% ) . direct causes accounted for 72.5% of maternal deaths where as 27.5% of maternal deaths were due to indirect causes.conclusion:there is a wide scope for improvement as a large proportion of the observed deaths are preventable .

INTRODUCTION Aims and objectives METHODS RESULTS DISCUSSION CONCLUSIONS

head and neck squamous cell carcinoma ( hnscc ) is the sixth most common cancer worldwide , with an average 5-year survival rate of around 50% ( 1 ) . despite improvements in the therapeutic modalities , the prognosis of these patients has not been significantly improved in the last three decades . alcohol and both smoked and smokeless tobacco use are associated with increased risk of developing malignancy of the oral cavity and pharynx ( 2 ) . recently , the role of oncogenic viruses especially hpv in the pathogenesis of scc has attracted the researchers attention . hpv viral proteins , e6 and e7 , play major roles in the cervical as well as orophararyngeal carcinogenesis . this proteins act through inactivation of tumor suppressor genes , p53 and retinoblastoma ( rb ) genes , resulting in cell immortalization and transformation ( 4 ) . perhaps one of the most important developments in the head and neck oncology over the past decade is the recognition of hpv status in oropharyngeal sccs ( opsccs ) . it is obvious that hpv - ralated opscc has unique epidemiological and clinical entity and it had better distinguishes from the ocsccs because of unknown role of hpv in non - oropharyngeal head and neck cancers ( 5 - 7 ) . ( 9 ) , and lingen et al . in 2013 ( 10 ) considered low etiologic role for hpv status in ocsccs . mentioned that probably other risk factors that are still unknown play roles in the pathogenesis of ocsccs . in recent years , much attention has been paid to the prognostic role of hpv status in hnscc . some researchers have found that patients with hpv detected in their cancers , have better prognosis compared to those without hpv ( 11 - 15 ) . nevertheless there are studies that have found no difference or poorer prognosis for hpv related tumors ( 16 - 22 ) . in this study , we have investigated the prevalence rate of hpv status in patients with ocscc , and its significance in the clinicopathological characteristics of tumors and patients prognosis . 132 patients with diagnosis of ocscc were included in this study between 2001 and 2013 . their records were collected from the files of department of pathology , faculty of dentistry and omid hospital , mashhad university of medical sciences , iran . age , gender , anatomical region , tnm stage , histological grade , therapeutic interventions , events including local , distant relapse and death were extracted from the medical records database . the haematoxylin and eosin stained sections from all specimens were reviewed by two pathologists to confirm the histology and grading . histological grading was assessed according to the who guidelines and tumor staging was reported according to the american joint committee on cancer staging criteria ( ajcc sixth edition , 2002 ) . cleaning off the microtome blade with xylene and ethanol after cutting each paraffin block was taken for preventing and monitoring contamination of samples . the tumor samples were deparaffinized using a series of xylene and ethanol washes . in this study , the tissue sample was mixed with 400 l digestion buffer which consisted of tris - cl ( 100 mm , ph 7.5 ) and tween-20 ( 0.055 ) and proteinase k 3l ( 20 mg / ml ; frementase , germany ) , followed by incubation at 56c for 3 hour . after the complete digestion of tissue , the mixture was incubated at 100c in hotblock ( accublock digital dry baths ; usa ) to inactivate proteinase k. the samples were then centrifuged at 5000 rpm , and the supernatant containing dna was used for pcr amplification ( 96-well thermal cycler ; applied biosystem , usa ) . the quality of dna was determined by amplification of housekeeping gene ( -globin ) using the gh20/pc04 primers ( cinnagen , iran ) . a consensus sequence from the l1 region of the hpv genome was amplified using gp5+/gp6 + primers ( cinnagen , iran ) ( 2 ) . gp5:5-ttt gtt act gtg gta gat act ac-3 and gp6:5-gaa aaa taa act gta aat cat att c-3 were used as the primer set . hpv typing ( hpv 6 , 11 , 16 , 18 and 31 ) was also performed using hpv type specific primers ( cinnagen , iran ) ( 19 ) . pcr products were separated in 2% agarose gels and visualized by green veiwer staining ( parstous , iran ) . all analyses were performed using data processing program spss / pc version 16.0 for windows ( spss inc . , chicago , il , usa ) . chi square , mann -whitney , student s t test and fisher s exact tests were used to assess the statistical significant differences between hpv status and clinicopathological parameters . the os and dfs rates were estimated by the kaplan - meier method , and differences in survival rates were analyzed with the log - rank test . 5 to 10 m sections were cut from each paraffin block . cleaning off the microtome blade with xylene and ethanol after cutting each paraffin block the tumor samples were deparaffinized using a series of xylene and ethanol washes . in this study , the tissue sample was mixed with 400 l digestion buffer which consisted of tris - cl ( 100 mm , ph 7.5 ) and tween-20 ( 0.055 ) and proteinase k 3l ( 20 mg / ml ; frementase , germany ) , followed by incubation at 56c for 3 hour . after the complete digestion of tissue , the mixture was incubated at 100c in hotblock ( accublock digital dry baths ; usa ) to inactivate proteinase k. the samples were then centrifuged at 5000 rpm , and the supernatant containing dna was used for pcr amplification ( 96-well thermal cycler ; applied biosystem , usa ) . the quality of dna was determined by amplification of housekeeping gene ( -globin ) using the gh20/pc04 primers ( cinnagen , iran ) . a consensus sequence from the l1 region of the hpv genome was amplified using gp5+/gp6 + primers ( cinnagen , iran ) ( 2 ) . gp5:5-ttt gtt act gtg gta gat act ac-3 and gp6:5-gaa aaa taa act gta aat cat att c-3 were used as the primer set . hpv typing ( hpv 6 , 11 , 16 , 18 and 31 ) was also performed using hpv type specific primers ( cinnagen , iran ) ( 19 ) . pcr products were separated in 2% agarose gels and visualized by green veiwer staining ( parstous , iran ) . all analyses were performed using data processing program spss / pc version 16.0 for windows ( spss inc . , chicago , il , usa ) . chi square , mann -whitney , student s t test and fisher s exact tests were used to assess the statistical significant differences between hpv status and clinicopathological parameters . the os and dfs rates were estimated by the kaplan - meier method , and differences in survival rates were analyzed with the log - rank test . moreover , eight patients were excluded because their clinicopathological databases were not complete . remaining 114 ocscc cases comprised of 58 men and 56 women . the mean age of patients was 58.6 years ( range 19 - 85 years).the follow - up period ranged from 6 months to 8 years . the hpv sequences were detected in 15 patients ( 13.16% ) : 5 cases with hpv11 ( 33.3% ) , one case with hpv6 ( 6.7% ) and two cases with both hpv6 and 11 ( 13.3% ) . in 7 patients ( 46.7% ) patients characteristics and tumor features of the hpv positive and negative ocscc cases are compared in table 1 . no significant differences were observed between the two groups in the variables of age , gender , tumor site , size and histological grading . fifty five hpv - negative patients ( 55.6% ) were n0 , while five hpv - positive patients ( 33.3% ) were n0 . in addition , while twenty two hpv - negatives ( 22.2% ) were n2 , eight hpv - positive patients ( 53.3% ) were n2 . the median lymph node involvement was significantly higher in hpv positive group compared to hpv negative group ( p = 0.045 ) . as a result , significantly more stage iva was seen among the hpv - positive tumors compared to the hpv - negative ones ( 66.7% vs. 35.4% , p = 0.04 ) . most of the hpv positive and negative cases were treated with surgery and adjuvant radiation therapy ( table 2 ) . thirty four out of 99 hpv - negative patients ( 34.3% ) died from their disease compared with 11 out of 15 in the hpv - positive cases ( 73.3% ) . chi square test revealed a significant relation between the rate of death and hpv positive status ( p = 0.004 ) . survival and hpv dna status : the median dfs was 13.9 months among the hpv - positive and 49.9 months among the hpv - negative patients with a significant difference ( p = 0.029 ) . also the median os of 15.7 months in the hpv - positive group compared to 49.6 months in the hpv - negative group was significantly better ( p = 0.017 ) . the kaplan - meier survival curves for the hpv - positive and negative cases are shown in figure 1 . we used multivariate analysis involving cox proportional hazards model to determine which factors were jointly predictive of os and dfs . the hpv status ( hr = 2.10 , ci = 1.02 to 4.28 , p = 0.041 ) and stage ( hr = 1.92 , ci = 1.36 to 2.70 , p < in addition , both hpv status ( hr = 2.80 , ci = 1.35 to 5.79 , p = 0.005 ) and stage of disease ( hr = 1.95 , ci = 1.37 to 2.79 , p < moreover , eight patients were excluded because their clinicopathological databases were not complete . remaining 114 ocscc cases comprised of 58 men and 56 women . the mean age of patients was 58.6 years ( range 19 - 85 years).the follow - up period ranged from 6 months to 8 years . the hpv sequences were detected in 15 patients ( 13.16% ) : 5 cases with hpv11 ( 33.3% ) , one case with hpv6 ( 6.7% ) and two cases with both hpv6 and 11 ( 13.3% ) . in 7 patients ( 46.7% ) patients characteristics and tumor features of the hpv positive and negative ocscc cases are compared in table 1 . no significant differences were observed between the two groups in the variables of age , gender , tumor site , size and histological grading . fifty five hpv - negative patients ( 55.6% ) were n0 , while five hpv - positive patients ( 33.3% ) were n0 . in addition , while twenty two hpv - negatives ( 22.2% ) were n2 , eight hpv - positive patients ( 53.3% ) were n2 . the median lymph node involvement was significantly higher in hpv positive group compared to hpv negative group ( p = 0.045 ) . as a result , significantly more stage iva was seen among the hpv - positive tumors compared to the hpv - negative ones ( 66.7% vs. 35.4% , p = 0.04 ) . most of the hpv positive and negative cases were treated with surgery and adjuvant radiation therapy ( table 2 ) . thirty four out of 99 hpv - negative patients ( 34.3% ) died from their disease compared with 11 out of 15 in the hpv - positive cases ( 73.3% ) . chi square test revealed a significant relation between the rate of death and hpv positive status ( p = 0.004 ) . survival and hpv dna status : the median dfs was 13.9 months among the hpv - positive and 49.9 months among the hpv - negative patients with a significant difference ( p = 0.029 ) . also the median os of 15.7 months in the hpv - positive group compared to 49.6 months in the hpv - negative group was significantly better ( p = 0.017 ) . the kaplan - meier survival curves for the hpv - positive and negative cases are shown in figure 1 . we used multivariate analysis involving cox proportional hazards model to determine which factors were jointly predictive of os and dfs . the hpv status ( hr = 2.10 , ci = 1.02 to 4.28 , p = 0.041 ) and stage ( hr = 1.92 , ci = 1.36 to 2.70 , p < in addition , both hpv status ( hr = 2.80 , ci = 1.35 to 5.79 , p = 0.005 ) and stage of disease ( hr = 1.95 , ci = 1.37 to 2.79 , p < the participation of hpv in the oral and oropharyngeal carcinogenesis was first proposed in 1983 by syrjanen et al . ( 23 ) and then supported by several other studies . according to many studies , the hpv prevalence rate in oral cancer is varied between 0% - 100% ( 6 , 19 , 24 - 26 ) . this widespread variability can be in part due to differences in methodology of hpv detection ways and differences in sample types , populations , and anatomic sites tested ( 27 , 28 ) . moreover , various studies have reported conflicting results for the probable role of hpv on the patient 's prognosis ( 15 , 16 , 29 ) . in the current study , regarding the sample size , the majority of small to medium sized studies ( < 100 patients ) have revealed a wide variation in hpv prevalence rates ( varying between 0% and 100% ) ; however , larger studies generally tend to show lower hpv prevalence rates ( varying between 1.4% and 48.8% ) . ( 27 ) have mentioned the sample size as an important factor in the reportedly heterogeneous prevalence rate of hpv , which is in accordance with our findings . the hpv positivity rate of 13.6% in our study is almost similar to those other reports of oral cavity cancers which are ranged from 12% to 70% for opscc ( 2 , 15 , 22 , 30 ) versus 3% to 15% for ocscc ( 2 , 20 , 31 ) . while there is another report that mentions a 25% incidence rate of hpv among tongue cancers in iran and yet all of them subtypes of 18 and 16 ( 32 ) , in the current study , 15 cases ( 13.16% ) were identified as hpv positive and the detected viral subtypes were subtypes 6 and 11 . however , it is not still clear whether these subtypes result in an infection driven carcinogenesis or they are just a transient infection ( 5 , 33 ) . pannone et al . mentioned that hpv 6 , which was known as low risk or non - oncogenic subtype to the cervix , was present in a larger number of head and neck cancers . hpv 6 and 11 have been found in some tonsillar and laryngeal carcinomas and in the malignant transformation of benign laryngeal papillomas , hpv 11 has been most commonly implicated . hpv 6 and 11 have also been on suspicion of malignancies such as verrucous carcinoma of the oral cavity ( 34 , 35 ) . hpv 6 and 11 were also observed in other studies on cancers of the head and neck ( 36 , 37 ) . studies on opscc often suggested that the incidence of hpv in this region is more prevalent ( 5 , 33 , 38 ) . it is also mentioned that hpv - mediated opsccs are more likely late stage , poorly differentiated , and with large lymph nodes in the neck , and in spite of that they have less risk of recurrence and death ( 2 , 5 , 24 , 33 , 39 - 41 ) . some studies indicate that such hpv - positive tumors are more radio - sensitive and have better overall survival rate ( 33 , 42 , 43 ) . these patients tend to be younger and often male with less consumption of alcohol and smoking ( 33 , 41 , 44 ) . there is also data indicating that patients with hpv - positive opscc are associated with a higher number of sexual partners ( 33 , 34 ) . in sccs limited to the oral cavity , usually the hpv prevalence rate is lower and hpv16 is also the most common type detected ( 2 , 28 ) . some studies have suggested that hpv does not play a significant role in the etiology of ocscc ( 8 - 10 ) and also does not play a role in the progression toward malignancy , even though some studies have demonstrated the role of hpv16 and 18 in oral carcinogenesis ( 45 - 47 ) . notably , because of relatively low frequency of hpv in ocsccs , only few studies have attamped to correlate hpv status with clinical outcome ( 5 , 24 , 46 , 47 ) . in most of the studies of hnsccs , the demonstrated clinicopatological features about hpv positive cases are usually related to the oropharyngeal samples ; therefore , little is known about the influence of hpv on clinical course of patients with the oral cavity cancer because of the lower hpv prevalence . no data currently supports the idea that hpv is significantly associated with improved outcome for patients with oral cancer . only few published studies on patients with oral cavity carcinoma specifically examined the impact of hpv on outcome . kaminagakura studied 114 patients and found a nonsignficant trend towards improved survival for 22 hpv - positive patients ( 47 ) . sugiyama et al . demonstrated a nonsignficant trend towards improved overall survival for hpvpositive oral cavity cancer patients ( 48 ) . smith found no association with hpv and outcome for patients with oral carcinoma , based on either serology or tumor hpv detection ( 49 ) . also in isayeva s cohort study of 89 patients with oral cavity carcinoma , no significant association was found for patients with either hpv16/18 e6e7 rna and time to disease progression or disease specific survival ( 5).therefore , future studies on oral cavity scc should be powered to address the important clinical issue of hpv status . in the present study , the mean age of the cases was 58.6 years , and there was no statistical difference between the hpv - positive and the hpv - negative groups based on age . however , in some studies , the hpv - associated patients who suffer from oropharyngeal cancers tend to be younger ( 33 , 39 , 42 , 44 ) . in our study , 53.3% of hpv - positive patients were males and 46.7% were females , and there was no meaningful relation between gender and the hpv status . such findings have been observed in some other studies ( 29 ) , although some studies on oropharynx lesions have reported that the hpv - positive cases are predominantly male ( 33 , 41 , 42 , 44 ) . in the present study , the most common tumor site was tongue , and there was no meaningful statistical relation between tumor location and the hpv status . similar findings have been reported in other studies ( 19 , 29 ) . according to the tumor grade , although 60 % of the hpv - positive patients were grade 2 and 59.6% of hpv - negative patients were grade1 , no meaningful statistical difference was found between hpv status and histological grading of the tumor ( 16 , 19 ) . although in most of the oropharyngeal cancer studies advanced tumor stages have been observed in the hpv - positive cases ( 18 , 33 , 39 , 41 , 44 , 50 ) , there are some studies which have not seen any difference ( 16 , 19 , 32 ) . in this study 55.6 % of the hpv - negative patients and only 33.3% of the hpv - positive patients were n0 . moreover , 66.7% of the hpv positive groups and only 35.4% of the hpv negative ones were stage iva . ( 51 ) have reported similar findings , although in some studies no difference have been observed ( 13 ) and in most of the hpv - related opscc cases higher lymph node involvement have been observed ( 18 , 33 , 39 ) . in the current study local recurrence was the most commen type of relapse , and there was no statistically significant difference according to hpv status . in most of the hpv - related opscc studies low reccurence rate have been observed ( 2 , 50 ) , although weinberger et al . have noted that hpv status has no prognostic value for local recurrence alone ( 52 ) . in our study , 34.4% of the hpv - negative patients and 73.3% of the hpv - positive patients died of disease . that statistically meaningful relation was observed between hpv status and patients death . less mortality risk in hpv - positive cases but as far as we know , no study on oral cavity cancers has especially dealt with the mortality rate of hpv - positive patients . we found that hpv - negative patients survived significantly longer than hpv - positive patients when both the os and the dfs were measured . similar findings have been reported in other studies based on tumors with higher stages of disease ( 16 , 22 ) . while the majority of oropharyngeal studies have reported an improved prognosis of hpv positive tumors ( 2 , 5 , 18 , 19 , 33 , 39 - 41 , 44 , 50 , 53 ) , there are still other studies which have reported no difference ( 2 , 5 , 18 , 19 ) . in our study there are other studies which indicate hpv - related opscc patients , have a lower intake of tobacco and alcohol ( 33 , 39 , 44 , 50 ) . currently , little is known about the influence of hpv on the clinical course and survival of patients with ocscc . although the findings of this study revealed relatively low prevalance of hpv dna in ocsccs and also the well known high risk subtypes were not observed , the carcinogenic role of this virus and its less common variant of hpv 6 and 11 , which we did see in the current study , can not be totally ignored in the oral cavity cancers . regarding high mortality rate of these variants of hpv - positive tumors , further investigations with higher sample volume using more developed methods currently , little is known about the influence of hpv on the clinical course and survival of patients with ocscc . although the findings of this study revealed relatively low prevalance of hpv dna in ocsccs and also the well known high risk subtypes were not observed , the carcinogenic role of this virus and its less common variant of hpv 6 and 11 , which we did see in the current study , can not be totally ignored in the oral cavity cancers . regarding high mortality rate of these variants of hpv - positive tumors , further investigations with higher sample volume using more developed methods are still required .

backgroundsquamous cell carcinoma ( scc ) is the most common malignancy of the oral cavity . a relationship between the human papilloma virus ( hpv ) infection and the prognosis of oral cavity scc ( ocscc ) has been discussed before.objectiveswe investigated the prevalence rate of hpv status in patients with ocscc , and its effects on clinicopathological characteristics of tumors and patients prognosis.patients and methodssections of formalin - fixed , paraffin - embedded tissue blocks from 114 histopathologically confirmed ocscc cases were investigated in this study . polymerase chain reaction ( pcr ) was applied to evaluate the hpv status in the samples.resultsfifteen ( 13.16% ) cases were identified as hpv positive . the detected viral subtypes in this study were the subtypes 6 and 11 . the stage and especially lymph node stage was significantly higher in the hpv positive group compared to the hpv negative group ( p = 0.04 ) . disease free survival ( dfs ) was remarkably lower in the hpv positive group compared to the hpv negative group ( 13.9 vs. 49.9 months , p = 0.02 ) . overall survival ( os ) was also significantly inferior in the hpv positive group ( 15.7 vs. 49.6 months , p = 0.01 ) . in the current study , no significant differences were observed between two groups in relation to the variables of age , gender , tumors site , tumor size , tumor grading and also the recurrence rate.conclusionsthe observed higher mortality rate among the hpv positive group indicates the poorer prognosis of this group in comparison with the hpv negative patients . the incidence rate of hpv infection was low in the studied samples ; however , interaction of subtypes 6 and 11 of hpv in poorer prognosis of the patients and a carcinogenic role of hpv in ocscc can not be ruled out .

1. Background 2. Objectives 3. Patients and Methods 3.1. Detection of HPV DNA Was Performed by PCR 3.2. Statistical Analysis 4. Results 4.1. Characteristics of Cases 5. Discussion 5.1. Conclusion

it is estimated that depression will become the second most common cause of disability , next to heart disease in a few decades . major depression affects 1 in 20 people during their lifetime . in many cultures , especially in developing eastern countries , talking about emotions somatic metaphors and complaints are usually expressed as substitutes for emotional discharge . such variability in depression rates , as noted by simon and his colleagues , may represent problems with definition and measurement rather than true differences in prevalence . it seems that western measures are not reliable sources for the estimation of the prevalence of affective disorders in culturally divergent populations . as kleinman noted , depression is not a universal psychiatric construct and the ways of understanding the body and the self are so different that this may lead to differences in psychopathology . neurasthenia refers to a bodily state and for the chinese it is easier to talk about somatic complaints rather than one s emotional status . one reason for this apparent disregard may be the stigma that is attached to psychiatric symptoms in chinese culture , compared to the relative acceptance of physical complaints . the who collaborative study assessed 573 patients , and showed that feelings of guilt and self - reproach were commonest in basle and montreal and least in tehran , where suicidal ideation was rare . in contrast , somatic symptoms were commonest in tehran and least in basle and montreal . in depressed chinese american patients , researchers found that the most common presenting complains were fatigue , insomnia , headache , cough and pain . in chinese culture in which psychiatric symptoms are usually stigmatized , somatic symptoms are accepted more than direct presentation of emotional symptoms . patients exhibited less psychiatric symptoms , when referred to a private physician , than those who were visited by a general primary care physician . luis caballero and his colleagues studied a population of spanish patients with major depressive disorders . they observed that 93% of patients had at least one somatic symptom which was fully or partially attributed to depression . bhui revealed in his study that south asians were more likely to visit their general practitioners ( gps ) , and exhibited somatic manifestations of mental distress more commonly than other groups . they were less likely to have a recognized mental disorder than white groups , and even with recognizable mental disorder , they were least likely to be referred to specialist care by gps . on the other hand , some surveys showed no differences in psychological and somatic symptoms between western and other countries . in one study two thirds of depressed patients in primary care presented with somatic symptoms . in another study , the complaints expressed by the patients as somatic entities , were actually depression symptoms in dsm - iv . in a study of 504 patients in tehran , researchers found a high rate of somatic symptoms in depressed patients . also , a similar study in kerman on 246 depressed patients , with frequent symptoms , showed that 40% of case suffered from somatic symptoms . according to the above - mentioned studies and our clinical experience , it seems that depression as a disorder has a different picture in eastern cultures compared to the one delineated by western researchers . in regard to the more frequent expression of somatic symptoms in eastern cultures , we assessed the probability of different presentations of depressive signs and symptoms in male and female patients and in cases with different cultural backgrounds and education . such studies can lead to a better understanding of such disorders and more accurate diagnosis of depression in developing countries . the definition and differentiation of psychiatric problems presented as somatic symptoms may prevent unnecessary interventions and expenses . mental health professionals working in such cultural atmosphere have frequently witnessed improvement in patients on antidepressant medications . these patients often had histories of traveling far and wide in search for a solution to their suffering which eventually has ended in the psychiatrist office . in this study , we have tried to answer some questions about the relationship between cultural background , level of education , age , marital status and gender on the one hand , and presenting symptoms of depressed patients on the other . this cross - sectional descriptive study assessed the presenting symptoms of 500 patients with major depressive disorder referred to a psychiatric clinic affiliated with shiraz university of medical sciences . diagnosis of the disorder was made based on the dsm - iv - tr ( diagnostic and statistical manual of mental disorders - iv - text revision ) criteria according to a face to face structured psychiatric interview based on the american psychiatric association criteria for the diagnosis of major depressive disorder . this is a standard tool for the diagnosis of such disorder in many countries including iran and conducted by a bilingual psychiatrist with more than 15 years of experience . the criteria for recruiting the patients in the study included no history of current substance abuse or dependency , patient s symptoms could not be justified by a general medical condition or other psychiatric entities such as anxiety or somatoform disorders , receiving no active psychiatric treatment , not visited by a psychiatrist during 3 months before evaluation , and not taking any psychiatric medications during this time . dsm - iv classifies the signs and symptoms of depression into two groups of vegetative and mental conditions . since pain is assessed as a frequent vegetative symptom , it was considered as a separate entity and eventually categorized into three groups including mental symptoms , pain , and physical symptoms other than pain . through psychiatric interview , we assessed the presenting complaints of these patients and considered such symptoms as guilt feeling , hypochondriac status , insomnia , and losses of appetite , weight , concentration , and interest , suicidal idea and attempt , hopelessness and crying . also the patients were asked about their chief complaints which were categorized into mental symptoms with pain , and physical symptoms without pain , the duration of the illness , description of the emotional or somatic disorders by the patients , causes of illness expressed by the patients as family problems including interpersonal or intergenerational difficulties , living problems such as occupational or financial difficulties , losses incurred , marital conflicts , and romantic breakups , and the clinician who first visited the patient namely general practitioner , psychiatrist or other specialist . these variables were compared by proper statistical analysis ( chi - square ) according to sex , age , marital status , cultural background ( rural or urban ) and education level . logistic regression analysis , backward method , was used to compare the association of different independent variables in patients with or without somatization as a symptom . of 500 patients studied , 380 ( 76.0% ) were females , 356 ( 71.2% ) married , 246 ( 46.2% ) had elementary school education , and 306 ( 61.2% ) belonged to the urban cultural background . demographic characteristics of the patients tables 2 and 3 demonstrate the descriptive features of patients chief complaints and symptoms of which headache ( 15.2% ) was the most frequent complaint , followed by irritability ( 10.6% ) , pain in the other areas of the body ( 10.4% ) , depression ( 8.0% ) and forgetfulness ( 7.2% ) . among physical ( organic ) complaints , the gastrointestinal ( 7.0% ) symptoms were more frequent than respiratory ( 3.4% ) and cardiac symptoms ( 3.2%).in this context , pain and physical complaints were more common in persons with lower education ( p<0.001 ) , rural cultural background ( p<0.001 ) , women ( p<0.001 ) and married patients ( p=0.007 ) . individuals with higher education ( p<0.001 ) and urban cultural background ( p<0.001 ) were more likely to visit a psychiatrist . there was no significant difference in the referral to psychiatrist between age , sex and marital status . frequency of chief complaints description of clinical variables hypochondriac obsessions were more common in the married ( p=0.024 ) , elder ( p<0.001 ) and lower educated people ( p<0.001).insomnia was more common in people with rural cultural background ( p=0.009 ) and in individuals with lower education ( p<0.001 ) . guilt feeling was more common in the older and younger people compared to middle aged patients ( p<0.001 ) . loss of appetite was more prevalent among older and younger people compared to middle aged patients ( p=0.017 ) and in people with rural cultural background ( p=0.003 ) . suicidal ideation were present more frequently among married persons ( p<0.001 ) , the lower age group ( p<0.001 ) and people with rural cultural background ( p=0.027 ) . suicidal attempt was more frequent in the lower age group ( p=0.009 ) and in the married ones ( p=0.047 ) . our study showed no difference in suicidal ideation and suicidal attempt between men and women . people with lower education ( p<0.001 ) and with rural cultural background ( p=0.001 ) related their symptoms to physical rather than emotional problems . in response to the question about the cause of illness , people with rural cultural background used more also , women pointed to marital problems and men to life difficulties as the main causes of their illness ( p<0.001).despair prevailed among the singles ( p=0.012 ) . in general , this study confirmed our initial assumption about a different profile of depressive symptoms in iranians compared to the western population . our patients showed a high frequency of somatic symptoms , especially pain , as a manifestation of depression . contrary to the common findings discussed in the western literature , we could not find a difference between men and women in suicidal ideation and attempt . however , some of our findings are in accordance with the profile of depressive symptoms in western countries . because of the large number of the variables and to prevent confusion , the depressive symptoms are discussed as separate entities in the following sections . pain and physical symptoms this study showed the importance of somatization in a group of depressed patients who seemed to have no word for their emotions . , men have a dominant role and women often have no knowledge about their rights and their interests . our finding also confirmed the previous observation in that somatic symptoms played an important role in the manifestation of depressive disorder in iranian patients . the importance of somatic symptoms in the depressed patients has also been shown in many studies especially those performed in eastern countries . nieuwsma pointed out that social stigma is the main factor to complain of somatic symptoms instead of depression . fear of stigmatization and reluctance to appear as psychiatric patients are important factor for expressing their emotional pain via somatic route . the pattern of somatization , as kleinman noted in his study on chinese patients , may be unfamiliar to western clinicians and may further complicate the concept of depression . referral to psychiatrist the general practitioners and other specialists are still primary physicians for persons with lower education and people with rural cultural background . this can be related to the unavailability of psychiatrists in rural areas . however , the factor of knowledge has to be considered as a contributory parameter for evaluating this condition . the study conducted by bhui and colleagues revealed that south asians are more likely to visit their general practitioners and less likely to have a recognized mental disorder than white groups , and even if this is recognized , they are least likely to be referred to a specialist by gps . higher prevalence of guilt feeling in patients with urban cultural background is a finding comparable with the results of who collaborative study which showed the higher prevalence of this symptom in western societies . one study showed that guilt feelings could be found in patients with both pakistani and austrian cultures , regardless of age and sex . in fact , guilt feeling was associated with the severity of psychomotor retardation in depression . in some patients , , the idea of original sin rooted in the christianity teachings plays a major role in the guilt feeling of depressed patients . the lesser prevalence of guilt feeling in the middle aged group can be related to the social activity and the functionality of these people compared to the elders and younger people . hypochondriac ideation according to the dsm - iv , hypochondriasis is a disorder with relatively similar prevalence in men and women . accordingly , in this study we could not find a significant difference between men and women in hypochondriac ideations . hypochondriasisas is an obsession about death and it can be an explanation for the higher prevalence of this symptom in the elderly . similarity of the prevalence of hypochondriac ideations in rural and urban areas may indicate that this symptom is less affected by the cultural backgrounds of patients than somatization . as a vegetative symptom , insomnia is also more frequent in the lower educated people and in rural areas and is associated with somatic symptoms . logistic regression analysis also showed the interplay of insomnia and somatic symptoms ( table 4 ) . whether this is representative of sleep disturbance due to somatic problems or vice versa remains to be clarified . factors associated with the chief complaint of somatization like insomnia , this can be affected by somatic symptoms , especially gastrointestinal manifestations . however , because of the similarity of weight loss in the rural and urban areas , this symptom can be a subjective feeling rather than a true anorexia leading to weight loss . loss of concentration this condition has frequently been reported by younger patients who may be more concerned about their memory problems than older people . however , the elders deny their defects in memory and do not complain about it . suicidal ideation and attempt according to the dsm - iv , suicidal ideation is more frequent in women and suicidal attempt is more prevalent in men . however , our study showed no difference in suicidal ideation and attempt between men and women . suicidal idea was more frequent among rural inhabitants , but no difference in suicidal attempt was found between them and urban population . perhaps this may be related to the hesitation of rural people in expressing their emotions . our findings of higher prevalence of suicidal attempt and ideation in married patients is in contrast with those of other studies . also , our study showed that both suicidal ideation and attempt are more frequent in the younger age group . this findings is in conflict with some studies that consider the old age as a risk factor for suicidal attempt . this difference can be related to the supportive care giving received by the older members of the family in eastern countries like iran . logistic regression analysis showed the interplay between suicidal ideation and somatic symptoms , but not between suicidal attempt and hopelessness with somatic symptoms . the higher prevalence of despair in singles is suggestive of the seriousness of the problem in this group . although a cause and effect relationship can not be derived from a cross - sectional study , the symptoms of hypochondriasis , suicidal idea , crying , irritability and insomnia were significantly associated with the complaint of somatization ( table 4 ) . one possible explanation is that somatic problems of patients disrupt their sleep and increase their vulnerability to environmental and emotional stimuli . this in turn , can aggravate the patients affective stability presented by more crying , irritability or thoughts of death . on the other hand , sleeping disorders may also lead to somatic complaints . considering the covariant variables in the regression analysis , only gender was associated with the complaint of somatization in patients with major depressive disorder . however , neither of the educational level and patients age are associated with the complaint of somatization . these results indicated that somatization as a complaint in the patients with major depressive disorder is independent from age and educational level . one explanation could be that somatization as a complaint in iranian culture is commonly expressed in both higher and lower educated class . the principal finding of this study is that somatic symptoms especially headache and pain in other areas of the body have a significant weight in the chief complaints of depressed patients . therapists need to pay attention to the various ways of presentation of this disorder in different cultures in order to understand the symptoms of patients with depression . hence , more extensive studies in other areas of the country are required to obtain a more reliable profile of depression symptoms in iranian population .

background : in some cultures , including ours , direct explanation of inner psychic world is inhibited and stigmatized , therefore finding alternative modes of expression . the aim of this cross - sectional study was to assess the frequency of somatization in the depressed patients . methods : the present study comprised 500 patients referred to the outpatient clinic of shiraz university of medical sciences , and diagnosed with major depressive disorders based on dsm - iv - tr . the presenting complaints of these patients were assessed through psychiatric interview . the presenting symptoms were divided into three main categories including mental symptoms , pain , and physical symptoms without pain . statistical analysis ( chi - square and logistic regression ) were performed to determine the relationship between presenting symptoms and some demographic variables such as age , gender , marital status , educational level and cultural background ( urban or rural ) . results : physical symptoms other than pain , mental symptoms , and pain were found in 193 ( 38.6% ) , 186 ( 37.2% ) , and in 121 ( 24.2% ) patients respectively . pain and physical complaints were more common in patients with rural cultural background , lower education , women and the married individuals . headache ( 15.2% ) , irritability ( 10.6% ) and pain in different parts of the body ( 10.4% ) were the most frequent chief complaints of the patients . hypochondriasis , suicidal idea , crying , irritability and insomnia were significant symptoms associated with the complaint of somatization . conclusion : somatic symptoms , especially pain , have a significant weight in the chief complaints of depressed patients . physicians need to pay particular attention to this important issue in order to better understand these patients .

Introduction Patients and Methods Results Discussion Conclusions None

non - hodgkin lymphomas ( nhls ) account for approximately 60% of all lymphomas in children and adolescents . childhood nhls are subdivided into burkitt 's lymphoma , diffuse large b - cell lymphoma ( dlbcl ) , lymphoblastic lymphoma , and anaplastic large - cell lymphoma . dlbcl is characterized by relatively more frequent extranodal presentation , seen in upto 40% of the cases . primary involvement of the lymphoma of the middle ear is rare , with only about 18 cases being reported in literature so far . here , we report a case of dlbcl , presented with features of facial palsy and otitis , who received initial symptomatic treatment and later chemotherapy after diagnosing dlbcl . a 2 years 8 months old boy visited our tertiary care hospital with complaints of ear ache ( left side ) and left facial palsy of 4 weeks duration . after initial 2 weeks of these symptoms , there was whitish serous discharge from the left ear . before referral to our hospital , he was treated for otitis media with antibiotics , details of which were not available . his complete blood picture and biochemistry investigations done in our hospital were within normal limits . as the symptoms persisted for 4 weeks , computed tomography of head and neck was done which was suggestive of solid mass lesion of 2 cm 2 cm size in the left mastoid with destruction of mastoid bone [ figure 1 ] . the disease was in stage 1 ( as per murphy 's staging ) and was completely resected . histopathological examination showed large cells of lymphoid cell proliferation immune histochemistry was positive for cd20 , bcl-2 and negative for cd3 , with low mib-1 , which confirmed dlbcl . his positron emission tomography for staging , bone marrow and cerebrospinal fluid revealed no abnormality . computed tomography head and neck suggestive of solid mass lesion of 2 cm 2 cm size in the left mastoid with destruction of mastoid bone computed tomography head and neck of right side showing no abnormality his initial clinical symptoms of earache and facial nerve palsy followed by ear discharge resolved after starting chemotherapy as per b - cell lymphoma protocol for 6 months . currently , he is 37 months off treatment , and no disease recurrence is seen clinically as well as radiologically . it is an aggressive form of lymphoma , usually curable with appropriate treatment and has high survival rate . rapid disease progression of dlbcl calls for an early , accurate diagnosis and appropriate treatment . however , unusual presentation can mislead the physician resulting in wrong diagnosis , which delays the treatment , thus promoting disease progression . extranodal presentations of childhood dlbcl are relatively uncommon in clinical practice , and much rarer are those primarily involving middle ear and mastoid . involvement of middle ear and mastoid can resemble the features of otitis media and unusual facial palsy mimicking mastoiditis . although these initial symptoms were suggestive of middle ear infection , the distinguishing factor was unresponsiveness to antibiotics . there have been very few reports of nhl with facial nerve involvement ; ogawa et al . mccabe et al . , reported a case of 2-year - old , an immunocompetent boy with spontaneous regression of an epstein - barr - virus - associated monoclonal lymphoid proliferation who presented with acute otitis media and facial palsy . have described a case of dlbcl with features of otitis media , mastoiditis , and facial palsy . as symptoms persisted even after 4 weeks of treatment , the patient was started on chemotherapy , to which he promptly responded . extranodal nhls of middle ear which is not a common clinical presentation may present as facial palsy , and misdiagnosed as otomastoiditis . there should be a high index of suspicion for primary neoplasms of the middle ear in patients with chronic otomastoiditis refractory to appropriate initial antibiotic therapy ; early diagnosis and appropriate treatment results in good therapeutic outcome and minimizes further complications .

extra nodal presentation of non hodgkins lymphoma ( nhl ) is a rare entity , and data available about the nhl that primarily involves of middle ear and mastoid is limited . we report a case of diffuse large b cell lymphoma ( dlbcl ) , in a 2 year 8 month old boy , who developed otalgia and facial palsy . computed tomography revealed a mass in the left mastoid . mastoid exploration and histopathological examination revealed dlbcl . this case highlights the importance of considering malignant lymphoma as one of the differential diagnosis in persistent otitis media and / facial palsy .

Introduction Case Report Discussion Financial support and sponsorship Conflicts of interest

the goal of these drug cocktail regimens is to achieve additive or synergistic effects among chemotherapeutics , thereby maximizing summation dose - intensity with resultant enhanced anticancer activities and increased patient survival . combinations have been identified and developed both through unbiased approaches and by rational design , and compounds that act on a single biochemical pathway are particularly strong candidates for synergy or potentiation . for example , inhibitors of poly(adp - ribose)polymerase-1 ( parp-1 ) , an enzyme that facilitates dna damage repair , potently synergize with dna damaging agents as demonstrated in cell culture and animal models . herein we describe an approach for potentiation not based on compounds acting on two targets within a single pathway , but rather with two compounds acting differentially to activate the same enzyme . during apoptosis , the zymogen procaspase-3 is activated via proteolysis to caspase-3 , and this active caspase-3 then cleaves scores of cellular substrates , executing the apoptotic program . as procaspase-3 protein levels are elevated in various tumor histologies , drug - mediated direct activation of procaspase-3 has gained significant interest as a selective anticancer strategy . furthermore , caspase-3 has been shown to play critical roles in cardiomyocyte hypertrophy , cellular differentiation , and remodeling . thus development of a strategy to magnify the timing and level of caspase-3 activation in a specific and direct manner could greatly aid the study of active caspase-3 in these nonapoptotic processes . to date , two major classes of compounds have been disclosed that enhance the activity and automaturation of procaspase-3 in vitro and induce apoptosis in cancer cells in culture . procaspase - activating compound-1 ( pac-1 , figure 1a ) enhances the activity of procaspase-3 in vitro via the chelation of inhibitory zinc ions , induces apoptosis in cancer cells in culture , and has shown efficacy in multiple murine tumor models . more recently , the compound 1541b ( figure 1a ) was discovered to promote the automaturation of procaspase-3 to caspase-3 in vitro and to induce apoptotic death of cancer cells in culture . compound 1541b appears to activate procaspase-3 via a binding - induced shift in the on off state equilibrium , or through formation of nanofibrils . pac-1 and 1541b exert their activating effect on procaspase-3 by distinct biochemical mechanisms , suggesting the potential for synergistic effects in cell culture and in vivo . ( b ) procaspase-3 ( 100 nm ) was incubated with pac-1 or 1541b in zinc - supplemented caspase activity buffer . data is normalized to procaspase-3 ( 100 nm ) activity in zinc - free caspase activity buffer as 100% activity . ( c ) procaspase-3 ( 350 nm ) in zinc - supplemented caspase activity buffer was incubated 1541b , pac-1 , or 1541b + pac-1 , and enzymatic activity was assessed with ac - devd - pna . data is normalized to 100% activity = 350 nm caspase-3 in zinc - free caspase activity buffer . error bars represent the sem of three replicates . in vitro , procaspase-3 has enzymatic activity that is dramatically lower than caspase-3 , with estimates ranging from reductions of 200 to 10 fold . low micromolar levels of zinc inhibit the activity of procaspase-3 and caspase-3 in vitro . pac-1 , a moderate affinity zinc chelator that has been shown to chelate the labile zinc pool in cells , allows procaspase-3 to once again process substrates , including itself . described herein is the combination use of the two small molecule activators of procaspase-3 , pac-1 and 1541b . these compounds act synergistically to enhance procaspase-3/caspase-3 activity in vitro , induce rapid procaspase-3 processing and caspase-3 activity in cell culture , potently and rapidly cause apoptotic death in a variety of cancer cell lines , and have efficacy in a murine tumor model . as zinc colocalizes with cellular procaspase-3/caspase-3 , it was of interest to determine if 1541b could activate recombinantly expressed procaspase-3 ( supporting figure s1a ) in the presence of zinc ; the activating effect of 1541b on procaspase-3 in vitro had previously only been evaluated under zinc - free conditions . as shown in figure 1b , compound 1541b does not enhance intrinsic procaspase-3 enzymatic activity in zinc - containing buffers , whereas pac-1 relieves zinc - mediated inhibition and induces the anticipated activating effect . this effect from pac-1 is reliant upon its zinc binding ability , as an analogue that does not bind zinc ( pac-1a ) is not active in this experiment . in addition , an inactive derivative of 1541b ( compound 1541d ) has no effect in this experiment ( structures and data in supporting figure s1b d ) . this orthogonal activity of pac-1 and 1541b on procaspase-3 ( pac-1 activating zinc - bound procaspase-3 , 1541b activating procaspase-3 with zinc removed ) suggests synergistic potential , and indeed , the combination of pac-1 and 1541b , when incubated with procaspase-3 in vitro , leads to dramatic caspase-3 activity far exceeding the effect of either compound alone ( figure 1c ) . consistent with the established structure activity relationships , pac-1a and 1541d as single agents and in combination were unable to facilitate procaspase-3 activation in vitro ( supporting figure s1d ) . to examine pac-1 synergy with 1541b for activation of procaspase-3 in cancer cells in culture , a panel of cancer cell lines were treated with combinations of pac-1 and 1541b and the caspase-3/-7 activity of the cell lysates was monitored with the fluorogenic caspase substrate ac - devd - afc . as seen in figure 2a , cotreatment of u-937 ( human lymphoma ) , bt-549 ( human breast cancer ) , and a549 ( human lung cancer ) cells with pac-1 and 1541b results in markedly more dramatic and rapid increases in devdase activity than treatment with either pac-1 or 1541b as single agents . the combination treatment stimulates caspase activity that rivals or surpasses that induced by staurosporine ( sts , 1 m ) , one of the most rapid inducers of cellular devdase activity known , and is dose dependent ( supporting figure s2 ) . pac-1/1541b combinations induce rapid and dramatic procaspase-3 activation and maturation in cancer cell lines in culture . ( a ) cancer cell lines were treated with pac-1 ( 30 m ) , 1541b ( 15 m ) , or the combination , cells were lysed at various time points , and caspase-3/-7 activity of the lysates was evaluated with the fluorogenic ac - devd - afc substrate . ( b ) western blot of various cancer cell lines after treatment with combinations of pac-1 ( 30 m ) and 1541b . treatment durations of 3 , 4 , and 8 h for u-937 , bt-549 , and a549 cells , respectively , were chosen on the basis of the timing of maximal caspase activity , as observed in cell lysate . to determine if the elevation of devdase activity was the result of enhanced cleavage of procaspase-3 to caspase-3 facilitated by compound cotreatment , cells were treated with pac-1 and 1541b combinations and assessed by western blotting . as shown in figure 2b , dramatic activation of procaspase-3 to caspase-3 was observed in u-937 , bt-549 , and a549 cells upon treatment with the pac-1/1541b combinations , whereas low / no procaspase-3 activation was observed with 1541b or pac-1 alone at the times and concentrations evaluated . analogous results in hl-60 ( human leukemia ) , hs578 t ( human breast cancer ) , and el4 ( murine lymphoma ) cells are shown in supporting figure s3 . apoptosis through the intrinsic pathway proceeds via an ordered series of events , with bcl-2 family proteins acting on the mitochondria , stimulating cytochrome c release , formation of the apoptosome , caspase-9 activation , and subsequent cleavage of procaspase-3 to caspase-3 . caspase-3 then cleaves scores of substrates , executing the apoptotic program . to examine these events in cells treated with pac-1 + 1541b , bt-549 cells were assessed for the timing of procaspase-3 activation relative to cytochrome c release from the mitochondria by western blot of fractionated cell lysate . as shown in figure 3a , analysis of cells treated with pac-1 + 1541b shows the appearance of cleaved caspase-3 bands in the cytosolic fraction before cytochrome c levels diminish in the mitochondria . at these compound concentrations and relatively short treatment times , 1541b as a single agent is considerably less effective at inducing procaspase-3 activation ( figure 3b ) , and pac-1 is not effective . importantly , as shown in figure 3c , staurosporine induces apoptosis through the canonical intrinsic pathway , with clear release of cytochrome c from the mitochondria prior to activation of procaspase-3 . rapid procaspase-3 activation is observed in cells treated with pac-1 + 1541b , occurring prior to release of cytochrome c from the mitochondria . ( a ) bt-549 breast cancer cells were treated with pac-1 ( 30 m ) and 1541b ( 10 m ) over 8 h and assessed in the cytosol for procaspase-3 activation , and in the mitochondria for cytochrome c levels . ( b ) bt-549 cells treated with 1541b ( 10 m ) and assessed for 8 h. ( c ) bt-549 cells treated with sts ( 1 m ) and assessed for 10 h. bt-549 cells were assessed by phase contrast microscopy and monitored for the phenotypic changes associated with caspase activation and apoptosis ( supporting figure s4 ) . consistent with the western blot and caspase activity data , only the combination of pac-1 and 1541b demonstrated substantial morphological changes indicative of apoptosis , such as blebbing . the combination of pac-1 and 1541b was evaluated for the capacity to induce apoptotic death in a variety of cancer cell lines in culture . these evaluations were performed at short incubation times , reflective of the timing of caspase activation observed in figure 2 and where neither compound exerts a significant effect as a single agent . pac-1 significantly synergizes with 1541b for potent proapoptotic activity in u-937 , bt-549 , and a549 cells ( figure 4a ) , and in hl-60 , hs578 t , u-87 , and el4 cells ( supporting figure s5 ; see supporting figure s6 for representative annexin v - fitc / propidium iodide histograms ) ; the dashed horizontal lines in each graph mark levels of cell death that would be observed from a strictly additive effect of pac-1 and 1541b . while the non - blood - brain barrier permeable analogue s - pac-1 possesses a comparable capacity for synergy as pac-1 ( supporting figure s7 ) , synergy was not observed when either pac-1a or 1541d were used in combination with 1541b or pac-1 ( supporting figures s8 and s9 ) . pac-1 and 1541b synergize to induce rapid cell death in cancer cell lines . ( a ) cancer cell lines were treated with the indicated concentrations of pac-1 ( 0 , 15 , and 30 m ) and 1541b ( 0 , 7.5 , 10 m ) for six hours , and apoptotic death was assessed using flow cytometry with annexin v - fitc / propidium iodide staining . the dotted horizontal lines represent the level of cell death expected from a mere additive effect of pac-1 and 1541b for each drug combination . ( b ) percent cell death observed after 6 h treatment of u-937 cells with pac-1 + 1541b combinations in matrix format . values are heat mapped with white equal to 0% cell death and red equal to 100% cell death . ( c ) combination index values calculated for each combination with combosyn software ( < 1 indicates synergistic interaction with values < 0.3 indicating strong synergism ) . values are heat mapped with lowest values in green and highest values in red . further assessment ( vs u-937 cells ) of a broad range of concentrations for both compounds clearly shows the dramatic synergy between these two agents . shown in figure 4b is the cell death induced by the compound matrix , with synergy apparent from simple comparisons of the percent cell death induced by the single agents versus the combinations ; this is true at both short ( 6 h , figure 4b ) and long ( 24 h , supporting figure s10 ) time points . synergism is frequently quantified in such experiments though calculation of combination indices ( ci ) . combinations describing synergistic interactions possess ci values < 1 , while antagonistic interactions have ci value > 1 ; additive effects occur when the ci value equals 1 . when u-937 cells were treated with a range of pac-1 and 1541b compound concentrations in combination , synergistic interactions were observed broadly , with several combinations considered strongly synergistic ( figure 4b and c and supporting figure s10 ) . the proapoptotic effect of the pac-1/1541b combination was markedly reduced with the pan - caspase inhibitor q - vd - oph , consistent with the involvement of caspases in the mode of cell death ( figure 5a ) . to further investigate the connection between activation of procaspase-3 and the cell death induced by the drug combination , mcf-7 cells were used , a cell line that does not express procaspase-3 , together with a matched mcf-7 cell line where procaspase-3 is expressed via a plasmid ( figure 5b ) . the combination of pac-1/1541b has minimal effect on mcf-7 cells transfected with control plasmid ( mcf-7 vrl ) , but dramatically induces apoptosis in procaspase-3 expressing mcf-7 cells ( mcf-7 c-3 ) ( figure 5c ) , an effect correlated with procaspase-3 activation ( figure 5d ) . pac-1 and 1541b induce rapid caspase - dependent cell death in cancer cell lines . ( a ) the pan - caspase inhibitor q - vd - oph ( 25 m ) protects against pac-1/1541b - mediated cell death in u-937 cells . ( b ) western blot analysis of mcf-7 cells transformed with empty plasmid ( mcf-7 vrl ) and mcf-7 cells transformed with a plasmid containing the gene for procaspase-3 ( mcf-7 c-3 ) . ( c ) mcf-7 vrl and mcf-7 c-3 cells were treated with the indicated concentrations of pac-1 and 1541b and apoptotic death was assessed using flow cytometry with annexin v - fitc / propidium iodide staining . the dotted horizontal lines represent the level of cell death expected from a mere additive effect of pac-1 and 1541b for each drug combination . ( d ) western blot of mcf-7 c-3 cells treated for 6 h with combinations of pac-1 ( 30 m ) and 1541b . ( e ) the combination of pac-1 and 1541b has an antitumor effect in vivo . el4 cells were injected subcutaneously into c57bl/6 mice ( 10 million cells per mouse ) , the animals were split into four groups and treated once - a - day for three days via ip injection with vehicle ( 2-hydroxypropyl--cyclodextrin , hpcd ) , 1541b ( 17.5 mg / kg in hpcd ) , pac-1 ( 125 mg / kg in hpcd ) , or 1541b + pac-1 ( 17.5 and 125 mg / kg , respectively , in hpcd ) . p - values are relative to vehicle control ; * , p < 0.005 ; * * , p < 5 10 . to explore the therapeutic utility of the dual procaspase-3 activation strategy , the combination of pac-1 and 1541b was assessed in vivo . prior to beginning efficacy studies , the tolerability and toxicity of 1541b as a single agent and in combination with pac-1 was determined ( supporting tables s1 , s2 , and s3 ) . on the basis of these results , a maximum combination dosage of 125 mg / kg of pac-1 followed by 17.5 mg / kg of 1541b was chosen for the efficacy model . the hematologic and nonhematologic toxicity of this treatment of pac-1 and 1541b was determined following 3 consecutive daily ip administrations of hpcd , pac-1 , 1541b , or combination ( pac-1 + 1541b ) . no clinically significant evidence for myelosuppression , renal injury , or hepatic toxicity was identified in any of the treatment cohorts ( supporting table s3 ) . for mice receiving hpcd , pac-1 , or 1541b alone , all collected target organs were devoid of histologic evidence for inflammation and necrosis . in mice receiving combination ip pac-1 and 1541b , pathologic changes were noted in the lung parenchyma of 2 out of 3 mice . these mice had eosinophilic and histiocytic perivascular and peribronchiolar infiltrates , which were mild to moderate in severity . the clinical significance of this finding is unclear , as mice in these groups were asymptomatic and did not demonstrate weight loss compared to controls . the pharmacokinetic parameters of each compound were then determined ( supporting table s4 ) . following a single ip injection at a dose of 125 mg / kg , pac-1 in hpcd was rapidly absorbed and achieved a maximal plasma concentration of 32720.0 ng / ml within 20 min postinjection . pac-1 was distributed quickly throughout the body with a mean distribution half - life of 20 min and a mean terminal elimination half - life of 4.2 h. likewise , 1541b in hpcd was absorbed and reached a maximal plasma concentration of 2699.9 ng / ml within 20 min of intraperitoneal administration . the distribution half - life of 1541b was 20 min and the terminal elimination half - life was 5.4 h. the el4 syngeneic murine lymphoma model was chosen to explore the therapeutic utility of pac-1 and 1541b combinations , as pac-1 and 1541b synergize to induce dramatic activation of procaspase-3 and cell death versus this cell line ( supporting figures s3c and s5d ) , and it is a challenging treatment model due to its rapid doubling time ( less than 24 h ) and uniformly high tumor formation rate ( 100% ) when inoculating greater than 1 10 cells subcutaneously in c57bl/6 mice . pac-1 was chosen over s - pac-1 for the combination studies as it has greater activity versus the el4 cell line in culture . c57bl/6 mice implanted with el4 cells were treated with pac-1 alone ( 125 mg / kg ) , 1541b alone ( 17.5 mg / kg ) , or sequential treatments of pac-1 + 1541b ( 125 mg / kg and 17.5 mg / kg , respectively ) once - a - day for three days , formulated in hpcd . once tumors in vehicle - treated mice had reached their maximum allowed size of 1500 mm ( 8 days ) , all mice were sacrificed , and tumors were excised and weighed . as shown in figure 5e , 1541b treatment had no effect , and pac-1 had a small but statistically significant effect on tumor growth in this model . while there is clear benefit to anticancer strategies utilizing combinations of drugs that act on different targets , the work described herein demonstrates that dramatic synergy can be observed with compounds that act through orthogonal mechanisms on the same biological target . this multitargeting approach may have particular advantages when activation of an enzyme is sought . pac-1 chelates the labile inhibitory zinc from procaspase-3/caspase-3 , thus priming the enzyme for robust and efficient activation by 1541b . in cell culture at concentrations where neither compound significantly induces death at 612 h , dramatic enhancement of cell death ( over the additive effect ) is observed with the pac-1/1541b combination . this cell death is tied to the ability of the pac-1/1541b combination to induce a rapid conversion of procaspase-3 to caspase-3 , as shown by the western blots and the caspase-3 enzymatic activity in cell lysates . pac-1 is safe in mice and dogs , and a derivative of pac-1 , s - pac-1 , showed promising activity in a phase i clinical trial of pet dogs with lymphoma , thus the observed synergy with 1541b could have clinical significance . pac-1 has been utilized as a tool for studying direct procaspase-3 activation in various systems , and data presented herein suggest that the pac-1/1541b combination will also be valuable as a tool for rapidly activating procaspase-3 . as interest in activating enzymes with small molecules is increasing rapidly and has considerable medicinal potential , our data suggest that targeting strategies using two small molecules with different activation mechanisms could be a general approach for dramatic enhancement of the intended biological effect . procaspase-3 and caspase-3 were expressed as described previously and purified with qiagen nickel - nta resin . increasing concentrations of pac-1 , 1541b , pac-1a and 1541d were assessed for their capacity to enhance activity of 100 nm procaspase-3 in an inhibitory zinc - based system ( 2 m znso4 , 50 mm hepes , 50 mm kcl , 1.5 mm tcep , 0.1% chaps , ph 7.6 ) . compounds were added to zinc - inhibited pc-3 , incubated for 30 min , and activity was assessed by cleavage of ac - devd - afc ( 50 m ) and was normalized to 100 nm procaspase-3 in zinc - free caspase activity buffer ( 50 mm hepes , 50 mm kcl , 1.5 mm tcep , 0.1% chaps , ph 7.6 ) . 1541b ( 20 and 25 m ) and pac-1 ( 25 and 50 m ) were evaluated separately and in combination for their capacity to activate 350 nm procaspase-3 over time in a mildly inhibitory zinc - based system ( 750 nm znso4 , 50 mm hepes , 50 mm kcl , 5 mm tcep , ph 7.6 , 0.1% tritonx-100 ) . at designated time points aliquots were assessed by cleavage of ac - devd - pna ( 200 m ) . 350 nm caspase-3 in a zinc - free caspase activity buffer ( 50 mm hepes , 50 mm kcl , 5 mm tcep , ph 7.6 , 0.1% tritonx-100 ) was used for 100% caspase-3 activity . u-937 ( 50 000 cells per well ) , bt-549 ( 10 000 cells per well ) and a549 ( 10 000 cells per well ) were plated in 96-well plates , allowed to adhere overnight ( bt-549 and a549 ) , and incubated with 1 m staurosporine ( sts ) , 1541b ( 7.5 , 10 , 12.5 , or 15 m ) , pac-1 ( 30 m ) , 1541b ( 7.5 , 10 , 12.5 , or 15 m ) and pac-1 ( 30 m ) or dmso ( final concentration 1% ) in phenol - red free rpmi media . plates were assessed for executioner caspase activity via addition of a 4 bifunctional lysis activity buffer ( 200 mm hepes , 400 mm nacl , 40 mm dtt , 0.4 mm edta , 1% tritonx-100 , 20 m ac - devd - afc ) . activity is expressed as normalized to minimal and maximal activity observed within the assay . for western analysis one million suspension ( u-937 , hl-60 and el4 ) or adherent cells at 75% confluency in 6-well plates were treated with increasing concentrations from 7.5 to 12.5 m 1541b in the presence or absence of 30 m pac-1 for a duration reflective of near maximal caspase activity as seen in the cell lysate experiment . at the conclusion of treatment , the medium and trypsin - aided detached cells were pelleted , lysed on ice in ripa buffer ( 50 mm tris base , 150 mm nacl , 1% tritonx-100 , 0.5% na - deoxycholate , 0.1% sds , ph7.4 , with a 1:100 dilution of protease inhibitor cocktail set iii ) , and clarified , and protein content was normalized by bca protein assay reagent ( pierce ) . samples were denatured , separated by sds - page ( 420% ) , and transferred to a membrane for western blot analysis of pro- and active caspase-3 ( cell signaling 9662 ) . blots were stripped and reprobed for -actin ( cell signaling 4970 ) as a loading control . bt-549 cells at 75% confluency ( allowed to adhere for > 24 h ) in 6-well plates were treated with 10 m 1541b and 30 m pac-1 , 10 m 1541b or 1 m sts for up to 10 h. at each time point , medium and trypsin - aided detached cells were pelleted , washed in pbs and suspended in cold digitonin permeabilization buffer ( 75 mm nacl , 1 mm sodium phosphate monobasic , 8 mm sodium phosphate dibasic , 250 mm sucrose , 200 g / ml of digitonin , protease cocktail inhibitor , ph 7.5 ) and placed on ice for 5 min . permeabilized cells were centrifuged ( 14 000 rcf , 5 min ) , and the supernatant ( cytosolic fraction ) was saved . the pellet ( mitochondrial fraction ) was washed with digitonin permeabilization buffer , lysed in ripa buffer , and clarified . samples were denatured , separated by sds - page and transferred to a membrane for western blot analysis probed for the presence of pro- and active caspase-3 ( cell signaling 9662 ) , and cytochrome c ( cell signaling 4272 ) . western blots were stripped and reprobed for -actin ( cell signaling 4970 ) and cox iv ( cell signaling 5247 ) as loading controls . the induction of apoptosis was measured by annexin v - fitc / propidium iodide staining and flow cytometry . either 1 000 000 suspension ( u-937 , hl-60 , el4 ) cells or adherent cells at 75% confluency in 6-well plates were treated with combinations of pac-1 and 1541b ( final concentration of dmso the entire contents of each well was then transferred to flow cytometry tubes , pelleted , and suspended in 450 l of annexin v - fitc binding buffer ( 10 mm hepes , 140 mm nacl , 2.5 mm cacl2 , 1% bsa , ph 7.4 ) , premixed with annexin v - fitc and propidium iodide dyes . staining was assessed by flow cytometry ( 10 000 events per sample ) . the annexin v - fitc ( )/propidium iodide ( ) population was confirmed to be greater than 90% viable and normalized as the live control . the role of caspases in the induction of apoptosis was assessed by cotreatment with 25 m q - vd - oph ( calbiochem ) . all experimental procedures were reviewed and approved by the university of illinois institutional animal care and use committee . 68 week old c57bl/6 mice were used in all experiments ( charles river ) . the maximum tolerated dose ( mtd ) of 1541b was determined . beginning at 5 mg / kg , ( formulated at 0.5 mg / ml in hpcd , ph 5.5 ) mice ( groups of three ) mtd was the highest dosage for which all mice survived and acceptable ( < 20% ) weight loss was observed . the mtds of pac-1 ( 200 mg / kg ) and 1541b ( 20 mg / kg ) were used as the base unit for determining the combined mtd . beginning with 1/10 of each mtd ( 20 mg / kg pac-1 + 2 mg / kg 1541b ) groups of 3 mice were treated with 1/10 , 1/6 , 1/4 , 1/3 , 1/2 and 3/4 of each single agent s mtd . given the tolerability of 1/2 and the toxicity of 3/4 , an additional treatment of 125 mg / kg pac-1 + 17.5 ten week old , c57bl/6 female mice ( n = 3/cohort ) were administered three consecutive daily intraperitoneal injections of hydroxypropyl--cyclodextrin ( vehicle ) , pac-1 ( 125 mg / kg ) , 1541b ( 17.5 mg / kg ) , or pac-1 + 1541b ( 125 mg / kg + 17.5 mg / kg , respectively ) , and then humanely sacrificed 24 h later . heparinized whole blood was collected for assessment of total white blood cells , neutrophils , lymphocytes , hematocrit , platelets , creatinine , blood urea nitrogen , albumin , alanine aminotransferase , alkaline phosphatase , and total bilirubin . mice were necropsied , and heart , lung , kidney , liver , spleen , gastrointestinal tract and brain were collected for histopathology . tissue samples were fixed overnight in 10% neutral buffered formalin , processed , and paraffin embedded for histopathology using routine methods . tissue blocks were sectioned into 3 m tissue sections and stained with hematoxylin and eosin . all slides were systematically evaluated by a single board certified veterinary pathologist ( lbb ) for evidence of acute or chronic inflammation and toxicity . all lesions were characterized , recorded , and scored for severity ( minimal = 1 , mild = 2 , moderate = 3 , and severe = 4 ) . ten week old , c57bl/6 female mice were administered a single intraperitoneal dose of pac-1 at 125 mg / kg or 1541b at 17.5 mg / kg , and sacrificed in cohorts of 3 at predetermined time points ( 0 , 10 , 20 , 30 , 40 , 60 , 120 , 240 , 360 , 720 , and 1440 min ) . blood was collected and centrifuged , and plasma separated for quantification of pac-1 or 1541b by hplc methods ( uiuc metabolomics center , urbana , il ) . pharmacokinetic analyses were performed , using a nonlinear regression program ( winnonlin , version 5.1 , pharsight corporation , cary , nc ) . ten million el4 cells were prepared in hbss and injected subcutaneously on the right flank of sedated ( ketamine / xylazine ) mice ( day 0 ) . by three hours postinjection the injection bleb was no longer evident , and soft tumors appeared the following morning . mice were randomized into four treatment groups : vehicle , 1541b alone , pac-1 alone , and 1541b + pac-1 . compounds were formulated in hpcd ( 1541b at 0.5 mg / ml at ph 5.5 and pac-1 at 12.5 mg / ml at ph 5.5 ) . all mice received two treatments , one hour apart : vehicle ( hpcd + hpcd ) , 1541b ( hpcd + 1541b ) , pac-1 ( pac-1 + hpcd ) and 1541b + pac-1 ( pac-1 + 1541b ) . after 8 days the largest tumors had achieved maximal size ; mice were sacrificed , and tumors were excised and weighed .

combination anticancer therapy typically consists of drugs that target different biochemical pathways or those that act on different targets in the same pathway . here we demonstrate a new concept in combination therapy , that of enzyme activation with two compounds that hit the same biological target , but through different mechanisms . combinations of procaspase-3 activators pac-1 and 1541b show considerable synergy in activating procaspase-3 in vitro , stimulate rapid and dramatic maturation of procaspase-3 in multiple cancer cell lines , and powerfully induce caspase - dependent apoptotic death to a degree well exceeding the additive effect . in addition , the combination of pac-1 and 1541b effectively reduces tumor burden in a murine lymphoma model at dosages for which the compounds alone have minimal or no effect . these data suggest the potential of pac-1/1541b combinations for the treatment of cancer and , more broadly , demonstrate that differentially acting enzyme activators can potently synergize to give a significantly heightened biological effect .

Introduction Results Discussion Methods

diabetic retinopathy is the name given to the changes in the retina , which develop over a period of time in diabetics . it remains one of the major causes of new - onset visual loss in developed countries . if the central part of the retina ( i.e. , the macula ) is involved , it is referred to as diabetic maculopathy . the traditional approach to diagnosis of diabetic maculopathy includes fundus ophthalmoscopy and fluorescein angiography ( fa ) . the early treatment diabetic retinopathy study ( etdrs ) identified stereoscopic slit - lamp biomicroscopy and stereo colour fundus photography as standard methods of macular thickness assessment utilized in order to determine whether the treatment should be commenced as they defined the clinically significant macular oedema ( etdrs report number 10 , 1991 ) . however , these methods are subjective and relatively insensitive to small changes in retinal thickness and , therefore , may be unable to identify mild or localized macular thickening . they also do not provide any data on retinal morphology and blood flow . on the other hand , fa is a highly effective test of evaluating retinal blood vessels , macular perfusion , and pattern of leakage causing the oedema . although very useful clinically , it also does not contribute much to the evaluation of retinal morphology and its thickness profile . in 1991 the researchers from massachusetts institute of technology and harvard university patented the technique of optical coherence tomography ( oct ) , which was a major breakthrough in ophthalmic diagnostics ( us5321501 a , swanson ea , huang d , fujimoto jg , puliafito ca , lin cp , schuman js . method and apparatus for optical imaging with means for controlling the longitudinal range of the sample ) . the first paper to present the potential of the new diagnostic method four years later the first paper was published which described the use of oct in diagnosis of macular diseases . the purpose of this paper is to provide an overview of clinical utility of oct in retinal assessment of diabetic patients . oct enables obtaining the high resolution ( few micrometres ) cross - sectional images ( tomograms ) of the human retina in a noninvasive manner . in contrast to classic fundus photography taken with fundus camera , oct also provides information on the depth that the scattered light comes from . if light is reflected by the deeper retinal layers , it has to go a longer way to return to the detector compared to the light reflected from more superficial layers . this feature makes it possible to precisely determine what retinal depth ( i.e. , layer ) the particular signal comes from . therefore , oct resembles ultrasound imaging with the only difference consisting in utilizing light instead of sound . the use of light gives oct higher axial resolution compared to any other imaging techniques currently used in clinical medicine . in classic oct setup the light emitted by the superluminescent diode is directed to the beam splitter which splits it into two equal beams . one of them is projected onto the reference mirror , the other one onto the retina , and is backscattered from its morphological elements . the wave interference may occur only when the optical path between the beam splitter and the mirror is equal to the distance between the splitter and one of the surfaces reflecting the light within the retina . in that case , the detector will record the change in the light intensity . in order to detect other reflecting surfaces , the position of the reference mirror is referred to as time domain oct ( tdoct ) due to the fact that the information on retinal morphology along the scanning beam is obtained by recording the optical signal when the mirror is moved . this technique required the reference arm to be held fixed , and the optical path length difference between sample and reference reflections is encoded by the frequency of the interferometric fringes as a function of the source spectrum . the first is spectral domain oct ( sdoct ) in which interferometric signal is detected using the spectrometer equipped with a line of light sensitive elements . the other method is a swept source oct ( ssoct ) utilizing swept tunable lasers and a standard photodiode detector . as in fdoct , the reference mirror remains fixed , the better mechanical stability of the system is achieved . additionally , the interferometric signal created by mixing the sample and reference light is sampled as a function of wavenumber and yields an entire depth scan at the same time . this makes it possible to achieve several hundred - fold increase in speed and sensitivity of scanning compared to tdoct . as a result , significant motion artefacts are avoided and multiple measurements can be taken in a short time enabling the three - dimensional retinal scanning . the oct images can be also acquired at the video - rate and the measured structures observed in real time . the first commercially available sdoct device ( copernicus , optopol sa , poland ) the term reflectivity is used in oct technique as an equivalent of echogenicity in ultrasonography . the areas showing reduced reflectivity are referred to as hyporeflective , whereas the increased reflectivity regions are referred to as hyperreflective . the reflectivity in a grey - scale is proportional to tissue brightness observed in oct . the higher the reflectivity is , the closer to white the colour will be . in order to effectively visualise subtle structures in oct scans , the false colour scale is often clinically used , in which the individual colours are purely conventional . usually white and red represent highest intensity signal , whereas black and blue correspond to the lowest intensity signal . however , such approach has a disadvantage , namely , possible occurrence of artefacts . if signal intensity changes , the colour of a given structure on oct scan may also change . the oct results are presented as an axial scan , referred to as an a - scan , similarly like in ultrasonography . it is acquired by presenting the amplitude of the back - scattered light as a function of echo delay time . as the scanning beam moves along the retina , many a - scans are acquired , which form the tomogram , that is a b - scan ( figure 1 ) . it presents the cross section of the retina in a plane perpendicular to its surface . the set of many consecutive b - scans is assembled into a 3d reconstruction of retinal structure ( figure 2 ) . the software in - built in commercially available oct devices makes it possible to carry out a quantitative data analysis . the results of such analyses primarily include total retinal and individual layers thickness maps as well as macular volume maps . retinal thickness is usually calculated for central fixation point , 9 etdrs - like macular regions , and total macular thickness ( figure 3 ) . retinal volume is displayed for 9 etdrs - like macular areas and total macular volume . the oct data is automatically segmented in order to generate the above maps ( figure 2 ) . when interpreting these maps , one should bear in mind that the artefacts may occur during segmentation , which will lead to improper retinal thickness measurements [ 10 , 11 ] . artefacts may arise as a result of poor image quality , eye movement during measurements , and retinal pathologies interfering with automated segmentation ( e.g. , retinal pigment epithelial detachment , subretinal fluid , fibrosis , or haemorrhage ) . oct maps may be compared to normative data , including age , sex , and race ( figure 3 ) [ 1214 ] . it should be noted that different oct devices have different in - built normative databases . therefore , direct comparisons of maps generated by different oct devices are pointless [ 15 , 16 ] . the quantitative monitoring of retinal thickness in a given patient requires using the same oct device model for all follow - up examinations . it is quite intuitive , but prior to discussing the aspects of the retinal architecture seen in oct , the physical foundations of the obtained images should be explained . that is why the oct images should not be interpreted indiscriminately as histologic specimens . in histopathological examination , different stains show the affinity to different morphological elements , for example , cell nuclei . on the other hand , particular colours on oct cross - sectional image correspond to different signal intensity levels . however , since retinal oct images highly resemble histologic specimens , despite the above difference , and oct is a noncontact examination , it can be seen as an optical biopsy , which does not require tissue excision in order to analyse its structure . histologically , the retina consists of 10 layers , 4 of them being cellular and 2 neuronal junctions . the axonal layers , that is , the nerve fibre layer and the plexiform layers , are capable of potent light scatter and appear yellow to red on false - colour oct images . the light scattering potential of nuclear layers is lower , so they are represented as blue and black areas . the first layer visible on oct images is the internal limiting membrane which appears as a hyperreflective line at the vitreoretinal interface . it is visualized in oct owing to the increased light scatter between the transparent vitreous and retinal surface . below , there lies the retinal nerve fibre layer , typically thicker in the nasal macula and capable of potent light scatter . subsequently , hyperreflective plexiform layers are imaged as well as the inner nuclear layer situated between them , which has a lower light scattering potential . then , the relatively thick hyporeflective outer nuclear layer is visible with a thin hyperreflective line underneath . a distinct bright stria that stretched in front of the rpe demarcates the junction between the inner and outer photoreceptor segments ( is / os ) . due to the increased length of outer cone segments in central fovea it contains melanin and is capable of very potent light scattering . on the other hand , below , choriocapilaries and the rest of the choroid are visible . in the centre of the macula reduced reflectivity is most often cause by intraretinal and subretinal fluid accumulation ( oedema , retinal detachment , serous rpe detachment ) . pathological features that can be hyperreflective are : hard exudates , calcification , epiretinal and thick vitreous membranes , fibrosis , haemorrhages , rpe hyperplasia , neovascular membranes , atrophy of the retina and rpe causing increased reflectivity of underlying choroid . thus , it facilitates detecting macular oedema which is the main pathologic feature of diabetic maculopathy . this is defined as any detectable retinal thickening due to fluid accumulation ( etdrs report number 10 , 1991 ) . the oedema may be symmetrical or involve only a sector of the macular area . it usually starts as a focal lesion and progresses towards a more diffuse form . in some cases , the macular edges may be thickened , even though the contour of the foveal centre remains normal . persistent retinal oedema resulting in muller cell necrosis leads to the formation of cystoid cavities , located mainly in the outer retina ( henle 's fibre and outer plexiform layer ) , and sometimes also in the inner plexiform layer . in the most advanced stages in eyes with well - established long term macular oedema , several central cysts can merge together forming large hyporeflective cavity which contributes to the significant thickening of the fovea ( figure 4 ) . therefore , the main characteristics of macular oedema in oct , apart from increased retinal thickness , include intraretinal spaces of reduced reflectivity , disintegration of the layered retinal structure , and usually also flattening of the central foveal depression . in some cases fluid can be seen under the neurosensory retina ( figures 4 and 5(a ) ) . they present as small hyperreflective deposits with posterior shadowing ( figures 5(c ) and 5(d ) ) . that is why koleva - gorgieva proposed the classification of cystoid diabetic macular oedema ( dme ) into mild , moderate , and severe according to the size of cystoid spaces . the cystoid spaces in eyes with intermediate and severe cystoid dme are mainly located in the outer layers , predominantly in the fovea . in some cases small cysts in the inner layers can also be found . if the cysts continue to increase , they may occupy the full thickness of the retina , leading to its atrophy and the profound vision loss . in the past few years , since the introduction of fdoct , it has become possible to accurately visualize the outer retinal layers . several authors have reported that the integrity of elm and is / os junction has a positive correlation with visual acuity [ 21 , 22 ] . shin et al . showed that the photoreceptor layer status is closely associated with final visual acuity in dme and that photoreceptor integrity prior to treatment can be predictive of potential visual recovery in dme . yohannan et al . demonstrated that disruption of is / os junction correlates well with a significant decrease in point sensitivity in eyes with dme . therefore , the assessment of outer retinal layer structure should be a part of a routine evaluation when performing oct in patients with dme . the ability to visualize the vitreoretinal interface ( figures 6 , 7 , and 8) is a unique feature of oct . it allows for macular traction imaging , which may play a role in dme development [ 25 , 26 ] . the traction may be induced by vitreoretinal interface abnormalities such as incomplete posterior vitreous detachment ( pvd ) or epiretinal membrane ( erm ) . if the posterior hyaloid is thin and only slightly detached from the surface of the macula , it is not visible in ophthalmoscopy , but can be easily detected by oct . the same is true for erm ; if it is thin and does not cause a significant retinal distortion , it can be only visualized using oct . that is why the assessment of vitreoretinal interface is an essential step in macular evaluation in patients with diabetic retinopathy . moreover , oct does not only work well as a diagnostic tool in macular traction but may also be used in order to monitor the postoperative morphological outcomes ( figure 9 ) . it can also help identify the postoperative complications of vitrectomy , such as retinal detachment , erm , and lamellar macular hole formation . the detached posterior vitreous face presents oct scans as a thin hyper - to - medium reflective horizontal or oblique line in the non - reflective vitreous cavity , above or inserting into the retina . in case of incomplete pvd it can lead to increase in macular thickness , loss of foveal depression , and formation of intraretinal cystoid spaces or pseudoholes ( figure 6 ) . the distinction between erms and a pvd is usually made on the basis of reflectivity . oct can also be used to document the opacity and thickness of erm , its distance from the surface of the retina , and such effect on the underlying retina as distortion , oedema , or neurosensory detachment . it should be noted that oct is complementary to ultrasound scanning in evaluation of vitreoretinal interface . ultrasound scans provide a more complete image of vitreous pathology but at the cost of lower resolution . fdoct , on the other hand , provides a more detailed image of vitreoretinal interface but limited to a relatively small area ( figure 7 ) . as oct uses light to acquire the images , if the optic media are opacified and fundus can not be visualized , the retinal cross sections will not be obtained . the limitations are similar to the ones associated with ophthalmoscopy and fa ( figure 10 ) . however , sometimes oct images can be acquired in cases where retinal assessment in ophthalmoscopy is impossible ( figure 11 ) . in some cases oct can even enable assessment of the space located posteriorly to the thin fibrovascular membrane in proliferative retinopathy ( figure 12 ) . another particular value of oct is the possibility of reliable and reproducible retinal thickness measurements ( figure 5 ) . using the retinal thickness maps , it is possible to monitor dme progression and assess treatment outcomes after laser photocoagulation , intravitreal injections of anti - vegf and steroids or , as mentioned before , vitrectomy . owing to retinal thickness maps not only oedema but also atrophy can be detected , which contributes to lack of improvement or even decreased vision after the oedema is resolved ( figure 3 ) . the treatment efficacy in dme should , then , be evaluated in terms of two outcomes : the functional one based on visual acuity measurements and anatomical one assessed in oct . the first oct classification of dme presented by otani et al . was based on retinal morphological changes : sponge - like swelling , cystoid oedema , and serous retinal detachment . along with the improving oct technology , it takes into account several quantitative and qualitative oct data : retinal thickness , retinal morphology , retinal topography , macular traction , and foveal photoreceptor status . normal macular morphology and thickness not reaching the criteria for subclinical dme;early subclinical macular oedema no clinically detected retinal thickening on ophthalmoscopy , oct measured retinal thickness exceeding normal + 2sds for central fixation point and fovea;established macular oedema retinal thickening and evident morphological characteristics of oedema . no macular oedema normal macular morphology and thickness not reaching the criteria for subclinical dme ; early subclinical macular oedema no clinically detected retinal thickening on ophthalmoscopy , oct measured retinal thickness exceeding normal + 2sds for central fixation point and fovea ; established macular oedema retinal thickening and evident morphological characteristics of oedema . this includes the following : simple noncystoid macular oedema increased retinal thickness , reduced intraretinal reflectivity , irregularity of the layered structure , and flattening of the foveal depression , without presence of cystoid spaces;cystoid macular oedema the above criteria , associated with presence of well - defined intraretinal cystoid spaces : ( a)mild cystoid macular oedema cystoid spaces with horizontal diameter < 300 m , ( b)intermediate cystoid macular oedema cystoid spaces with horizontal diameter 300 m < 600 m , ( c)severe cystoid macular oedema cystoid spaces with horizontal diameter 600 m , or large confluent cavities with retinoschisis appearance;serous macular detachment any of the above , associated with serous macular detachment ( hyporeflective area under the detached neurosensory retina and over the hyperreflective line of the rpe ) . simple noncystoid macular oedema increased retinal thickness , reduced intraretinal reflectivity , irregularity of the layered structure , and flattening of the foveal depression , without presence of cystoid spaces ; cystoid macular oedema the above criteria , associated with presence of well - defined intraretinal cystoid spaces : ( a)mild cystoid macular oedema cystoid spaces with horizontal diameter < 300 m , ( b)intermediate cystoid macular oedema cystoid spaces with horizontal diameter 300 m < 600 m , ( c)severe cystoid macular oedema cystoid spaces with horizontal diameter 600 m , or large confluent cavities with retinoschisis appearance ; mild cystoid macular oedema cystoid spaces with horizontal diameter < 300 m , intermediate cystoid macular oedema cystoid spaces with horizontal diameter 300 m < 600 m , severe cystoid macular oedema cystoid spaces with horizontal diameter 600 m , or large confluent cavities with retinoschisis appearance ; serous macular detachment any of the above , associated with serous macular detachment ( hyporeflective area under the detached neurosensory retina and over the hyperreflective line of the rpe ) . this includes the following : nonsignificant macular oedema;clinically significant macular oedema , as defined by etdrs and evaluated on the oct retinal topography map . nonsignificant macular oedema ; clinically significant macular oedema , as defined by etdrs and evaluated on the oct retinal topography map . this includes the following : no macular traction presence of complete pvd ( weiss ring detected on ophthalmoscopy ) , or no pvd ( no visible posterior hyaloid line on fdoct ) , and no erm;questionable macular traction incomplete pvd with perifoveal or peripapillary adhesion and/or globally adherent erm without detectable distortion of retinal surface contour at the points of adhesion;definite macular traction incomplete pvd with perifoveal adhesion and/or focal erm with detectable distortion of retinal contour at the points of adhesion . no macular traction presence of complete pvd ( weiss ring detected on ophthalmoscopy ) , or no pvd ( no visible posterior hyaloid line on fdoct ) , and no erm ; questionable macular traction incomplete pvd with perifoveal or peripapillary adhesion and/or globally adherent erm without detectable distortion of retinal surface contour at the points of adhesion ; definite macular traction incomplete pvd with perifoveal adhesion and/or focal erm with detectable distortion of retinal contour at the points of adhesion . this includes the following : is / os and elm intact;is / os and elm with disrupted integrity . is / os and elm intact ; is / os and elm with disrupted integrity . apart from the acquisition of morphological images , oct can also detect a doppler frequency shift of reflected light , which provides information on flow and movement [ 3537 ] . wang et al . reported that reproducible and repeatable measurements of total blood flow can be obtained using doppler oct . in another study , wang et al . compared blood flow in a patient with diabetes and no retinopathy with another patient with treated proliferative retinopathy . the first subject showed a total blood flow value at the lower level of the normal range , whereas the same value in a patient with diabetic retinopathy was lower compared to healthy population . these results clearly indicate that doppler oct may play a role in noninvasive assessment of retinal blood flow in diabetic patients . the oct can also be used for visualizing the tiny blood vessels within the macula . during the american academy of ophthalmology annual meeting 2012 we presented a novel method ( oct angiography ) for the noninvasive visualization of three - dimensional retinal microcapillary network using intensity - based oct and also validated its clinical usefulness in retinal vascular diseases including diabetic retinopathy ( sikorski bl , szkulmowski m , malukiewicz g , kowalczyk a , wojtkowski m. noninvasive visualization of 3d retinal microcapillary network using oct . oct angiography proved to be capable of showing 10 micron blood vessels , revealing the vascular nonperfusion and identifying microexudates which were not otherwise visible on clinical examination and fundus photography . moreover , oct angiography can show even more capillaries in the pericentral macula than fa and allows discerning and visualizing separately the superficial and deep capillary plexus . therefore , we believe that classic structural oct examination together with oct angiography may provide a comprehensive solution in a single imaging modality in patients with diabetic maculopathy . oct can perform micrometre - resolution , cross - sectional imaging of the retina that closely approximates its histological layers . one of the huge advantages of oct is that patients find this procedure very comfortable because it is noncontact and the measurement time is very short . in patients with diabetic retinopathy oct can be successfully utilized as an objective monitoring technique of the macular thickening before and after therapy . oct is also very useful for vitreous assessment , showing whether it is attached or detached from the macula . it is helpful in detecting vitreoretinal traction that may not have been identified clinically . to summarize , oct may assist in patient selection with diabetic maculopathy who can benefit from treatment , identify what treatment is indicated , guide its implementing , and allow precise monitoring of treatment response . it seems to be the technique of choice for the early detection of macular oedema and for the followup of diabetic maculopathy .

diabetic maculopathy ( dm ) is one of the major causes of vision impairment in individuals with diabetes . the traditional approach to diagnosis of dm includes fundus ophthalmoscopy and fluorescein angiography . although very useful clinically , these methods do not contribute much to the evaluation of retinal morphology and its thickness profile . that is why a new technique called optical coherence tomography ( oct ) was utilized to perform cross - sectional imaging of the retina . it facilitates measuring the macular thickening , quantification of diabetic macular oedema , and detecting vitreoretinal traction . thus , oct may assist in patient selection with dm who can benefit from treatment , identify what treatment is indicated , guide its implementing , and allow precise monitoring of treatment response . it seems to be the technique of choice for the early detection of macular oedema and for the followup of dm .

1. Introduction 2. OCT Principles and Interpretation 3. OCT Findings in Diabetic Maculopathy 4. OCT Classification of DME 5. Technological Advances in OCT 6. Conclusion

the design of the china peace - retrospective ami study has been published previously.8 in brief , we developed a nationally representative sample of hospitalizations for ami in 2001 , 2006 , and 2011 using a 2-stage random sampling design . in the first stage , we identified hospitals using a simple random sampling procedure within 5 geographical - economic strata of china : eastern - rural ; central - rural ; western - rural ; eastern - urban ; and central / western - urban regions . we used these strata because hospital volumes and clinical capacities differ between urban and rural areas as well among the 3 official geographical regions ( eastern , central , and western ) of mainland china . we combined central and western urban regions given their similar per capita income and health services capacity . in the 3 rural strata , the sampling framework consisted of the central hospital in each of the predefined rural regions ( 2010 central hospitals in 2010 rural regions ) . in the 2 urban strata , the sampling framework consisted of the highest - level hospitals in each of the predefined urban regions ( 833 hospitals in 287 urban regions ) . we randomly selected representative hospitals from 2011 to assess current practices and traced this cohort backward to 2006 and 2001 to describe temporal trends . in the second stage , using systematic random sampling procedures , we drew cases from each sampled hospital using the local hospital database for patients with ami in 2001 , 2006 , and 2011 . ami cases were identified using a principal discharge diagnosis of ami based on international classification of diseases versions 9 or 10 , given that hospitals in china are mandated by the ministry of health to list this information on the first page of the medical record , and in rare cases when such information was not available , we confirmed the diagnosis through medical record review . data abstraction quality was monitored by randomly auditing 5% of the medical charts , with overall accuracy exceeding 98% . the central ethics committee at the china national center for cardiovascular diseases , or local internal ethics committees approved the china peace - retrospective ami study . the funder of the study had no role in study design , data collection , data analysis , data interpretation , or writing of the report . we limited study samples to patients potentially eligible for spironolactone , namely , those with known hf or diabetes at discharge . patients who had a length of hospital stay shorter than 24 hours were excluded to ensure that all patients had sufficient opportunity to receive spironolactone . subsequently , we classified patients into 1 of 4 groups : the ideal group consisted of patients with a documented lvef 40% and no contraindication to spironolactone ; the contraindicated group consisted of patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; the not indicated group consisted of patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; and the unknown indications group consisted of patients whose lvef was not measured during the hospitalization . data elements collected included demographic information , medical history , patient characteristics at presentation , hospital characteristics , laboratory parameters , concomitant therapy , and documented diagnosis . cardiovascular risk ( cvr ) factors were coded as present if diagnosed before or during admission ; clinical characteristics , including vital signs , represented those recorded on admission . to capture the laboratory values likely to influence the decision about spironolactone therapy in patients who received the drug , we used the last potassium and creatinine values before administration of medication . for patients who ultimately did not receive spironolactone , we used the highest lab value recorded during hospitalization to ensure identification of any possible contraindication . lvef values were based on echocardiography , radionuclide angiography , or computerized tomography coronary angiography . to examine temporal trends in spironolactone therapy , we used the cochran - armitage tests and applied weights proportional to the inverse sampling fraction of hospitals to account for differences in the sampling fraction for each time period . when exploring patient and hospital characteristics associated with the use of spironolactone , categorical variables were expressed as frequencies and percentages and analyzed using chi - square tests . among all the variables , missing data were rare and occurred only for the age variable ( 0.1% ) , which was imputed to the overall median to avoid case - wise deletion . we used logistic regression models to identify predictors independently associated with spironolactone use in different patient groups by indications . variables in models include demographic characteristics , cvr factors , medical history , conditions and vital signs on admission , estimated glomerular filtration rate ( egfr ) , ami type , economic - geographical region , rural / urban region , and years . in addition , we also included hospital characteristics , such as teaching status and percutaneous coronary intervention ( pci ) capability ( table 1 ) . a generalized estimating equation model was developed to account for clustering of patients within hospitals . all variables in the bivariate model were included in the multivariable model except those with frequencies under 1% . bivariate analysis of characteristics associated with spironolactone therapy among ideal patients ace inhibitor indicates angiotensin - converting enzyme inhibitor ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; egfr , estimated glomerular filtration rate ; nstemi , non - st - segment elevation myocardial infarction ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; stemi , st - segment elevation myocardial infarction . all comparisons were 2-tailed , with p<0.05 considered statistically significant . all statistical analyses were performed using sas software ( version 9.2 ; sas institute , cary , nc ) and r software ( version 3.0.2 ; r foundation for statistical computing , vienna , austria ) . the design of the china peace - retrospective ami study has been published previously.8 in brief , we developed a nationally representative sample of hospitalizations for ami in 2001 , 2006 , and 2011 using a 2-stage random sampling design . in the first stage , we identified hospitals using a simple random sampling procedure within 5 geographical - economic strata of china : eastern - rural ; central - rural ; western - rural ; eastern - urban ; and central / western - urban regions . we used these strata because hospital volumes and clinical capacities differ between urban and rural areas as well among the 3 official geographical regions ( eastern , central , and western ) of mainland china . we combined central and western urban regions given their similar per capita income and health services capacity . in the 3 rural strata , the sampling framework consisted of the central hospital in each of the predefined rural regions ( 2010 central hospitals in 2010 rural regions ) . in the 2 urban strata , the sampling framework consisted of the highest - level hospitals in each of the predefined urban regions ( 833 hospitals in 287 urban regions ) . we randomly selected representative hospitals from 2011 to assess current practices and traced this cohort backward to 2006 and 2001 to describe temporal trends . in the second stage , using systematic random sampling procedures , we drew cases from each sampled hospital using the local hospital database for patients with ami in 2001 , 2006 , and 2011 . ami cases were identified using a principal discharge diagnosis of ami based on international classification of diseases versions 9 or 10 , given that hospitals in china are mandated by the ministry of health to list this information on the first page of the medical record , and in rare cases when such information was not available , we confirmed the diagnosis through medical record review . data abstraction quality was monitored by randomly auditing 5% of the medical charts , with overall accuracy exceeding 98% . the central ethics committee at the china national center for cardiovascular diseases , or local internal ethics committees approved the china peace - retrospective ami study . the funder of the study had no role in study design , data collection , data analysis , data interpretation , or writing of the report . we limited study samples to patients potentially eligible for spironolactone , namely , those with known hf or diabetes at discharge . patients who had a length of hospital stay shorter than 24 hours were excluded to ensure that all patients had sufficient opportunity to receive spironolactone . subsequently , we classified patients into 1 of 4 groups : the ideal group consisted of patients with a documented lvef 40% and no contraindication to spironolactone ; the contraindicated group consisted of patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; the not indicated group consisted of patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; and the unknown indications group consisted of patients whose lvef was not measured during the hospitalization . data elements collected included demographic information , medical history , patient characteristics at presentation , hospital characteristics , laboratory parameters , concomitant therapy , and documented diagnosis . cardiovascular risk ( cvr ) factors were coded as present if diagnosed before or during admission ; clinical characteristics , including vital signs , represented those recorded on admission . to capture the laboratory values likely to influence the decision about spironolactone therapy in patients who received the drug , we used the last potassium and creatinine values before administration of medication . for patients who ultimately did not receive spironolactone , we used the highest lab value recorded during hospitalization to ensure identification of any possible contraindication . lvef values were based on echocardiography , radionuclide angiography , or computerized tomography coronary angiography . to examine temporal trends in spironolactone therapy , we used the cochran - armitage tests and applied weights proportional to the inverse sampling fraction of hospitals to account for differences in the sampling fraction for each time period . when exploring patient and hospital characteristics associated with the use of spironolactone , categorical variables were expressed as frequencies and percentages and analyzed using chi - square tests . among all the variables , missing data were rare and occurred only for the age variable ( 0.1% ) , which was imputed to the overall median to avoid case - wise deletion . we used logistic regression models to identify predictors independently associated with spironolactone use in different patient groups by indications . variables in models include demographic characteristics , cvr factors , medical history , conditions and vital signs on admission , estimated glomerular filtration rate ( egfr ) , ami type , economic - geographical region , rural / urban region , and years . in addition , we also included hospital characteristics , such as teaching status and percutaneous coronary intervention ( pci ) capability ( table 1 ) . a generalized estimating equation model was developed to account for clustering of patients within hospitals . all variables in the bivariate model were included in the multivariable model except those with frequencies under 1% . bivariate analysis of characteristics associated with spironolactone therapy among ideal patients ace inhibitor indicates angiotensin - converting enzyme inhibitor ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; egfr , estimated glomerular filtration rate ; nstemi , non - st - segment elevation myocardial infarction ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; stemi , st - segment elevation myocardial infarction . all statistical analyses were performed using sas software ( version 9.2 ; sas institute , cary , nc ) and r software ( version 3.0.2 ; r foundation for statistical computing , vienna , austria ) . the nationally representative samples described in the china peace - retrospective ami study consisted of 16 100 patients hospitalized for ami in 162 hospitals across china ( figure 1a ) , with the 2011 sample representing 245 720 patients across china . after excluding patients with a length of stay shorter than 24 hours , and those without hf or diabetes when discharged , we identified 6906 patients ( 12.2% in 2001 , 27.7% in 2006 , and 60.1% in 2011 ) who were potentially eligible for spironolactone ( figure 1b ) . across all years , median age was 69 years ( interquartile range , 59 to 76 ) and 35.8% were female . among these patients , 44.2% had diabetes , and almost three quarters of patients ( 73.9% ) had hf - among them cvr factors were common : 57.9% had hypertension ( htn ) , 29.8% were current smokers , and 28.0% had coronary artery disease . a , flow diagram showing the process used to produce a nationally representative sampling of hospitals in china . b , flow diagram showing the approach to classify patients into 4 groups according to their indications for spironolactone . n represents number of patients . there were notable changes in the relative proportion of the 4 patient groups over time ( figure 2 ) . for example , the proportion of ideal patients doubled from 2001 to 2006 ( 4.5% to 9.1% ) and remained stable thereafter ( 10.2% in 2011 ) . in contrast , the proportion of contraindicated patients varied little across the 3 years ( 13.0% , 11.9% , and 10.2% in 2001 , 2006 , and 2011 , respectively ) . patients increased markedly over the years ( from 15.6% in 2001 to 46.8% in 2011 ; p<0.001 for trend ) , whereas that of unknown indications acute myocardial infarction patients with heart failure or diabetes grouped by their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . overall , the weighted rate of spironolactone use in 2011 differed among patients in each group : 72.4% in ideal ; 27.5% in contraindicated ; 38.3% in not indicated ; and 35.1% in unknown indications . spironolactone use increased in all groups over the past decade : among ideal patients , the weighted rate of use increased from 28.6% in 2001 to 68.5% in 2006 and to 72.4% in 2011 ( p<0.001 for trend ) , whereas for contraindicated patients it increased from 11.4% in 2001 to 22.4% in 2006 and to 27.5% in 2011 ( p=0.002 for trend ) . similar increases were observed among not indicated patients ( p=0.007 for trend ) and unknown indications patients ( p<0.001 for trend ; figure 3 ) . given that spironolactone can also be used to treat htn or as a concomitant therapy in hf with reduced lvef , we performed a post - hoc analysis describing spironolactone use in a specific subgroup of not indicated patients , namely , those with neither htn nor hf . in this subgroup of patients in 2011 , 17.2% received spironolactone . spironolactone use ( weighted ) among different groups of acute myocardial infarction patients with heart failure or diabetes according to their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . bivariate analysis of the factors associated with the use of spironolactone among ideal patients is shown in table 1 . all characteristics were entered into the multivariable model to determine independent predictors of use ( figure 4 ) . specifically , older patients ( 65 year ) were more likely to be treated than younger patients ( 72.2% vs. 55.1% ; or , 2.07 ; 95% ci , 1.30 to 3.30 ) . patients with htn or with symptoms of hf at admission were both more likely to receive spironolactone ( 70.2% vs. 61.1% ; or , 1.57 ; 95% ci , 1.11 to 2.23 and 72.9% vs. 52.1% ; or , 2.00 ; 95% ci , 1.18 to 3.36 , respectively ) than those without these comorbidities . patients in pci - capable hospitals were more likely to be treated ( 71.1% vs. 51.6% ; or , 2.12 ; 95% ci , 1.12 to 4.02 ) . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; pci , percutaneous coronary intervention . characteristics in multivariable analysis that remained independently associated with the use of spironolactone in the other 3 groups are shown in figures5 through 7 . contraindicated and unknown indications groups ( or , 1.84 ; 95% ci , 1.15 to 2.94 and or , 1.74 ; 95% ci , 1.31 to 2.29 , respectively ) . patients > 65 years old and patients with htn had a high likelihood of receiving spironolactone among both not indicated and unknown indication groups . across all groups , patients with symptoms of hf at admission to the hospital were more likely to be treated with spironolactone ( contraindicated : or , 3.26 ; 95% ci , 2.05 to 5.16 ; unknown indications : or , 2.60 ; 95% ci , 2.12 to 3.18 ; not indicated : or , 2.16 ; 95% ci , 1.73 to 2.69 ) . factors associated with spironolactone therapy among contraindicated patients in multivariable model . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among not indicated patients in the multivariable model . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among unknown indications patients in the multivariable model . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; sbp , systolic blood pressure . the nationally representative samples described in the china peace - retrospective ami study consisted of 16 100 patients hospitalized for ami in 162 hospitals across china ( figure 1a ) , with the 2011 sample representing 245 720 patients across china . after excluding patients with a length of stay shorter than 24 hours , and those without hf or diabetes when discharged , we identified 6906 patients ( 12.2% in 2001 , 27.7% in 2006 , and 60.1% in 2011 ) who were potentially eligible for spironolactone ( figure 1b ) . across all years , median age was 69 years ( interquartile range , 59 to 76 ) and 35.8% were female . among these patients , 44.2% had diabetes , and almost three quarters of patients ( 73.9% ) had hf - among them cvr factors were common : 57.9% had hypertension ( htn ) , 29.8% were current smokers , and 28.0% had coronary artery disease . a , flow diagram showing the process used to produce a nationally representative sampling of hospitals in china . b , flow diagram showing the approach to classify patients into 4 groups according to their indications for spironolactone . n represents number of patients . there were notable changes in the relative proportion of the 4 patient groups over time ( figure 2 ) . for example , the proportion of ideal patients doubled from 2001 to 2006 ( 4.5% to 9.1% ) and remained stable thereafter ( 10.2% in 2011 ) . in contrast , the proportion of contraindicated patients varied little across the 3 years ( 13.0% , 11.9% , and 10.2% in 2001 , 2006 , and 2011 , respectively ) . patients increased markedly over the years ( from 15.6% in 2001 to 46.8% in 2011 ; p<0.001 for trend ) , whereas that of unknown indications acute myocardial infarction patients with heart failure or diabetes grouped by their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . overall , the weighted rate of spironolactone use in 2011 differed among patients in each group : 72.4% in ideal ; 27.5% in contraindicated ; 38.3% in not indicated ; and 35.1% in unknown indications . spironolactone use increased in all groups over the past decade : among ideal patients , the weighted rate of use increased from 28.6% in 2001 to 68.5% in 2006 and to 72.4% in 2011 ( p<0.001 for trend ) , whereas for contraindicated patients it increased from 11.4% in 2001 to 22.4% in 2006 and to 27.5% in 2011 ( p=0.002 for trend ) . similar increases were observed among not indicated patients ( p=0.007 for trend ) and unknown indications patients ( p<0.001 for trend ; figure 3 ) . given that spironolactone can also be used to treat htn or as a concomitant therapy in hf with reduced lvef , we performed a post - hoc analysis describing spironolactone use in a specific subgroup of not indicated patients , namely , those with neither htn nor hf . in this subgroup of patients in 2011 , 17.2% received spironolactone . spironolactone use ( weighted ) among different groups of acute myocardial infarction patients with heart failure or diabetes according to their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . bivariate analysis of the factors associated with the use of spironolactone among ideal patients is shown in table 1 . all characteristics were entered into the multivariable model to determine independent predictors of use ( figure 4 ) . specifically , older patients ( 65 year ) were more likely to be treated than younger patients ( 72.2% vs. 55.1% ; or , 2.07 ; 95% ci , 1.30 to 3.30 ) . patients with htn or with symptoms of hf at admission were both more likely to receive spironolactone ( 70.2% vs. 61.1% ; or , 1.57 ; 95% ci , 1.11 to 2.23 and 72.9% vs. 52.1% ; or , 2.00 ; 95% ci , 1.18 to 3.36 , respectively ) than those without these comorbidities . patients in pci - capable hospitals were more likely to be treated ( 71.1% vs. 51.6% ; or , 2.12 ; 95% ci , 1.12 to 4.02 ) . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; pci , percutaneous coronary intervention . characteristics in multivariable analysis that remained independently associated with the use of spironolactone in the other 3 groups are shown in figures5 through 7 . contraindicated and unknown indications groups ( or , 1.84 ; 95% ci , 1.15 to 2.94 and or , 1.74 ; 95% ci , 1.31 to 2.29 , respectively ) . patients > 65 years old and patients with htn had a high likelihood of receiving spironolactone among both not indicated and unknown indication groups . across all groups , patients with symptoms of hf at admission to the hospital were more likely to be treated with spironolactone ( contraindicated : or , 3.26 ; 95% ci , 2.05 to 5.16 ; unknown indications : or , 2.60 ; 95% ci , 2.12 to 3.18 ; not indicated : or , 2.16 ; 95% ci , 1.73 to 2.69 ) . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among not indicated patients in the multivariable model . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among unknown indications patients in the multivariable model . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; sbp , systolic blood pressure . in this national quality assessment analyzing spironolactone use among patients with ami in china , we found that spironolactone use increased over time . however , suboptimal patient identification and selection were detected throughout the study period and persisted in 2011 . although more patients underwent lvef assessment , which is necessary to determine their eligibility for spironolactone , one third of patients did not have an lvef assessment during their hospitalization for ami in 2011 . spironolactone use among patients who may not benefit and those with contraindications was common and such use increased significantly over time . our findings indicate that the chinese health care system rapidly responded to new information that highlighted the utility of spironolactone , but also appears to have driven increased use among patients who lack a strong indication , albeit at a lower rate than in ideal patients . these findings highlight an opportunity for hospitals in china to improve the translation of evidence into clinical practice . to our knowledge , this is the first comprehensive , nationally representative quality assessment of spironolactone use in ami in china . previous studies evaluating the use of aldosterone antagonists among ami patients in other countries have focused only on patients with definite indications for treatment.911 in contrast , our study describes to what extent patients with ami are evaluated for spironolactone and shows that spironolactone is used not only among patients with indications , but also those with contraindications , without indications , and with indications unknown . additionally , the use of a nationally representative sample ensures that the findings of this analysis are broadly applicable across china and can serve as the basis for future quality improvement initiatives . lvef assessment among patients with ami improved over the past decade , which created more opportunities to consider spironolactone therapy ; however , further improvement is possible . in 2001 , indications for two thirds of patients were unknown and only 4.5% of the cohort was classified as ideal patients increased to 10% ; however , one third of patients still lacked an lvef assessment . in comparison , among patients in the united states with ami from 2007 to 2009 , the rate of lvef assessment was reported to be 91.0%.12 it should be noted that lvef assessment is a critical component of the care of patients with ami because it enables risk stratification and guides the prescription of other therapies as well , for instance , inhibitors of angiotensin converting enzyme ( ace ) . the increasing use of spironolactone among ideal patients in china over time is encouraging . in 2001 , spironolactone use among ideal patients was no better than other groups , but it increased sharply thereafter , possibly in response to the ephesus study , which was published after 2001 and clearly supported the use of aldosterone antagonists in this patient population.6 there are no previous studies in china with which to compare our results . however , a registry - based study in spain reported that 54.8% patients with ami and hf received aldosterone antagonist in hospital between 2006 and 20089 ; and in the united states , the prescription rate of aldosterone antagonists at discharge among patients with ami and reduced lvef was only approximately 15% from 2009 to 2010.10,11 although the utilization seems to be better in china than in other countries , there remains room for further improvement , given the potential benefit of this agent . the substantial use of spironolactone among patients with contraindications nearly 1 in 4 patients was concerning and indicates a gap in the patient selection process that can expose patients to potential harm , such as worsening hyperkalemia or significant renal dysfunction.4,13 it must be noted that there may be circumstances in which spironolactone use is contraindicated according to the guidelines , yet clinicians perceive that the benefit of the drug will outweigh its risks . for example , some recently published studies demonstrate that the benefit of aldosterone antagonists may offset its risk in the setting of moderate hyperkalemia.14,15 nonetheless , the rising proportion of contraindicated patients being treated with spironolactone is consistent with a study in the united states and suggests that the growth in use may , at times , be indiscriminate.16 spironolactone was frequently used among patients without clear indications , namely , those with lvef > 40% . it should be noted that the recent treatment of preserved cardiac function heart failure with an aldosterone antagonist study found no benefit of spironolactone use in patients with hf and a preserved lvef.17 in the subgroup of patients without any identifiable indication , including htn or systolic hf , 17.2% were treated , suggesting that a substantial number of patients are receiving a drug that has limited benefit for their condition , and with a potential for adverse effects . in all 4 groups , such a pattern indicates that the presence of hf was a common and important trigger for spironolactone prescription by clinicians and implies a wide acceptance of the drug in hf . additionally , the steady rate of loop diuretic use among patients with hf indicates that spironolactone was not being systematically substituted for loop diuretics , and that other factors are driving spironolactone uptake . the selection issues identified in this analysis may impede the transfer of benefit to appropriate patients . quality improvement initiatives emphasizing the appropriate use of spironolactone are warranted to improve patient selection and avoid adverse events . however , to ensure accurate abstraction , we had strict definitions for all variables and employed quality control procedures to insure that the abstraction accuracy reached 98% . second , we may not have captured all patients with contraindications owing to inadequate physician documentation or unmeasured serum chemistry , which may lead to errors in the estimation of ideal patients and contraindicated patients . third , the benefit of aldosterone antagonists for ami patients was demonstrated in the ephesus study , which studied eplerenone , rather than spironolactone , which is the only aldosterone antagonist available in china . however , the structural similarity of spironolactone and eplerenone suggests that they may have similar efficacy , and guidelines endorse the use of both drugs for patients with ami . last , because data were abstracted from deidentified medical charts , we were unable to determine whether some patients were included in multiple study years , or had multiple admissions during the same year ; however , given the 5-year difference between the 3 time points , and the random sampling method , the number of such patients is likely to be minimal . first , our analysis used data abstracted from medical records . however , to ensure accurate abstraction , we had strict definitions for all variables and employed quality control procedures to insure that the abstraction accuracy reached 98% . second , we may not have captured all patients with contraindications owing to inadequate physician documentation or unmeasured serum chemistry , which may lead to errors in the estimation of ideal patients and contraindicated patients . third , the benefit of aldosterone antagonists for ami patients was demonstrated in the ephesus study , which studied eplerenone , rather than spironolactone , which is the only aldosterone antagonist available in china . however , the structural similarity of spironolactone and eplerenone suggests that they may have similar efficacy , and guidelines endorse the use of both drugs for patients with ami . last , because data were abstracted from deidentified medical charts , we were unable to determine whether some patients were included in multiple study years , or had multiple admissions during the same year ; however , given the 5-year difference between the 3 time points , and the random sampling method , the number of such patients is likely to be minimal . we identified opportunities to optimize the use of spironolactone post - ami in chinese clinical practice , including wider lvef assessment , more - careful selection of patients , and increasing the utilization among ideal patients . our findings shed light on existing practice patterns in the treatment of ami in china , serve as the basis for future quality assessment efforts , and illuminate the barriers to more - appropriate use of evidence - based therapies for all countries seeking opportunity to optimize care . this project was partly supported by the research special fund for public welfare industry of health ( 201202025 ) from the national health and family planning commission of china . dr krumholz is supported by grant u01hl105270 - 05 ( center for cardiovascular outcomes research at yale university ) from the national heart , lung , and blood institute . dr ross is supported by the national institute on aging ( k08ag032886 ) and by the american federation for aging research through the paul b. beeson career development award program . the funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript . dr krumholz reports being the recipient of research grants from medtronic and johnson & johnson , through yale university , to develop methods of clinical trial data sharing , and the chair of a cardiac scientific advisory board for unitedhealth . dr ross reports that he is a member of a scientific advisory board for fair health , inc . dr masoudi receives salary support from the american college of cardiology for his role as the senior medical officer of the national cardiovascular data registries . appendix s1 . members of the china peace collaborative group - retrospective ami study site investigators and china peace study consultants .

backgroundspironolactone , the only aldosterone antagonist available in china , improves outcomes in acute myocardial infarction ( ami ) among patients with systolic dysfunction and either diabetes or heart failure ( hf ) . however , national practice patterns in the use of spironolactone in china are unknown.methods and resultsfrom a nationally representative sample of ami patients from in 2001 , 2006 , and 2011 , we identified 6906 patients with either diabetes or hf and classified them into 1 of 4 groups according to their eligibility for spironolactoneideal(left ventricular ejection fraction [ lvef ] 40% and without contraindications ) , contraindicated , not indicated ( neither ideal nor contraindicated ) , and unknown indications ( lvef unmeasured)to determine how frequently patient eligibility for this drug is assessed in the hospital , how it is used in several groups , and to identify factors associated with the use in these groups . from 2001 to 2011 , the proportion of patients whose eligibility for spironolactone was not assessed decreased ( 66.9% in 2001 to 32.8% in 2011 ) . spironolactone use significantly increased among ideal patients over this period ( 28.6% to 72.4% ; p<0.001 for trend ) , but also in contraindicated patients ( 11.4% to 27.5% ; p=0.002 for trend ) and in other patients groups ( not indicated : 27.5% to 38.3% ; unknown indications : 21.3% to 35.1% ; both p<0.01 for trend ) . in all 4 groups , patients presenting with hf on admission were more likely to receive spironolactone.conclusionsalthough the appropriate use of spironolactone and assessment of eligibility increased in china over the past decade , there remains marked opportunities for improvement.clinical trial registrationurl : http://www.clinicaltrials.gov unique identifier : nct01624883 .

Clinical Trial Registration Methods Study Design of China PEACE-Retrospective AMI Study Study Samples Variables Statistical Analysis Results Study Cohort Use of Spironolactone Therapy Among Different Groups Patient and Hospital Characteristics Associated With Spironolactone Therapy Discussion Limitations Conclusion Sources of Funding Disclosures Supporting Information

to our knowledge the most recent article on celiac dissection was published in 2015 and reported 24 known cases of spontaneous isolated celiac trunk dissection . in our literature review we did not identify a single case of isolated celiac artery dissection that presented during a hypertensive emergency . we present a case of hypertensive emergency induced spontaneous isolated celiac trunk dissection , which was managed nonoperatively with a labetalol drip . the definition of an arterial dissection is the cleavage of the arterial wall by an intramural hematoma located between two elastic layers , . as we are reporting the 25th known case of celiac artery dissection many of the other reported cases involve other arteries such as the common hepatic , splenic , superior mesenteric , and gastroduodenal , , , , , , . additionally , while some of those cases reported hypertension as a risk factor , no other case presents as hypertensive emergency with an isolated celiac artery dissection . the first case of visceral artery dissection involved the superior mesenteric artery ( sma ) and was reported in 1947 , while the first celiac artery dissection was not reported until 1959 . when they occur in the visceral arteries , the most common location is within the sma , . celiac artery dissection has been described in blunt abdominal trauma , but it only accounts for 12 percent of all visceral vascular lesions . to our knowledge the most recent article on celiac dissection was published in 2015 and reported 24 known cases of spontaneous isolated celiac trunk dissection . in our literature review we did not identify a single case of isolated celiac artery dissection that presented during a hypertensive emergency . we present a case of hypertensive emergency induced spontaneous isolated celiac trunk dissection , which was managed nonoperatively with a labetalol drip . the definition of an arterial dissection is the cleavage of the arterial wall by an intramural hematoma located between two elastic layers , . as we are reporting the 25th known case of celiac artery dissection many of the other reported cases involve other arteries such as the common hepatic , splenic , superior mesenteric , and gastroduodenal , , , , , , . additionally , while some of those cases reported hypertension as a risk factor , no other case presents as hypertensive emergency with an isolated celiac artery dissection . the first case of visceral artery dissection involved the superior mesenteric artery ( sma ) and was reported in 1947 , while the first celiac artery dissection was not reported until 1959 . when they occur in the visceral arteries , the most common location is within the sma , . celiac artery dissection has been described in blunt abdominal trauma , but it only accounts for 12 percent of all visceral vascular lesions . a 43 year - old man with a past medical history of uncontrolled hypertension , for which he had reportedly been non - compliant with follow - up , presented with complaints of severe , sudden - onset epigastric pain which was non - radiating and constant for 1 h prior to arrival . he reported moving his bowels prior to the onset of symptoms , and noted passing flatus after the onset of symptoms . the patient denied a history of recent abdominal trauma . on initial examination his vital signs consisted of a temperature of 98.1 fahrenheit , heart rate of 78 , respiratory rate of 16 , blood pressure of 234/144 , and 99% oxygen saturation on room air . his hypertensive emergency in the setting of severe abdominal pain necessitated an emergent vascular surgery consultation . examination of the abdomen revealed a soft , non - distended , and obese abdomen . the patient was diffusely tender to palpation , with maximum tenderness overlying the epigastric region . there was no evidence of voluntary guarding or rebound , and he did not display peritoneal symptoms . pulse examination was positive for 2 + radial , femoral , dorsalis pedis , and posterior tibial pulses bilaterally . there was a well - healed , vertical , surgical scar in the left groin from an unspecified procedure secondary to a remote history of traumatic stab wound . laboratory analyses ( complete blood count , coagulation parameters , comprehensive metabolic panel , lactic acid , and troponin i ) were all within normal limits . contrast enhanced ( oral and intravenous ) computed tomography of the chest and abdomen / pelvis demonstrated normal course and caliber of the great vessels , ascending aorta , descending aorta , and abdominal aorta without aneurysmal dilatation . there was no evidence of displaced intimal calcification , intramural hematoma , or dissection flap to suggest an aortic dissection . an intimal flap was noted within the celiac trunk , starting at the origin and extending into the left gastric , splenic , and the common hepatic arteries . the true and false lumens of the celiac trunk appeared well perfused . from the level of the bifurcation of the celiac trunk , perfusion of the true lumen of the left gastric , splenic , and common hepatic artery was noted . the superior mesenteric artery , bilateral renal arteries , inferior mesenteric artery , common iliac arteries , and internal / external iliac arteries were patent without aneurysmal dilatation . there were no signs of ischemic change of the liver and stomach , and the pancreas was of normal size and contour . the patient was treated with 8 mg of morphine with only slight relief from his symptoms . the patient was also treated with 10 mg labetalol iv and his systolic blood pressure improved into the 140s . an additional 6 mg of morphine was administered for persistent abdominal pain and an additional 10 mg labetalol was prescribed . the patient was admitted to the cardiac intensive care unit for aggressive blood pressure control and was initiated on a labetalol drip to maintain systolic blood pressure less than 120 mm hg . with aggressive blood pressure control throughout the evening of hospital day 0 and hospital day 1 , the patient s abdominal pain decreased in intensity . he was then started on a regular diet , which he consumed without nausea , vomiting , or exacerbation of his pain . on hospital day 1 , the patient was weaned off of the labetalol drip , and his blood pressure medication regimen was transitioned to a by mouth route of hydralazine , labetalol , and amlodipine . the most common symptom in patients with celiac artery dissection is acute or chronic epigastric or abdominal pain , , , . in many of the case studies the patient presented to the emergency department with sudden onset epigastric pain , , . other manifestations include obstructive jaundice , pancreatitis , intestinal angina , tachycardia , hemorrhage , and hypertension , , . interestingly , patients do not usually present with nausea , vomiting , or peritoneal symptoms , , . another case study had a patient present with hematemesis , melena , and postprandial pain . one of the more unique presentations was in a patient who had a history of long - term energy drink consumption and intense exercise routines . patients with celiac artery dissection may also have a self - limited course of these aforementioned symptoms . extensive of the dissection may occur into adjacent arterial walls , including those of the splenic artery and proximal hepatic arteries . renal arteries and the splenic artery may also infarct in the first week after celiac dissection . the hepatic artery may develop an aneurysm . there was one patient who experienced a sequential sma dissection in the subsequent week during his hospital stay our patient did not experience any of these complications .. the crux of the diagnosis of this condition relies on contrast enhanced ct ( ct ) . other modalities that can be used are ct angiography ( cta ) , magnetic resonance imaging ( mri ) , magnetic resonance angiography ( mra ) , and doppler ultrasonography ( us ) . cta is used for definitive diagnosis as it allows precise determination of collateral circulation , . us is of some efficacy because one can assess areas of abnormal flow in the proper habitus . other cases have found infiltration of the fat surrounding the celiac artery or celiac artery aneurysm , . intramural hematoma has been identified on ct and may lead to moderate narrowing of the vessel . another reported a length of 812 millimeters . in the one patient that presented with upper gastrointestinal hemorrhage ct showed enhancement of perigastric and gastric intramural vascular collaterals due to chronic ischemia secondary to celiac artery dissection . another case study repeated the ct scan as early as 12 h later . regardless of the timeline chosen , it is imperative to have serial ct scans to monitor for the potential serious complications of celiac artery dissection . there are many identifiable and accepted risk factors for celiac artery dissection , , , , , , . hypertension is an identified risk factor , though celiac artery dissection as the presenting symptom for hypertensive emergency has yet to be described until now . in addition to hypertension , the other known risk factors are atherosclerotic disease , abdominal aortic aneurysm , cystic medial necrosis , fibromuscular dysplasia , connective tissue disorders such as ehlers - danlos syndrome , trauma , previous abdominal surgery , stenosis or occlusion of a major artery , syphilis , polyarteritis nodosa , and pregnancy , , , , . one case series noted that the two most common risk factors for celiac artery dissection were hypertension followed by vasculitis ( particularly polyarteritis nodosa ) . anti - inflammatory drugs and steroids may be indicated to treated underlying conditions such as polyarteritis nodosa . the duration of anticoagulant and antiplatelet therapy is variable but it is thought 36 months is sufficient . , for anticoagulation heparin or lovenox bridging to warfarin is recommended , . anticoagulation is essential in order to prevent thromboembolic complications , . in order to reduce the risk of propagation of the dissection and rupture , one retrospective review of 19 patients with celiac artery dissection , published in 2015 , notes that long term anticoagulation is not needed in patients undergoing endovascular stenting . complications such as occlusive lesions , aneurysm formation , arterial rupture , hemodynamic instability , persistent pain , uncontrolled hypertension , ischemia , end organ damage , or dissection extensive are indications for operative treatment , , , . when surgical intervention is necessary , a vascular surgeon should be consulted emergently . operative treatment may entail resection and anastomosis , prosthetic bypass , or endovascular stenting , , . follow - up for the patient population with celiac artery dissection should include ct scan with iv contrast at 3- and 6-month intervals to evaluate for stability of the dissection . earlier imaging may be obtained should patients become increasingly symptomatic on a more accelerated timeline . in patients with contraindications to iv contrast injection , ultrasound in conjunction with mri may suffice as an alternative option . it is reported that celiac artery dissection has a poor prognosis . this prognosis , though , depends on the extent of involvement of subsegmental branches , . involvement of the renal , hepatic , or splenic arteries can cause detrimental end - organ damage . going forward his prognosis will ultimately depend on control of his blood pressure . in the event that his symptoms recur , he will be a surgical candidate ( fig . 1 , fig . 2 , fig . 3 , fig . to the best of our knowledge , we present the 25th case of isolated celiac artery dissection . this is the first case of hypertensive emergency induced spontaneous isolated celiac trunk dissection in literature . visceral artery dissection is a rare commodity , and celiac artery dissection is less common than dissection in the sma . it is a difficult diagnosis to make on history and physical alone , necessitating contrast enhanced ct imaging . in patients who present with acute onset abdominal pain in the setting of hypertension , we recommend an immediate contrast enhanced ct scan . in patients with contraindications no research conducted on patient and he has given approval - steven d. kozusko on behalf of all authors . written informed consent was obtained from the patient for publication of this case report and accompanying images - steven d. kozusko on behalf of all authors . swergold data collection dr . swergold data analysis or interpretation dr . swergold writing the paper dr . kozusko oversight dr . sturt steven d. kozusko on behalf of all authors .

highlightsto the best of our knowledge , we present the 25th case of isolated celiac artery dissection.this is the first case of hypertensive emergency induced spontaneous isolated celiac trunk dissection in literature.visceral artery dissection is a rare commodity , and celiac artery dissection is less common than dissection in the sma.it is a difficult diagnosis to make on history and physical alone , necessitating contrast enhanced ct imaging.our patient was managed nonoperatively with a labetalol drip and did well .

Introduction Rationale Background Case study Discussion Conclusion Conflicts of interest Funding Ethical approval Consent Author contribution Guarantor

in 2008 , a 30-year - old primigravida physician experienced intrauterine fetal demise ( iufd ) at 20 weeks gestation , as established by last menstrual period and confirmed by ultrasound at 8 weeks . the patient spent most of her pregnancy providing direct patient care . in october 2007 , during gestational weeks 26 , she was exposed to at least 2 patients with presumed influenza . she reported symptoms of fever , myalgia , headache , and cough during gestational week 4 . she had no testing for influenza , did not receive antiviral therapy , and took acetaminophen ; her symptoms resolved . an ultrasound at 18 weeks demonstrated early growth restriction and oligohydramnios with normal appearing fetal kidneys and head . the patient had an unremarkable medical history , did not smoke , and was normotensive throughout pregnancy . microscopically , histiocytes were abundant in the maternal space ( chronic intervillositis ) and were noted within the fetal chorionic villi ( hofbauer cells ; figure 1 , panel a ) . histologic analysis of fetal autopsy specimens showed scattered luminal histiocytes of the lung and gut . no other inflammatory changes or viral inclusions were identified in the placenta or fetal organs . tissue sample from 30-year - old primigravida patient exposed to seasonal influenza ( h1n1 ) . a ) intervillous ( maternal ) spaces with clusters / sheets of histiocytes ( chronic intervillositis ) and fibrotic fetal chorionic villi with hofbauer cells histiocytic inflammation ( hematoxylin and eosin stain , original magnification 200 ) . b ) dual - stained immunofluorescent assay showing antibodies to influenza a virus ( h1n1 ) ( tetramethylrhodamine isothiocyanate , red ) and cytokeratin ( fluorescein isothiocyanate , green ) in intravillous ( fetal ) and intervillous ( maternal ) space . chronic intervillositis was evident in the placental histology and has been previously described with nonspecific changes in miscarriage and recurrent pregnancy loss ( 4 ) . histiocytes identified from the maternal intervillous space and fetal chorionic villi demonstrated characteristics of viral production ( figure 2 ) . several well - formed viral capsids were noted within the cytoplasm , each containing regularly spaced projections along the surface of the virion corresponding to the hemagglutinin and neuraminidase spikes . tissue sample from 30-year - old primigravida patient exposed to seasonal influenza ( h1n1 ) . electron microscopy ( original magnification 70,000 ) of maternal intervillous space and fetal chorionic villi , showing intranuclear viral transcription aligning along the nuclear envelope electron hypodensities ( asterisks ) , and intracytoplasmic viral production in varying stages shown by numerous electron densities ( v and ^ ) . pcr for m1 capsid protein confirmed influenza a. immunohistochemical testing was performed on tissue from the placenta and on a sampling of fetal organs by using influenza a virus ( h1n1)specific antibody . in the placenta , stippled immunofluorescence ( figure 1 , panel b ) was noted in the histiocytes of the intervillous space ( maternal ) and fetal intravillous histiocytes . in the fetal organs , immunofluorescence for subtype h1n1 was notable in the surface epithelial cells and scattered luminal histiocytes of the fetal respiratory and gastrointestinal tracts . no immunofluorescence was noted within the fetal organs , which implied transamnionic passage of virus ( amnionic fluid infection ) . y - specific immunofluorescent stain with histiocyte counterstaining was performed . because only the intravillous ( fetal ) histiocytes stained positively , maternal and fetal inflammatory responses were confirmed . harris reported higher rates of miscarriage after exposure and infection in the earliest months of pregnancy in the spanish influenza outbreak of 1918 ( subtype h1n1 ) ( 5 ) . hardy et al . reported a similar observation following the asian influenza outbreak of 195758 ( 6 ) , as did investigators in the united kingdom during the 198586 influenza season ( 7 ) . yawn et al . documented influenza a transplacental transfer from the mother to amniotic fluid and fetal heart ( 8) . . summarized placental findings associated with influenza infections in publications from 1987 and 1994 , describing perivillous and villous inflammation with concurrent amniotic fluid immunofluorescence for influenza virus ( 9 ) . in another study ( 10 ) , researchers from beijing , people s republic of china , reported autopsy findings for 2 patients ( 1 of whom was 4 months pregnant ) who died of avian influenza a ( h5n1 ) . viral antigens were present in the maternal pulmonary system , and virus was detected in placental hofbauer cells ( histiocytes ) , cytotrophoblast , fetal lung , circulating mononuclear cells , and liver macrophages ( 10 ) . their documentation of tropism of the influenza virus to placental tissue is similar to the histopathologic features in our report . our unexpected discovery of viral production in placental , maternal , and fetal histiocytes is another example of transplacental passage of influenza . adverse fetal outcomes have reportedly included congenital malformation and schizophrenia , but factors that determine these effects remain unclear ( 11,12 ) . fetal consequences of influenza exposure may be an effect of systemic inflammatory response to the infection , which could represent direct action of the virus on the placenta , the maternal fetal interface , or the fetus . reported the inflammatory response of decidual and fetal tissue in the presence of influenza virus infection and demonstrated expression of mrna for a set of proinflammatory cytokines such as interleukin-6 , tumor necrosis factor- , and granulocyte macrophage colony - stimulating factor , secreted in substantial amounts in response to exposure to influenza virus in vitro ( 13 ) . as our case suggests , a complex interaction of maternal and fetal responses at the interface is a more likely explanation . a total of 15%20% of pregnancies end in spontaneous miscarriage , most before gestational week 12 ( 3 ) . approximately 50% of miscarriages are associated with chromosomal defects , which leaves many other miscarriages unexplained ( 14 ) . examined first trimester miscarriages not associated with chromosomal abnormality , observing histologic changes of chronic intervillositis and increased perivillous fibrin ( 15 ) . the mechanism of loss was thought to be related to a maternal inflammatory response to infectious antigen or an autoimmune phenomenon . whether these findings can be extrapolated to second trimester losses histopathologic features of these early second trimester miscarriages are not well studied , and descriptions are usually included in reports of fetal losses during 1427 weeks , which include iufd ( > 20 weeks ) and miscarriage . consequently , a direct causal relationship between influenza a and fetal demise has not been well established . in our report , a retrospective investigation of the patient s products of conception with ultrastructural , immunohistochemical , and molecular methods that identified subtype h1n1 implies an exposure during her first trimester . type - specific serologic analysis was not possible because she received influenza vaccine several weeks after presumed exposure . the maternal and fetal inflammatory responses ( chronic intervillositis ) after placental infection may have reduced villous surface area for oxygen and nutrient transport , resulting in growth restriction and ultimately second trimester loss . investigation of the effects of influenza in early pregnancy is needed to evaluate potential pathophysiological relationships between maternal exposure , infection , and fetal loss or adverse outcome . during the 200910 influenza pandemic , the centers for disease control and prevention ( atlanta , ga , usa ) collected information from populations exposed to and infected with pandemic ( h1n1 ) 2009 virus . we recommend that future influenza outbreak evaluations of women exposed to influenza with first or early second trimester pregnancy loss include collections of placental and fetal tissue samples .

a second trimester fetal demise followed influenza - like illness in early pregnancy . influenza a virus ( h1n1 ) was identified in maternal and fetal tissue , confirming transplacental passage . these findings suggested a causal relationship between early exposure and fetal demise . management of future influenza outbreaks should include evaluation of products of conception associated with fetal loss .

The Case Conclusions

trauma and especially multiple trauma including traumatic brain injury ( tbi ) are the most common killers in children and adults before age 50 around the world [ 13 ] . mortality of multiple trauma including tbi following traffic accidents ranges between 18% and 25% [ 1 , 4 ] . but not only the first hours and days are critical ; for years , more and more multiple trauma patients have been surviving the initial accident and acute care period but only to develop systemic inflammatory response syndrome ( sirs ) , sepsis , or multiple organ dysfunction syndrome ( mods ) in the course of the few weeks of critical care [ 2 , 5 , 6 ] . for 5000 multiple trauma patients in germany , the german trauma registry showed an in - hospital mortality of 12% in 2010 . in the same report , critical care complications such as sirs , sepsis , and mods are reported for 15% of patients . other groups report similar rates of sirs , sepsis , and mods [ 2 , 6 , 7 ] . it is generally known that these complications again deteriorate the prognosis and outcome of the initially surviving trauma patient and increase late mortality to up to 50% if manifest mods is developed [ 2 , 7 , 8 ] . to understand pathophysiological processes leading to multiple organ failure , sirs , and sepsis and taking place during this devastating illness , profound pathophysiological knowledge on the effects of the initial trauma ( first hit ) is essential to minimize additional harm by the following surgical or critical care treatment ( potential second hit ) [ 911 ] . therefore , numerous neurotrauma as well as shock research studies with many different animal and clinical models showed inflammatory changes after trauma [ 1214 ] . only recently , semple et al . reported on the important role of ccl2 in c57bl / j6 mice , peaking at 4 to 12 h following closed head injury and leading to improved functional outcome and lesion size reduction as well as less secondary brain damage . another group showed that the substance minozac may attenuate the effect of proinflammatory cytokines after closed tbi in mice . in the same study , the authors discussed if minozac may also increase long - term neurofunction after tbi . on the other hand , femur fracture and shock in mice are known to trigger an inflammatory response for years , and recently several groups discussed therapeutic strategies to modulate this inflammatory response [ 12 , 14 ] . however , little attention has been paid to the immune reaction following combined trauma and the interaction of the brain and the other organs of the body . this is even more important , since the injury pattern of closed tbi , lower extremity fracture , and shock is very common in multiply injured patients treated in trauma centers around the world . therefore , we investigated the humoral and cellular inflammatory changes of the combined tbi , shock , and femur fracture in a polytrauma model of the mouse . the study was approved by the animal research committee of the hannover medical school ( medizinische hochschule hannover , mhh ) and the county government of lower saxony ( bezirksregierung niedersachsen ) , no . 03/672 . 45 male c57bl / j6 mice aged 810 weeks with a mean weight of 27.4 g ( 3.7 g ) were used for the study . all animals were handled at room temperature for 7 days before treatment . throughout the study period , analgetic therapy during the whole time of the study was provided by metamizol 25 mg/1000 ml ( novaminsulfon ratiopharm , ratiopharm gmbh , ulm ) in the drinking water of the animals ad libitum . all surgical and trauma procedures were performed at a level of deep surgical anesthesia during maintained spontaneous breathing . for anesthesia , subcutaneous injection of ketamine ( ketanest , parke - davis gmbh , karlsruhe , germany ) with a dose of 100 mg / kg body weight and 2% xylazine ( rompun , bayer vital gmbh , leverkusen , germany ) with a dose of 16 the eyes of the animals were covered by dexpanthenol ( bepanthen , hoffmann - la roche ag , grenzach - wyhlen , switzerland ) . 30 of the animals underwent a standard wd - tbi following a model published previously by shapira et al . with modification according to chen et al . . under a surgical level of anesthesia , the animal was positioned in a stereotaxic frame , the head was shaved , and the exposed scalp disinfected by softasept n ( braun , melsungen , germany ) . next , the 3 mm diameter probe was positioned on the planned impact point at the left of the sagittal suture and caudad to the coronal suture . the trauma was now performed with a mean velocity of 3 m / s by a software - guided process ( labview , national instruments , austin , tx , usa ) . afterwards , the animals remained under continuous observation and warming until full recovery was reached . femur fracture was applied by a blunt guillotine device according to van griensven et al . , we used a weight of 400 g falling out of a height of 160 mm on the fixed femur . success of the fracture mechanism was controlled clinically ( crepitation and instability ) , while the animal was still in anesthesia . the guillotine procedure resulted in an a - type femur fracture combined with a moderate soft tissue injury . after completion of the injury , we performed a closed reduction and splinting of the fracture by a piece of wood in proper length that was tied to the fractured extremity proximally and distally of the fracture . after both traumata were set , we draw 60% of the blood volume of the respective mouse via an orbital puncture with a heparinized capillary . volume was restored after 1 hour of shock with ringer 's solution four times the extracted blood volume into the tail vein . this resulted in an individual resuscitation procedure . only after this resuscitation was completed and the animal showed 36c of body temperature , anesthesia was terminated and the animal awoke . subjects were sacrificed 96 hours after trauma by exsanguination in deep surgical anaesthesia , while blood samples for cytokines were extracted . lymphocytes were analyzed using standard flow cytometry ( facs analysis ) for cd-4 , cd-8 , cd-4-cd-8 , and cd-56 cells ( facs calibur , bd biosciences , heidelberg , germany ) . serum cytokines ( tumor necrosis factor , tnf ; chemokine cc2 ligand , ccl2 ; interleukins 6 and 12 , il-6 and il-12 ) were analyzed by cytometric bead array technique ( bdtm cytometric bead array mouse inflammation kit , bd biosciences , pharmingen , germany ) and facs as described earlier . results are reported as mean standard error of the mean ( sem ) . for statistical analysis , the polytraumatized animals showed a significantly higher mortality than the animals of both singular trauma groups ( figure 1 ) . tnf , ccl2 , and il-6 showed significantly higher concentrations at 96 h after trauma in the co - tx group , while the single - trauma groups seem to raise their cytokine levels clearly less and without significant differences between groups wd - tbi and fx - sh ( figure 2 ) . il-12 showed no difference in plasma concentration between the groups at 96 h after trauma . the cd-8 ratio was significantly lower , and the cd-56 ratio was significantly higher in the fx - sh mice than in each of the other groups . while the intracranial effects of traumatic brain injury have been a subject of multiple studies , the aim of this study was to investigate the interaction of the brain injury and systemic shock after lower extremity trauma in regard to changes in the inflammatory response . our main results were : increased levels of tnf , ccl2 , and il-6 in the co - tx group , lower rates of cd-8 and higher rates of cd-56 cells in the fx - sh group , an increased mortality in the combined trauma animal group . however , some weaknesses of our study have to be kept in mind . as always in animal studies , nonetheless , mouse models are very common due to their availability , ease of handling , and practicability . anyhow , a correlation with similar large animal models and additional studies in humans are required to complete our findings and value the clinical relevance . furthermore , the injury pattern may seem artificial in its constellation since one huge challenge of human polytrauma studies is the diversity of injury patters requiring complex inclusion and exclusion procedures to gain comparable populations in terms of injury severity and injury pattern . however , in our point of view , this strengthens our study : the injury pattern and severity of our individuals is highly standardized , and the model and both its components are robust and reproducible . so comparability of our injuries and populations is granted , even more so with regard to the complex hypothesis of a combined trauma being more than only the sum of its parts . this holds even true for standard parameters such as weight , size , sex , and age , that usually also affect human studies of polytrauma patients . our first finding supports any injury severity - related changes in inflammatory response reported from humans [ 2 , 2022 ] . our co - tx group simply suffered more severe injuries than the fx - sh and wd - tbi groups . looking at each monotrauma , our plasma cytokine levels are comparable to those reported earlier in literature [ 12 , 14 ] . however , the levels of the proinflammatory cytokines are elevated not only by factor 2 but by factors 3 to 10 , respectively , even as late as 96 hours after trauma . likewise , in a murine trauma - haemorrhage model , other authors reported tnf elevations [ 23 , 24 ] . the same holds true for tbi in humans [ 25 , 26 ] and clinically relevant models of tbi . while these groups reported elevated tnf levels 1 to 4 h after trauma , later publications also found tnf effects on neuronal recovery and prevention of secondary brain damage . so , while the importance of tnf after peripheral or brain injury remains rather clear and reciprocal effects were described to some extent , we found no earlier report on the extent of tnf plasma level rise after combined tbi and trauma haemorrhage . reported a 3 - 4 fold increase in circulating plasma levels after combined fracture and fluid percussion tbi in rats . in humans , il-6 was classified as the most important secondary cytokine in trauma , not only because of its multitude of mainly proinflammatory effects and its association to the severity of trauma , but also because of its prognostic relevance differing survivors and nonsurvivors . so , highest il-6 levels in the most severe trauma group and the highest mortality are in line with earlier results in literature . similarly , ccl-2 is increased in the brain after tbi and leads to increased migration of intravascular leukocytes across a damaged blood - brain barrier [ 31 , 32 ] . as well ccl-2 was found to play a key role in the development of organ dysfunction after trauma by activating monocytes / macrophages to migrate from the intravascular space into peripheral tissues . while thibeault et al . showed no association between il-6 and ccl-2 after trauma , they could show such an association between tnf and ccl-2 . so , while being in line with recent literature , our findings also remain conclusive within themselves with respect to the inflammatory cascades described earlier . with respect to proinflammatory il-12 , our nonsignificantly low serum levels are backed up by recent literature . in tbi , one author described differences in il-12 after 24 h but not later . in human multiple trauma patients suffering from chest trauma , furthermore , il-12 seems to exert some major effects via cd56 + -(nk- ) cells . however , depressed il-12 production in trauma patients was associated with a shift towards a cd4+(th2 ) type pattern of adverse clinical outcome . while numerous reports focus on intravascular lymphocyte populations and function after trauma , the populations crucial for mods to develop are those in peripheral tissues . the significant decrease of the cd-8 + ratio in the splenic suspension of our fx - sh group is supported by the earlier finding of a decreased ratio of cd-8 + lymphocytes in peripheral tissues after trauma hemorrhage compared to controls . however , we found no report on lymphocyte populations in peripheral tissues after traumatic brain injury . yet , the wd - tbi group might be comparable to controls for peripheral trauma and hemorrhage ( our fx - sh group ) and therefore shows higher cd-8 + ratios . still , this hypothesis does not explain the cd-8 + ratio of the co - tx group . restoration of the low cd-8 + ratio after fx - sh by wd - tbi might be speculated on but so far no theory on responsible mechanisms is known . since this result was rather surprising for our group , detailed evaluation of key processes for lymphocyte populations after combined neuro- and peripheral trauma and hemorrhage must be left for future experiments . cd-56-(nk- ) cells were found in a significantly higher concentration in the splenic suspension of our fx - sh group compared to our wd - tbi and co - tx groups . while the same reasons as above for the physiologic cd-56 ratio in our wd - tbi group may be discussed , in the co - tx group one more aspect needs consideration : raised tnf levels in a murine sepsis model were associated with an increased ratio of nk cells [ 35 , 36 ] . however , for cd-56 + cells as well as for cd-8 + cells , the detailed mechanisms and reasons underlying the differences found in our study can not readily be explained . further experiments should focus on lymphocyte populations in peripheral tissues after tbi and combined tbi and trauma hemorrhage . mortality may just reflect the inflammatory changes and/or trauma load [ 1 , 12 ] . in humans , injury severity clearly is associated with mortality . likewise , injury severity was associated with an increased inflammatory response as measured by increased il-6 levels . so was mortality , an increased il-6 level in multiply injured patients was associated with increased mortality . il-6 even was suggested to be used for prognosis in severe trauma [ 2 , 30 ] . in murine models , other groups support the findings in humans and of our study [ 12 , 14 , 37 ] . our animal model of combined extremity fracture , shock , and closed traumatic brain injury clearly showed that the inflammatory response of the combined trauma is severalfold elevated as compared to the respective monotrauma in terms of humoral parameters . the almost physiological findings for peripheral tissue lymphocyte populations in the isolated tbi and in the combined trauma group as compared to the significantly elevated ratios of the trauma - hemorrhage group are not readily explained and need further evaluation . mortality in our combined trauma model is clearly associated with trauma load and inflammation and such parallels the findings in multiple trauma patients .

objective . despite broad research in neurotrauma and shock , little is known on systemic inflammatory effects of the clinically most relevant combined polytrauma . experimental investigation in an animal model may provide relevant insight for therapeutic strategies . we describe the effects of a combined injury with respect to lymphocyte population and cytokine activation . methods . 45 male c57bl/6j mice ( mean weight 27 g ) were anesthetized with ketamine / xylazine . animals were subjected to a weight drop closed traumatic brain injury ( wd - tbi ) , a femoral fracture and hemorrhagic shock ( fx - sh ) . animals were subdivided into wd - tbi , fx - sh and combined trauma ( co - tx ) groups . subjects were sacrificed at 96 h. blood was analysed for cytokines and by flow cytometry for lymphocyte populations . results . mortality was 8% , 13% and 47% for fx - sh , wd - tbi and co - tx groups ( p < 0.05 ) . tnf ( 11/13/139 for fx - sh / wd - tbi / co - tx ; p < 0.05 ) , ccl2 ( 78/96/227 ; p < 0.05 ) and il-6 ( 16/48/281 ; p = 0.05 ) showed significant increases in the co - tx group . lymphocyte populations results for fx - sh , wd - tbi and co - tx were : cd-4 ( 31/21/22 ; p = n.s . ) , cd-8 ( 7/28/34 , p < 0.05 ) , cd-4-cd-8 ( 11/12/18 ; p = n.s . ) , cd-56 ( 36/7/8 ; p < 0.05 ) . conclusion . this study shows that a combination of closed tbi and femur - fracture/ shock results in an increase of the humoral inflammation . more attention to combined injury models in inflammation research is indicated .

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

acute pancreatitis due to antipsychotic treatment is rare but sometimes causes a fatal adverse effect . some atypical antipsychotic agents , including clozapine , olanzapine , quetiapine , and risperidone , are associated with acute pancreatitis.1,2 ) among them , acute pancreatitis caused by risperidone is the rarest.3,4 ) although most cases of acute pancreatitis due to atypical antipsychotic agents occur within 6 months of starting antipsychotic administration,1 ) we experienced a schizophrenic patient suffering from pancreatitis after more than 6 months of risperidone therapy . a 69-year - old japanese woman was diagnosed with schizophrenia at the age of 30 years and received outpatient care at another mental hospital . her positive symptoms were not prominent , but her cognitive level was so impaired that she could not regulate her appetite and consumed about 2,000 kcal / day in addition to three ordinary meals . she had never smoked , did not drink alcohol , and did not take any illegal drugs . blood tests ( table 1 ) , abdominal ultrasonography , and a computed tomography ( ct ) scan were performed . clinical features were accompanied by laboratory findings of hyperamylasemia ( amylase , 1,191 u / l ) , hyperlipasemia ( lipase , 1,514 u / l ) , and mild liver enzyme elevations . results of the abdominal ultrasonography were positive for gallstones in the gallbladder and distention of the common bile duct . subsequently , the amylase and lipase titers remained high ( 461 u / l and 804 u / l , respectively ) , although alanine and aspartate aminotransferases decreased gradually to normal levels . at this point , we felt that it was safe for her to start taking the risperidone again . two days after starting the risperidone , serum lipase and amylase increased again to 1,275 u / l and 745 u / l , respectively , and ck also increased ( 766 u / l ) . we decided to suspend the risperidone and introduced 10 mg intravenous haloperidol injections once per day . two days after discontinuing the risperidone , the serum amylase decreased ( 605 u / l ) , but the serum lipase level remained elevated ( 1,654 u / l ) . one week after discontinuing the risperidone , the levels of amylase and lipase decreased gradually ( 309 u / l and 542 u / l , respectively ) , and ck dropped to the normal range . as her general clinical condition and biochemical markers were stable , we changed the haloperidol injection to an oral solution of 6 mg / day aripiprazole because her mental condition worsened after stopping the risperidone treatment . her mental status improved with the aripiprazole treatment , and she was discharged without positive laboratory findings . the patient 's monthly blood tests continue to be normal , including amylase , lipase , and blood cell counts . although atypical antipsychotic - induced pancreatitis has been reported in conjunction with hyperglycemia,5 ) the pathophysiological mechanism of these adverse events remains unclear . most antipsychotic - induced pancreatitis occurs within 6 months after administration1 ) ; however , our case developed pancreatitis more than 6 months after the start of risperidone treatment . risperidone is a 5-ht2a antagonist and ameliorates diet - induced necrotic pancreatitis in mice,6 ) and reduced serum pancreatic amylase levels is observed after endoscopic retrograde cholangiopancreatography.7 ) however , there is no evidence of an association between risperidone treatment and acute pancreatitis . a thorough evaluation for pancreatitis , such as alcohol , tumor , and autoimmune causes , gallstones were present , which were due to an adverse effect of risperidone because the two separate risperidone administrations elevated serum amylase and lipase independently . aripiprazole is currently used in such cases , as aripiprazole is thought to have fewer effects on metabolism , including saccharometabolism , than other atypical antipsychotic agents . lifestyle was also a risk factor in this case . thus , it is necessary to monitor pancreatic function in addition to hyperglycemia in such cases .

acute pancreatitis with antipsychotic treatment is rare but sometimes causes a fatal adverse effect . most cases of acute pancreatitis due to atypical antipsychotic agents are reported to occur within six months of starting antipsychotic administration . acute pancreatitis caused by risperidone is rare . the patient had a high fever , stomachache and vomiting . the results of the abdominal computed tomograhpy scan were negative . the results of the abdominal ultrasonography were positive for gallstones in gallbladder and distention of the common bile duct . she had been fasting and received antibiotic intravenous injections . amylase and lipase titers were high . after risperidone discontinuation , both the levels of the amylase and the lipase were gradually decreased . three months later , the patient still maintains a good clinical balance . although atypical antipsychotic - induced pancreatitis has been reported in conjunction with hyperglycemia , the pathophysiologic mechanism of these adverse events remains unclear . this case got pancreatitis 6 month after risperidone treatment . using the antipsychotic agents , it is necessary to monitor pancreas function .

INTRODUCTION CASE DISCUSSION

polycystic ovary syndrome ( pcos ) , the most common female endocrine disorder , is a heterogeneous endocrine and metabolic disorder , affecting 6 - 10% of women of reproductive age ( 1 ) . features of pcos may manifest at any age , ranging from childhood ( premature puberty ) , teenage years ( hirsutism , menstrual abnormalities ) , early adulthood and middle life ( infertility , glucose intolerance ) to later life ( diabetes mellitus and cardiovascular diseases ) ( 2 ) . several of these features increase the risk of cardiovascular diseases ( cvd ) in women ( 3 ) and the prevalence of hypertension in women with pcos is about 40% in comparison with a prevalence of about 25.8 in the general population ( 4 ) . pcos is also associated with a higher risk of myocardial infarction ( relative risk ) ( 5 ) and with a compromised cardiovascular profile independent from obesity in young women ( 6 ) . hyperandrogenism and insulin resistance or deficiency was linked to pcos , as early as 1921 , when achard and thiers published their classic description of a bearded woman with diabetes ( 7 ) . the polycystic ovary syndrome was then called the stein - leventhal syndrome , which was first described in 1935 . originally , diagnosis required pathognomonic ovarian findings and the clinical triad of hirsutism , amenorrhea and obesity ( 8) . experimental induction of a polycystic ovarian syndrome ( pcos ) in rodents by brower in 1996 was made possible by the use of a single intramuscular ( i.m . ) the rats ceased ovulation and developed characteristics of human pcos , including large cystic follicles in the ovaries and altered concentrations of luteinizing hormone ( 9 ) . roman chamomile or chamaemelum nobile ( l. ) , ( synonym anthemis nobilis l. from asteraceae family ) , is a perennial herb cultivated in western europe and north africa . in traditional medicine , chamomile flowers are used as an anti - spasmolytic and anti - inflammatory tea for stomach disorders . in women , the antispasmodic effects of chamomile ease menstrual cramps and lessen the possibility of premature labor . chamomile extract 's stimulating effect on leukocytes ( macrophages and b lymphocytes ) is used in skin irritations and eczema ( 11 ) . the tranquilizer/ sedative effects of chamomile depress the central nervous system making it useful for curing insomnia ( 12 ) . studies on chamomile extract have shown that both lipophilic and hydrophilic compounds take part in its therapeutic activity . the most characteristic constituents of this plant species are volatile oil , sesquiterpene lactones and phenols including flavonoids . the main constituents of chamomile flowers include several phenolic compounds , primarily the flavonoids , apigenin , quercetin , patuletin , luteolin and their glucosides . flavonoids are chemical phenylbenzopyrones , which are usually conjugated with sugars and are present in all vascular plants . the benzopyranonring system is a molecular scaffold which is found in flavonoid natural products and has weak aromatase inhibitory activity ( 13 ) . several biological properties have been ascribed to flavonoids , which among them are the wellknown anti - inflammatory , antioxidant , antihepatotoxic and antiviral activities together with their vasculo - protector and spasmolitic effects . one of the 6 major subgroups of flavenoids is the subgroup of flavone including flavon , apigenin and luteolin , which all three exist in chamomile . the effects of flavonoids on the central nervous system have been considered just in the past 10 years . in particular , the studies performed by medina 1989 have demonstrated the capacity of some flavonoids for binding to the central type benzodiazepine ( bzd ) receptors ( 14 ) . apigenin , one of the predominant flavonoids in chamomile , was found to competitively inhibit the binding of flunitrazepam ( a benzodiazepine receptor blocker of ) . at doses up to 30 mg / kg , apigenin was shown to have a clear anxiolytic activity without the sedative or muscle relaxing effects noted for benzodiazepines ( e.g. , valium ) without noticeable anticonvulsant properties ( 14 ) . effects of phytoestrogens on dna synthesis and tyrosine kinase activity show that chrysin , quercetin , apigenin and luteolin inhibit estradiol ( e2)-induced dna synthesis or have antiproliferative properties ( antimutagenic ) , growth inhibitory properties ( 15 ) and inhibitory effects on aromatase activity ( 13 ) . due attention to pcos which is the most common female endocrine disorder in women of reproductive age and the therapeutic activity of chamomile ( flavonoids ) , we saught to ( 1 ) compare the circulating levels of gonadotropins and gonadal steroids before and after injection of chamomile extract in pco induced rats ( 2 ) , study the signs of pcos in the ovarian tissue and ( 3 ) change in the number of dominant follicles upon chamomile extract administration . thirty virgin adult cycling wistar rats , weighting 200 - 220 g were divided into two groups and housed every six mice into a cage under standard conditions ( 212c , 12-hour light/ 12-hour dark cycles ) for at least one week before and throughout the study , with free access to standard chow and tap water ad libitum . the study and all the procedures were carried out in accordance with the guidelines for the care and use of laboratory animals of tehran university of medical sciences ethics committee and that of the national institutes of health guide for care and use of laboratory animals ( publication no . estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours , and assessed by light microscopy for the relative proportion of leukocytes , epithelial and cornified cells found in daily vaginal lavages , which characteristically change during different stages of the estrous cycle . the rat estrous cycle ( estrus , diestrus 1 , diestrus 2 , and proestrus ) usually lasts about 4 days , in both control and pco rats ( 16 ) . chamomile flowers were collected from natural resources in ahvaz , iran . after grinding the dried flowers , after one week of acclimatization , 8-week - old rats ( n = 30 ) were divided into two groups of control and pco rats . the control group received 0.2 ml corn oil and all the rates assigned to the pco group received an i.m . injection of 0.2 mg estradiol valerate ( ev ) ( aburaihan co. , iran ) , in 0.2 ml of corn oil , to induce pco as described by brawer 1996 ( 9 ) . all the ev - treated rats were evaluated 60 days after the injection , when follicular cysts are first detected . subsequently , pco rats were subdivided into four groups : one group did not receive chamomile extract and other three groups received different doses ( 25 , 50 and 75 mg / kg ) of chamomile alcoholic - extract intraperitoneally for ten days . blood samples were collected and serum luteinizing hormone ( lh ) , follicular stimulation hormone ( fsh ) and estradiol levels were determined by elisa method . , costa mesa , u.s.a . ) , and estradiol kit ( drg international , gmbh , u.s.a . ) . ovaries of the controls and ev - treated rats were removed , cleaned from adherent fat and connective tissue , and fixed in 10% formaldehyde buffer for at least 24 hours . statistical analysis was done by using spss software , version 13 ( spss inc , illinois , u.s.a . ) . comparisons between the controls and ev - treated rats were made by anova , followed by the student - newman - keuls post - hoc test . thirty virgin adult cycling wistar rats , weighting 200 - 220 g were divided into two groups and housed every six mice into a cage under standard conditions ( 212c , 12-hour light/ 12-hour dark cycles ) for at least one week before and throughout the study , with free access to standard chow and tap water ad libitum . the study and all the procedures were carried out in accordance with the guidelines for the care and use of laboratory animals of tehran university of medical sciences ethics committee and that of the national institutes of health guide for care and use of laboratory animals ( publication no . thirty virgin adult cycling wistar rats , weighting 200 - 220 g were divided into two groups and housed every six mice into a cage under standard conditions ( 212c , 12-hour light/ 12-hour dark cycles ) for at least one week before and throughout the study , with free access to standard chow and tap water ad libitum . the study and all the procedures were carried out in accordance with the guidelines for the care and use of laboratory animals of tehran university of medical sciences ethics committee and that of the national institutes of health guide for care and use of laboratory animals ( publication no . estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours , and assessed by light microscopy for the relative proportion of leukocytes , epithelial and cornified cells found in daily vaginal lavages , which characteristically change during different stages of the estrous cycle . the rat estrous cycle ( estrus , diestrus 1 , diestrus 2 , and proestrus ) usually lasts about 4 days , in both control and pco rats ( 16 ) . chamomile flowers were collected from natural resources in ahvaz , iran . after grinding the dried flowers , after one week of acclimatization , 8-week - old rats ( n = 30 ) were divided into two groups of control and pco rats . the control group received 0.2 ml corn oil and all the rates assigned to the pco group received an i.m . injection of 0.2 mg estradiol valerate ( ev ) ( aburaihan co. , iran ) , in 0.2 ml of corn oil , to induce pco as described by brawer 1996 ( 9 ) . all the ev - treated rats were evaluated 60 days after the injection , when follicular cysts are first detected . subsequently , pco rats were subdivided into four groups : one group did not receive chamomile extract and other three groups received different doses ( 25 , 50 and 75 mg / kg ) of chamomile alcoholic - extract intraperitoneally for ten days . blood samples were collected and serum luteinizing hormone ( lh ) , follicular stimulation hormone ( fsh ) and estradiol levels were determined by elisa method . , costa mesa , u.s.a . ) , and estradiol kit ( drg international , gmbh , u.s.a . ) . ovaries of the controls and ev - treated rats were removed , cleaned from adherent fat and connective tissue , and fixed in 10% formaldehyde buffer for at least 24 hours . statistical analysis was done by using spss software , version 13 ( spss inc , illinois , u.s.a . ) . comparisons between the controls and ev - treated rats were made by anova , followed by the student - newman - keuls post - hoc test . a p - value less than 0.05 was considered statistically significant . estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours , and assessed by light microscopy for the relative proportion of leukocytes , epithelial and cornified cells found in daily vaginal lavages , which characteristically change during different stages of the estrous cycle . the rat estrous cycle ( estrus , diestrus 1 , diestrus 2 , and proestrus ) usually lasts about 4 days , in both control and pco rats ( 16 ) . chamomile flowers were collected from natural resources in ahvaz , iran . after grinding the dried flowers , after one week of acclimatization , 8-week - old rats ( n = 30 ) were divided into two groups of control and pco rats . the control group received 0.2 ml corn oil and all the rates assigned to the pco group received an i.m . injection of 0.2 mg estradiol valerate ( ev ) ( aburaihan co. , iran ) , in 0.2 ml of corn oil , to induce pco as described by brawer 1996 ( 9 ) . all the ev - treated rats were evaluated 60 days after the injection , when follicular cysts are first detected . subsequently , pco rats were subdivided into four groups : one group did not receive chamomile extract and other three groups received different doses ( 25 , 50 and 75 mg / kg ) of chamomile alcoholic - extract intraperitoneally for ten days . blood samples were collected and serum luteinizing hormone ( lh ) , follicular stimulation hormone ( fsh ) and estradiol levels were determined by elisa method . , costa mesa , u.s.a . ) , and estradiol kit ( drg international , gmbh , u.s.a . ) . ovaries of the controls and ev - treated rats were removed , cleaned from adherent fat and connective tissue , and fixed in 10% formaldehyde buffer for at least 24 hours . statistical analysis was done by using spss software , version 13 ( spss inc , illinois , u.s.a . ) . comparisons between the controls and ev - treated rats were made by anova , followed by the student - newman - keuls post - hoc test . in the present study , we examined the effects of dried chamomile flowers alcoholic extract on the ovaries and uteri of pco - induced rats ( figure 1 ) . the ovary . in the ovarian tissue , the cysts were mainly appeared by a single intramuscular dose of estradiol valerate , 2 mg in 0.2 ml of corn oil rats with pcos which had been treated by 50 mg / kg of chamomile extract showed recovery demonstrated by macroscopic and microscopic morphological examination in the ovarian and uterine tissues . in the ovarian tissue , the cysts had mainly disappeared ( figure 2 ) and the number of dominant follicles had increased ( figure 3 ) and better endometrial tissue arrangements were observable ( figure 4 ) . mean hormonal changes in pco - induced animals , injected with 50 mg / kg of chamomile extract , showed statistically significant differences in comparison with the controls ( p < 0.05 ) , ( table 1 ) . serum levels of estradiol and gonadotropins , lh and fsh , were significantly decreased in the chamomile group relevant to the control group ( p < 0.05 ) . the cysts were mainly disappeared by chamomile administration . in uterus , better endometrial tissue arrangements were also observed upon chamomile administration a schematic illustration of how gaba neurons are involved in the regulation of lh surge . an inhibitory gaba tone induced by estrogen is disrupted by the circadian clock information carried by avp neurons . this results in the disinhibition of gnrh neurons by inhibitory gaba neurons , and in turn it induces the surge of gnrh secretion . experimental polycystic ovary ( pco ) in rodents resembling some aspects of human pco syndrome , for example changes in serum levels of gonadotropin - releasing hormones ( gnrh ) and appearance of cysts , was induced by injecting a long - acting estradiol valerate ( ev ) . classical neuroendocrinological studies indicate that in female rats , the neuronal component responsible for the induction of the lh surge is located in the preoptic area ( poa ) ( 17 , 18 ) . in fact , it has been reported that gonadotropine releasing hormone neurons in the poa express the immediate early gene , c - fos , at the time of the lh surge suggesting that such gnrh neurons in the poa are responsible for the generation of the gnrh surge ( 19 ) . to understand the mechanism underlying the generation of the lh surge , ( i.e. , the gnrh surge ) accumulating evidence suggests that gamma amino butyric acid ( gabaergic ) regulation of gnrh neurons is profoundly involved in the regulation of lh surge ( 20 , 21 ) , ( figure 4 ) . it has been suggested that a decrease in the inhibitory tone of gabaa on gnrh neurons causes lh to surge ( 22 ) . consistently , kimura ( 1994 ) showed that intravenous infusion of bicuculline , a gabaa receptor antagonist , on the morning of proestrus induced a premature surge - like secretion of lh ( 23 ) . studies on chamomile extract have shown that both lipophilic and hydrophilic compounds take part in its therapeutic activity . the most characteristic constituents of this plant species are volatile oil , sesquiterpene lactones and phenolics including flavonoids . the effects of flavonoids on the central nervous system have been considered only in the past 10 years . particularly , the studies performed by medina 1989 have demonstrated the capacity of some flavonoids for binding to the central type benzodiazepine ( bzd ) receptors . apigenin , one of the predominant flavonoids in chamomile , was found to competitively inhibit the binding of flunitrazepam , a benzodiazepine derivative , and was fitted into a pharmacophore model for ligands binding to the gabaa receptor benzodiazepine site . this reflected the affinities of the compounds in the [ ( 3 ) h]-flumazenil binding assay ( 14 , 2426 ) . therefore , the effects of chamomile extract in rats which were shown in the present study can be attributed to the direct activation of the central benzodiazepine site . extract of dried matricaria chamomilla l. flowers not only can induce recovery from an induced pco state in rats , which is probably due to the interaction of the gaba system combined with the effects of chamomile in the regulation of lh surge secretion , it but also can increase dominant follicles . in the uterus , it causes better endometrial tissue arrangements .

introductionpolycystic ovary syndrome ( pcos ) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction . presently , little is known about the primary factors that initiate pcos . chamomile flowers are used in alternative medicine for its anti - spasmolytic and anti - inflammatory effects . antispasmodic properties of chamomile ease menstrual cramps and lessen the possibility of premature labor . this medicinal herb also stimulates menstruation . in this study , we evaluated the effects of chamomile alcoholic - extract on the biochemical and clinical parameters in a rat model of pcos.materials and methodsestrous cyclicity of 30 virgin adult cycling rats was monitored by vaginal smears obtained between 0800 and 1200 hours . after about 4 days , each rat received an i.m . injection of estradiol valerate ( aburaihan co. , iran ) , 2 mg in 0.2 ml of corn oil , to induce pco . corn oil was injected to the rats in the control group . all the rats in the experimental group were evaluated for follicular cysts 60 days after the injection . rats with pcos were treated by multiple doses ( 25 , 50 , 75 mg / kg ) of intraperitoneal injections of chamomile alcoholic - extract for ten days . the data were statistically analyzed at a significance level of p<0.05 by anova , followed by the student newman - keuls post hoc test.resultsthe histological and hormonal results showed that chamomile can decrease the signs of pcos in the ovarian tissue and help lh secretion in rats ( p<0.05).conclusionthe alcoholic - extract of dried matricaria chamomilla l. flowers can not only induce recovery from a pco induced state in rats , but also increase dominant follicles . additionally better endometrial tissue arrangements can be regarded as another therapeutic effect of chamomile .

Introduction Materials and Methods None Animals and Care Experiment Design Vaginal Smears Chamomile Extract Preparation Hormonal Treatment and Study Procedures Measuring Circulating Gonadotropins and Gonadal Steroids Ovarian Morphology Statistical Analysis Results Discussion Conclusion

phylloquinone , also referred to as vitamin k1 , is a compound present in all photosynthetic plants ( 1 ) . phylloquinone is the primary dietary source of vitamin k. in general , green , leafy vegetables contain the highest known phylloquinone concentrations and contribute approximately 60% of total phylloquinone intake ( 2 , 3 ) . as indicated in table 1 , spinach and collards , which have concomitant high concentrations of chlorophyll associated with the photosynthetic process , hence , dark leaf color have substantially higher concentrations of phylloquinone compared to the more commonly consumed iceberg lettuce , which is substantially paler , hence , lower chlorophyll concentrations . the other plant sources of phylloquinone are certain plant oils including soybean , canola ( also known as rapeseed ) , cottonseed , and olive ( table 1 ) . margarine , spreads , and salad dressings derived from these plant oils are important dietary sources of phylloquinone ( 4 , 5 ) . plant oils are used for preparation of multiple mixed dishes , hence many commercially prepared foods including baked goods also contain small amounts of phylloquinone . although multiple databases now exist that contain some phylloquinone contents of foods ( 6 , 7 ) , the most extensive analysis of phylloquinone in common foods using established food sampling protocols ( 8) are found in the united states department of agriculture ( usda ) nutrient database for standard reference ( http://www.ars.usda.gov/sp2userfiles/place/12354500/data/sr22/nutrilistsr22a430.pdf ; accessed 05.22.10 ) . however , numbers in this table are indicative of median concentrations of all analysis for a given food in the author 's laboratory and may differ from reference because of the inclusion of unpublished data . the commercial hydrogenation of phylloquinone - rich oils results in a transformation of phylloquinone into a hydrogenated form , 2,3-dihydrophylloquinone ( 9 ) . as expected , trans fatty acid concentrations are highly correlated with dihydrophylloquinone concentrations ( 10 ) . in the us food supply commercial hydrogenation of plant oils was a common practice that prolonged shelf life of the oil - based products . many foods sold in fast - food restaurants and frozen prepared products such as fast - food french fries , doughnuts , and breaded fish sticks contained high concentrations of 2,3-dihydrophylloquinone ( 5 ) ( table 1 ) . however , with current controversy regarding the health consequences of trans fatty acid intake ( 11 ) , there have been regional trans fat bans in the us food supply . it is anticipated that the decrease in hydrogenation of plant oils will also reduce the presence of dihydrophylloquinone in the us food supply , hence monitoring of this form of vitamin k in the food supply is warranted . unlike trans fatty acids , dihydrophylloquinone is only present in hydrogenated plant oils , hence monitoring this form of vitamin k in the food supply is a robust approach to monitoring the practice of commercial hydrogenation of plant oils . menaquinones are the other category of vitamin k present in the food supply ( fig . menaquinones are often referred to as vitamin k2 , which is somewhat misleading given that all menaquinones are not alike in their origin or their function . menaquinones are primarily of bacterial origin , and differ in structure from phylloquinone in their 3-substituted lipophilic side chain . the major menaquinones contain 410 repeating isoprenoid units indicated by mk-4 to mk-10 ; forms up to 13 isoprenoid groups have been identified . 1 ) is unique among the menaquinones in that it is not of bacterial origin . instead , mk-4 is formed by a realkylation step from menadione present in animal feeds or is the product of tissue - specific conversion directly from dietary phylloquinone ( 12 , 13 ) . in the united states , menadione is the synthetic form of vitamin k used in poultry feed . as such , mk-4 formed from menadione is present in poultry products in the us food supply ( 14 ) . however , mk-4 formed from phylloquinone is limited to organs not commonly consumed in the diet including kidney . the exceptions are dairy products with mk-4 found in milk , butter , and cheese , albeit in modest amounts . therefore it is unlikely that mk-4 is an important dietary source of vitamin k in food supplies that do not use menadione for poultry feed nor are rich in dairy products . there is growing interest in the health benefits of longer - chain menaquinones , which are limited to certain foods in the food supply . menaquinone-7 ( mk-7 ) is primarily the product of fermentation using bacillus subtilis natto and is present in a traditional japanese soybean - based product called natto . natto contains approximately 2.5 times more mk-7 compared to the phylloquinone content of spinach ( table 1 ) . natto also contains mk-8 and phylloquinone ( 84 and 35 g/100 g , respectively ) , although both are modest in concentration compared to mk-7 ( 6 ) . some cheeses also contain mk-8 and mk-9 ( 6 ) , but these are dependent on cheese production practices , hence the food composition databases are limited in their ability to characterize menaquinone intake across different food supplies . phylloquinone , also referred to as vitamin k1 , is a compound present in all photosynthetic plants ( 1 ) . phylloquinone is the primary dietary source of vitamin k. in general , green , leafy vegetables contain the highest known phylloquinone concentrations and contribute approximately 60% of total phylloquinone intake ( 2 , 3 ) . as indicated in table 1 , spinach and collards , which have concomitant high concentrations of chlorophyll associated with the photosynthetic process , hence , dark leaf color have substantially higher concentrations of phylloquinone compared to the more commonly consumed iceberg lettuce , which is substantially paler , hence , lower chlorophyll concentrations . the other plant sources of phylloquinone are certain plant oils including soybean , canola ( also known as rapeseed ) , cottonseed , and olive ( table 1 ) . margarine , spreads , and salad dressings derived from these plant oils are important dietary sources of phylloquinone ( 4 , 5 ) . plant oils are used for preparation of multiple mixed dishes , hence many commercially prepared foods including baked goods also contain small amounts of phylloquinone . although multiple databases now exist that contain some phylloquinone contents of foods ( 6 , 7 ) , the most extensive analysis of phylloquinone in common foods using established food sampling protocols ( 8) are found in the united states department of agriculture ( usda ) nutrient database for standard reference ( http://www.ars.usda.gov/sp2userfiles/place/12354500/data/sr22/nutrilistsr22a430.pdf ; accessed 05.22.10 ) . however , numbers in this table are indicative of median concentrations of all analysis for a given food in the author 's laboratory and may differ from reference because of the inclusion of unpublished data . the commercial hydrogenation of phylloquinone - rich oils results in a transformation of phylloquinone into a hydrogenated form , 2,3-dihydrophylloquinone ( 9 ) . as expected , trans fatty acid concentrations are highly correlated with dihydrophylloquinone concentrations ( 10 ) . in the us food supply commercial hydrogenation of plant oils was a common practice that prolonged shelf life of the oil - based products . many foods sold in fast - food restaurants and frozen prepared products such as fast - food french fries , doughnuts , and breaded fish sticks contained high concentrations of 2,3-dihydrophylloquinone ( 5 ) ( table 1 ) . however , with current controversy regarding the health consequences of trans fatty acid intake ( 11 ) , there have been regional trans fat bans in the us food supply . it is anticipated that the decrease in hydrogenation of plant oils will also reduce the presence of dihydrophylloquinone in the us food supply , hence monitoring of this form of vitamin k in the food supply is warranted . unlike trans fatty acids , dihydrophylloquinone is only present in hydrogenated plant oils , hence monitoring this form of vitamin k in the food supply is a robust approach to monitoring the practice of commercial hydrogenation of plant oils . menaquinones are the other category of vitamin k present in the food supply ( fig . menaquinones are often referred to as vitamin k2 , which is somewhat misleading given that all menaquinones are not alike in their origin or their function . menaquinones are primarily of bacterial origin , and differ in structure from phylloquinone in their 3-substituted lipophilic side chain . the major menaquinones contain 410 repeating isoprenoid units indicated by mk-4 to mk-10 ; forms up to 13 isoprenoid groups have been identified . 1 ) is unique among the menaquinones in that it is not of bacterial origin . instead , mk-4 is formed by a realkylation step from menadione present in animal feeds or is the product of tissue - specific conversion directly from dietary phylloquinone ( 12 , 13 ) . in the united states , menadione is the synthetic form of vitamin k used in poultry feed . as such , mk-4 formed from menadione is present in poultry products in the us food supply ( 14 ) . however , mk-4 formed from phylloquinone is limited to organs not commonly consumed in the diet including kidney . the exceptions are dairy products with mk-4 found in milk , butter , and cheese , albeit in modest amounts . therefore it is unlikely that mk-4 is an important dietary source of vitamin k in food supplies that do not use menadione for poultry feed nor are rich in dairy products . there is growing interest in the health benefits of longer - chain menaquinones , which are limited to certain foods in the food supply . menaquinone-7 ( mk-7 ) is primarily the product of fermentation using bacillus subtilis natto and is present in a traditional japanese soybean - based product called natto . natto contains approximately 2.5 times more mk-7 compared to the phylloquinone content of spinach ( table 1 ) . natto also contains mk-8 and phylloquinone ( 84 and 35 g/100 g , respectively ) , although both are modest in concentration compared to mk-7 ( 6 ) . some cheeses also contain mk-8 and mk-9 ( 6 ) , but these are dependent on cheese production practices , hence the food composition databases are limited in their ability to characterize menaquinone intake across different food supplies . the current us dietary guidelines for intakes of vitamin k are 90 and 120 g / day for women and men , respectively ( 15 ) . these guidelines are termed adequate intakes ( ai ) because the institute of medicine concluded in 2001 that there were insufficient data available to generate a precise recommendation for vitamin k. the ai values for vitamin k were generated from the third national health and nutrition examination survey ( nhanes iii , 19881994 ) and based on the median phylloquinone intake in the united states for each age and gender category ( 15 ) . in the absence of abnormal bleeding associated with low vitamin k intakes among adults , it was assumed that the current intakes are adequate . however , the adequacy of intake defined by an absence of bleeding is controversial . furthermore , the elderly report median intakes below the current ai for adults . as reviewed elsewhere ( 16 ) , there is controversy regarding biochemical measures of subclinical vitamin k deficiency and as a consequence , the true dietary requirement of vitamin k is unknown . for the purpose of this review , dietary intakes of vitamin k will be presented relative to the current ai . by comparison , the guidelines in the united kingdom are 1 g / kg body / day ( 17 ) and are set at 75 g / day for adult men , 60 g / day for women , aged 1829 year , and 65 g / day for women 30 years and over in japan ( 18 ) . estimates of phylloquinone intake in various populations are probably more accurate than intakes of other forms of vitamin k and there is a substantial difference in the reported intakes from a number of countries , which seems to be related to food consumption practices in different areas ( 1823 ) . for example , the mean reported vitamin k intake of young japanese women ( mean 21.2 years , n=124 ) was about 230 g / day ( 18 ) . the estimated phylloquinone intakes in northern china , england , and scotland were 247 , 103 , and 70 g / day , respectively ( 23 , 24 ) . of note , phylloquinone intakes have been decreasing over the last two decades in the united kingdom , consistent with a concomitant decline in leafy green vegetable consumption ( 25 ) . based on the nhanes iii data , the elderly ( defined as older as 71 years of age ) have median phylloquinone intakes lower than the current ai ( 89 and 79 g / day for women and men , respectively ) . this observation supports the findings of others who have reported very low phylloquinone intakes among those in nursing homes ( 26 ) and those with alzheimer 's disease ( 27 ) . given emerging association of low vitamin k intakes with risk of certain diseases such as cardiovascular disease and osteoarthritis ( 16 ) , the elderly present a potentially vulnerable subgroup of the population with respect to vitamin k. very little is known about the contribution of dietary menaquinones to overall vitamin k nutrition and although it has been stated that approximately 50% of the daily requirement for vitamin k is supplied by the gut flora through the production of menaquinones , there is little evidence to support this estimate ( 28 ) . in one study among adults with acute bacterial overgrowth as induced by omeprazole , menaquinones produced by these bacteria had some contribution to vitamin k status during dietary phylloquinone restriction , but not enough to restore biochemical measures of vitamin back to normal range ( 29 ) . as previously indicated , there are regional differences in the forms and content of menaquinones in the food supply . for example , natto is unique to a traditional japanese diet whereas the cheeses that contain high concentrations of mk-8 and mk-9 appear to be most prevalent in european dairy producing food supplies . although there are reported menaquinone intakes , these are limited to studies from japan ( 18 ) and the netherlands ( 21 ) , and are low compared to phylloquinone intakes . in the united states , menaquinones are limited in the food supply and have not been systematically assessed . there are surprisingly little data on the relative biological availability of different forms of vitamin k among different food sources . furthermore , there is a growing body of literature to suggest that our understanding of vitamin k is still rudimentary in light of new developments regarding different forms . as previously stated , phylloquinone is of plant origin , with absolute intakes being predominantly from green leafy vegetables . phylloquinone is tightly bound to the membranes of plant chloroplasts , and is less bioavailable compared to phylloquinone obtained from plant oils and/or dietary supplements ( 30 ) . some estimates place the absorption of phylloquinone to be 10% from plants compared to supplements ( 31 ) . however there appear to be differences in absorption compared to the plant species , with phylloquinone obtained from broccoli and collards having greater absorption compared to spinach ( 31 , 32 ) . similarly , and not unexpected because vitamin k is lipophilic , addition of a fat source to the meal results in higher absorption . for a more comprehensive review of vitamin k absorption phylloquinone differs from 2,3-dihydrophylloquinone by a saturation of a single bond at the 2,3 position of the side chain ( 9 ) . surprisingly this single substitution results in a lower absorption of dihydophylloquinone compared to an equimolar amount of phylloquinone ( 34 ) . there is also indirect evidence of lower activity of dihydrophylloquinone as an enzyme cofactor , which currently is the only known function of vitamin k. the implications of this poor bioavailability and activity are currently unknown , although at least one study suggests a detrimental effect on bone mineral density among older adults in a community - based cohort ( 35 ) . the potential impact of poor utilization of dihydrophylloquinone on bone health will be of little importance in the future should hydrogenated oils be removed from the food supply . mk-7 , when administered in the form of natto in equimolar amounts to phylloquinone administered in the form of spinach , has a peak height difference of more than 10-fold compared to phylloquinone , with a half - life of 56 hours , compared to 7.5 hours for phylloquinone ( 36 ) . whereas all forms of vitamin k appear to be initially associated with triglyceride - rich lipoproteins ( trl ) , the longer chain menaquinones including mk-7 and mk-9 are also associated with low - density lipoprotein ( ldl ) . these preliminary data suggest that the menaquinones have different transport pathways and distribution , which has implications for transport to extra - hepatic tissue such as bone ( 33 ) . emerging studies on mk-4 challenge our current understanding of vitamin k. as demonstrated using stable isotopes , mk-4 is a conversion product of phylloquinone via the intermediate , menadione ( 37 ) . there appears to be both local and systemic conversion to mk-4 , with the local conversion being the predominant pathway . to add complexity to the interpretation is the observation that this conversion does not occur in all tissues . whereas the liver contains primarily phylloquinone and very long - chain menaquinones , mk-4 is the predominant form in the brain , pancreas , and glands ( 38 ) . in terms of dietary intakes , mk-4 intakes are low compared to other forms of vitamin k. however , dietary phylloquinone converts to mk-4 in those tissues where mk-4 appears to be required so it is likely that low mk-4 intakes are of little consequence to health when there is adequate dietary phylloquinone available for conversion to mk-4 . in conclusion , much of our understanding of vitamin k nutrition has focused on the primary dietary source , phylloquinone . in contrast , the menaquinones are present in the food supply , but there are limited food composition data available . as a corollary , estimates of intakes of menaquinones are very limited and our understanding of their role in vitamin k nutrition is not well understood . based upon work supported by the us department of agriculture , agricultural research service under cooperative agreement no . any opinions , findings , conclusions , or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the us department of agriculture .

vitamin k is present in the diet in the forms of phylloquinone and menaquinones . phylloquinone , which is the major dietary source , is concentrated in leafy plants and is the vitamin k form best characterized in terms of food composition and dietary intakes . in contrast , menaquinones are the product of bacterial production or conversion from dietary phylloquinone . food composition databases are limited for menaquinones and their presence in foods varies by region . dietary intakes of all forms of vitamin k vary widely among age groups and population subgroups . similarly , the utilization of vitamin k from different forms and food sources appear to vary , although our understanding of vitamin k is still rudimentary in light of new developments regarding the menaquinones .

Food sources Phylloquinone 2,3-Dihydrophylloquinone Menaquinones Dietary intakes Are all forms of vitamin K the same? Conflict of interest and funding

adult articular cartilage has insufficient ability to repair after either erosion or damage and it is unlikely to be reformed to normal condition once it has been impaired . articular cartilage repair has been based on finding and resolving the primary reason for the injury , but it has been recognized that only treating the damage site is not adequate to gain good long - term functionality . the matrix environment of hyaline cartilage is different from that of subchondral bone in biological , physical and chemical characteristics . this led to the proposal that in order to gain positive results in tissue repair , the microenvironment in which the repair process takes place should be retouched , such as those elements that play a pro - angiogenic , conductive , cell differentiation and chemotactic role . this tendency has been the reason behind a growth in using genetic and tissue engineering in the examination of articular cartilage repair . a number of natural and artificial biomaterials have been used as scaffolds for cartilage reformation , but their biological effectiveness as well as safety remains unresolved . therefore , scaffold biomaterials extracted from the patients own body have long been a tempting choice . platelet - rich plasma ( prp ) , which can be easily harvested from autologous blood , has been used in clinical experiments for decades . prp is rich in growth factors , containing those that help the proliferation of chondrogenic cells and discharging of cartilaginous matrix , such as platelet - derived growth factor ( pdgf ) , transforming growth factor ( tgf - b ) , vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) and insulin - like growth factor ( igf ) . a new member in platelet concentrated products is platelet - rich fibrin ( prf ) , which can be prepared easily without biochemical blood handling . prf is similar to prp except in biochemical architectures because of slow polymerization during prf preparation . such a network leads to a more competent cell mobilization and repopulation and consequently healing . stromal cell - derived factor-1 ( sdf1 ) , a chemokine factor expressed by bmscs , regulates the trafficking , homing and migration of stem cell by mediating chemokinesis and chemotaxis into the injury site . we believe that autologous prp and prf could be used alone as a 3-d scaffold capable of cartilage tissue regeneration because of releasing endogenous growth factors . sdf1 stimulates cartilage mesenchymal stem cells ( c - mscs ) , synovial membrane mesenchymal stem cells ( sm - mscs ) , and bone marrow stromal cells ( bmscs ) to migrate to the subchondral bone in full - thickness defects to make cartilage . the aim of the study was to evaluate tissue repair along with prp and prf or without treatment . the secondary objective was to evaluate the efficacy of prp and prf used with sdf1 . the 9 ml of whole blood collected in a tube containing 1 ml an anticoagulant ( 3.8% sodium citrate solution ) was centrifuged for 5 minutes at 2,000 rpm and the supernatant was placed in a new tube . the plasma at the top was discarded and the remaining 1ml of liquid prp precipitate was collected . after mixing prp ( 200 l / defect ) and 40 mmol / l calcium chloride ( cacl2 ) , a gel was formed . the prp gel bonded to the adjacent cartilage about 10 minutes after implantation . the 10 ml blood samples collected from the hearts of six rabbits without anticoagulant were immediately centrifuged at 3,000 rpm for 10 minutes . in the middle of the tube , a fibrin clot was formed between supernatant acellular plasma and the lower red corpuscles . before transplantation , 600 l of rhsdf1 alpha ( 120 ng / ml peprotech ) was incubated with prp gel , prf and gelatin separately for 2 hours at 37c . a total of 18 adult male white japanese rabbits ( weighing 2.5 - 3.0 kg ) were used in this study . the animals were housed in metal wire cages , in a temperature - controlled room under a 12:12 h light - dark cycle at 2224c and 5060% relative humidity . animal interventional experiments were carried out at animal experiments of tehran university of medical sciences under the rules and regulation of the animal care and use committee of shiraz university school of medicine . the 36 bilateral knees of the 18 rabbits were randomly divided into 6 experimental groups of 6 knees per group . each animal was sedated by intramuscular injection of ketamine hydrochloride 60 mg / kg and xylazine 6 mg / kg . in sterile conditions , a full - thickness cylindrical cartilage defect of 4 mm in diameter and 3 mm in depth was created in the patellar groove using a standard size stainless biopsy punch . i , the defects were left unfilled ( control group ) . in group ii , the defects were filled with platelet - rich plasma only ( prp group ) . in group iii , the defects were filled with platelet - rich fibrin only ( prf group ) . in group iv , the defects were filled with gelatin containing stromal cell - derived factor-1 ( gel+sdf1 group ) . in group v , the defects were filled with platelet - rich plasma containing stromal cell - derived factor-1 ( prp+sdf1 group ) . in group vi , the defects were filled with platelet - rich fibrin containing stromal cell - derived factor-1 ( prf+sdf1 group ) in a randomized manner . the distal parts of the femurs were excised , photographed and graded for cartilage repair , according to the international cartilage repair society score ( icrs ) macroscopic assessment scores ( table 1 ) . icrs macroscopic evaluation of cartilage repair after gross evaluation , samples were fixed in 4% paraformaldehyde for 7 days , decalcified in 10% edta for 3 weeks and then embedded in paraffin and cut perpendicularly into 5 m sections . then , the sections were stained for general histology with hematoxylin and eosin ( h&e ) and with toluidine blue to estimate the cartilaginous matrix distribution . the re - created tissue was scored blindly according to the icrs scale ( table 2 ) . icrs visual histological assessment scale immunofluorescent analysis was performed using a monoclonal antibody against collagen ii ( cat number : ma1 - 37493 ) . heat - induced epitope retrieval was done in citrate buffer ( sodium citrate , 10 mm ; ph 6.0 ) in a pressure cooker for 4 minutes at full pressure . subsequent to cooling , endogenous peroxidase was blocked using a 3% hydrogen peroxide solution for 20 minutes . the slides were then washed with phosphate buffer solution ( 10 mm ; ph 7.4 ) and incubated with primary antibodies overnight at 4c . the slides were washed again three times with pbs and subsequently incubated with an fitc - conjugated secondary antibody ( cat number : 11555100 ) according to the respective manufacturer s instructions . total rna extraction was performed using rnx - plus kit ( cinnagen ) according to the protocol . the concentration and quantification of total rna were measured with spectrophotometer , with the od260/od280 ratio of all rna samples 1.92.0 and od260/od230 ratio up to 2 . the first strand cdna was synthesized with the first - strand cdna synthesis kit ( bioneer kit , k-2101 ) . for each reaction , 1 g ( 1 l ) rna was used for reverse transcription , in a mixture of 20 pmoles ( 1 l ) random primer , and 18 l depc - d.w with a final volume of 20 l . the mixture was incubated at 15c for 1 min , 50c for 60 minutes and heated at 95c for 5 minutes to terminate the reaction . qpcr was performed in a volume 1 l of primer and 1 l from template also add 3 l depc.d.w with 5 l master mix ( accupower 2x greenstartmqpcr master mix , bioneer kit ) . all pcr reactions were in the following condition : initial 95c for 15 minutes , followed by 35 cycles at 95c for 15 s and 60c for 30 s. after completing the qpcr , their products run on agarose gels for quality check and real - time data analyzed by livak rules . the sequences of primers used in real - time pcr analyses were as follows : aggrecan forward gagaccaagtcctcaagccc ; reverse ctctgtctcctggcaggttc . a comparison between groups for each outcome was carried out with the kruskal - wallis test . the 9 ml of whole blood collected in a tube containing 1 ml an anticoagulant ( 3.8% sodium citrate solution ) was centrifuged for 5 minutes at 2,000 rpm and the supernatant was placed in a new tube . the plasma at the top was discarded and the remaining 1ml of liquid prp precipitate was collected . after mixing prp ( 200 l / defect ) and 40 mmol / l calcium chloride ( cacl2 ) , a gel was formed . the prp gel bonded to the adjacent cartilage about 10 minutes after implantation . the 10 ml blood samples collected from the hearts of six rabbits without anticoagulant were immediately centrifuged at 3,000 rpm for 10 minutes . in the middle of the tube , a fibrin clot was formed between supernatant acellular plasma and the lower red corpuscles . before transplantation , 600 l of rhsdf1 alpha ( 120 ng / ml peprotech ) was incubated with prp gel , prf and gelatin separately for 2 hours at 37c . a total of 18 adult male white japanese rabbits ( weighing 2.5 - 3.0 kg ) were used in this study . the animals were housed in metal wire cages , in a temperature - controlled room under a 12:12 h light - dark cycle at 2224c and 5060% relative humidity . animal interventional experiments were carried out at animal experiments of tehran university of medical sciences under the rules and regulation of the animal care and use committee of shiraz university school of medicine . the 36 bilateral knees of the 18 rabbits were randomly divided into 6 experimental groups of 6 knees per group . each animal was sedated by intramuscular injection of ketamine hydrochloride 60 mg / kg and xylazine 6 mg / kg . in sterile conditions , a medial para - patellar arthrotomy was made in both knees . a full - thickness cylindrical cartilage defect of 4 mm in diameter and 3 mm in depth was created in the patellar groove using a standard size stainless biopsy punch . ii , the defects were filled with platelet - rich plasma only ( prp group ) . in group iii , the defects were filled with platelet - rich fibrin only ( prf group ) . in group iv , the defects were filled with gelatin containing stromal cell - derived factor-1 ( gel+sdf1 group ) . in group v , the defects were filled with platelet - rich plasma containing stromal cell - derived factor-1 ( prp+sdf1 group ) . in group vi , the defects were filled with platelet - rich fibrin containing stromal cell - derived factor-1 ( prf+sdf1 group ) in a randomized manner . the distal parts of the femurs were excised , photographed and graded for cartilage repair , according to the international cartilage repair society score ( icrs ) macroscopic assessment scores ( table 1 ) . after gross evaluation , samples were fixed in 4% paraformaldehyde for 7 days , decalcified in 10% edta for 3 weeks and then embedded in paraffin and cut perpendicularly into 5 m sections . then , the sections were stained for general histology with hematoxylin and eosin ( h&e ) and with toluidine blue to estimate the cartilaginous matrix distribution . the re - created tissue was scored blindly according to the icrs scale ( table 2 ) . icrs visual histological assessment scale immunofluorescent analysis was performed using a monoclonal antibody against collagen ii ( cat number : ma1 - 37493 ) . heat - induced epitope retrieval was done in citrate buffer ( sodium citrate , 10 mm ; ph 6.0 ) in a pressure cooker for 4 minutes at full pressure . subsequent to cooling , endogenous peroxidase was blocked using a 3% hydrogen peroxide solution for 20 minutes . the slides were then washed with phosphate buffer solution ( 10 mm ; ph 7.4 ) and incubated with primary antibodies overnight at 4c . the slides were washed again three times with pbs and subsequently incubated with an fitc - conjugated secondary antibody ( cat number : 11555100 ) according to the respective manufacturer s instructions . total rna extraction was performed using rnx - plus kit ( cinnagen ) according to the protocol . the concentration and quantification of total rna were measured with spectrophotometer , with the od260/od280 ratio of all rna samples 1.92.0 and od260/od230 ratio up to 2 . the first strand cdna was synthesized with the first - strand cdna synthesis kit ( bioneer kit , k-2101 ) . for each reaction , 1 g ( 1 l ) rna was used for reverse transcription , in a mixture of 20 pmoles ( 1 l ) random primer , and 18 l depc - d.w with a final volume of 20 l . the mixture was incubated at 15c for 1 min , 50c for 60 minutes and heated at 95c for 5 minutes to terminate the reaction . qpcr was performed in a volume 1 l of primer and 1 l from template also add 3 l depc.d.w with 5 l master mix ( accupower 2x greenstartmqpcr master mix , bioneer kit ) . all pcr reactions were in the following condition : initial 95c for 15 minutes , followed by 35 cycles at 95c for 15 s and 60c for 30 s. after completing the qpcr , their products run on agarose gels for quality check and real - time data analyzed by livak rules . the sequences of primers used in real - time pcr analyses were as follows : aggrecan forward gagaccaagtcctcaagccc ; reverse ctctgtctcctggcaggttc . a comparison between groups for each outcome was carried out with the kruskal - wallis test . the macroscopic evaluation of the treated areas in the prf+sdf1 group revealed complete repair and integrated well with the surrounding cartilage as compared with other groups ( figure 1 ) . a post - hoc analysis showed that icrs scores of all experimental groups were significantly higher than those in the control group and the scores of the prf+sdf1 group were greater than those of the other groups ( p<0.001 ) ( figure 2 ) . gross appearance of defects in the trochlear groove at four weeks after surgery , respectively . b ) prp group : healing tissue is thinner than normal surrounding cartilage and shows a large fissure . d ) gel+sdf1 group : repaired tissue covers the defect almost completely , but is thin and distinguishable from the surrounding cartilage . e ) prp+sdf1 group : healing tissue covers defect with smooth white tissue and distinguishable from the surrounding cartilage . f ) prf+sdf1 group : repaired tissue covers the defect completely and no obvious margin was notable . a ) icrs macroscopic and b ) icrs microscopic scores in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) ; * * significant difference with rpp group ( p<0.05 ) . four weeks after surgery , the regenerated regions were evaluated by h&e and toluidine blue staining . the regenerated regions of the prf+sdf1 group were entirely filled by the repaired tissue and showed better results than the other groups ( figures 3 and 4 ) . a post - hoc analysis showed that icrs scores of all groups , except for the prp group , were significantly higher than the control group ( p<0.001 ) . the scores of the prf+sdf1 group were greater than the other groups , but were not significant when compared with the gel+sdf1 and prp+sdf1 groups . the scores of the prp group were significantly lower than the other groups except for the prf group , which was not significant ( figure 2 ) . the qualitative study of immunofluorescent staining of collagen ii demonstrated that neo - cartilages were minimally positive in the control , prp , and prf groups . in contrast , the gel+sdf1 , prp+sdf1 and prf+sdf1 groups exhibited a remarkable distribution of type ii collagen ( figure 5 ) . the microscopic appearance of h&e staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . a ) control group : the defect is concave and a noticeable thin layer of non - cartilaginous tissue . b ) prp group : the defect is filled by fibrous tissue with a crack . d ) gel+sdf1 group and ( e ) prp+sdf1 group : the defects in both groups are filled with a repaired tissue , which are thinner than the normal surrounding cartilage . f ) prf+sdf1 group : the defect is entirely filled by the repaired tissue , resembling the healthy cartilage surrounding the tissue . histological appearance of toluidine blue staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . b ) prp group , and c ) prf group : the defects in both groups have poor staining fibrocartilagous tissues and poorly organized ecm . d ) gel+sdf1 group , and e ) prp+sdf1 group : the repaired tissues show a good staining in both groups . a ) control group , b ) prp group , c ) prf group , d ) gel+sdf1 group , e ) prp+sdf1 group , and f ) prf+sdf1 group . the neo - cartilages are minimally positive in a , b and c. the neo - cartilages are positive in d and e. a remarkable distribution of type ii collagen is seen in f. four weeks after surgery , a real - time pcr analysis revealed that the mrna expression of sox9 was significantly greater in the regenerated tissue of all groups than the untreated control group ( p<0.001 ) . the level of gene expression of aggrecan in all groups was significantly higher than the control group , except for the prp group that was not significant ( p<0.001 ) ( figures 6 and 7 ) . the amount of gene expression of sox9 and aggrecan in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) . the quality checking of rt - pcr for sox9 and aggrecan from the regenerated cartilage of defects in the trochlear groove at four weeks after surgery . lane a : control group where transplantation was not performed ; lane b : prp group ; lane c : prf group ; lane d : gel+sdf1 group ; lane e : prp+sdf1 group , and lane f : prf+sdf1 group . the macroscopic evaluation of the treated areas in the prf+sdf1 group revealed complete repair and integrated well with the surrounding cartilage as compared with other groups ( figure 1 ) . a post - hoc analysis showed that icrs scores of all experimental groups were significantly higher than those in the control group and the scores of the prf+sdf1 group were greater than those of the other groups ( p<0.001 ) ( figure 2 ) . gross appearance of defects in the trochlear groove at four weeks after surgery , respectively . b ) prp group : healing tissue is thinner than normal surrounding cartilage and shows a large fissure . d ) gel+sdf1 group : repaired tissue covers the defect almost completely , but is thin and distinguishable from the surrounding cartilage . e ) prp+sdf1 group : healing tissue covers defect with smooth white tissue and distinguishable from the surrounding cartilage . f ) prf+sdf1 group : repaired tissue covers the defect completely and no obvious margin was notable . a ) icrs macroscopic and b ) icrs microscopic scores in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) ; * * significant difference with rpp group ( p<0.05 ) . four weeks after surgery , the regenerated regions were evaluated by h&e and toluidine blue staining . the regenerated regions of the prf+sdf1 group were entirely filled by the repaired tissue and showed better results than the other groups ( figures 3 and 4 ) . a post - hoc analysis showed that icrs scores of all groups , except for the prp group , were significantly higher than the control group ( p<0.001 ) . the scores of the prf+sdf1 group were greater than the other groups , but were not significant when compared with the gel+sdf1 and prp+sdf1 groups . the scores of the prp group were significantly lower than the other groups except for the prf group , which was not significant ( figure 2 ) . the qualitative study of immunofluorescent staining of collagen ii demonstrated that neo - cartilages were minimally positive in the control , prp , and prf groups . in contrast , the gel+sdf1 , prp+sdf1 and prf+sdf1 groups exhibited a remarkable distribution of type ii collagen ( figure 5 ) . the microscopic appearance of h&e staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . a ) control group : the defect is concave and a noticeable thin layer of non - cartilaginous tissue . b ) prp group : the defect is filled by fibrous tissue with a crack . d ) gel+sdf1 group and ( e ) prp+sdf1 group : the defects in both groups are filled with a repaired tissue , which are thinner than the normal surrounding cartilage . f ) prf+sdf1 group : the defect is entirely filled by the repaired tissue , resembling the healthy cartilage surrounding the tissue . histological appearance of toluidine blue staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . b ) prp group , and c ) prf group : the defects in both groups have poor staining fibrocartilagous tissues and poorly organized ecm . d ) gel+sdf1 group , and e ) prp+sdf1 group : the repaired tissues show a good staining in both groups . a ) control group , b ) prp group , c ) prf group , d ) gel+sdf1 group , e ) prp+sdf1 group , and f ) prf+sdf1 group . the neo - cartilages are minimally positive in a , b and c. the neo - cartilages are positive in d and e. a remarkable distribution of type ii collagen is seen in f. four weeks after surgery , a real - time pcr analysis revealed that the mrna expression of sox9 was significantly greater in the regenerated tissue of all groups than the untreated control group ( p<0.001 ) . the level of gene expression of aggrecan in all groups was significantly higher than the control group , except for the prp group that was not significant ( p<0.001 ) ( figures 6 and 7 ) . the amount of gene expression of sox9 and aggrecan in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) . the quality checking of rt - pcr for sox9 and aggrecan from the regenerated cartilage of defects in the trochlear groove at four weeks after surgery . lane a : control group where transplantation was not performed ; lane b : prp group ; lane c : prf group ; lane d : gel+sdf1 group ; lane e : prp+sdf1 group , and lane f : prf+sdf1 group . this study considered the effects of prp and prf with or without sdf1 in full - thickness lesions in rabbits knees . a literature review indicated that the effects of prf+sdf1 and prp+sdf1 were not studied so far . we demonstrated that the implantation of prf+sdf1 led to the greatest yield scores of full - thickness lesions in rabbits knee . therefore , it can be concluded that the scaffolds containing sdf1 have a better result on cartilage repair . zhang w. et al . implanted a combination of collagen scaffold to mimic the subchondral bone matrix environment containing sdf1 in order to repair partial - thickness defects in rabbits knee . the results demonstrated that collagen scaffold containing sdf1 improved the repairing process by a better support for c - mscs and sm - mscs migration and adhesion six weeks after transplantation . some researchers used ultra - purified alginate gel containing sdf1 to improve osteochondral lesions in rabbits knee . according to their studies , the repairing effect of osteochondral defects is elevated , because sdf1 increases the migration of host cells ( mainly bmscs ) to the site of injury , and this makes sdf1 as a potential factor in cartilage repair . they studied the length of the hypertrophic zone by immunohistochemical analysis of tibia cartilaginous growth plates of chicken embryos , which were incubated with sdf1 for 2 , 4 , and 6 days in organ culture . the results showed the length of the hypertrophic zone was significantly elevated after 4 and 6 days . in injured tissue with the permissive matrix environment , endogenous stem cells can participate in corrective process . some studies have introduced populations of stem / progenitor cells in the synovial membrane and articular cartilage , which show characteristic of stem cells and have the potential for cartilage repair . the findings of the above studies are in agreement with our results that show sdf1 can facilitate the migration of c - mscs and sm - mscs to full - thickness defect sites . although sdf1 has been notified to promote inflammatory cell migration , we could not find inflammatory cells in the newly formed tissue in rpf , prp and gel containing sdf1 groups . in parallel with our data , a study on tendon repair by exogenous sdf1 showed improvement of injury site and diminishing accumulation of inflammatory cells . in prf , the fibrin formation meshes , which are similar to biochemical architectures of the natural one , can trap a significant amount of circulating cytokines . this method increases the lifespan of these cytokines ( as a long - term effect ) , without which , it will be only released and used at the time of the primary phase of matrix remodeling . as a result , when the cells begin extracellular matrix remodeling to reconstruct the injured site , the cytokines will be available in the affected area for the required longer period . cytokines are important factors in the delicate balance of tissue homeostasis . in a pilot study , . showed that all patients with osteochondral defects who received bm - mscs transplanted on platelet - rich fibrin glue ( pr - fg ) had a significant improvement in their functional knee scores and mri findings after 6 months and remained stable 12 months post operation . in agreement with this clinical study , choukroun j. et al . revealed that filling a tooth socket by prf can enhance the healing process without any complication . in a supplementary study , they reported that adding prf to bone allograft in sinus elevation surgery can lead to 4-month reduction in healing time by increasing the speed of bone allograft maturation . however , in contrast to our results , an in - vivo study by shao x.x . et al . on osteochondral defects in rabbits knee showed that bmscs seeded in fibrin glue matrix forms a poor - quality cartilage - like tissue . it can be concluded that the reason for varying results can be due to the differences in biomechanical conditions of defect types . et al . reported that differentiation of bone - marrow derived stem cells can be affected by mechanical signals . moreover , suitable recurring compressive stress significantly increases chondrocyte proliferation in addition to aggrecan and collagen synthesis by chondrocytes . some researchers reported that cell adhesion and migration can facilitate by some elements such as different cell type and substrate stiffness . most importantly , in a separate study , wang h. et al . had compared the amount of bounded bovine chondrocytes on various surfaces of middle deep zone bone or calcified cartilage or hyaline cartilage . they reported that different contact surface stiffness give different ability to cells for migration and attachment . activated prp can be used in different forms like gel as a scaffold , liquid form injection or can be infiltrated in the tissue thickness . the selection of each form relies upon the type of tissue and the existing problem . by degradation of the fibrin skeleton of prp growth factors play a significant role in tissue repair , especially in the regenerative process of tissues with low cellular density and vascularization , such as cartilage . a study showed that if prp was applied in full - thickness chondral lesions , the signs of healing will be observed , but it did not show healthy or functional behavior . wu w. et al . reported cartilage formation into the prp scaffold after implanting prp gel as a carrier for cultured autologous chondrocytes in the subcutaneous tissue of rabbits knees . another study suggested that transplantation of prp gel along with synovial membrane derived mesenchymal stem cells ( sdscs ) can facilitate the repair of osteochondral lesions in rabbits knees . perhaps the underlying reason for different results is various compositions of this plasma fraction in different species , including rabbits . prf was harvested from an autologous blood sample that had a limited volume and prf tissue banks were not achievable . in the present study , in addition to using prp and prf as scaffolds for cell seeding , we used a mixture of sdf1 and prp and prf scaffolds to enhance the migration of host cells . in our research , the effects of prp and prf alone on the full - thickness defects were also studied separately . we found that the added sdf1 stimulated cell or the combination of prp , prf , and sdf1 stimulated the observed improvement .

background : the purpose of this study was to create biomaterial scaffolds like platelet - rich plasma ( prp ) and platelet - rich fibrin ( prf ) containing stromal cell - derived factor-1 ( sdf1 ) as a chemokine to induce hyaline cartilage regeneration of rabbit knee in a full thickness defect.methods:we created a full thickness defect in the trochlear groove of thirty - six bilateral knees of eighteen mature male rabbits . the knees were randomly divided into six groups ( group i : untreated control , group ii : prp , group iii : prf , group iv : gelatin+sdf1 , group v : prp+sdf1 , and group vi : prf+sdf1 ) . after four weeks , the tissue specimens were evaluated by macroscopic examination and histological grading , immunofluorescent staining for collagen type ii , and analyzed for cartilage marker genes by real - time pcr . the data were compared using statistical methods ( spss 20 , kruskal - wallis test , bonferroni post hoc test and p<0.05).results : macroscopic evaluations revealed that international cartilage repair society ( icrs ) scores of the prf+sdf1 group were higher than other groups . microscopic analysis showed that the icrs score of the prp group was significantly lower than other groups . immunofluorescent staining for collagen ii demonstrated a remarkable distribution of type ii collagen in the gel+sdf1 , prp+sdf1 and prf+sdf1 groups compared with other groups . real - time pcr analysis revealed that mrna expression of sox9 and aggrecan were significantly greater in the prf+sdf1 , prp+sdf1 , gel+sdf1 and prf groups than the control group ( p<0.05).conclusion : our results indicate that implantation of prf scaffold containing sdf1 led to the greatest evaluation scores of full - thickness lesions in rabbits .

Introduction Materials and Methods None None None None None None None None Results None None None Discussion Conclusion

it was initially developed for angiography , but it has also been found useful in oral and maxillofacial radiology ( omfr ) . the introduction of cbct has made three - dimensional imaging possible in omfr . cbct offers a 3d approach to data acquisition , image reconstruction , image display , and image interpretation . cbct scan can be achieved in a relatively short period of time , making it ideal for imaging children and claustrophobic adults . the patient finds the examination comfortable as it can be performed with the patient in a standing or sitting position . the technique is characterized by fast image acquisition with relatively high three - dimensional resolution , lower radiation exposure to patients , and fewer artifacts when compared with conventional computed tomography ( ct ) . the voxels are isotropic and the resolution varies from 0.07 to 0.25 mm depending on the manufacturer . these values exceed most high - grade multi - slice ct units in terms of spatial resolution , real time image analysis , and three - dimensional display . cbct technology has been used for preoperative implant planning , evaluation of the jaws , and presurgical assessment of the bone volume required for orthognathic surgery . as cbct systems have become more prevalent , its use has gained attention in the field of otolaryngology . cbct of the inner ear [ figures 1 and 2 ] is effective in the diagnoses of most malformations , dysplasia , traumatic lesions , thin osseous labyrinthine wall erosion , or dehiscence . the advantages and diagnostic value of cbct have been demonstrated in the analysis of the position of cochlear implants in the temporal bone , the free visualization of alloplastic middle ear titanium , gold , or platinum implants . it is also possible that the smallest bone structure or position of the prosthesis can be defined with high precision contributing valuable information to the diagnosis , preoperative planning , and prevention of intra - operative complications . 60-year - old female patient with clinical history of hearing loss diagnosed with sensorineural hearing loss . cbct of temporal bone three - dimensional image with air cavity volume rendering ( green ) , shows normal inner ear : semicircular canals ( white arrows ) , vestibule ( white star ) , cochlea ( yellow arrow ) and internal auditory canal ( red star ) . 28-year - old male patient with clinical history of bilateral hearing loss and semicircular canal dehiscence diagnosed with conductive hearing loss due to bilateral fixing of the malleus . bilateral cbct of temporal bone three - dimensional image with osseous volume rendering demonstrates normal superior semicircular canal . a semicircular canal dehiscence ( scd ) is characterized by a pathological opening in a small section of the bony wall of the semicircular canal ( sc ) of the inner ear . an scd can be found in three different places according to different etiologies : the superior [ figures 3 and 4 ] , lateral , and posterior scs . regardless of differences in scd location , dehiscence patients have complained of similar symptoms such as vertigo , oscillopsia , and/or hearing loss . 35-year - old male patient with clinical history of vertigo and autophony diagnosed with left superior semicircular canal dehiscence . cbct of temporal bone ( a and d ) coronal ( b ) sagittal ( c ) oblique sagittal images show a dehiscence of the left superior semicircular canal ( white arrow ) . 17-year - old patient with clinic history of progressive hearing loss , without vertigo and oscillopsia diagnosed with sensorineural hearing loss and right superior semicircular canal dehiscence . cbct of temporal bone , air cavity volume rendering ( green ) images ( a ) sagittal view , ( b ) endocranial view , ( c ) osseous volume rendering oblique sagittal view and ( d ) osseous volume rendering superior view show superior semicircular canal dehiscence ( yellow arrow in a ) , ( yellow arrow in b ) , ( white arrow in c ) , and ( white arrow in d ) . superior semicircular canal dehiscence ( sscd ) syndrome is characterized by sound and/or pressure - induced vertigo and oscillopsia due to a dehiscence of bone overlying the superior sc in the roof of the temporal bone or in the neighborhood of the epidural space . minor et al . , first described this in 1998 , and it has become a new and exciting concept in the field of otology . vertigo as a result of loud noises ( tullio phenomenon ) , autophony , oscillopsia , disequilibrium due to sound changes in middle ear pressure , or changes in intracranial pressure were observed in these patients . sscd may present with a variety of atypical vestibular and/or auditory symptoms , which can be suggestive of otosclerosis . sscd is relatively rare , the reported cases in the literature range from 0.4% to 0.7% with the majority of patients being asymptomatic . several etiologies have been linked to hereditary , developmental , intracranial hypertension , and trauma . other authors have suggested that sscd may be due to a congenital or a genetic condition . sscd most commonly occurs along the apex or the roof of the sc , termed the arcuate eminente . however , approximately 9.1% of sscd may also occur more posteriorly or medially , involving the groove of the superior petrosal sinus [ figure 5 ] . 42-year - old male patient , with clinical history of cranial trauma and initial diagnosis of left temporal bone fracture diagnosed with left temporal bone fracture and ipsilateral superior semicircular canal dehiscence . cbct of temporal bone shows incidental find of dehiscence of the superior semicircular canal ( black arrow ) in superior petrosal sinus canal area ( white arrow ) , cochlea ( white star ) , and ossicle chain ( black star ) . diagnosis of sscd is based on the presence of vestibular or auditory symptoms and radiologic findings . the dehiscence is confirmed with high resolution computed tomography ( hrct ) scanning of the temporal bones using bone window settings . this is a key component in the diagnosis of sscd , especially when a surgical intervention is contemplated . minor reported a sensitivity of 100% , a specificity of 99% , a positive predictive value of 99% , and a negative predictive value of 100% in the identification of sscd . a semicircular canal dehiscence ( scd ) is characterized by a pathological opening in a small section of the bony wall of the semicircular canal ( sc ) of the inner ear . an scd can be found in three different places according to different etiologies : the superior [ figures 3 and 4 ] , lateral , and posterior scs . regardless of differences in scd location , dehiscence patients have complained of similar symptoms such as vertigo , oscillopsia , and/or hearing loss . 35-year - old male patient with clinical history of vertigo and autophony diagnosed with left superior semicircular canal dehiscence . cbct of temporal bone ( a and d ) coronal ( b ) sagittal ( c ) oblique sagittal images show a dehiscence of the left superior semicircular canal ( white arrow ) . 17-year - old patient with clinic history of progressive hearing loss , without vertigo and oscillopsia diagnosed with sensorineural hearing loss and right superior semicircular canal dehiscence . cbct of temporal bone , air cavity volume rendering ( green ) images ( a ) sagittal view , ( b ) endocranial view , ( c ) osseous volume rendering oblique sagittal view and ( d ) osseous volume rendering superior view show superior semicircular canal dehiscence ( yellow arrow in a ) , ( yellow arrow in b ) , ( white arrow in c ) , and ( white arrow in d ) . superior semicircular canal dehiscence ( sscd ) syndrome is characterized by sound and/or pressure - induced vertigo and oscillopsia due to a dehiscence of bone overlying the superior sc in the roof of the temporal bone or in the neighborhood of the epidural space . minor et al . , first described this in 1998 , and it has become a new and exciting concept in the field of otology . vertigo as a result of loud noises ( tullio phenomenon ) , autophony , oscillopsia , disequilibrium due to sound changes in middle ear pressure , or changes in intracranial pressure were observed in these patients . sscd may present with a variety of atypical vestibular and/or auditory symptoms , which can be suggestive of otosclerosis . sscd is relatively rare , the reported cases in the literature range from 0.4% to 0.7% with the majority of patients being asymptomatic . several etiologies have been linked to hereditary , developmental , intracranial hypertension , and trauma . other authors have suggested that sscd may be due to a congenital or a genetic condition . sscd most commonly occurs along the apex or the roof of the sc , termed the arcuate eminente . however , approximately 9.1% of sscd may also occur more posteriorly or medially , involving the groove of the superior petrosal sinus [ figure 5 ] . 42-year - old male patient , with clinical history of cranial trauma and initial diagnosis of left temporal bone fracture diagnosed with left temporal bone fracture and ipsilateral superior semicircular canal dehiscence . cbct of temporal bone shows incidental find of dehiscence of the superior semicircular canal ( black arrow ) in superior petrosal sinus canal area ( white arrow ) , cochlea ( white star ) , and ossicle chain ( black star ) . diagnosis of sscd is based on the presence of vestibular or auditory symptoms and radiologic findings . the dehiscence is confirmed with high resolution computed tomography ( hrct ) scanning of the temporal bones using bone window settings . this is a key component in the diagnosis of sscd , especially when a surgical intervention is contemplated . minor reported a sensitivity of 100% , a specificity of 99% , a positive predictive value of 99% , and a negative predictive value of 100% in the identification of sscd . teymoortash et al . , analyzed radiological imaging of 43 structures of the temporal bone by cbct and ct in 38 human temporal bones . the frequency of visualization of these anatomic structures were studied and statistically analyzed : 9 structures ( 20.1% ) of axial scans and 5 structures ( 11.6% ) of coronal scans could be identified as statistically significantly ( p < 0.05 ) more often in cbct than in ct . they determined that anatomical structures of the temporal bone can be identified significantly better in cbct than in ct scans . a dehiscent - appearing superior canal was seen in 4.0% of cases , while published pathologic studies reported that only 0.5% of temporal bone sscs have a dehiscence ( p < 0.001 ) . they established that the prevalence of dehiscent - appearing superior canal on thin - section temporal bone scanning with reformation in the ssc plane is much higher than anticipated by pathologic studies . the results were compared with intra - operative findings to evaluate the diagnostic value of cbct in cases of erosion of the scs . they determined the presence of a fistula of the scs in 100% of the cases . in conclusion , when compared with multi detector computed tomography , cbct imaging for the inner ear is an excellent alternative . it results in lower radiation to patients , fewer artifacts , and equal or higher resolution depending on the unit used .

superior semicircular canal dehiscence is a relatively new syndrome in the field of otology . it is of unknown etiology presenting with a variety of vestibular and auditory symptoms and radiologic findings play a crucial role in its diagnosis . cone beam computed tomography has been shown to be a powerful tool in the field of otolaryngology . it is a three dimensional technique that uses lower radiation resulting in fewer artifacts and offers higher resolution when compared with multi - slice computed tomography . it is considered to be an excellent imaging modality for radiological exploration of the ear .

INTRODUCTION Superior semicircular canal dehiscence DISCUSSION CONCLUSION

primary cysts have a lined epithelium and secondary cyst could be secondary to trauma . epidermoid splenic cysts are example of primary congenital cysts that contain an epithelial lining , unlike secondary cysts , which are composed of fibrous tissues , when the cyst is large they have nonspecific abdominal symptoms like pain , nausea , or a palpable mass usually in the left upper quadrant . epidermoid cysts and parasitic cysts are examples of primary cysts and usually have a classic presentation on imaging . despite imaging modalities and the patient s history , it can be difficult to diagnose an epidermoid cyst without a histological examination . the purpose of this paper is to discuss variable and atypical radiological presentation of primary splenic cysts including epidermoid cysts . a 51-year - old female presented to the ed using private transportation with complaints of left upper quadrant abdominal pain , nausea and vomiting for a couple days . she had significant medical history of hypertension , gerd , and hyperlipidemia with a bmi of 37.5 and no previous surgical history . patient had quit smoking cigarettes two years prior , does not drink alcohol or use any illicit drugs . physical exam was significant to being mildly tender to palpation over her left upper quadrant . vitals , complete blood count , basic metabolic panel , liver function tests , and urine analysis were all within normal limits . abdominal ultrasound ( fig . 1 ) visualized a 7.7 cm complex mass in the medial aspect of the spleen . 2 ) revealed a hypodense cystic mass in the medial aspect of the spleen measuring 10.4 cm 8.2 cm with a hounsfield attenuation of 15 ; containing multiple foci of fat with hounsfield attenuation of 102 . patient was assumed to have a confined cystic tumor of unknown etiology without any acute complications at this point . after long discussion with the patient with this large cystic mass of the spleen of unknown pathology , splenectomy was offered to the patient . she underwent uneventful laparoscopic hand - assisted splenectomy 6 weeks after presentation to the ed . she continued to have an uneventful recovery on follow - up with no recurrence of painful symptoms . 4 ) , it weighed 404 g , and was 17 11 5 cm in dimensions and was partially cystic . 11 10 7 cm smooth walled yellow tan cyst was present . on microscopic examination , sections demonstrated unremarkable splenic parenchyma and cyst walls lined by stratified squamous epithelium . immunohistochemical stains demonstrated an adequate distribution of cd20 + b - lymphocytes and cd3 + t - lymphocytes . . examples of primary benign neoplasms involving the spleen include haemangiomas , lymphangiomas , angiomas and splenic mesenchymal tumors such as lipomas , angiomyolipomas , fibromas , fibrosarcomas , and leiomyosarcomas . . they could be a pseudocyst secondary to trauma or a true cyst , which might be infectious or congenital such as an epidermoid cyst . epidermoid splenic cysts are primary congenital cysts that contain an epithelial lining , unlike secondary cysts which are composed of fibrous tissues , . patients usually do not present with specific symptoms if the cyst is small , and when the cyst is large , they have nonspecific abdominal symptoms like pain , nausea , or a palpable mass usually in the left upper quadrant . on imaging , epidermoid and parasitic cysts , which are also primary cysts , can be similar in appearance showing up as well - defined solitary lesions with septations . on computed tomography , septations , calcifications and internal debris . when findings are consistent with multiple neighboring cysts or daughter cysts , parasitic cysts are in favor as a diagnosis . epidermoid cysts of the spleen usually have a thin - walled anechoic appearance on ultrasound with no evidence of calcification on computed tomography . on t2-weighted magnetic resonance imaging , the cyst is hyper - intense , and on t1-weighted imaging , it is hypo - intense but can be increased depending on cyst contents . we used computed tomography to accurately identify the location , size , and adjacent structures of the cyst with an understanding that a definitive diagnosis would only be made by histopathological examination . we identified a complex mass on ultrasound and a hypodense lesion on computed tomography with multiple foci of fat , which were very different demonstrations from what is expected of a classic epidermoid or parasitic cyst on imaging . when examined , an epidermoid cyst usually has a white or grey smooth internal wall with fibrous trabeculations . under microscopic examination , an epithelium is visualized that is stratified squamous , which was consistent with our findings , and the contents can be serous , turbid , or thick and vary in color . surgical treatment is recommended for a symptomatic splenic cyst and for cysts larger than 5 cm , , . marsupialization or fenestration as treatments are indicated for superficial cysts , but they have a high recurrence rate , . in our case a large cyst , greater than 5 cm was identified and extended behind the stomach and into the medial aspect of the spleen . a total splenectomy was our indicated diagnostic and treatment of choice due to size of the cyst , presentation with symptoms , and also as a preventative intervention against future rupture , hemorrhage , infection , or recurrence . it is important to recognize that epidermoid splenic cysts may not always present in a classic fashion on imaging so it is trivial to include them as a differential diagnosis when warranted . the exact nature of a cyst will not be known prior to histological examination as in our case when imaging is not accurate or definitive in differentiation . therefore , the cyst was treated based on symptomology , size , and location and a total splenectomy which was indicated , was performed . bilal kharbutli , md and sharmilee vuyyuru , do have contributed in the study concept and writing .

highlightssplenic tumors are not common , many can have minimal to no symptoms , and they can be found incidentally.splenic tumors can be primary or metastatic . they can be benign or malignant.splenic cysts can be infectious , traumatic or congenital.epidermoid splenic cysts may not always present in a classic fashion on imaging , making diagnosis challenging.many splenic masses and complex cysts would require surgical pathological diagnosis .

Introduction Case presentation Pathology Discussion Conclusion Funding Ethical approval Consent Author contribution Guarantor

fungal infections are well - recognized life threatening problems in solid organ transplant ( sot ) , namely in liver transplant recipients . host and environmental factors are critical and important determinants regarding the epidemiology of fungal infections in transplantation . the incidence and aetiology of fungal infections differ dramatically between hematopoietic stem cell transplantation and sot . in the last group , those receiving lung grafts have the highest incidence ( 7.9% ) followed by heart ( 3.4% ) , liver ( 3.1% ) , kidney ( 1.1% ) , and pancreas ( 0.7% ) . in liver transplanted patients , the incidence of invasive fungal infections ranges from 4 to 50% and candida spp . and aspergillus spp fungal cutaneous and subcutaneous infections are frequently associated with people with professional activities related to agriculture or forestry work and the entry of the fungus is facilitated by penetrating injuries caused by thorns , splinters , nails , etc . the limbs are the most affected , but other body parts can also be involved . alternaria general characteristics include the production of dark - coloured phaeodictyospores in chains , and a beak of tapering apical cells [ 6 , 7 ] . some species are recognized to cause cutaneous phaeohyphomycoses and are described as emerging pathogens in immunocompromised patients , being transplantation the most common risk factor and also associated to the use of prednisone . a 65-year - old male caucasian was observed in a dermatology consultation of a central hospital in lisbon on 16th april 2014 . the patient presented with two painless , fast - growing nodules which had developed within 4-months . one of the nodules was located on the right leg and the other one on the dorsum of the left hand ( figs . 1 and 2 ) . he had a clinical history of 6-months liver transplantation and at the time of observation was under immunosuppressive therapy with tacrolimus 8 mg once a day , mycophenolate mofetil 500 mg twice a day and prednisone 20 mg once a day . the patient referred a previous history ( previous to the transplantation ) of agricultural activities with frequent skin injuries . physical examination revealed a well - defined erythematous nodule with 1.4 cm of maximum diameter , with central haematic crusts and a peripheral scali collarette on the dorsum of the left hand and a violaceous nodule with 1.5 cm with central haematic crusts and serous exudate on the right leg . skin biopsies were collected on that day from both nodules using a 4 mm biopsy punch and sent for histological and mycological examination . histological sections of the biopsy were stained with periodic acid - schiff and results retrieved on day + 13 revealed an inflammatory infiltration of the dermis and the presence of multiple fungal spores and hyphae . the mycological analysis of the biopsy material was performed using with calcofluor - white staining , followed by observation under fluorescence microscopy . clinical specimens were cultured on sabouraud 's dextrose agar containing chloramphenicol at 30 c and 35 c . cultures yielded a grey velvety fungus , with a dark - brown reverse on day + 14 . when sub - cultured both on malt extract agar for identification , the microscopic examination of the moulds performed by teased mount and lacto - phenol cotton blue preparation , showed different macroscopic features : green ( leg biopsy ) to grey ( hand biopsy ) colonies both with dark reverse . microscopic examination showed chains of brown conidia with rounded base and beaked apex , with transverse and oblique septation . there were a slight difference between microscopy from leg and hand biopsy , in the first one , conidial chains were more branched than in the second one , and conidia were less tubular ( figs . 3 and 4 ) . from the above microscopic and macroscopic features , the isolates were identified as alternaria spp . on day + 20 and sequenced for speciation . the lesions were excised on day + 34 and the patient started antifungal therapy on the same day with oral itraconazole ( 100 mg daily ) for 3 months with adjustment of immunossupressive therapy . total genomic dna was extracted from purified colonies and the internal transcribed spacer ( its ) region of ribosomal dna ( rdna ) of these isolates was amplified using primer set its1 ( 5tccgtaggtgaacctgcgg3 ) and its4 ( 5tcctccgcttattgatatgc3 ) . sequencing of both strands was performed and nucleotide sequences were edited using the programme chromas2 and aligned using the programme clustal x2 . the sequences obtained were compared with sequences deposited in the genbank and cbs - knaw fungal biodiversity centre databases . molecular sequencing resulted in the identification of alternaria infectoria from the culture obtained from the patient 's hand and alternaria alternata from the culture obtained from the patient 's leg . the sequencing coverage was high ( 99100% ) , as well as the homologies with reference strains ( 98% ) , which is the value considered as an acceptable identification to species level . in fact , we obtained a sequence with a homology of 98% and 99.5% with alternaria infectoria and alternaria alternata , respectively . nucleotide sequences of the isolates were deposited in the genbank database under the accession numbers kp171632 ( alternaria infectoria ) and kp171633 ( alternaria alternata ) . phaeohyphomycosis can be caused by different species , being a. alternata and a. infectoria the most commonly isolated . dematiaceous fungi have melanin in their cell walls , which represent a virulence factor for several pathogenic fungi . the raise of patients under immunosuppressive drug therapy has increased and so the risk of fungal infections is likely to increase . the patient of this case report lives in a rural area , but did not refer trauma after transplantation . however , after a detailed questionnaire , the patient referred that before the transplantation he carried out different agricultural activities and had skin injuries . cutaneous fragility induced by hypercorticism could be an important co - factor permitting direct inoculation from the environment . nevertheless , several fungi can also remain latent for long periods of time and the clinical symptoms of fungal infection may appear several years after the inoculation of the etiological agent . there are currently no standardised treatments for cutaneous infections caused by alternaria spp . in those cases and when it is possible , the reduction of the immunosuppression level can be enough to resolve the lesions , although the long - term use of systemic itraconazole is often recommended . co - existent fungal infections associated with alternaria spp are sometimes referred [ 1723 ] , despite infrequently . furthermore , most of the published cutaneous fungal infection cases refer infection due to only one species as etiological agent . to our knowledge , this is the first case described of a cutaneous co - infection with two different species of alternaria . infection with different species can be under diagnosed , since when multiple lesions are observed , biopsy material is normally taken from only one of them . early detection and appropriate antifungal therapy are essential in the treatment of this emergent infection , molecular identification is a useful tool to distinguish mixed fungal infection .

fungal invasive infections are rare in general population but are an emergent cause of infection in the immunocompromized population , especially in the solid organ transplant recipients . herein the authors report a clinical case of a liver transplanted patient suffering a cutaneous co - existent infection with a. alternata as well as a. infectoria.to our knowledge this is the first case of cutaneous concomitant infection due to those two species reported not only in portugal but also worldwide . the patient was treated with surgical excision of the lesions and oral itraconazol without relapse .

Introduction Case report Discussion Conflict of interest

prospective studies have shown the superiority of laparoscopic appendectomy in the treatment of acute appendicitis . however , this technique has not yet gained widespread acceptance because of its longer operative time and lower cost effectiveness when compared with the open approach . moreover , although recent retrospective studies have shown that laparoscopic appendectomy is associated with a significantly shorter hospital stay , other reports demonstrate no significant difference in hospital stay when the laparoscopic and the open approach were compared . the minilaparoscopic approach is associated with less abdominal wall trauma , a lower infection rate at the trocar site and abdominal wall hernia , and produces excellent cosmetic results . in this study , our goal was to confirm the safety and show the feasibility of the minilaparoscopic approach for the treatment of acute appendicitis . between may 1999 and june 2001 , a minilaparoscopic appendectomy was attempted in 37 patients with a preoperative diagnosis of acute appendicitis and without a previous history of abdominal surgeries . the pneumoperitoneum was created by insertion of a veress needle through a small incision in the umbilicus . a 2.2-mm trocar was placed laterally to the left rectus abdominis muscle and served to manipulate a 2.2-mm , 0-degree laparoscope . another 5-mm trocar was placed just below the umbilical crease and was used for the ultrasonic scalpel . a third 2.2-mm trocar was placed suprapubically in the midline and was used for grasper access . exploration of the right lower abdominal cavity identified either an inflamed appendix or ruled out appendicial pathology . in the presence of appendicial pathology , the appendix was held with an atraumatic grasper and dissected with a 5-mm ultrasonic scalpel . an endoloop was positioned at the base of the appendix and tied 1 cm distally to its base . the appendix was then dissected by using an ultrasonic scalpel and placed in a sterile 8 and 1/2-size glove finger used as a specimen bag and removed through the 5-mm trocar site . all specimens were sent to the pathology laboratory for analysis . at the end of the procedure , all trocars were removed and the fascial defect at the 5-mm trocar site was repaired with absorbable sutures . all skin wounds were closed with steri - strips , and sterile dressings were applied . all patients were discharged with written instructions regarding diet , physical activity , use of oral nonsteroidal anti - inflammatory drugs ( nsaids ) for pain control , and follow - up clinic visits were scheduled for 1 week , 1 month , and 6 months postoperatively . the study population consisted of 37 patients , of which 7 were males and 30 were females . the average age of the patients was 26 years ( range , 7 to 48 ) . minilaparoscopic appendectomy was initially attempted in 37 patients ; however , it was only completed in 31 ( 84% ) . in 2 patients ( 5.3% ) , the procedure was completed with the conventional laparoscopic approach , because both patients had a perforated appendix . both patients were operated on with larger trocars and instruments because this made it safer to manipulate and resect the appendix and to wash the abdominal cavity . in 1 patient ( 2.7% ) , the procedure was converted to an open approach through a midline incision due to diffuse fecal peritonitis as a consequence of a ruptured appendix . in 3 patients ( 8% ) , an appendectomy was not performed because of pathology confined to the ovary . one wound infection occurred at the umbilical trocar site , which was treated conservatively . during the extraction of the specimen through the 2.2-mm umbilical trocar , the specimen bag ruptured and the inflamed appendix contaminated the abdominal wall . of the 31 patients , four patients ( 13% ) had a normal appendix , 26 patients ( 84% ) had acute appendicitis , and 1 patient ( 3% ) had a perforated appendix . the average operating time was 34 minutes ( range , 15 to 80 ) , and the median length of stay was 1.2 days ( range , 1 to 5 ) . postoperative pain management consisted of 2 doses of 10 mg of ketorolac tromethamine given intravenously . in all patients , wounds resulting from trocar placement healed well . previous studies have confirmed the superiority of laparoscopic appendectomy in the treatment of acute appendicitis . it is associated with decreased postoperative pain , a shorter length of hospitalization , a faster return to normal activity , an early resumption of dietary intake , and fast resolution of postoperative ileus . however , this technique has not yet gained widespread acceptance because of its longer operative time and higher cost compared with the open approach . in a prospective , randomized study of 50 patients , minn et al showed that the laparoscopic approach did not offer any improved benefits compared with the open approach for the routine patient with acute appendicitis . nevertheless , we believe that the laparoscopic approach allows good exploration of the abdominal cavity and may be helpful in ruling out different pathological states . in our experience , minilaparoscopic exploration of the abdominal cavity diagnosed pathology of the ovary in 8% of patients . it has been shown that 80% of women who underwent an open appendectomy , compared with 10% of women treated with laparoscopic appendectomy , may develop adhesions that in fact may be responsible for the infertility or chronic abdominal pain , or both . based on our experience , the procedure was completed with the conventional laparoscopic approach in 2 patients ( 5.3% ) because of a perforated appendix . we have learned that it was safer to insert bigger trocars and instruments to manipulate and resect the appendix , and also wash the abdominal cavity . we believe that converting to conventional laparoscopy was not a technical regression because the outcome did not differ from the outcome in those who underwent the minilaparoscopic appendectomy . it is generally true that the cost of laparoscopic appendectomy is higher than that of open appendectomy . although shorter hospitalization , less pain medications , and better patient compliance are well - established benefits associated with laparoscopic treatment , the financial burden associated with it in the long - term is also well established . sterile use of finger cut gloves instead of sterile specimen containers and use of nondisposable trocars and instruments may contribute greatly to the reduction in cost . it can be used multiple times ; it is versatile and can be used for other minimally invasive procedures , thus lowering its overall cost . the benefits of the laparoscopic approach in terms of better diagnostic accuracy and safety , especially for women of childbearing age , outweighs the disadvantage of a longer average operative time of about 17 minutes . in terms of advantages associated with the use of the minilaparoscopic approach , a reduction in abdominal wall trauma and a decrease in the number of complications , such as infection of the trocar site and abdominal wall hernia have been reported elsewhere . moreover , the smaller dimension of the trocars accounts for approximately a 70% reduction in postoperative pain , compared with that for the conventional laparoscopic approach . one patient , however , developed a wound infection at the umbilical trocar site that resulted from direct contact of the inflamed appendix with the abdominal wall after rupture of the finger bag . to avoid such a complication in the future , we modified the technique by extracting the bag though the 5-mm suprapubic incision , and since then no similar complications have occurred . the wounds from trocar placement healed well , and the general condition of all patients was excellent at 6-month follow - up . this study shows that the minilaparoscopic approach has the same advantages as the conventional approach in terms of better diagnostic accuracy and safety , especially for women of childbearing age .

background : minilaparoscopic appendectomy for appendicitis is not a well - established procedure . this approach provides less abdominal wall trauma , fewer complications , and excellent cosmetic results . our aim was to show the feasibility and safety of the minilaparoscopic approach.methods:minilaparoscopic appendectomy was performed in 37 patients . two 2.2-mm trocars were used to manipulate a 2.2-mm , 0-degree laparoscope and for grasper access . a 5-mm trocar was used for the ultrasonic scalpel.results:no deaths occurred . in 3 patients ( 8% ) , appendectomy was aborted due to pathology of the ovary . conversion to the open approach occurred in 2.7% of patients . the average operating time was 34 minutes ( range , 15 to 80 ) , and the median length of hospital stay was 1.2 day ( range , 1 to 5).conclusions : the minilaparoscopic approach a ) has the same advantages as the conventional laparoscopic approach in terms of better diagnostic accuracy and safety ; b ) a low incidence of complications ; and c ) yields excellent cosmetic results .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

mental health problems , such as depression and anxiety disorders , are often underrecognized and untreated . . showed that the prospect of being treated increases with the severity of the illness , but also that half of those affected by a serious mental illness remained untreated . it is easy to understand that a serious condition needs treatment to avoid complications such as suicide , need of inpatient care , and disability . however , studies have shown that the risk of such complications did not differ significantly between mild forms of mental illness compared to moderate forms . in several studies , around half of those affected by psychological distress or psychiatric diagnoses however , even if they seek , the detection rate of psychiatric symptoms is low . a recent meta - analysis of studies regarding general practitioners ability to recognize mild depression showed a detection sensitivity of 56.5% . this emphasizes the importance of further increasing the awareness of mild cases of mental illness . in primary care , a vast majority of patients affected by depression and anxiety present with somatic symptoms [ 11 , 12 ] . somatic complaints include changes in appetite and libido , lack of energy , sleep disturbances , dizziness , palpitations , dyspnoea , and general aches , and pains such as headache , back and other musculoskeletal pain , and gastrointestinal disturbances . identifying persons affected by mental illness , but seeking care for somatic symptoms , is a major difficulty especially in the primary care setting , due to both patient - related issues as well as physician - related issues . it is of importance that somatic symptoms associated with mental health disorders are not confused with somatoform disorders ( i.e. , conversion , somatization , hypochondriasis , and somatization disorder ) . the knowledge of factors influencing care seeking in persons affected by depressive and anxiety disorders in the population is limited . hence , it is important to elucidate factors associated with care seeking in these groups , over all , and factors associated with not seeking care for psychological symptoms . this study aims to describe the prevalence of care seeking among persons with depression and anxiety disorders using data from a population - based study in sweden . first , we aim to study whether affected persons seek care and if care seeking is associated with socioeconomic factors and health status . further , we aim to study if those who seek care for psychological symptoms at the general practitioners differ compared to those who seek care from other health care facilities or do not seek at all . this study is based on the part study ( an acronym in swedish for mental ill - health , work , and relationships ) . part is a longitudinal population - based study of mental health conducted in the stockholm county , sweden . in 1998 - 1999 , 19742 randomly selected swedish citizens aged 2064 years , residing in the stockholm county , were invited to participate and 10441 persons ( response rate 53% ) responded to the self - administrated questionnaire ( baseline ) that included questions on demographic and socioeconomic characteristics , somatic and psychiatric health , and use of drugs . three years after they had answered the first questionnaire ( baseline ) those who answered were reassessed with another similar questionnaire including questions on health care seeking ; 8700 persons participated ( retention rate 83% ) . psychiatrists performed interviews using schedules for clinical assessment in neuropsychiatry ( scan ) , in order to validate the answers of the questionnaires . a comparison between depressions according to the major depression inventory ( mdi ) used in the questionnaire and scan showed good compliance . nonparticipation analysis , using national registers , performed after the first two waves , revealed that the association between gender , age , income , education , country of birth , and psychiatric diagnoses in the national registers was similar among participants and nonparticipants [ 16 , 17 ] . for detailed information about the part study see the technical report . for the purpose of this study we restricted our analyses to the 8387 subjects that participated in both baseline and the first followup , with information on symptoms of depression and anxiety . definitions of anxiety and depressive disorders were made according to dsm - iv criteria , including research criteria , using validated diagnostic scales based on the questionnaire . the included scales were the sheehan patient - rated ( panic ) anxiety scale and the major ( icd-10 ) depression inventory , mdi . social phobia was assessed using the avoidance part of an instrument developed by marks and mathews and for obsessive - compulsive disorders screening questions suggested by the swedish psychiatric association and swedish institute for health services development were used . anxiety disorders included panic syndrome with agoraphobia , agoraphobia without panic syndrome , social phobia , obsessive - compulsive disorder , panic syndrome without agoraphobia , anxiety syndrome due to somatic cause , specific phobia , posttraumatic stress syndrome , general anxiety disorder , and acute stress syndrome . some of the persons affected by major depressive disorder may have a bipolar disorder since there was no scale for manic episodes in the questionnaire . three mutually exclusive groups were created : any depressive disorder ( n = 465 ) , any anxiety disorder ( n = 751 ) , and coexistent depressive and anxiety disorder ( n = 810 ) . in total 2 026 persons ( 24% ) fulfilled the criteria for any depressive or anxiety disorder . health care is accessible to everyone living in sweden , and because of tax subsidies , costs are limited for individuals . both private- and public - funded outpatient clinics are under the same regulations and the patient can choose their preference for the same cost , with exception for those private clinics without affiliation to the public health care system . with regards to psychologists and psychotherapists , there are also private practices without affiliation , and thus not subsidized , a more expensive alternative for the patient . the health care system is organized with a broad base of easy - accessible primary care in health centers , where a variety of health professionals ( doctors , nurses , physiotherapists , psychologists , counsellors , and other staff members ) work . the usual path to seek care is to turn to the health centre to see a specialist in general medicine ( general practitioner , gp ) . the major part of patients is taken care of at this level , but in case the patient needs to see another specialist , he or she is referred by the gp . the gp can also refer the patient to a psychologist or likewise . within the psychiatric sector , it is also possible to directly take contact with an outpatient psychiatric clinic , if it is obvious that the mental problems are severe enough to belong to the psychiatric care . if not , the patient will be redirected to the primary health care centre . the first was have you , due to sleeping problems , personal problems or psychological symptoms , been in contact with one or more of the following during the last 12 months ? psychologist / psychotherapist public or private , general practitioner public or private , other medical / psychological treatment , and alternative medical treatment . seeking care for psychological symptoms was defined as having checked one or more of the response alternatives . the second question was have you , due to bodily symptoms or somatic illness , been in contact with one or more of the following during the last 12 months ? with the following response alternatives : general practitioner public or private , specialist public or private , other medical treatment , and alternative medical treatment . data on country of origin and education was derived from the baseline questionnaire , and all other data was retrieved from the followup . hazardous alcohol use was evaluated using audit ( alcohol use disorders identification test ) [ 26 , 27 ] . education was categorized into three groups : basic compulsory education ( 9 years ) , upper secondary education ( 1012 years ) , and higher education ( college / university , 13 years ) . data from the second wave included household composition , children in household ( permanently or more than half of the time were considered as living with children ) . labour market position included employment / own business , on leave ( studies and parental leave ) , unemployed or in labour market policy measures , disability pension or sick leave for more than a month , and retirement . having a close friend included the answers entirely or fairly true to the question if there was a special person the person felt he / she could get support from . disability last 30 days included those who had been so affected by psychological symptoms / problems that they had not been able at all to pursue the ordinary tasks . somatic illness was measured by a list of 26 somatic disorders , and only those currently treated by a doctor were considered as exposed to somatic illness . the statistical analyses aimed to describe presence of care seeking and possible factors associated with such among persons affected with depression or anxiety disorders . also , the analyses describe what factors could be associated with seeking different types of care . this was done by using cross - tabulation in ibm spss statistics 19.0 on different kinds of care seeking to describe the prevalence of care seeking by demographic , socioeconomic , and psychiatric factors . pearson chi - square tests were used to test for statistical significance . additionally , to analyse differences between persons seeking different kinds of care , one - way analysis and bonferoni tests were used . partially missing answers were treated as missing values in the analyses ( varying from 0.04% on born abroad to at most 2.4% for seeking somatic care ) . a description of the study sample , stratified by depressive and/or anxiety disorders , is presented in table 1 . persons with depression were more often female , young , single , living without children , less often having a close friend and less educated . they reported more often to be on sick leave / disability pension , unemployed , treated for somatic illness , having hazardous alcohol use and were more often affected by disability , compared with those without depression and anxiety . persons with anxiety were more often female , younger and more often had hazardous alcohol use , compared with those without depression and anxiety . persons with comorbid depression and anxiety showed similar differences as those affected by depression and also reported more often having country of origin outside sweden . of those affected by depression and/or anxiety , 47.1% of the persons stated that they had been in contact with some type of health care facility within the last year due to psychological symptoms ; see table 2 . persons who had been seeking help for psychological symptoms were more often female , older , singles , born abroad , or outside the labour market . additionally , they more often had comorbidity factors such as somatic illness , or both depression and anxiety , were more severely affected by depression and more often disabled due to psychological symptoms . when it comes to type of care , 30.4% of the persons affected with depression and/or anxiety had been seeking help for their psychological symptoms at a gp , and 33.5% had been seeking help at other caregivers ; see table 3 . about thirteen percent had reported a gp as their only care provider . in the group that went to the gp , there was an overrepresentation of persons with both depression and anxiety , and disability due to psychological symptoms as well as somatic illness compared to those that did not seek care . this was applicable for both those who had the gp as their only provider , as well as those who also had seen a psychiatrist , psychologist , or other ( alternative / other medical or psychological ) . seeking care to a greater extent to psychiatrists or psychologists was also the case for those with comorbid anxiety and depression , and disability due to psychological symptoms . when it came to those with hazardous alcohol use , there was an overrepresentation among those who had seen both a gp and a psychiatrist / psychologist , compared to the persons that had only attended gp or had not been seeking at all . those that had been seeking gp were also older and more often they had less education , than those who were more likely to not seek care at all , or to seek only a psychiatrist or psychologist . at the gps , persons outside the labour market , on sick leave or disability pension were overrepresented , as well as persons born in another country , both among those who had the gp as their only care provider or in combinations . persons born abroad were also less likely to only have a psychiatrist or psychologist as their only provider . regarding the group that turn only to alternative care or other medical / psychological treatment , there seemed to be no differences among groups except for persons born abroad and less educated persons that were underrepresented . in the present study we found that 52.5% of those affected by depression and/or anxiety disorders did not seek care for psychological symptoms . among those not seeking care for psychological symptoms , two - thirds one reason for seeking for somatic symptoms might be that they primarily have identified the somatic symptoms that often accompanies depression and anxiety , which has been reported in several previous studies [ 12 , 29 , 30 ] . comparison with other studies is somewhat difficult due to different measures on both mental health and of outcomes such as care seeking or treatment . in our study , the proportion seeking care was 47.1% . several other studies have showed prevalence for seeking care for psychological distress or a variety of psychiatric diagnoses ( such as depression , dysthymia , gad , panic disorder , phobias ) , ranging from 36 to 60% [ 39 ] . this shows that the problem with people in need who does not seek help is widely spread . in the present study , persons less likely to seek help were male , younger , born in sweden , living with a partner , employed / on leave for studies or parental leave , retired or had higher education . several studies have reported that prejudices in the general population against male persons affected by mental disorders are higher than against affected females . having a job , being a student , or on parental leave might imply less daytime available in to be spent seeking care . when it comes to labour market position , being on sick leave might be a promoting factor but also a result of care seeking per se and an indicator of severity . summarizing , in the group of persons less likely to seek there is an overrepresentation of individuals that perhaps are less likely to be suspected of being affected of mental problems due to lower load of risk factors as well as assumed to be well adjusted in society . those with milder symptoms and less disability due to psychological symptoms were also less likely to seek . evidently this could be due to less need of care , and it could be argued that minor depression and distress could be resolved without professional help [ 32 , 33 ] . however , mild disorders are increasingly considered clinically significant and detecting them in an early stage might prevent them from turning into serious cases in the future [ 2 , 35 , 36 ] . having been seeking care both at the gps and at the psychiatrists or psychologist / psychotherapists could mean , with regards to how the health care system is organized in sweden , that the gp has referred the patient . this is especially the case when it comes to persons with a hazardous alcohol use that more often had been seeing a gp and a psychiatrist / psychologist . in this study we lacked information on type of clinical specialization but it is likely that it was referrals from gps to clinics specializing in alcohol dependence . there was no gender difference for the category that had seen a gp and a psychiatrist / psychologist / therapist . this could possibly stand for that there is no gender difference when it comes to proportional referrals from the gp , which is gratifying . persons with higher education were less likely to seek care at all , and if they did , they were more likely to turn to a psychiatrist / psychologist . this could possibly stand for a perceived need for a more specific treatment , higher ability to interpret their symptoms as psychological , or more knowledge on possible places to go . that persons under the age of 35 years show the same pattern could maybe stand for partly the same , as in a perceived need for a more specific treatment , but perhaps also for less stigmata surrounding mental problems . the opposite is shown for persons born abroad ; characteristics of migrants ' pathways to psychiatric care have been reported to be delays in seeking professional help , a lower probability of medical referral , frequent involvement of the police and emergency services , and high proportions of compulsory and secure - unit admissions . persons on sick leave , with a disability pension , or unemployed were more likely to see a gp , alone or in combination with psychiatrist / psychologist or other and more likely to seek care . it could be argued that having a long - term psychological health problem might be preceding poorer social functioning resulting in unemployment or sick leave / disability pension . also , contact with a gp or a psychiatrist is a necessity for the medical certificates needed for the social insurance system initiating a sick leave or disability pension , which in part could explain the overrepresentation among these groups . but also , it could stand for a more severe psychological health status or a greater need of treatment . studies have shown that unemployment , as well as sick leave or disability pension per se , can have a negative effect on psychological health . an important factor for not seeking care for psychological symptoms seems to be not having any treatment for somatic illness . this could be an important finding ; if a person has treatment for any somatic problems , he or she already established a relationship to the physician or care - giving facility , which might make bringing up psychological problems easier . older people might also have an easier access to care due to a prior relationship with their gp based on somatic illness or plainly longer experience of care seeking . the category turning only to alternative care seemed to have less to do with the mental illness per se , not varying with severity of illness or disability , but instead with socioeconomic factors that could be argued possibly related to limitations such as high cost or less knowledge of such . in the present population - based study , validated diagnostic scales for assessing anxiety and depression were used [ 4042 ] . one limitation is the cross - sectional design , which limits the possibilities to draw causal conclusions . the self - reported care seeking was measured retrospectively one year back from filling in the questionnaire . the scales measuring symptoms of depression cover the last 14 days and for anxiety the last 30 days , respectively . it could therefore be argued that persons might have symptoms but not yet contacted health care or that persons might fall out of the depression and/or anxiety group population because they have had symptoms previously but not during the last month . however , when examining reports of the duration of symptoms , we found that , of those having any form of depression , one - third had it more than two years , one - third since more than six months , and one - third between two weeks and six months . among those with anxiety , as a general practitioner , it is of great importance to further increase awareness of mild cases of mental illness , especially among groups that might be less expected to be affected by mental illness .

background . in primary care , a vast majority of patients affected with depression and anxiety present with somatic symptoms . detection rate of psychiatric symptoms is low , and knowledge of factors influencing care seeking in persons affected by depressive and anxiety disorders on a population level is limited . objective . this study aims to describe if persons , affected by depression and anxiety disorders , seek care and which type of care they seek as well as factors associated with care seeking . method . data derives from a longitudinal population - based study of mental health conducted in the stockholm county in 19982010 and the present study includes 8387 subjects . definitions of anxiety and depressive disorders were made according to dsm - iv criteria , including research criteria , using validated diagnostic scales . 2026 persons ( 24% ) fulfilled the criteria for any depressive or anxiety disorder . results . forty - seven percent of those affected by depression and/or anxiety had been seeking care for psychological symptoms within the last year . a major finding was that seeking care for psychological symptoms was associated with having treatment for somatic problems . conclusions . as a general practitioner , it is of great importance to increase awareness of mild mental illness , especially among groups that might be less expected to be affected .

1. Introduction 2. Objectives 3. Material and Methods 4. Results 5. Discussion 6. Study Strengths and Limitations 7. Conclusions

despite global reductions in fertility rates , parenthood remains a central life goal in most societies . in recent years , progress in medical technology has offered hope to many infertile couples , especially in the developed world . the us is one of the countries with the highest rate of multiple births in the world . this rate is directly attributed to the increased use of assisted reproductive technologies ( art ) in achieving pregnancy . the number of babies born to gestational surrogates increased to 89% in just 4 years , from 2004 to 2008 . iranian culture generally considers children as divine gifts , and producing children is the fundamental reason for marriage among many couples . having children is generally regarded as strengthening the institution of the family and as a sign of commitment to iranian cultural values . infertility is a social onus for women who are expected to produce children early within marriage in iran . according to the world health organization , over 80 million people ( about 10 - 15% ) of the families experience infertility . in iran , this practice of transferring an embryo or fetus from one womb to another is not forbidden in shiite jurisprudence . most researches have focused on a number of specific issues such as attachment and disclosure to surrogate offspring ; experiences , characteristics , and motivations of surrogate mothers ; and changes in profiles of the commissioning mothers . while some researchers have shown that clients experienced powerful emotions as a result of infertility and its treatment , and women experienced more stress than their partners , and this factor may delay their adaptation with the parenting process , in a retrospective qualitative study in uk , mccallum et al . showed that throughout the pregnancy period of a surrogate mother , couples recalled their levels of anxiety as low and their experience as positive , and in pashmi 's quantitative study , most of the surrogate and commissioning mothers did not consider surrogacy a problematic issue . klock and greenfeld found that in in vitro fertilization ( ivf ) , women experienced more satisfaction with being able to get pregnant than the women who conceived naturally and reported decreased anxiety as the pregnancy progressed . although surrogacy is essentially done with ivf technique , the key difference between surrogacy with other methods of ivf is that commissioning mothers in surrogacy will not experience the physical changes of pregnancy . most of the studies in iran have focused on defining surrogacy , demonstrating the legal aspects , ethical aspects , survey of cultural attitudes about the use of this technique , and ethnography of volunteer surrogate women . but studies about the third - party reproduction in iran are few , and the impacts of these methods on recipients , donors , and children are unknown . we found only one quantitative study , conducted by pashmi et al . in iran , on commissioning mothers experiences . evaluated the experiences of the 30 women involved in surrogacy ( 15 surrogate mothers and 15 commissioning mothers ) and compared them with 30 normal mothers ( women who conceived spontaneously ) in isfahan , a province of iran , using a questionnaire and structured interviews . results showed that the commissioning mothers had happy experiences as well as anxious feelings during the pregnancy of the surrogate mother . they were anxious about the health of both the surrogate mother and the baby . based on mercer , identifying a mother 's unique concerns and available resources to address these concerns and reinforcing her caretaking skills are important in fostering the mother 's sense of competence as she works on gaining a maternal identity . nurses are in a unique position to have long - term positive effects on mothers during this transition , and have a leadership role in providing anticipatory guidance and education that is grounded in the unique experiences of primigravid and multigravid women . there are a few studies that investigated the quality of commissioning mothers experiences such as their anxiety and stress . since the number of spouses treated by art such as surrogacy is growing worldwide , it is crucial to understand their emotional and psychological reactions toward surrogacy during the 9 months of the waiting period . this is the first qualitative study in iran , which assessed the experiences of commissioning mothers . with their diverse culture and social contexts , developing countries demand much more context - dependent studies this study has aimed to explore and explain the nature of concerns ( experiences ) of commissioning mothers . the aim of this study was to explore and explain the nature of concerns ( experiences ) of commissioning mothers . the aim of this study was to explore and explain the nature of concerns ( experiences ) of commissioning mothers . content analysis is a research method that has come into wide use in health studies in recent years . this type of design is usually appropriate when an existing theory or research literature on a phenomenon is limited . royan research centre consists of six departments ( endocrinology and female infertility , epidemiology and reproductive health , reproductive genetics , reproductive imaging , andrology , and embryology ) and one clinic actively working on different aspects of infertility and the development of new methods for infertility treatment . our participants were first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) . they had to be able to speak persian and willing to participate in the research . participants were selected from gestational surrogacy cases ( the conceptions were with own gametes ) , as gestational surrogacy is more prevalent in iran than partial surrogacy and we had a better access to them . women who were in a waiting list for finding a surrogate by the infertility centers or those who were waiting to know the result of embryo implantation and the women whose embryo was aborted after implantation were not included in this study . it means for selection , at first , we clarified mothers who had the first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) , and then , interviews were conducted . the first author ( mz ) conducted all the interviews from november 2010 to august 2011 . of the 10 commissioning mothers suggested by royan research centre , 7 agreed to participate in the study and the other 3 mothers refused due to a fear of disclosure . finally , this research was conducted with 24 interviews , and 16 people including 12 commissioning mothers , 2 surrogate mothers , and 2 of the infertility center staffs who directly and continuously dealt with the mothers ( social workers ) . the interviews were continued to obtain richer data and stopped when no new information was obtained . the analysis starts with reading all data repeatedly to achieve immersion and obtain a sense of the whole . then , data are read word by word to derive the codes by first highlighting the exact words from the text that appear to capture key thoughts or concepts . as this process continues , these labels often come directly from the text and then become the initial coding scheme . then , the codes are sorted into categories based on how different codes are related and linked . coding and analysis was done by a single researcher who was the first author ( mz ) , and the other researcher checked them again and confirmed them as it is necessary for credibility . the interviews were recorded and typed ( in persian ) as soon as possible in the exact words , along with all nonverbal gestures such as crying , laughter , or silence . then , they were entered in maxqda2007 software that makes it easier to organize and keep an overview over memos and contents . they were read and reviewed several times and , eventually , disintegrated into meaning units . after reviewing each meaning unit , next , the primary open codes were classified based on conceptual and meaning similarities and put in the main categories which were more general and more conceptual . the categories were finally reduced to the two main themes of cultural dilemma and uncertain waiting . in order to maximize credibility of the findings in this study , prolonged engagement with data ( 1 year ) , members check , externals check , and triangulation recourses ( commissioning mothers , surrogates , and infertility center staffs ) were used . in order to keep confidentiality , as some of the commissioning mothers did not have any opportunity to express their emotions and concerns , they found the interviews so relaxing since they could release their pressures and this matter increased the credibility of the study . the study process was carefully recorded step by step so that conformability , validity , transferability , and audit of the study were achieved and another researcher could follow this research . before data collection , an approval was obtained from the ethics committee of tarbiat modarres university and royan institute . by a phone call in the beginning , the research purpose , interviewing techniques , confidentiality of information , and the right to withdraw from the study were explained to the participants . a suitable time and place for an interview interviews were conducted individually in a quiet environment like home , the park near participants homes , or at royan research centre . royan research centre consists of six departments ( endocrinology and female infertility , epidemiology and reproductive health , reproductive genetics , reproductive imaging , andrology , and embryology ) and one clinic actively working on different aspects of infertility and the development of new methods for infertility treatment . our participants were first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) . they had to be able to speak persian and willing to participate in the research . participants were selected from gestational surrogacy cases ( the conceptions were with own gametes ) , as gestational surrogacy is more prevalent in iran than partial surrogacy and we had a better access to them . women who were in a waiting list for finding a surrogate by the infertility centers or those who were waiting to know the result of embryo implantation and the women whose embryo was aborted after implantation were not included in this study . it means for selection , at first , we clarified mothers who had the first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) , and then , interviews were conducted . the first author ( mz ) conducted all the interviews from november 2010 to august 2011 . of the 10 commissioning mothers suggested by royan research centre , 7 agreed to participate in the study and the other 3 mothers refused due to a fear of disclosure . from the infertility centers of other provinces , finally , this research was conducted with 24 interviews , and 16 people including 12 commissioning mothers , 2 surrogate mothers , and 2 of the infertility center staffs who directly and continuously dealt with the mothers ( social workers ) . the interviews were continued to obtain richer data and stopped when no new information was obtained . the analysis starts with reading all data repeatedly to achieve immersion and obtain a sense of the whole . then , data are read word by word to derive the codes by first highlighting the exact words from the text that appear to capture key thoughts or concepts . as this process continues , these labels often come directly from the text and then become the initial coding scheme . then , the codes are sorted into categories based on how different codes are related and linked . coding and analysis was done by a single researcher who was the first author ( mz ) , and the other researcher checked them again and confirmed them as it is necessary for credibility . the interviews were recorded and typed ( in persian ) as soon as possible in the exact words , along with all nonverbal gestures such as crying , laughter , or silence . then , they were entered in maxqda2007 software that makes it easier to organize and keep an overview over memos and contents . they were read and reviewed several times and , eventually , disintegrated into meaning units . after reviewing each meaning unit , next , the primary open codes were classified based on conceptual and meaning similarities and put in the main categories which were more general and more conceptual . the categories were finally reduced to the two main themes of cultural dilemma and uncertain waiting . in order to maximize credibility of the findings in this study , prolonged engagement with data ( 1 year ) , members check , externals check , and triangulation recourses ( commissioning mothers , surrogates , and infertility center staffs ) were used . in order to keep confidentiality , as some of the commissioning mothers did not have any opportunity to express their emotions and concerns , they found the interviews so relaxing since they could release their pressures and this matter increased the credibility of the study . the study process was carefully recorded step by step so that conformability , validity , transferability , and audit of the study were achieved and another researcher could follow this research . before data collection , an approval was obtained from the ethics committee of tarbiat modarres university and royan institute . by a phone call in the beginning , the research purpose , interviewing techniques , confidentiality of information , and the right to withdraw from the study were explained to the participants . a suitable time and place for an interview interviews were conducted individually in a quiet environment like home , the park near participants homes , or at royan research centre . the demographic characteristics of commissioning mothers and surrogate mothers are shown in tables 2 and 3 , respectively . demographic characteristics of mothers demographic characteristics of surrogates the experiences of the participants were classified in two main themes : cultural dilemma with three subthemes : social taboo , concerns about disclosure to others and child , concerns about altered maternal and child 's identityuncertain waiting with three subthemes : concerns about health of fetus and surrogate , concerns about unknown surrogate , and concerns about lack of preparation for maternal role [ table 4 ] . cultural dilemma with three subthemes : social taboo , concerns about disclosure to others and child , concerns about altered maternal and child 's identity uncertain waiting with three subthemes : concerns about health of fetus and surrogate , concerns about unknown surrogate , and concerns about lack of preparation for maternal role [ table 4 ] . social taboos , 2 . concerns about disclosure to others and child , and 3 . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : social taboos have rooted in the cultural barriers of surrogacy usage in the society . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : well , for the things people say . my in - laws for instance , my mother in - law , for example , was saying that one of our relatives had an implantation ! although she is an educated person , supposedly , they should know better , but still their reaction was so bad . these all were too much for me to take , too much to take . ( m7 ) 2.mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : i mean i was anxious all t he time about this idea what should i do if somebody finds out? ( m1 ) all mothers also were worried about disclosure to their child and its revelation by relatives or young children . they believed this can not be done suddenly without preparation and should be planned to be revealed by parents themselves . they were afraid of the child to be informed when he / she does not have proper understanding of the events yet and the ability to analyze the truth , so that disclosure by others may lead to huge psychological trauma and his / her blaming parents as liars . one of the mothers said : i feel that telling this subject to the child has to be done at a certain age . i mean , we should reveal the truth when she is mature enough to be able to understand what you are telling her . if she is told all of a sudden , she would think of me as a liar because i did nt tell her the truth , she would not believe me and i might struggle with her a lot . ( m2 ) 3.another concern in this category was altering the maternal and child 's identity . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . these mothers even suffer more after the child 's birth . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . what if someone does nt respect my child , treat him like a foster kid , what should i do ? ( m6 ) statements of social workers in the infertility center confirmed this kind of emotions in mothers : they are worried about the relationship between their relatives and their newborn babies . concerns about altering maternal identity means that they were worried about not being accepted as a real mother by others and even by the baby . mothers were worried that in case of a disclosure , they may not be accepted as the genetic mother . i do nt know whether they will accept me as a mother now if they know this is my baby . i have this kind of baby.(m6 ) they were also concerned about not being assumed as a real mother by the child , for example : i am afraid that in future my baby would tell me that we found him in the street and adopted him or got him from a foster home . 1.mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . well , one thing that disturbs me is that i sometimes tell myself maybe because the baby is injected until 9 months long , i have heard many times about premature babies birth in the 7 or 8 months , or miscarriage , or the baby born with deformities . even before birth in the 9th month ( m4 in waiting period ) based on most of the mothers thoughts , it was clear that they had always been worried to hear horrible news about their fetus . i thought all the time something would happen to them ( her three fetuses ) . what would happen now ? really , every night , i have this nightmare that the surrogate mother is sending me an sms . ( m9 ) mothers concerns were increased in critical conditions such as a threatened abortion or decreasing fluid of amniotic sac partially during delivery . during the last month after check - up , the dr . told me that i was dehydrated , my amniotic fluid was too little , and the mother was so horrified . ( sm2 ) since the physical and psychological relaxations of pregnant women directly affect the baby 's physical and psychological well - being , mothers were worried about the surrogates comfort : when i call her up , the first thing i ask her is how she is doing . and then , i ask about the baby . for example , i ask her , is everything ok in your home ? are you relaxed? ( m4 in waiting period ) in addition to the effect of surrogate 's health on fetus , mothers had also had an altruistic feeling to the surrogate and they were worried about the possibility of a physical damage or even death because of their child 's delivery . ( m2 ) 2.another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : i told my husband , do nt take me home tonight. he asked me how can i leave my baby in her womb and go home? i just could nt . ( m3 ) 3.the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : during these 9 months , you wish to have what you have always wanted in one thing , the baby . now , when you got what you wanted for so long , you found out you are not ready . my god when this child is born , i am not even able to touch him . how can i make a relationship with him? i think those 9 months of pregnancy makes you prepared for that . social taboos , 2 . concerns about disclosure to others and child , and 3 . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : social taboos have rooted in the cultural barriers of surrogacy usage in the society . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : well , for the things people say . my in - laws for instance , my mother in - law , for example , was saying that one of our relatives had an implantation ! although she is an educated person , supposedly , they should know better , but still their reaction was so bad . these all were too much for me to take , too much to take . ( m7 ) 2.mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : i mean i was anxious all t he time about this idea what should i do if somebody finds out? ( m1 ) all mothers also were worried about disclosure to their child and its revelation by relatives or young children . they believed this can not be done suddenly without preparation and should be planned to be revealed by parents themselves . they were afraid of the child to be informed when he / she does not have proper understanding of the events yet and the ability to analyze the truth , so that disclosure by others may lead to huge psychological trauma and his / her blaming parents as liars . one of the mothers said : i feel that telling this subject to the child has to be done at a certain age . i mean , we should reveal the truth when she is mature enough to be able to understand what you are telling her . if she is told all of a sudden , she would think of me as a liar because i did nt tell her the truth , she would not believe me and i might struggle with her a lot . ( m2 ) 3.another concern in this category was altering the maternal and child 's identity . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . these mothers even suffer more after the child 's birth . what if someone does nt respect my child , treat him like a foster kid , what should i do ? ( m6 ) statements of social workers in the infertility center confirmed this kind of emotions in mothers : they are worried about the relationship between their relatives and their newborn babies . concerns about altering maternal identity means that they were worried about not being accepted as a real mother by others and even by the baby . mothers were worried that in case of a disclosure , they may not be accepted as the genetic mother . i do nt know whether they will accept me as a mother now if they know this is my baby . i have this kind of baby.(m6 ) they were also concerned about not being assumed as a real mother by the child , for example : i am afraid that in future my baby would tell me that we found him in the street and adopted him or got him from a foster home . concerns about health of the fetus and surrogate , 2 . unknown surrogate , and 3 . 1.mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . well , one thing that disturbs me is that i sometimes tell myself maybe because the baby is injected until 9 months long , i have heard many times about premature babies birth in the 7 or 8 months , or miscarriage , or the baby born with deformities . even before birth in the 9th month ( m4 in waiting period ) based on most of the mothers thoughts , it was clear that they had always been worried to hear horrible news about their fetus . i thought all the time something would happen to them ( her three fetuses ) . really , every night , i have this nightmare that the surrogate mother is sending me an sms . ( m9 ) mothers concerns were increased in critical conditions such as a threatened abortion or decreasing fluid of amniotic sac partially during delivery . during the last month after check - up , the dr . told me that i was dehydrated , my amniotic fluid was too little , and the mother was so horrified . ( sm2 ) since the physical and psychological relaxations of pregnant women directly affect the baby 's physical and psychological well - being , mothers were worried about the surrogates comfort : when i call her up , the first thing i ask her is how she is doing . and then , i ask about the baby . for example , i ask her , is everything ok in your home ? are you relaxed? ( m4 in waiting period ) in addition to the effect of surrogate 's health on fetus , mothers had also had an altruistic feeling to the surrogate and they were worried about the possibility of a physical damage or even death because of their child 's delivery . ( m2 ) 2.another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : i told my husband , do nt take me home tonight. he asked me how can i leave my baby in her womb and go home? i just could nt . ( m3 ) 3.the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : during these 9 months , you wish to have what you have always wanted in one thing , the baby . now , when you got what you wanted for so long , you found out you are not ready . i kept telling myself , my god when this child is born , i am not even able to touch him . how can i make a relationship with him? i think those 9 months of pregnancy makes you prepared for that . mothers participating in this study had chosen surrogacy as their last hope , after years of infertility and sometimes after repeated unsuccessful treatments , and this turns them to sensitive people as the stress during the waiting period can intensify the remaining effects of infertility in these individuals . study findings have shown semantic elements of commissioning mothers concerns ( experiences ) in the two themes of cultural dilemma and uncertain waiting . these themes suggested that the experience of becoming a mother through surrogacy is highly stressful for mothers , and during the waiting period , they experience such a stressful situation in which according to one of the mothers , 9 months of waiting period passes like nine centuries . the study findings have shown that the traditional society of iran has not universally accepted surrogacy and cultural rejection is a great stressful element for commissioning families . in pashmi 's study in iran , most of the concerns were related to the social attitude toward surrogacy . abbasi - shvazy et al . showed that women who receive donor oocytes believe that the iranian community has not accepted art yet , as in the case of a disclosure , infertility stigma , which they have been attributed to , is transferred to their children as an illegitimate baby . mothers in the above - mentioned study also expressed their fear of disclosure of surrogacy to their children , and that their children might feel being a different human being in comparison with their friends . in hershberger . 's phenomenological study on received donor oocytes in california , women addressed social acceptance of this method as a social support . they expressed their concern as disclosing the nature of conception may have the potentiality to cause discordances in the relationships and identities within the family , maternal identity , and maternal insecurity . fear of social stigma and rejection by the society were the other concerns of these women . the predominant traditional cultural and social context and lack of cultural preparation were the major effective factors concerning mothers emotions , which , based on our findings , led to the concerns about the others reactions and altered maternal identity . it should also be considered that full surrogacy is a completely different condition from oocyte donation in which mothers do not experience pregnancy , which is important in collation of findings of different studies . also , in kirkman 's study , women who became mothers through egg donation mentioned concerns about their maternal identity . in contrast with the present study , the retrospective study of mccallum et al . in uk showed a low level of anxiety in couples throughout the pregnancy of a surrogate mother . in their study , the majority of the couples families reacted either positively or neutrally to the news , with only 7% reporting any negative reactions from the family . also , in contrast to our findings , studies in uk showed that families using surrogacy ( complete and partial ) acted openly in exposing the truth to the child . it seems that the main reason for this difference is different cultural contexts in iran and uk . iran is in a historical transition phase from tradition to modernism , which simultaneously causes special conflicts in utilization of new treatment modalities such as surrogacy by families . so , giving the public sufficient information about surrogacy and the other supplemental therapies for infertility by media such as tv and newspapers can improve the negative attitude toward surrogacy and assist most of the surrogate mothers and infertile couples . concerns about fetus 's and surrogate 's health , lack of preparation for motherhood role , and an unfamiliar surrogate were all aspects of uncertain waiting , so that the mothers imagined an uncertain future for them during this period . being anxious about the health of both the surrogate mother and van den akker reported fear of the treatment failure and a normal baby worries in 15% and 8% of the mothers , respectively . it is clear that even in natural pregnancies , mothers are concerned about the child 's health , the consequences of the waiting period , and lack of their preparation for a maternal role , but based on commissioning mothers , their unawareness about fetus was an important factor that intensified their concerns . reported a high level of anxiety about pregnancy security and fetal health among the participants , which is similar to our findings . uncertainty about the outcome had a great impact on the stress level of the women experiencing ivf , which is consistent with the study of ardent et al . all findings about these mothers must be interpreted cautiously , as this method is different from other techniques of mothering , with no waiting period . also , in another study comparing the women with a natural pregnancy with ivf , the results showed that both ivf mothers and ivf partners were more anxious about losing the pregnancy compared to couples who conceived naturally , possibly as they themselves had a difficulty in getting pregnant successfully . ivf partners had a high degree of anxiety about the expected baby concerning not being healthy or normal , but their anxiety decreased as the pregnancy progressed to similar levels of those who conceived naturally . in the present study , only one mother reported that her anxiety decreased as the pregnancy progressed in the surrogate mother . meanwhile , other participants felt more anxious in the later stages of the waiting period . this difference may be related to perceiving the movements of fetus through pregnancy progression of the mothers with other infertility treatments , which makes mothers more relaxed and confident about their fetus 's health , while this can not be felt by surrogacy commissioning mothers , so they become more concerned about losing the baby and missing their dreams of becoming a mother while waiting . van den akker also found that commissioning mothers anxiety ( in full or partial surrogacy ) could be significantly intensified in the final stages of surrogate pregnancy . lack of knowledge about the surrogate , relying on the surrogate mother , and fear of not delivering the baby after birth were the other stated concerns of those mothers , which were also partially found in van den akker 's study . in her study , 23% of commissioning mothers were worried that relinquishing the baby may become emotionally difficult for the surrogate mother . other types of worries included emotional difficulties for commissioning ( 3% ) or surrogate mother ( 19% ) . keen ( as cited in teman ) stated that 99% of the surrogate mothers willingly relinquished the child as they had contractually agreed to do . less than 0.1% of surrogacy cases end up in court battles . based on mccallum et al . 's report , only one of the surrogate mothers had doubts about relinquishing the baby and the others did not have any problem to do that . in the present study and in pashmi 's study in iran , all of the surrogates gave the baby to the commissioning mother immediately after delivery and there was not any problem reported . van den akker believes that the surrogate agency information appears to be quite successful in assisting the surrogates to achieve a cognitively consonant state . in conclusion , the study results found that mothers experienced a continuous stressful waiting period and psychological and social insecurity . this study suggests being more supportive for commissioning mothers through psychological consultations , application of relaxation techniques , and family education classes . in these classes , even though this study tried to collect in - depth information by utilizing various resources on one hand and the participating mothers were studied in both waiting period and after child birth on the other hand , there were some limitations . due to unavailability of partial surrogate samples , this research has been conducted only on gestational / full surrogacy , while partial surrogacy may yield different results . in addition , cultural and , particularly , religious differences in women can influence their experiences and perceptions . the result of this study can be used as a practical guide for nurses and other team members of art in iran and other muslim countries where art is not forbidden . in addition , in the countries with similar culture , where these technologies are not permitted , it can be used in decision making about this matter . provision of the best health care , as the purpose of nursing , requires careful assessment and evaluation of people 's experiences about diseases and treatment methods . in order to provide sophisticated care , art nurses should first know about clients experiences , and then , plan nursing care accordingly . the study results showed that psychological support is necessary before a successful intervention during the waiting period and after child birth . it is recommended that since surrogacy is considered as a high - risk psychological experience , these mothers should be cared for and receive special consultations before making such a decision . these findings help the mothers through nurses interventions in health care clinics or wherever they provide nursing care . nurses who work in art field should identify their roles and abilities to positively affect mothers experiences . increasing the use of art necessitates nurses planning and preparation to improve the health of the families who use this technology and are the fundamental institutions in the society .

background : there are a few studies about commissioning mothers understanding from the surrogacy during 9 months of waiting for delivery in iran and other countries . this study was conducted with an aim to explore and explain the nature of concerns ( experiences ) of commissioning mothers.materials and methods : a qualitative design with a conventional content analysis approach was used to gather and analyze the experiences of commissioning mothers . they were selected from royan research centre and other infertility centers in iran . after purposive sampling for the selection of the participants , unstructured interviews were held for data collection . twenty - four unstructured interviews were conducted with 12 commissioning mothers , 2 surrogate mothers , and 2 infertility center social workers who directly and continuously dealt with these mothers.results:two main themes emerged from the data analysis : 1 . cultural dilemma ( consisting of three subthemes : social taboo , concerns about disclosure to others and the child , concerns about altering maternal and child 's identity , and 2 . uncertain waiting ( consisting of three subthemes : concerns about health of fetus and surrogate , concerns about an unfamiliar surrogate , and concerns about lack of preparation for maternal role).conclusions : the study reveals the importance of maternal emotional care in this group and introduces a new arena for nurses activity . these findings help the mothers by nurses activities in health care clinics and anywhere they deliver nursing care .

I Aim of the study M Setting Participants Sampling strategy Data collection Data management and data analysis Consideration of rigor Ethical considerations R Cultural dilemma Uncertain waiting D C

characterization of the bioactive constituents from the black pigmented ink has resulted in a handful of chemical elucidations . the ink from the molluscs has created a great interest towards its bioactive molecules with promising antibacterial , antitumour , antileukemic , and antiviral activities . the ink is ejected from the ink gland of the squid loligo duvauceli through the ink duct to escape from its predators . the black pigment was found to be melanin and the process of melanogenesis was explained in the ink gland of sepia sp . . the ink is a complex mixture of organelles , premelanosomes , melanosomes , granules , proteic material ( enzymes ) , glucosamine , and phospholipids in suspension . at the moment of extraction the ink gland has been also shown to contain a variety of melanogenic enzymes as tyrosinase , dopachrome tautomerase , and peroxidase . the ink has also various primary roles in the world of alternative medicine and has the widest range of therapeutic application . despite these reports there is no considerable interest shown towards the purification procedures of the l. duvauceli 's ink . potential chemical cues in squid ink have been identified and quantified using reverse - phase , high - performance , liquid chromatography ( rp - hplc ) . so this study is aimed at exploring the active bioconstituents of the ink by silica gel column chromatography and the gas chromatography - mass spectroscopic analysis ( gc - ms ) for its antimicrobial constituents . the collection of ink and the crude solvent extraction of the constituents from l. duvauceli ink was done by the method followed earlier . the crude extracts were subjected to sterility checking after exposing the extracts under uv light for 2 hrs . 5 mg of each extract was mixed in sterile nutrient broth and was incubated for 2 hrs which was plated onto nutrient agar for checking the sterility of the extracts . the antimicrobial activity of the crude extracts was performed by conventional agar well diffusion method . the n - hexane extract has scored in our earlier reports a high antimicrobial property against the clinical bacterial and fungal isolates . thus the n - hexane extract was chosen for the further fractionation by silica gel column chromatography . separation of the active biomolecules from the crude n - hexane extract was done by silica gel column chromatography . briefly , 10 gm of the crude n - hexane extract was subjected for fractionation using silica gel column . the crude extract was adsorbed on to silica gel ( 100200 mesh , sisco ) and chromatographed employing a step gradient solvent system from low to high polarity . the starting solvent system was 100% n - hexane and subsequently the polarity was increased by varying the solvent concentration with ethyl acetate ( etoac ) . the solvent gradient for the chromatogram is 100% hexane , 20 : 80 etoac / hex , 40 : 60 etoac / hex , 60 : 40 etoac / hex , 80 : 20 etoac / hex , 100% etoac , 100% diethyl ether , 20 : 80 ethanol / etoac , 40 : 60 ethanol / etoac , 60 : 40 ethanol / etoac , 80 : 20 ethanol / etoac , 100% ethanol , 20 : 80 water / etoac , 50 : 50 water / etoac , and 100% water . in order to select the best mobile phase for eluting the fractions , 5 l of each eluted fraction the fractions that showed the elution of similar compounds were pooled and concentrated under vacuum below 40c using heidolph , ve-11 rotaevaporator for 30 min . high performance thin layer chromatography ( hptlc ) analysis combined fractions were kept under air current to facilitate drying . the concentrated fraction was obtained and subjected to sterility checking as mentioned earlier . the active fraction was stored at 4c in sterile glass brown bottles until used for the bioactivity studies . the antimicrobial activity of isolated fractions was checked against escherichia coli ( atcc 25922 ) , klebsiella pneumoniae ( atcc 10031 ) , staphylococcus aureus ( atcc 25923 ) , candida albicans ( atcc 10231 ) , and lactobacillus acidophilus [ mtcc 447 ] . briefly , mueller hinton agar plate was divided into two halves and 50 l of inoculum of each test organism was spread as lawn cultures on the same plate to achieve a confluent growth at each half . the agar plates were allowed to dry and wells or cups of 8 mm were made with a sterile agar borer on the inoculated agar plates . 10 mgs of the pooled fraction was mixed with dmso and was made ready for the study . a 50 l volume of the active fraction was propelled directly into the wells of the inoculated specific media agar plates for each test and control organism . erythromycin ( 30 g ) and amphotericin b ( 100 u ) were used as the positive controls for the bacteria and the yeast , respectively . the plates were allowed to stand for 10 minutes for diffusion of the extract to take place and were incubated at 37c for 24 h. after incubation the plates were observed for the zone of inhibition around the wells and the zone of inhibition was measured using an antibiotic sensitivity measuring scale ( himedia , mumbai ) . determination of mic value for the active antimicrobial fraction was determined by microbroth dilution method . serial dilutions of the active fraction were done in a 96-well microtitre plate with dmso . the dilution factor was 5 , 2.5 , 1.25 , 0.625 , 0.312 , and 0.156 mg / ml . to each dilution 100 l of the culture broths of the test organisms was added in their respective wells and the plate was incubated at 37c for 24 hrs . after incubation the spectrophotometric analysis was performed and the od values were recorded . the mbc value was confirmed by microbial spot checker board method where 3 l of each dilution was spotted onto mueller hinton agar plates and incubated at 37c for 24 hrs . after incubation the spot showing the complete absence of microbial growth indicates the minimum bactericidal value and the spot showing the visible decrease in the number of colonies indicates the minimum inhibitory concentration ( mic value ) . the active fraction was then subjected to gc - ms analysis with the split injection ratio of 1 : 20 in hexane in shimadzu gc - ms qp 2010 ( japan ) gas chromatograph with capillary column length of 30 m , 0.25 mm diameter , and 0.25 m of film thickness . gc - ms operating conditions were as follows : injector temperature at 280 , column oven temperature at 45 , flow control mode at linear velocity , and column flow of helium ( 99.9% purity ) at 1.40 ml / min . oven temperature programme was maintained at 45c for 2 min and 300c for 10 min with overall holding time of 36.5 min . mass spectra conditions applied were as follows : electron impact at 40 ev , ion source temperature at 200c , and interface temperature at 240c . the n - hexane extract scoring a high antimicrobial activity upon column fractionation over silica gel yielded a total of 8 fractions . elution with hex / etoac in the ratio of 4 : 1 yielded the active fraction . the active fraction was subjected to tlc , hptlc , and gc - ms analysis . tlc profile showed a single spot with a retention factor of 0.76 ( figure 1 ) . the same plate was subjected to hptlc analysis , with a scanning wavelength of 254 nm . the chromatogram showed a single peak obtained as calibration spectrum data with a noise level at 0.072 mv , camag software , and scanned with scanner ii with area normalization of 83.91% that indicated the maximum extraction with hex / etoac . antimicrobial bioassay revealed that the active fraction possesses a high antibacterial activity against the test organisms ( table 1 ) . the zone size was measured as 18 mm for e. coli and k. pneumoniae , 16 mm for s. aureus , 23 mm for c. albicans , and 18 mm for l. acidophilus ( figure 2 ) . the mbc value was determined as an average of 2.5 mg / ml for e. coli , k. pneumoniae , and c. albicans , 5 mg / ml for s. aureus and l. acidophilus . the microbial spot checker ( figure 3 ) board method yielded complete absence of the growth at the spot inoculated with the determined mbc value . the previous dilution that showed the visible decrease in the number of colonies was determined as the mic and was deduced as 1.25 mg / ml for e. coli , k. pneumoniae , and c. albicans and 2.5 mg / ml for s. aureus and l. acidophilus . the bioactive fraction upon gc - ms analysis revealed a chromatogram showing nine peaks with bis(2-ethylhexyl ) phthalate [ behp ] possessing the highest percentage of area normalisation ( 91% ) ( figure 4 ) . the mass spectrum was found to be superimposable ( > 93 ) with that of the authentic compound from the gc - ms library . based on the gc - ms analysis the active fraction was structurally elucidated as bis(2-ethylhexyl ) phthalate . the chromatogram also showed the presence of other minor compounds such as octadecane ( 0.29% ) , naphthalene ( 0.13% ) , tetradecane ( 0.41% ) , pentadecane ( 0.58% ) , hexadecane ( 1.02% ) , heptadecane ( 0.53% ) , and cholesterol ( 5.07% ) ( table 2 ) . the analysis report reveals the presence of phthalate derivative and other minor volatile essential oils as potent antimicrobial agents extracted from the squid ink . marine natural products have been a strong source for novel drug products , or have been a model for introducing a commercial drug . squid ink is not the most elusive and enigmatic pigment found in nature but just a particle waiting for a rational study . in recent years , the problem of antimicrobial ( drug ) resistance is emerging and many diseases are increasingly difficult to treat because of the emerging drug - resistant organisms . the design of effective and novel dosing regimens that suppress the emergence and proliferation of resistant microbial populations is crucial . as resistance has increased to alarming proportion , a safe and cheaper source can always be an alternative to the routine therapeutics . as mollusk has been reported to possess various active molecules , the ink from the squid l. duvauceli previous associated studies state that the crude extraction of the squid ink is achieved using various solvents . successful prediction of natural bioactive molecules from natural sources is largely dependent on the type of solvent used in the extraction procedure and in many studies it was found that extracts in organic solvents provided more consistent antimicrobial activity . the crude extraction in our earlier studies is thus achieved by parallel solvent extraction method , where the ink is mixed with various solvents individually and is not added sequentially . tlc and hptlc analysis are the simplest and cheapest method of detecting any natural constituent because the method is easy to run and reproducible and requires little equipment . active crude extracts are chosen for column chromatography due to its relatively low complexity as seen with tlc , bioautography , and disc diffusion method . the solvent gradient used for elution of the biomolecules has been successfully standardized and is best achieved with n - hexane and ethyl acetate gradient . the selection of the solvent gradients can be rationalized in terms of the polarity of the compounds being extracted by each solvent and in addition to their intrinsic bioactivity , by their ability to dissolve or diffuse in the different media used in the assay . for the antimicrobial bioassay , the concentrated fractions were dissolved with dmso and were employed for the agar well diffusion bioassay . the choice of dmso as a solvent is due to its solvency for a wide range of chemicals , its low antibacterial activity at concentrations less than 2% , and its low toxicity . the findings of the antimicrobial bioassay report that the active fraction has antimicrobial efficacy against the gram positive cocci s. aureus , gram negative bacilli e. coli and k. pneumoniae , gram positive bacilli l. acidophilus , and the pathogenic yeast c. albicans . the active fraction scores a high activity against c. albicans that indicates its potent antifungal activity . the mbc value has also been deduced and is determined as 2.5 to 5 mg / ml against the tested organisms . microbial spot checker board also yields the same result with the absence of growth at the spot inoculated with the determined mbc value . gc - ms analysis of the bioactive fraction shows the presence of the bioactive compounds which are further confirmed with the library data . using mass spectroscopy the molecular mass of a compound and its elemental composition further this method involves very little amount of the test sample and gives the molecular weights accurately . gc - ms analysis has showed bis(2-ethylhexyl ) phthalate as the major constituent with large area normalization of 91.43% . it correlates with the mass spectrum of behp reported earlier from a phthalate isolated from a marine bacterial strain . the other minor constituents with low area normalization were also identified by the molecular mass from the library database . bis(2-ethylhexyl ) phthalate is found in low levels in the environment as it is subjected to biodegradation . this derivative has also been reported to be present in the fish and lipid tissues and also as a pollutant in marine environment . the bioactivity of the phthalate derivatives has already been reported in many plants , algae , and marine microorganisms and also from many marine species . few reports are available for the antibacterial potential of phthalate derivatives from plants and from flowers . bis(2-ethylhexyl ) phthalate extracted from streptomyces bangladheshiensis has been reported to be a potent antibacterial agent against gram positive bacteria . the other volatile minor compounds identified by gc - ms analysis have also been found to be potent antibacterial agents . the antibacterial activities of pentadecane and heptadecane compounds extracted from sea urchin have also been reported to possess potent activity against gram positive and gram negative bacteria . behp has been reported to possess a potent antifungal activity against major pathogenic fungi like candida , cryptococcus , and aspergillus sp . . the other minor constituents analyzed by gc - ms have also been reported to possess antifungal activity . naphthalene derivative has been reported to possess the same against c. albicans and aspergillus sp . . the antifungal activity of cholesterol hydrazone derivative has also been studied against c. albicans at a concentration of 1.5 g / ml . the antifungal activity of tetradecane and octadecane has been reported against c. albicans . gc - ms analysis of a natural cure concoction epa - ijebu showed the presence of natural alkanes such as hexadecane , heptadecane , and octadecane with potent antifungal activity . in correlation with these reports , the study results reveal that the ink has potent antimicrobial constituents which owes for its antibacterial and antifungal properties . the promising antimicrobial activity of these bioactive constituents needs a further multipronged research to implement its use as a novel therapeutic agent in near future for treating ailments with the drug resistant microbial pathogens . this study has suggested the presence of antimicrobial bioconstituents in the squid ink by column fractionation studies . gc - ms analysis has also aided the evaluation of the major and minor compounds present in it through its mass spectrum data . the study thus concludes the synergistic effects of an array of compounds in the squid ink towards its potent antimicrobial property . a novel therapeutic compound from a new marine source like the squid ink would be of much use in eradicating the microbial pathogens and it would definitely aid in the control and emergence of drug resistant strains .

chromatographic characterization and the gc - ms evaluation of the black pigmented ink of loligo duvauceli in the present study have yielded an array of bioactive compounds with potent antimicrobial property . facing an alarm of antimicrobial resistance globally , a need for elucidating antimicrobial agents from natural sources will be the need for the hour . in this view , this study is aimed at characterizing the black pigmented ink of the indian squid l. duvauceli . the squid ink was subjected to crude solvent extraction and was fractionated by silica gel column chromatography . tlc and hptlc profiles were recorded . antimicrobial bioassay of the squid ink fractions was done by agar well diffusion method . the antimicrobial fraction was then characterized using gc - ms analysis . the results showed that the n - hexane extract upon column fractionation yielded a total of 8 fractions with the mobile phase of hex / etoac in different gradients . tlc and hptlc profiles showed a single spot with a retention factor of 0.76 . fraction 1 showed significant antibacterial activity against escherichia coli , klebsiella pneumoniae , staphylococcus aureus , and lactobacillus acidophilus and a promising antifungal activity against candida albicans . the antimicrobial fraction upon gc - ms analysis of bis(2-ethylhexyl ) phthalate ( behp ) possesses the highest percentage of area normalisation ( 91% ) with other few minor constituents . the study is concluded by stating that the antimicrobial efficacy of the squid ink might be due to the synergistic effects of the phthalate derivative and the other minor volatile compounds analysed in the squid ink .

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

in recent years , drug - eluting stents ( des ) have been demonstrated to dramatically reduce the rate of restenosis and the need for repeat revascularization.1 - 3 ) despite these promising results , stent thrombosis seems to occur more frequently with des and often seems to be associated with clopidogrel resistance.4 ) we report a case of recurrent stent thrombosis associated with clopidogrel resistance in a patient with acute myocardial infarction . a 63-year - old female was transferred to the emergency department complaining of squeezing chest pain that had increased over the past twelve hours . she underwent emergent coronary angiography ( cag ) , which revealed critical stenosis in the proximal and middle left anterior descending coronary artery ( lad ) . she was successfully treated with two paclitaxeleluting stents ( 3.012 mm and 2.528 mm taxus stents , boston scientific , reading , pa , usa ) in the proximal and middle lad ( fig . she was treated with triple antiplatelet therapy ( aspirin 100 mg , clopidogrel 75 mg , and cilostazol 200 mg daily ) because she was found to be hyporesponsive to clopidogrel when tested for adenosine diphosphase ( adp)-induced platelet aggregation utilizing the verifynow p2y12 point - of - care assay ( 181/0 p2y12 reaction unit/% ) . three days after discharge , she again developed chest pain and presented to the emergency department with mental confusion associated with acute pulmonary edema . kinase - mb 34.0 u / l , troponin - i 67.01 ng / ml , troponin - t 5.5 ng / ml ) . emergency cag , after intubation due to hypoxemia , showed total occlusion of the mid - lad due to stent thrombosis ( fig . 4 ) . after intravenous administration of a glycoprotein iib / iiia receptor blocker ( reopro ) , balloon angioplasty was carried out multiple times using a 3.0 mm balloon at 10 - 12 atm because of recurrent , immediate thrombus formation and coronary occlusion . the next day , however , she complained of severe chest pain again , and the ecg showed marked st - segment elevation in v1-v6 , i , and avl ; and new - onset complete right bundle branch block with left anterior fascicular block ( fig . she was successfully treated with balloon angioplasty and a final angiogram revealed improved flow over stented lad without intraluminal filling defect . despite the cilostazol medication , adp - induced platelet aggregation showed that she was still hyporesponsive to clopidogrel ( 171/0 p2y12 reaction unit/% ) . she was discharged after uneventful recovery with triple anti - platelet therapy using an increased dose of aspirin ( aspirin 200 mg , clopidogrel 75 mg , cilostazol 200 mg daily ) . we report this case to draw more attention to stent thrombosis associated with clopidogrel resistance after des implantation . this case shows that a des patient with clopidogrel resistance can be vulnerable to stent thrombosis even if treated with triple anti - platelet therapy , which in recent studies has been shown to be more effective in preventing stent thrombosis than conventional dual anti - platelet therapy . stent thrombosis is an uncommon but serious complication of coronary artery stents that often presents as myocardial infarction ( mi ) or death . over several trials , the incidence of stent thrombosis was 0.58 - 1.3% in des.5)6 ) several factors have been associated with stent thrombosis , including older age , black race , diabetes mellitus , bifurcation lesion , in - stent restenosis lesion , procedure - related factors such as stent malposition , greater stent length , postprocedure acute renal failure , non - compliance to anti - platelet agent and anti - platelet resistance.7 - 9 ) anti - platelet resistance as an independent predictor of stent thrombosis , even several years after implantation of des , increases the risk of stent thrombosis . in this patient , stent thrombosis may have been caused by several risk factors , especially anti - platelet resistance . several therapeutic approaches ( the addition of cilostazol or a glycoprotein iib / iiia inhibitor , increased dosage of clopidogrel and aspirin ) might be taken for a patient with anti - platelet resistance . in our patient , although cilostazol ( 200 mg daily ) was added to conventional dual anti - platelet therapy , recurrent stent thrombosis occurred . subsequently , the daily dose of aspirin was increased from 100 mg to 200 mg . in the des era , stent thrombosis is a fatal complication and anti - platelet therapy has been shown to be very important in preventing stent thrombosis . thus , assessment of the patient 's responsiveness to anti - platelet agents may be a crucial factor in monitoring these drugs ' therapeutic efficacy and improving clinical outcomes after implantation of des . recent studies have shown that adequate anti - platelet effects are not achieved in 5% to 45% of the patients taking aspirin and in 4% to 30% of patients taking clopidogrel10)11 ) and therefore suggest that many patients are resistant or only partially responsive to the anti - platelet agents . currently , however , routine screening for anti - platelet resistance remains a persistent , unresolved issue and further evidence is necessary before it will be possible to recommend this testing as part of standard assessment of pci candidates . in addition , further prospective studies are needed to set guidelines for optimal treatment of patients with antiplatelet resistance who are at increased risk of stent thrombosis , a catastrophic complication of des implantation .

drug - eluting stents ( des ) are considered the treatment of choice for most patients with obstructive coronary artery disease when percutaneous intervention ( pci ) is feasible . however , stent thrombosis seems to occur more frequently with des and occasionally is associated with resistance to anti - platelet drugs . we have experienced a case of recurrent stent thrombosis in a patient with clopidogrel resistance . a 63-year - old female patient suffered from acute myocardial infarction and underwent successful pci of the left anterior descending coronary artery ( lad ) with two dess . she was found to be hyporesponsive to clopidogrel and was treated with triple anti - platelet therapy ( aspirin 100 mg , clopidogrel 75 mg , and cilostazol 200 mg daily ) . three days after discharge , she developed chest pain and was again taken to the cardiac catheterization laboratory , where coronary angiography ( cag ) showed total occlusion of the mid - lad where the stent had been placed . after intravenous administration of a glycoprotein iib / iiia inhibitor , balloon angioplasty was performed , resulting in thrombolysis in myocardial infarction ( timi ) iii antegrade flow . the next day , however , she complained of severe chest pain , and the electrocardiogram showed marked st - segment elevation in v1-v6 , i , and avl with complete right bundle branch block . emergent cag revealed total occlusion of the proximal lad due to stent thrombosis . she was successfully treated with balloon angioplasty and was discharged with triple anti - platelet therapy .

Introduction Discussion

abnormal knee kinematics are considered to be an important factor influencing the long - term outcomes after cruciate ligament injuries1,2,3 . in order to improve long - term outcomes of treatments for the patients with cruciate ligament injuries , therefore , it is important to precisely evaluate the kinematics of knees with cruciate ligament injuries . the motion of a knee joint with ligament injury is commonly evaluated by static methods such as the lachman test , posterior drawer test , valgus stress test , and arthrometric measurements , e.g. kt-1000 . however , patients with knee ligament deficiencies usually complain about their symptoms under dynamic conditions . therefore , there is a possibility that the instability of the knee joint evaluated in static conditions does not reflect the functional disabilities of its ligament injury1 , 3,4,5 . three - dimensional motion analysis using optical skin marker systems has been widely used as an evaluation method of body kinematics in dynamic conditions with muscular activations . this approach provides six - degrees - of - freedom motion of the kinematics of the knee during activities of daily living without restricted conditions in vivo . andriacchi et al . developed a point cluster technique ( pct ) , which employs an overabundance of markers ( a cluster ) placed on each segment to minimize the effects of skin movement artifacts6 . the pct can be extended to minimize skin movement artifact by optimal weighting of the markers according to their degree of deformation . the criteria of the normal range of three - dimensional knee kinematics during walking remain uncertain . side - to - side difference in the range of knee motion is widely used as a criterion of the normal range of knee motion1 . to detect an abnormal knee motion during walking , it is useful to determine the normal range of the side - to - side difference in knee kinematics during walking based on the data using the pct . to the best of our knowledge , however , no study has examined this ; therefore , the purpose of the present study was to determine the normal range of the side - to - side difference in knee kinematics using pct during walking based on three dimensional kinematics of the knee of healthy subjects . the study was approved by the research ethics committee of hakodate national college of technology and informed consent was obtained from each subject before the test . twenty - one healthy normal volunteers without knee pain or an episode of injury to the legs were recruited for the study . the subjects were 11 males and 10 females with an average age of 27.0 ( sd 7.2 ) years , and ranging from 21 to 47 years , and an average body mass index of 20.6 ( sd 2.4 ) kg / m , ranging from 16.8 to 27.7 kg / m . the subjects were tested bilaterally at a self - selected normal walking speed during their walking for 8 m after the first four strides . the subjects walked barefoot on a hard surface at an average walking speed of 1.25 m / s . the subjects took a rest before each trial until they had recovered from fatigue . the kinematic data were collected by a three - dimensional motion analysis system using four infrared light cameras ( rrorflex , qualisys ab inc . , ground reaction forces during the tests were also measured using two multi - component force plates ( or6 , advanced mechanical technology inc . , three - dimensional motion data were processed using qualisys track manager ( qualisys track manager 3d , qualisys ab inc . , force data were used to identify the time of heel strike in each gait cycle . a gait cycle was defined as the interval between heel strike to heel strike on the ipsilateral side . light - reflective markers were placed by physical therapists well - trained in the pct and body surface anatomy6 . kinematic measurements were made of 21 light - reflective markers arranged on two limb segments , creating separate cluster groups of 11 markers on the thigh and 10 markers on the shank . the pct employs an overabundance of markers ( a cluster ) placed on each segment to minimize the skin motion artifact due to segmental form change by muscle contraction and marker - oscillation6 . the pct is based on a cluster of points uniformly distributed on the limb segment . each point is assigned an arbitrary mass , which can be varied at each time step . the center of mass and the inertia tensor of the cluster of points are calculated as described below . the eigenvalues and the eigenvectors of the inertia tensor are the principal moments of inertia and the principle axes of the point cluster . the eigenvalues are invariant to movement if the segment is behaving as a rigid body . if the eigenvalues change from their value in the rest position , then the segment where the markers are placed has deviated from rigid body movement . the individual weighting is adjusted by redistributing the weight factors at each time step to minimize the eigenvalue changes . examined the pct using simulation trials with systematic and random components of deformation error introduced into marker position vectors7 . they also tested the pct in vivo with a subject fitted with an external fixation device ( ilizarov ) . the average location and orientation measurement errors of the pct were 0.6 mm ( sd 0.6 mm , maximum approximately 3 mm ) and 0.5 degrees ( sd 0.5 degrees , maximum approximately 4 degrees ) respectively7 . another study has also reported an accuracy of 0.79 degrees ( sd 0.48 degrees ) for flexion - extension , 1.79 degrees ( sd 0.93 degrees ) for internal - external rotation , and 0.40 degrees ( sd 0.12 degrees ) for abduction - adduction8 . intra - class correlations coefficients ( 1 , 5 ) and standard errors of measurement of the pct have been calculated using the data of a previous study and indicate substantial or almost perfect reliability9 . in the present study , the position coordinate data were converted to flexion - extension ( fe ) , abduction - adduction ( aa ) , internal - external ( ie ) rotation , and the anterior - posterior ( ap ) , medial - lateral ( ml ) , superior - inferior ( si ) translation of the tibia with respect to a gait cycle6 . data from three trials were averaged and the average was used for the analysis of each limb . the value of the right leg subtracted from that of the left leg was defined as the side - to - side difference in rotation / translation in each stage of the gait cycle . we then determined the average side - to - side differences and their 95% confidence intervals ( cis ) . for statistical analysis , one - way analysis of variance and post - hoc bonferroni tests were performed to compare the average side - to - side difference of each motion among 0% , 20% , 40% , 60% and 80% of the time of a gait cycle . differences were considered statistically significant at p < 0.05 with bonferroni adjustment for post - hoc multiple comparisons . based on the ground reaction forces of the force plates , the stance and swing phases during walking were determined . as a result , 0% to mean 61.6% ( sd 1.3% ) of the gait cycle was regarded as the stance phase . therefore , 0% , 20% , 40% , 60% and 80% of the gait cycle were considered as the early stance phase , the middle stance phase , the late stance phase , the early swing phase and the late middle swing phase , respectively . the average knee kinematics pattern of the right and left legs during a gait cycle are shown in fig . 1.the average knee kinematics pattern of the right and left legs during a gait cyclea ) flexion - extension ; b ) adduction - abduction ; c ) internal - external rotation of the tibia . regarding fe of the knee during walking , the average values ( 95% cis ) of side - to - side differences at each time frame of normal walking were 4.3 degrees ( 3.0 to 5.7 degrees ) , 4.9 degrees ( 3.5 to 6.3 degrees ) , 4.4 degrees ( 2.9 to 5.9 degrees ) , 7.4 degrees ( 5.2 to 9.6 degrees ) and 5.4 degrees ( 3.4 to 7.3 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . 2.the 95% confidence intervals ( cis ) of side - to - side differences of the normal subjectsa ) flexion - extension ( fe ) ; b ) abduction - adduction ( aa ) ; c ) internal - external ( ie ) rotation ; d ) anterior - posterior ( ap ) translation ; e ) medial - lateral ( ml ) translation ; f ) superior - inferior ( si ) translation , table 1table 1.the average of side - to - side differences and their 95% confidence intervals in each stage of the gait cyclewalking cycle0%20%40%60%80%rotation ( degrees)flexion - extension4.3 ( 3.05.7)4.9 ( 3.56.3)4.4 ( 2.95.9)7.4 ( 5.29.6)5.4 ( 3.47.3)abduction - adduction3.8 ( 2.65.1)4.5 ( 2.76.4)3.2 ( 2.14.3)5.4 ( 3.67.2)8.9 ( 5.911.9)internal - external7.4 ( 5.59.4)5.7 ( 3.77.8)4.7 ( 3.36.1)6.2 ( 4.08.4)7.4 ( 4.710.2)translation ( mm)anterior - posterior9.7 ( 6.712.7)7.7 ( 4.510.9)6.8 ( 4.19.6)9.2 ( 5.013.4)12.8 ( 6.818.8)medial - lateral6.3 ( 3.79.0)5.3 ( 3.47.1)5.5 ( 3.77.3)6.7 ( 4.98.5)5.9 ( 3.78.1)superior - inferior7.7 ( 4.510.8)8.1 ( 4.211.9)8.2 ( 4.112.4)8.9 ( 4.413.5)9.8 ( 9.818.8)(mean , 95% ci ) . a : post - hoc bonferroni tests ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . b : post - hoc bonferroni test ( vs. 40% p<0.001 ) ) . there were no significant differences in the side - to - side differences of fe among 0% , 20% , 40% , 60% , and 80% of the gait cycle ( p=0.086 ) . the average side - to - side differences ( 95%cis ) of aa were 3.8 degrees ( 2.6 to 5.1 degrees ) , 4.5 degrees ( 2.7 to 6.4 degrees ) , 3.2 degrees ( 2.1 to 4.3 degrees ) , 5.4 degrees ( 3.6 to 7.2 degrees ) and 8.9 degrees ( 5.9 to 11.9 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . the side - to - side difference of aa at 80% of the walking cycle was significantly greater than those at 0% , 20% , 40% and 60% of the gait cycle ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . the average side - to - side differences ( 95%cis ) of ie rotation were 7.4 degrees ( 5.5 to 9.4 degrees ) , 5.7 degrees ( 3.7 to 7.8 degrees ) , 4.7 degrees ( 3.3 to 6.1 degrees ) , 6.2 degrees ( 4.0 to 8.4 degrees ) and 7.4 degrees ( 4.7 to 10.2 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . there were no significant differences in the side - to - side differences of ie rotation among 0% , 20% , 40% , 60% , and 80% of the gait cycle ( p=0.332 ) . at 0% , 20% , 40% , 60% , and 80% of the gait cycle , the average values ( 95% cis ) of side - to - side differences of ap translation were 9.7 mm ( 6.7 to 12.7 mm ) , 7.7 mm ( 4.5 to 10.9 mm ) , 6.8 mm ( 4.1 to 9.6 mm ) , 9.2 mm ( 5.0 to 13.4 mm ) and 12.8 mm ( 6.8 to 18.8 mm ) , respectively ( fig . the side - to - side difference of ap translation at 80% of the gait cycle was significantly greater than that at 40% of the gait cycle ( p=0.001 ) . the average side - to - side differences ( 95%cis ) of ml translation were 6.3 mm ( 3.7 to 9.0 mm ) , 5.3 mm ( 3.4 to 7.1 mm ) , 5.5 mm ( 3.7 to 7.3 mm ) , 6.7 mm ( 4.9 to 8.5 mm ) and 5.9 mm ( 3.7 to 8.1 mm ) , respectively ( fig . 2-e , table 1 ) . there were no significant differences in the side - to - side differences of ml translation among 0% , 20% , 40% , 60% , and 80% of the walking cycle ( p=0.875 ) . similarly the average side - to - side differences ( 95%cis ) of si translation were 7.7 mm ( 4.5 to 10.8 mm ) , 8.1 mm ( 4.2 to 11.9 mm ) , 8.2 mm ( 4.1 to 12.4 mm ) , 8.9 mm ( 4.4 to 13.5 mm ) and 9.8 mm ( 9.8 to 18.8 mm ) , respectively ( fig . there were no significant differences in the side - to - side differences of sl translation among 0% , 20% , 40% , 60% , and 80% of the walking cycle ( p=0.947 ) . the average knee kinematics pattern of the right and left legs during a gait cycle a ) flexion - extension ; b ) adduction - abduction ; c ) internal - external rotation of the tibia the 95% confidence intervals ( cis ) of side - to - side differences of the normal subjects a ) flexion - extension ( fe ) ; b ) abduction - adduction ( aa ) ; c ) internal - external ( ie ) rotation ; d ) anterior - posterior ( ap ) translation ; e ) medial - lateral ( ml ) translation ; f ) superior - inferior ( si ) translation ( mean , 95% ci ) . a : post - hoc bonferroni tests ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . b : post - hoc bonferroni test ( vs. 40% p<0.001 ) for clinical application , the knee kinematics of a case with the posterior cruciate ligament injury ( pcl ) were also evaluated . a 37 year - old male suffered a knee injury while playing rugby football . the case showed grade-3 positive in the posterior drawer test , and posteromedial rotatory instability11 . the mri images indicated a pcl injury . at two months after injury , evaluation of 3-d kinematics during walking based on the side - to - side differences of kinematic data during each stage of gait , the tibia of the injured leg was shifted posteriorly in ap translation during 20% to 60% walking cycle , and shifted internally in ie rotation during 0% to 60% gait cycle ( fig . 3fig . 3the side - to - side difference of the femur relative to the tibia in the case with pcl and pms injuries with the 95% confidence intervals ( cis ) of the normal subjects . a ) anterior - posterior ( ap ) translation ; b ) internal - external ( ie ) rotation ) . the side - to - side difference of the femur relative to the tibia in the case with pcl and pms injuries with the 95% confidence intervals ( cis ) of the normal subjects . a ) anterior - posterior ( ap ) translation ; b ) internal - external ( ie ) rotation the present study investigated the side - to - side differences in kinematics of the knee joint during walking by healthy subjects . the side - to - side differences in adduction - abduction rotation and anterior - posterior translation of the tibia were found to be highly dependent on the stage of gait , and they were significantly larger in the swing phase than in the stance phase . therefore , our findings suggest that it is important to know the normal ranges of the side - to - side differences in knee kinematics in each stage of the gait cycle , in particular those of adduction - abduction and anterior - posterior translation of the knee , in order to evaluate the knee kinematics of subjects during walking . the reason why the side - to - side differences of adduction - abduction rotation and anterior - posterior translation of the tibia during walking are larger in the swing phase than in the stance phase remain unknown . one possible reason is mechanical restraint of the motion of the tibia during walking . the leg is not subject to weight bearing in the swing phase , and the tibia can move with less mechanical restraint in the swing phase than in the stance phase . without weight bearing , the knee is relaxed and the joint kinematics are likely to be influenced by ligament balance , rather than joint morphology . the knee kinematics during walking of a pcl injury case are also presented . for this case , an abnormal side - to - side difference in internal rotation of the tibia during walking as well as an abnormal side - to - side difference in posterior translation of the tibia were found . li et al . reported that pcl deficiency significantly increases posterior translation of the tibia during knee flexion under a weight - bearing condition using a dual - orthogonal fluoroscopic system12 . however , there was no significant difference in internal - external rotation between pcl - injury and normal knees throughout the range of flexion . in our present study , the case might have had associated injuries of posteromedial structures of the knee . previous cadaver studies have shown that additional injuries of posteromedial structures significantly increase internal rotation of the tibia as well as posterior translation of the tibia in pcl deficient knees13,14,15 . regarding clinical relevance , our findings indicate that the evaluation of the side - to - side difference of knee - joint kinematics provides useful information for clinical practice . however , caution is required in the clinical application of the evaluation of the side - to - side difference of knee - joint kinematics during walking because , there is a possibility that the contralateral leg would functionally adapt to the condition after trauma or disease . some investigators have reported that the contralateral leg as well as the injured leg functionally adapt to the condition after the injury of the acl and show the so - called quadriceps - avoidance gait16,17,18,19 . therefore , the evaluation of the side - to - side difference of knee joint kinematics may underestimate the knee kinematics of an injured leg . while this approach can measure in vivo kinematics of the knee in a dynamic condition , namely in a more physiological condition , it has been reported to have low accuracy due to skin movement artifacts20 , 21 . the recently described pct employs an overabundance of markers ( a cluster ) placed on each segment to minimize the effects of skin movement artifact6 . the pct can be extended to minimize skin movement artifacts by optimal weighting of the markers according to their degree of deformation6 . furthermore pct permits direct in vivo measurement of the complete six - degrees - of - freedom motion of the kinematics of the knee during activities of daily living . pct has , therefore , recently attracted attention , since the data of three dimensional kinematics of the knee during activities of daily living including walking are considered to reflect function of the knee joint . walking speed , surface condition , and shoes presumably influence the results of gait analysis . the knee kinematics measured in this study during walking and gait speed were similar to those reported by other investigators22,23,24,25,26 . first , since the point cluster technique is based on skin makers , the accuracy of the relative positions of the femur with respect to the tibia depends on the marker attachment technique of the examiner . second , the sample size of the present study , 21 , was not large enough to determine the normal range of the side - to - side - difference of the knee kinematics during walking . third , the effect of age or gender differences was not investigated in the present study . however , no significant differences have been reported between age groups and gender in previous knee kinematics studies27,28,29 , suggesting their effects in this study would have been small . despite the above - noted limitations , the mean side - to - side differences and their 95% cis as objective criteria of normal knee kinematics are useful information for the functional evaluation of subjects with disorders of the knee joint . in conclusion , the knee kinematics of healthy subjects during walking were measured using the pct , and the normal range of side - to - side differences in knee kinematics were determined for five stages of the gait cycle . the side - to - side differences in aa rotation and ap translation were highly dependent on the stage of gait ; therefore , the normal ranges of the side - to - side differences in knee kinematics of each stage of gait , in particular aa rotation and ap translation of the tibia , are useful information for the evaluation of the normal and pathological knee kinematics during walking .

[ purpose ] the purpose of this study was to determine the normal range of the side - to - side difference in three dimensional knee kinematics measured by the point cluster technique ( pct ) . [ subjects ] the subjects were twenty - one healthy normal volunteers without knee pain or an episode of injury to the legs . [ methods ] the subjects were tested bilaterally at a self - selected normal walking speed and six degrees of freedom knee kinematics were measured using the pct , and the 95% confidence intervals of the average side - to - side differences in flexion - extension ( fe ) , adduction - abduction ( aa ) , internal - external ( ie ) rotation , and anterior - posterior ( ap ) , medial - lateral ( ml ) , superior - inferior ( si ) translation in each stage of the gait cycle were determined . [ results ] the average side - to - side differences and their 95% confidence intervals in rotation / translation in each stage of the gait cycle were determined . the side - to - side differences in aa rotation and ap translation of the tibia were significantly larger in the swing phase than in the stance phase . [ conclusion ] the side - to - side differences in aa rotation and ap translation were highly dependent on the stage of the gait cycle . therefore , the normal ranges of the side - to - side differences in knee kinematics in each stage of the gait cycle , in particular aa rotation and ap translation of the tibia , is useful information for evaluating knee kinematics during walking .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor channels mediate most of the fast synaptic excitatory transmission in the brain . importantly , the glua2 subunit with its edited q / r site ( sommer et al . , 1992 ) ensures that ampa receptors containing this subunit are largely ca - impermeable ( hollmann et al . , 1991 ; burnashev et al . , 1992 ; jonas and burnashev , 1995 ; isaac et al . , 2007 ) . by contrast , glua2-lacking ampa receptor channels , i.e. , any homomeric or heteromeric assembly of glua1 , a3 , or a4 , are ca - permeable and subject to voltage - dependent block by intracellular polyamines , resulting in a characteristic doubly rectifying current - voltage ( i v ) relationship . imbalance in favor of ca permeable ampars can cause cns dysfunction ( liu and zukin , 2006 ) . in synaptic ampa channels , sensitivity to endogenous polyamines and the degree of rectification can be lessened by interaction with tarps , the transmembrane ampar regulatory proteins ( soto et al . , 2007 ) . thus , a low extent of rectification of synaptic ampar - mediated currents need not reflect a particular ampar composition , but may also indicate interaction with regulatory factor(s ) . as gleaned from the properties of ampa receptors in different neuronal populations , the current consensus would state that principal excitatory neurons operate with glua2-containing ampa receptors ( adesnik and nicoll , 2007 ; lu et al . , 2009 ) , whereas gaba - ergic inhibitory neurons often additionally feature glua2-lacking synaptic ampa receptors ( geiger et al . , 1995 ; toth and mcbain , 1998 ; liu and cull - candy , 2002 ) . while there is firm consensus , 1992 ; lu et al . , 2009 ) , conflicting evidence surrounds the possible presence of glua2-lacking ampar channels in hippocampal schaffer collateral / commissural synapses in ca1 pyramidal cells , arguably the best - studied synapses in the brain . several groups observed that the molecular identity of ampars in ca1 cell synapses is altered upon induced changes in synaptic strength . plant et al . ( 2006 ) reported that induction of long - term potentiation ( ltp ) elicits a transient increase in the proportion of polyamine sensitive ampars at the potentiated synapses , revealed by an increased rectification index ( ri ) . , adesnik and nicoll ( 2007 ) failed to obtain evidence for the insertion of synaptic ca - permeable ampa receptors upon ltp induction , concluding that all synaptic ampars in ca1 cells contain glua2 subunits . thus , whether glua2-lacking ampars exist at glutamatergic ca1 cell synapses and contribute to fast transmission clearly needs re - evaluating . the most common approach to detect the participation of glua2-lacking ampars in synaptic transmission is to test sensitivity to the wasp polyamine toxin , philanthotoxin-433 ( phtx-433 ) . glua2-lacking channels can be strongly blocked by phtx-433 in an activity dependent manner , while glua2 containing ampars are insensitive to the drug ( washburn and dingledine , 1996 ) . however , bath - applied phtx-433 may target a glua2-lacking channel population already blocked by endogenous polyamines ( pa ) , and thus , a moderate contribution of glua2-lacking channels could be overlooked . in this context , it is important to note that virtually all glua2-lacking ampar channels contain polyamine in the closed state , regardless of the membrane potential . this results in the characteristic doubly rectifying shape of their i v curves , which reflects the speed of relief from polyamine block . this speed is dependent on the absolute transmembrane electric field , and is faster when the cell is hyperpolarized ( rozov et al . , 1998 ) . even at physiological resting membrane potentials ( 70 mv ) , a substantial portion of glua2-lacking channels could still be blocked by polyamines during single unitary epscs , and repetitive high - frequency stimulation is required to relieve the block ( rozov and burnashev , 1999 ) . hence , to get an accurate readout of the phtx effect on ampar - mediated currents the influence of endogenous polyamines should first be minimized . to resolve the issue of ca - permeable ampa channels in ca1 pyramidal cells of wild - type ( wt ) , we took recourse to gene - targeted mice lacking either glua2 ( shimshek et al . , 2006 ) or glua1 ( zamanillo et al . , 1999 ) or expressing glua1 in a q / r site - edited version ( sanchis - segura et al . , 2006 ) . this allowed us to analyze the artificial situations of ca1 cell synapses having ampa receptors of maximal ( glua2 mutant mice ) or minimal ( glua1 and glua1(r ) mutants ) sensitivity to polyamines . the presence of ca - permeable ampa channels in wt should then be revealed by increased sensitivity to phtx-433 relative to ampa channels in glua1 and glua1(r ) mouse mutants . no such increase in phtx-433 sensitivity should be observed if wt ca1 pyramidal cell synapses indeed lack ca - permeable ampa channels , as posited by a prevalent view ( adesnik and nicoll , 2007 ) . our data clearly demonstrate that in wt ca1 synapses , 810% of the ampa channels are ca - permeable . transverse hippocampal 250 m slices were prepared from the brains of 4256 day - old wt , glua1 ( zamanillo et al . , 1999 ) , glua1(r ) ( sanchis - segura et al . , 2006 ) and glua2 mice ( shimshek et al . , 2006 ) , killed by decapitation . the slicing chamber contained an oxygenated ice - cold solution [ modified from ( dugue et al . , 2005 ) ] composed of ( in mm ) : k - gluconate , 140 ; n-(2-hydroxyethyl ) piperazine - n-ethanesulfonic acid ( hepes ) , 10 ; na - gluconate , 15 ; ethylene glycol - bis ( 2-aminoethyl)-n , n , n,n-tetraacetic acid ( egta ) , 0.2 ; and nacl , 4 ( ph 7.2 ) . slices were incubated for 30 min at 35c before being stored at room temperature in artificial csf ( acsf ) containing ( in mm ) : nacl , 125 ; nahco3 , 25 ; kcl , 2.5 ; nah2po4 , 1.25 ; mgcl2 , 1 ; cacl2 , 2 ; and glucose , 25 ; bubbled with 95%o2 and 5%co2 . patch electrodes were pulled from hard borosilicate capillary glass ( sutter instruments flaming / brown micropipette puller ) . these were filled with either a polyamine - free or a polyamine - containing solution . the polyamine - free solution consisted of ( in mm ) cs - gluconate , 100 ; cscl , 10 ; hepes , 10 ; nacl , 8 ; egta , 0.2 ; mgatp , 2 ; na3gtp , 0.3 ; phosphocreatine , 10 ; and k2atp , 20 ( ph 7.3 with csoh ) . the polyamine - containing solution was identical except for the addition of 0.5 mm spermine , yielding a free spermine concentration of 40 m ( watanabe et al . , 1991 ) . whole - cell recordings from these neurons were made in voltage - clamp mode using a heka epc-9 amplifier ( list elektronik ) . glass electrodes filled with acsf were placed in the dendritic region of stratum radiatum , 50100 m from the cell body , to stimulate schaffer collateral inputs at inter - stimulus intervals of 6 s. the stimulation intensity was adjusted to produce an epsc with an amplitude of 50 pa at the beginning of each recording . inhibitory synaptic transmission was blocked during recordings by adding 10 m gabazine to the perfusion acsf . for analysis , five subsequent responses were averaged and normalized to the mean epsc amplitude obtained during 2030 min of recording . the degree of blockade was calculated as a ratio of the average steady - state current amplitude before and after phtx-433 application . series resistance was monitored , and data from cells in which series resistance varied by > 15% during recording were discarded from analysis . glutamate ( 1 mm ) was applied using a piezo - controlled ( p 245.70 , physik instrumente , waldbronn , germany ) fast application system with a double - barrel application pipette onto outside - out patches of hek293 cells constitutively expressing homomeric glua1 receptors . for statistical analysis , student 's t - test was used , and data are presented as mean sd . we compared the effect of phtx-433 on epscs evoked in ca1 pyramidal cell synapses in hippocampal slices of glua2 mice in two distinct experimental conditions , one ensuring chelation of intracellular polyamines and the other having buffered spermine added to the intracellular solution . as in this mouse mutant all ampa channels are polyamine sensitive , we could evaluate the maximal effect of the applied concentration of phtx-433 ( 10 m ) on epscs mediated solely by glua2-lacking synaptic ampa channels . this should be useful for revealing their existence and estimating their contribution in wt , in which they would co - exist with glua2-containing ampars . additionally , we evaluated the sensitivity of synaptic ampars to intracellular polyamines in glua2 mice and compared it to that of wt . to this end , we measured the synaptic current - voltage ( i v ) relationship in ca1 pyramidal neurons dialyzed with polyamine - containing intracellular solution . evoked epscs from patched ca1 pyramidal cells of glua2 mice were recorded by stimulating schaffer collaterals at interstimulus intervals of 6 s with either polyamine - containing or -free patch - pipette solution . the washout of endogenous polyamines led to a three to fourfold increase in epsc amplitudes ( 0.29 0.27 relative to the steady - state values ; figures 1a , b ) . subsequent application of phtx-433 ( 10 m ) reduced the amplitude of responses to 0.34 0.15 compared to maximal amplitudes after polyamine washout ( n = 7 from 4 mice ; p < 0.001 ) . thus , 10 m of phtx-433 blocked 70% of the glua2-less ampa channels . however , when 0.5 mm of spermine was included in the pipette solution we typically observed a slight , though not significant , reduction in the epsc amplitude as whole - cell dialysis was progressing . initial amplitudes were 1.12 0.28 relative to the steady - state level reached after 30 min of dialysis ( p > 0.05 ; n = 7 from 5 mice ) . reduction of postsynaptic responses by subsequently applied phtx-433 was significantly less pronounced when compared to polyamine - free conditions ( 0.63 0.09 ; p < 0.01 ; figures 1a , b ) . maximal and polyamine - attenuated levels of phtx-433 blockade in ca1 pyramidal cells of glua2 mice . ( a ) the time course of epsc amplitude changes during washout of polyamine and subsequent phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were dialyzed with polyamine - free intracellular solution ( green circles ) . also shown is the effect of exogenously added spermine on epsc amplitudes and the masking effect of spermine on phtx-433 suppression ( black circles ) . ( b ) representative averaged traces at the indicated times of recording in polyamine - free conditions are shown in green , in polyamine - containing condition in black . ( c ) effect of intracellular polyamines on ampar - mediated synaptic currents in glua2 and wt mice . ampar - mediated synaptic i v curves measured with polyamine - containing intracellular solution in glua2 and wt mice ( left panel ) . rectification properties of synaptic ampar channels in glua2 and wt mice ( right panel ) . ris were calculated as the ratio of epsc amplitudes measured at 30 and 70 mv ( epsc70/epsc30 ) . collectively , the data from glua2 mice suggest that endogenous or exogenously added polyamines constitutively block glua2-lacking channels and attenuate the effect of bath - applied phtx-433 . strong sensitivity of ampars to polyamines in glua2 mice was further demonstrated by measuring the synaptic i blockade in these mutants of the glua2-less ampars by polyamines resulted in a characteristic inwardly rectifying i figure 1c compares the rectification properties of ampar channels mediating synaptic transmission at schaffer collateral / commissural synapses in ca1 pyramidal cells of glua2 and wt mice . obviously , glua2 ablation leads to enhanced polyamine sensitivity relative to wt , as reflected by an increased ri ( glua2 : ri = 10.3 0.85 , n = 5 ; wt : ri = 3.9 0.77 n = 5 ; p < 0.01 ) . we next determined how the duration of agonist application affects the phtx-433 blocking potency and to what extent the current suppression by the toxin depends on intracellular polyamine content . we employed fast agonist application on outside - out patches of hek293 cells expressing homomeric glua1 receptor channels in presence of 50 m cyclothiazide to exclude an influence of channel desensitization . application of 1 mm glutamate for 100 ms at 70 mv evoked stable inward currents , which were drastically reduced in presence of 10 m phtx-433 , reaching a steady - state level of 3 1% of the initial amplitude ( figure a1a ; however , at 5 ms agonist applications , the steady - state level reached upon phtx-433 application was significantly higher ( 28 11% of control amplitudes ; figure a1b ; n = 5 ; p < 0.01 ) . similar experiments with a polyamine - free pipette solution demonstrated a reduction of glutamate - evoked currents ( 5 ms ; 1 mm ) that was stronger ( 11 7% relative to control values ; figure a1c ; n = 5 ) than the 28 11% observed with the pa - containing solution ( p < 0.05 ) . thus , usage of phtx-433 as a tool to probe the expression of glua2-lacking ampars requires the washout of endogenous polyamines . we performed similar experiments on glua1 and glua1(r ) mouse mutants , in which the likelihood for the presence in ca1 pyramidal cells of synaptic glua2-less , ca - permeable ampa channels is low and hence , sensitivity to polyamines and phtx-433 should be lacking . indeed , prolonged whole - cell dialysis with polyamine - free intracellular solution did not result in a significant increase in epsc amplitudes in ca1 pyramidal cells in either glua1 or glua1(r ) mice . we did record a moderate enhancement of ampar - mediated responses ( 0.78 0.1 and 0.81 0.11 , n = 7 cells , for 45 mice of each mutant line ) , which is most likely due to frequency facilitation or augmentation ( thomson , 2000 ; zucker and regehr , 2002 ) . as expected , application of phtx-433 ( 10 m ) did not affect epsc amplitudes in the glua1 ( 0.98 0.13 ; n = 7 from 4 mice ) or glua1(r ) mutants ( 1.07 0.16 ; n = 7 from 5 mice ) . synaptic ampar channels in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants are polyamine and phtx-433 insensitive . ( a ) the time course of epsc amplitude changes recorded in ca1 pyramidal neurons of glua1 ( green circles ) and glua1(r ) ( blue circles ) mutant mice during washout of polyamine and subsequent phtx-433 ( 10 m ) application . ( b ) representative averaged traces at the indicated times of recording from glua1 mice shown in green , and for glua1(r ) mutants in blue . ( c ) effect of intracellular polyamines on rectification properties of ampar - mediated synaptic currents in glua1 and wt animals . synaptic ampar - mediated i v curves measured with polyamine - containing intracellular solution in glua1 and wt mice ( left panel ) . rectification indices of synaptic ampar channels in glua2 and wt mice ( right panel ) . addition of 0.5 mm spermine to the intracellular solution did not cause any inward rectification of ampar - mediated synaptic currents in glua1 mice , and the i v curve became slightly outwardly rectifying ( figure 2c ) . however the shape of the i v curve and the ri measured in glua1 mice differed significantly from those in wt mice under the same experimental conditions ( figure 2c ; glua1 : ri = 2.32 0.42 , n = 5 and wt : ri = 3.9 0.77 n = 5 ; p < 0.05 ) . we conclude that absence of glua1 ( or expression of glua1 in its r - form ) greatly diminish the presence of synaptic ca - permeable ampa channels . according to previous studies , epscs recorded from ca1 pyramidal cells are insensitive to phtx-433 ( adesnik and nicoll , 2007 ; lu et al . , 2009 ) . as intracellular polyamines can occlude block by phtx-433 ( figure 1 ) , we first determined how endogenous polyamine washout changes the potency of exogenously applied phtx-433 on epsc amplitudes in wt ca1 cells . when the cells were dialyzed with polyamine - free solution , the amplitudes of evoked ampar - mediated responses increased during the first 1520 min of recording , finally reaching a steady - state level ( figures 3a , b ) . n = 9 from 6 mice ) , when normalized to the steady - state values ( 2030 min of recording ) . subsequent application of phtx-433 ( 10 m ) caused a gradual reduction of the postsynaptic responses , reaching a plateau level ( 5060 min of recording ) of 0.69 0.17 ( p < 0.01 ) , as compared to values prior to phtx-433 application ( figures 3a , b ) . ( a ) scatter plot shows epsc amplitude dynamics during prolonged whole - cell dialysis and following phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were patched with either polyamine - free ( green circles ) or polyamine - containing ( black circles ) intracellular solution . ( b ) representative averaged traces at the indicated times of recording in polyamine - free ( green ) or polyamine - containing ( black ) solution . ( c ) dependence of ampar synaptic channel rectification properties in wt ca1 pyramidal cells on intracellular polyamine content . synaptic ampar - mediated i v curves measured with polyamine - containing and -free intracellular solutions ( left panel ) . dependence of rectification indices of synaptic ampar channels on the presence of polyamines in wt mice ( right panel ) . by contrast , when wt ca1 neurons were patched with polyamine - containing solution , the amplitudes of responses during the initial phase of dialysis were either stable or underwent a slight , but not significant reduction ( 1.18 0.14 , p > 0.05 , n = 7 from 5 mice ) . moreover , application of phtx-433 caused no significant changes in epsc amplitudes ( 1.12 0.14 ; p > 0.05 ) . thus , in wt ca1 pyramidal cells , washout of intracellular polyamines resulted in an increase in the amplitude of evoked postsynaptic responses , which was in large part counteracted by subsequent application of phtx-433 ( figures 3a , b ) . this remarkable difference to the results from synaptic ampars in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants constitutes unequivocal evidence for the presence of synaptic ca - permeable ampa receptors in wt ca1 cells . to further substantiate our finding on the existence of glua2-lacking ampars at schaffer collateral / commissural synapses in wt ca1 pyramidal cells , we evaluated the effect of the presence of intracellular spermine on the shape of ampar - mediated i v curves and on the ri . v relation recorded with polyamine - free solution was nearly linear and deviated strongly at positive potentials from that observed with polyamine - containing solution . also , the ri in presence of spermine was significantly higher compared to that in polyamine - free conditions [ wt ( sp ) : 3.9 0.77 , n = 5 and wt ( no sp ) : 2.86 0.28 , n = 5 ; p < 0.05 ] . we compared the effect of phtx-433 on epscs evoked in ca1 pyramidal cell synapses in hippocampal slices of glua2 mice in two distinct experimental conditions , one ensuring chelation of intracellular polyamines and the other having buffered spermine added to the intracellular solution . as in this mouse mutant all ampa channels are polyamine sensitive , we could evaluate the maximal effect of the applied concentration of phtx-433 ( 10 m ) on epscs mediated solely by glua2-lacking synaptic ampa channels . this should be useful for revealing their existence and estimating their contribution in wt , in which they would co - exist with glua2-containing ampars . additionally , we evaluated the sensitivity of synaptic ampars to intracellular polyamines in glua2 mice and compared it to that of wt . to this end , we measured the synaptic current - voltage ( i v ) relationship in ca1 pyramidal neurons dialyzed with polyamine - containing intracellular solution . evoked epscs from patched ca1 pyramidal cells of glua2 mice were recorded by stimulating schaffer collaterals at interstimulus intervals of 6 s with either polyamine - containing or -free patch - pipette solution . the washout of endogenous polyamines led to a three to fourfold increase in epsc amplitudes ( 0.29 0.27 relative to the steady - state values ; figures 1a , b ) . subsequent application of phtx-433 ( 10 m ) reduced the amplitude of responses to 0.34 0.15 compared to maximal amplitudes after polyamine washout ( n = 7 from 4 mice ; p < 0.001 ) . thus , 10 m of phtx-433 blocked 70% of the glua2-less ampa channels . however , when 0.5 mm of spermine was included in the pipette solution we typically observed a slight , though not significant , reduction in the epsc amplitude as whole - cell dialysis was progressing . initial amplitudes were 1.12 0.28 relative to the steady - state level reached after 30 min of dialysis ( p > 0.05 ; n = 7 from 5 mice ) . reduction of postsynaptic responses by subsequently applied phtx-433 was significantly less pronounced when compared to polyamine - free conditions ( 0.63 0.09 ; p < 0.01 ; figures 1a , b ) . maximal and polyamine - attenuated levels of phtx-433 blockade in ca1 pyramidal cells of glua2 mice . ( a ) the time course of epsc amplitude changes during washout of polyamine and subsequent phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were dialyzed with polyamine - free intracellular solution ( green circles ) . also shown is the effect of exogenously added spermine on epsc amplitudes and the masking effect of spermine on phtx-433 suppression ( black circles ) . ( b ) representative averaged traces at the indicated times of recording in polyamine - free conditions are shown in green , in polyamine - containing condition in black . ( c ) effect of intracellular polyamines on ampar - mediated synaptic currents in glua2 and wt mice . ampar - mediated synaptic i v curves measured with polyamine - containing intracellular solution in glua2 and wt mice ( left panel ) . rectification properties of synaptic ampar channels in glua2 and wt mice ( right panel ) . ris were calculated as the ratio of epsc amplitudes measured at 30 and 70 mv ( epsc70/epsc30 ) . collectively , the data from glua2 mice suggest that endogenous or exogenously added polyamines constitutively block glua2-lacking channels and attenuate the effect of bath - applied phtx-433 . strong sensitivity of ampars to polyamines in glua2 mice was further demonstrated by measuring the synaptic i blockade in these mutants of the glua2-less ampars by polyamines resulted in a characteristic inwardly rectifying i figure 1c compares the rectification properties of ampar channels mediating synaptic transmission at schaffer collateral / commissural synapses in ca1 pyramidal cells of glua2 and wt mice . obviously , glua2 ablation leads to enhanced polyamine sensitivity relative to wt , as reflected by an increased ri ( glua2 : ri = 10.3 0.85 , n = 5 ; wt : ri = 3.9 0.77 n = 5 ; p < 0.01 ) . we next determined how the duration of agonist application affects the phtx-433 blocking potency and to what extent the current suppression by the toxin depends on intracellular polyamine content . we employed fast agonist application on outside - out patches of hek293 cells expressing homomeric glua1 receptor channels in presence of 50 m cyclothiazide to exclude an influence of channel desensitization . application of 1 mm glutamate for 100 ms at 70 mv evoked stable inward currents , which were drastically reduced in presence of 10 m phtx-433 , reaching a steady - state level of 3 1% of the initial amplitude ( figure a1a ; however , at 5 ms agonist applications , the steady - state level reached upon phtx-433 application was significantly higher ( 28 11% of control amplitudes ; figure a1b ; n = 5 ; p < 0.01 ) . similar experiments with a polyamine - free pipette solution demonstrated a reduction of glutamate - evoked currents ( 5 ms ; 1 mm ) that was stronger ( 11 7% relative to control values ; figure a1c ; n = 5 ) than the 28 11% observed with the pa - containing solution ( p < 0.05 ) . thus , usage of phtx-433 as a tool to probe the expression of glua2-lacking ampars requires the washout of endogenous polyamines . we performed similar experiments on glua1 and glua1(r ) mouse mutants , in which the likelihood for the presence in ca1 pyramidal cells of synaptic glua2-less , ca - permeable ampa channels is low and hence , sensitivity to polyamines and phtx-433 should be lacking . indeed , prolonged whole - cell dialysis with polyamine - free intracellular solution did not result in a significant increase in epsc amplitudes in ca1 pyramidal cells in either glua1 or glua1(r ) mice . we did record a moderate enhancement of ampar - mediated responses ( 0.78 0.1 and 0.81 0.11 , n = 7 cells , for 45 mice of each mutant line ) , which is most likely due to frequency facilitation or augmentation ( thomson , 2000 ; zucker and regehr , 2002 ) . as expected , application of phtx-433 ( 10 m ) did not affect epsc amplitudes in the glua1 ( 0.98 0.13 ; n = 7 from 4 mice ) or glua1(r ) mutants ( 1.07 0.16 ; n = 7 from 5 mice ) . synaptic ampar channels in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants are polyamine and phtx-433 insensitive . ( a ) the time course of epsc amplitude changes recorded in ca1 pyramidal neurons of glua1 ( green circles ) and glua1(r ) ( blue circles ) mutant mice during washout of polyamine and subsequent phtx-433 ( 10 m ) application . ( b ) representative averaged traces at the indicated times of recording from glua1 mice shown in green , and for glua1(r ) mutants in blue . ( c ) effect of intracellular polyamines on rectification properties of ampar - mediated synaptic currents in glua1 and wt animals . synaptic ampar - mediated i v curves measured with polyamine - containing intracellular solution in glua1 and wt mice ( left panel ) . rectification indices of synaptic ampar channels in glua2 and wt mice ( right panel ) . addition of 0.5 mm spermine to the intracellular solution did not cause any inward rectification of ampar - mediated synaptic currents in glua1 mice , and the i v curve became slightly outwardly rectifying ( figure 2c ) . however the shape of the i v curve and the ri measured in glua1 mice differed significantly from those in wt mice under the same experimental conditions ( figure 2c ; glua1 : ri = 2.32 0.42 , n = 5 and wt : ri = 3.9 0.77 n = 5 ; p < 0.05 ) . we conclude that absence of glua1 ( or expression of glua1 in its r - form ) greatly diminish the presence of synaptic ca - permeable ampa channels . according to previous studies , epscs recorded from ca1 pyramidal cells are insensitive to phtx-433 ( adesnik and nicoll , 2007 ; lu et al . , 2009 ) . as intracellular polyamines can occlude block by phtx-433 ( figure 1 ) , we first determined how endogenous polyamine washout changes the potency of exogenously applied phtx-433 on epsc amplitudes in wt ca1 cells . when the cells were dialyzed with polyamine - free solution , the amplitudes of evoked ampar - mediated responses increased during the first 1520 min of recording , finally reaching a steady - state level ( figures 3a , b ) . the average initial amplitude was 0.45 0.21 ( n = 9 from 6 mice ) , when normalized to the steady - state values ( 2030 min of recording ) . subsequent application of phtx-433 ( 10 m ) caused a gradual reduction of the postsynaptic responses , reaching a plateau level ( 5060 min of recording ) of 0.69 0.17 ( p < 0.01 ) , as compared to values prior to phtx-433 application ( figures 3a , b ) . ( a ) scatter plot shows epsc amplitude dynamics during prolonged whole - cell dialysis and following phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were patched with either polyamine - free ( green circles ) or polyamine - containing ( black circles ) intracellular solution . ( b ) representative averaged traces at the indicated times of recording in polyamine - free ( green ) or polyamine - containing ( black ) solution . ( c ) dependence of ampar synaptic channel rectification properties in wt ca1 pyramidal cells on intracellular polyamine content . synaptic ampar - mediated i v curves measured with polyamine - containing and -free intracellular solutions ( left panel ) . dependence of rectification indices of synaptic ampar channels on the presence of polyamines in wt mice ( right panel ) . by contrast , when wt ca1 neurons were patched with polyamine - containing solution , the amplitudes of responses during the initial phase of dialysis were either stable or underwent a slight , but not significant reduction ( 1.18 0.14 , p > 0.05 , n = 7 from 5 mice ) . moreover , application of phtx-433 caused no significant changes in epsc amplitudes ( 1.12 0.14 ; p > 0.05 ) . thus , in wt ca1 pyramidal cells , washout of intracellular polyamines resulted in an increase in the amplitude of evoked postsynaptic responses , which was in large part counteracted by subsequent application of phtx-433 ( figures 3a , b ) . this remarkable difference to the results from synaptic ampars in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants constitutes unequivocal evidence for the presence of synaptic ca - permeable ampa receptors in wt ca1 cells . to further substantiate our finding on the existence of glua2-lacking ampars at schaffer collateral / commissural synapses in wt ca1 pyramidal cells , we evaluated the effect of the presence of intracellular spermine on the shape of ampar - mediated i v curves and on the ri . v relation recorded with polyamine - free solution was nearly linear and deviated strongly at positive potentials from that observed with polyamine - containing solution . also , the ri in presence of spermine was significantly higher compared to that in polyamine - free conditions [ wt ( sp ) : 3.9 0.77 , n = 5 and wt ( no sp ) : 2.86 0.28 , n = 5 ; p < 0.05 ] . the controversy concerning the presence of glua2-less , ca - permeable ampa channels in hippocampal ca1 pyramidal cell synapses ( plant et al . , 2006 ; 2010 ) prompted this study , for which we employed , in addition to wt mice , mouse mutants lacking either the major ampar subunits glua1 or glua2 or expressing the glua1(r ) subunit , which , like glua2 , drastically attenuates the ca - permeability of ampars containing it ( in preparation ) . polyamine sensitivity of glua2-lacking ampars is often used to evaluate the relative contribution of these channels to the net ampar - mediated synaptic current . endogenous or exogenously added polyamines can block these receptors , resulting in strong current rectification , which can be measured as ri . alternatively , washout of polyamines should increase epsc amplitudes in synapses in which glua2-lacking channels contribute to synaptic transmission ( rozov and burnashev , 1999 ) . the magnitude of this enhancement would depend on the endogenous polyamine concentration and the ratio between glua2-lacking and glua2-containing channels ( figures 4a , b ) . in all our experiments , we observed epsc augmentation upon polyamine washout during the initial 2025 min of recording , but the scale of this enhancement strongly depended on the particular mouse mutant . as expected , the increase was largest in glua2 mice and smallest in glua1 or glua1(r ) mutants . in the latter two mutant lines the augmentation of postsynaptic responses is unlikely to reflect the washout of polyamines , since the ampars in these mutants should be polyamine insensitive . a possible explanation for the epsc amplitude rise in these mutants might be activity dependent presynaptic phenomena , such as frequency facilitation or augmentation ( thomson , 2000 ; zucker and regehr , 2002 ) , which could also contribute to the epsc enhancement by polyamine washout in wt and glua2 mice this assumption allows us to correct the data from wt and glua2 mice and thus estimate better the putative impact of polyamine washout on fast excitatory synaptic transmission at schaffer collateral to ca1 pyramidal cell synapses ( figure 4c ) . ( a ) polyamines enter and block glua2-lacking ampars in the closed state . in glua2 mice almost all channels the channel conductance is strongly attenuated , resulting in reduced amplitudes of the net response ( middle panel ) . in contrast , washout of polyamines results in an increased ampar conductance , leading to enhanced epsc amplitudes ( right panel ) . ( b ) when glua2-lacking and glua2-containing ampars are co - expressed at the same synapse , the effect of polyamine washout will depend on the ratio between the two channel types . representative traces show enhancement of ampar - mediated epscs during polyamine washout ( wt in red , glua2 in black ) . ( c ) the plot summarizes data on the initial amplitude enhancement observed in wt , glua2 and glua1 mice . the dashed line shows the exponential fits of the time dependent amplitude augmentation during polyamine washout ( wt in red , glua2 in black and glua1 in blue ) . since the vast majority of ca1 cell ampa channels in glua1 mice should be polyamine insensitive , the slight epsc enhancement represents another intrinsic property of schaffer collateral / commisural to ca1 pyramidal cells synapses ( e.g. , frequency facilitation ) . thus , by subtracting the glua1 data from those obtained from wt or glua2 mice , we can calculate the putative effect of endogenous polyamine washout on epsc amplitudes in wt ( red line ) and glua2 ( black line ) mice . we also employed the sensitivity of synaptic ampa currents to phtx-433 to detect the presence of glua2-lacking ampars . the accuracy of this phtx-433 probing test is sensitive to intracellular polyamines . indeed , as we show , the presence of 40 m of free spermine reduces the phtx-433 suppression of ampar - mediated epscs by 50% in glua2 mice , in which all ampa channels should be sensitive to the wasp toxin . the use of glua2 mice allowed us to determine that after polyamine washout , 10 m phtx-433 blocks 70% of the epscs mediated by glua2-lacking ampa channels . the epscs mediated by ampa channels in glua1 or glua1(r ) mutants were insensitive to phtx-433 , consonant with the predicted lack of ca - permeable ampa channels in these mutant mice , and thus demonstrating that 10 m phtx-433 exerts no unspecific effects on synaptic transmission . notably , following polyamine washout , 10 m phtx-433 blocked 31% of the epscs in wt ca1 pyramidal cell synapses , clearly demonstrating the presence of ca - permeable glua2-less ampa channels . while in glua2 mice , where all ampars are polyamine sensitive , phtx blocked 66% of the epsc . by solving a simple rule of proportion we can calculate that in wt ca1 pyramidal cells , under artificial polyamine - free conditions , glua2-lacking channels mediate 47% of the net epsc . to estimate the proportion of such channels in the entire synaptic ampar population , one needs to consider that the conductance of glua2-lacking ampars is 510 times higher than that of glua2-containing channels . these data allow us to estimate the relative contribution of glua2-lacking channels to synaptic transmission at schaffer collateral ca1 cell synapses . in polyamine - free conditions , recombinant glua2-lacking channels have a maximal conductance of 1925.5 ps , whereas the conductance of glua2-containing ampars is 2.5 ps ( oh and derkach , 2005 ) . we thus estimate the population of glua2-lacking ampars to be in the range of 810% . under native conditions , the contribution of glua2-lacking channels to the synaptic response depends on the intracellular polyamine concentration and should , therefore , be < 47% . however , during repetitive activity , the impact of these channels will increase due to the activity - dependent relief from polyamine block . also , the degree of the block could be significantly attenuated by interaction of glua2-lacking ampars with tarps . thus , the role of polyamine - sensitive ampars at schaffer collateral / commissural synapses might be differentially regulated during shorter and longer activity epochs , thus providing additional ways to control the synaptic gain . our analyses might resolve the apparent disagreement in the field regarding glua2-lacking ampars in ca1 pyramidal cells . the phtx-433 test becomes less sensitive in the presence of intracellular polyamines , since the toxin can only block channels , which are not occupied by internal polyamines . crucially , polyamines block glua2-lacking channels in the closed state , and the block itself is not voltage dependent . therefore , to maximize the measurable effect of phtx-433 , the washout of endogenous polyamines becomes essential , lest the contribution of these channels to synaptic transmission is underestimated , as demonstrated here by fast agonist application experiments on recombinant glua1 homomeric ampars . the presence of ca - permeable ampa channels in ca1 cell synapses prompts the question as to their functional role . plant et al . ( 2006 ) observed transient incorporation of such ampa channels following induction of hippocampal ltp and reported that the transient ca - influx is essential for the subsequent maintenance phase of ltp . indeed , the contribution of the ca - influx via gated ca - permeable ampa channels is very low relative to that of nmda channels , considering an approximately 10-fold lower fractional ca - current and a > 10-fold lesser envelope of charge transfer of the ampa channels . notably , our data indicate no need for the transient incorporation of ca - permeable ampa channels into ca1 cell synapses upon ltp induction ( plant et al . , 2006 ) , given that such channels already constitute a small subpopulation of the synaptic ampa receptors in wt ca1 pyramidal neurons . rather , it appears that ltp induction may facilitate the participation in excitatory transmission of the synaptic ca - permeable ampa channels , conceivably . via phosphorylation of intracellular subunit domains , with the additional negative charges close to the inner channel mouth accelerating the unblocking of polyamines . 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 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 glua2 subunit in heteromeric alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor channels restricts ca2 + permeability and block by polyamines , rendering linear the current - voltage relationship of these glutamate - gated cation channels . although glua2-lacking synaptic ampa receptors occur in gaba - ergic inhibitory neurons , hippocampal ca1 pyramidal cell synapses are widely held to feature only glua2 containing ampa receptors . a controversy has arisen from reports of glua2-lacking ampa receptors at hippocampal ca3-to - ca1 cell synapses and a study contesting these findings . here we sought independent evidence for the presence of glua2-lacking ampa receptors in ca1 pyramidal cell synapses by probing the sensitivity of their gated cation channels in wild - type ( wt ) mice and gene - targeted mouse mutants to philanthotoxin , a specific blocker of glua2-lacking ampa receptors . the mutants either lacked glua2 for maximal philanthotoxin sensitivity , or , for minimal sensitivity , expressed glua1 solely in a q / r site - edited version or not at all . our comparative electrophysiological analyses provide incontrovertible evidence for the presence in wild - type ca1 pyramidal cell synapses of glua2-less ampa receptor channels . this article is part of a special issue entitled calcium permeable ampars in synaptic plasticity and disease .

Introduction Methods Results PhTx-433 suppression of EPSCs in absence of GluA2 depends on intracellular polyamine content Synaptic AMPARs in CA1 pyramidal cells of PhTx-433 sensitive AMPA channels in WT CA1 pyramidal cell synapses Discussion Conflict of interest statement

lignocellulosic materials represent an abundant and inexpensive source of sugars and can be microbiologically converted to industrial products . xylitol ( c5h12o5 ) , a sugar alcohol obtained from xylose , is generated during the metabolism of carbohydrates in animals and humans . xylitol was present in fruits and vegetables , at low concentration , which makes its production from these sources economically unfeasible . its sweetening power was comparable to that of sucrose and is higher than that of sorbitol and mannitol . in addition to reducing dental caries , xylitol also promotes tooth enamel remineralization by reversing small lesions . this happens because , when in contact with xylitol , the saliva seems to be favorably influenced ; the chemical composition of xylitol induces the calcium ions and phosphate . for these characteristics , xylitol was a feed stock of great interest to food , odontological , and pharmaceutical industries . however it was expensive and it requires several steps of xylose purification before the chemical reaction [ 4 , 9 , 10 ] . xylitol production through bioconversion has been proposed as for utilizing microorganism such as yeast , bacteria , and fungi [ 11 , 12 ] . among these , yeast has shown to possess desirable properties for xylitol production [ 13 , 14 ] . therefore , for the present study , yeast strain pachysolen tannophilus was selected for xylitol production . furthermore studies have shown that nutritional factors including sources of carbon and nitrogen can influence xylitol production . corncob is a large volume solid waste for using sweet corn processing industry in india . they are currently used as animal feed or returned to the harvested field for land application . corncob contains approximately over 40% of the dry matter in residues and thus has value has a raw material for production of xylose , xylitol , arabinose , xylobiose , and xylo oligosaccharides . these carbohydrates mainly consist of the xylose and other minor pentose [ 1820 ] . among various agricultural wastes , corncob was regarded as promising agricultural resources for microbial xylitol production . in microbial production of xylitol from corncob , the cobs were first hydrolysed to produce from hemicelluloses by acid hydrolysis and the corncob hydrolysate is then used as the medium for xylitol production . the bioconversion of xylitol is influenced by the concentration of various ingredients in culture medium . this study also investigates the effect of process variables such as ph , temperature , substrate concentration , inoculum size , and agitation speed on xylitol yield . response surface methodology ( rsm ) is a mathematical and statistical analysis , which is useful for the modeling and analysis problems that the response of interest is influenced by several variables . rsm was utilized extensively for optimizing different biotechnological process [ 22 , 23 ] . in the present study , the screening and optimization of medium composition and process variables for xylitol production by pachysolen tannophilus using plackett - burman and rsm were reported . the plackett - burman screening design was applied for knowing the most significant nutrients enhancing xylitol production . then , box - behnken design and central composite design ( ccd ) were applied to determine the optimum level of significant nutrients and process variables , respectively . the yeast strain pachysolen tannophilus ( mtcc 1077 ) was collected from microbial type culture collection and gene bank , chandigarh . the lyophilized stock cultures were maintained at 4c on culture medium supplemented with 20 g of agar . the medium composition ( g / l ) was compressed of the following : malt extract : 3.0 ; yeast extract : 3.0 ; peptone : 5.0 ; glucose : 10.0 at ph : 7 . corncob was collected from perambalur farms , tamil nadu , india , and was dried in sunlight for 2 days , crushed , and sieved for different mesh size ranging from 0.45 mm to 0.9 mm ( 2040 mesh ) and used for further studies . the composition of the corncob ( g / l ) : xylose : 28.7 , glucose : 5.4 , arabinose : 3.7 , cellobiose : 0.5 , galactose : 0.7 , mannose : 0.4 , acetic acid : 2 , furfural : 0.8 , hydroxymethyl furfural : 0.2 was used for xylitol production . 2 g of corncobs at a solid loading of 10% ( w / w ) was mixed with dilute sulfuric acid ( 0.1% ( w / v ) ) and pretreated in an autoclave at 120c with residence time of 1 hour . the liquid fraction was separated by filtration and the unhydrolysed solid residue was washed with warm water ( 60c ) . hemicellulose acid hydrolysate was heated at 100c for 15 min to reduce the volatile components . the hydrolysate was overlined with solid ca(oh)2 up to ph 10 , in combination with 0.1% sodium sulfite , and filtered to remove the insoluble materials . activated charcoal treatment was an efficient and economic method of reduction in the amount of phenolic compounds , acetic acid , aromatic compounds , furfural , and hydroxymethylfurfural normally found in hemicellulosic hydrolysates . after centrifugation , the solutions were mixed with powdered charcoal at 5% ( w / v ) for 30 and stirred ( 100 rpm ) at 30c . the liquor was recovered by filtration , chemically characterized , and used for culture media . fermentation was carried out in 250 ml erlenmeyer flasks with 100 ml of pretreated corncob hemicelluloses hydrolysate is adjusted to ph 7 with 2 m h2so4 or 3 m naoh and supplemented with different nutrients concentration for tests according to the selected factorial design , were used for fermentation medium and sterilized at 120c for 20 mins . the flasks were maintained at 30c for agitation at 200 rpm for 48 hours . after the optimization of medium composition , the fermentation was carried out with different parameter levels ( table 5 ) with the optimized media for tests according to the selected factorial design . during the preliminary screening , the experiments were carried out for 5 days and the maximum production was obtained in 48 hours . sugar and sugar alcohol in the culture broth were measured by high - performance liquid chromatography ( hplc ) , model lc-10-ad ( shimadzu , tokyo , japan ) equipped with a refractive index ( ri ) detector . the chromatography column used was a aminex hpx-87h ( 300 7.8 mm ) column at 80c with 5 mm h2so4 as mobile phase at a flow rate of 0.4 ml / min , and the injected sample volume was 20 l . the rsm has several classes of designs , with its own properties and characteristics . central composite design ( ccd ) , box - behnken design , and three - level factorial design are the most popular designs applied by the researchers . a prior knowledge with understanding of the related bioprocesses is necessary for a realistic modeling approach . it assumes that there are no interactions between the different variables in the range under consideration . plackett - burman experimental design is a fractional factorial design and the main effects of such a design may be simply calculated as the difference between the average of measurements made at the high level ( + 1 ) of the factor and the average of measurements at the low level ( 1 ) . to determine which variables significantly affect xylitol production , nine variables were screened in 12 experimental runs ( table 1 ) , and insignificant ones are eliminated in order to obtain a smaller , manageable set of factors . the low level ( 1 ) and high level ( + 1 ) of each factor ( 1 , + 1 ) g / l ) : k2hpo4 ( 6.6 , 7 ) , yeast extract ( 1.5 , 5 ) , peptone ( 2 , 5 ) , kh2po4 ( 1.2 , 3.6 ) , xylose ( 9.8 , 10.2 ) , ( nh4)2so4 ( 1 , 4 ) , mgso47h2o ( 0.7 , 1.3 ) , malt ( 2.8 , 3.2 ) , and glucose ( 9.8 , 10.2 ) , and they were coded with a , b , c , d , e , f , g , h , i , respectively . the statistical software package minitab 16 is used for analyzing the experimental data . once the critical factors are identified through the screening , the box - behnken design was used to obtain a quadratic model after the central composite design ( ccd ) was used to optimize the process variables and obtain a quadratic model . the box - behnken design and ccd was used to study the effects of the variables towards their responses and subsequently in the optimization studies . this method was suitable for fitting a quadratic surface , and it helps to optimize the effective parameters with a minimum number of experiments , as well as to analyze the interaction between the parameters . in order to determine the relationship between the factors and response variables , a regression design was employed to model a response as a mathematical function ( either known or empirical ) for few continuous factors , and good model parameter estimates are desired . the coded values of the process parameters are determined by the following equation : ( 1)xi = xix0x , where xi is coded value of the ith variable , xi is uncoded value of the ith test variable , and x0 is uncoded value of the ith test variable at center point . the regression analysis is performed to estimate the response function as a second - order polynomial : ( 2)y=0+i=1kixi+i=1kiixi2i=1 , i < jk1 j=2kijxixj , where y is the predicted response , 0 constant , and i , ii , ij are coefficients estimated from regression . they represent the linear , quadratic , and cross - products of xi and xj on response . the regression and graphical analysis are carried out using design - expert software ( version 7.1.5 , stat - ease , inc . , the adequacy of the models is further justified through analysis of variance ( anova ) . lack - of - fit is a special diagnostic test for adequacy of a model that compares the pure error , based on the replicate measurements to the other lack of fit , based on the model performance . f value , calculated as the ratio between the lack - of - fit mean square and the pure error mean square , is the statistic parameter used to determine whether the lack - of - fit is significant or not , at a significance level . the statistical model was validated with respect to xylitol production under the conditions predicted by the model in shake - flask level . samples were drawn at the desired intervals and xylitol production was determined as described above . plackett - burman experiments ( table 1 ) showed a wide variation in xylitol production . this variation reflected the importance of optimization to attain higher productivity . from the pareto chart shown in figure 1 the variables , namely , peptone , xylose , mgso47h2o , and yeast extract , were selected for further optimization to attain a maximum response . the levels of factors and the effect of their interactions on xylitol production were determined by box - behnken design of rsm . the design matrix of experimental results by tests was planned according to the 29 full factorial designs . twenty - nine experiments were performed at different combinations of the factors shown in table 2 , and the central point was repeated five times . the predicted and observed responses along with design matrix are presented in table 3 , and the results were analyzed by anova . the second - order regression equation provided the levels of xylitol production as a function of peptone , xylose , mgso47h2o , and yeast extract , which can be presented in terms of coded factors as in the following equation : ( 3)y=0.70 + 0.053a+0.018b+0.057c+0.054d + 0.092ab(2.500e003)ac + ( 1.000e002)ad+0.028bc+0.077bd 0.040cd0.083a20.16b20.076c20.11d2 , where y is the xylitol yield ( g / g ) and a , b , c , and d were peptone , xylose , mgso47h2o , and yeast extract , respectively . there is only a 0.01% chance that a model f - value this large could occur due to noise . values of prob > f less than 0.05 indicate that model terms are significant . values greater than 0.1 indicate that model terms are not significant . in the present work , linear terms of a , c , and d and all the square effects of a , b , c , and d and the combination of ab , bd , and cd were significant for xylitol production . the coefficient of determination ( r ) for xylitol production was calculated as 0.9634 , which is very close to 1 and can explain up to 96.00% variability of the response . the predicted r value of 0.7898 was in reasonable agreement with the adjusted r value of 0.9267 . the adequate precision value of 16.010 indicates an adequate signal and suggests that the model can navigate the design space . the above model can be used to predict the xylitol production within the limits of the experimental factors that the actual response values agree well with the predicted response values . experimental conditions for optimization of the process variables for xylitol yield were determined by ccd of rsm . the design matrix of experimental results by tests was planned according to the 50 full factorial designs , and the central point was repeated eight times . the predicted and observed responses along with design matrix are presented in table 6 and the results were analyzed by anova . the second - order regression equation provided the levels of xylitol production as a function of temperature , substrate concentration , ph , agitation speed , and inoculums size , which can be presented in terms of coded factors as in the following equation : ( 4)y=0.79 + 0.025a+0.043b+0.049c + 0.030d+0.038e0.029ab(4.063e003)ac + 0.018ad+(2.187e003)ae(9.688e003)bc + 0.014bd+(5.312e003)be+(1.562e003)cd + ( 5.312e003)ce(3.125e004)de0.040a2 0.041b20.046c20.027d20.034e2 , where y was the xylitol yield ( g / g ) and a , b , c , d , and e are temperature , substrate concentration , ph , agitation speed , and inoculums size , respectively . there is only a 0.01% chance that a model f - value this large could occur due to noise . values of prob > f less than 0.05 indicate that model terms are significant . values greater than 0.1 indicate that model terms are not significant . in the present work , linear terms and all the square effects of a , b , c , d , and e and the coefficient of determination ( r ) for xylitol production was calculated as 0.9148 , which is very close to 1 and can explain up to 91.00% variability of the response . the predicted r value of 0.6867 was in reasonable agreement with the adjusted r value of 0.8561 . the adequate precision value of 12.951 indicates an adequate signal and suggests that the model can navigate the design space . in both designs the interaction effects of variables on xylitol production were studied by plotting 3d surface curves against any two independent variables , while keeping another variable at its central ( 0 ) level . the 3d curves of the calculated response ( xylitol yield ) and contour plots from the interactions between the variables were obtained . this evidence from above figures shows the dependency of xylose , mgso47h2o , yeast extract on xylitol production . the optimal operation conditions of peptone , xylose , mgso47h2o , and yeast extract for maximum xylitol production were determined by response surface analysis and also estimated by regression equation . the predicted values from the regression equation closely agreed with that obtained from experimental values . the xylitol production increased with increase in temperature to about 36c , and thereafter xylitol production decreased with further increase in temperature . this evidance from above figures shows the dependency of ph , substrate concentration , agitation speed , and inoculum size on xylitol production . the optimal operation conditions of temperature , substrate concentration , ph , agitation speed , and inoculum size for maximum xylitol production were determined by response surface analysis and also estimated by regression equation . the predicted values from the regression equation closely agreed with that obtained from experimental values . validation of the experimental model was tested by carrying out the batch experiment under optimal operation conditions which are ( g / l ) : peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , yeast extract : 4.66 established by the regression model . under optimal process variables levels are temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 g / l ) , inoculum size ( 3.69 ml ) , and agitation speed ( 194.44 rpm ) . the xylitol production ( 0.80 g / g ) obtained from experiments was very close to the actual response ( 0.78 g / g ) predicted by the regression model , which proved the validity of the model . in this work , plackett - burman design was used to test the relative importance of medium components on xylitol production . among the variables , peptone , xylose , mgso47h2o , and yeast g / l ) : peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , and yeast extract : 4.66 . then the influence of various process variables , namely , temperature , ph , substrate concentration , agitation speed , and inoculum size on the xylitol production was evaluated by ccd . the optimum levels of process variables are temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 g / l ) , inoculum size ( 3.69 ml ) , and agitation speed ( 194.44 rpm ) . this study showed that the corncob is a good source for the production of xylitol . using the optimized conditions , the xylitol yield reaches 0.80 g / g .

optimization of the culture medium and process variables for xylitol production using corncob hemicellulose hydrolysate by pachysolen tannophilus ( mttc 1077 ) was performed with statistical methodology based on experimental designs . the screening of nine nutrients for their influence on xylitol production was achieved using a plackett - burman design . peptone , xylose , mgso47h2o , and yeast extract were selected based on their positive influence on xylitol production . the selected components were optimized with box - behnken design using response surface methodology ( rsm ) . the optimum levels ( g / l ) were peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , yeast extract : 4.66 . the influence of various process variables on the xylitol production was evaluated . the optimal levels of these variables were quantified by the central composite design using rsm , for establishment of a significant mathematical model with a coefficient determination of r2 = 0.91 . the validation experimental was consistent with the prediction model . the optimum levels of process variables were temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 g / l ) , inoculum size ( 3.69 ml ) , and agitation speed ( 194.44 rpm ) . these conditions were validated experimentally which revealed an enhanced xylitol yield of 0.80 g / g .

1. Introduction 2. Materials and Methods 3. Results and Discussions 4. Conclusion

osteoporosis is a systematic bone disease characterized by low bone mass and deterioration of microarchitecture of the bone , leading to bone fragility and eventually fractures 1 . the gold standard method recommended by the world health organization in the diagnosis of osteoporosis is dual - x - ray absorptiometry ( dxa ) . using this method , osteoporosis is defined by a bone mineral density ( bmd ) lower than -2.5 standard deviations ( sd ) of the reference bmd of caucasian women aged 20 - 29 years 2 . undeniably , this simplified definition of osteoporosis eases the physicians in diagnosing and initiating treatment for osteoporotic patients . however , there are several limitations of dxa which prevent it from being used in the mass screening of osteoporosis , which is currently a rising healthcare medical condition in the developing countries 3 . quantitative ultrasound ( qus ) is a bone health assessment technique which has gained much popularity in recent years since its introduction in 1984 . compared to dxa , qus offers wider accessibility to the public because it is portable , easier to handle , lower in cost and does not emit ionizing radiation 4 . this technology has been used to determine the bone health status in women 5 , men 6 , children 7 and in certain cases , infants 8 . the ultrasound is a type of sound wave with a frequency exceeding the normal auditory range of humans ( > 20 khz ) . the sound waves produced by unique piezoelectric probes are emitted and travelled longitudinally or horizontally through the bone under study . there are usually two probes on the qus device : the emission and receiver probes . the segment of bone under study will be placed between these probes and the ultrasound waves emitted from the emission probes through the bone will be sensed by the receiver probe 9 . there are two types of qus depending on the axis the ultrasound waves take to travel through the bone . horizontal transmission uses probes that measure the speed of sound on the cortical layer of the bone at a fixed distance . longitudinal transmission is more often used and the bone segment measured is the calcaneus 4 . according to the international society of clinical densitometry ( iscd ) , calcaneal qus is the only recognized measurement of qus as the determinant of bone health status because more research has been performed on the calcaneus as compared to the other bone segments 10 . besides , the calcaneus consists of 95% trabecular bone and possesses two lateral surfaces 11 , which facilitates the movement of ultrasound through it . therefore , this discussion will emphasize on this measurement technique , which is the calcaneal qus . two parameters commonly generated by qus are the speed of sound ( sos ) and the broadband ultrasound attenuation ( bua ) . the speed of sound refers to the division of transmission time of the sound waves by the length of the body part studied . the unit used in the measurement of sos is meter per second ( m / s ) . broadband attenuation of sound refers to the slope between attenuation of sound signals and its frequency , and the unit used is db / mhz . attenuation occurs because the energy is absorbed by the soft tissue and bone when the sound waves travel through them . currently , more sophisticated qus indices derived from these two basic measurements are available , such as amplitude - depend sos ( ad - sos ) , stiffness index ( si ) , quantitative ultrasound index ( qui ) and estimated bmd ( ebmd ) . some researchers suggested that these composite parameters are more useful in the determination of subjects with low bone health status 9 . previous in vitro studies examining the relationship between calcaneal qus and bone properties found that sos was closely related to bmd 12 - 16 . toyras et al . indicated that this relationship was strong , with a coefficient of correlation ( r ) of 0.888 17 . significant correlations between sos with microarchitecture indices of the bone , such as bone volume ( bv / tv ) , bone surface ( bs / tv ) , number of nodes ( n.nd . ) , trabecular number ( tb.n . ) , trabecular thickness ( tb.th . ) and trabecular separation ( tb.sp . ) there were opinions that these correlations were mediated by the bone mass , and if bmd was controlled , these relationships would revert to become non - significant 18 . however , a computer simulation study performed by haat et al . showed that after adjusting for bmd , bv / tv remained significantly associated with sos 20 . this was confirmed by later studies using excised samples , whereby microarchitecture of the bone was significantly associated with sos and contributed to the variation of sos apart from bmd 12 , 15 . bone biomechanical studies revealed that young 's modulus , compressive modulus , ultimate strength and elasticity of bone were significantly associated with sos 12 , 16 , 21 . indicated that the combination of bone density and young 's modulus could explain 93.34% of the in vitro variation of sos 12 . studies also showed that bua was significantly associated with biomechanical parameters 21 , but toyras et al . indicated that this was only true in low - density bone samples 16 , 17 . in high - density bovine samples , these in vitro experiments ( summarized in table 1 ) showed that qus indices are able to reflect the two principal constituents of bone health , which are the bone quantity ( bmd and bone mass ) and the bone quality ( bone microarchitecture and strength ) . the stronger association between qus indices and bmd indicates that bone quantity contributes to most of the variation in qus 22 . in humans , there were significant correlations between qus indices and bmd values at various body sites assessed cross - sectionally . reported that all three qus indices , bua , sos and si were significantly correlated to bmd at lumbar spine and femur in postmenopausal women , but only sos correlated significantly to bmd at lumbar spine and femur in premenopausal women 23 . in a study by mszros et al . in men , bua correlated significantly and moderately with bmd at lumbar spine , femoral neck and radius midshaft . however , sos did not correlate with the bmd at the aforementioned sites 6 . in a longitudinal study by trimpau et al . involving 80 swedish women aged 53 - 73 years , bua and sos were significantly correlated with bmd at multiple skeletal sites at the first screening and the after seven years later . furthermore , the changes of dxa and qus measurements during the follow - up period were also significantly correlated 24 . the ability of qus to predict fractures were also validated in several human cohort studies . hernandez et al . examined 5195 spanish postmenopausal women 65 years and found that all qus indices ( bua , sos , ebmd and qui ) were significantly different between subjects with and without history of fractures . logistic regression analysis also confirmed that these qus indices were significantly associated with previous fractures 25 . the study of varenna et al . in 4832 italian men aged 60 - 80 years found that qus indices ( bua , sos , si ) were significant associated with history of hip fracture and non - spinal fracture 26 . these observations from cross - sectional studies were further validated by prospective studies . in the norfolk cohort study involving 14824 men and women discovered that one sd decrease in ultrasound velocity translated to a 60% increase in fracture risk in both genders . they also found that the risk increased for older subjects and doubled for subjects with history of fractures 27 . in the asian population , fujiwara et al . showed that sos , bua and si significantly predicted hip , wrist and non - spinal fractures in japanese men and women followed for 5 years 28 . in a recent meta - analysis , moayyeri et al . concluded that sos , bua , si and qui significantly predicted fractures after reviewing 21 independent studies 29 ( table 2 ) . several studies showed that the fracture prediction by qus was equal and sometimes better than dxa . in cross - sectional studies by mszros et al . 6 and gonnelli et al.30 involving male subjects , both studies also revealed that sos had better or the same discriminatory ability than bmd 6 , 30 . in a longitudinal study by chan et al . , the combination of bua and femoral neck bmd predicted hip , vertebral or any fractures better than individual indices in postmenopausal women but not in men followed for 13 years 31 . on the other hand , some studies reported that bmd had better discriminability than qus , and the combination of dxa and qus did not improve predictability . hong kong chinese men followed for 6.5 years demonstrated that bua , qui and bmd ( hip and spine ) significantly predicted major fragility fractures and non - vertebral fractures , but bmd was better in predicting the former . in addition , the combination of bmd and qus did not improve fracture predictability 32 . reported that only lumbar spine bmd predicted vertebral fractures in postmenopausal women but qus did not 33 . . showed similar results in pre- and postmenopausal women in their study 23 . in view of the heterogeneity of the results on the comparison , it was revealed that the predictability of qus in non - spinal fractures was similar to dxa , but dxa was more superior in predicting hip fractures 34 ( table 2 ) . according to the official position of iscd on qus bone assessment , several aspects concerning the use of t - score , reference range , precision and inter - device comparison were addressed 10 . in the classification of low bone density using dxa , t - score with cut - off points of -1.0 sd for osteopenia and -2.5 sd for osteoporosis however , the use of the same cut - off points in qus measurement is not recommended because qus and dxa essentially employ different technology in assessing bone health 10 . several studies also showed that simply applying the conventional dxa cut - offs in qus measurement significantly underestimates the true prevalence of osteoporosis 35 , 36 . a number of cut - off points for bone health classifications for qus had been suggested previously , but they were specific to the device used 37 - 39 . as an example , frost et al . reported that t - score cut off values for osteoporosis were -1.61 , -1.94 and -1.90 for bua , sos and ebmd measured using hologic sahara ultrasonometer , and -1.45 and -2.10 for bua and sos measured using osteometer dtuone 38 . the use of an appropriate reference range is important for accurate classification of bone health using qus and dxa . for example , a caucasian reference range , where bone density outcomes are typically higher than asian bone density outcomes , will eventuate in asian subjects being classified as having low bone density . chin et al . reported that even the use of references from different asian countries caused significant discrepancies in the classification of subjects with low bone health 40 . the normative values for different populations around the world had been generated for various qus devices 36 , 40 - 44 . both the population of interest and the device used should be considered when incorporating the respective normative values in qus device . due to the fact that numerous qus devices have been developed by many manufactures , each with its own designed logarithm for the calculation and interpretation of qus indices , inter - device comparison of the results of bone health assessment the precision of qus devices was reported to be poorer compared to dxa devices 45 . this may be one of the reason qus devices are not recommended for patient follow - up in the treatment of osteoporosis unless dxa is inaccessible 10 . the precision values of sos and bua are different due to the effect of a large denominator of the former , hence sos tends to have smaller precision values 45 . an example for precision values reported for the cuba mccue instrument were 2.4% for bua and 0.3% for sos 46 . we suggest that for the use of qus in the screening of bone health in local community , an ultrasonometer validated against dxa should be used . the ultrasonometer should also be equipped with the local reference curve ( or a reference curve from a population with the most similar background ) for the purpose of bone health classification . short - term and long - term in vivo precision of the device should be established for the purpose of the follow - up of subjects . furthermore , qus results should be interpreted with clinical risk factors for maximal detection of subjects with osteoporosis . several studies also indicated that it can predict fractures for both genders . in developing countries with poor accessibility to dxa , qus could be used as an effective screening tool for early detection of osteoporosis . an early detection would allow preventive measures to be taken to hinder the progression of the osteoporosis .

quantitative ultrasound ( qus ) has emerged as a convenient and popular screening tool for osteoporosis . this review aimed to provide basic information on the principle of qus measurement and discuss the properties of bone reflected by qus indices . qus employed high frequency sound waves generated by the device to determine bone health status in humans . in vitro studies showed that qus indices were significantly associated with bone mineral density ( bmd ) , bone microarchitecture and mechanical parameters . in humans , qus indices were found to be associated with bmd as well . in addition , qus could discriminate subjects with and without fracture history and predict risk for future fracture . in conclusion , qus is able to reflect bone quality and should be used in the screening of osteoporosis , especially in developing countries where dual - x - ray absorptiometry devices are less accessible to the general population .

Introduction QUS parameters and the bone properties they reflect The Comparison of Fracture Prediction between QUS and DXA Controversy in QUS Bone Assessment Summary

thrombocytosis is commonly seen in clinical practice and often the cases reported are incidental . the differential diagnosis for thrombocytosis is broad and the diagnostic process can be challenging . in general , causes of thrombocytosis can be described as spurious , reactive , or clonal in nature . a number of population studies have examined the degree of thrombocytosis as well as the frequency of various etiologies of thrombocytosis when it occurs . reactive causes are by far the most common etiology of thrombocytosis in these population studies , comprising 8897% of cases in adults in two large case series and 100% of pediatric cases in a single case series . extreme thrombocytosis , defined as a platelet count > 1,000 10/l is quite rare , as only 25.8% of patients demonstrate this degree of thrombocytosis upon presentation . although often thought to be more common in clonal processes , extreme thrombocytosis can also be due to reactive causes , with 82% of cases of extreme thrombocytosis in one series being reactive in nature . a 26-year - old male patient was scheduled for surgery of right indirect inguinal hernia with right sided encysted hydrocele of the cord . the preoperative laboratory tests reported normal coagulation and biochemical parameters , haemoglobin values of 15.1 g% , no leukocytosis and a platelet count of 2.60 lakhs / cumm . patient was operated , right sided mesh hernioplasty with excision of hydrocele of the cord was done . there were no adverse incidents during surgery , the patient was smoothly extubated in the surgery room and transferred to the post anaesthesia care unit . a routine postoperative analytical blood control was performed , showing high platelets count ( 1,100,000/cumm ) , hemoglobin 11.6 mg / dl and leukocytes 18,400/cumm , with normal values of coagulation and biochemistry tests . another blood analysis was extracted to verify these data , which again returned a high platelet count ( 1,300,000/cumm ) . we suggested monitoring platelet count throughout the postoperative period by serial analytical , with introduction of thromboembolic prophylaxis and control of bleeding in the face of the probability of some degree of platelet dysfunction . the immediate period after surgery was uneventful and the patient was discharged to the ward with hemodynamic stability . later , on the next postoperative day , patient developed huge scrotal haematoma with ecchymosis around the incision line . scrotal haematoma was explored , clots were drained , but no obvious bleeder could be identified . postoperative antithrombotic prophylaxis and bleeding monitoring were maintained until the patient was discharged after 2 weeks of admission , to follow up with outpatient treatment . analytical blood controls showed that the platelet count was down to normal in 20 days . thrombocytosis is an incidental finding in 3550% of cases and determination of the cause creates a diagnostic challenge . the causes of thrombocytosis can be described as spurious , reactive , or clonal in nature as shown in table 1 . causes of thrombocytosis the reactive thrombocytosis , also called secondary thrombocytosis is the most common type and appears after acute inflammatory , infectious , neoplastic and stress processes . in these scenarios the levels of thrombopoietin , interleukin-6 and catecholamines are very high , and are thought to be responsible for the increased number of platelets . it is always necessary to distinguish between clonal ( also known as primary or essential thrombocytosis ) and reactive ( or secondary ) thrombocytosis , because their treatment and prognosis differ . distinguishing clinical features for primary and secondary thrombocytosis although the diagnostic tests to differentiate essential and reactive thrombocytosis are not easy to perform , laboratory tests that show increased acute phase reactants such as c - reactive protein , fibrinogen , erythrocyte sedimentation rate and interleukin-6 may be useful in the diagnosis of reactive thrombocytosis . it is accepted that upto 1,000,000 l platelets level are a benign condition , although it remains unclear if these findings are associated with an increased postoperative thromboembolic or haemorrhagic risk . prophylactic treatment with platelet inhibitors in these situations is controversial , although some authors do consider management of low - dose acetylsalicylic acid appropriate . reactive thrombocytosis is generally felt thought to be a self - limited process which resolves with resolution of the underlying disorder when possible . the risk of thrombotic complications with reactive thrombocytosis is felt to be low , as 1.6% of patients with reactive thrombocytosis had thrombotic complications in one large case series . all of these thrombotic events were venous in location and occurred in patients with other risk factors ( postoperative setting or underlying malignancy ) . even in cases of extreme reactive thrombocytosis the appearance of thrombocytosis after surgery needs to be diagnosed to establish the type of thrombocytosis ( clonal or reactive ) , as treatment and prognosis are quite different between them . and at all times , the surgeon must remain vigilant due to the possible risk of bleeding or thromboembolic complications . the work described in this paper was supported jointly by department of pathology and department of surgery , himsr , new delhi . the authors would like to thank hamdard institute of medical sciences for the administrative support . the authors also thank the editor and the reviewers for their valuable comments and suggestions that have led to the substantial improvement of the paper . the work described in this paper was supported jointly by department of pathology and department of surgery , himsr , new delhi . the authors would like to thank hamdard institute of medical sciences for the administrative support . the authors also thank the editor and the reviewers for their valuable comments and suggestions that have led to the substantial improvement of the paper .

thrombocytosis is often an incidental finding seen in 3550% of cases and the cause determination creates a diagnostic challenge . extreme thrombocytosis is rare and seen in 25.8% patients only . among the various causes of increased platelet count , surgical procedures have attracted much attention in both experimental and clinical domain . the appearance of thrombocytosis after surgery needs to be diagnosed to establish the type of thrombocytosis ( clonal or reactive ) , as treatment and prognosis are quite different between them . this case report is vital because of two reasons : first , the increase in platelet count is difficult to rationalize than many of the other thrombocytoses , such as those related to primary augmentation of the function of the bone marrow ; second , the association of platelets with the clotting process has led to the belief that their increase after a surgical procedure is connected with the occurrence of postoperative thrombosis . this case presents an interesting finding from a patient who has undergone major abdominal surgery and has shown an unexpected perpetual increase in platelet count .

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patients attending the haydarpasa numune hospital neurology clinic , istanbul , turkey , were asked to volunteer for the study if they fulfilled the inclusion criteria for ad . the control group included age- and sex - matched individuals who had no cognitive decline . all patients and their carers read the information sheet provided before giving verbal and written consent to participate in the study . the echocardiographic examinations were performed in siyami ersek cardiovascular and thoracic surgery center , istanbul , turkey . the diagnosis of ad was made according to the probable ad criteria proposed by the national institute of neurological and communicative disorders and stroke and the alzheimer s disease and related disorders association ( nincds - adrda),13 as well as the diagnostic and statistical manual of mental disorders , fourth edition.14 the control group participants were defined as having no demonstrable cognitively based limitations of activities of daily living , including employment , when applicable . rigorous criteria were used to exclude anyone with any type of symptomatic or severe brain - related neurologic or psychiatric illness . this was done by prospective interview of the participant and careful scrutiny of the medical records . examples of excluded conditions were mental retardation , epilepsy , cerebral infarction or hemorrhage , multiple sclerosis , brain tumors , major depressive disorders ( unipolar or bipolar ) , schizophrenia , traumatic brain injuries , and substance abuse . patients with more than mild mitral or aortic regurgitation and aortic or mitral stenosis were also excluded from the study . the nondemented ( nd ) participants were defined as having a mini - mental state examination13,14 score of 28 or higher , a clinical dementia rating of 0 , and functional assessment staging score of 2 or lower . all echocardiographic evaluations were performed with a transducer of 2.53.5 mhz and a ge vivid 3 echocardiography device ( general electric company , milwaukee , wi , usa ) . each patient was evaluated with m - mode , two - dimensional doppler , and tissue doppler echocardio graphy . the end - diastolic and end - systolic diameters of the left ventricle ( lv ) as well as the end - systolic diameter of the left atrium ( la ) were measured with m - mode echocardiography on the parasternal long axis view , in accordance with the american society of echocardiography guidelines.15 la volume was calculated with the ( a1a2a30.524 ) formula using the left atrial diameters measured from the parasternal long axis ( anteroposterior - a1 ) and apical four - chamber ( mediolateral - a2 , apicobasal - a3 ) views . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . diastolic fillings were classified initially on the basis of the peak mitral flow velocity of the early rapid filling wave ( e ) , peak velocity of the late filling wave caused by atrial contraction ( a ) , e / a ratio , and deceleration time ( dt ) , which is the time interval for the peak e velocity to reach zero baseline . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . the longitudinal motion of the mitral annulus has been shown to correlate with the rate of myocardial relaxation.15 the velocity of the mitral annulus can be recorded by the lv - pulsed tissue doppler imaging , which is an essential part of evaluation of diastolic function . tissue doppler imaging was performed in the apical four - chamber view using a 5 mm pulsed doppler sample volume with as minimal optimal gain as possible to obtain the best signal - to - noise ratio . the signal filter of the spectral pulsed doppler was adjusted until the nyquist limit was 1520 cm / s using a transducer of 3.54.0 mhz in frequency and we used minimal optimal gain to perform the tissue doppler echocardiography . the monitor sweep speed was set at 50100 mm / s to optimize the spectral display of myocardial velocities . on the apical four - chamber view , the pw doppler cursor was placed on the lateral mitral annulus of the left ventricle , septal mitral annulus , and right ventricle s tricuspid annulus , respectively , to obtain tissue doppler spectral images . the myocardial peak early ( ea ) and late diastolic ( aa ) velocities were obtained from these areas . the time passing from the beginning of the p wave on ecg to the aa wave on tissue doppler trace was accepted as the atrial conduction time . the atrial conduction times measured from mitral lateral ( mt ) , mitral septal ( ms ) , and tricuspid lateral ( tl ) annuli were named as ml - emd ( electromechanical delay ) , ms - emd , and tl - emd , respectively . the time difference between ml - emd and tl - emd we measured aortic diameters at a level 3 cm above the aortic cusps in long axis from the parasternal view . m - mode diameter measurements were made in systole ( point of maximal anterior motion of aorta ) and at end - diastole ( q wave on ecg ) . the means of three diameter measurements in sequential cardiac cycles were used for data analysis . systemic arterial blood pressure ( bp ) was measured at the right brachial artery by manual sphygmomanometer with the patient supine using an adequately sized cuff . pulse pressure ( pp ) was calculated by subtracting the diastolic bp from the systolic bp . the elastic properties of the aorta were indexed by calculation of aortic distensibility ( d ) , stiffness index ( si ) , and pressure - strain elastic modulus ( ep ) , and were as follows : ( 1)d=2(asad)/(ad[pspd ] ) , ( 2)si = ln(ps / pd)/([asad]/ad),and ( 3)ep=(pspd)/([asad]/ad),where as is aortic diameter at end - systole , ad is aortic diameter at end - diastole , ps is systolic bp , pd is diastolic bp , and ln is natural logarithm.1618 the continuous variables within group data were calculated as average standard deviation , and categorical variables as numbers and percentages . the student s t - test was used for comparison of parametric variables and the categorical variables were tested by pearson s test and fisher s exact test . binary logistic regression analysis was used to find the possible independent association between ad and study parameters . in multivariate regression analysis , p - values are two sided , and values < 0.05 were considered statistically significant . all statistical studies were carried out using statistical package for social sciences ( spss ) software ( v 16.0 for windows ; spss inc . , chicago , il , usa ) . all echocardiographic evaluations were performed with a transducer of 2.53.5 mhz and a ge vivid 3 echocardiography device ( general electric company , milwaukee , wi , usa ) . each patient was evaluated with m - mode , two - dimensional doppler , and tissue doppler echocardio graphy . the end - diastolic and end - systolic diameters of the left ventricle ( lv ) as well as the end - systolic diameter of the left atrium ( la ) were measured with m - mode echocardiography on the parasternal long axis view , in accordance with the american society of echocardiography guidelines.15 la volume was calculated with the ( a1a2a30.524 ) formula using the left atrial diameters measured from the parasternal long axis ( anteroposterior - a1 ) and apical four - chamber ( mediolateral - a2 , apicobasal - a3 ) views . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . diastolic fillings were classified initially on the basis of the peak mitral flow velocity of the early rapid filling wave ( e ) , peak velocity of the late filling wave caused by atrial contraction ( a ) , e / a ratio , and deceleration time ( dt ) , which is the time interval for the peak e velocity to reach zero baseline . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . the longitudinal motion of the mitral annulus has been shown to correlate with the rate of myocardial relaxation.15 the velocity of the mitral annulus can be recorded by the lv - pulsed tissue doppler imaging , which is an essential part of evaluation of diastolic function . tissue doppler imaging was performed in the apical four - chamber view using a 5 mm pulsed doppler sample volume with as minimal optimal gain as possible to obtain the best signal - to - noise ratio . the signal filter of the spectral pulsed doppler was adjusted until the nyquist limit was 1520 cm / s using a transducer of 3.54.0 mhz in frequency and we used minimal optimal gain to perform the tissue doppler echocardiography . the monitor sweep speed was set at 50100 mm / s to optimize the spectral display of myocardial velocities . on the apical four - chamber view , the pw doppler cursor was placed on the lateral mitral annulus of the left ventricle , septal mitral annulus , and right ventricle s tricuspid annulus , respectively , to obtain tissue doppler spectral images . the myocardial peak early ( ea ) and late diastolic ( aa ) velocities were obtained from these areas . the time passing from the beginning of the p wave on ecg to the aa wave on tissue doppler trace was accepted as the atrial conduction time . the atrial conduction times measured from mitral lateral ( mt ) , mitral septal ( ms ) , and tricuspid lateral ( tl ) annuli were named as ml - emd ( electromechanical delay ) , ms - emd , and tl - emd , respectively . the time difference between ml - emd and tl - emd was defined as the interatrial emd . we measured aortic diameters at a level 3 cm above the aortic cusps in long axis from the parasternal view . m - mode diameter measurements were made in systole ( point of maximal anterior motion of aorta ) and at end - diastole ( q wave on ecg ) . the means of three diameter measurements in sequential cardiac cycles were used for data analysis . systemic arterial blood pressure ( bp ) was measured at the right brachial artery by manual sphygmomanometer with the patient supine using an adequately sized cuff . pulse pressure ( pp ) was calculated by subtracting the diastolic bp from the systolic bp . the elastic properties of the aorta were indexed by calculation of aortic distensibility ( d ) , stiffness index ( si ) , and pressure - strain elastic modulus ( ep ) , and were as follows : ( 1)d=2(asad)/(ad[pspd ] ) , ( 2)si = ln(ps / pd)/([asad]/ad),and ( 3)ep=(pspd)/([asad]/ad),where as is aortic diameter at end - systole , ad is aortic diameter at end - diastole , ps is systolic bp , pd is diastolic bp , and ln is natural logarithm.1618 the continuous variables within group data were calculated as average standard deviation , and categorical variables as numbers and percentages . the student s t - test was used for comparison of parametric variables and the categorical variables were tested by pearson s test and fisher s exact test . binary logistic regression analysis was used to find the possible independent association between ad and study parameters . in multivariate regression analysis , p - values are two sided , and values < 0.05 were considered statistically significant . all statistical studies were carried out using statistical package for social sciences ( spss ) software ( v 16.0 for windows ; spss inc . , a total of 53 individuals were enrolled : 29 in the ad group ( 12 male and 17 female ; mean age standard deviation , 76.85.2 years ) and 24 in the nd control group ( 14 female and ten male ; mean age 77.16.7 years ) . the age difference between the ad and nd groups was not statistically significant ( p=0.65 ) . there were no significant differences between alzheimer patients and controls regarding age , sex , body mass index , and risk factors , including hypertension , diabetes mellitus , and smoking status . lv diastolic and systolic diameters , lv ejection fraction , and lv mass were comparable between the two groups . la anteroposterior diameters and la volumes were significantly higher in the ad group ( p=0.01 , p=0.01 , respectively ) . mitral valve pw doppler parameters were significantly different between the two groups , with lower e velocities and higher a velocities , dt , and ivrt in alzheimer patients ( p=0.01 for each comparison ) . mitral annular ea velocity was significantly lower in alzheimer patients , whereas aa velocity and e / ea ratio were similar between the groups . aortic diameters , parameters of aortic stiffness , and atrial conduction times are reported in table 3 . systolic aortic diameter was comparable between the groups ; however , diastolic aortic diameter was significantly higher in the alzheimer patients . ( 11.85.7 versus 3.31.1 ; p=0.01 ) and ep ( 139.538.7 versus 46.312.1 kpa ; p=0.01 ) were significantly higher , and d was significantly lower ( 1.510.75 versus 4.551.12 10 cm / dyn ; p=0.01 ) in the ad group . the atrial conduction times measured from the mitral valve s lateral ( ml ) and septal ( ms ) annuli were significantly higher in the alzheimer patients . however , the conduction time measured from the tl annulus was similar between the ad and nd groups . the interatrial emd was significantly higher in the ad group ( 16.111.1 versus 10.56.4 ms ; p=0.03 ) . in univariate binary logistic regression analysis , pp ( odds ratio [ or ] : 1.18 , 95% confidence interval [ ci ] : 1.071.306 ; p=0.01 ) , la volumes ( or : 1.20 , 95% ci : 1.081.35 ; p=0.01 ) , interatrial emd ( or : 1.07 , 95% ci : 1.011.146 ; p=0.04 ) , si ( or : 4.46 , 95% ci : 1.7111.64 ; p=0.01 ) , d ( or : 0.08 , 95% ci : 0.020.32 ; p=0.01 ) , and ep ( or : 1.12 , 95% ci : 1.041.21 ; p=0.01 ) were correlated with presence of ad . in multivariate regression analysis , using a model adjusted for age , sex , presence of hypertension , la volumes , and interatrial emd , d ( or : 0.09 , 95% ci : 0.020.41 ; p=0.01 ) and ep ( or : 1.12 , 95% ci : 1.021.23 ; p=0.02 ) were found to be independent correlates of ad . however , age , sex , presence of hypertension , la volume , interatrial emd , and si did not remain as independent correlates of the disease . the main findings of our study were that patients with ad have abnormal aortic stiffening compared to age- and sex - matched controls . even though lv diastolic parameters and atrial conduction times in ad patients were significantly different from control subjects , these parameters were not independent correlates of ad . in this study , we observed that patients with ad have altered diastolic function when compared to a control group . the intergroup differences of the e and a wave velocity , e / a ratio , dt , ivrt , la volume , and atrial conduction times measured from ml and ms annuli were statistically significant . mitral valve pw parameters were significantly different between the two groups , with lower e velocities and higher a velocities , dt , and ivrt in alzheimer patients . mitral annular ea velocity was significantly lower in alzheimer patients , whereas aa velocity and e / ea ratio were similar between the groups . our results from both the ad and control groups were consistent with borderline low e and borderline high a components of the diastolic filling velocities , resulting in an e / a ratio of less than 1.0 . this level of diastolic relaxation impairment is consistent with the age range of our study participants . however , our findings were associated with statistically significant impairment of diastolic parameters in the ad group when compared with the nd group . similar to our findings , belohlavek et al19 found that patients with ad have altered mitral inflow parameters and vortex formation time . however , they did not evaluate for the more sensitive tissue doppler parameters for diastolic functions that we observed in our study . in previous studies , it was shown that ad was associated with vascular risk factors and cardiovascular disease process.5,2023 hypertension , diabetes , stroke , and atherosclerosis have been shown to increase ad risk.7,24,25 during the aging process , heart , arteries , and brain suffer the continuous cumulative consequences of hemodynamic and mechanical stresses;19,2628 further , the myocardium undergoes structural changes including interstitial fibrosis and cellular disarray , which affects the diastolic functions negatively as a result of aging.2931 in this study , we observed that intraventricular blood transport rheologic processes are suboptimal and altered in patients with ad . although no causal relationship can be deduced from our data , it may be further speculated that the suboptimal hemodynamic efficiency could be the pathophysiologic link between the more pronounced lv relaxation dysfunction and ultimate brain cognitive dysfunction in patients with ad . however , these are preliminary observations and need to be taken cautiously because of the small number of patients involved in this study and the limitations in the accuracy of the clinical diagnosis of ad . in addition to conventional diastolic parameters we observed that patients with ad had increased atrial conduction times . yavuz et al32 found that atrial conduction times increase in hypertensive patients with diastolic dysfunction . also in yavuz et al s study,32 the atrial conduction times measured from ml and ms and also interatrial emd were longer in the ad group , which can be associated with more impaired diastolic function . in our study , we observed that pp , ep , d , and si were correlated with presence of ad . the measurements in our study were made by conventional echocardiography , and this is the key difference of our study from others.10,11,28,33 similar to our findings , hanon et al10 observed that pw velocity ( pwv ) was significantly higher in subjects with vascular dementia or ad than in those without cognitive impairment , and they pointed out that arterial stiffness appeared to be an independent determinant not only of vascular dementia , but also of ad . a previous study by bateman et al28 showed that early ad is characterized by reduced blood flow and reduced compliance . in contrast to our observations , a previous study by dhoat et al11 which compared vascular compliance and arterial stiffness between vascular dementia , ad , and control groups by measuring central arterial compliance , augmentation index , and pwv , concluded that arterial stiffness in patients with ad is no different from that in controls . several mechanisms may explain our findings of an association between increased aortic stiffness and ad . aortic stiffness can cause an increase in central pp , which may influence arterial remodeling at the site of the extracranial and intracranial arteries . indeed , pp and arterial stiffness have been related to atherosclerosis or arteriosclerosis in large34,35 and small36 vessels . an increased pp has been associated with the prevalence and severity of cerebral white matter lesions,37 and , in a recent study , aortic stiffness appeared as an independent predictor of stroke in patients with essential hypertension.38 our study indicates an independent correlation between aortic stiffness and ad , supporting previous studies suggesting that functional changes of the arterial system10,11,28,33 could play a role in the pathogenesis of alzheimer dementia . this study was a cross - sectional study without long - term follow - up , and the prognostic importance of these echocardiographic parameters is not known . in this study , we observed that patients with ad have altered diastolic function when compared to a control group . the intergroup differences of the e and a wave velocity , e / a ratio , dt , ivrt , la volume , and atrial conduction times measured from ml and ms annuli were statistically significant . mitral valve pw parameters were significantly different between the two groups , with lower e velocities and higher a velocities , dt , and ivrt in alzheimer patients . mitral annular ea velocity was significantly lower in alzheimer patients , whereas aa velocity and e / ea ratio were similar between the groups . our results from both the ad and control groups were consistent with borderline low e and borderline high a components of the diastolic filling velocities , resulting in an e / a ratio of less than 1.0 . this level of diastolic relaxation impairment is consistent with the age range of our study participants . however , our findings were associated with statistically significant impairment of diastolic parameters in the ad group when compared with the nd group . similar to our findings , belohlavek et al19 found that patients with ad have altered mitral inflow parameters and vortex formation time . however , they did not evaluate for the more sensitive tissue doppler parameters for diastolic functions that we observed in our study . in previous studies , it was shown that ad was associated with vascular risk factors and cardiovascular disease process.5,2023 hypertension , diabetes , stroke , and atherosclerosis have been shown to increase ad risk.7,24,25 during the aging process , heart , arteries , and brain suffer the continuous cumulative consequences of hemodynamic and mechanical stresses;19,2628 further , the myocardium undergoes structural changes including interstitial fibrosis and cellular disarray , which affects the diastolic functions negatively as a result of aging.2931 in this study , we observed that intraventricular blood transport rheologic processes are suboptimal and altered in patients with ad . although no causal relationship can be deduced from our data , it may be further speculated that the suboptimal hemodynamic efficiency could be the pathophysiologic link between the more pronounced lv relaxation dysfunction and ultimate brain cognitive dysfunction in patients with ad . however , these are preliminary observations and need to be taken cautiously because of the small number of patients involved in this study and the limitations in the accuracy of the clinical diagnosis of ad . in addition to conventional diastolic parameters we observed that patients with ad had increased atrial conduction times . yavuz et al32 found that atrial conduction times increase in hypertensive patients with diastolic dysfunction . also in yavuz et al s study,32 the atrial conduction times measured from ml and ms and also interatrial emd were longer in the ad group , which can be associated with more impaired diastolic function . in our study , we observed that pp , ep , d , and si were correlated with presence of ad . the measurements in our study were made by conventional echocardiography , and this is the key difference of our study from others.10,11,28,33 similar to our findings , hanon et al10 observed that pw velocity ( pwv ) was significantly higher in subjects with vascular dementia or ad than in those without cognitive impairment , and they pointed out that arterial stiffness appeared to be an independent determinant not only of vascular dementia , but also of ad . a previous study by bateman et al28 showed that early ad is characterized by reduced blood flow and reduced compliance . in contrast to our observations , a previous study by dhoat et al11 which compared vascular compliance and arterial stiffness between vascular dementia , ad , and control groups by measuring central arterial compliance , augmentation index , and pwv , concluded that arterial stiffness in patients with ad is no different from that in controls . several mechanisms may explain our findings of an association between increased aortic stiffness and ad . aortic stiffness can cause an increase in central pp , which may influence arterial remodeling at the site of the extracranial and intracranial arteries . indeed , pp and arterial stiffness have been related to atherosclerosis or arteriosclerosis in large34,35 and small36 vessels . an increased pp has been associated with the prevalence and severity of cerebral white matter lesions,37 and , in a recent study , aortic stiffness appeared as an independent predictor of stroke in patients with essential hypertension.38 our study indicates an independent correlation between aortic stiffness and ad , supporting previous studies suggesting that functional changes of the arterial system10,11,28,33 could play a role in the pathogenesis of alzheimer dementia . this study was a cross - sectional study without long - term follow - up , and the prognostic importance of these echocardiographic parameters is not known . our findings suggest that patients with ad , the most common form of dementia , are more likely to have diastolic dysfunction and longer atrial conduction times . more importantly , our study has shown that d and ep , which are the basic parameters of aortic stiffness , were independently correlated with presence of ad . although we could not detect a causal relationship in this study , subclinical cardiac involvement in ad warrants further investigation . further prospective studies are needed to clarify the role of these findings during disease development , progress , and management .

backgroundalzheimer s disease ( ad ) is closely linked to cardiovascular risk factors.methodsechocardiographic studies were performed , including left ventricular diastolic functions , left and right atrial conduction times , and arterial stiffness parameters , namely stiffness index , pressure - strain elastic modulus , and distensibility , on 29 patients with ad and 24 age - matched individuals with normal cognitive function.resultsthe peak mitral flow velocity of the early rapid filling wave ( e ) was lower , and the peak velocity of the late filling wave caused by atrial contraction ( a ) , deceleration time of peak e velocity , and isovolumetric relaxation time were higher in the ad group . the early myocardial peak ( ea ) velocity was significantly lower in ad patients , whereas the late diastolic ( aa ) velocity and e / ea ratio were similar between the two groups . in alzheimer patients , stiffness index and pressure - strain elastic modulus were higher , and distensibility was significantly lower in the ad group compared to the control . interatrial electromechanical delay was significantly longer in the ad group.conclusionour findings suggest that patients with ad are more likely to have diastolic dysfunction , higher atrial conduction times , and increased arterial stiffness compared to the controls of same sex and similar age .

Introduction Methods Echocardiography Statistical analyses Results Discussion Diastolic functions and atrial conduction time Aortic stiffness Limitations Conclusion

the subplate forms a transient layer in the developing cerebral cortex and consists of migratory and postmigratory neurons , dendrites , axons , growth cones , synapses and glial cells . the subplate is located between the intermediate zone and the cortical plate , which during further development differentiates into the neocortical layers ii to vi ( figure 1a ) . kostovic and molliver were the first who identified the subplate as a distinct layer in the embryonic human cerebral cortex ( kostovic and molliver , 1974 ) . a subplate can be identified in all mammals although its relative thickness , developmental profile and persistence in adulthood vary among species . anatomical data indicate an evolutionary difference in the ontogenetic fate of the subplate . in the rat and other rodents many subplate cells survive into adulthood forming layer vib or vii ( aboitiz and montiel , 2007 ) . prominent species differences also exist in the relative thickness of the subplate , which increased during evolution . in humans the subplate develops to approximately six times the thickness of the cortical plate around 29 weeks of gestation ( mrzljak et al . , 1990 ) , whereas in rodents it remains a relatively thin layer during development ( uylings et al . , 1990 ) . the majority of the subplate cells are born early before the first cortical plate neurons ( luskin and shatz , 1985 ; valverde et al . , 1989 ) . in rodents subplate neurons may be also generated later in development ( hevner et al . , 2004 ) . a substantial proportion of subplate cells are not born in the ventricular neuroepithelium , but instead originate in the medial ganglionic eminence and follow a tangential migratory route to their positions in the developing cortex ( lavdas et al . , 1999 ) . subplate cells represent a rather heterogeneous neuronal population according to their morphology , neurotransmitter identity and connectivity ( for review allendoerfer and shatz , 1994 ) . ( a ) prenatal development of the human cerebral cortex as suggested by bystron et al . ( 2008 ) . drawing from pasko rakic . photograph shows coronal brain section stained with cresyl violet of a gestational week 18 human neocortex . approximate embryonic day ( e ) and gestational week ( gw ) are given for each developmental stage . cp , cortical plate ; iz , intermediate zone ; mz , marginal zone ; pp , preplate ; sp , subplate ; svz , subventricular zone ; ( sg ) , subpial granular layer ( part of mz ) ; vz , ventricular zone . ( b ) morphological properties of biocytin - stained subplate neurons in newborn rat cerebral cortex . ( b1 ) postnatal day ( p ) three horizontal bitufted cell with large fusiform soma and primary dendrites oriented parallel to the pial surface . ( b3 ) p3 multipolar cell with extensive dendritic arborization within sp and layers v / vi . ( b4 ) p2 inverted pyramidal neuron with triangular soma and apical dendrite oriented towards white matter . scale bar in ( b4 ) corresponds to ( b1b4 ) and pial surface is located toward the top in all photomicrographs of ( b ) . reproduced and modified with permission from ( bystron et al . , 2008 ) ( a ) , ( kostovic et al . , 2002 ) [ photograph in ( a ) ] and from ( hanganu et al . , 2002 ) ( b ) . subplate cells play important roles in the structural and functional organization of the cerebral cortex and in early necortical plasticity . axons arising from subplate neurons pioneer the corticofugal pathway and have been proposed to form a cellular scaffold for guiding thalamocortical axons ( mcconnell et al . 1990 ) ( for review allendoerfer and shatz , 1994 ; molnr , 1998 ) . thalamic axons ( lund and mustari , 1977 ; rakic , 1977 , 1983 ) and early deletion of subplate neurons in kitten visual cortex prevents the segregation of thalamocortical axons within layer iv and the formation of ocular dominance columns ( ghosh and shatz , 1992 ; kanold et al . , 2003 ) . furthermore , subplate cells regulate the maturation of gabaergic synaptic transmission and establish the balance between excitation and inhibition in the developing neocortical network ( kanold and shatz , 2006 ) . subplate neurons reveal a large variety of morphologies ( figure 1b ) ( hanganu et al . , 2002 ) . inverted pyramidal - like and horizontal cells as well as polymorphic neurons with different shapes and spiny or smooth dendrites have been classified as subplate neurons ( kostovic and rakic , 1980 ; wahle et al . , 1987 ; valverde et al . , 1989 ; kostovic and rakic , 1990 ) due to their earlier generation and more mature developmental stage , subplate neurons show a relatively extensive dendritic tree when compared to the more immature pyramidal neurons of the cortical plate ( mrzljak et al . , 1992 ) . descending dendrites from subplate neurons may invade the underlying intermediate zone and ascending dendrites may extend into the cortical plate ( del ro et al . , 2000 ) . subplate neurons reveal markers for gaba or glutamate and may co - express various peptides ( chun et al . , 1987 ; the morphological and neurochemical heterogeneity also explains , why various attempts failed to identify a specific marker for subplate neurons ( wahle et al . , 1994 ) . ( 2009 ) succeeded in identifying a number of novel markers for murine subplate cells , which may soon allow the definition of different subpopulations of subplate neurons . subplate cells participate in local and long - distance axonal connections indicating that these neurons may function as local circuit as well as projection neurons . they show a dense axonal arborization within the subplate , but also project to the marginal zone / layer i ( clancy and cauller , 1999 ) and to the cortical plate , where they form axonal collaterals within layer iv ( friauf et al . , the majority of the subplate neurons projecting into the cortical plate reside in the upper half of the subplate and provide a glutamatergic synaptic input to the developing cortical plate , including the layer iv neurons ( friauf et al . , 1990 ; long - distance axons from subplate neurons invade the thalamus during early stages of corticogenesis and form an axonal scaffold for the establishment of cortical efferent and afferent projections ( mcconnell et al . , 1989 , 1994 ; kim et al . , 1991 ; de carlos and o'leary , 1992 ) . beside these local and long - distance projections arising from glutamatergic subplate neurons , gabaergic subplate cells also project to both neighbouring and more distant neocortical regions and form a corticocortical synaptic network ( tomioka et al . , 2005 ; higo et al . , 2007 ) . since gaba may act as an excitatory neurotransmitter during early cortical development ( for review ben - ari et al . , 2007 ) , the postsynaptic action of gabaergic subplate neurons may be also depolarizing ( figure 6 in hanganu et al . , 2002 ) . ultrastructural studies of subplate cells in various species have demonstrated symmetrical as well as asymmetrical synapses with relatively mature properties ( kostovic and rakic , 1980 , 1990 ; chun and shatz , 1988 ; herrmann et al . , 1994 ) , indicating that subplate neurons receive gabaergic as well as glutamatergic synaptic inputs . as suggested by kostovic and rakic ( 1980 ) , glutamatergic inputs onto subplate neurons may arise from the thalamus and other neocortical areas , whereas gabaergic synaptic inputs may originate from gabaergic interneurons in the subplate . thalamocortical synaptic contacts with spines and shafts of subplate neuron dendrites have been demonstrated in the neonatal ferret ( herrmann et al . , 1994 ) and a dense network of corticocortical fibers have been reported in the subplate of the embryonic mouse ( crandall and caviness , 1984 ) . n - methyl - d - aspartate ( nmda ) , -amino-3-hydroxy-5-methylisoxazole-4-propionic acid ( ampa ) and kainate receptors and the essential subunits for their receptor function have been demonstrated in the subplate of various species , suggesting the presence of functional glutamatergic synapses in subplate neurons ( herrmann , 1996 ; aoki , 1997 ; catalano et al . , gabaa receptors ( huntley et al . , 1990 ) and gabaa receptor subunits ( meinecke and rakic , 1992 ) in the subplate indicate that functional gabaergic synaptic inputs should also be present in subplate neurons . these morphological , ultrastructural and immunohistochemical data are complemented by functional studies on the properties of the subplate and single subplate cells in different mammalian species . in contrast to the heterogeneity in morphological and chemical appearance , electrophysiological recordings from single subplate neurons in rodents demonstrate rather homogeneous functional properties . whole - cell patch - clamp recordings from subplate cells in newborn rodent neocortical slices revealed resting membrane potentials and input resistances in the range of 55 mv and 11.2 g , respectively ( luhmann et al . , hirsch and luhmann , 2008 ) . in response to sustained depolarization by intracellular current injection , subplate neurons are capable of firing overshooting and repetitive action potentials at frequencies exceeding 40 hz ( figures 2c and 3a ) . similar results were obtained from subplate neurons in acute neocortical slices harvested postmortem from human fetal brain at gestational week 1622 ( moore et al . , 2009 ) . ( a ) intracellular biocytin staining of one subplate neuron ( yellow arrow ) in the p3 mouse neocortex results in columnar labeling of several dye - coupled cells in the subplate and in the cortical plate.(b ) photograph of two simultaneously recorded p1 subplate neurons , which were both stained with biocytin . ( c ) paired current - clamp recordings of the cells shown in ( b ) . black traces were obtained from the cell marked by black arrow in ( b ) and red traces are from the cell marked by red arrow . ( c1 ) injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by black arrow causes voltage deflections in the gap junction coupled neuron . ( c2 ) vice versa , injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by red arrow causes voltage deflections in the other neuron . cortical surface in ( a ) and ( b ) is up , action potentials in ( c ) are truncated . the hyperpolarizing responses in the lower traces are averages of four recordings . reproduced and modified with permission from ( dupont et al . , 2006 ) . ( a ) typical action potential firing pattern of a subplate neuron in the cerebral cortex of a p1 rat . ( b ) excitatory postsynaptic currents ( epscs ) recorded in a p3 subplate cell at a holding potential of 70 mv and elicited by selective electrical stimulation of the thalamocortical input ( see c ) . synaptic responses were obtained under control conditions in normal extracellular bathing solution and after application of 10 m cnqx to block ampa / kainate receptors . the inset shows the unimodal latency distribution of 50 thalamocortical epscs recorded from one spn . ( c ) photomicrograph of biocytin - stained thalamocortical projections in a 400-m thick coronal slice from a p1 rat . asterisk shows extracellular injection site of biocytin crystal and white circles mark position of bipolar electrode to selectively activate the thalamocortical input . ( d ) schematic diagram illustrating the distribution of postsynaptic receptors on a subplate neuron for monosynaptic inputs arising from the thalamus ( thal . ) , the cortical plate ( cp ) including layers v / vi and within the subplate ( sp ) . note that synaptic inputs from other subplate neurons can lead to activation of depolarizing gabaa receptors and nmda receptors activated at negative membrane potentials . reproduced and modified with permission from ( luhmann et al . , 2000 ) ( a ) ( hanganu et al . , 2002 ) ( b d ) . in comparison to other neurons in the immature cerebral cortex , subplate cells also reveal the most mature properties in action potential characteristics and in the biophysical properties of voltage - dependent sodium and calcium currents . these observations have been made in developing rodent ( luhmann et al . , 2000 ) as well as in human cerebral cortex ( moore et al . , 2009 ) , indicating that these relatively mature functional properties enable subplate cells to transmit afferent neuronal activity faithfully to the developing cortical plate . intracellular labeling of single subplate cells with fluorescent dyes or biocytin revealed an extensive neuronal network of dye - coupled neurons in the subplate and cortical plate ( figure 2a ) . in newborn rats , on average about nine neurons are dye - coupled to a single subplate cell and these gap junction coupled networks are often organized in a columnar manner ( dupont et al . , 2006 ) . whole - cell patch - clamp recordings from pairs of dye - coupled subplate neurons ( figure 2b ) allowed a more detailed electrophysiological characterization of the electrical synapses connecting subplate neurons ( figure 2c ) . the average coupling conductance amounted to about 1.2 ns ( dupont et al . , 2006 ) . these data indicate that subplate cells are strongly coupled via electrical synapses and form a functional columnar syncytium with neurons located in the cortical plate . it is tempting to speculate that this early columnar organization results from the radial , column - like neuronal migration of newly generated neurons into the developing neocortex ( noctor et al . , 2004 ) , which is also controlled by gap junctional coupling ( elias et al . , 2007 ) electrophysiological and optical imaging recordings further support the hypothesis that subplate neurons are well integrated in the developing cerebral cortex . in vitro intracellular recordings and current - source density analyses in late embryonic and early postnatal kitten visual cortex demonstrated that subplate neurons receive functional excitatory synaptic inputs from axons that course in the developing white matter ( friauf et al . subplate cells in newborn rat somatosensory cortical slices reveal a substantial amount of spontaneous postsynaptic currents ( spscs ) with different kinetics and pharmacological profile demonstrating that subplate neurons receive functional synaptic inputs mediated by ampa , nmda and gabaa receptors ( hanganu et al . , 2001 ) . a more detailed analysis of the synaptic inputs onto subplate cells could be obtained by electrical stimulation of specific afferent axonal projections ( figures 3b , c ) ( staiger et al . , 1999 ) . brief electrical stimulation of the thalamocortical projection elicits in immature rat and cat subplate neurons a fast and reliable excitatory postsynaptic potential / current ( epsp / c ) , which can blocked by the ampa / kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione ( cnqx ) ( friauf et al . , 1990 ; hanganu et al . , elicits a cnqx - insensitive component , which can be blocked by the nmda receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ( cpp ) , demonstrating that the thalamic input also activates functional nmda receptors on subplate cells ( hanganu et al . , 2002 ) . these electrophysiological observations from single subplate neurons are further supported by current source - density analyses performed in late embryonic and early postnatal kitten ( friauf and shatz , 1991 ) and rat cerebral cortex ( molnr et al . , 2003 ) , demonstrating that subplate cells in these species receive a functional thalamic input at earliest stages of neocortical development . recording of optical images with voltage - sensitive dyes in neocortical slices of prenatal rats have shown that there is a few days delay between the arrival of thalamocortical axons at the subplate at e16 and the appearance of functional thalamocortical synaptic transmission at e19 ( higashi et al . , 2002 ) . beside a glutamatergic thalamocortical input with relatively mature functional properties ( short delay , fast kinetics , reliable responses ) , subplate cells in newborn rodents receive additional intracortical synaptic inputs from various presynaptic sources ( figure 3d ) . local glutamatergic synaptic inputs arise from pyramidal neurons in the cortical plate and from glutamatergic subplate cells , which both activate postsynaptic ampa / kainate and nmda receptors ( hanganu et al . , this intra - subplate glutamatergic input differs from the thalamocortical input . whereas the intra - subplate input reveals a pronounced facilitation when repetitively activated at 1040 hz , the thalamocortical input is rather stable or suppressed at these stimulation frequencies ( figure 4 ) ( hirsch and luhmann , 2008 ) . the intra - subplate synaptic input can sustain high stimulation frequencies and may boost the thalamocortical input , thereby enhancing activity in the developing neocortical circuit ( hirsch and luhmann , 2008 ) . anatomical studies in rodent and human immature cerebral cortex indicate that glutamatergic synaptic inputs most likely also arise from other neocortical sources via corticocortical connections ( ivy and killackey , 1981 ; kostovic and jovanov - milosevic , 2006 ) , but the functional properties of these long - distance synaptic inputs onto subplate cells are currently unknown . input - specific differences in temporal summation of nmda receptor - mediated epsps recorded in subplate neurons of the newborn mouse cerebral cortex . recordings were performed in the presence of cnqx and bicuculline methiodide to isolate nmda receptor - mediated epsps . ( a ) five successive stimuli to the thalamic ( left ) or subplate ( right ) input reveals prominent temporal summation and repetitive action potentials ( truncated ) of the subplate input at frequencies between 10 and 40 hz . ( b ) synaptic response to 20-hz subplate stimulation at higher resolution [ same trace as in ( a ) ] . ( c ) quotient between nmda receptor - epsp integral and control integral ( determined at 0.03 hz ) for 13 subplate neurons . reproduced with permission from hirsch and luhmann ( 2008 ) . subplate cells in rodents receive a gabaergic synaptic input from neighbouring gabaergic neurons located in the subplate and probably also in the cortical plate ( figure 3d ) ( hanganu et al . , 2002 ) . however , as in other immature brain structures , this gabaergic input most likely has a pure excitatory postsynaptic effect ( figure 6 in hanganu et al . an excitatory effect has been demonstrated in subplate cells also for glycine and taurine ( kilb et al . , 2008 ) . subplate neurons are not only well integrated in the developing cortical circuit , but during certain developmental periods they also receive a very selective input from neuromodulatory brain structures . both in primates ( for review ( rakic , 1995 ) as well as in rodents ( calarco and robertson , 1995 ; mechawar and descarries , 2001 ) the subplate is specifically innervated by cholinergic fibers arising from the basal forebrain and from monoaminergic inputs . the activation of postsynaptic nicotinic acetylcholine receptors elicits in subplate cells a marked depolarization which is largely mediated by the activation of alpha4/beta2 receptors ( hanganu and luhmann , 2004 ) . the responsiveness to activation of muscarinic acetylcholine receptors ( machr ) is more complex and reveals a remarkable oscillatory discharge mode of subplate cells ( hanganu et al . , 2009 ) . application of muscarine to neocortical slices from newborn rats induces in subplate cells repetitive burst discharges with burst frequencies in the range of 20 hz ( figure 5a ) ( hanganu et al . , 2009 ) . similar burst patterns can be recorded from subplate neurons when the tissue concentration of acetylcholine is raised by application of the cholinesterase inhibitor neostigmine ( figure 5b ) . these electrophysiological data are further supported by calcium imaging experiments , which demonstrate that activation of muscarinic receptors induces repetitive ca - transients which are highly coordinated within the subplate ( figure 1 in hanganu et al . , 2009 ) . subplate cells , which express the m1m5 subunits of the machr , are instantly and massively excited upon activation of postsynaptic muscarinic receptors and switch into an oscillatory burst firing mode ( figure 5 ) . since subplate cells are densely coupled via electrical ( figure 2 ) and chemical synapses ( figure 3d ) , these cellular oscillations are synchronized and amplified within the intra - subplate network . gap junctional coupling , depolarizing gaba actions and a tonic non - synaptic gaba release contribute to the generation and maintenance of the cholinergic network oscillations ( figure 9 in hanganu et al . , 2009 ) . subplate neurons in newborn rat cerebral cortex reveal repetitive burst discharges upon activation of postsynaptic cholinergic receptors . ( a ) current - clamp whole - cell recording from a p3 subplate cell at a holding potential of 70 mv . note the long - lasting burst discharge after a single 30-s long bath application of muscarine ( marked by a black bar ) . frequency power spectrum of the action potential discharge within one representative burst from the trace above . ( b ) current - clamp whole - cell recording from a p2 subplate neuron at a holding potential of 70 mv displaying the effects of 10 m neostigmine , which blocks endogenous acetylcholine esterase and thereby increases the concentration of acetylcholine in the neocortical slice . frequency power spectrum of the action potential discharge within burst from the upper trace in ( b ) . the crucial role of the subplate in generating cholinergic network oscillations is supported by experiments on thick ( 8001000 m ) neocortical slices from newborn rodents . only slices with an intact subplate reveal network oscillations upon activation of muscarinic receptors with carbachol ( dupont et al . , 2006 ) . when the subplate is removed , slices do not show any carbachol - induced network oscillations ( figure 6 ) . furthermore , selective electrical stimulation of the subplate in thick neocortical slices from newborn mice using a multi - electrode array ( mea ) also evokes large - scale network oscillations ( figure 7 ) ( sun and luhmann , 2007 ) . in a recent study on mouse neocortical slice cultures , moody and coworkers suggested that the subplate may act as a pacemaker region to generate propagating waves of spontaneous activity in the neonatal cerebral cortex ( lischalk et al . , 2009 ) . , 2006 ; sun and luhmann , 2007 ) or by the sensory input , i.e. the retina ( hanganu et al . , 2006 ) or the whiskers ( yang et al . , 2009 ) . at early developmental stages , the sensory input from the periphery firstly reaches the subplate and is subsequently transmitted to the cortex ( friauf and shatz , 1991 ) . during this developmental period , role of the subplate in triggering carbachol - induced network oscillations in the neonatal mouse cerebral cortex . ( a ) field potential response to carbachol application in a coronal 800-m thick cortical slab preparation with an intact subplate as shown in nissl stained section below . ( b ) lack of carbachol - induced activity in a cortical slab preparation , in which the subplate was eliminated . local electrical stimulation of the subplate elicits propagating network oscillations in the developing cerebral cortical network of the newborn mouse . ( a ) photograph of p3 mouse nissl - stained coronal slice with cortical layers and position of 60-channel multi - electrode array ( mea ) . ( b ) mea recordings from the neocortical slice shown in ( a ) with site of bipolar stimulation in the subplate ( 80 a , 200-s duration , 50 hz , 10 times ) . spacing between electrodes ( c ) recording at electrode number 84 [ dotted red rectangle in ( a ) ] shown at higher resolution with corresponding wavelet analysis and fourier spectrum . reproduced with permission from sun and luhmann ( 2007 ) . electrophysiological and calcium imaging recordings in acute neocortical slices from newborn rodents ( hanganu et al . , 2009 ) as well as calcium imaging data obtained in neocortical cell cultures ( voigt et al . , 2001 ) indicate that gabaergic subplate cells play a central role in generating this neuronal network activity . voigt et al . ( 2001 ) estimated that a minimal number of two gabaergic subplate neurons per square millimeter are required for the occurrence of synchronous network activity . the unique structural and functional properties enable subplate cells to receive , synchronize and amplify the afferent and intrinsic synaptic inputs . it is tempting to speculate on the downstream effects of these early network oscillations in cortical maturation ? it has been demonstrated that brain derived neurotrophic factor ( bdnf ) is released from synaptically localized secretory granules following burst stimulation at 2050 hz ( balkowiec and katz , 2000 ; hartmann et al . , 2001 ) ( for review lessmann et al . , 2003 ) . if subplate - driven oscillations contribute to the local secretion of bdnf , the released bdnf may strengthen the synaptic connectivity between neurons in these early ensembles . beside strengthening synaptic connections via local bdnf release , we recently demonstrated that the rate of apoptosis in organotypic slice cultures of the neonatal mouse cerebral cortex is regulated by electrical activity patterns , which resemble in many aspects the subplate - driven activity ( heck et al . , 2008 ) . this activity - dependent regulation of neuronal apoptosis was at least partly mediated by activation of the bdnf receptor trkb , indicating that subplate - driven network oscillations may also influence programmed cell death via activity - dependent bdnf release . a large proportion of newly generated gabaergic interneurons arising from the medial ganglionic eminence and pyramdial neurons from the ventricular zone must migrate through the subplate on their way to the developing cortical plate ( kriegstein and noctor , 2004 ) . gabaergic and glutamatergic subplate neurons partly release their neurotransmitter in a paracrine , non - synaptic manner ( hanganu et al . , 2009 ) , thereby regulating the neuronal migration pattern ( reiprich et al . , 2005 ; manent et al . , 2005 , 2006 ; . neurotransmitter release in the subplate may rise substantially during oscillatory network activity , thereby activating low affinity or extrasynaptic receptors causing alterations in neuronal migration ( denter et al . , 2009 ) . on the basis of the currently available data we suggest the following model ( figure 8) : during early cortical development , in most mammals before birth , subplate cells with relatively mature structural properties ( elaborated dendritic tree , complex axonal projections , mature symmetrical and asymmetrical synapses ) receive a functional glutamatergic synaptic input from specific thalamic nuclei and a selective input from neuromodulatory systems ( e.g. cholinergic inputs from the basal forebrain ) . in rodents , subplate neurons are densely interconnected via electrical and chemical synapses and upon activation of muscarinic ( and probably also other metabotropic receptors ( wagner and luhmann , 2006 ) discharge in repetitive 20 hz bursts ( lower inset in figure 8) . gabaergic subplate neurons releasing gaba in a synaptic ( 1 in figure 8) and tonic non - synaptic manner ( 2 in figure 8) facilitate the generation of oscillatory network activity ( hanganu et al . , 2009 ) . intra - subplate connections arising from glutamatergic subplate cells may boost the subplate activity in the 1040 hz frequency range ( hirsch and luhmann , 2008 ) . subplate cells are dye - coupled to cortical plate neurons in a columnar manner ( dupont et al . , 2006 ) and faithfully transmit synchronized 1020 hz network oscillations generated and amplified in the subplate to the cortical plate and marginal zone ( upper inset in figure 8) ( dupont et al . , 2006 ; wagner and luhmann , 2006 ; the subplate receives a transient and selective synaptic input from neuromodulatory systems , from the sensory periphery via specific thalamic nuclei and via corticocortical fibers from other neocortical areas . subplate cells are capable to fire repetitive action potentials ( figure 3a ) and upon activation of cholinergic postsynaptic receptors discharge in repetitive bursts ( lower inset and figure 5 ) . gabaergic subplate neurons release gaba in an activity - dependent ( 1 ) and tonic ( 2 ) manner thereby inducing a postsynaptic excitatory response . subplate neurons are not only coupled via these excitatory synaptic circuits , but also via gap junctions ( figure 2 ) . chemical and electrical synapses contribute to the generation of subplate - driven oscillatory activity which is transmitted to the gap junctional coupled network in the cortical plate ( figure 2a ) and marginal zone and induces 1020 hz neocortical network oscillations ( upper inset ) . elimination or interruption of the subplate prevents the generation of the subplate - driven synchronized activity patterns and disturbs the maturation of the columnar architecture ( ghosh and shatz , 1992 ; kanold et al . , 2003 ; kanold and shatz , 2006 ) . under normal conditions and with further development , subplate cells disappear by apoptosis ( ferrer et al . , 1990 ; price et al . , 1997 ; arias et al . , 2002 ) or transform into white matter interstitial cells ( valverde and facal - valverde , 1988 ; valverde et al . , 1995 ) or layer vib ( layer vii ) neurons ( woo et al . , 1991 ; reep , 2000 ) in the normal mature cerebral cortex , subplate neurons have lost their capabilities to receive and amplify incoming neuronal activity and to generate synchronized network oscillations . however , as already suggested by jones ( 1995 ) , disturbances in the pattern of programmed cell death in the subplate may cause a failure to establish normal patterns of connections in the overlying cerebral cortex , leading to long - term neurological deficits such as schizophrenia . abnormal placement of ( surviving subplate ? ) neurons in the white matter and atypical circuits have been observed in the prefrontal cortex of schizophrenic patients ( akbarian et al . , 1996 ; it has been further suggested that subplate - like neurons may persist in cortical dysplasia and contribute to the manifestation of pharmacoresistant epilepsy in adults ( cepeda et al . , 2007 ) . interestingly in the resectioned human tissue gaba application induced depolarizing postsynaptic responses and spontaneous gabaergic synaptic potentials even elicited action potentials ( cepeda et al . , 2007 ) . in summary , experimental data indicate that subplate cells , most likely gabaergic subplate neurons , play an important role in the generation of early synchronized network activity and in the normal development of the neocortical network and columnar architecture . clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits , pathological activities and long - term neurological deficits . the authors declare that the research presented in this paper was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

due to their unique structural and functional properties , subplate cells are ideally suited to function as important amplifying units within the developing neocortical circuit . subplate neurons have extensive dendritic and axonal ramifications and relatively mature functional properties , i.e. their action potential firing can exceed frequencies of 40 hz . at earliest stages of corticogenesis subplate cells receive functional synaptic inputs from the thalamus and from other cortical and non - cortical sources . glutamatergic and depolarizing gabaergic inputs arise from cortical neurons and neuromodulatory inputs arise from the basal forebrain and other sources . activation of postsynaptic metabotropic receptors , i.e. muscarinic receptors , elicits in subplate neurons oscillatory burst discharges which are transmitted via electrical and chemical synapses to neighbouring subplate cells and to immature neurons in the cortical plate . the tonic non - synaptic release of gaba from gabaergic subplate cells facilitates the generation of burst discharges . these cellular bursts are amplified by prominent gap junction coupling in the subplate and cortical plate , thereby eliciting 1020 hz oscillations in a local columnar network . thus , we propose that neuronal networks are organized at earliest stages in a gap junction coupled columnar syncytium . we postulate that the subplate does not only serve as a transient relay station for afferent inputs , but rather as an active element amplifying the afferent and intracortical activity .

Introduction Structural Properties of Subplate Neurons Functional Properties of Subplate Neurons Subplate Neurons are Well Integrated in the Developing Neocortical Network Oscillatory Properties of Subplate Cells GABAergic Subplate Cells Drive Early Cortical Networks Conflict of Interest Statement

reflection is growing in importance as a means to promote a learner - centered environment where the learner is encouraged to learn through the practice of nursing with subsequent reflection . when students are in uncertain new situations , they are expected to use focused thought to apply learned principles to clinical situations and critically evaluate their performance and decisions , rather than focusing on technical knowledge in isolation . the national league for nursing has reiterated the importance of both reflective and critical thinking through their designation as core competencies for nurse educators . clinical journals are one of the strategies used throughout the undergraduate nursing curricula to facilitate and guide students ' reflective thinking processes . reflective journals are considered by ( most ) faculty members to be essential in fostering an understanding of course concepts and application of concepts to clinical practice ; however , students continually question their worth in their anecdotal comments and in formal student course evaluations . is student dissatisfaction based on reflective journaling in general , the format within an individual course or modality , or some other characteristic of the individual student ? it is possible that reflective journals are not consistently reaching the anticipated critical reflection / thinking outcomes that are desired for all students . the purposes of the study were to determine ( a ) the critical attributes of a reflective journal and relative levels of each attribute , ( b ) the relative importance of each attribute as perceived by the students , and ( c ) the perception of relative importance for student subgroups . the reflective learning process for one entering a profession , particularly one entering the health care field , is a very personal experience . the day - to - day experiences in the clinical setting often challenge strongly held beliefs . educators rightfully focus on developing learning tools that include reflection on learned course content , on clinical experiences , on personal values and on their role as the novice nurse . at the foundation of the journaling process reflection , via journal writing , has been an integral component of clinical learning for many years . most educators agree that reflective journaling is valuable and contributes to learning ; however , they do not agree on how best to structure the reflective journal and if the format of the journal should vary for different student subgroups . first , journaling can be viewed as a transformative learning tool for the nursing instructor , challenging the attitudes and the practices of the learner . educators strive to awaken the novice to the perspective of the client , the family , the role of evidence , or the impact of the multifaceted health care system . students are encouraged to share the thoughts surrounding their actions and focus on the meaning of their experiences . the focus moves from accomplishing a list of behaviors correctly to the process of learning . through learning journaling can also be viewed as a skill requiring reasoning and writing expertise . with guidance and support , the clinical reasoning skills seen in the reflective journal can be further developed . in the epp review of the literature , tanner 's clinical judgment model lends structure to the clinical reasoning process of the nurse and the nursing student . reflection is guided using a consistent method , which is designed to promote competence of the baccalaureate nursing student . clinical reasoning is viewed as a skill , that is , structured , practiced , and ultimately greater proficiency is achieved . another aspect to consider is adding structure to the reflective journal , which is thought to promote reflection in nursing students . in an effort to promote learning and promote proficient reflection , nurse educators have developed models or guidelines to assist students to analyze their clinical experiences . guided reflection has been shown to move students past simply describing events toward more thorough introspection and concept synthesis regarding clinical experiences . by directing students to collect pertinent information , by probing and asking pointed questions , educators direct students toward thinking exercises that foster a deeper understanding . the process of reflection is guided rather than left open to free - form , unstructured comments . finally , the reflective journal should be a secure place that encourages the student to question and to elaborate on emotional responses or challenges to existing beliefs . fink defined optimal instructor feedback as being timely , caring , and discriminant . the lasater clinical judgment rubric describes the components of clinical decision making in a tool that structures the evaluative feedback from an instructor . as online educational systems develop , instructors have begun to use group discussion boards to postreflective journals to promote interactive group learning . there is a paucity of research on the use of online group discussion boards for reflective journaling . students collaborating on journals have demonstrated higher levels of reflection , suggesting benefit from a group process [ 12 , 13 ] . on the other hand , collaborative reflective journals have shown promise for some students and present a barrier to others as the journal requires expressing personal thoughts and receiving feedback in a setting that is not private . the research has shown that many students value reflective journals , but that is not consistent for all students . in one study , a group of students indicated that they were skeptical of the benefits of reflective journaling , the act of reflecting did not improve their practice , yet they recognized that there were positive impacts on practice for students who successfully reflected upon their practice . there have been no studies of student perception of the format of reflective journal or studies of student factors that may facilitate or hinder journaling effectiveness . no study has systematically analyzed the perceptions of students related to learning through reflective journals . what is the relative importance of each attribute involved in the journaling experience?what are changes that would increase satisfaction of students with the journaling experience?do differences in student characteristics lead to changes in their preferences ? do differences in student characteristics lead to changes in their preferences ? after receiving irb approval from university x , students in three undergraduate nursing classes were given the opportunity to voluntarily complete a confidential survey without an instructor present . the survey was offered to second semester junior students ( n = 41 ) , first semester senior students ( n = 40 ) , and final semester senior students ( n = 38 ) . within this convenience sample , 13 , 24 , and 19 completed surveys , respectively , were received from these classes for a response rate of 55.5% . delphi or expert panel of nurse educators convened to try and determine which attributes of reflective journaling would be viewed by students as the most important characteristics of the journaling process . the panel was comprised of four doctoral , and two masters prepared faculty members with 86 cumulative years of experience educating nursing students in the clinical setting . after extensive discussion and consideration of many possible components of reflective journaling , the panel determined that there were five major attributes contained within the student experience of reflective journaling . after determining these attributes the attributes and levels are described in table 1 . additionally , we asked four demographic questions to see if there were relationships between these characteristics and the preferences expressed for the five journaling attributes . these demographic variables are experience , sex , grade point average ( gpa ) , and primary language ( esl ) . gpa is divided into greater than 3.49 ( gpa 1 ) , between 2.5 and 3.5 ( gpa 2 ) , and less than 2.5 ( gpa 3 ) . esl is a self - reporting variable specifying whether english is their primary language , and students answer either yes ( esly ) or no ( esln ) . experience relates to the number of classes they have already completed where journaling was a component of the coursework . the students were either three trimesters from graduation ( exp 1 ) , two trimesters from graduation ( exp 2 ) , or in the final trimester before graduation ( exp 3 ) . in other words , the more coursework each student had completed , the greater exposure they had had to reflective journaling , as each trimester contains a journaling component in at least one course . conjoint analysis is a market - based research analysis model where potential and/or actual customers make choices based on their preferences . these choices are based on the individual attributes of the product under consideration and are considered jointly ( conjoint ) . for example , all else equal , most consumers would choose the cheapest computer . however , they can not have both of these attributes simultaneously because customers are forced to pay additional money for the faster computer should they desire more speed . people make tradeoffs every day as they try and balance the partial worth of many attributes like speed , color , size , power , convenience , durability , prestige , necessity , and price ( among others ) . a similar model exists in education . students , like computer purchasers , have preferences about what is important to them and are , in effect , buyers of nursing education . like the manufacturers of computers who care greatly about the preferences of their customers , the sellers of nursing education should also care about the preferences of their customers . for example , in reflective journaling , students might desire a short time to complete their journaling requirement and , at the same time , want a transformative experience which requires more time . each attribute and its corresponding levels were entered into the conjoint value analysis ( cva ) software program which automatically generates a survey . the survey began with the four demographic questions previously described and followed with twenty pair - wise discrete choice questions . each question presented the student respondent with a choice of two journaling experiences from which they had to choose one . ( scale 3 ) to indifferent ( scale 5 ) to somewhat prefer right each attribute is present in all choices , but the levels within each attribute are varied , and these variations are shuffled with each succeeding question . additionally , the cva questionnaire generator automatically moves attribute levels from left to right as the respondent proceeds through the questionnaire . a typical conjoint question is in table 2 , and the complete survey is available upon request . after data entry , the cva software runs multiple ordinary least squares ( olss ) functions by performing simultaneous regressions of the five independent attribute variables on the dependent variable of preference . for a detailed explanation of cva the results are presented as average utilities ( preferences ) , and the average importance of each attribute is estimated , and an r is calculated . the results are then exported to the sawtooth marketing simulation ( smrt ) software which runs a series of simulation algorithms where the different levels of the same attribute are compared to each other while holding all other attributes the same . these results estimate the share preferences for each attribute level relative to other levels . through multiple iterations , all preferences for each level are estimated in relationship to each other by simply dividing one into the next . finally , to estimate the changes ( if any ) that the demographic variables have on the preferences of attributes levels within reflective journaling , the marketing simulation software takes the attribute preference data and filters it through the four demographic questions and provides a result containing product shares of preference and standard errors . through these two statistics , confidence intervals and p values ( z scores ) the importance ( table 3 ) of the five attributes varies from a low of 16.75% ( format ) to a high of 23.58% ( time ) . the lack of any one attribute being extremely high or low relative to the other attributes suggests that all five attributes were considered and paid attention to by the respondents . this is further reinforced by an r squared ( see table 4 ) of 0.777 . that is , this model explains 77.7% of the variability of journaling preferences among nursing students and is clearly superior to a random predictor of this variation . the utilities ( see table 4 ) are interval - scaled values without a real zero . therefore , utilities represent weights relative to each other , and the sum of the three levels equals zero . it is clear that students prefer a shorter time , complete confidentiality , recognition of their behavior , one - time complete feedback , and a semistructured format . however , because these are interval values , they provide preferences only . for these data to be useful in the real world of developing courses and , more specifically , journaling experiences , there must be a real zero . additionally , to accurately compare different levels of any single attribute , all other attributes must be held constant . the smrt uses a randomized first choice simulation algorithm to accomplish this . for a detailed explanation of the smrt software package , please refer to the sawtooth research paper on market simulations referenced in the bibliography . students prefer a weekly time commitment of 15 minutes over 30 minutes ( p < 0.05 ) , and they prefer 30 minutes over 45 minutes ( p < 0.05 ) . by decreasing the time commitment from 45 to 30 minutes , student satisfaction increases by 15% and if students are currently spending 45 minutes a week and an instructor reduces that to 15 minutes ( p < 0.05 ) , student satisfaction increases by 54% . ( nonanonymous ) option were rejected by students . by moving the journaling experience from open to either completely confidential ( p < 0.05 ) or to shared content ( p < 0.05 ) however , it is less clear whether students have a problem sharing their thoughts anonymously . however , by taking the square root of the squared sum of the two separate standard error terms and dividing that into the difference of the partial shares , a p value is easily found in a z table ( 0.07 ) . therefore , students also prefer complete confidentiality to anonymous sharing ( p = 0.07 ) , but their satisfaction only increases by 9% by moving from the latter to the former . students want results from their journaling efforts though they are relatively indifferent whether they simply learn to recognize their behaviors or have those behaviors transformed . in both iterations in fact , by changing the journaling experience from one where the students perceive that no change has occurred to one where they either recognize their behaviors ( p < 0.05 ) or have them transformed ( p < 0.05 ) , student satisfaction increases by 41% and 38% , respectively . however , the two 95% c.i . that compared recognition to transformation almost completely overlap . so although instructors may think that it is a higher level of learning for students to be transformed versus simply recognizing positive and negative behaviors , students do not value that difference . students value feedback over simply checking their work for completeness although they are indifferent whether that feedback comes only once or multiple times . by moving from a model of checking for completeness to one where students receive feedback once ( p < 0.05 ) , student satisfaction increases by 11% . however , the 95% intervals for checking for completeness versus multiple feedbacks have overlapping tails and the p value ( 0.06 ) is calculated ( as above ) using the z table . finally , students are indifferent to a single feedback or multiple feedbacks . the 95% c.i . for both of these levels almost completely overlap , and there is no difference . students do not value a completely structured format . by moving from a completely structured format to either a semi - structured ( p < 0.05 ) or open format ( p = 0.06 ) student satisfaction increases by 10% and 9% , respectively . however , students did not perceive much difference between the semi - structured or open format . there was no difference . finally , to estimate the changes ( if any ) that the demographic variables have on the preferences of attributes levels within reflective journaling , the attribute preference data was filtered through the four demographic questions . most of the demographic categories had no significant effect on student preferences , and these are not reported though the data is available upon request . the sex and gpa of the respondents had no significant effect on student preferences for journaling attributes . that is , all calculated confidence intervals that compared these two demographic variables had a great deal of overlap . the experience level of the students did have an effect on the value of time and the preference of format . those students with less experience ( exp 1 ) had a much greater preference for less time spent journaling ( p < 0.023 ) and a much greater preference for a structured format ( p < 0.39 ) . conversely , those students with the most experience ( exp 3 ) had a much greater preference for a free - form format ( p < 0.39 ) when compared to students with less journaling experience . students that self - reported english as their second language ( esly ) viewed the results of journaling differently . that is , although the no effect level of result was perceived negatively by all students , it was perceived much more negatively by non - esl students . those students whose first language was english ( esln ) preferred feedback to no feedback , and esl students did not . the results for the esl demographic should be viewed with some caution as there were only 2 students reporting themselves as esl . although the weakness of a small cohort is captured in the standard error calculation , which leads directly to the p value , it still gives pause . we report those results here because mathematically the differences were significant but will leave it up to the reader to decide . question 1 : what is the relative importance of each of the five attributes involved in the journaling experience ? in descending order , the most important characteristics of journaling are time , confidentiality , results , feedback and format ( table 1 ) . question 2 : what are the changes that would most increase the satisfaction of students with the journaling experience ? to increase satisfaction for students , instructors should strive to reduce the time students spend journaling , maintain anonymity ( though content might be shared ) , and instill a perception in students that reflective journaling will have a tangible effect on each student 's ability to transform or simply recognize their behaviors ( table 5 , far right column ) . question 3 : do differences in student characteristics lead to changes in their preferences about the journaling experience . however , students with less experience in journaling value time more than the entire cohort and students with more experience prefer an open format with fewer instructor cues . additionally , esl students placed less value on instructor feedback and behavior recognition or transformation . however , because of the small sample size , these results should be viewed with some caution ( table 6 ) . the delphi panel included this dimension as an attribute of the journal recognizing that it is a paramount concern of nursing students . nursing instructors will probably read this and recognize the challenge of creating journaling exercises that are both time efficient and produce the requisite level of reflection . however , students would highly value new journaling exercises that prove to be more time efficient . online , discussion systems should be developed that include areas for confidential discussion and individual feedback , as well as open discussions areas where students can benefit from interaction and collaboration . in the age of the electronic classroom , anonymity and collaboration it is interesting to note the undergraduate student 's comments on the structured journal considering the development of various models or guidelines [ 7 , 8 ] . students preferred either a semi - structured or an open , free - form journal . educators working on models that structure student responses should be advised to insure that exercises include areas for students to write freely without predetermined structure . there is evidence that using a model or structured journal early in a nursing program and moving to a predominantly unstructured journal later in the program will be valued more highly by students . instructors should insure that whether using a model or not , students value substantive feedback after they have prepared reflective journals . esl students viewed journaling differently compared to non - esl students . c. kuo developed an online writing system to support nonnative students with writing exercises . the system offers a supportive environment encouraging writing practice , peer review , and use of an e - portfolio , online dictionary , phrase lists , and so forth . research findings are beginning to reveal more about the kinds of support that will benefit this group of students . therefore , there exists a risk that a significant variable was overlooked by the delphi panel and not included in the survey . the omission of significant variables can lead to specification error which has the potential to bias coefficients and results . the students in our sample attend the same school , are taught by the same teachers , and live in the state of x. journaling experiences nationwide or worldwide might be quite different than those experienced by the students at our institution and , therefore , these results might not generalize to other populations in other institutions across the country . for example , there were only 2 esl students , 6 male students , and 3 who reported a gpa under 2.5 . all calculations in this study make use of the standard error term , and standard errors take into account small sample sizes . however , we would still view all of the small sample conclusions with some caution . similarly , regardless of sample size , a p value of 0.05 still means that we are only correct 19 out of 20 times and , therefore , could be wrong . marketing professionals have surveyed their customers and potential customers in an effort to provide the product or service that is most desired by them . we also wanted to find out what aspects of journaling our customers ( students ) value . this study applied the statistical method of conjoint value analysis , a sound quantitative modeling method that has been used in the field of marketing for many years . results indicated that all students value time , confidentiality , results , feedback , and format , in that order . the less - experienced students valued a structured journal , while the more advanced students valued a more free - form journal . nurse educators should consider these attributes when developing and refining the journals in their courses . while journaling is a process that supports learning , it is also very personal . students must see that the journaling system is confidential and that they will receive thoughtful feedback from the instructors . this study helps us better understand nursing students , our customers , and to better design journaling experiences that future students might find more satisfying . more research is needed to test new journaling systems as well as to explore the unique needs of student subgroups , particularly esl students .

this study used a statistical technique , conjoint value analysis , to determine student perceptions related to the importance of predetermined reflective journaling attributes . an expert delphi panel determined these attributes and integrated them into a survey which presented students with multiple journaling experiences from which they had to choose . after obtaining irb approval , a convenience sample of 66 baccalaureate nursing students completed the survey . the relative importance of the attributes varied from a low of 16.75% ( format ) to a high of 23.58% ( time ) . the model explained 77% of the variability of student journaling preferences ( r2 = 0.77 ) . students preferred shorter time , complete confidentiality , one - time complete feedback , semistructured format , and behavior recognition . students with more experience had a much greater preference for a free - form format ( p < .05 ) when compared to students with less journaling experience . additionally , the results of english as a second language students were significantly different from the rest of the sample . in order to better serve them , educators must consider the relative importance of these attributes when developing journaling experiences for their students .

1. Introduction 2. Review of Literature 3. Discussion 4. Conclusions

acute pancreatitis is an inflammatory disease characterized by the presence of acute abdominal pain and/or increased levels of serum pancreatic enzymes . more importantly , takeda et al . have indicated that the mortality rate was of approximately 20% when acute pancreatitis developed to severe acute pancreatitis ( sap ) . similarly , several lines of evidence have demonstrated that systemic inflammatory response syndrome ( sirs ) and multiple - organ dysfunction syndrome ( mods ) are the 2 most common causes of death in the early stage ( within approximately 2 weeks ) of patients with sap . previous research has suggested that acute respiratory distress syndrome ( ards ) , which is a clinical manifestation of lung injury , is one of the earliest manifestations of functional deterioration in human organs and is one of the most common and serious complications of sap . the most severe clinical manifestations of ards exhibit significant hypoxemia with bilateral pulmonary infiltrates consistent with edema . currently , the criteria for the diagnosis of ards are mainly based on the berlin definition , which includes the original diagnosis of acute lung injury ( ali ) and ards : acute onset of respiratory distress , hypoxemia ( ali : pao2/fio2 300 mmhg , ards : pao2/fio2 200 mmhg ) , bilateral consolidation of chest radiograph , and absence of clinical findings of cardiogenic pulmonary edema . in practice , the complexity of multiple - item criteria of ards often causes inconvenience for physicians in the management of patients with sap . if ards also causes changes in several related bio - markers , similar to the characteristic elevation of troponin in myocardial infarction , this bio - marker might be useful to help diagnose of sap - induced ards . previous reports indicate that pulmonary surfactant consists principally of various lipids and proteins ( surfactant proteins ) . furthermore , the major constituent of surfactant proteins is sp - a , and the remainder includes sp - b , sp - c , and sp - d . recently , a study suggested that sp - a is a large multimeric protein found in the airways and alveoli . several lines of evidence have shown that sp - a not only mediates the inflammatory response , but also controls vascular permeability , which are considered to be important factors in the development of ards . a large number of studies have shown that sp - a levels in bronchoalveolar lavage fluid ( balf ) are significantly lower in patients with bleomycin - induced ards compared with healthy volunteers . in contrast , the serum levels of sp - a were raised in patients with bleomycin - induced ards . thus , the serum levels of sp - a may be suggested as a sensitive and specific serum marker on the diagnosis of sap - induced ards . it has been suggested that the presence of qualitatively assessed ( yes or no ) diffuse alveolar damage ( dad ) should be considered the criterion standard for the diagnosis of ards . to improve the early detection and diagnosis of sap - induced ards , determination of serum levels of sp - a in sap - induced ards rats was used . in the present study , we therefore evaluated serum sp - a levels according to the criterion standard for the diagnosis of ards to evaluate the qualitative assessment of presence of diffuse alveolar damage ( dad ) in the diagnosis of sap - induced ards in an animal model . one hundred male wistar rats weighing 250300 g were purchased from cavens laboratory animal company , changzhou , jiangsu , china . six rats were housed per cage with food and water available ad libitum and maintained on a 12-h light / dark cycle ( lights on at 07:00 am ) . eighty rats were randomly divided into 2 groups ( n=40 each ) : the sham - operated ( so group ) and the severe acute pancreatitis ( sap group ) and then randomly subdivided into 1-h , 2-h , 3-h , and 4-h subgroups , with 10 rats in each subgroup . the rats in the 1-h , 2-h , 3-h , and 4-h subgroups of the sap group , a total of 40 rats , were subdivided into a sap - induced ards group ( ards group ) and a sap without ards group ( non - ards group ) according to the diagnosis of ards for lung tissue , including the presence of dad , qualitatively assessed ( yes or no ) after the operation . the other 20 rats were regarded as the baseline and euthanized without any operation in initial experiments . intra - peritoneal injection of 1% sodium pentobarbital ( 50 mg / kg ; sigma , st . louis , mo , usa ) was used for abdominal cavity anesthesia to all rats of the 1-h , 2-h , 3-h , 4-h subgroups . animal care was in accordance with the national institutes of health guide for the care and use of laboratory animals , and was approved by the animal use and protection committee of soochow university . the abdominal surgery of the so group ( n=40 ) consisted of opening the abdomen , flipping the pancreas , returning them to their original position , then closing the abdominal cavity with 2 layers of sutures . louis , mo , usa ) was administered into the common biliopancreatic duct of the sap group ( n=40 ) by retrograde injection . we identified the duodenal papilla inside the duodenum duct wall , and then used a no . a segmental epidural catheter was inserted into the duodenum cavity through the hole , and inserted retrogradely into the biliary - pancreatic duct through the papilla . this was followed by retrograde transfusion of 4.5% sodium taurocholate solution ( 4.5% ; 0.1 ml/100 g ) by a microinjection pump at 0.1 ml / min . all rats of the 2 groups were injected with normal saline ( 20 ml / kg ) through the vena caudalis to compensate for loss of fluid during surgery . the rats in all groups were euthanized at 1 , 2 , 3 and 4 hours , according to the subgroup , after the operation . blood samples ( 5 ml ) were collected from the heart , and were mixed with edta anticoagulative liquor , then were centrifuged at 3000 sera of their blood samples were collected and maintained at 4c for the detection of sp - a . serum levels of sp - a were determined by the elisa method by using the rat sp - a elisa kit purchased from mybiosource ( san diego , california , usa ) . the antigens were measured by elisa inhibition assays using antibodies raised against alveolar proteinosis - derived sp - a . briefly , in order to free the sp - a from any associated plasma or surfactant components , aliquots were first treated with edta , sds , and triton x-100 . serial dilutions of the samples in pbst containing 0.25% bsa ( w / v ) were incubated in an elisa plate with aliquots of the respective antibody . free antibody was captured using a second elisa plate coated with purified sp - a ( 1 g / ml ) and the amount measured using alkaline phosphatase conjugated igg against rabbit immunoglobulins and 15 mm disodium p - nitrophenyl phosphate in 1.0 m diethanolamine and 0.5 mm mgcl 2 as a substrate . after 1 h , the absorbance of the substrate was measured at 405 nm using a dynatech mr5000 reader ( dynatech laboratories , chantilly , va , usa ) . the assayzap program ( biosoft , ferguson , mo , usa ) was used to generate a standard curve and to compute the concentration of sp - a in each sample . standards , assayed in quadruplicate , were included in each elisa plate at 8 serial dilutions ( range 1.95250 ng / ml for sp - a , r>0.99 ) . the antibodies used do not react with any other known antigens and the assays have coefficients of variance of ~6% . rat pancreatic and lung tissues were collected , fixed in 10% formalin , embedded in paraffin , and sectioned into 4- sections , after which they were stained by hematoxylin and eosin ( h&e ) staining for histopathological observation . histopathology of the pancreas and lungs was scored and classified by 2 professional pathologists using a double - blind method . each group randomly selected 3 slices ; for each slice , 10 high - power fields of vision were again randomly selected , and finally the extent of pancreatic tissue damage by edema , infection , hemorrhage , and necrosis were evaluated . the pathological score for pancreatic tissue was calculated according to rongiones standards as a reference , with a minimum score of 0 and the highest score of 4 . the pathological grade for lung tissue was according to lei as a reference , with a minimum grade of 0 and the highest grade of iii . criteria for the diagnosis of ards for lung tissue included the presence of diffuse alveolar damage ( dad ) , qualitatively assessed ( yes / positive result or no / negative result ) as : intra - alveolar edema , alveolar type i cell necrosis , alveolar type ii cell ( cuboidal cells ) proliferation progressively covering the denuded alveolar - capillary membrane , interstitial proliferation of fibroblasts and myofibroblasts , or organizing interstitial fibrosis . values of sp - a for ards were determined by the receiver operating characteristic curve ( roc ) . the comparison among multiple groups was analyzed with 1-dimensional analysis of variance after data conversion . ards group and so group + non - ards group was tested with nonparametric rank sum test . true- positive ( tp ) was defined as sp - a values higher than the cut - off value in rats with sap induced - ards groups ( ards group ) . false - negative ( fn ) included rats with ards but who scored negative . sensitivity and specificity formulas for calculation were : sensitivity = tp/(tp+fn)100% , specificity = tn/(tn+fp)100% , diagnostic accuracy=(tp+tn)/(tp+fp+tn+fn)100% , positive predictive value = tp/(tp+fp ) 100% , and negative predictive value = tn/(tn+fn)100% . one hundred male wistar rats weighing 250300 g were purchased from cavens laboratory animal company , changzhou , jiangsu , china . six rats were housed per cage with food and water available ad libitum and maintained on a 12-h light / dark cycle ( lights on at 07:00 am ) . eighty rats were randomly divided into 2 groups ( n=40 each ) : the sham - operated ( so group ) and the severe acute pancreatitis ( sap group ) and then randomly subdivided into 1-h , 2-h , 3-h , and 4-h subgroups , with 10 rats in each subgroup . the rats in the 1-h , 2-h , 3-h , and 4-h subgroups of the sap group , a total of 40 rats , were subdivided into a sap - induced ards group ( ards group ) and a sap without ards group ( non - ards group ) according to the diagnosis of ards for lung tissue , including the presence of dad , qualitatively assessed ( yes or no ) after the operation . the other 20 rats were regarded as the baseline and euthanized without any operation in initial experiments . intra - peritoneal injection of 1% sodium pentobarbital ( 50 mg / kg ; sigma , st . louis , mo , usa ) was used for abdominal cavity anesthesia to all rats of the 1-h , 2-h , 3-h , 4-h subgroups . animal care was in accordance with the national institutes of health guide for the care and use of laboratory animals , and was approved by the animal use and protection committee of soochow university . the abdominal surgery of the so group ( n=40 ) consisted of opening the abdomen , flipping the pancreas , returning them to their original position , then closing the abdominal cavity with 2 layers of sutures . louis , mo , usa ) was administered into the common biliopancreatic duct of the sap group ( n=40 ) by retrograde injection . we identified the duodenal papilla inside the duodenum duct wall , and then used a no . a segmental epidural catheter was inserted into the duodenum cavity through the hole , and inserted retrogradely into the biliary - pancreatic duct through the papilla . this was followed by retrograde transfusion of 4.5% sodium taurocholate solution ( 4.5% ; 0.1 ml/100 g ) by a microinjection pump at 0.1 ml / min . all rats of the 2 groups were injected with normal saline ( 20 ml / kg ) through the vena caudalis to compensate for loss of fluid during surgery . the rats in all groups were euthanized at 1 , 2 , 3 and 4 hours , according to the subgroup , after the operation . blood samples ( 5 ml ) were collected from the heart , and were mixed with edta anticoagulative liquor , then were centrifuged at 3000 sera of their blood samples were collected and maintained at 4c for the detection of sp - a . serum levels of sp - a were determined by the elisa method by using the rat sp - a elisa kit purchased from mybiosource ( san diego , california , usa ) . the antigens were measured by elisa inhibition assays using antibodies raised against alveolar proteinosis - derived sp - a . briefly , in order to free the sp - a from any associated plasma or surfactant components , aliquots were first treated with edta , sds , and triton x-100 . serial dilutions of the samples in pbst containing 0.25% bsa ( w / v ) were incubated in an elisa plate with aliquots of the respective antibody . free antibody was captured using a second elisa plate coated with purified sp - a ( 1 g / ml ) and the amount measured using alkaline phosphatase conjugated igg against rabbit immunoglobulins and 15 mm disodium p - nitrophenyl phosphate in 1.0 m diethanolamine and 0.5 mm mgcl 2 as a substrate . after 1 h , the absorbance of the substrate was measured at 405 nm using a dynatech mr5000 reader ( dynatech laboratories , chantilly , va , usa ) . the assayzap program ( biosoft , ferguson , mo , usa ) was used to generate a standard curve and to compute the concentration of sp - a in each sample . standards , assayed in quadruplicate , were included in each elisa plate at 8 serial dilutions ( range 1.95250 ng / ml for sp - a , r>0.99 ) . the antibodies used do not react with any other known antigens and the assays have coefficients of variance of ~6% . rat pancreatic and lung tissues were collected , fixed in 10% formalin , embedded in paraffin , and sectioned into 4- sections , after which they were stained by hematoxylin and eosin ( h&e ) staining for histopathological observation . histopathology of the pancreas and lungs was scored and classified by 2 professional pathologists using a double - blind method . each group randomly selected 3 slices ; for each slice , 10 high - power fields of vision were again randomly selected , and finally the extent of pancreatic tissue damage by edema , infection , hemorrhage , and necrosis were evaluated . the pathological score for pancreatic tissue was calculated according to rongiones standards as a reference , with a minimum score of 0 and the highest score of 4 . the pathological grade for lung tissue was according to lei as a reference , with a minimum grade of 0 and the highest grade of iii . criteria for the diagnosis of ards for lung tissue included the presence of diffuse alveolar damage ( dad ) , qualitatively assessed ( yes / positive result or no / negative result ) as : intra - alveolar edema , alveolar type i cell necrosis , alveolar type ii cell ( cuboidal cells ) proliferation progressively covering the denuded alveolar - capillary membrane , interstitial proliferation of fibroblasts and myofibroblasts , or organizing interstitial fibrosis . values of sp - a for ards were determined by the receiver operating characteristic curve ( roc ) . the comparison among multiple groups was analyzed with 1-dimensional analysis of variance after data conversion . ards group and so group + non - ards group was tested with nonparametric rank sum test . true- positive ( tp ) was defined as sp - a values higher than the cut - off value in rats with sap induced - ards groups ( ards group ) . false - negative ( fn ) included rats with ards but who scored negative . sensitivity and specificity formulas for calculation were : sensitivity = tp/(tp+fn)100% , specificity = tn/(tn+fp)100% , diagnostic accuracy=(tp+tn)/(tp+fp+tn+fn)100% , positive predictive value = tp/(tp+fp ) 100% , and negative predictive value = tn/(tn+fn)100% . the glandular architecture of the pancreas in the so group was entirely normal ( h&e ) ( figure 1 ) . the architecture of the lung in the so group was entirely normal ( h&e ) ( figure 2 ) . histopathologic features of the pancreas in the sap group included various degrees of edema , minimal necrosis , and inflammatory infiltration ( h&e ) ( figure 3 ) . according to the criteria for the diagnosis of ards , the presence of dad was qualitatively assessed ( yes or no ) ; in the 1-h , 2-h , 3-h , and 4-h subgroups we found 1 , 5 , 9 , and 10 cases of ards , respectively . therefore , the sap group was then subdivided into the ards group ( 25 rats ) and non - ards group ( 15 rats ) according to the criteria for the diagnosis of ards . histopathologic features of lung in the ards group included the presence of dad , intra - alveolar edema , and organizing interstitial fibrosis ( h&e ) ( figure 4 ) . serum sp - a levels ( means sd ) in the baseline , so , and sap groups were 43.1514.29 , 51.9116.99 , and 193.435.37 . there was no statistical difference between so group and baseline in serum sp - a levels ( p>0.05 ) . serum sp - a levels of the sap group were significantly increased compared to baseline and so group ( * p<0.05 , p<0.05 ) ( figure 5 ) . serum sp - a levels ( means sd ) in the so group , ards group , and non - ards group were 51.9116.99 , 198.029.73 , and 185.743.21 ug / ml , respectively . serum sp - a levels were significantly increased from the ards group to the so group ( * p<0.05 ) and from the non - ards group to the so group ( p<0.05 ) . there was no statistical difference between the ards group and non - ards group in serum sp - a levels ( p>0.05 ) ( figure 6 ) . according to the analysis of roc curves , the best cut - off value for the serum sp - a level for the diagnosis of sap - induced ards was 150 ug / ml . in analysis of all sap - induced ards rats , the area under the roc curve of sp - a was 0.88 ( figure 7 ) . the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of sp - a in the diagnosis of sap - induced ards were 100.0% , 81.8% , 71.4% , 100.0% , and 87.5% , respectively ( table 1 ) . the glandular architecture of the pancreas in the so group was entirely normal ( h&e ) ( figure 1 ) . the architecture of the lung in the so group was entirely normal ( h&e ) ( figure 2 ) . histopathologic features of the pancreas in the sap group included various degrees of edema , minimal necrosis , and inflammatory infiltration ( h&e ) ( figure 3 ) . according to the criteria for the diagnosis of ards , the presence of dad was qualitatively assessed ( yes or no ) ; in the 1-h , 2-h , 3-h , and 4-h subgroups we found 1 , 5 , 9 , and 10 cases of ards , respectively . therefore , the sap group was then subdivided into the ards group ( 25 rats ) and non - ards group ( 15 rats ) according to the criteria for the diagnosis of ards . histopathologic features of lung in the ards group included the presence of dad , intra - alveolar edema , and organizing interstitial fibrosis ( h&e ) ( figure 4 ) . serum sp - a levels ( means sd ) in the baseline , so , and sap groups were 43.1514.29 , 51.9116.99 , and 193.435.37 ug / ml , respectively . there was no statistical difference between so group and baseline in serum sp - a levels ( p>0.05 ) . serum sp - a levels of the sap group were significantly increased compared to baseline and so group ( * p<0.05 , p<0.05 ) ( figure 5 ) . serum sp - a levels ( means sd ) in the so group , ards group , and non - ards group were 51.9116.99 , 198.029.73 , and 185.743.21 ug / ml , respectively . serum sp - a levels were significantly increased from the ards group to the so group ( * p<0.05 ) and from the non - ards group to the so group ( p<0.05 ) . there was no statistical difference between the ards group and non - ards group in serum sp - a levels ( p>0.05 ) ( figure 6 ) . according to the analysis of roc curves , the best cut - off value for the serum sp - a level for the diagnosis of sap - induced ards was 150 ug / ml . in analysis of all sap - induced ards rats , the area under the roc curve of sp - a was 0.88 ( figure 7 ) . the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of sp - a in the diagnosis of sap - induced ards were 100.0% , 81.8% , 71.4% , 100.0% , and 87.5% , respectively ( table 1 ) . in the present study , we evaluated serum sp - a levels for the diagnosis of sap - induced ards in a rat model . importantly , the sap - induced ards rat model is mostly suitable for investigating the effectiveness and specificity of a particular therapeutic approach . the model that we chose had to be similar to the etiology and convenient for the experimental operations . some models focus on etiology , such as the duct ligation model and the taurocholate - induced pancreatitis model . but the latter is more simple , and less damaging to the animals , which mimic biliary pancreatitis in the clinical setting better than the duct ligation model . several lines of evidence have indicated that the taurocholate - induced pancreatitis model induced by ards was successfully 34 h after the operation . thus , we constructed this model by using sodium taurocholate solution injected into the common biliopancreatic duct . according to the criteria for the diagnosis of ards , 1 , 5 , 9 , and 10 cases of ards were determined as positive results in the 1-h , 2-h , 3-h , and 4-h subgroups , respectively . the results of the present study are consistent with previous findings that sap leading to a systemic inflammatory response was characterized by widespread leukocyte activation and distant organ injury ( e.g. , ards ) [ 2628 ] . our previous study showed that a definite correlation exists between pathological changes ( pancreas and lung ) and progression of sap . in the present study , we found that the glandular architecture of the pancreas and the architecture of the lung in the so group were entirely normal ( h&e ) ( figures 1 and 2 ) . furthermore , histopathologic features of the pancreas , various degrees of edema , minimal necrosis , and inflammatory infiltration ( h&e ) were detected in glandular architecture of the pancreas in the sap group ( figure 3 ) . the sap group was subdivided into the ards group ( 25 rats ) and non - ards group ( 15 rats ) according to criteria including presence of dad , intra - alveolar edema , and organizing interstitial fibrosis ( h&e ) ( figure 4 ) . these results are similar to findings of the present study , suggested that the cases of positive results , which included the presence of dad , was qualitatively assessed ( yes or no ) , showing an increasing trend associated with longer time in the preceding 4 h in the rat model of sap - induced ards . this gap in serum levels of sp - a has previously been reported in many studies , with the results being similar to those in the present study baseline , 43.1514.29 ug / ml vs. so group 51.9116.99 ug / ml vs. sap group 193.435.37 ug / ml ( figure 5 ) ; so group , 51.9116.99 ug / ml vs. ards group 198.029.73 ug / ml vs. non - ards group 185.743.21 ug / ml ( figure 6 ) . these findings demonstrated that ards , the most common and serious complications of sap , was induced in the sap group and contributed to an elevation of sp - a in serum in this study . however , the underlying mechanism has not yet been fully elucidated and may be related to multiple factors . we propose that the possible mechanisms include epithelial injury and leak increased secretion of sp - a per type ii cell , increased leakage from the airspace to the interstitium , an increase in total number of type ii cells per lung due to diffuse hyperplasia , and decreased clearance from the vascular compartment . in addition , the degree of lung injury and sp - a levels leaked from the airspace to the serum was similar in the ards group and non - ards group . moreover , the presence of dad was not observed in the non - ards group because of multiple factors , such as the time of lung injury , individual differences , and the degree of lung injury , rather than the absence of lung injury . found that serum sp - a levels are upregulated by an acute inflammatory stress ; therefore , we used determination of serum levels of sp - a to diagnose sap - induced ards in the rat model . our data analysis showed that sp - a>150 ug / ml statistically discriminates between sap - induced ards and other groups ( so group and non - ards group ) with 100.0% sensitivity and 81.8% specificity . at this cut - off value , the diagnostic accuracy was the highest , and the area under the roc curve of sp - a was 0.88 ( figure 7 ) . furthermore , sp - a>150 ug / ml was diagnosed as sap - induced ards with 71.4% positive predictive value , 100.0% negative predictive value , and 87.5% accuracy in this study . these data demonstrated that the accuracy of diagnosis of sp - a for sap - induced ards is relatively high . therefore , the sp - a , as lung - specific bioactive surface proteins , are valuable parameters for the prediction of sap - induced ards . interestingly , because it is impossible to collect the human pancreatic and lung tissues in the clinical course of treatment , there is little data on this value . we , therefore consider that our findings can be extrapolated to other animals and to humans . although further clinical studies are required to confirm this , rats with sp - a greater than 150 ug / ml and high pulmonary vascular permeability index may be typical for sap - induced ards . in addition , ellsa can detect small changes in sp - a content and serum sp - a level can be easily , quickly , and repeatedly measured at the bedside . serum sp - a level correlates with the progression of lung injury and predicts progression to ards in patients with increased risk . thus , serum sp - a was chosen in this study as the first validated quantitative measure for sap - induced ards and we believe that monitoring serum sp - a level in peripheral blood of sap patients is helpful to evaluate whether sap induces ards . the serum levels of sp - a may be useful as a sensitive and specific serum marker for the diagnosis of sap - induced ards , as confirmed in the present study . however , a great limitation of the present study is that we did not observe the long - term clinical significance , and future studies are required to investigate this .

backgroundthe complexity of multiple - item criteria in acute respiratory distress syndrome ( ards ) often causes inconvenience for physicians in the management of patients with severe acute pancreatitis ( sap ) . we evaluated whether serum sp - a levels in the presence of diffuse alveolar damage ( dad ) can be qualitatively assessed for diagnosis of sap - induced ards.material/methodseighty rats were randomly divided into 2 groups ( n=40 each ) the sham - operated ( so ) group and the sap group and then randomly subdivided into 4 subgroups in a time - course manner . furthermore , rats in the sap group were subdivided into the sap induced - ards group ( ards group ) and the sap without ards group ( non - ards group ) according to the diagnostic standard of ards . the diagnostic cut - off values of sp - a for sap - induced ards were determined by the receiver operating characteristic curve ( roc).resultsserum sp - a levels in baseline , so group , sap group , ards group , and non - ards group were 43.1514.29 , 51.9116.99 , 193.435.37 , 198.0 + 29.73 , and 185.743.21 ug / ml , respectively . the best cut - off value for the serum sp - a level for the diagnosis of sap - induced ards was 150 ug / ml and the area under the roc curve of sp - a was 0.88 . the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of sp - a in the diagnosis of sap - induced ards were 100.0% , 81.8% , 71.4% , 100.0% , and 87.5% , respectively.conclusionsserum sp - a levels may allow the detection of sap - induced ards and may help to support the clinical diagnosis of ards . the optimal serum sp - a cut - off value to discriminate sap - induced ards and other groups ( so group and non - ards group ) is around 150 ug / ml .

Background Material and Methods Animals and groups SAP and ARDS induction Determination of serum SP-A levels Pathologic criteria for pancreas and lung tissues Statistical analysis Results Expression of pathology in pancreas and lung tissues Expression of Serum SP-A levels Diagnosis of SP-A according to ROC curves Discussion Conclusions

mucormycosis , also known as zygomycosis , is an opportunistic and lethal mycological infection caused by a fungus of the order mucorales . it is mostly observed in immunocompromised patients such as those with hematological malignancy or those who have solid organ transplantation ( sot ) or bone marrow transplants . the infection usually starts in the middle nasal meatus and spreads to the paranasal sinuses , the orbit and the intracranial structures by direct extension or through blood vessels . early diagnosis , initiating prompt surgical and medical therapy and correcting the underlying conditions play a vital role in eliminating this infection , but mortality remains high . mucormycosis is uncommon in sot recipients and most cases have been reported after liver or renal transplantation . a 52-year - old male patient was transferred to our department from another center for retroorbital pain , headache and decreased vision accuracy in the left eye for the past 36 h. five months earlier , he had undergone orthotopic heart transplantation under the bicaval technique due to ischemic heart failure . his medical history was significant for type ii diabetes mellitus , poorly controlled by insulin . he also had hypertension and dyslipidemia ( both under treatment ) and nondialysis - dependent chronic kidney disease . his baseline immunosuppressive regimen consisted of prednisone , tacrolimus and mycophenolic acid and he was also taking prophylactic antibiotics : cotrimoxazole , valganciclovir and isoniazid with pyridoxine . physical examination on admission showed that the patient was alert , orientated , afebrile and his vital signs were stable . anterior rhinoscopy , endoscopy with 0 rigid endoscope and examination of the oral cavity were unremarkable . although the nasal exploration was normal , in view of the symptoms and the previous transplant , two samples endoscopically directed , one of the nasal fossa by swabbing and the second of the middle meatus were collected . blood tests showed glucose levels of 248 mg / dl ( 13.8 mmol / l ) and creatinine of 3.63 mg / dl . computed tomography ( ct ) scan revealed soft tissue opacification of left anterior ethmoid sinus and inflammatory changes in the apex of the left orbit [ figure 1a ] . ( a ) computed tomography scan showed soft tissue opacification of left ethmoid sinus ( white arrows ) and inflammatory changes in the apex of the left orbit ( black arrows ) . ( b ) fundus of the left eye with edema and papillary pallor ( * ) , posterior pole retinal edema ( white arrows ) and arteriolar sclerosis ( black arrows ) . ( c ) left nasal endoscopy with 0 rigid endoscope , showing black necrotic eschar ( white arrows ) through the left middle meatus and the middle turbinate ( black arrows ) . nasal septum ( * ) , ( d ) lactophenol cotton blue culture showing rhizopus fungus . sporangiophore ( black arrow ) ( lactophenol cotton blue stain , x1000 ) the metabolic derangement was treated , intravenous antibiotics ( piperacillin / tazobactam 2000/250 mg 8 hourly ) was started and insulin was administered on a sliding scale . on the second day of hospitalization , the patient 's clinical status worsened dramatically . left eye movements were limited in all directions ( frozen eye ) and the left pupil was mydriatic . signs of central retinal artery occlusion were found on the left fundoscopy : edema and papillary pallor , posterior pole retinal edema and arteriolar sclerosis [ figure 1b ] . a second nasal 0 rigid endoscope showed necrotic tissue in the left middle meatus and the middle turbinate [ figure 1c ] . the lactophenol cotton blue preparation revealed a sporangium located on the columella at the apical end of the sporangiophore [ figure 1d ] . following the confirmation of rm , intravenous liposomal amphotericin b the magnetic resonance imaging showed that infection had progressed in the apex of the left orbit , involving the cavernous sinus . we proposed extensive surgical treatment via left paralateral rhinotomy with orbital exenteration and total ethmoidectomy . its incidence has increased over the last 20 years due to increasing use of immunosuppressive drugs . it appears most frequently in the first 6 months after transplantation due to the higher immunosuppression . the incidence ranges from 0.4% to 16% depending on the sot type and is 0%0.6% in heart transplant recipients . other significant predisposing conditions to mucormycosis in sot recipients are poorly controlled diabetes mellitus and renal failure . the fungus spreads quickly in a medium of elevated glucose and acid ph and it is unusual in patients with metabolically controlled diabetes . the main protection against hyphae is neutrophils , but low serum ph decreases their pathogenic and chemotactic capacity . our patient had three predisposing conditions : immunosuppression for sot , poorly controlled diabetes mellitus and renal failure . three main ways of transmission of mucormycosis in transplant recipients have been reported : inhalation in approximately 70% , ingestion in 2% and percutaneous introduction in 13% . it then extends to the orbit and periorbital area where it can produce the orbital apex syndrome with ophthalmoplegia , ptosis , mydriasis and decreased visual accuracy . finally , it disseminates to the cavernous sinus and then to the meninges and brain . clinical presentation of the disease usually involves symptoms of sinusitis and periorbital cellulitis with facial pain and decreased vision . a nasal endoscopy and a biopsy or a culture of the infected area are essential for the definitive diagnosis . a culture gives us more information about the species of mucorales , but it has a higher rate of false negatives than the biopsy . yohai et al . reported nasal ulceration or necrosis in 48% and 78% respectively in rm . in our case , the initial nasal endoscopy was unremarkable , but we performed a middle meatus culture in view of the high suspicion of rm and the culture revealed a fungus of the order mucorales . the contrast - enhanced sinus ct is helpful to delimit the extension of rm and reveal signs of invasion . magnetic resonance is more sensitive than ct to evaluate intradural and intracranial involvement , the cavernous sinus thrombosis and thrombosis of the cavernous portions of the internal carotid artery . radical resection removing all necrotic tissue should be done and various debridements are usually required . lateral rhinotomy , orbital exenteration and occasionally intracranial surgery are performed depending on the extension of rm . orbital exenteration is necessary when the orbit is infected with limited eye movements in all directions ( frozen eye ) and loss of vision . the surgery that we proposed to our patient was a left paralateral rhinotomy with orbital exenteration and total ethmoidectomy removing all the unviable tissue . amphotericin b is the drug of selection , but it is limited because of its renal and systemic toxic effect . this lipid formulation improves circulation time and concentration in the infected area of the associated amphotericin b. the duration of the treatment is not explicitly established . survival rates in diabetic patients with rm are higher than those in patients with other predisposing factors such as renal failure , hematologic malignancies or autoimmune diseases . a delay in diagnosis and treatment , brain involvement , hemiparesis , bilateral sinus involvement and facial necrosis is also associated with poor survival . rm is a rare and aggressive fungal infection in heart transplant recipients and carries a high mortality rate .

mucormycosis is an opportunistic acute fungal infection with a high mortality rate seen in immunocompromised patients . it is extremely rare in heart transplant recipients . rhinocerebral mucormycosis ( rm ) is the most frequently observed presentation . we report a case of rm in a heart transplant recipient 5-month after the procedure , with a fatal outcome .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

dental erosion is a clinical condition that is currently being studied since researchers have reported an increase in its prevalence . furthermore , dentists are becoming more aware about the etiology and early detection of this condition . acid exposure causes enamel surface demineralization , and long - term demineralization leads to erosive enamel wear as a consequence of progressive mineral loss . dentin erosion presents a different histology lesion , since the progression of demineralization is modulated by the presence of the demineralized organic matrix ( dom ) . the dom may be affected by enzymatic and chemical degradation , which may enhance the progression of dentin wear over time . the primary preventive measure for dental erosion is to reduce the frequency and duration of acid exposure . as it is difficult to control the behavior of the patient , such as the frequency of acid intake or special drinking habits , other strategies have been proposed to control dental erosion that are rather less dependent on the compliance of the patient . the most tested alternative is to increase the acid resistance of the teeth through the application of fluoride . accordingly , highly concentrated fluoride applications such as oral rinses , gels and varnishes have been tested . the application of naf at high concentration is able to promote a caf2-like layer precipitation on enamel . the caf2 globules behave as a physical barrier , inhibiting the contact of the acid with enamel and/or acting as a fluoride reservoir . however , this layer presents low acid resistance , thus , its efficacy in controlling dental erosion is restricted , at least under in vitro conditions . on this basis , recent studies have focused on other fluorides , such as those containing polyvalent metals , which may have a higher efficacy than naf due to surface precipitation or incorporation of ions into sound and demineralized tissue . accordingly , the tif4 has shown a promising erosion - inhibiting effect both in vitro and in situ compared to other fluorides . it is hypothesized that titanium may conflate with phosphate groups , producing an acid - resistant surface coating . most studies have applied fluoride between the erosive challenges , showing a positive effect of different fluoride formulations on the progression of tooth erosion . recently , our research group has shown that an experimental tif4 varnish can be more or at least as effective as a naf varnish in reducing erosive tooth wear when applied only once on a previously sound surface . however , there have been no data published regarding the effect of tif4 and naf varnishes on the reduction of tooth erosion progression when applied on pre - eroded tooth surfaces . patients are usually treated with fluoride to prevent erosion when they have signs of the lesion . therefore , the present study aimed to analyze and compare the effect of tif4 and naf varnishes on enamel and dentin erosion progression in vitro . the null hypothesis tested was that there is no significant difference between tif4 and naf varnishes on the progression of erosive tooth wear when compared to the control . bovine teeth were prepared to obtain 48 enamel and 60 root dentin samples ( 4x4x3 mm ) . the teeth were stored in 0.1% buffered thymol solution ( ph 7.0 ) at 4c . the specimens were cut using an isomet low - speed saw cutting machine ( buehler ltd . , lake bluff , il , usa ) with two diamond discs ( extec corp . , eneld , ct , usa ) , which were separated by a 4 mm thick spacer . the surfaces of the specimens were ground at using water - cooled silicon carbide discs ( 320 , 600 and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , removing about 200 m of the surface of the tooth . thereafter , the samples were cleaned in an ultrasonic device with deionized water for 5 min . the removal of the cement from the root dentin was verified using a microscope ( x40 magnification ) . two - thirds of the outer surface ( untreated area ) of the specimens were covered with nail varnish in order to create sound areas on both sides of a central band of eroded enamel and dentin . an initial erosive demineralization was created by immersion of the specimens in 0.1% citric acid ( ph 2.5 ) for 30 min at room temperature under gentle agitation , using a shaking bath with horizontal movements ( 60 rpm ) . after acid exposure , enamel and dentin wear ( m ) were quantitatively determined using contact profilometry ( mahr perthometer , mahr ltda , gttingen , germany ) . for the baseline profile measurement ( initial erosive wear ) , the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) . during the measurement , the dentin specimens were maintained in water ( 100% humidity ) to avoid the dom from shrinking . the diamond stylus was moved from the first reference to the exposed area and then over the other reference area ( 2.5 mm long and 2 mm wide ) . the vertical distance between the horizontal line drawn on the reference areas and the one drawn on the experimental ( eroded ) area was defined as tooth wear using mahr surf xt20 ( software mahr surf xt20 , 2009 , mahr ltda , gttingen , germany ) . the values were averaged ( m ) and used for the random allocation of the samples into the groups ( treatments ) . twelve enamel and 15 dentin specimens were randomly distributed according to the baseline wear of each one of the 4 groups : a 4% tif4 varnish ( 2.45% f- , ph 1.0 ) , a 5.42% naf varnish ( 2.45% f- , ph 5.0 ) , a placebo varnish ( without fluoride ) and no varnish ( control ) . all varnishes contained colophonium , synthetic resin , thickening polymer , essence , artificial sweetener and ethanol . the varnishes were applied in a thin layer using a microbrush , and the specimens were stored in artificial saliva . after 6 h , the varnishes were carefully removed with acetone solution ( 1:1 ) and a scalpel blade , avoiding to touch the enamel surface . the samples were submitted to an erosive demineralization by immersion in 0.1% citric acid ( ph 2.5 ) for 90 s 4 times a day for 5 d. after each demineralization , the specimens were rinsed with deionized water ( 10 s ) and transferred into artificial saliva ( ph 6.8 , 30 ml / specimen , unstirred , 25c ) for 2 h. after the last daily erosive treatment , the specimens were also stored in artificial saliva overnight . the citric acid was renewed at each erosive challenge , and the artificial saliva was replaced daily . the artificial saliva consisted of 0.2 mm glucose , 9.9 mm nacl , 1.5 mm cacl2.2h2o , 3 mm nh4cl , 17 mm kcl , 2 mm nascn , 2.4 mm k2hpo4 , 3.3 mm urea , 2.4 mm nah2po4 , and 11 m ascorbic acid ( ph 6.8 ) . after 5 days of ph cycling , the nail varnish was removed , and 5 profiles were recorded as done at the baseline profile measurement . the values were averaged ( m ) , and the increment value ( final wear baseline wear ) was calculated for enamel and dentin specimens ( with and without the dom in the case of dentin ) and then submitted to statistical analysis . after the profilometric analysis , the dentin samples were submitted to degradation of the collagen fibrils in order to check the influence of the dom on the profile measurement and comparison among the groups . a collagenase enzyme ( type vii from clostridium histolyticum , product no . louis , mo , usa ) , with a collagen digestion activity of 1.98 u/g , solid at 25c , ph 7.5 , in the presence of calcium ions , was used to remove the dom . the collagen layer was removed by adding 100 u / ml of enzyme into artificial saliva containing 20 mm hepes , 0.70 mm cacl2 , 0.20 mm mgcl2.6h2o , 4 mm kh2po4 , 30 mm kcl , 0.30 mm nan3 , and edta - free protease inhibitor cocktail ( complete tm protease inhibitor of cocktail , santa cruz biotechnology , santa cruz , ca , usa ) for 5 d at 37c . the software graphpad instat ( graphpad software , san diego , ca , usa ) was used . the assumptions of equality of variances and normal distribution of data were verified for all the variables tested using bartlett and kolmogorov - smirnov tests , respectively . as the equality of variances was satisfied for enamel , the differences among treatments were analyzed using anova followed by tukey s test . for dentin , the equality of variances was not satisfied , and the differences among treatments were analyzed using kruskal - wallis followed by dunn s test . the sample size was 12 for enamel and 15 for dentin , and the level of significance was set at 5% . bovine teeth were prepared to obtain 48 enamel and 60 root dentin samples ( 4x4x3 mm ) . the teeth were stored in 0.1% buffered thymol solution ( ph 7.0 ) at 4c . the specimens were cut using an isomet low - speed saw cutting machine ( buehler ltd . , lake bluff , il , usa ) with two diamond discs ( extec corp . , eneld , ct , usa ) , which were separated by a 4 mm thick spacer . the surfaces of the specimens were ground at using water - cooled silicon carbide discs ( 320 , 600 and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , removing about 200 m of the surface of the tooth . thereafter , the samples were cleaned in an ultrasonic device with deionized water for 5 min . the removal of the cement from the root dentin was verified using a microscope ( x40 magnification ) . two - thirds of the outer surface ( untreated area ) of the specimens were covered with nail varnish in order to create sound areas on both sides of a central band of eroded enamel and dentin . an initial erosive demineralization was created by immersion of the specimens in 0.1% citric acid ( ph 2.5 ) for 30 min at room temperature under gentle agitation , using a shaking bath with horizontal movements ( 60 rpm ) . after acid exposure , enamel and dentin wear ( m ) were quantitatively determined using contact profilometry ( mahr perthometer , mahr ltda , gttingen , germany ) . for the baseline profile measurement ( initial erosive wear ) , the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) . during the measurement , the dentin specimens were maintained in water ( 100% humidity ) to avoid the dom from shrinking . the diamond stylus was moved from the first reference to the exposed area and then over the other reference area ( 2.5 mm long and 2 mm wide ) . five profile measurements were performed at intervals of 0.5 mm . the vertical distance between the horizontal line drawn on the reference areas and the one drawn on the experimental ( eroded ) area was defined as tooth wear using mahr surf xt20 ( software mahr surf xt20 , 2009 , mahr ltda , gttingen , germany ) . the values were averaged ( m ) and used for the random allocation of the samples into the groups ( treatments ) . twelve enamel and 15 dentin specimens were randomly distributed according to the baseline wear of each one of the 4 groups : a 4% tif4 varnish ( 2.45% f- , ph 1.0 ) , a 5.42% naf varnish ( 2.45% f- , ph 5.0 ) , a placebo varnish ( without fluoride ) and no varnish ( control ) . all varnishes contained colophonium , synthetic resin , thickening polymer , essence , artificial sweetener and ethanol . the varnishes were applied in a thin layer using a microbrush , and the specimens were stored in artificial saliva . after 6 h , the varnishes were carefully removed with acetone solution ( 1:1 ) and a scalpel blade , avoiding to touch the enamel surface . the samples were submitted to an erosive demineralization by immersion in 0.1% citric acid ( ph 2.5 ) for 90 s 4 times a day for 5 d. after each demineralization , the specimens were rinsed with deionized water ( 10 s ) and transferred into artificial saliva ( ph 6.8 , 30 ml / specimen , unstirred , 25c ) for 2 h. after the last daily erosive treatment , the specimens were also stored in artificial saliva overnight . the citric acid was renewed at each erosive challenge , and the artificial saliva was replaced daily . the artificial saliva consisted of 0.2 mm glucose , 9.9 mm nacl , 1.5 mm cacl2.2h2o , 3 mm nh4cl , 17 mm kcl , 2 mm nascn , 2.4 mm k2hpo4 , 3.3 mm urea , 2.4 mm nah2po4 , and 11 m ascorbic acid ( ph 6.8 ) . after 5 days of ph cycling , the nail varnish was removed , and 5 profiles were recorded as done at the baseline profile measurement . the values were averaged ( m ) , and the increment value ( final wear baseline wear ) was calculated for enamel and dentin specimens ( with and without the dom in the case of dentin ) and then submitted to statistical analysis . after the profilometric analysis , the dentin samples were submitted to degradation of the collagen fibrils in order to check the influence of the dom on the profile measurement and comparison among the groups . a collagenase enzyme ( type vii from clostridium histolyticum , product no . louis , mo , usa ) , with a collagen digestion activity of 1.98 u/g , solid at 25c , ph 7.5 , in the presence of calcium ions , was used to remove the dom . the collagen layer was removed by adding 100 u / ml of enzyme into artificial saliva containing 20 mm hepes , 0.70 mm cacl2 , 0.20 mm mgcl2.6h2o , 4 mm kh2po4 , 30 mm kcl , 0.30 mm nan3 , and edta - free protease inhibitor cocktail ( complete tm protease inhibitor of cocktail , santa cruz biotechnology , santa cruz , ca , usa ) for 5 d at 37c . the software graphpad instat ( graphpad software , san diego , ca , usa ) was used . the assumptions of equality of variances and normal distribution of data were verified for all the variables tested using bartlett and kolmogorov - smirnov tests , respectively . as the equality of variances was satisfied for enamel , the differences among treatments were analyzed using anova followed by tukey s test . for dentin , the equality of variances was not satisfied , and the differences among treatments were analyzed using kruskal - wallis followed by dunn s test . the sample size was 12 for enamel and 15 for dentin , and the level of significance was set at 5% . the baseline erosive wear means were 12.9 and 11.4 m for enamel and dentin , respectively . the mean increment of enamel wear ( m ) was similar between tif4 and naf varnish . both fluoride varnishes showed significantly less erosion ( 67% and 53% decrease in enamel wear , respectively ) compared to the placebo varnish and the control group ( p<0.0001 ) , which did not differ from each other . table 1meanstandard deviation of the baseline , final and increment enamel erosive wear ( m ) according to each treatment tif nafplacebocontrolbaseline13.31.712.82.712.82.312.52.7final14.72.114.82.016.72.517.02.7increment1.51.1a2.11.7a3.91.1b4.50.9b*distinct lower case letters indicate significant differences among the treatment groups ( anova and tukey s test , p<0.0001 , n=12 ) * distinct lower case letters indicate significant differences among the treatment groups ( anova and tukey s test , p<0.0001 , n=12 ) table 2baseline , final and increment dentin erosive wear means ( m ) , with and without dom ( interquartile range ) , respectively , according to each treatment tif4 nafplacebocontrolbaseline11.311.211.211.2final12.3/13.112.0/13.214.6/16.014.8/15.4increment0.97 ( 0.49 ) 1.87 ( 0.95 ) 1.03 ( 0.68 ) 2.13 ( 0.73 ) 3.53 ( 1.42 ) 4.47 ( 1.91 ) 3.53 ( 1.62 ) 4.36 ( 2.18 ) * distinct lower case letters indicate significant differences among the treatment groups ( kruskal - wallis and dunn , p<0.0001 , n=15 ) * distinct lower case letters indicate significant differences among the treatment groups ( kruskal - wallis and dunn , p<0.0001 , n=15 ) regarding dentin , tif4 and naf presented similar ability to reduce the progression of erosive wear ( 72.5% and 71% decrease in dentin wear , respectively ) , regardless of the presence or absence of the dom , compared to placebo varnish and control group ( p<0.0001 ) , which did not differ from each other . investigations of the potential of tif4 to prevent tooth erosion have been performed since 1997 . it has been speculated that its effect against erosion is due to a high incorporation of fluoride into enamel and a formation of an acid surface precipitation composed by tio2 or hydrated titanium phosphate . our group showed that the tif4 incorporated in an experimental varnish presented a higher protective potential than the tif4 solution on enamel erosion , enamel erosion - abrasion and dentin erosion - abrasion in vitro . we speculate that the better ability of the varnish to adhere to the tooth surface , allowing an increased contact time with the tooth , is responsible for its positive effect . however , our previous studies have focused on the prevention of dental erosion rather than on the control of its progression . the question of the present study was whether tif4 or naf varnishes were also able to reduce the progression of tooth wear when applied on previously eroded surfaces . in our study , we produced a baseline enamel and dentin erosive lesion by aggressive acid exposure done under agitation . fast stirring can increase the five - fold of the enamel wear compared to constant conditions . clinically , this procedure simulates drinking habits such as rinsing , which may increase the risk of erosive wear . the idea behind the procedure was also to simulate a patient with a history of erosive lesions , for whom a fluoride application would be indicated , according to the basic erosive wear examination ( bewe ) guidelines . the varnishes were then applied on the eroded surface only once and removed after 6 h in order to simulate the clinical professional application . in the present study , all fluoridated varnishes had a similar significant potential to reduce enamel and dentin progression , therefore , the null hypothesis can be rejected . however , there was no significant difference between both fluoride varnishes . the results observed for dentin agree with recent data from our group , in which all fluoridated varnishes tested ( experimental tif4 and naf , duraphat and duofluorid varnishes ) were similarly able to significantly reduce dentin wear compared to the placebo varnish , the fluoridated solutions ( tif4 and naf ) and the control group . a limitation of analyzing erosion in dentin is the complexity of the lesion histology , which is characterized by centripetal mineral loss starting in the peritubular areas that progresses to the intertubular area , leading to a bulk tissue loss and the presence of a completely demineralized dentin surface zone that is stable while hydrated . therefore , erosive wear is difficult to quantify since the quality of the remaining organic layer may interfere in the measurement . the limitation of the wear measurement is due to the shrinkage of the dom that may occur under different environments . therefore , to generate reliable data , the profiles must be measured with the samples immersed in water . on the other hand , schlueter , et al . ( 2011 ) advise that the dom should be removed before the profilometric measurement to prevent this problem from occurring . we found that the thickness of the dom of our samples was about 1 m , regardless of the treatment . it is important to highlight that , in the present study , the results were maintained when the wear was measured on dentin without the dom . some studies have shown that the preventive effect of fluorides on dentin erosion is lower compared to enamel and highly dependent on the presence of the organic matrix . dentin erosion is much more complex than enamel erosion because the organic matrix plays an important role in the progression of wear . the dom has a buffering capacity that prevents further demineralization , especially in the presence of fluoride . at least for naf , the enzymatic removal of the organic matrix substantially decreases its effect . considering that it is still unclear if what extent the organic matrix is retained under clinical conditions , it would be interesting to analyze the effect of tif4 varnish applied on eroded dentin with or without dom . in respect to the results of enamel , the present data are not in accordance with a previous study from our group , in which we found significantly less erosion for the enamel samples treated with a tif4 varnish than those treated with a naf ( duraphat ) or naf / caf2 ( duofluorid ) varnish . based on the experimental conditions , we speculate that the glaze - like surface layer produced by tif4 may be thinner and with lower amounts of ti on demineralized enamel surfaces compared to sound ones , as previously shown by chevitarese , et al . , we suggest that the effect of other preventive measures ( professional products ) should be tested on previously eroded tooth surfaces in future studies , since fluoride application is indicated only for patients with a high risk of erosion , according to bewe guidelines . the effect of the tif4 varnish on eroded dentin surfaces in the absence of the dom is another important topic to be further considered . finally , the interaction of this polyvalent metal fluoride in terms of glaze surface layer formation on an eroded tooth should be tested under conditions as close as possible to the clinical situation by simulating general tooth wear ( e.g. erosion and abrasion ) and using in situ models . under the conditions of the present study , it can be concluded that the tif4 varnish was as effective as the naf varnish to reduce the enamel and dentin erosion progression in vitro .

objective this study assessed the effect of fluoride varnishes on the progression of tooth erosion in vitro . material and methods : forty - eight enamel and 60 root dentin samples were previously demineralized ( 0.1% citric acid , ph 2.5 , 30 min ) , leading to a baseline and erosive wear of 12.9 and 11.4 m , respectively . the samples were randomly treated ( 6 h ) with a 4% tif4 varnish ( 2.45%f- , ph 1.0 ) , a 5.42% naf varnish ( 2.45%f- , ph 5.0 ) , a placebo varnish and no varnish ( control ) . the samples were then subjected to erosive ph cycles ( 4x90 s / day in 0.1% citric acid , intercalated with artificial saliva ) for 5 days . the increment of the erosive tooth wear was calculated . in the case of dentin , this final measurement was done with and without the demineralized organic matrix ( dom ) . enamel and dentin data were analyzed using anova / tukey s and kruskal - wallis / dunn tests , respectively ( p<0.05).results the tif4 ( means.d : 1.51.1 m ) and naf ( 2.11.7 m ) varnishes significantly reduced enamel wear progression compared to the placebo varnish ( 3.91.1 m ) and control ( 4.50.9 m ) . the same differences were found for dentin in the presence and absence of the dom , respectively : tif4 ( average : 0.97/1.87 m ) , naf ( 1.03/2.13 m ) , placebo varnish ( 3.53/4.47 m ) and control ( 3.53/4.36 m).conclusion the tif4 and naf varnishes were equally effective in reducing the progression of tooth erosion in vitro .

INTRODUCTION MATERIAL AND METHODS Sample preparation Initial erosive lesion and profile measurement Fluoride treatment and pH cycling Final profile measurement Statistical analysis RESULTS DISCUSSION CONCLUSIONS

the hla - a locus exon 2 region from position 73346 , allele a*01011 , was used for the design of consensus oligos ( figure 1 ) . allele - specific ( variant ) oligos were designed based on single or double nucleotide variants according to alignment to the arbitrarily selected consensus sequence ( hla - a*0201 ) . variant and consensus oligos consisted of 18-mers with a 5 ' amino - modifier having a six - carbon spacer for immobilization ( operon technologies , inc , alameda , ca ) . melting temperatures of oligo probes were maintained as close as possible to a range of 5660c . 350400 pmol/ l oligo probes synthesized in 96-well format were dried and re - suspended in 3 ssc for printing . probes were spotted onto a 3d - link activated slide for covalent immobilization ( motor roller ) using omnigrid robotic printer ( genemachine ) with four printing pins picking up 0.25 l of probe solution and depositing 0.6 nl per spot ( telechem international , inc . ) . spot diameter was 90100 m , spaced at 250 m to prepare 4 16 6 spot / arrays . after printing , slides were kept in a sealed humidifier chamber at room temperature overnight and blocked with 50 mm ethanolamine , 0.1 m tris , ph9 and 0.1% sds at 50c for 15 minutes followed with rinsing in water twice , washing with 4 ssc/0.1% sds 50c for 45 minutes , rinsing with water briefly and centrifuging at 800 rpm for 3 minutes with micro - plate carriers . 12 heterozygous or homozygous samples were tested for oligo nucleotide array hybridization and confirmed by sequence - based typing using the abi prism 3700 - 96 analyzer . in order to generate single strand dna , pcr products from exon 1 to exon 5 of the hla - a locus were amplified with an attachment of t7 promoter to the 5 ' end using 5 ' t7-ex1a-6 primer 5'aaacgacggccagtgaatacgactcactataggcgccagacgccgaggatggcc3 ' ) and three 3 ' primers ( 3 ' ex 5-a 993 - 1 cat tgc tgg cct ggt tct cc ; 3 ' ex 5-a 993 - 2 cat tgc tgg cct ggt tct ctt ; 3 ' ex 5-a 993 - 3 cat tgc tgg cct agt tct ctt ) . pcr reaction was mixed with 25 l of hotstart pcr reagents ( qiagen , ca ) , 5 ng-0.5 g of genomic dna , 5 l of 15 m 5'primer , 5 l of 15 m 3'primer mix and h2o to a 50 l final volume . the reaction was cycled at 95c for 10 minutes , 96c for 35 seconds , 65c for 45 seconds , 72c for 3 minutes , 4 cycle ; 96c for 30 seconds , 60c for 40 seconds , 72c for 3 minutes , 19 cycles and 96c for 30 seconds , 55c for 40 seconds , 72c for 2 minutes , 9 cycles . one l of pcr product from each sample was analyzed using a bioanalyzer on dna7500 chip ( agilent biotechnology ) . the pcr products were then precipitated with etoh at room temperature and re - suspended in depc - treated h2o at 0.1 g/l concentration . in vitro transcription ( ivt ) for each sample , the following reaction mixture was made : 4 l of each 75 mm ntp ( a , g , c and utp ) , 4 l reaction buffer , 4 l enzyme mix ( rnase inhibitor and t7 phage polymerase ) and 1 g purified pcr product in 16 l depc - treated h2o . amplified rna was then purified using trizol reagent according to manufacture instruction ( gibcobrl ) and re - suspended in 40 l of depc water . rna concentrations were estimated by using a bioanalyzer on rna 6000 chip ( agilent biotechnology ) . fluorescence - labeled single strand cdna was generated by reverse transcription ( rt ) and used for hybridization . in the rt reaction , 4 l of first strand buffer , 1 l random hexmer ( 8 g/l ; boehringer mannheim ) , 2 l 10 low t - dntp ( 5 mm a , c and gtp , 2 mm dttp ) , 2 l 1 mm cy - dutp ( cy3 for reference sample or cy5 for test sample unless otherwise specified ) , 2 l 0.1 m dtt , 1 l rnasin and 1.2 g amplified rna in 8 l depc h2o were mixed and heated for five minutes at 65c . this was followed by addition of 1 l superscript ii ( life technology , ) , 40 minutes of incubation at 42c , another 1 ul of superscript ii and 50 more minutes continued incubation at 42c . reactions were stopped by addition of 2.5 l 500 mm edta , 5 l 1 m naoh and heated to 65c for 15 minute to hydrolyze the rna . tris buffer ( 12.5 l of 1 m ) was added immediately to neutralize the ph , and the volume risen to 70 l by adding 35 l of 1 te . the flow through mixed with 200 l 1 te was concentrated to 2040 l using microcon ym-30 column ( millipore ) and further concentrated to 8 ul using speed - vacuum . cy3- and cy5-labeled probes were combined ( 1:1 ratio ) and 5 l 20 ssc , 0.5 l 10% sds and 0.5 l of 4 mg / ml salmon sperm dna were added to the probe for hybridization . prepared probe mixture was applied to an array slide with cover slid and hybridized at 47c for different amounts of time as described in the text . slides were washed with 4 ssc , 2 ssc with 0.1 % sds , 1 ssc , 0.2 ssc and 0.05 ssc sequentially for one minute each step and dried by centrifugation at 800 rpm for 3 minutes . the slides were then scanned for fluorescent signal using a genepix 4000b scanner and the results analyzed using genepix pro3 software ( axon instruments , inc . ) . the hla - a locus exon 2 region from position 73346 , allele a*01011 , was used for the design of consensus oligos ( figure 1 ) . allele - specific ( variant ) oligos were designed based on single or double nucleotide variants according to alignment to the arbitrarily selected consensus sequence ( hla - a*0201 ) . variant and consensus oligos consisted of 18-mers with a 5 ' amino - modifier having a six - carbon spacer for immobilization ( operon technologies , inc , alameda , ca ) . melting temperatures of oligo probes were maintained as close as possible to a range of 5660c . 350400 pmol/ l oligo probes synthesized in 96-well format were dried and re - suspended in 3 ssc for printing . probes were spotted onto a 3d - link activated slide for covalent immobilization ( motor roller ) using omnigrid robotic printer ( genemachine ) with four printing pins picking up 0.25 l of probe solution and depositing 0.6 nl per spot ( telechem international , inc . ) . spot diameter was 90100 m , spaced at 250 m to prepare 4 16 6 spot / arrays . after printing , slides were kept in a sealed humidifier chamber at room temperature overnight and blocked with 50 mm ethanolamine , 0.1 m tris , ph9 and 0.1% sds at 50c for 15 minutes followed with rinsing in water twice , washing with 4 ssc/0.1% sds 50c for 45 minutes , rinsing with water briefly and centrifuging at 800 rpm for 3 minutes with micro - plate carriers . 12 heterozygous or homozygous samples were tested for oligo nucleotide array hybridization and confirmed by sequence - based typing using the abi prism 3700 - 96 analyzer . in order to generate single strand dna , pcr products from exon 1 to exon 5 of the hla - a locus were amplified with an attachment of t7 promoter to the 5 ' end using 5 ' t7-ex1a-6 primer 5'aaacgacggccagtgaatacgactcactataggcgccagacgccgaggatggcc3 ' ) and three 3 ' primers ( 3 ' ex 5-a 993 - 1 cat tgc tgg cct ggt tct cc ; 3 ' ex 5-a 993 - 2 cat tgc tgg cct ggt tct ctt ; 3 ' ex 5-a 993 - 3 cat tgc tgg cct agt tct ctt ) . pcr reaction was mixed with 25 l of hotstart pcr reagents ( qiagen , ca ) , 5 ng-0.5 g of genomic dna , 5 l of 15 m 5'primer , 5 l of 15 m 3'primer mix and h2o to a 50 l final volume . the reaction was cycled at 95c for 10 minutes , 96c for 35 seconds , 65c for 45 seconds , 72c for 3 minutes , 4 cycle ; 96c for 30 seconds , 60c for 40 seconds , 72c for 3 minutes , 19 cycles and 96c for 30 seconds , 55c for 40 seconds , 72c for 2 minutes , 9 cycles . one l of pcr product from each sample was analyzed using a bioanalyzer on dna7500 chip ( agilent biotechnology ) . the pcr products were then precipitated with etoh at room temperature and re - suspended in depc - treated h2o at 0.1 g/l concentration . in vitro transcription ( ivt ) for each sample , the following reaction mixture was made : 4 l of each 75 mm ntp ( a , g , c and utp ) , 4 l reaction buffer , 4 l enzyme mix ( rnase inhibitor and t7 phage polymerase ) and 1 g purified pcr product in 16 l depc - treated h2o . amplified rna was then purified using trizol reagent according to manufacture instruction ( gibcobrl ) and re - suspended in 40 l of depc water . rna concentrations were estimated by using a bioanalyzer on rna 6000 chip ( agilent biotechnology ) . fluorescence - labeled single strand cdna was generated by reverse transcription ( rt ) and used for hybridization . in the rt reaction , 4 l of first strand buffer , 1 l random hexmer ( 8 g/l ; boehringer mannheim ) , 2 l 10 low t - dntp ( 5 mm a , c and gtp , 2 mm dttp ) , 2 l 1 mm cy - dutp ( cy3 for reference sample or cy5 for test sample unless otherwise specified ) , 2 l 0.1 m dtt , 1 l rnasin and 1.2 g amplified rna in 8 l depc h2o were mixed and heated for five minutes at 65c . this was followed by addition of 1 l superscript ii ( life technology , ) , 40 minutes of incubation at 42c , another 1 ul of superscript ii and 50 more minutes continued incubation at 42c . reactions were stopped by addition of 2.5 l 500 mm edta , 5 l 1 m naoh and heated to 65c for 15 minute to hydrolyze the rna . tris buffer ( 12.5 l of 1 m ) was added immediately to neutralize the ph , and the volume risen to 70 l by adding 35 l of 1 te . the flow through mixed with 200 l 1 te was concentrated to 2040 l using microcon ym-30 column ( millipore ) and further concentrated to 8 ul using speed - vacuum . cy3- and cy5-labeled probes were combined ( 1:1 ratio ) and 5 l 20 ssc , 0.5 l 10% sds and 0.5 l of 4 mg / ml salmon sperm dna were added to the probe for hybridization . prepared probe mixture was applied to an array slide with cover slid and hybridized at 47c for different amounts of time as described in the text . slides were washed with 4 ssc , 2 ssc with 0.1 % sds , 1 ssc , 0.2 ssc and 0.05 ssc sequentially for one minute each step and dried by centrifugation at 800 rpm for 3 minutes . the slides were then scanned for fluorescent signal using a genepix 4000b scanner and the results analyzed using genepix pro3 software ( axon instruments , inc . ) .

detection of unknown single nucleotide polymorphism ( snp ) relies on large scale sequencing expeditions of genomic fragments or complex high - throughput chip technology . we describe a simplified strategy for fluorimetric detection of known and unknown snp by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes . the array consists of two sets of 18-mer oligonucleotide probes . one set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected " consensus " sequence ( consensus - oligos ) , the other includes oligos specific for known snp within the same genomic region ( variant - oligos ) . fluorescence - labeled dna amplified from a homozygous source identical to the consensus represents the reference target and is co - hybridized with a differentially - labeled test sample . lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant - oligos designates a known allele . lack of hybridization to consensus- and variant - oligos indicates a new allele . detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal . this method can identify unknown snp in heterozygous conditions with a sensitivity of 82% and specificity of 90% . this strategy should dramatically increase the efficiency of snp detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials .

Material and methods Oligo nucleotide probe design Oligonucleotide array printing and post process of slides Test and consensus reference samples Preparation of target nucleic acids Target labeling and hybridization

cancer is one of the most common malignancies worldwide ; it is the leading cause of death in economically developed countries and the second leading cause of death in developing countries . approximately 12.7 million new cases of cancer and 7.6 million cancer - related deaths were reported in 2008 . despite the efforts exerted by many researchers to elucidate the mechanism of carcinogenesis , environmental factors , diet , lifestyle , and smoking and drinking habits have been implicated in the development of cancer [ 3 , 4 ] . various epidemiological studies have revealed that inflammation - associated factors , such as interleukin- ( il- ) 1 , il-6 , il-10 , and tumor necrosis factor- , are associated with cancer tumorigenesis . interleukin-17 ( il-17 ) is a proinflammatory cytokine that serves important functions in inflammation , autoimmune disorders , and cancer . the il-17 cytokine family consists of six members ( il-17a to il-17f ) and five receptors ( il-17ra to il-17rd and sef ) [ 7 , 8 ] . these cytokines are primarily produced from a subset of cd4 + effector cells known as th17 cells [ 9 , 10 ] . clinical studies have shown increased il-17 expression in malignant tumors [ 1114 ] . single nucleotide polymorphisms ( snps ) can alter gene functions and protein expression , which influence cell proliferation and increase cancer risk . the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms are the most common loci associated with il-17 activity and cancer risk . in 2009 , shibata et al . conducted the first study and reported a positive relationship between gastric cancer and the il-17a rs2275913g > a polymorphism in a japanese population . but no significant association was found between gastric cancer and polymorphisms of il-17f rs763780 t > c . many epidemiological studies have focused on the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk therefore , we performed a meta - analysis to clarify the possible association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk . the pubmed , embase , and chinese national knowledge infrastructure databases were searched using the terms cancer , tumor , the related articles option was also used in each research article to find potential relevant studies on the same topic . only studies published in english or chinese the inclusion criteria in this meta - analysis were as follows : ( a ) researches that focused on population , ( b ) studies that evaluated the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk , ( c ) case - controls studies , and ( d ) studies that contain available genotype frequency to estimate the odds ratio ( or ) and 95% confidence intervals ( cis ) . the largest or the most recent publication was selected when some data were the same or overlapped . two investigators ( yu - ming niu and hua yuan ) independently extracted the following information from each eligible publication : first author 's name , publication year , country of origin , ethnicity of the individuals involved ( categorized as asian or caucasian ) , source of controls , number of cases and controls , number of genotypes cases and controls , hardy - weinberg equilibrium ( hwe ) and minor allele frequency ( maf ) , and cancer category . the strength of the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk was assessed by calculating crude ors with 95% cis . heterogeneity was calculated based on the i statistic with low , moderate , and high i values of 25% , 50% , and 75% , respectively [ 16 , 17 ] . when i 50% ( which indicated a lack of heterogeneity ) , the or estimation of each model was calculated by using the fixed - effects model ( mantel - haenszel method ) ; otherwise , the random - effects model ( dersimonian and laird method ) was used . sensitivity analyses were assessed by deleting each study to reflect the influence of individual datasets on the pooled ors . statistical analysis was performed using stata version 11.0 ( stata corporation , college station , tx , usa ) with two - sided p values . a total of 185 relevant studies were found with the research words and manual research . after careful review , 10 published case - control studies involving 4,516 cases and 5,645 controls met our inclusion criteria [ 15 , 2028 ] . we found 10 and 7 eligible studies with adequate genotype and research subjects according to il-17a rs2275913g > a and il-17frs763780t > c polymorphism . nine studies involved asian populations ( seven involved the chinese population ) , and one study involved a caucasian population . diverse genotyping methods were used , including polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) [ 23 , 27 ] , taqman [ 21 , 24 , 28 ] , polymerase chain reaction - sequence specific primers ( pcr - sscp ) [ 15 , 25 ] , massarray [ 20 , 22 ] , and snapshot snp assay methods in eligible publications . the genotypic distribution of controls in only two and three studies deviated from hwe in the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms , respectively . the il-17a rs2275913g > a polymorphism showed significant associations with cancer risk in all populations ( for a versus g : or = 1.28 , 95% ci : 1.161.41 , p < 0.001 , i = 61.1% ; for ga versus gg : or = 1.12 , 95% ci : 1.021.23 , p = 0.015 , i = 27.8% ; for aa versus gg : or = 1.71 , 95% ci : 1.382.41 , p < 0.001 , i = 69.6% ; for ga + aa versus gg : or = 1.23 , 95% ci : 1.131.34 , p < 0.001 , i = 6.4% ( figure 2 ) ; and for aa versus gg + ga : or = 1.62 , 95% ci : 1.272.07 , p < 0.001 , i = 81.4% ) . the succeeding stratified analysis according to hwe , ethnicity , and study design subgroup also presented that the il-17a rs2275913g > a polymorphism may be a strong risk factor in the development of cancer , especially gastric cancer , in the chinese population . statistical analysis also indicated that the il-17f rs763780t > c polymorphisms were significantly associated with cancer risk , particularly gastric cancer ( for c versus t : or = 1.29 , 95% ci : 1.141.46 , p < 0.001 , i = 0% ; for tc versus tt : or = 1.33 , 95% ci : 1.131.55 , p < 0.001 , i = 21.6% ; for cc versus tt : or = 1.40 , 95% ci : 1.041.88 , p = 0.026 , i = 0% ; for tc + cc versus tt : or = 1.34 , 95% ci : 1.161.55 , p < 0.001 , i = 16% ( figure 3 ) ) . a single study involved in the meta - analysis was deleted each time to reflect the influence of the individual data set on the pooled ors , and the corresponding pooled ors were not qualitatively altered . this indicated that the results about the association between il-17 gene polymorphisms and cancer risk were statistically robust ( figures 4 and 5 ) . funnel plot and egger 's test were performed to estimate the publication bias of literature . publication bias was detected in the meta - analyses on the allele contrast , homozygote ( aa versus gg ) , dominant , and recessive models of the il-17a rs2275913g > a polymorphism ( figure 6 for ga + aa versus gg model ) , except for the ga versus gg model ( p = 0.652 ) . stratified analyses were conducted only with the hwe and chinese population , but the results were not substantially different . for the il-17f rs763780t > c polymorphism , the funnel plots did not show any asymmetrical evidence in all genetic models ( figure 7 ) . the result was further supported by analysis using egger 's tests ( p = 0.102 for c versus t ; p = 0.185 for tc versus tt ; p = 0.382 for cc versus tt ; p = 0.114 for tc + cc versus tt ( figure 7 ) ; p = 0.792 for cc versus tt + tc ) . carcinogenesis is a multistep process that involves numerous factors , such as smoking , drinking , xenobiotics infections , nutrition deficiency , and host genetic factor . cancer - related inflammation factors have been recently confirmed to increase the risk of developing malignant tumors . il-17 is a relatively novel cytokine family that is connected with adaptive and innate immune systems . il-17a and il-17f are members of the il-17 cytokine family that are responsible for the pathogenic activity of il-17 cells , the lineage of cd4 effector cells , and multiple proinflammatory mediators . genetic polymorphisms of the il-17a and il-17f cytokines could change the function and expression of cytokines , which influence the activity of ils [ 12 , 30 , 31 ] . several studies have revealed that il-17a and il-17f polymorphisms are associated with gastric cancer , breast cancer , and so on . however , the results of these studies are inconsistent . in 2009 , shibata et al . were the first to report that the aa homozygote was significantly correlated with the development of gastric cancer compared with the common homozygous genotype ( gg ) in a japanese population ( or = 3.02 , 95% ci : 1.864.91 ) . one year later , chen et al . also found a positive relationship between gastric cancer and the il-17a rs2275913g > a polymorphism in a chinese han population with a drinking habit ( for a versus g : or = 1.37 , 95% ci : 1.071.76 ) . , rafiei et al . , zhang et al . , and zhuet al . in gastric cancer . furthermore , the mutation of il-17a rs2275913g > a locus was also demonstrated as tumorigenic for bladder cancer , breast cancer , and cervical cancer . however , another article detected no significant association between the il-17a rs2275913g > a polymorphism and gastric cancer risk . regarding the il-17f rs763780t > c polymorphism , wu et al . found that the ct and cc genotypes are associated with an increased risk of gastric cancer compared with the tt genotype ( or = 1.51 , 95% ci : 1.221.87 for ct ; or = 1.61 , 95% ci : 1.032.51 for cc ) . by contrast , zhou et al . showed that bladder cancer patients have significantly higher frequencies of t allele than controls . this result indicates that this t allele is significantly associated with bladder cancer ( or = 1.46 , 95% ci : 1.072.00 ) . furthermore , other researches did not find any significant association between the il-17f rs763780t > c polymorphism and cancer risk [ 15 , 20 , 22 , 24 , 26 ] . to the best of our knowledge , this meta - analysis is the first to determine the association of il-17 polymorphisms with cancer risk . this study focused on two common il-17 polymorphisms , namely , il-17a rs2275913g > a ( 10 studies with 4,516 cases and 5,645 controls ) and il-17f rs763780t > c ( 7 studies with 2,863 cases and 3,773 controls ) . significant associations were found between the il-17a rs2275913g > a polymorphism and cancer risk in all five genotype models of total populations . besides , we also detected some association between the il-17f rs763780t > c polymorphism and the risk of asians ( chinese ) , population - based and hospital - based controls , and gastric cancer or other cancers in the subgroup analysis by hwe publications , ethnicity , control design , and cancer category . interestingly , nine researches focused on asian population ; the results of our meta - analysis demonstrated that the il-17a rs2275913g > a polymorphism may be a stranger canner risk for asian ethnicity ( including chinese ) . moreover , the category of control design did not influence the results ; not only the population - based but also the hospital - based controls all showed that significant association existed between il-17a rs2275913g > a polymorphism and cancer risk . in all selected publications , seven studies focused on gastric cancer and the results also indicated that il-17a rs2275913g > a polymorphism plays an important role during the development of gastric cancer . for il-17f rs763780t > c polymorphism , all selected studies were conducted in asian population and significant association only was found in codominant model ( cc versus tt ) in total population . furthermore , subgroup analyses revealed a significantly increased risk of gastric cancer with il-17f rs763780t > c polymorphism in four models . a combination of relative genotypes may be a higher risk factor than a single locus genotype . linkage disequilibrium and haplotype analyses of the two polymorphisms were not conducted because of the lack of original data on the individual genotypes from the included studies . third , these results were based on unadjusted estimates , and the evaluations were limited without the effects of gene - gene and gene - environment interactions . finally , most of the included studies had been conducted on asians but not on caucasians and africans , and the association between ethnicity variation and cancer risk could not be explored deeply . in conclusion , despite these limitations , the present meta - analysis demonstrates that the il-17a rs2275913g > a polymorphism is associated with cancer development . furthermore , the il-17f rs763780t > c polymorphism may be a potential risk factor in the development of gastric cancer . in the future , large - scale , case - control , and well - designed studies must be conducted to validate the findings of our meta - analysis and to comprehensively understand the potential gene - gene and gene - environment interactions between il-17 polymorphisms and cancer risk .

epidemiological studies have suggested that interleukin-17 ( il-17 ) polymorphisms are associated with cancer risk . however , the results of these studies are inconsistent . therefore , we performed a meta - analysis to obtain a precise conclusion . odds ratios ( ors ) with 95% confidence intervals ( cis ) were used to assess the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk . publication bias and sensitivity analyses were performed to ensure the statistical power . overall , 10 relevant case - control studies involving 4,516 cases and 5,645 controls were included . the pooled ors with 95% cis indicated that the il-17a rs2275913g > a polymorphism was significantly associated with increased cancer risk ( for a versus g : or = 1.28 , 95% ci : 1.161.41 , p < 0.001 , i2 = 61.1% ; for ga versus gg : or = 1.12 , 95% ci : 1.021.23 , p = 0.015 , i2 = 27.8% ; for aa versus gg : or = 1.71 , 95% ci : 1.382.41 , p < 0.001 , i2 = 69.6% ; for ga + aa versus gg : or = 1.23 , 95% ci : 1.131.34 , p < 0.001 , i2 = 6.4% ; for aa versus gg + ga : or = 1.62 , 95% ci : 1.272.07 , p < 0.001 , i2 = 81.4% ) . succeeding analysis of hwe and stratified analysis of gastric cancer and the asian ( and chinese ) population revealed similar results . the il-17f rs763780t > c polymorphism was also significantly associated with gastric cancer development . overall , the present meta - analysis suggests that il-17 polymorphisms increase the risk of developing cancer , particularly gastric cancer , in the asian ( and chinese ) population .

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

vibrissae provide input to specific thalamic nuclei and these nuclei have well - defined connections with the somatosensory cortical region where sensory information is processed ( woolsey and van der loos , 1970 ) . these connections have a somatotopic organization , and sensory information from each vibrissa reaches a specific barreloid in the thalamic ventrobasal ( vb ) complex and is relayed to a specific cortical barrel field in the primary somatosensory cortex . during their developmental outgrowth , thalamocortical axons are guided by a variety of cues ( molnr et al . , 2012 ) . after crossing the subpallial - pallial border , thalamocortical axons advance within the intermediate zone ( iz ) and , approaching the cortex , they accumulate below the cortical plate ( cp ) at the subplate ( ghosh et al . , the interaction of thalamocortical axons with the subplate is one of the most enigmatic processes in the development of thalamocortical fibers . even in reeler or p35 knockout ( ko ) mice , where the subplate is aberrantly located , thalamocortical axons cross the cp toward the misplaced subplate before connecting to their final targets ( hoerder - suabedissen and molnr , 2015 ) . however , the molecular mechanisms that control thalamocortical axon - subplate interaction and ultimately the correct targeting of thalamic projections are largely unknown . in the developing brain , bioactive phospholipids like lysophosphatidic acid ( lpa ) play important roles in cortical migration ( fukushima et al . , 2000 ) and neuronal apoptosis ( kingsbury et al . , 2003 ) . these effects are mediated by lpa receptors , namely lpa1-r and lpa2-r , which are expressed in the developing brain ( kingsbury et al . , 2003 ) . lpa is a well - described repellent factor for axons , eventually leading to growth cone ( gc ) collapse ( campbell and holt , 2003 ) . however , while in vitro experiments suggested an involvement of lpa1-r in lpa - mediated axonal retraction , deletion of specific lpa receptors did not lead to significant alterations of fiber tracts in the brain ( contos et al . , 2002 ) and did not affect inhibitory lpa effects on retinal gcs ( birgbauer and chun , 2010 ) . lpa is enzymatically inactivated by dephosphorylation via lipid phosphate phosphatases ( lpps ) , which are cell - surface lipid enzymes and constitute a large family of phosphatidic acid phosphatases ( pap2 ) . recently , it was shown that lpa signaling at the synapse is controlled by prg-1/lppr4 ( trimbuch et al . , 2009 ) , which belongs to a new class of lpa - interacting molecules ( plasticity - related genes , prgs ) sharing transmembrane features of the lpps ( bruer et al . , 2003 ) but which seem to have a different mode of action ( mcdermott et al . , 2004 ) . among these , prg-2/lppr3 is expressed in developing thalamocortical axons and has a high expression level in gcs . using both prg-2 animals and a thalamus - specific prg-2/gbx2/r26 mouse line , we set out to analyze the role of this new lpa regulatory mechanism in outgrowing axons . prg-2/lppr3 is prominently expressed in the thalamus ( as shown by in situ hybridization by http://www.genepaint.org ; figure s1b , available online ) and in developing thalamocortical fibers ( figures 1a and s1a ; for antibody specificity see figures s1g s1i ) , where prg-2 co - localized with the thalamic axonal marker l1 ( robichaux et al . , 2014 ) . according to this expression pattern , we hypothesized that prg-2 plays a role in the developing thalamocortical projection . we therefore performed biocytin tracing studies in thalamocortical brain slices of wild - type ( wt ) and prg-2 mice at embryonic day ( e)17 ( figures 1d , 1e , 1h , and 1i ) , a time point when thalamocortical axons accumulate below the subplate ( molnr et al . , 1998 ) . slices were injected with alexa 488-labeled biocytin into the vb complex of the thalamus and were incubated for 6 hr to allow for anterograde biocytin transport . this procedure combines the advantages of maintaining slice integrity with precise tracer application in the vb . while wt thalamocortical fibers were restricted to the iz ( figures 1d and 1h ) , as described by others ( molnr et al . , 1998 ) , prg-2 fibers aberrantly protruded into the cp ( figures 1e and 1i ) . to further substantiate this finding , we used a conditional prg-2 mouse line ( figures s1e and s1f ) , which was cross - bred with a gbx2 line , resulting in thalamus - specific creer expression ( chen et al . 2013 ) , and an r26 line ( madisen et al . , 2010 ) , which produces red fluorescent protein ( rfp ) upon cre - mediated recombination . using thalamus - specific prg-2-deficient mice ( prg-2/gbx2/r26 ) , we analyzed the effects of thalamus - specific prg-2 deletion induced by tamoxifen treatment at e12.5 ( prg-2 ; see also figures s1c and s1d ) , as described ( normand et al . , 2013 ) . here , we found a similar aberrant phenotype as observed in constitutive prg-2 mice ( figures 1f and 1j ) . to prove that not only a fiber subpopulation aberrantly protruded into the cp , we analyzed the full thalamocortical projection , which was displayed by the rfp reporter , and confirmed the aberrant phenotype ( figure 1 g ) . quantitative assessment of these slices revealed a significant disruption of the thalamocortical projection in constitutive prg-2 mice as well as in prg-2 mice ( figure 1k ) . to demonstrate that misrouting was also present in vivo , we applied neurovue tracer in the thalamic vb at e17 in fixed whole brains and analyzed thalamocortical axons . while thalamocortical fibers formed well - defined projections restricted to the iz in wt animals ( figure 1l ) , in prg-2 mice , thalamocortical fibers invaded the cp , showing a similar misrouting as observed in biocytin - traced thalamocortical slices ( figure 1 m ) . to avoid remote effects , e.g. , by prg-2 deletion in the cortex , we analyzed the thalamocortical projection in vivo in prg-2 mice , where the rfp reporter delineated the thalamocortical fibers with high accuracy , as described by others ( normand et al . , 2013 ) . here , prg-2-deficient thalamocortical fibers aberrantly invaded the cp , while prg-2-expressing fibers were restricted to the iz ( figures 1b and 1c ) . interestingly , in prg-2 animals , the subplate had a normal appearance ( figures s1j and s1k ) and the cortical organization , as well as the synaptic connectivity , was not altered ( figures s1m s1 t ) , ruling out possible subplate or cortical defects that could induce mistargeting of thalamocortical fibers ( ghosh et al . , since thalamocortical projection defects observed at prenatal stages might be a temporary feature that eventually disappears after formation of thalamocortical connections , we analyzed the thalamocortical fiber tract at postnatal day ( p)5 , a time point at which the connections of these fibers are established ( molnr et al . we applied lipophilic tracers in different brain areas ( motor cortex , somatosensory cortex , and visual cortex ) at p5 and found that retrogradely labeled thalamocortical fibers derived from their corresponding thalamic nuclei grossly maintained their termination pattern ( figures s2a s2c ) . to analyze whether the thalamocortical projection to the somatosensory cortex follows the well - established somatotopic termination pattern to the appropriate barrels , we inserted lipophilic tracers in the vb and visualized the thalamic projection in the corresponding barrels in tangential cut cortical slices . as shown in figure 1n , in the somatosensory cortex of wt animals lipophilic tracer - stained axons were restricted to their corresponding barrels . however , in prg-2 mice , thalamic axons readily crossed barrel borders aberrantly invading neighboring barrels ( figures 1o , 1p , and s1l ) . to further assess misrouting in prg-2 mice , we performed retrograde tracing of thalamocortical fibers by paired injection of retrogradely transported fluorescent latex microbeads into adjacent cortical barrels with 400 m separation between injection sites . ( 1995 ) , even injections placed in close proximity ( 75120 m apart ) resulted in segregated neuronal clusters of retrogradely labeled cells in the thalamic vb ( agmon et al . , 1995 ) . while in wt animals , even nearby injections of differently labeled fluorescent beads ( red and green fluorescence ; see figure s2d ) resulted in labeling of distinct subpopulations in the corresponding vb thalamic nuclei ( figures 1q and 1s ) , in prg-2 mice , injections even at distant sites ( see figure s2e ) resulted in double labeling of the corresponding subpopulations ( figures 1r and 1 t ; exemplarily retrograde stained neurons in the thalamus are shown in figures s2f and s2 g ) . quantitative assessment of double - labeled neurons revealed a significantly higher number in prg-2 animals , while the total number of retrogradely traced neurons did not differ between genotypes ( figures 1u and 1v ) . thus , prg2 depletion in mice leads to a misrouting in thalamocortical projections . to test whether the aberrant thalamic projection in prg-2 mice results from absence of prg-2 in thalamocortical axons , we used thalamocortical slice cultures of e15 wt and prg-2 mice at the peak of thalamocortical outgrowth . the thalamocortical projection was visualized by co - electroporation of a gfp - expressing construct into the thalamic vb at e15.5 . after 48 hr , slice cultures were fixed and the axonal projection was analyzed ( figure 2a ) . in wt slices , thalamocortical fibers were restricted to the iz ( figure 2b ) , while prg-2 thalamocortical axons aberrantly invaded the cp ( figure 2c ) . however , when prg-2 was re - expressed ( together with gfp ) in thalamocortical axons , these fibers were again restricted to the iz and did not show obvious differences to wt fibers ( figures 2d and s3a ) . quantitative analysis of thalamocortical cultures confirmed the significant amount of aberrant thalamocortical projections and the rescued targeting of these axons after prg-2 re - expression ( figure 2e ) . in sum , re - expression of prg-2 in thalamocortical axons was sufficient to completely abrogate aberrant targeting of the thalamocortical fiber projection in slices from prg-2 mice . since prg-2 is a member of the family of lpa - interacting molecules ( trimbuch et al . , 2009 ) , we analyzed the presence of autotaxin ( atx , enpp-2 , lysopld ) , the lpa - synthesizing enzyme , in the somatosensory cortex at e16 . atx is expressed by subplate neurons , as has been shown on the mrna level ( hoerder - suabedissen et al . , 2013 ) , and by a gfp reporter at corresponding age ( figures s4a and s4b ) . due to its secreted and diffusible nature , we found strong atx expression at the lower border of the cp and at the upper border of the iz ( figure 3a ) . here , atx immunoreactivity overlaps with that of lpa and prg-2 , which was expressed in fasciculated thalamocortical fibers in the iz ( figures 3b3d ; higher magnification in figures 3e3h ) . these findings are in line with a putative role of atx / lpa in restricting prg-2-expressing thalamocortical fibers to the iz . moreover , lpa - degrading enzymes like lpp1 and lpp3 are strongly expressed in the iz ( figures s6n and s6o ) , which suggests that proper regulation of phospholipids allows thalamocortical axons to occupy a corridor in the iz , possibly contributing to the fasciculated appearance of the thalamocortical projection . we therefore perturbed phospholipid signaling and analyzed the effect of inhibited lpa synthesis on the correct targeting of thalamocortical axons . we applied a recently characterized atx blocker ( pf8380 ) ( gierse et al . , 2010 ) , which was shown to effectively decrease lpa concentrations ( vogt et al . , 2015 ) , after atx inhibition ( 0.1 m ) , wt thalamocortical fibers aberrantly invaded the cp ( figure 3j ) , showing a similar phenotype as observed in slices from prg-2 mice ( figures 1e and 1i ) . quantitative analysis revealed that this phenotype was observed in more than 92% of all pf8380-treated slices ( figure 3l ) . to further study whether this effect was due to cortical lpa production inhibition of atx in the cp was sufficient to induce an aberrant targeting of thalamocortical fibers ( figure 3k ) . in contrast , pf8380 injection into the thalamus did not significantly alter the thalamocortical targeting , supporting a role for cortical - derived lpa in thalamocortical guidance ( figure 3l ) . taken together , our data provide strong evidence that correct guidance of thalamic fibers to the cortex depends on subplate - derived lpa , produced by atx , and on the presence of prg-2 on thalamic fibers . the role of lpa in gc collapse is well described ( campbell and holt , 2003 ) ; however , the molecular mechanism transducing lpa action from the membrane to the cytoskeleton is not well understood . while specific , g protein - coupled lpa receptors were reported not to affect lpa - induced collapse of retinal gcs ( birgbauer and chun , 2010 ) , authors of the same group showed that downstream inhibition of the g12/13-rho - rock pathway was capable of inhibiting lpa - induced retinal gc collapse ( fincher et al . , 2014 ) . this is in line with reports of downstream lpa signal transduction acting on the cytoskeleton ( campbell and holt , 2003 ) . to analyze the role of axonal prg-2 in lpa - mediated thalamic axon guidance , we embedded thalamic explants in matrigel with a polarized concentration of lpa ( distance to tf - lpa - containing matrigel was 401.6 53.63 m for wt explants and 475 146.80 m for prg-2 explants ) and on one side , the matrigel contained 10 m fluorescent - labeled lpa ( tf - lpa ) , while the adjacent side did not contain lpa ( see figures 4a s5e , and 4d2 for the border of the tf - lpa - containing matrigel region at higher magnification ) . the 3d matrigel structure , where fibers are able to radially expand , allows analysis of fiber growth into the lpa - rich zone and the adjacent control zones . however , while wt axons were not able to invade this lpa - rich zone , prg-2 axons grew far inside it ( figures 4c and 4d1 ) . quantitative analysis of fibers entering the lpa - rich zone at different distances revealed significantly higher invasion by fibers from prg-2 thalamic slices ( figure 4e ) , while no difference between wt and prg-2 axon fiber outgrowth was observed in the adjacent , non - lpa - containing control side ( figure 4f ) . direct comparison of lpa - containing and control sides revealed that wt axons were significantly repelled by the lpa - rich zone , while prg-2 axons were not affected ( figure 4 g ) . since prg-2 deficiency allowed axons to enter the lpa - rich zone , which is repulsive for wt axons , these data suggest that prg-2 is critically involved in mediating the repulsive lpa sensitivity in thalamic axons . during normal development , thalamocortical axons do not collapse at the border to the cp but rather turn away from this border and continue to grow in the iz until reaching their final target . this indicates that lpa provided by the subplate induces the turning of axon gcs when thalamocortical fibers approach lpa - rich areas . to mimic this situation in our in vitro outgrowth assay , we exposed thalamocortical explants to lower lpa concentrations ( 1 m lpa ; figure 4h ) . here , wt axons displayed a typical turning behavior in front of the lpa - rich zone ( figures 4i and 4k ) . to confirm this observation axon gcs were exposed to a low , but continuous , lpa concentration ( 500 nm lpa ) . to avoid bias by perfusion flow , which may repel axons , gcs were exposed to an lpa - covered pipette tip at 40 m distance ( see figure s5f for experimental overview ) . while wt axons stopped growing and turned away from the lpa source , this behavior was not observed for prg-2 axons , which even seemed to be attracted by lpa ( figures 4l4n ; movies s1 and s2 ) . quantitative analysis revealed a significant repulsion of wt axons by 500 nm lpa , which was not observed in prg-2 axons ( figure 4o ) . however , prg-2 axons were still repelled by other guidance cues like sema3a ( figures s5 g and s5h ) , which are well - described repulsive cues ( mintz et al . , 2008 ) . in sum , these data indicate that prg-2 at the gc mediates the repellent effect of lpa for axonal guidance . prg-2/lppr3 has an intracellular 400 aa - long c - terminal tail , which is putatively involved in signal transduction . to determine interaction partners of this intracellular domain , we first performed a y2h screen using this prg-2 c - terminal tail as a bait and found a positive interaction with the ferm domain of radixin ( rdx ) ( fehon et al . , 2010 ) . using immunoprecipitation ( ip ) on e17 brain lysate this interaction is specific for prg-2 since only prg-1 and prg-2 possess an intracellular c terminus , and prg-1 is not expressed at embryonic ages ( figure s6j ) . to further elucidate the role of this interaction in proper guidance of thalamocortical fibers to the developing cortex , we performed co - localization studies at e16 and found co - expression of prg-2 and rdx in the thalamocortical tract ( figures 5b , 5c , s6k , and s6i ) . expression analysis of prg-2 and rdx showed a similar expression pattern , where expression for both molecules increased from e11.5 , a time point where thalamic neurons are already present and start to differentiate ( figure s6f ) . to prove that the rdx / prg-2 interaction in fact occurs in the axon gc , we performed subcellular immunofluorescent co - localization studies of rdx and prg-2 and found a strong signal in gcs ( figure 5d ) . here , prg-2 is expressed at the gc membrane and co - localizes with rdx , which shows an asymmetric distribution , as described by others ( mintz et al . , 2008 ) . rdx belongs to the ezrin , radixin , and moesin ( erm ) protein family . however , due to their embryonal expression pattern at e14.5 , ezrin and moesin do not seem to be involved in the outgrowth of the thalamocortical projection ( figures s6l and s6 m ) . the c terminus of rdx can directly bind to filamentous actin ( f - actin ) , thereby acting as a crosslinker between the cortical f - actin cytoskeleton and the plasma membrane ( fehon et al . , the activation of rdx involves the phosphorylation of a critical c - terminal threonine residue ( t564 ) , which is highly conserved in the erm protein family . therefore , we analyzed the role of prg-2 in rdx activation using a specific antibody that recognizes the phosphorylation of t564 in rdx , t567 in ezrin , and t558 in moesin ( hausrat et al . , 2015 ) . interestingly , we found significantly lower phosphorylated erm ( perm ) levels in prg-2 neurons ( figures 5e and 5f ) , indicating diminished activation of erm proteins . to prove the role of lpa in erm activation , we assessed the effect of different lpa concentrations . while 100 nm lpa already revealed an increase in erm phosphorylation , 1 m lpa induced a significant and robust increase in perm levels ( figures 5 g and 5h ) . we further analyzed perm levels over time and found a fast and constant increase in erm phosphorylation in the course of lpa stimulation ( figures 5i , 5j , and s6a s6c ) . to understand the functional significance of the lpa - dependent perm expression , we analyzed the rdx / prg-2 interaction under lpa - free conditions and after 1 m lpa stimulation for 15 min . here , we used rdx - gfp transfected , stable prg-2-expressing hek cells , which have comparable prg-2 expression , and performed coips of rdx and prg-2 using an anti prg-2 antibody ( figure 5k ) . quantitative analysis ( rdx levels were adjusted according to the prg-2 ip signal ) revealed a significantly increased rdx / prg-2 interaction upon 1 m lpa stimulation ( figure 5l ) . interestingly , analysis of the ip product revealed that phosphorylation of the prg-2-interacting rdx was significantly higher upon lpa stimulation when compared to serum - starved cells ( figures 5 m and 5n ) . since we could show that rdx binds to the intracellular c terminus of prg-2 ( figure s6d ) , to further analyze whether rdx phosphorylation is a prerequisite for prg-2 binding and to determine the prg-2 binding sites , we performed a systematic peptide scan using a celluspot array format ( spot ) and mapped the rdx / prg-2 interaction ( figure 5o ) . here , prg-2 intracellular c - terminal overlapping peptides ( 69 overlapping peptide fragments , 14 aa in length ) were analyzed regarding their binding affinity to rdx - gst or to rdx - gst , its non - phosphorylated form . we detected six putative interaction sites that showed significant difference in their binding to rdx - gst or to rdx - gst , pointing to a lower binding affinity of the non - phosphorylated rdx - gst to prg-2 ( see also figure s6p ) . we have therefore performed ips from stable prg-2-expressing hek293 cells transfected either with wt rdx - gfp ( rdx ) or non - phosphorylated rdx - gfp ( rdx ) using the described prg-2 antibody . in line with data from the peptide - microarray - based mapping , although binding of rdx was present at lower levels when compared to rdx , prg-2/rdx binding could be clearly detected ( figures 5p and 5q ) . this points to the fact that rdx phosphorylation , although not a prerequisite for rdx / prg-2 interaction , significantly increases rdx / prg-2 interaction . to answer the question of whether rdx / prg-2 association at the membrane is important for lpa - dependent rdx phosphorylation in neurons , we analyzed the effect of lpa stimulation on perm expression in wt and prg-2 neurons . here , lpa stimulation of wt neurons resulted in a significant increase in perm levels , while prg-2 neurons failed to show any significant change ( figures 5r and 5s ) . these results point to a yet unknown , but critical role of prg-2 in lpa - induced rdx activation . rdx is concentrated below the plasma membrane , a critical position linking transmembrane signaling to the actin cytoskeleton . since phosphorylation influences rdx activity in connecting the actin - cytoskeleton with the membrane ( fehon et al . , 2010 ) , transmembrane signaling events leading to rdx activation are optimally suited to act in extracellular cue mediated axon guidance . we therefore analyzed the effect of extracellular lpa in inducing perm changes at the gc membrane in wt and prg-2 neurons . in wt gcs , perm was concentrated in a compartment directly adjacent to the gc membrane ( figures 6a and 6b ) and strongly increased upon lpa stimulation ( figures 6e , 6f , and 6i ) . in contrast , no change of perm was observed in prg-2 gcs ( figures 6c , 6d , and 6g6i ) . moreover , direct comparison of perm levels in the center of the gc and in the periphery revealed a shift of the perm signal toward the membrane of wt gcs , but not of prg-2 gcs ( figure 6j ) . to prove whether rdx is dynamically recruited to the membrane via prg-2 binding induced by a local extracellular lpa source , we transfected wt or prg-2 neurons with gfp - tagged rdx and performed live imaging studies . while in wt neurons , local lpa stimulation rapidly induced a translocation of rdx - gfp to the membrane next to the lpa source ( figures 6k and 6l ) , prg-2 neurons did not show such a change ( figures 6 m and 6n ) . in sum , these data show that prg-2 is the mediator of lpa - induced localization of rdx to the gc membrane , where it is present in its phosphorylated form , in turn increasing binding to prg-2 . to prove that erm phosphorylation and activation play a role in lpa - induced gc turning , we analyzed the subcellular localization of perm in gcs of thalamic explants exposed to an lpa - rich environment ( 1 m lpa ) , as shown in figure 4i . detailed analysis of turning gcs , exemplarily encircled in figure 7a , revealed strong perm signal co - localized with f - actin at the tip of gcs turning away from the lpa - rich zone ( figures 7b7e ) . to confirm that prg-2 interacts with rdx at the gc , we performed a proximity ligation assay ( pla ) , a method that allows the study of in situ protein - protein interactions at resolutions < 40 nm ( rhett et al . , 2011 ) . here , we found a strong signal at the gc tip , confirming the prg-2/rdx interaction at the site of lpa action ( figures 7f and 7 g ) . taken together , these results confirm the critical role of prg-2/rdx interaction for the induction of rdx activation in gcs by extracellular lpa , which eventually leads to axon turning . to further substantiate the role of rdx in lpa - induced gc turning , we analyzed the thalamocortical projection in rdx mice . using biocytin tracing in living e17 thalamocortical slices , we observed misrouted thalamocortical axons in rdx slices that aberrantly invaded the cp ( figures 7i7k),while in slices from control littermates , the thalamocortical projection was confined to the iz ( figures 7h and 7k ) . in order to better understand the functional role of the prg-2/rdx interaction in thalamocortical axon guidance , we analyzed the effect of partial reduction of these proteins in transheterozygous mice ( prg-2/rdx ) in thalamocortical slices at e17 . here , the thalamocortical projection aberrantly invaded the cp , suggesting that it is indeed the interaction of prg-2 and rdx that is important for correct guidance of these fibers by an lpa gradient ( figures 7l7o ) . these data corroborate the idea that the lpa - prg-2-rdx / perm signal transduction axis is critical for correct guidance and restriction of thalamocortical axons to the iz . moreover , in line with decreased rdx expression after e16 ( figures s6 g and s6h ) , these data could explain how thalamocortical axons are finally able to enter into their target cortical region . to assess the functional outcome of altered thalamocortical projections in prg-2 mice , we first measured intrinsic optical responses in the corresponding barrel upon single - whisker back - and - forth deflection . this approach measures blood - flow changes in the representation of single whiskers in the barrel cortex upon activation . using this method , we found that the response area upon single - whisker stimulation , which corresponds to the stimulated barrel ( for morphological barrel assessment , see figure s7a ) , was larger in prg-2 mice . these data point to a more diffuse and broader cortical response extending beyond the border of the activated barrel ( figures 8a8c ) . moreover , it reflects our findings using retrograde tracing of thalamocortical fibers in prg-2 mice showing a clear overlap in the termination pattern of neurons from neighboring thalamic barreloids in the vpm ( figures 1r , 1 t , and 1u ) . this is in agreement with enlargement of functional barrel columns in mice due to impaired precision of the thalamocortical projection , as reported in other mouse models ( lokmane et al . , 2013 ) . to get a layer - specific resolution of neuronal activity , we performed in vivo electrophysiological recordings using multi - channel electrodes . we observed in prg-2 mice a slight decrease in multi - unit activity ( mua ) upon single - whisker deflection in layer iv and a significant decrease in layers ii / iii for prg-2 mice compared to wt controls ( figures 8d8f ) , which are the main target layers of the thalamocortical projection ( meyer et al . recent data suggest that layers ii / iii contain neurons specifically responsive for texture coarseness ( garion et al . , 2014 ) . we therefore assessed the ability of prg-2 mice to distinguish texture coarseness using a modified radial arm maze , where arms were covered with sandpaper of two slightly different grades ( 60 grade [ grain size , 265 m ] and 80 grade [ grain size , 190 m ] ; figure 8 g ) . only arms covered with the coarser ( 60 grade ) sandpaper were rewarded by accessible food pellets and counted as correct entrance . since experiments were performed in darkness and no other cues were presented , mice had to orient by sensing sandpaper roughness . while wt mice showed a significant improvement in their ability to choose the correct arm , prg-2 mice did not improve beyond chance levels even after 9 days of continuous training ( figure 8h ) . to exclude bias by an impaired learning ability , we tested prg-2 mice in the morris water maze ( mwm ) . escape latency for visible and hidden platforms was indistinguishable between prg-2 and wt littermates ( figures s7e was not impaired in prg-2 mice , we conclude that their performance at chance level in the texture discrimination task is a direct result of impaired thalamocortical axon projection precision . in line with unaltered learning ability , motor performance was also unaltered , as shown by analysis in the mwm or supporting limb responses ( figures s7h s7k ) . to exclude an effect of prg-2 deletion outside the thalamus , e.g. , in the cortex , which could affect cortical processing of somatosensory information , we also tested the above described thalamus - specific prg-2-deficient mouse line ( prg-2 ) . as shown in figures s8l s8o using retrograde tracing of thalamocortical fibers projecting to the cortical barrels , these animals display a similar phenotype as described for the constitutive prg-2 animals , where we could show that different barrels were aberrantly innervated by the same neuron in the thalamic vpm . furthermore , staining for prg-2 revealed that fibers originating from gbx-2-expressing thalamic neurons ( identified by the rfp reporter ) did not express prg-2 ( figures s7p s7s ) . behavioral analysis of the prg-2 mice in the sandpaper radial maze revealed that these mice displayed the same phenotype as the constitutive prg-2 mice and were not able to distinguish between the two sandpapers of different coarseness ( figure 8h ) . together with our in vivo imaging and electrophysiological data , these findings strongly indicate that misguided thalamocortical axons in prg-2 mice alter cortical information processing , resulting in a specific deficit in sensory discrimination at adult ages . an intriguing phenomenon in cortical development is the specific outgrowth of thalamocortical fibers that reach the appropriate cortical area before their target cells migrate to their final position in the cp ( kostovic and rakic , 1990 ) . as shown in other systems , thalamocortical axons accumulate in the iz below the cp ( ghosh et al . , , they interact with a transient neuronal population , the subplate neurons , which keep them in place and are thereby critically important for correct patterning of the thalamocortical connections . although the role of subplate neurons has long been recognized and gene expression patterns of subplate neurons have been described ( for review , see hoerder - suabedissen and molnr , 2015 ) , molecular mechanisms regulating the subplate neurons / thalamocortical fiber interaction are far from being understood . here , we analyzed the role of prg-2/lppr3 , a phospholipid - interacting molecule strongly expressed in thalamocortical axons during development ( figure 1a ) . using a constitutive prg-2 mouse line as well as a thalamus - specific prg-2 ko line ( prg-2/gbx2/r26 ) , where tamoxifen application at e12.5 leads to specific deletion of prg-2 in the thalamus ( prg-2 ; normand et al . , 2013 ) , we discovered that prg-2-deficient thalamocortical axons prematurely and aberrantly entered the cp . this altered the precise somatotopic termination of thalamocortical fibers in the somatosensory barrel field cortex and led to a deficit in sensory discrimination . molecular analysis revealed a signal transduction axis in the gc leading from the lpa - synthesizing molecule atx to lpa , prg-2 , and finally to phosphorylated rdx , a well - described linker between the gc membrane and the actin cytoskeleton . we demonstrate this atx / lpa / prg-2/prdx signaling axis on thalamocortical axon gc turning and suggest that this mechanism is responsible for keeping thalamocortical axons in the iz before entering the cp . the role of subplate cells was concluded from ablation experiments , where thalamocortical fibers , no longer able to contact subplate cells , escaped the subcortical waiting period and extended beyond their target cortical regions . this specific phenotype relies mechanistically on the lost sensitivity of thalamocortical axons toward extracellular lpa gradients synthesized by atx , a diffusible molecule expressed in subplate neurons ( hoerder - suabedissen et al . , 2013 ) . eventually , this leads to loss of proper axon guidance and to an invasion of inappropriate cortical compartments . since we also analyzed thalamus - specific prg-2 ko animals ( normand et al . , 2013 ) , we could rule out that this misrouting is due to defects , e.g. , derived from cortical prg-2 deficiency . interestingly , the same phenotype was detected when the catalytic function of atx was blocked , thereby impeding lpa production . our data suggest a thalamocortical guidance mechanism with a mutual dependence on lpa / prg-2 interaction . the critical importance of prg-2-mediated extracellular lpa effects on thalamic axon guidance was further clarified by single - axon analysis . while downstream mechanisms mediating lpa gc collapse have been described ( campbell and holt , 2003 ) , direct and specific lpa - interacting molecules involved in this mechanism remain unclear . in line with live imaging data , wt axons were repelled / collapsed when in contact with 10 m lpa , while lower lpa concentrations ( 1 m ) induced a turning behavior in gcs . however , prg-2 axons were not affected even by high lpa concentrations ( 10 m ) . searching for downstream interacting partners , we found rdx to directly interact and be co - expressed with prg-2 in thalamocortical axons . moreover , subcellular analyses revealed rdx to interact with prg-2 in gcs as shown by pla . importantly , ip experiments revealed that the prg-2/rdx binding was highly increased upon lpa stimulation . the role of this prg-2/rdx interaction was functionally confirmed , showing that prg-2 is critically important for rdx activation by extracellular lpa . while in wt neurons , concentrations as low as 100 nm lpa induced clear rdx phosphorylation , in prg-2 neurons , rdx phosphorylation did not increase , even upon 15 min of 1 m lpa stimulation . our data showing rdx phosphorylation upon lpa stimulation in full - length prg-2-expressing hek293 cells suggest that prg-2/rdx association increased rdx phosphorylation upon lpa stimulation . however , when analyzing binding to prg-2 to either wt rdx - gfp or its non - phosphorylated form ( rdx - gfp ) , we detected binding under both conditions , albeit with a significant reduction of binding to prg-2 of the non - phosphorylated form . these data show that rdx phosphorylation is not a prerequisite for prg-2/rdx binding ; however , it influences this binding . our finding that transheterozygous loss of prg-2 and rdx already results in altered thalamocortical fiber projection indicates an important role of the lpa / prg-2/rdx signal axis . since the active form of erms ( perm ) is critical for gc guidance ( mintz et al . , 2008 ) , we investigated the role of this signal axis at the gc , the critical location for axon guidance . here , lpa stimulation induced strong erm phosphorylation at the gc membrane of wt axons , while prg-2 gcs failed to show these changes . in line , rdx was rapidly translocated to the membrane directly next to a local lpa source , indicating an active role of lpa / prg-2/rdx signaling in gc guidance . together with the detection of perm adjacent to f - actin at the tip of turning gcs , this signaling axis appears to directly control lpa - induced axon turning . using in vivo imaging and high - density multi - electrode recordings in the barrel cortex of prg-2 mice and performing behavioral tests both in prg-2 mice and prg-2 animals , we elucidated the functional consequences of prg-2 deficiency and subsequent altered thalamocortical connectivity in cortical layers iv and ii / iii as well as in the characteristic discrimination abilities associated with these layers ( garion et al . , 2014 ) . it appears that rather specific deficits can be observed even upon drastic developmental defects resulting in grossly impaired cortical lamination and subsequent changes in the thalamocortical projection ( wagener et al . , 2016 ) , as shown by preserved functional sensory maps in the somatosensory barrel cortex observed in reelin - deficient mice ( guy et al . , 2015 ) . ( 2015 ) suggest microcircuits in layers ii / iii are important for specific stimulus orientation ( cossell et al . , 2015 ) , supporting the idea that layer ii / iii neurons mediate rather specific functions . this implies that functional changes in layers ii / iii seen in prg-2 mice can best explain the sensory discrimination deficits of the animal . taken together , we describe a novel molecular guidance mechanism involving phospholipid signaling influencing thalamocortial axon guidance at a critical time point , at which axons exhibit a waiting period at the cortical subplate . the alteration of the lpa / prg-2/prdx signal transduction pathway resulted in distinct electrophysiological and behavioral deficits in adult animals and highlighted the importance of this pathway for proper thalamocortical connectivity . all experiments were conducted in accordance with the national laws for the use of animals in research and with the european communities council directive 86/609/eec , and approved by the local ethical committee ( landesuntersuchungsamt rheinland - pfalz 23 . details of the generation and breeding of prg-2 , prg-2/gbx2/r26 , and rdx mice are provided in the supplemental experimental procedures . immunofluorescent ( if ) stainings , pla for detection of in situ protein interaction , and anterograde and retrograde tracing experiments were performed following standard protocols . organotypic slices were prepared as described in the supplemental experimental procedures . for biocytin tracing experiments , pressure injections were performed into e17 embryonic slices using glass capillaries attached to a toohey spritzer ( toohey company ) to induce biocytin alexa fluor 488 ( thermo fisher scientific ) . slices were then incubated for 6 hr and fixed with 4c pfa ( 4% ) . for electroporation experiments , corresponding plasmids were injected into organotypic slices from e15.5 brains that were electroporated by needle tungsten electrodes and an nepa21 super electroporator ( nepa gene ) . . , 2012 ) , cultivated for 2 days in vitro ( div ) and used for assessment of perm levels in the gc . if staining and perm assessment were performed as described in the supplemental experimental procedures . ip studies were performed on e17 brain material or on cell lysate from stably prg-2-expressing hek293 cells according to standard protocols . intrinsic signal optical imaging ( ioi ) and in vivo electrophysiological recordings in the barrel cortex of adult mice were performed according to standard protocols as described in the supplemental experimental procedures . sandpaper discrimination test and mwm were performed according to standard procedures . for animal experiments , mice from the same litter or of similar age following experiments , results were analyzed , animals were re - genotyped , and corresponding final statistical analyses were performed . briefly , after assessing for normal distribution ( using the kolmogorov - smirnov test ) , comparison between two groups , if not otherwise stated , was performed using a two - tailed unpaired t test for normal distributed data or a mann - whitney test for nonparametric data . when data were normalized to controls ( set as 100% ) , a one - sample t test was used . for quantitative assessment of aberrant fibers in traced or electroporated slices , a fisher s exact test was applied . comparison between more than two groups was performed using a one - way anova for parametric data or a kruskal - wallis test for nonparamatric data . variance was similar between groups and was estimated using either the f - test or the brown - forsythe test and bartlett s test . post hoc analysis for more than two groups for parametric data was performed using the bonferroni adjustment for multiple comparisons and dunn s test for nonparametric data . for behavioral analyses assessing genotype effects and time effects , as well as for comparisons of the multiple binding sites of prg-2 , h.j . , n.s . , h.m.m . , a.h .- s . , p .- h.p . , w.f . , and t.t . provided reagents ; writing original draft , j.v . ; review and editing , j.v . , r.n . , j.c .

summaryprecise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information . here , we show that prg-2 , a phospholipid - interacting molecule , is important for thalamocortical axon guidance . developing thalamocortical fibers both in prg-2 full knockout ( ko ) and in thalamus - specific ko mice prematurely entered the cortical plate , eventually innervating non - corresponding barrels . this misrouting relied on lost axonal sensitivity toward lysophosphatidic acid ( lpa ) , which failed to repel prg-2-deficient thalamocortical fibers . prg-2 electroporation in the prg-2/ thalamus restored the aberrant cortical innervation . we identified radixin as a prg-2 interaction partner and showed that radixin accumulation in growth cones and its lpa - dependent phosphorylation depend on its binding to specific regions within the c - terminal region of prg-2 . in vivo recordings and whisker - specific behavioral tests demonstrated sensory discrimination deficits in prg-2/ animals . our data show that bioactive phospholipids and prg-2 are critical for guiding thalamic axons to their proper cortical targets .

Introduction Results Discussion Experimental Procedures Author Contributions

vitamin a deficiency is the leading cause of preventable childhood blindness in the developing world . although rare in the united states , vitamin a deficiency has been known to occur as a result of poor dietary intake , liver diseases , and gastrointestinal malabsorption [ 2 , 3 , 4 , 5 ] . vitamin a is a fat - soluble vitamin ingested in the diet in two forms : as retinol itself from animal sources , such as milk , meat , fish , liver , and eggs , or as the provitamin carotene from plant sources , such as green leafy vegetables , yellow fruits , and red palm oil . on the ocular surface , vitamin a deficiency has a wide range of ocular manifestations including conjunctival and corneal xerosis , keratomalacia , retinopathy , visual loss , and nyctalopia , also called night blindness , which is the earliest and most common symptom . we report a case of bilateral sequential corneal ulceration in a patient with severe vitamin a deficiency in the context of eosinophilic gastroenteropathy . a 29-year - old man was referred to our cornea unit with a left corneal ulcer of 6 weeks ' duration . he was previously diagnosed with eosinophilic gastroenteropathy ( biopsy proven ) , skin atopy and atopic keratoconjunctivitis . he had been treated with peroral steroids 15 mg / day and methotrexate 6 mg / week since the age of 11 years for his gastroenteropathy . he had surgery for steroid - induced cataracts with toric intraocular lens implantation at the age of 27 years . in addition , he had a number of food intolerances and subsisted mostly on a diet of potatoes . prior to his presentation , he had noted irritation , itchiness , and a decrease in vision in his left eye for 6 weeks and was treated with topical anti - allergic and topical steroid drops ; however , no improvement could be observed . polymerase chain reaction testing for herpes simplex virus was negative , and the cultures did not show any growth . acuity without correction was 20/80 od , improved by pinhole to 20/50 , and 20/150 os with a manifest refraction of 0.25/-2.5/5 , not improved by pinhole . the intraocular pressure was 19 mm hg od and 21 mm hg os by tonopen . slit - lamp examination showed temporal wrinkling of the conjunctiva in both eyes , with a dry granular pattern and loss of transparency ( fig . the left temporal cornea showed an epithelial defect overlying a partial - thickness ulcer with approximately 50% thinning and adjacent limbitis . in both eyes , the anterior chambers were quiet and there were well - centered posterior chamber iols . our patient was initially diagnosed with peripheral ulcerative keratitis in the left eye in the presence of atopic keratoconjunctivitis . his steroids were increased to 50 mg / day and tapered every 3 days by 10 mg / day . he was put on preservative - free artificial tears . a vasculitic workup ( c - reactive protein , erythrocyte sedimentation rate , antinuclear antibody , antineutrophil cytoplasmic antibody , rheumatoid factor , syphilis serology ) was unremarkable . serum was sent for analysis of vitamin a and retinol - binding protein ( rbp ) levels , with a processing time of several weeks at our location . the ulcer in the left eye epithelialized over the following 2 weeks and subsequently vascularized from the temporal conjunctiva . two weeks later , the patient presented to our clinic again with significant right eye pain . he had a corneal ulcer in his right eye , complicated by a small corneal perforation , with the iris plugging the wound ( fig . serendipitously , at that time , the results of his serum vitamin a levels were available , revealing that the levels were significantly decreased to 0.2 mol / l ( normal range 0.9 - 2.5 ) . the rbp level was also reduced to 12 mg / l ( normal 30 - 92 ) . the patient was treated with iv multivitamin ( containing 10,000 iu vitamin a , c , d , e and b complex ) for 5 consecutive days while in hospital , and then with oral vitamin a 200,000 iu once a week for 2 weeks . 2b ) and the corneal keratinization in both eyes resolved over the following 6 weeks ( fig . we present a rare case of vitamin a deficiency secondary to a malnutrition syndrome leading to bilateral xerosis and sequential corneal ulceration . similar reports from developed populations are rare , but have been described in association with disorders such as chronic alcoholism , cystic fibrosis , and anorexia nervosa . the initial treatment with an increase of systemic steroids improved the ulceration and seemed to suggest an immunogenic process . however , the involvement of the second eye while on steroid treatment forced consideration of other causes . clinical suspicion of xerophthalmia at the time of presentation prompted testing for vitamin a and serum rbp . it was unfortunate in our case that the progression of the disease occurred in the time taken for results to be reported . the earliest ocular symptom of vitamin a deficiency is nyctalopia , secondary to depletion of photopigments . unfortunately , we were not able to obtain an electroretinogram ( erg ) before the treatment . an abnormal erg at presentation in association with the anterior segment findings would have helped to establish a diagnosis of vitamin a deficiency before serum levels were available . it has been shown that eosinophilic gastroenteropathy can cause malabsorption or protein - losing enteropathy [ 11 , 12 ] , but in our case , a gastroenterology consult confirmed inactive disease and showed no other signs of malabsorption ( normal albumin levels and international normalized ratio ) . the most likely reason was felt to be malnutrition due to lack of vitamin a uptake secondary to the patient 's multiple food intolerances that accompany eosinophilic gastroenteropathy . the long - lasting success of oral vitamin a supplementation further favors previous malnutrition over malabsorption . the oral dosage regimen is 200,000 iu vitamin a in oil , followed the next day with an additional dose of 200,000 iu . if patients have severe corneal disease or malabsorption , the preferred dose is 100,000 iu water - miscible vitamin a administered intramuscularly . it is important to note that if vitamin a deficiency is associated with a protein - energy malnutrition syndrome , protein levels must be corrected at the same time as vitamin supplementation . our case serves as a reminder to corneal specialists in developed populations of the potentially devastating effects of vitamin a deficiency on the eye . vitamin a deficiency should be suspected in any practice in the presence of corneal ulceration in combination with malabsorption and malnutrition syndromes .

purpose : vitamin a deficiency is a very rare condition in the developed world and can lead to a variety of ocular changes from xerosis and xerophthalmia to corneal ulcer and perforation . the treatment of this devastating disease is simple and inexpensive . it is therefore important to recognize and treat accordingly , especially in the event of ulcers unresponsive to treatment or in the presence of severe malnutrition / malabsorption syndromes . the purpose of this case report is to remind physicians of the potentially devastating effects of vitamin a deficiency on the eyes and to demonstrate outcomes after vitamin a treatment . methods : single observational case report . results : a 29-year - old male with known eosinophilic gastroenteropathy was treated with oral steroids for peripheral ulcerative keratitis . two weeks after resolution , the patient suffered from peripheral ulcerative keratitis in his other eye , with a self - sealing perforation . vitamin a deficiency was confirmed and successfully treated , leading to subsequent resolution of signs and symptoms . conclusions : vitamin a deficiency can be present in patients with malabsorption and malnutrition syndromes and should be considered as cause of corneal ulceration .

Introduction Case Report Results Discussion Disclosure Statement

grounding or earthing refers to direct skin contact with the surface of the earth , such as with bare feet or hands , or with various grounding systems . subjective reports that walking barefoot on the earth enhances health and provides feelings of well - being can be found in the literature and practices of diverse cultures from around the world.1 for a variety of reasons , many individuals are reluctant to walk outside barefoot , unless they are on holiday at the beach . various grounding systems are available that enable frequent contact with the earth , such as while sleeping , sitting at a computer , or walking outdoors . these are simple conductive systems in the form of sheets , mats , wrist or ankle bands , adhesive patches that can be used inside the home or office , and footwear . these applications are connected to the earth via a cord inserted into a grounded wall outlet or attached to a ground rod placed in the soil outside below a window . for the footwear applications , a conductive plug is positioned in the shoe sole at the ball of the foot , under the metatarsals , at the acupuncture point known as kidney 1 . from a practical standpoint , these methods offer a convenient and routine , user - friendly approach to grounding or earthing . they can also be used in clinical situations , as will be described in the section entitled summary of findings to date.1 recently , a group of about a dozen researchers ( including the authors of this paper ) has been studying the physiological effects of grounding from a variety of perspectives . this research has led to more than a dozen studies published in peer - reviewed journals . while most of these pilot studies involved relatively few subjects , taken together , the research has opened a new and promising frontier in inflammation research , with broad implications for prevention and public health . the findings merit consideration by the inflammation research community , which has the means to verify , refute , or clarify the interpretations we have made thus far . grounding reduces or even prevents the cardinal signs of inflammation following injury : redness , heat , swelling , pain , and loss of function ( figures 1 and 2 ) . rapid resolution of painful chronic inflammation was confirmed in 20 case studies using medical infrared imaging ( figure 3).2,3 our main hypothesis is that connecting the body to the earth enables free electrons from the earth s surface to spread over and into the body , where they can have antioxidant effects . specifically , we suggest that mobile electrons create an antioxidant microenvironment around the injury repair field , slowing or preventing reactive oxygen species ( ros ) delivered by the oxidative burst from causing collateral damage to healthy tissue , and preventing or reducing the formation of the so - called inflammatory barricade . we also hypothesize that electrons from the earth can prevent or resolve so - called silent or smoldering inflammation . if verified , these concepts may help us better understand and research the inflammatory response and wound healing , and develop new information on how the immune system functions in health and disease . grounding appears to improve sleep , normalize the day night cortisol rhythm , reduce pain , reduce stress , shift the autonomic nervous system from sympathetic toward parasympathetic activation , increase heart rate variability , speed wound healing , and reduce blood viscosity . a summary has been published in the journal of environmental and public health.4 one of the first published grounding studies examined the effects of grounding on sleep and circadian cortisol profiles.5 the study involved 12 subjects who were in pain and had problems sleeping . they slept grounded for 8 weeks using the system shown in figure 4 . during this period , their diurnal cortisol profiles normalized , and most of the subjects reported that their sleep improved and their pain and stress levels declined . the results of the experiment led to these conclusions : 1 ) grounding the body during sleep yields quantifiable changes in diurnal or circadian cortisol secretion levels that , in turn , 2 ) produce changes in sleep , pain , and stress ( anxiety , depression , and irritability ) , as measured by subjective reporting . the cortisol effects described by ghaly and teplitz5 are particularly significant in the light of recent research showing that prolonged chronic stress results in glucocorticoid receptor resistance.6 such resistance results in failure to downregulate inflammatory responses , which can thereby increase risks of a variety of chronic diseases . this effect complements the findings described in the effects on pain and the immune response section . a pilot study on the effects of grounding on pain and the immune response to injury employed delayed - onset muscle soreness ( doms).7 doms is the muscular pain and stiffness that takes place hours to days after strenuous and unfamiliar exercise . the phase of contraction that occurs when a muscle shortens , as in lifting a dumbbell , is referred to as concentric , whereas the phase of contraction as a muscle lengthens , as in lowering a dumbbell , is referred to as eccentric . eight healthy subjects performed an unfamiliar , eccentric exercise that led to pain in their gastrocnemius muscles . this was done by having them perform two sets of 20 toe raises with a barbell on their shoulders and the balls of their feet on a 2-inch 4-inch wooden board.7 all subjects ate standardized meals at the same time of day , and adhered to the same sleep cycle for 3 days . at 5.40 pm on each day , four of the subjects had conductive grounding patches adhered to their gastrocnemius muscles and the bottoms of their feet . they remained on the grounded sheets except for visits to the bathroom and meals . as controls , four subjects followed the same protocol except that their patches and sheets were not grounded . the following measurements were taken before the exercise and 1 , 2 , and 3 days thereafter : pain levels , magnetic resonance imaging , spectroscopy , cortisol in serum and saliva , blood and enzyme chemistry , and blood cell counts.7 pain was monitored with two techniques . the subjective method involved morning and afternoon use of a visual analog scale . in the afternoon , a blood pressure cuff was positioned on the right gastrocnemius and inflated to the point of acute discomfort . the grounded subjects experienced less pain , as revealed with both the analog soreness scale ( figure 5 ) and by their ability to tolerate a higher pressure from the blood pressure cuff ( figure 6).7 the doms grounding study report7 contains a summary of the literature on the changes in blood chemistry and content of formed elements ( erythrocytes , leukocytes , and platelets ) expected after an injury . the immune system detects pathogens and tissue damage and responds by initiating the inflammation cascade , sending neutrophils and lymphocytes into the region.812 as expected , the white cell counts increased in the ungrounded or control subjects . white cell counts in the grounded subjects steadily decreased following the injury ( figure 7).7 previous research has shown increases in neutrophils following injury.1316 this happened in both grounded and ungrounded subjects ( figure 8) , although neutrophil counts were always lower in the grounded subjects.7 as the number of neutrophils increases , lymphocytes are expected to decrease.1719 in the doms study , the lymphocyte count in the grounded subjects was always below the ungrounded subjects ( figure 9).7 normally , neutrophils rapidly invade an injured region8,2022 in order to break down damaged cells and send signals through the cytokine network to regulate the repair process . oxidative burst.21 while ros clear pathogens and cellular debris so that the tissue can regenerate , ros can also damage healthy cells adjacent to the repair field , causing so - called collateral damage . the fact that the grounded subjects had fewer circulating neutrophils and lymphocytes could indicate that the original damage resolved more quickly , collateral damage reduced , and the recovery process accelerated . this would explain the reduction in the cardinal signs of inflammation ( redness , heat , swelling , pain , and loss of function ) following acute injury , as documented , for example , in figures 1 and 2 , and the rapid reduction of chronic inflammation documented in figure 3 . our working hypothesis features this scenario : mobile electrons from the earth enter the body and act as natural antioxidants;3 they are semi - conducted through the connective tissue matrix , including through the inflammatory barricade if one is present;23 they neutralize ros and other oxidants in the repair field ; and they protect healthy tissue from damage . the fact that there are fewer circulating neutrophils and lymphocytes in the grounded subjects may be advantageous because of the harmful role these cells are thought to play in prolonging inflammation.24 we also raise the possibility that the inflammatory barricade is actually formed in ungrounded subjects by collateral damage to healthy tissue , as was suggested by selye in the first and subsequent editions of his book the stress of life ( figure 10).25 while there may be other explanations , we suggest that rapid resolution of inflammation takes place because the earth s surface is an abundant source of excited and mobile electrons , as described in our other work.1 we further propose that skin contact with the surface of the earth allows earth s electrons to spread over the skin surface and into the body . the meridians are known to be low resistance pathways for the flow of electrical currents.2628 another pathway is via mucous membranes of the respiratory and digestive tracts , which are continuous with the skin surface . sokal and sokal29 found that the electrical potential on the body , on the mucosal membrane of the tongue , and in the venous blood rapidly drop to approximately 200 mv . these effects reveal changes in the internal electrical environment within the body.29 selye30 studied the histology of the wall of the inflammatory pouch or barricade ( figure 10 ) . our hypothesis is that electrons can be semi - conducted across the barrier , and can then neutralize reactive oxygen species ( free radicals).30 a semiconducting collagen pathway or corridor may explain how electrons from the earth quickly attenuate chronic inflammation not resolved by dietary antioxidants or by standard medical care , including physical therapy ( figure 3 ) . taken together , these observations indicate that grounding or earthing the human body significantly alters the inflammatory response to an injury . one of the first published grounding studies examined the effects of grounding on sleep and circadian cortisol profiles.5 the study involved 12 subjects who were in pain and had problems sleeping . they slept grounded for 8 weeks using the system shown in figure 4 . during this period , their diurnal cortisol profiles normalized , and most of the subjects reported that their sleep improved and their pain and stress levels declined . the results of the experiment led to these conclusions : 1 ) grounding the body during sleep yields quantifiable changes in diurnal or circadian cortisol secretion levels that , in turn , 2 ) produce changes in sleep , pain , and stress ( anxiety , depression , and irritability ) , as measured by subjective reporting . the cortisol effects described by ghaly and teplitz5 are particularly significant in the light of recent research showing that prolonged chronic stress results in glucocorticoid receptor resistance.6 such resistance results in failure to downregulate inflammatory responses , which can thereby increase risks of a variety of chronic diseases . this effect complements the findings described in the effects on pain and the immune response section . a pilot study on the effects of grounding on pain and the immune response to injury employed delayed - onset muscle soreness ( doms).7 doms is the muscular pain and stiffness that takes place hours to days after strenuous and unfamiliar exercise . the phase of contraction that occurs when a muscle shortens , as in lifting a dumbbell , is referred to as concentric , whereas the phase of contraction as a muscle lengthens , as in lowering a dumbbell , is referred to as eccentric . eight healthy subjects performed an unfamiliar , eccentric exercise that led to pain in their gastrocnemius muscles . this was done by having them perform two sets of 20 toe raises with a barbell on their shoulders and the balls of their feet on a 2-inch 4-inch wooden board.7 all subjects ate standardized meals at the same time of day , and adhered to the same sleep cycle for 3 days . at 5.40 pm on each day , four of the subjects had conductive grounding patches adhered to their gastrocnemius muscles and the bottoms of their feet . as controls , four subjects followed the same protocol except that their patches and sheets were not grounded . the following measurements were taken before the exercise and 1 , 2 , and 3 days thereafter : pain levels , magnetic resonance imaging , spectroscopy , cortisol in serum and saliva , blood and enzyme chemistry , and blood cell counts.7 pain was monitored with two techniques . the subjective method involved morning and afternoon use of a visual analog scale . in the afternoon , a blood pressure cuff was positioned on the right gastrocnemius and inflated to the point of acute discomfort . the grounded subjects experienced less pain , as revealed with both the analog soreness scale ( figure 5 ) and by their ability to tolerate a higher pressure from the blood pressure cuff ( figure 6).7 the doms grounding study report7 contains a summary of the literature on the changes in blood chemistry and content of formed elements ( erythrocytes , leukocytes , and platelets ) expected after an injury . the immune system detects pathogens and tissue damage and responds by initiating the inflammation cascade , sending neutrophils and lymphocytes into the region.812 as expected , the white cell counts increased in the ungrounded or control subjects . white cell counts in the grounded subjects steadily decreased following the injury ( figure 7).7 previous research has shown increases in neutrophils following injury.1316 this happened in both grounded and ungrounded subjects ( figure 8) , although neutrophil counts were always lower in the grounded subjects.7 as the number of neutrophils increases , lymphocytes are expected to decrease.1719 in the doms study , the lymphocyte count in the grounded subjects was always below the ungrounded subjects ( figure 9).7 normally , neutrophils rapidly invade an injured region8,2022 in order to break down damaged cells and send signals through the cytokine network to regulate the repair process . oxidative burst.21 while ros clear pathogens and cellular debris so that the tissue can regenerate , ros can also damage healthy cells adjacent to the repair field , causing so - called collateral damage . the fact that the grounded subjects had fewer circulating neutrophils and lymphocytes could indicate that the original damage resolved more quickly , collateral damage reduced , and the recovery process accelerated . this would explain the reduction in the cardinal signs of inflammation ( redness , heat , swelling , pain , and loss of function ) following acute injury , as documented , for example , in figures 1 and 2 , and the rapid reduction of chronic inflammation documented in figure 3 . our working hypothesis features this scenario : mobile electrons from the earth enter the body and act as natural antioxidants;3 they are semi - conducted through the connective tissue matrix , including through the inflammatory barricade if one is present;23 they neutralize ros and other oxidants in the repair field ; and they protect healthy tissue from damage . the fact that there are fewer circulating neutrophils and lymphocytes in the grounded subjects may be advantageous because of the harmful role these cells are thought to play in prolonging inflammation.24 we also raise the possibility that the inflammatory barricade is actually formed in ungrounded subjects by collateral damage to healthy tissue , as was suggested by selye in the first and subsequent editions of his book the stress of life ( figure 10).25 while there may be other explanations , we suggest that rapid resolution of inflammation takes place because the earth s surface is an abundant source of excited and mobile electrons , as described in our other work.1 we further propose that skin contact with the surface of the earth allows earth s electrons to spread over the skin surface and into the body . the meridians are known to be low resistance pathways for the flow of electrical currents.2628 another pathway is via mucous membranes of the respiratory and digestive tracts , which are continuous with the skin surface . sokal and sokal29 found that the electrical potential on the body , on the mucosal membrane of the tongue , and in the venous blood rapidly drop to approximately 200 mv . these effects reveal changes in the internal electrical environment within the body.29 selye30 studied the histology of the wall of the inflammatory pouch or barricade ( figure 10 ) . our hypothesis is that electrons can be semi - conducted across the barrier , and can then neutralize reactive oxygen species ( free radicals).30 a semiconducting collagen pathway or corridor may explain how electrons from the earth quickly attenuate chronic inflammation not resolved by dietary antioxidants or by standard medical care , including physical therapy ( figure 3 ) . taken together , these observations indicate that grounding or earthing the human body significantly alters the inflammatory response to an injury . the concept that the inflammatory barricade forms from collateral damage to healthy tissue surrounding an injury site is supported by selye s classic studies published along with his description of the granuloma or selye pouch ( figure 10).25,30 moreover , research in cell biology and biophysics reveals the human body is equipped with a system - wide collagenous , liquid crystalline semiconductor network known as the living matrix,31 or in other terms , a ground regulation system32,33 or tissue tensegrity matrix system ( figure 11).34 this body - wide network can deliver mobile electrons to any part of the body and thereby routinely protect all cells , tissues , and organs from oxidative stress or in the event of injury.23,31 the living matrix includes the extracellular and connective tissue matrices as well as the cytoskeletons of all cells.31 integrins at cell surfaces are thought to allow for semi - conduction of electrons to the cell interior , and links across the nuclear envelope enable the nuclear matrix and genetic material to be part of the circuitry.23 our hypothesis is that this body - wide electronic circuit represents a primary antioxidant defense system . the extracellular part of the matrix system is composed mainly of collagen and ground substances ( figures 11 and 12 ) . this concept was introduced by albert szent - gyrgyi in the kornyi memorial lecture in budapest , hungary in 1941 . his talk was published in both science ( towards a new biochemistry?)35 and nature ( the study of energy levels in biochemistry).36 the idea that proteins might be semiconductors was immediately and firmly rejected by biochemists . many modern scientists continue to reject semi - conduction in proteins , because living systems only have trace amounts of silicone , germanium , and compounds of gallium that are the most widely used materials in electronic semiconductor devices . however , there are many ways of making organic semiconductors without using metals . one of the sources of confusion was the widely held belief that water was a mere filler material . we now know that water plays crucial roles in enzymatic activities and semi - conduction . hydrated proteins actually are semiconductors , and have become important components in the global microelectronics industry . organic microcircuits are preferred for some applications , because they can be made very small , self - assemble , are robust , and have low energy consumption.37,38 one of the leaders in the field of molecular electronics , ns hush , has recognized albert szent - gyrgyi and robert s mulliken for providing two concepts fundamental to the industrial applications : theories of biological semi - conduction , and molecular orbital theory , respectively.39 in recent studies , given awards by the materials research society in both europe and the usa , scientists from israel made flexible biodegradable semiconductor systems using proteins from human blood , milk , and mucus.40 silicon , the most widely used semiconducting material , is expensive in the pure form needed for semiconductors , and is inflexible and environmentally problematic . organic semiconductors are predicted to lead to a new range of flexible and biodegradable computer screens , cell phones , tablets , biosensors , and microprocessor chips . we have come a long way since the early days when semi - conduction in proteins was so thoroughly rejected.41,42,43 ground substance polyelectrolyte molecules associated with the collagenous connective tissue matrix are charge reservoirs ( figure 12 ) . the glycosaminoglycans have a high density of negative charges due to the sulfate and carboxylate groups on the uronic acid residues . the matrix is therefore a body - wide system capable of absorbing and donating electrons wherever they are needed to support immune functioning.44 the interiors of cells including the nuclear matrix and dna are all parts of this biophysical electrical storage and delivery system . the time - course of the effects of grounding on injury repair can be estimated in various ways . first , we know from medical infrared imaging that inflammation begins to subside within 30 minutes of connecting with the earth via a conductive patch placed on the skin.2,3 secondly , metabolic activity increases during this same period . specifically , there is an increase in oxygen consumption , pulse rate , and respiratory rate and a decrease in blood oxygenation during 40 minutes of grounding.45 we suspect that the filling of the charge reservoirs is a gradual process , possibly because of the enormous number of charged residues on the polyelectrolytes , and because they are located throughout the body . when charge reservoirs are saturated , the body is in a state we refer to as inflammatory preparedness this means that the ground substance , which pervades every part of the body , is ready to quickly deliver antioxidant electrons to any site of injury via the semiconducting collagenous matrix ( see figure 16b ) . these considerations also imply anti - aging effects of earthing or grounding , since the dominant theory of aging emphasizes cumulative damage caused by ros produced during normal metabolism or produced in response to pollutants , poisons , or injury.46 we hypothesize an anti - aging effect of grounding that is based on a living matrix reaching every part of the body and that is capable of delivering antioxidant electrons to sites where tissue integrity might be compromised by reactive oxidants from any source.47,48 molecules generated during the immune response were also followed in the doms study.7 parameters that differed consistently by 10% or more between grounded and ungrounded subjects , normalized to baseline , included creatine kinase , phosphocreatine / inorganic phosphate ratios , bilirubin , phosphorylcholine , and glycerolphosphorylcholine . bilirubin is a natural antioxidant that helps control ros.4953 while bilirubin levels decreased in both grounded and ungrounded groups , the margin between the subjects was large ( figure 13).7 the inflammatory markers changed at the same time that the pain indicators were changing . this was revealed by both the visual analog pain scale and by the pressure measurements on the right gastrocnemius ( figures 5 and 6 ) . the authors of the doms study suggested that bilirubin may have been used as a source of electrons in the ungrounded subjects.7 it is possible that the lower decline in circulating bilirubin in the grounded subjects was due to the availability in the repair field of free electrons from the earth . other markers encourage the hypothesis that the grounded subjects more efficiently resolved tissue damage : the pain measures , inorganic phosphate - phosphocreatine ratios ( pi / pcr ) , and creatine kinase ( ck ) . muscle damage has been widely correlated with ck.5456 as figure 14 shows , ck values in the ungrounded subjects were consistently above those in the grounded subjects.7 differences between pi / pcr of the two groups were monitored by magnetic resonance spectroscopy . these ratios are indicative of metabolic rate and cellular damage.5760 inorganic phosphate levels are indicative of hydrolysis of pcr and adenosine triphosphate . the ungrounded subjects had higher levels of pi , while the grounded subjects showed higher levels of pcr . these findings indicate that mitochondria in the grounded subjects are not producing as much metabolic energy , probably because there is less demand due to more rapid achievement of homeostasis . the pilot study7 on the effects of earthing in speeding recovery from the pain of doms provides a good basis for a larger study . the concepts presented here are summarized in figure 16 as a comparison between mr shoes ( an ungrounded individual ) and mr barefoot ( a grounded individual ) . a search for inflammation in the national library of medicine database ( pubmed ) reveals over 400,000 studies , with more than 34,000 published in 2013 alone . the most common cause of death and disability in the united states is chronic disease . seventy - five percent of the nation s health care spending , which surpassed us$2.3 trillion in 2008 , is for treating chronic disease . heart disease , cancer , stroke , chronic obstructive pulmonary disease , osteoporosis , and diabetes are the most common and costly chronic diseases.61 others include asthma , alzheimer s disease , bowel disorders , cirrhosis of the liver , cystic fibrosis , multiple sclerosis , arthritis , lupus , meningitis , and psoriasis . osteoporosis affects about 28 million aging americans.61,62 however , there are few theories on the mechanisms connecting chronic inflammation with chronic disease . the research on grounding or earthing summarized here provides a logical and testable theory based on a variety of evidence . the textbook description of the immune response describes how large or small injuries cause neutrophils and other white blood cells to deliver highly ros and rns to break down pathogens and damaged cells and tissues . classical textbook descriptions also refer to an inflammatory barricade that isolates injured tissues to hinder the movement of pathogens and debris from the damaged region into adjacent , healthy tissues . selye described how the debris coagulates to form the inflammatory barricade ( figure 10 ) . this barrier also hinders the movements of antioxidants and regenerative cells into the blocked - off area . repair can be incomplete , and this incomplete repair can set up a vicious inflammatory cycle that can persist for a long period of time , leading to so - called silent or smoldering inflammation that in turn , over time , can promote the development of chronic disease . remarkable as it may seem , our findings suggest that this classical picture of the inflammatory barricade may be a consequence of lack of grounding , and of a resultant electron deficiency . wounds heal very differently when the body is grounded ( figures 1 and 2 ) . healing is much faster , and the cardinal signs of inflammation are reduced or eliminated . those who research inflammation and wound healing need to be aware of the ways grounding can alter the time - course of inflammatory responses . they also need to be aware that the experimental animals they use for their studies may have very different immune systems and responses , depending on whether or not they were reared in grounded or ungrounded cages . it is standard research practice for investigators to carefully describe their methods and the strain of the animals they use so that others can repeat the studies if they wish . an assumption is that all wistar rats , for example , will be genetically and physiologically similar . however , a comparison of neoplasms in sprague dawley rats ( originally outbred from the wistar rat ) from different sources revealed highly significant differences in the incidences of endocrine and mammary tumors . the frequency of adrenal medulla tumors also varied in rats from the same suppliers raised in different laboratories . the authors stressed the need for extreme caution in evaluation of carcinogenicity studies conducted at different laboratories and/or on rats from different sources.63 from our perspective , these variations are not at all surprising . animals will differ widely in the degree to which their charge reservoirs are saturated with electrons . are their cages made of metal , and if they are , is that metal grounded ? how close are their cages to wires or conduits carrying 60/50 hz electricity ? from our research , those factors will have measurable impacts on immune responses . hidden variable that could have affected the outcomes of countless studies , and also could affect the ability of other investigators to reproduce a particular study . dominant lifestyle factors such as insulating footwear , high - rise buildings , and elevated beds separate most humans from direct skin connection with the earth s surface . an earth connection was an everyday reality in past cultures that used animal skins for footwear and to sleep on . we suggest that the process of killing pathogens and clearing debris from injury sites with ros and rns evolved to take advantage of the body s constant access to the virtually limitless source of mobile electrons the earth provides when we are in contact with it . antioxidants are electron donors , and the best electron donor , we strongly believe , is right under our feet : the surface of the earth , with its virtually unlimited storehouse of accessible electrons . electrons from the earth may in fact be the best antioxidants , with zero negative secondary effects , because our body evolved to use them over eons of physical contact with the ground . our immune systems work beautifully as long as electrons are available to balance the ros and reactive nitrogen species ( rns ) used when dealing with infection and tissue injury . our modern lifestyle has taken the body and the immune system by surprise by suddenly depriving it of its primordial electron source . this planetary separation began accelerating in the early 1950s with the advent of shoes made with insulating soles instead of the traditional leather . the disconnection from the earth may be an important , insidious , and overlooked contribution to physiological dysfunction and to the alarming global rise in non - communicable , inflammatory - related chronic diseases . a lack of electrons can also de - saturate the electron transport chains in mitochondria , leading to chronic fatigue and slowing the cellular migrations and other essential activities of the cells of the immune system.64 at this point , even a minor injury can lead to a long - term health issue . when mobile electrons are not available , the inflammatory process takes an abnormal course . cells of the immune system may fail to distinguish between the body s diverse chemical structures ( called self ) and the molecules of parasites , bacteria , fungi , and cancer cells ( called non - self ) . this loss of immunologic memory can lead to attacks by some immune cells on the body s own tissues and organs . an example is the destruction of insulin - producing beta cells of the islets of langerhans in the diabetic patient . lupus erythematosus is an extreme example of an auto - immune condition caused by the body s immune system attacking host tissues and organs . lupus , for example , can affect many different body systems , including skin , kidneys , blood cells , joints , heart , and lungs . with time , the immune system becomes weaker and the individual more vulnerable to inflammation or infections that may not heal , as often seen with the wounds of diabetic patients . specifically , which part or parts of the body the weakened immune system will attack first depends on many factors such as genetics , habits ( sleep , food , drinks , exercise , etc ) , and toxins in the body and in the environment.65,66 a repeated observation is that grounding , or earthing , reduces the pain in patients with lupus and other autoimmune disorders.1 accumulating experiences and research on earthing , or grounding , point to the emergence of a simple , natural , and accessible health strategy against chronic inflammation , warranting the serious attention of clinicians and researchers . the living matrix ( or ground regulation or tissue tensegrity - matrix system ) , the very fabric of the body , appears to serve as one of our primary antioxidant defense systems . as this report explains , it is a system requiring occasional recharging by conductive contact with the earth s surface

multi - disciplinary research has revealed that electrically conductive contact of the human body with the surface of the earth ( grounding or earthing ) produces intriguing effects on physiology and health . such effects relate to inflammation , immune responses , wound healing , and prevention and treatment of chronic inflammatory and autoimmune diseases . the purpose of this report is two - fold : to 1 ) inform researchers about what appears to be a new perspective to the study of inflammation , and 2 ) alert researchers that the length of time and degree ( resistance to ground ) of grounding of experimental animals is an important but usually overlooked factor that can influence outcomes of studies of inflammation , wound healing , and tumorigenesis . specifically , grounding an organism produces measurable differences in the concentrations of white blood cells , cytokines , and other molecules involved in the inflammatory response . we present several hypotheses to explain observed effects , based on current research results and our understanding of the electronic aspects of cell and tissue physiology , cell biology , biophysics , and biochemistry . an experimental injury to muscles , known as delayed onset muscle soreness , has been used to monitor the immune response under grounded versus ungrounded conditions . grounding reduces pain and alters the numbers of circulating neutrophils and lymphocytes , and also affects various circulating chemical factors related to inflammation .

Introduction Summary of findings to date Effects on sleep Effects on pain and the immune response Anatomical and biophysical aspects Discussion Conclusion

food chain information ( fci ) is one of the innovations introduced by the hygiene package to enhance the concept of food security and ensure welfare and animal health . food chain information can be used by the operator of the slaughterhouse within the haccp plan in order to organise the slaughtering and by the official veterinarian to take decisions on the carcass . information should cover what follows ( european commission , 2004 ) : i ) status of provenance or the regional animal health status ; veterinary medicinal products or other treatments administered within a relevant period and with a withdrawal period > 0 , together with their dates of administration and withdrawal periods ; ii ) occurrence of diseases that may affect the safety of the meat ; iii ) the results , if they are relevant to the public health , of any analysis carried out on samples taken from the animals or other samples taken to diagnose diseases that may affect the safety of the meat , including samples taken in a framework of the monitoring and control of zoonoses and residues ; iv ) relevant reports about previous ante- and post - mortem inspections of animals from the same holding of provenience , including reports from the official veterinarian production data when they may indicate the presence of diseases ; v ) name and address of the private veterinarian normally attending the holding of provenience . fci must be provided to the slaughterhouse operators at least 24 h before the arrival of the animals . it is also possible to send fci simultaneously in the following cases : for pigs , poultry or farmed game that have passed the antemortem inspection at the farm and are accompanied by a veterinary certificate declaring the animals as healthy ; for slaughtering of domestic solipeds or other animals subject to emergency slaughter if accompanied by a veterinary certificate attesting the successful antemortem inspection ; for animals that have not been sent directly from the holding of provenance to the slaughterhouse . within the council regulation ( ec ) n. 2076/2005 ( european commission , 2005 ) there is another exception to regulation ( ec ) n. 853/2004 ( european commission , 2004 ) , which provides for the simultaneous transmission of fci and animals regardless of species , subject to agreements between food business operator ( fbo ) and the official veterinary . if the veterinary decides for the slaughtering , even in the absence of fci , the document must be provided within 24 h , otherwise the entire carcass will be destroyed . several eu member states drafted guidelines for the correct application of the document . unfortunately in italy only few regions did it . the aim of the present work is to compare the different european guidelines and to analyse the situation in piedmont after few years of the entry into force of this provision in order to assess the presence of potential problems and propose solutions . we analysed the guidelines of : france , united kingdom , belgium , the netherlands , spain and germany ( affsa , 2008 ; spanish government , 2009 ; afsca , 2009 ; pve , 2014 ) . for each country we evaluated the guidelines and compared information contained within the fci for the different animal species . in order to analyse the situation in the piedmont year we collected for one year and compared the fci in 11 slaughterhouses differing among them for capacity and slaughtered species ( cattle , pigs , small ruminants , poultry , rabbits and horses ) . the fci models were classified as : fci negative ( compiled form with one or more items not completed ) ; fci absent ( module of not completed for any item ) ; fci positive ( compiled for each item ) . in the end we analysed which information was lacking in the fci negative class the different guidelines proposed in the european union , despite being similar in many parts , have undergone adaptations depending on the epidemiological situation of each state . all these adaptations have occurred as the results of inspections carried out by the central and/or the food veterinary office ( fvo ) . in particular , these adaptations took into consideration the diseases occurring in each country . the forms varied from very detailed on specific diseases to forms in which only symptoms are requested ( affsa , 2008 ) . considering the animal specificity of the models , the majority of the examined countries only spain and germany have a single model regarding all species ( spanish government , 2009 ; german republic , 2004 ) . in general , the fci model of the poultry industry is the most detailed in all countries . most of the countries have paper forms ; but holland , uses computerised systems for small ruminants , for example . the fci model is sent the day before slaughtering with few exceptions . in france fci for poultry and rabbits is sent 48 h before slaughtering . in holland for sheep , goats and cattle it is possible to send fci simultaneously with the animals if few animals are sent for the slaughtering . a common finding is the fact that the fci models are collected by simple questions with direct answer ( yes / no ) and in the forms blanks for any additional information are provided . this situation results in large discrepancies such as : undifferentiated modules or modules differentiated by species , modules integrated or non - integrated to other mandatory documents ( e.g. mod . iv ) , modules with space or without space for additional data ( symptoms , pharmaceutical treatments ) . in general , almost all of the piedmont slaughterhouses considered do not require the document 24 h before the arrival of the animals . for all the species considered , the number of fci forms classified as negative is the most representative group ( from 78.89 to 99.1% ) and the models classified as positive are the smallest group ( from 0 to 7.14% ) . an important consideration must be ruled out related to the slaughtering capacity : the less the number of slaughtered animals , the more attention is paid by the veterinary service to check the forms . in addition to these general considerations our analysis highlighted some critical issues related to individual species . for cattle in the category fci negative , the missing data concerns , mainly , forms where pathologies were signaled but symptoms were not reported . in particular , those animals were sent for an emergency slaughtering or were accompanied by health certificates compiled by the private veterinarian . for the category fci absent the high percentage ( 8.25% ) recorded in a slaughterhouse was due to agreements between farmers and the fbo as a part of the supplier s accreditation process . it is clear that this is not just an arbitrary interpretation of the concept of the fci , but also improper extension of the accreditation process . another finding for cattle is that the fci is often considered data related to the farm and not the animals sent to the slaughterhouse . for this reason sometimes cattle from the same farm had more models n. iv and only one fci model . considering pigs , a peculiarity is due to the fact that some slaughterhouses export to australia , russian federation and china . for this slaughter plants , the information contained in the fci module gives additional health information being a mandatory request of the importing nation . considering the equine sector , a critical common point is the fact that many documents are written in the language of the country of origin only . as for small ruminants furthermore , the high percentage of parasitosis often found in post - mortem are not linked to an appropriate record neither of treatments nor of symptoms on the fci form . for poultry the possibility to perform ante - mortem inspection in the holding of provenance makes compulsory to fill out a health certificate in addition to the accompanying document . only in poultry slaughterhouses the fci form this agreement with the veterinary service enables the fbo to avoid the compilation of the health certificate representing a charge for him . in rabbits slaughtering the form was sent 24 h before the arrival of the animals due to the higher percentage of drug treatments . in the end , our analisys stressed out a certain number of criticities common for all the species considered . the fci negative is due mostly to the lack of information about symptoms even in the presence of health certificates and drug treatments . sometimes drug treatments are completely omitted on fci modules and on model n. iv or sometimes declared on the model n. iv and absent on fci modules . the information of livestock is almost always reported in reference to compulsory eradication plans for domestic cattle , pigs and small ruminants , while data are never reported to voluntary schemes or specific accreditation programmes . finally , we infer how in our country the real benefit derived from fci in terms of better health quality of food as a consequence of the uniformity and timely completion of this model is not yet fully understood ( franchini , 2004 ) . however , these findings are completely in line with what has been shown by the audit of the fvo in other european countries , such as ireland , lithuania and cyprus ( european commission , 2011 , 2012a , 2012b ) . given the large differences emerged , in our opinion it is essential to standardise the system by the aid of training courses and of new official fci forms , with guidelines for the compilation ( appendix ) . the eu reiterates the importance of increasing the level of food safety through the collection and transmission of information along the entire food chain . fci is a reliable tool to achieve it . from the data collected in this investigation the importance of fci seems not to be perceived by the fbo and also by the public veterinary service , which is often limited to assess the presence of the form , without checking its content . we believe that the issue of guidelines by the state - regions conference could in italy , like in other eu countries , give effectively increase the veterinary checks at slaughter and therefore favour the safety of the meat .

food chain information ( fci ) is an innovation of the new european regulation . its purpose is to enhance the concept of food security . fci includes specifications such as : health status , information on treatments and diseases , analytical reports on control plans , zoonoses or environmental contaminants , production performance , etc . the aim of this article is to compare the different european guidelines and analyse the situation in piedmont in order to assess potential problems and propose solutions . european guidelines are similar one another , but they have been tailored to the epidemiological situations of each state . except for spain and germany , fci models are different for each species and the poultry sector is the most detailed . unfortunately , italy has not provided guidelines yet , and this has generated considerable differences . overall , the number of fci models with incomplete information is the largest group compared to the models not completed for each entry . the main deficiencies are related to pharmacological treatments . the health status of the farm is listed consistently regarding the compulsory eradication plans , but other national voluntary or accreditation plans are rarely mentioned . the situation is similar in other european countries . in conclusion , fci is an effective tool if applied with consistency and reason . only in this way the collection of data will be effective and representative of the food chain .

Introduction Materials and Methods Results and Discussion Conclusions

abdominal pregnancies ( both primary and secondary ) have a reported incidence of 1 in 10,000 . worldwide , the incidence of ectopic pregnancies is increasing , with significant contribution from assisted reproductive technologies ( art ) . however , the incidence of abdominal pregnancies have remained constant or reduced , and maternal mortality reduced from 20% to less than 5% in last 20 years due to early diagnosis and management . early diagnosis has enabled laparoscopic management in many of these cases . in this study , we report a case of primary abdominal pregnancy following intra - uterine insemination ( iui ) managed laparoscopically . y.p , a 31-year - old female , attended our opd on 22 august 2010 , with chief complaints of amenorrhea of 2 months , following an iui cycle . she had primary infertility of 3 years , and was undergoing iui for mild - to - moderate male factor coupled with mild pelvic endometriosis . this was her first iui cycle ( cc1 00 mg d2-d6 , hcg trigger , and dydrogestrone as luteal support ) . the undisturbed ectopic gestation sac seen on the anterior surface of the uterus and anterior leaf of broad ligament the ectopic pregnancy site which started to bleed , on manipulation of the uterus bleeding ectopic sac separating from peritoneal surface protruding gestational sac bleeding from the implantation site after gestational sac was sucked out implantation site after removal of ectopic sac and hemostasis her previous menstrual cycles were regular . on transvaginal sonography , the uterus was bulky , very thick regular echogenic endometrium , and no gestational sac in cavity [ figure 7 ] . both ovaries were normal with a corpus luteum in the left ovary [ figures 8 and 9 ] . there was a 1.6 cm mass with strongly echogenic rim , close to the uterus , in the left side suggestive of an ectopic pregnancy sac . serum b - hcg in the same day was 9470 miu / ml [ figures 10 and 11 ] . uterus with empty endometrial cavity transvaginal scan showing left ovary with a corpus luteum image of the normal right ovary transvaginal sonography showing ectopic gestational sac in the region of the left adnexa transvaginal sonography picture showing both the empty uterus and adjacent ectopic sac laparoscopy was offered to the patient , who however , desperately requested for the conservative approach ( she had undergone diagnostic hystero - laparoscopy 2 months back during her infertility workup , and also did not want to loose a fallopian tube ) . she was given one intramascular injection of methotrexate ( 50 mg ) on 22.08.2010 following routine blood tests . however , follow - up of serial b - hcg on 26.08.2010 showed persisting levels of 9300 miu / ml and transvaginal sonography showed a size of the mass increased to 2 cm . was bulky , deviated to the right side with a few old and new endometriotic deposits on the antero - superior surface . the pregnancy sac was implanted on the peritoneal surface of the broad ligament , between the round ligament and the utero - vesical fold . initial appearance was a mound - like elevation , with yellowish brown margins [ figures 1 and 2 ] . there was significant bleeding from the base [ figure 5 ] , which was controlled with bipolar coagulation and superficial infiltration of pitressin [ figure 6 ] . -hcg fell to 203 iu within 4 days , and subsequently declined to non - pregnant levels . incidence has been reported as approximately 1 in 10,000 live births , and 9.2 per 1000 ectopic pregnancies . most of these are secondary abdominal pregnancies , which means the embryo had primarily implanted in the fallopian tube , extruded , or expelled and then secondarily implanted itself on another intra - abdominal surface . in primary abdominal pregnancy , which is the rarest type of ectopic gestation , the conceptus implants on the peritoneal surface . studdiford 's criteria used to diagnose primary abdominal pregnancy are described as : the presence of normal bilateral tubes and ovaries with no evidence of recent or past pregnancy.no evidence of a uteroperitoneal fistula.the presence of pregnancy related exclusively to the peritoneal surface , early enough to eliminate the possibility of secondary implantation after primary tubal nidation . the presence of normal bilateral tubes and ovaries with no evidence of recent or past pregnancy . the presence of pregnancy related exclusively to the peritoneal surface , early enough to eliminate the possibility of secondary implantation after primary tubal nidation . alternatively , a classification relevant from diagnosis and treatment defines early peritoneal pregnancy ( 20 weeks of gestation ) or advanced ( 20 weeks of gestation ) . primary abdominal pregnancies have been reported from omentum , sigmoid colon , posterior peritoneum of pelvis , spleen , liver , diaphragm , obturator foramen , posterior surface of uterus , retroperitoneum , and pancreas.[721 ] primary peritoneal pregnancy has been reported in relation to foci of endometriosis . rare and new varieties of ectopics such as heterotopic pregnancies , cervical , cesarean scar pregnancies have surfaced . however , the incidence of abdominal pregnancies has not changed , may be reduced due to early diagnosis . presentation of patents with primary abdominal pregnancy varies greatly , however , with early diagnosis like our case , patients may be totally asymptomatic . in earlier literature abdominal pregnancies were typically diagnosed late . now with serial b - hcg , excellent ultrasonography , and mri facilities , these pregnancies are diagnosed much earlier . our case is unique in some aspects . to our knowledge , this is the only case reported of primary peritoneal pregnancy in the anterior peritoneal leaf of the broad ligament following intrauterine insemination . the presence of uterine surface endometriosis was documented by a routine diagnosis laparoscopy only 2 months earlier . prognosis for future fertility also appears good following early diagnosis , as the tubes , ovaries are not directly involved in pregnancy , nor removed during the operative procedure and are not involved in postoperative adhesion formation .

primary abdominal pregnancy is an extremely rare type of extrauterine pregnancy . it has been reported from many unusual intra - abdominal sites . we report a case of primary abdominal pregnancy following intra - uterine insemination ( not reported earlier to our knowledge ) . implanted on the anterior surface of the uterus possibly related to an endometriotic foci . early diagnosis enabled laparoscopic management of this case .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSIONS

nursing science as well as mental health care in general has , in fact , comprised both the humanistic paradigm that can be seen as a bottom - up approach , where the patient 's individual experiences are considered , and the biomedical model that can be regarded as a top - down approach toward the patient , where the professionals are viewed as the experts ( lindsey & hartrick , 1996 ) . the organizational processes such as deinstitutionalization and integration of patients in community - based mental health services have made the challenge to create a clear definition of health at all levels in mental health care even more essential ( magnusson , hgberg , ltzen , & severinsson , 2004 ) . an important distinction has been made between illness prevention , which can be described as the avoidance of disease , and health promotion that comprises a number of activities seeking to expand positive potentials for health ( pender , 1996 ) . it has been maintained that , although a more holistic health perspective has been developed in nursing science in recent decades , nursing research sometimes still tends to characterize health as the absence of symptoms of disease or handicap ( hwu , coates , & boore , 2001 ) . a divergence has , however , been recognized in the nursing discipline between these two contrasting health care paradigms ( malin & teasdale , 1991 ) . a focus in health care that remains mainly on illness and deficits tends to reinforce the patient 's experience of illness and disability ( simmons , 1989 ; svedberg , jormfeldt , & arvidsson , 2003 ) . however , the main focus in previous research regarding health in mental health care has been to alleviate and reduce mental illness and symptoms of disease , while frequently overlooking the patients needs of support for their health from a subjective perspective ( berg & sarvimki , 2003 ) . psychiatric symptoms are , from the perspective of pathogenesis , viewed as signs of a malfunctioning in the brain and the main goal of health care is to cure or prevent illness . from this perspective the concept of health is viewed as normality and disease is viewed as a deviation from this normality . according to this perspective , interventions such as prescribing a pill aiming at alleviating illness and symptoms of disease are often simultaneously viewed as health promoting as health and illness are seen as endpoints of the same continuum . however , health can not sufficiently be explained in a negative way as absence of symptoms because such an explanation tends to neglect the positive aspects of health ( downie et al . , 1996 ; hedelin & strandmark , 2001 ; naidoo & wills , 2000 ) . thus it is of great importance that the staff in mental health care develops a willingness to respect the dignity of patients and to become aware of the patients possibilities and resources ( jormfeldt , svedberg , fridlund , & arvidsson , 2007 ; svedberg et al . , 2003 ) . the adoption of a positive attitude and focus on possibilities has been proposed to motivate people with long - term mental health problems to help them engage in and to take an active part in their own care ( repper , ford , & cooke , 1994 ) . the perspective of salotogenesis is built on knowledge about health , how it is achieved , and incorporates a continuous view of health and illness . the individual is viewed as a whole person in relation to his or her environment and the main goal from this perspective is to strengthen and promote health . from a holistic perspective health care of today seems to take for granted that interventions aiming at alleviating symptoms of disease automatically promote processes of health as well . however , clinical experiences from mental health nursing show that this is not necessarily the case . perhaps the promotion of health processes reduces the process of illness to a greater extent than the prevailing , mainly biomedical , clinical praxis of today is able to promote those health processes . as an example from the biomedical perspective , the diagnosis of schizophrenia is often described as incurable and the patient is often instructed to follow doctors prescriptions very carefully because medication is seen as the foundation for treatment and the most superior intervention to alleviate the symptoms ( van os et al . , 2006 ) . this intervention may place our patient in the position where the symptoms are partly alleviated but the patient is still at great risk of becoming ill in the event of stressful situations . it is easy to understand that the patient 's motivation to maintain hope and follow prescriptions can sometimes be reduced if his or her problems are primarily viewed from this perspective . from a health perspective , the goals and desires of the individual are vital and serve as guidance for the care . the process of reaching individual goals is seen as the superior pathway for a cure from mental illness . an intervention to support our imagined patient to reach his individual goals may place him / her in a position where he / her has a good chance of achieving a good life and a feeling of well - being . if or when medication is used , from this perspective , it is to support the patient in his / her process of reaching his / her goals from a salutogenic perspective of health . valid and reliable measures of a positive multidimensional concept of health in mental health services need to address autonomy , social involvement , and comprehensibility ( jormfeldt , svensson , arvidsson , & hansson , 2008 ) . autonomy incorporates the ability to function in daily life , self - esteem , and to feel positively about oneself . perceptions among nurses and patients in mental health services have been shown to be very much alike ( jormfeldt , 2010 ; jormfeldt , svedberg , & arvidsson , 2003 ; svedberg , jormfeldt , fridlund , & arvidsson , 2004 ) . however , one difference between nurses and patients expressions of health is the importance of feelings of being equal and important to other people . these feelings were related to a feeling of self - worth and health , which nurses working in mental health services do not always express an awareness of ( jormfeldt et al . , 2003 ) . patients in mental health services have described the importance of being looked upon just as anyone else as well as the need for help to reduce the shame of having to request and receive psychiatric care in order to reduce stigmatization and enhance health ( schrder , ahlstrm , & larsson , 2006 ) . ( 2002 ) found that overall self - esteem was predicted by being satisfied with family relationships and that having at least one close friend was a predictor of positive self - esteem , while being able to cope without friends was associated with superior self - esteem . self - esteem has shown to be strongly associated with health while psychiatric symptoms to a much lesser extent have shown to be negatively associated to health ( jormfeldt , arvidsson , svensson , & hansson , 2008 ) . social involvement contains experiences of attachment to others and feelings of being both a recipient and a donor of social support . mental health patients emphasize the importance of experiences of participation in social contexts and being able to give support to others , while the nurses more often speak of the importance of patients experiencing trust and being part of a social context ( jormfeldt et al . , 2007 ) . hedelin and jonsson ( 2003 ) describe mutuality in relationships between people as a major element in their experience of mental health . the essence of mental health has been defined as the experience of confirmation by means of being noticed , respected , and regarded as a valuable person by others ( hedelin & strandmark , 2001 ) , and one very important aspect of health is to feel included in a social network . ( 1999 ) have studied clinical characteristics that may be related to quality of life without finding any associations with global subjective quality of life , while one objective indicator , i.e. , to have at least one close friend , was associated with superior self - rated quality of life . unmet needs in the domain of social relationships are related to a worse quality of life ( hansson et al . , 2003 ) . involuntary loneliness and the experience of not belonging to anyone have been described as a lack of participation in the world ( dahlberg , 2007 ) . comprehensibility includes the understanding and awareness of one 's own situation , how the situation has emerged , and a view of how to get the situation changed if desired . comprehensibility could be recognized as meaningfulness , being at peace , and looking forward to something positive , as well as personal growth and seeing suffering as a motivating force to change ( jormfeldt et al . , 2003 ) . health as a process includes the view of suffering as a motivator for development toward change and increased awareness ( jormfeldt et al . , 2007 ) as described by several previous researchers ( herberts & eriksson , 1995 ; lindsey , 1996 ; long , 1998 ) . jones and meleis ( 1993 ) maintained that increased self - esteem , which is often enhanced through empowerment processes , is essential in order to gain the energy that is needed to be able to fully use one 's own personal health capacity . a number of earlier researchers have depicted health as an increased awareness and a foundation for increased well - being ( eriksson , 1984 ; long , 1998 ; moch , 1998 ; parse , 1990 ) . the process toward increased awareness and greater personal meaning has been labeled transition and could be encouraged through communication of thoughts and needs ( skrster & willman , 2006 ) . this awareness can , in turn , lead to more constructive behavior in accordance with individual wishes related to the environment . positive mental health has to be promoted at the individual level ( mezzich , 2005 ) and include factors such as acceptance , faith , hope , meaningfulness , and meaningful relationships ( edward , welch , & chater , 2009 ) . according to this reasoning , the concept of health is far beyond the limits of the wellness illness continuum ( hwu , coates , boore , & bunting , 2002 ; moch , 1998 ) . health promotion interventions are determined by the definition of health . to be able to truly promote health in mental health care , we have to define health in a positive manner and not only as an absence of illness or disease . the construct of remission can be seen as an example of measuring absence of symptoms of illness as a measure of health . the symptomatic remission criteria among patients diagnosed with bipolar disorder or schizophrenia is used as an assessment of reduction of symptoms . the underlying presumption is that remission is a prerequisite for everyday functioning among these patients . remission is suggested to be a good measure of treatment outcome and it is also proposed that remission of clinical symptoms is a required first step toward functional recovery ( van os et al . , 2006 ) . on the other hand , the construct of remission pays little attention to the subjective experience of the patient and the focus is entirely on the absence of symptoms of illness even though the concept of remission often is described as a positive concept related to recovery , health , and quality of life . the difficulties in measuring subjectively experienced health in individuals or in populations as well as in mental health services without a clear definition of the positive dimension of health has been highlighted by jormfeldt et al . the overall goal of nursing in general health care as well as in mental health care is to promote the patient 's subjective experience of health . an important measure of quality of care is thus to assess to what extent this goal is reached . the more ambiguous the definition of health is , the less importance the concept will be given and other more clarified concepts , as symptoms of illness or disease , will be given greater significance when mental health care is evaluated . in health care it has traditionally been easier and more common to measure absence of symptoms of disease as these symptoms often are more clearly defined then subjectively experienced health . studies evaluating questionnaires measuring patients subjectively experienced health in mental health services are rare , and most questionnaires used to measure health in health care have , in fact , been measuring absence of illness in terms of symptoms and disabilities in line with the biomedical view of health ( christiansen & kooiker , 1999 ; whitehead , 2003 ) . consequently it is often the negative dimension of the concept of health that is being measured , evaluated , and used as a base for evidence . most evidence - based models appear to understate the importance of non - pharmacological interventions and a lack of knowledge about such interventions is recognized ( hayman - white & happel , 2007 ) . nursing care and caring science requires humanistic knowledge with a hermeneutical dimension to achieve real progress in caring ( eriksson , 2002 ) , the challenge is to shape an evidence base for this kind of knowledge . health is to a great extent associated with self - esteem and to a lesser extent with clinical characteristics and psychiatric symptoms . mental health care must consequently involve interventions , relationships , and contexts primarily aiming to support the patient 's self - esteem and empowerment if it is to promote health . the key to enhanced health among patients in mental health services is to acquire support regarding self - esteem , empowerment , and quality of life . to view the goal of mental health care mainly as reducing symptoms should not be sufficient when mental health as well as overall health is to such a low level explained purely by psychiatric symptoms . subjectively experienced health is one of the most important outcome measures of the quality of care provided to patients in mental health care irrespective of type of symptom , illness , or disability . advanced analysis of the concept of health may contribute to enhanced empowerment strategies in health care . further research of importance would be to investigate the effects and effectiveness of empowerment approaches in mental health services and to consider every possible causal relationship between psychiatric symptoms and health in order to develop and implement more constructive intervention approaches in mental health services . clinical interventions aiming at strengthening positive dimensions of health and self - esteem are required in mental health care in order to meet the patients needs of enhanced health and to make further investigations possible regarding effects of such interventions . the author has not received any funding or benefits from industry or elsewhere to conduct this study .

this paper will explore two contrasting paradigms in mental health care and their relationship to evidence - based practice . the biomedical perspective of pathogenesis and the health perspective of salotogenesis are two major diverse views in mental health care . positive dimensions of health are traditionally viewed as software not suitable for statistical analysis , while absence of symptoms of disease are regarded as measurable and suitable for statistical analysis and appropriate as a foundation of evidence - based practice . if the main goal of mental health care is to enhance subjectively experienced health among patients , it will not be sufficient to evaluate absence of symptoms of disease as a measure of quality of care . the discussion focuses on the paradox of evidence - based absence of illness and disease versus subjectively experienced health and well - being as criterions of quality of care in mental health care .

The importance of defining health in mental health care Positive dimensions of health Do we need to measure health in mental health care? Conclusions Implications for further research and relevance in clinical practice Conflict of interest and funding

glycemic abnormalities and diabetes are on the rise globally ( 1 ) . according to the most recent statistics , 9.3% of the u.s . population , 29.1 million individuals , live with diabetes , and the level of glycemia in the general public ( mean fasting plasma glucose ) since 1980 has risen by 2.5 mg / dl per decade in women , and by 3.2 mg / dl per decade in men ( 1,2 ) . hyperglycemia is common after stressful events , such as myocardial infarction , stroke , and sepsis , or in the postoperative setting , after cardiac surgery ( 3 ) . stress - induced hyperglycemia is a transient phenomenon , distinct from the chronic glucose dysregulation brought about by diabetes ( 3 ) . studies ( 47 ) have shown that stress hyperglycemia after cardiac surgery , which occurs in patients both with and without diabetes , is associated with a higher risk of complications , including major infections , and increased mortality . the management of stress hyperglycemia in patients receiving critical care is a matter of great controversy ( 8) . the rationale of glucose control management rests on the hypothesis that the relationship between hyperglycemia and adverse outcomes is one of causation . trials assessing the potential benefits of strict glycemic control ( target range 80110 mg / dl ) ( 912 ) have produced conflicting results , with early studies reporting decreased mortality and morbidity , and subsequent studies showing a lack of benefits or even worse outcomes , along with an increased risk of hypoglycemia . these trials included a heterogeneous selection of patients , which may have influenced the response to short - term changes in glucose levels . given the uncertainty about the effectiveness of different protocols targeting normoglycemia , most medical societies have endorsed a moderate approach to glucose control in perioperative and critical care settings , recommending that patients , regardless of their diabetes status , have their serum glucose levels maintained at < 180 mg / dl ( 6,13 ) . more recently , due the ongoing debate , the surgical care improvement project , a national program undertaken to improve outcomes in surgery whose measures are publicly reported on the centers for medicare and medicaid hospital website and affect reimbursement , has suspended its recommendation on maintaining postoperative glucose levels at < 180 mg / dl ( 14 ) . an increasing body of evidence shows that the association between stress hyperglycemia and adverse outcomes varies depending on the pre - existence of diabetes ( 3,1517 ) . although diabetes is a heterogeneous disease with a broad spectrum of manifestations and symptom severity ( 18 ) , most of the previous studies have analyzed the impact of stress hyperglycemia in diabetes without further stratification by prior treatment . however , prior treatment history and degree of glycemic control may be important effect modifiers ( 19 ) . consideration of these factors would permit the selection of more appropriate glucose targets for specific groups of patients , particularly in intensive care , where complications can be life threatening and costs are the highest . the purpose of this study is to assess the clinical and economic outcomes associated with postoperative hyperglycemia among patients without and with diabetes with different treatment histories who have undergone cardiac surgery . between february and october 2010 , the cardiothoracic surgical trials network conducted a multicenter prospective cohort study to assess the incidence of hospital - acquired infections . all adult cardiac surgery patients ( 18 years old ) without pre - existing infection on hospital admission were eligible to participate ( n = 5,158 ) ( 20 ) . of the 10 participating centers ( 9 american and 1 canadian ) , only patients from u.s . centers ( n = 4,614 ) were included in order to avoid the confusion of mixing data from different health care systems with very different reimbursement methods . billing data for these nine centers were obtained from the university healthsystem consortium , an alliance of u.s . academic medical centers with the goal of promoting improvements in the quality , safety , and efficiency of health care . the final study population included 4,316 patients in whom glucose was measured within 48 h after surgery . the study protocol was approved by the institutional review boards of each of the participating study centers . baseline variables prior to surgery included the following : demographics , anthropometrics , laboratory results , and comorbid conditions . pre - hospital admission diabetes status was defined by prior therapy with oral antidiabetes medication only , a history of non insulin - treated diabetes mellitus ( nitdm ) , or a history of insulin - treated diabetes mellitus ( itdm ) . the latter group included patients treated with insulin only or a combination of insulin and oral antidiabetic medications . ( hba1c ) was assessed preoperatively in patients with diabetes . in 83 nitdm patients ( 15% ) and 66 itdm patients ( 16% ) glomerular filtration rate ( gfr ) was estimated from serum creatinine using the chronic kidney disease epidemiology collaboration equation ( 22 ) . surgical parameters included sternotomy , hospital admission type ( elective , urgent , emergent ) , procedure type ( isolated valve , isolated coronary artery bypass graft [ cabg ] , transplantation or ventricular assist device , cabg with valve , thoracic aortic , other ) , and surgery duration . the study protocol included blood glucose measurements every 6 h for 48 h after surgery . based on previous research using maximum blood glucose level as a measure of blood glucose control ( 23 ) , we used the highest value among these measurements . hyperglycemia was defined as having at least one measurement > 180 mg / dl . to convert glucose values to millimoles per liter , multiply by 0.0555 . participating centers provided their protocol for managing glucose in the intensive care unit ( icu ) after cardiac surgery . the standard protocol of each icu was then used to approximate the actual treatment that the individual patients of that hospital received . among the centers , three had guidelines recommending a target range for blood glucose concentration between 80 and 120 mg / ml , whereas the other six centers had a target range between 140 and 180 mg / ml . the protocols were grouped into the following two categories for the analysis : tight outcomes related to resource use included hospitalization costs and hospital length of stay ( los ) . clinical outcomes during the hospital stay were defined as death , major infection , cardiac complications , and respiratory complications . this method of approximating cost is widely used and provides reasonably accurate estimates of actual costs ( 24 ) . major infections were classified using definitions adapted from the centers for disease control and prevention / national healthcare safety network ( 20 ) . complications other than infections were identified through icd-9 codes , and were defined as cardiac complications ( code 9971 ) and respiratory failure ( codes 5185 , 51881 , 51882 , 51884 , and 7991 ) , with exclusion of those flagged as present on admission . previous studies related to the existence of possible differences between patients with and without diabetes , with respect to outcomes associated with stress hyperglycemia , were searched on pubmed using the terms critical care , critically ill , icu , hyperglycemia , and diabetes mellitus , and english language and human species were used as filters . the search retrieved 653 citations , including 254 review articles . after reviewing titles and abstracts , we divided the study population in subgroups of patients with no diabetes , nitdm , and itdm . differences in baseline characteristics were assessed using kruskal - wallis one - way anova for continuous variables and tests for categorical variables . the relationship between maximum glucose levels and outcomes models included all baseline variables and interactions between a variable indicating diabetes status ( no diabetes , nitdm , and itdm ) and maximum glucose levels when analyzed continuously , or hyperglycemia when analyzed dichotomously . predictors were selected using costs as outcome and the akaike information criterion , which calculates the log - likelihood penalized for the number of parameters included . final regression models included the maximum glucose - diabetes treatment interaction term , age , sex , race ( white vs. other ) , bmi , white blood cell count , gfr , hemoglobin level , ejection fraction , renal insufficiency , lung disease , congestive heart failure , prior cardiac surgery , corticosteroid use , hospital admission type ( elective / urgent / emergent ) , procedure type , surgery duration , thoracic approach , and medical center . within an analysis restricted to patients with diabetes missing hba1c values were imputed by multiple imputation with a multivariable algorithm including all predictors , costs , hospital los , and the composite end point of complications . nonlinear associations of continuous variables were modeled by restricted cubic spline functions with four knots ( 25 ) . for estimating costs , we used a distribution with a log link function ; for hospital los , we used a negative binomial distribution with a log link function ; and for complications , we used logistic regression . to demonstrate the adjusted change in each outcome with varying glucose levels , we predicted the outcome of interest in each patient by using the regression equation and varying blood glucose concentrations ( between 120 and 300 mg / dl when continuous , and > 180 vs. 180 mg / dl when dichotomous ) while keeping the other parameters fixed at their observed value . to take into account parameter uncertainty , regression models were refitted in 1,000 bootstrap data sets , and subsequently predictions were made in each bootstrap . after ordering the 1,000 bootstrap estimates , 2.5% and 97.5% quantiles were used for calculating 95% cis . to examine how a routine implementation of the recommended threshold < 180 mg / dl would affect our different patient subgroups , we analyzed the expected outcomes above and below this threshold . differences in the individual complications were adjusted for age , sex , and type of procedure only . each scenario represented the implementation of a different glucose control threshold ranging from 120 to 300 mg / dl , and assumed the perfect situation in which patients glucose levels were maintained below this threshold . per scenario , patients were reassigned a maximum glucose value equal to the threshold when their actual value was higher . at lower thresholds , more patients have a glucose value above the threshold and will be reassigned to the threshold value ; whereas , at higher thresholds , more patients will keep their actual glucose value . finally , we graphically depicted the difference between the expected outcome of each scenario and the originally observed outcome . this difference will tend to zero at higher thresholds , given that more patients will keep their actual glucose levels . all analyses were performed using r version 3.1.0 ( r foundation for statistical computing [ http://www.r-project.org/ ] ) . between february and october 2010 , the cardiothoracic surgical trials network conducted a multicenter prospective cohort study to assess the incidence of hospital - acquired infections . all adult cardiac surgery patients ( 18 years old ) without pre - existing infection on hospital admission were eligible to participate ( n = 5,158 ) ( 20 ) . of the 10 participating centers ( 9 american and 1 canadian ) , only patients from u.s . centers ( n = 4,614 ) were included in order to avoid the confusion of mixing data from different health care systems with very different reimbursement methods . billing data for these nine centers were obtained from the university healthsystem consortium , an alliance of u.s . academic medical centers with the goal of promoting improvements in the quality , safety , and efficiency of health care . the final study population included 4,316 patients in whom glucose was measured within 48 h after surgery . the study protocol was approved by the institutional review boards of each of the participating study centers . baseline variables prior to surgery included the following : demographics , anthropometrics , laboratory results , and comorbid conditions . pre - hospital admission diabetes status was defined by prior therapy with oral antidiabetes medication only , a history of non insulin - treated diabetes mellitus ( nitdm ) , or a history of insulin - treated diabetes mellitus ( itdm ) . the latter group included patients treated with insulin only or a combination of insulin and oral antidiabetic medications . ( hba1c ) was assessed preoperatively in patients with diabetes . in 83 nitdm patients ( 15% ) and 66 itdm patients ( 16% ) glomerular filtration rate ( gfr ) was estimated from serum creatinine using the chronic kidney disease epidemiology collaboration equation ( 22 ) . surgical parameters included sternotomy , hospital admission type ( elective , urgent , emergent ) , procedure type ( isolated valve , isolated coronary artery bypass graft [ cabg ] , transplantation or ventricular assist device , cabg with valve , thoracic aortic , other ) , and surgery duration . the study protocol included blood glucose measurements every 6 h for 48 h after surgery . based on previous research using maximum blood glucose level as a measure of blood glucose control ( 23 ) , we used the highest value among these measurements . hyperglycemia was defined as having at least one measurement > 180 mg / dl . to convert glucose values to millimoles per liter , participating centers provided their protocol for managing glucose in the intensive care unit ( icu ) after cardiac surgery . the standard protocol of each icu was then used to approximate the actual treatment that the individual patients of that hospital received . among the centers , three had guidelines recommending a target range for blood glucose concentration between 80 and 120 mg / ml , whereas the other six centers had a target range between 140 and 180 mg / ml . the protocols were grouped into the following two categories for the analysis : tight outcomes related to resource use included hospitalization costs and hospital length of stay ( los ) . clinical outcomes during the hospital stay were defined as death , major infection , cardiac complications , and respiratory complications . hospitalization costs were calculated from the billing data using medicare cost center specific cost - to - charge ratios . this method of approximating cost is widely used and provides reasonably accurate estimates of actual costs ( 24 ) . major infections were classified using definitions adapted from the centers for disease control and prevention / national healthcare safety network ( 20 ) . complications other than infections were identified through icd-9 codes , and were defined as cardiac complications ( code 9971 ) and respiratory failure ( codes 5185 , 51881 , 51882 , 51884 , and 7991 ) , with exclusion of those flagged as present on admission . previous studies related to the existence of possible differences between patients with and without diabetes , with respect to outcomes associated with stress hyperglycemia , were searched on pubmed using the terms critical care , critically ill , icu , hyperglycemia , and diabetes mellitus , and english language and human species were used as filters . the search retrieved 653 citations , including 254 review articles . after reviewing titles and abstracts , we divided the study population in subgroups of patients with no diabetes , nitdm , and itdm . differences in baseline characteristics were assessed using kruskal - wallis one - way anova for continuous variables and tests for categorical variables . the relationship between maximum glucose levels and outcomes models included all baseline variables and interactions between a variable indicating diabetes status ( no diabetes , nitdm , and itdm ) and maximum glucose levels when analyzed continuously , or hyperglycemia when analyzed dichotomously . predictors were selected using costs as outcome and the akaike information criterion , which calculates the log - likelihood penalized for the number of parameters included . final regression models included the maximum glucose - diabetes treatment interaction term , age , sex , race ( white vs. other ) , bmi , white blood cell count , gfr , hemoglobin level , ejection fraction , renal insufficiency , lung disease , congestive heart failure , prior cardiac surgery , corticosteroid use , hospital admission type ( elective / urgent / emergent ) , procedure type , surgery duration , thoracic approach , and medical center . within an analysis restricted to patients with diabetes missing hba1c values were imputed by multiple imputation with a multivariable algorithm including all predictors , costs , hospital los , and the composite end point of complications . nonlinear associations of continuous variables were modeled by restricted cubic spline functions with four knots ( 25 ) . for estimating costs , we used a distribution with a log link function ; for hospital los , we used a negative binomial distribution with a log link function ; and for complications , we used logistic regression . to demonstrate the adjusted change in each outcome with varying glucose levels , we used the recycled prediction method ( 26 ) . in brief , we predicted the outcome of interest in each patient by using the regression equation and varying blood glucose concentrations ( between 120 and 300 mg / dl when continuous , and > 180 vs. 180 mg / dl when dichotomous ) while keeping the other parameters fixed at their observed value . to take into account parameter uncertainty , regression models were refitted in 1,000 bootstrap data sets , and subsequently predictions were made in each bootstrap . after ordering the 1,000 bootstrap estimates , how a routine implementation of the recommended threshold < 180 mg / dl would affect our different patient subgroups , we analyzed the expected outcomes above and below this threshold . differences in the individual complications were adjusted for age , sex , and type of procedure only . each scenario represented the implementation of a different glucose control threshold ranging from 120 to 300 mg / dl , and assumed the perfect situation in which patients glucose levels were maintained below this threshold . per scenario , patients were reassigned a maximum glucose value equal to the threshold when their actual value was higher . at lower thresholds , more patients have a glucose value above the threshold and will be reassigned to the threshold value ; whereas , at higher thresholds , more patients will keep their actual glucose value . finally , we graphically depicted the difference between the expected outcome of each scenario and the originally observed outcome . this difference will tend to zero at higher thresholds , given that more patients will keep their actual glucose levels . all analyses were performed using r version 3.1.0 ( r foundation for statistical computing [ http://www.r-project.org/ ] ) . the average age in the study population was 65.5 years , and 66% were male . among patients with diabetes , 43% were itdm patients . these patients generally had glucose levels that were less well controlled than nitdm patients , as indicated by their hba1c levels . moreover , patients with itdm had lower renal function , and a higher prevalence of congestive heart failure and chronic lung disease compared with the other patient groups ( table 1 ) . hyperglycemia during the first 48 h after surgery was reported in 70% of patients with diabetes and 36% of patients without diabetes . hyperglycemia was less frequent in hospitals with a tight glucose control protocol compared with hospitals with nontight glucose control . hypoglycemia , conversely , was more frequent in hospitals with tight glucose control , particularly in itdm patients ( table 1 ) . these results were confirmed in multivariable analyses , where the presence of a tight glucose control standard protocol in the hospital icu increased the likelihood of the development of hypoglycemia in itdm patients compared with a nontight protocol ( odds ratio of hypoglycemia in itdm patients = 2.39 [ 95% ci 1.64 to 3.48 ] ) . patients baseline characteristics continuous variables are reported as the median ( interquartile range ) , and categorical variables as n ( proportion ) . the test was performed for comparing categorical variables , and kruskal - wallis one - way anova was performed for comparing continuous variables . dm , diabetes mellitus ; na , not applicable ; vad , ventricular assist device implantation or explantation ; wbc , white blood cell . * the standard protocol of the icu had a blood glucose target range within 140180 mg / dl ( n = 2,816 ) . * * the standard protocol of the icu had a blood glucose target range within 80120 mg / dl ( n = 1,500 ) . itdm patients had , on average , higher cost and longer hospital los than patients with no diabetes or nitdm . there were 29 ( 0.9% ) , 5 ( 0.9% ) , and 7 ( 1.7% ) in - hospital deaths , respectively , among patients with no diabetes , nitdm , and itdm . furthermore , there were remarkable differences in the relationship of increasing glucose levels with resource use and clinical outcomes among patients with no diabetes , nitdm , and itdm ( fig . 1 ) . in patients without diabetes , the cost of hospitalization , hospital los , and the risk of complications increased with increasing glucose levels . in nitdm patients , cost , hospital los , and risk of complications peaked at 220 mg / dl , and slightly decreased with further glycemic increases . in contrast , in itdm patients , cost , hospital los , and the risk of complications during the hospital stay were highest at glucose concentrations < 180 mg / dl , and decreased with increasing glucose values , reaching a minimum between 180 and 240 mg / dl . persistent hyperglycemia , which was defined as two or more measurements > 180 mg / ml , occurred in 12.3% of patients without diabetes and 45.9% of patients with diabetes . in itdm patients , an increase in the number of hyperglycemic measurements was associated with reduced cost , number of complications , and hospital los , compared with patients with no hyperglycemia . conversely , in patients with no diabetes and patients with nitdm , such an increase correlated with higher cost , more complications , and prolonged hospital los ( supplementary fig . hba1c was significantly higher in itdm patients than in nitdm patients ( 7.5 vs. 6.7% [ 58 vs. 50 mmol / mol ] ) on average ( table 1 ) . in patients with diabetes , outcomes improved with increasing hba1c values . however , these improvements were not statistically significant in the multivariable complete case analysis . relative changes were 0.91 ( 95% ci 0.80 to 1.04 ) for the odds of complications , 0.98 ( 95% ci 0.95 to 1.00 ) for hospital los , and 0.98 ( 95% ci 0.95 to 1.00 ) for total costs . estimates based on multiple imputation were not different from those based on complete case analysis . among patients without diabetes , the cost of those with hyperglycemia was , on average , nearly $ 10,000 higher than the cost of those without hyperglycemia ; whereas , among itdm patients , the cost of those patients with hyperglycemia was on average approximately $ 15,000 lower ( table 2 ) . after adjustment for baseline and procedure variables , the change in cost with hyperglycemia was approximately an extra $ 3,000 in patients without diabetes and a decrease of $ 6,000 in patients with itdm . in patients with nitdm , the change in cost with hyperglycemia was positive but not significant . adjusted changes in hospital los associated with hyperglycemia followed a pattern similar to that of cost , with 0.8 additional days ( 95% ci 0.4 to 1.3 ) in patients without diabetes , and a decrease of 1.6 days ( 95% ci 3.7 to 0.4 ) in itdm patients , although the latter did not reach statistical significance . clinical and economic outcomes associated with postoperative hyperglycemia dm , diabetes mellitus . * adjusted for age , sex , race , bmi , white blood cell count , gfr , hemoglobin , history of heart failure , renal insufficiency , ejection fraction , prior cardiac surgery , history of lung disease , corticosteroids , surgery time , sternotomy performed yes / no , surgery type , procedure , and study site . * * adjusted for age , sex , and procedure . in patients without diabetes , after adjustment , hyperglycemia was associated with a 1.6% ( 95% ci 0.5 to 2.8 ) increased risk of major infections , a 2.6% ( 95% ci 0.0 to 5.3 ) increased risk of respiratory complications , and a trend toward increased cardiac complications ( table 2 ) . in nitdm patients , the risk of such complications associated with hyperglycemia increased as well , but without reaching statistical significance . conversely , in patients with itdm , hyperglycemia was associated with a reduced risk of adverse outcomes , particularly with respect to respiratory complications ( 12.5% [ 95% ci 22.4 to 3.0 ] ) and major infections ( 4.1% [ 95% ci 9.1 to 0.0 ] ) . in the analysis of hypothetical scenarios of different glucose thresholds , the outcomes after cardiac surgery predicted in patients without diabetes and patients with ntidm would improve with lowering postoperative glucose values . the expected benefits comprised an approximate $ 2,000 cost reduction and a 515% reduction in complications . in contrast , in patients with itdm , thresholds < 180 mg / dl would be harmful , although in this range outcomes were uncertain ( fig . the average age in the study population was 65.5 years , and 66% were male . among patients with diabetes , 43% were itdm patients . these patients generally had glucose levels that were less well controlled than nitdm patients , as indicated by their hba1c levels . moreover , patients with itdm had lower renal function , and a higher prevalence of congestive heart failure and chronic lung disease compared with the other patient groups ( table 1 ) . hyperglycemia during the first 48 h after surgery was reported in 70% of patients with diabetes and 36% of patients without diabetes . hyperglycemia was less frequent in hospitals with a tight glucose control protocol compared with hospitals with nontight glucose control . hypoglycemia , conversely , was more frequent in hospitals with tight glucose control , particularly in itdm patients ( table 1 ) . these results were confirmed in multivariable analyses , where the presence of a tight glucose control standard protocol in the hospital icu increased the likelihood of the development of hypoglycemia in itdm patients compared with a nontight protocol ( odds ratio of hypoglycemia in itdm patients = 2.39 [ 95% ci 1.64 to 3.48 ] ) . patients baseline characteristics continuous variables are reported as the median ( interquartile range ) , and categorical variables as n ( proportion ) . the test was performed for comparing categorical variables , and kruskal - wallis one - way anova was performed for comparing continuous variables . dm , diabetes mellitus ; na , not applicable ; vad , ventricular assist device implantation or explantation ; wbc , white blood cell . * the standard protocol of the icu had a blood glucose target range within 140180 mg / dl ( n = 2,816 ) . * * the standard protocol of the icu had a blood glucose target range within 80120 mg / dl ( n = 1,500 ) . itdm patients had , on average , higher cost and longer hospital los than patients with no diabetes or nitdm . there were 29 ( 0.9% ) , 5 ( 0.9% ) , and 7 ( 1.7% ) in - hospital deaths , respectively , among patients with no diabetes , nitdm , and itdm . furthermore , there were remarkable differences in the relationship of increasing glucose levels with resource use and clinical outcomes among patients with no diabetes , nitdm , and itdm ( fig . 1 ) . in patients without diabetes , the cost of hospitalization , hospital los , and the risk of complications increased with increasing glucose levels . in nitdm patients , cost , hospital los , and risk of complications peaked at 220 mg / dl , and slightly decreased with further glycemic increases . in contrast , in itdm patients , cost , hospital los , and the risk of complications during the hospital stay were highest at glucose concentrations < 180 mg / dl , and decreased with increasing glucose values , reaching a minimum between 180 and 240 mg / dl . persistent hyperglycemia , which was defined as two or more measurements > 180 mg / ml , occurred in 12.3% of patients without diabetes and 45.9% of patients with diabetes . in itdm patients , an increase in the number of hyperglycemic measurements was associated with reduced cost , number of complications , and hospital los , compared with patients with no hyperglycemia . conversely , in patients with no diabetes and patients with nitdm , such an increase correlated with higher cost , more complications , and prolonged hospital los ( supplementary fig . hba1c was significantly higher in itdm patients than in nitdm patients ( 7.5 vs. 6.7% [ 58 vs. 50 mmol / mol ] ) on average ( table 1 ) . in patients with diabetes , outcomes improved with increasing hba1c values . however , these improvements were not statistically significant in the multivariable complete case analysis . relative changes were 0.91 ( 95% ci 0.80 to 1.04 ) for the odds of complications , 0.98 ( 95% ci 0.95 to 1.00 ) for hospital los , and 0.98 ( 95% ci 0.95 to 1.00 ) for total costs . estimates based on multiple imputation were not different from those based on complete case analysis . among patients without diabetes , the cost of those with hyperglycemia was , on average , nearly $ 10,000 higher than the cost of those without hyperglycemia ; whereas , among itdm patients , the cost of those patients with hyperglycemia was on average approximately $ 15,000 lower ( table 2 ) . after adjustment for baseline and procedure variables , the change in cost with hyperglycemia was approximately an extra $ 3,000 in patients without diabetes and a decrease of $ 6,000 in patients with itdm . in patients with nitdm , adjusted changes in hospital los associated with hyperglycemia followed a pattern similar to that of cost , with 0.8 additional days ( 95% ci 0.4 to 1.3 ) in patients without diabetes , and a decrease of 1.6 days ( 95% ci 3.7 to 0.4 ) in itdm patients , although the latter did not reach statistical significance . clinical and economic outcomes associated with postoperative hyperglycemia dm , diabetes mellitus . * adjusted for age , sex , race , bmi , white blood cell count , gfr , hemoglobin , history of heart failure , renal insufficiency , ejection fraction , prior cardiac surgery , history of lung disease , corticosteroids , surgery time , sternotomy performed yes / no , surgery type , procedure , and study site . * * adjusted for age , sex , and procedure . in patients without diabetes , after adjustment , hyperglycemia was associated with a 1.6% ( 95% ci 0.5 to 2.8 ) increased risk of major infections , a 2.6% ( 95% ci 0.0 to 5.3 ) increased risk of respiratory complications , and a trend toward increased cardiac complications ( table 2 ) . in nitdm patients , the risk of such complications associated with hyperglycemia increased as well , but without reaching statistical significance . conversely , in patients with itdm , hyperglycemia was associated with a reduced risk of adverse outcomes , particularly with respect to respiratory complications ( 12.5% [ 95% ci 22.4 to 3.0 ] ) and major infections ( 4.1% [ 95% ci 9.1 to 0.0 ] ) . in the analysis of hypothetical scenarios of different glucose thresholds , the outcomes after cardiac surgery predicted in patients without diabetes and patients with ntidm would improve with lowering postoperative glucose values . the expected benefits comprised an approximate $ 2,000 cost reduction and a 515% reduction in complications . in contrast , in patients with itdm , thresholds < 180 mg / dl would be harmful , although in this range outcomes were uncertain ( fig . in this study , we demonstrated a significant association of postoperative stress hyperglycemia with economic and clinical outcomes , which varies with the presence of diabetes and its treatment . in patients with diabetes treated with insulin , glucose levels below the generally recommended threshold of 180 mg / dl conversely , in patients without and with diabetes not treated with insulin , adverse outcomes were decreased or unchanged . in these patient groups , the association between glucose levels and outcomes was characterized by a dose - response relationship with increasing complication rates at higher glucose levels . the results of this study suggest that targeting a glucose range below the threshold of 180 mg / dl , an approach in line with current guidelines , may be harmful in patients with diabetes treated with insulin . there are several possible explanations for a differential response among patients with and without itdm . first , metabolic homeostasis is altered in the cells of patients with diabetes , particularly in itdm patients ( 27 ) . the appropriate concentration of glucose for survival under stress - related conditions may be much higher in patients with itdm to provide energy for wound repair and to recover from major physiological insults ( 28 ) . the long - term exposure to higher blood glucose levels could result in the downregulation of glucose transporters limiting the influx of glucose across the cellular membrane ( 29 ) . independent of glucose level , higher hba1c levels decreased the likelihood of complications in patients with diabetes , as assessed with our composite end point . although it has been shown that higher hba1c values may decrease the risk of atrial fibrillation ( 30 ) and mortality in patients with acute hyperglycemia ( 31 ) , we found only a nonsignificant trend for decreasing the risk of complications . the protective effect mentioned above and a decreased risk of hypoglycemic events could be an explanation for this phenomenon . there is accumulating evidence that patients without a previous diagnosis of diabetes face a worse prognosis than patients with diabetes when stress hyperglycemia occurs ( 15,32 ) . in a large retrospective analysis ( 33 ) , the association between mortality risk and hyperglycemia in critically ill patients without diabetes was significantly stronger than in patients with diabetes . more recently , an increased risk of postoperative adverse events , such as myocardial infarction , stroke , transient ischemic attack , infectious complication , and renal insufficiency , was linked to hyperglycemia in patients without diabetes , but not in patients with diabetes ( 34 ) . in contrast with our findings , we found earlier reports ( 6,35,36 ) showing that lower glucose levels in patients with diabetes were linked to lower mortality and lower infection rates . however , the mode of insulin delivery in these earlier studies may not be directly comparable to the current glucose management practice . moreover , in these studies , previous treatment of diabetes was not considered , and results could have been driven by a majority of patients not previously being treated with an insulin regimen . randomized trials comparing intensive insulin therapy to more moderate approaches have reported mixed results ( 37 ) . such persisting discrepancies in the effects of intensive insulin therapy likely result from the high variability existing across centers in the multiple components of glucose control ( i.e. , monitoring , feeding , and glucose targets ) and the changes in standard practice that have occurred over time . these historic changes have modified the difference in insulin protocol between the intensive treatment and the control group in randomized clinical trials . however , in randomized controlled trials that did report a survival benefit from tight glycemic control , patients with diabetes have been the exception , demonstrating no benefit from the intervention ( 10,38 ) . our data expand this body of evidence in two crucial aspects . by considering the type of prior diabetes treatment , we show that the association between glycemic control and outcomes is different for those patients with a history of insulin therapy compared with patients without diabetes and patients with nitdm , after adjustment for baseline clinical variables . in addition , we report the economic burden associated with stress hyperglycemia for patients with and without diabetes . however , our analysis was based on an earlier , observational study ( 20 ) whose original aim was to evaluate the incidence of hospital - acquired infections after cardiac surgery , and therefore a number of limitations need to be mentioned . consequently , patients with undiagnosed diabetes may have been misclassified as not having diabetes , and for these patients diabetes control before hospital admission could not be assessed . studies ( 39 ) have reported a 10% rate of latent diabetes in icu patients with hyperglycemia . however , such misclassification , if present , would not affect the findings related to the group of patients with diabetes . second , insulin usage and parenteral nutrition protocols differed among centers , and data on adherence to these protocols were not collected . therefore , we could not include this information in our analysis . on the other hand , we adjusted for the standard insulin protocol used in the icus of the study centers , which reflects to some extent the differences in glucose management among different icus . third , the glucose measurements data , which were only collected during the 48 h after surgery ( at 6-h intervals ) , likely did not capture all the measurements that were performed in the icu after the surgery . however , such a limited sampling is unlikely to have biased the results differentially for patients with no diabetes , nitdm , and itdm . fourth , we used icd-9 codes to define complications other than infection , which did not allow us to evaluate the sequence of events that occurred , limiting conclusions about causal pathways . nevertheless , the consistency in the associations of glucose across economic and clinical outcomes further supports our conclusions . although we adjusted for a broad range of demographic characteristics and illness - related confounders , residual confounding may still have biased our findings to some extent . as is the case for observational studies , our observations only support an association between postoperative glucose levels and clinical outcomes , and not a causal relationship . likewise , we can not infer from our results whether previous insulin therapy has a causative role or is merely a marker of severity of illness associated with differential outcomes . the pandemic of diabetes calls for improved management of hyperglycemia , both outside and inside the hospital . roughly 20% of cardiac surgery patients have pre - existing diabetes , with a large proportion having more advanced disease requiring insulin therapy . more than 60% of these patients have at least one blood glucose measurement > 180 mg / dl , the glycemic threshold recommended by current clinical guidelines ( 6,40 ) . in the context of findings by others , our results support conducting a randomized controlled trial to evaluate a stratified approach to glucose control based on diabetes history and prior use of insulin . in summary , our findings suggest that current recommendations , which use a single maximum glucose threshold for the control of stress hyperglycemia after cardiac surgery , may not achieve the intended benefits in all patient subgroups . such a blanket approach could instead be harmful to patients with more advanced diabetes . given the substantial clinical and economic benefits that may be attained , patient stratification with indicators of chronic glucose dysregulation should be investigated .

objectivethe management of postoperative hyperglycemia is controversial and generally does not take into account pre - existing diabetes . we analyzed clinical and economic outcomes associated with postoperative hyperglycemia in cardiac surgery patients , stratifying by diabetes status.research design and methodsmulticenter cohort study in 4,316 cardiac surgery patients operated on in 2010 . glucose was measured at 6-h intervals for 48 h postoperatively . outcomes included cost , hospital length of stay ( los ) , cardiac and respiratory complications , major infections , and death . associations between maximum glucose levels and outcomes were assessed with multivariable regression and recycled prediction analyses.resultsin patients without diabetes , increasing glucose levels were associated with a gradual worsening of outcomes . in these patients , hyperglycemia ( 180 mg / dl ) was associated with an additional cost of $ 3,192 ( 95% ci 1,972 to 4,456 ) , an additional hospital los of 0.8 days ( 0.4 to 1.3 ) , an increase in infections of 1.6% ( 0.5 to 2.8 ) , and an increase in respiratory complications of 2.6% ( 0.0 to 5.3 ) . however , among patients with insulin - treated diabetes , optimal outcomes were associated with glucose levels considered to be hyperglycemic ( 180 to 240 mg / dl ) . this level of hyperglycemia was associated with cost reductions of $ 6,225 ( 12,886 to 222 ) , hospital los reductions of 1.6 days ( 3.7 to 0.4 ) , infection reductions of 4.1% ( 9.1 to 0.0 ) , and reductions in respiratory complication of 12.5% ( 22.4 to 3.0 ) . in patients with non insulin - treated diabetes , outcomes did not differ significantly when hyperglycemia was present.conclusionsglucose levels < 180 mg / dl are associated with better outcomes in most patients , but worse outcomes in patients with diabetes with a history of prior insulin use . these findings support further investigation of a stratified approach to the management of patients with stress - induced postoperative hyperglycemia based on prior diabetes status .

Introduction Research Design and Methods Study Population Clinical and Economic Variables Literature Review Statistical Analysis Results Study Population Outcomes by Diabetes Status Impact of Glucose Thresholds Conclusions Supplementary Material

gastroesophageal reflux disease ( gerd ) is a common gastrointestinal disease in asia as well as in the west ( 1 , 2 ) . studies have reported that 10%-20% of the adult western population experience typical gerd symptoms ( heartburn and/or regurgitation ) at least once per week ( 1 ) . the prevalence of gerd in adult koreans is reported to be 3.5%-8.5% ( 3 - 5 ) . this increase has been possibly attributed to changes in diet , an increasing aged population , the increasing frequency of endoscopic examinations , and the widely spreading knowledge on gerd . the montreal definition describes gerd as a condition that develops when the reflux of gastric contents causes troublesome symptoms and/or complications ( 6 ) . a number of regional and international guidelines and recommendations for the management of gerd have been published . the genval workshop report on reflux disease management , published in 1999 ( 7 ) , and the asia - pacific consensus on the management of gerd , published in 2008 ( 8) , previous studies show that proton pump inhibitors ( ppis ) are the most effective drug for patients with gerd ( 7 , 8) . actually , ppis are known to be most commonly prescribed for the treatment of gerd . however , the prescription pattern of ppis in patients with gerd may be different between countries , which is partly attributed to the economic situation or the health insurance system . in korea , national health insurance covers the cost for the medication prescribed in hospitals and clinics according to the insurance reimbursement guideline . accordingly , the prescription pattern of ppis used for the treatment of gerd is substantially influenced by the insurance reimbursement guideline . thus , the aim of this study was to document practice pattern of gastroenterologists for the management of gerd patients under the minimal influence of the insurance reimbursement guideline . fifty - six gastroenterologists at 51 secondary and tertiary care hospitals representing most regions of korea participated in the study . all participating gastroenterologists were required to recruit about 20 consecutive eligible patients who complained of typical gerd symptoms ( heartburn and/or regurgitation ) . patients were eligible for inclusion in this study if they were aged 18 yr and had typical gerd symptoms ( heartburn and/or regurgitation ) at least once per week over the previous 7 days with or without esophagitis . patients were excluded from participation if they had taking ppis or histamine 2 receptor blocking agents within the previous 4 weeks . the other exclusions were hypersensitivity to the active ingredient of ppis and the presence of organic diseases such as gastrointestinal cancer , severe liver disease , pancreatic disease , peptic ulcer , inflammatory bowel disease , severe renal impairment , or severe respiratory disease . thus , we tried to reduce the influence of the study on the routine clinical practice for enrolled patients . the only thing that was affected by the study protocol was to choose a ppi . in order to minimize the influence of the insurance reimbursement guideline on the use of ppis , we recommended the use of rabeprazole ( pariet , janssen korea ltd . , seoul , korea ) for patients in whom ppi treatment should be considered for the management of gerd symptoms , because rabeprazole is the only ppi of which both full - dose and half - dose are permitted for the initial and maintenance treatment of gerd , irrespective of the presence of erosive esophagitis , in the national insurance reimbursement guideline . the others associated with the treatment for patients enrolled in the study had not been set by the study protocol . , gastroenterologists followed their usual practice of patient care ; the severity of symptoms and medications prescribed were recorded at each visit , using a web - based electronic data capture system . at the visit for inclusion , demographic data and information on the patient 's symptoms , previous investigations for gerd , and treatment received were documented . in addition , the frequency ( days / week ) and severity ( 0 , none ; 1 , mild ; 2 , moderate ; 3 , severe ) of symptoms over the previous 7 days were assessed . when esophagogastroduodenoscopy ( egd ) or 24-hr esophageal ph monitoring had been performed for the evaluation of gerd symptoms , its results were reviewed . since this study was not a clinical trial , there was no hypothesis for the determination of the adequate sample size . nonetheless , a sample size of 1,120 patients ( 20 patients per a gastroenterologist ) was planned . a subgroup analysis was carried out using the chi - squared test and student 's t - test . the statistical analysis was performed using spss for windows version 11 ( spss inc . , the study was approved by each of the institutional review boards of the hospitals where the principal investigator ( approval number : 4 - 2008 - 0201 ) and other investigators belonged . each patient received information on the study design such as data handling and aims of the study . this study was an observational study and designed to have as little impact as possible on the management of patients . the study was approved by each of the institutional review boards of the hospitals where the principal investigator ( approval number : 4 - 2008 - 0201 ) and other investigators belonged . each patient received information on the study design such as data handling and aims of the study . this study was an observational study and designed to have as little impact as possible on the management of patients . fifty - six gastroenterologists at 32 secondary and 19 tertiary care hospitals participated in the study . a total of 1,197 patients ( 523 males , 44% ) of mean age 53.1 ( 13.6 ) yr were recruited . fifty - seven percent of them were enrolled at secondary care hospitals and the others ( 43% ) at tertiary care hospitals . at the time of enrollment , 479 patients ( 40% ) had previous egd results and 382 patients ( 32% ) underwent egd before the start of medications . thirty - six percent of those who had egd results showed erosive esophagitis , and 3% had barrett 's esophagus . at the time of enrollment , 6 patients had previous results of 24-hr esophageal ph monitoring . seven patients underwent 24-hr esophageal ph monitoring during the evaluation period before the start of medications , all of whom had atypical gerd symptoms and history of previous treatment for gerd symptoms . baseline data and symptoms on patients enrolled in the study are shown in table 1 and 2 , respectively . the most common accompanying symptom was chest pain , followed by postprandial fullness , globus , belching , nausea , abdominal pain , early satiety , sore throat , chronic cough , hoarseness , dysphagia , vomiting , and odynophagia in the order of prevalence . during the 16-week study period , the mean total number of visits per patient was 3.4 ( 1.4 , ranged 1 - 6 ) . the total duration of ppi treatment during the study period was 2 weeks in 2% , > 2 weeks and 4 weeks in 9% , > 4 weeks and 8 weeks in 10% , > 8 weeks and 12 weeks in 5% , and > 12 weeks and 16 weeks in 74% . the total number of visits and the total duration of ppi treatment did not significantly differ between patients with and without erosive esophagitis ( table 3 ) . at the start of treatment , gastroenterologists prescribed rabeprazole 20 mg ( full - dose ) daily to 94% of the patients who received ppi treatment and rabeprazole 10 mg ( half - dose ) daily to the remaining 6% . at the second visits , rabeprazole 20 mg daily was prescribed to 89% of those who were followed , and 10 mg daily to the remainder . at the third visit , the participating gastroenterologists prescribed rabeprazole 20 mg daily to 70% of those who were followed and 10 mg daily for the remaining 30% . during ppi treatment , prokinetics , mucosal protectives , antacids and histamine 2 receptor blocking agents were concomitantly used in 31% , 5% , 4% , and 1% of the patients , respectively . fifty - six gastroenterologists at 32 secondary and 19 tertiary care hospitals participated in the study . a total of 1,197 patients ( 523 males , 44% ) of mean age 53.1 ( 13.6 ) yr were recruited . fifty - seven percent of them were enrolled at secondary care hospitals and the others ( 43% ) at tertiary care hospitals . at the time of enrollment , 479 patients ( 40% ) had previous egd results and 382 patients ( 32% ) underwent egd before the start of medications . thirty - six percent of those who had egd results showed erosive esophagitis , and 3% had barrett 's esophagus . at the time of enrollment , 6 patients had previous results of 24-hr esophageal ph monitoring . seven patients underwent 24-hr esophageal ph monitoring during the evaluation period before the start of medications , all of whom had atypical gerd symptoms and history of previous treatment for gerd symptoms . baseline data and symptoms on patients enrolled in the study are shown in table 1 and 2 , respectively . the most common accompanying symptom was chest pain , followed by postprandial fullness , globus , belching , nausea , abdominal pain , early satiety , sore throat , chronic cough , hoarseness , dysphagia , vomiting , and odynophagia in the order of prevalence . during the 16-week study period , the mean total number of visits per patient was 3.4 ( 1.4 , ranged 1 - 6 ) . the total duration of ppi treatment during the study period was 2 weeks in 2% , > 2 weeks and 4 weeks in 9% , > 4 weeks and 8 weeks in 10% , > 8 weeks and 12 weeks in 5% , and > 12 weeks and 16 weeks in 74% . the total number of visits and the total duration of ppi treatment did not significantly differ between patients with and without erosive esophagitis ( table 3 ) . at the start of treatment , gastroenterologists prescribed rabeprazole 20 mg ( full - dose ) daily to 94% of the patients who received ppi treatment and rabeprazole 10 mg ( half - dose ) daily to the remaining 6% . at the second visits , rabeprazole 20 mg daily was prescribed to 89% of those who were followed , and 10 mg daily to the remainder . at the third visit , the participating gastroenterologists prescribed rabeprazole 20 mg daily to 70% of those who were followed and 10 mg daily for the remaining 30% . during ppi treatment , prokinetics , mucosal protectives , antacids and histamine 2 receptor blocking agents were concomitantly used in 31% , 5% , 4% , and 1% of the patients , respectively . several international guidelines for the management of gerd are reported ( 6 - 8 ) , but no domestic guidelines on clinical practice for patients with gerd have been issued yet in korea . in the present study , we followed patients with typical gerd symptoms for 16 weeks , and observed gastroenterologists ' treatment patterns . the current study showed that ppis are prescribed for the majority of patients with typical gerd symptoms and that korean gastroenterologists preferred a full - dose ppi for both initial and maintenance treatment of gerd , irrespective of the presence of erosive esophagitis , rather than a half - dose ppi . primary care clinics were excluded , because the aim of the study was to document gastroenterologists ' treatment patterns . patients with typical gerd symptoms who visit secondary and tertiary care hospitals participating in the study are treated by gastroenterologists . the institutions involved were selected on a nationwide scale in consideration of the number of regional population . egd is relatively cheap in korea because its cost is covered by national medical insurance . seventy - two percent of patients enrolled in the study had the results of egd . erosive esophagitis was observed in 36% of them , the majority of which was of grade la - a or la - b . longitudinal studies conducted in asia show an increase in the prevalence of gerd symptoms and reflux esophagitis ( 9 - 11 ) . the ratio of erosive reflux disease ( erd ) to nonerosive reflux disease ( nerd ) in asia is believed to be lower than in the west ( 12 ) . the ratio of erd found in the present study was 36% , which seems to be getting comparable to that reported in the west . this may be partly attributed to the fact that the participating institutions were secondary and tertiary care hospitals . since studies on the sensitivity and specificity of heartburn for predicting the presence of gerd are lacking in korea , it is uncertain whether heartburn is a definite marker of gerd in the korean population . functional heartburn is believed to be a different entity from nerd , particularly in terms of acid reflux patterns and the responsiveness to ppis ( 13 ) . functional heartburn can be diagnosed when there are no abnormal acid or nonacid esophageal reflux , no symptom correlation with reflux , and no response to ppis ( 14 ) . abnormal acid or nonacid reflux can be determined using a 24-hr esophageal ph / impedance monitoring . esophageal ph monitoring was available in 28 institutions ( 55% of the participating institutions ) . however , only 1% of the study patients underwent 24-hr ambulatory esophageal ph monitoring , which suggests that this test is not commonly performed even at secondary and tertiary care hospitals . in general , ppi trial is recommended to be preceding esophageal ph monitoring , particularly in patients with typical gerd symptoms . in the present study , ambulatory esophageal ph monitoring was performed mainly in patients with atypical gerd symptoms and history of previous ppi treatment for gerd symptoms , which is in keeping with the current guidelines . the asian - pacific experts for gerd reported a general consensus that ppis provide the most effective treatment for erd and nerd ( 8) . the consensus is based on previous studies showing that ppis are superior to histamine 2 receptor blocking agents in terms of healing esophageal mucosa and relieving heartburn symptoms in patients with gerd ( 15 - 17 ) . in the present study , most of patients enrolled in the study received ppi treatment ( 87% of the study patients ) . prokinetics were the most commonly used concomitant medications , which can be possibly supported by the positive value of prokinetics in the treatment of gerd ( 18 , 19 ) and substantial overlap between gerd symptoms and dyspepsia ( 5 , 20 ) . our findings of the present study that 27% and 15% of the patients with typical gerd symptoms had postprandial fullness and early satiety , respectively , demonstrate overlap between gerd symptoms and dyspepsia . the asian - pacific consensus includes statements on the duration of ppi treatment for erd and nerd as follows ; " nerd patients require more than 4 weeks of initial continuous ppi therapy and erd patients need a minimum of 4 - 8 weeks of initial continuous ppi therapy " ( 8) . it is difficult to determine timing to change initial treatment to maintenance treatment . according to the guidelines , maintenance treatment usually begins after 4 weeks of initial treatment in nerd patients and after 8 weeks of initial treatment in patients with erd . the influence of national health insurance system on the use of drugs is too big in korea , because it covers all patients visiting hospitals due to gerd symptoms . based on the national health insurance reimbursement guideline , full - dose of most ppis , except rabeprazole , is permitted only for the initial treatment of erd , but not for the initial treatment of nerd and the maintenance treatment of erd and nerd . according to that guideline , only half - dose ppi can be used for the initial treatment in patients with gerd symptoms who have no endoscopic findings and in nerd patients , and for the maintenance treatment of gerd . rabeprazole is the only ppi of which both full - dose and half - dose are permitted for the initial and maintenance treatment of erd and nerd under the korean health insurance system . so , in order to reduce distortion or variability in the clinical practice for gerd patients due to the national health insurance reimbursement guideline , we made a decision to recommend rabeprazole when ppi treatment is required for the enrolled patients . since our main aims were to investigate the prescription pattern on the dosage of ppis used for the treatment of gerd and to know whether korean gastroenterologists keep the international guideline for the treatment of gerd , we tried to reduce the influence of the national health insurance reimbursement guideline and the study protocol . thus , treatment for patients enrolled in the study had not been set by the study protocol , and participating gastroenterologists carried out their practice at their own decision . given that rabeprazole 20 mg daily was used as initial treatment in 94% of the patients who received ppi treatment , the majority of korean gastroenterologists appear to prefer a full - dose ppi for the initial treatment of gerd , irrespective of the presence of erosive esophagitis . gastroenterologists participating in this study tended to use a full - dose ppi without step - down of the dose . these points are not in keeping with the insurance reimbursement criteria , which might be attributed to a high proportion of gerd patients requiring a long - term treatment of a fulldose ppi . in the present study , the mean duration of continuous ppi therapy during the 16-week observational period was 96 and 95 days in patients with and without erosive esophagitis , respectively . in the present study , 62% of the study patients were continuously treated with ppis until the end of the study period . participating gastroenterologists were allowed to prescribe only rabeprazole , either 10 mg or 20 mg , if they want to use ppi . rabeprazole is the only ppi that both dosages are permitted to be used in the maintenance therapy according to the reimbursement guideline . accordingly , they appear to choose the ppi dosage mainly based on the treatment efficacy . if other ppis would have selected for this study , they might have prescribed according to the reimbursement guideline , irrespective of the treatment efficacy . one of our aims was to investigate drug compliance , but we found that drug compliance was impossible to investigate , because many patients were not followed during the study period . since this was an observational study , follow - up was not controlled by the study protocol . so , follow - up loss was not prevented , and drug compliance could not be investigated . according to the study design , participating gastroenterologists were able to make a decision for tests and treatment for the enrolled patients at their discretion . thus , our findings suggest that many korean patients with typical gerd symptoms need a long - term treatment of a full - dose ppi . in conclusion , endoscopy is commonly performed for the evaluation of gerd symptoms , but 24-hr esophageal ph monitoring is not . a full - dose ppi is preferred for the initial and maintenance treatment of gerd under the minimal influence of the insurance reimbursement guideline , which may reflect a high proportion of gerd patients requiring a long - term treatment of a full - dose ppi . practice pattern of gastroenterologists for the management of gerd under the minimal influence of the insurance reimbursement guideline : a multicenter prospective observational study kwang jae lee , jin il kim , ju sang park , byung sik moon , sang - gyun kim , jae hee chun , hoon - yong jung , chang hwan choi , seong woo chun , geun am song , myung gyu choi and hoon jai chun we studied practice pattern of gastroenterologists for the management of gerd under the minimal influence of the insurance reimbursement guideline in korea . a full - dose proton pump inhibitor ( ppi ) is preferred for the initial and maintenance treatment of gerd , which may reflect a high proportion of gerd patients requiring a long - term treatment of a full - dose ppi .

the objective of the study was to document practice pattern of gastroenterologists for the management of gastroesophageal reflux disease ( gerd ) under the minimal influence of the insurance reimbursement guideline . data on management for 1,197 consecutive patients with typical gerd symptoms were prospectively collected during 16 weeks . in order to minimize the influence of reimbursement guideline on the use of proton pump inhibitors ( ppis ) , rabeprazole was used for the ppi treatment . a total of 861 patients ( 72% ) underwent endoscopy before the start of treatment . ppis were most commonly prescribed ( 87% ) . at the start of treatment , rabeprazole 20 mg daily was prescribed to 94% of the patients who received ppi treatment and 10 mg daily to the remaining 6% . at the third visits , rabeprazole 20 mg daily was prescribed to 70% of those who were followed and 10 mg daily for the remaining 30% . continuous ppi treatment during the 16-week period was performed in 63% of the study patients . in conclusion , a full - dose ppi is preferred for the initial and maintenance treatment of gerd under the minimal influence of the insurance reimbursement guideline , which may reflect a high proportion of gerd patients requiring a long - term treatment of a full - dose ppi .

INTRODUCTION MATERIALS AND METHODS Ethics statement RESULTS Baseline data Treatment patterns DISCUSSION AUTHOR SUMMARY

a case report of a 56-year - old male farmer who sought medical attention after a month - long evolution of irritative symptoms in his right eye , accompanied by visual acuity ( va ) impairment . the patient received topical and oral broad - spectrum antibiotic treatment with no improvement before being referred to a cornea specialist , where he was found to have va of 20/150 and was noted on biomicroscopy to have endothelial feathery coalescent lesions . the patient was admitted to the hospital for an aqueous humor sample and intravenous voriconazole . however , clinical evidence of improvement was confirmed after 5 days of antimycotic intravenous therapy . complete clinical resolution was achieved at 1 month after treatment completion with oral voriconazole , as evidenced by va of 20/20 and disappearance of endothelial lesions . endothelial involvement by fungi is a rare condition . in this case , no microbes were isolated , but the characteristic morphology of the lesions , the history of onychomycosis , and the spectacular response to voriconazole turn this case into a valid presumptive diagnosis . the cornea is the ocular structure most commonly affected by fungi . most often , the source of infection is exogenous , such as vegetal trauma , among others . in addition , it is associated with any disease affecting the eye s normal surface , such as persistent epithelial defects , neurotrophic ulcers , use of contact lenses , and secondary attenuation of cell defense mechanisms after topical steroids.15 infectious endothelial involvement of the cornea has been mainly attributed to different viral species.6 however , herein we present the case of a patient with a clear endothelial infectious involvement that successfully responded to systemic antifungal therapy with voriconazole . a 56-year - old male banana grower sought medical attention after a month - long evolution of irritative symptoms in his right eye accompanied by visual acuity ( va ) impairment . his medical history confirmed excision of bilateral pterygium 15 years earlier , but no recent ocular trauma was related by the patient . the general medical practitioner ( nonophthalologist ) initiated treatment with topical prednisolone acetate 1% and gentamicin sulfate 0.3% every 6 hours for 1 week for a presumed bacterial conjunctivitis , but the patient was referred to the ophthalmologist after lack of success with this therapy . an initial diagnosis of infectious keratitis ( with unaffected epithelium but endothelial involvement ) was made , and treatment with topical moxifloxacin chlorhydrate 0.5% four times per day and gentamicin sulfate 0.3% three times per day plus oral ciprofoxacin 750 mg every 12 hours was prescribed . after 6 days on this therapeutic regimen and with lack of clinical improvement , the patient was referred to a cornea specialist . he was found with right eye va of 20/150 and normal va in his left eye . the biomicroscopy performed on the affected eye confirmed the integrity of the epithelium with an unruptured bulla over the pupillary area , presence of folds on descemet s membrane , anterior chamber cells 2+(standardization of uveitis nomenclature working group),7 and multiple coalescent circular lesions of feathery and whitish appearance on the endothelium ( figure 1a ) . fundoscopic examination with an indirect ophthalmoscope under midriasis was normal . based on these findings , therefore , the patient was admitted to hospital to further explore the etiology of the lesion . for this , an aqueous humor sample was examined for fungi detection by culture and polymerase chain reaction ( pcr ) , and systemic antimycotic therapy was initiated immediately after sample taking . by recommendation of the infectologist , the patient was empirically treated with voriconazole 200 mg intravenously ( iv ) every 12 hours ( diluted in 250 ml of 0.9% saline solution administrated over 2 hours ) and topical gatifloxacin 0.3% every 6 hours for 1 week ( as post puncture antibacterial prophylaxis ) . after 3 days on iv therapy with voriconazole , the patient showed significant clinical improvement , evidenced by an important decline in the number of cells within the anterior chamber and some reduction on the feathery endothelial lesions described previously . aqueous humor microbiologic study was negative ( gram , potassium hydroxide staining , aerobic , anaerobic , and fungal cultures ) . pcr , venereal disease research laboratory , herpes simplex , and hiv testing were also negative . after 5 days on this regimen , the patient was discharged with voriconazole 200 mg orally every 12 hours . after a week , the patient was found to have va of 20/30 , considerable improvement of endothelial lesions , and faint residual corneal edema . the same treatment regimen was administered for a further week , and fluorometholone 0.1% every 12 hours was added to therapy for edema . examination 2 weeks after discharge confirmed further edema reduction and almost complete resolution of endothelial lesions . after 4 weeks of antifungal treatment ( 3 weeks of those were as an outpatient ) , the patient showed full edema resolution , the presence of a few small endothelial scars , and 20/20 va ( figure 1c ) . fluorometholone was then withheld and oral voriconazole was continued until completion of 2 months with antifungal therapy . infectious endothelial involvement of the cornea has been mainly attributed to different viral species.6 however , we report an interesting case of a patient whose endothelial lesions suggest mycotic etiology that successfully responded to systemic antifungal therapy with voriconazole . usual manifestations of fungal keratitis include epithelial and stromal ulceration , abscessing with stromal necrosis , epithelial or stromal edema and , rarely , cell reaction in the anterior chamber , hypopyon , and fibrin deposits within the anterior chamber and endothelial plates.1,5 nevertheless , the most striking finding in this case was the confinement of damage to endothelial plates with subsequent central corneal edema , bulla formation , folds on descemet s membrane , and cell reaction in the anterior chamber . therefore , we propose an endogenous source of infection , and postulate the presence of recurrent onychomycosis as a risk factor in the absence of prior trauma or epithelial disruption . however , microbiological isolates turned out to be negative both in blood and in aqueous humor . a characteristic clinical sign of fungal keratitis is the irregular nature of infiltrates , traditionally described as feathery or cotton wool.1,4,8 this feature prompted the suspicion of fungal origin in our patient , as this pattern was observed on the endothelium . although gram and potassium hydroxide staining , as well as pcr , were negative , our diagnosis was established on clinical grounds and confirmed by the success of empirical therapy with voriconazole . it is important to point out that laboratory diagnosis in this case has been limited by the low volume of sample taken , which was destined for multiple studies ( stains , cultures , pcr ) . in addition , experience with these tests and available primers in our country are also limited . some authors have reported on the good efficacy of intracameral injection with amphotericin b and voriconazole in cases of endophthalmitis secondary to keratitis.911 although this is an adequate therapeutic alternative , it was not required , as the patient showed an excellent response to systemic therapy with oral voriconazole . an interesting case of a presumed endogenous mycotic endotheliitis is herein reported in a patient with a history of recurrent onychomycosis who develops endothelial lesions of mycotic morphology . the patient responds to a systemic iv and oral scheme with voriconazole , and thus an endogenous and mycotic etiology is presumed . however , this could not be confirmed by microbiological studies , due to the limitations that we have cited .

purposeto report an interesting case of infectious endotheliitis of presumed mycotic origin.methodsa case report of a 56-year - old male farmer who sought medical attention after a month - long evolution of irritative symptoms in his right eye , accompanied by visual acuity ( va ) impairment . the patient received topical and oral broad - spectrum antibiotic treatment with no improvement before being referred to a cornea specialist , where he was found to have va of 20/150 and was noted on biomicroscopy to have endothelial feathery coalescent lesions . the patient was admitted to the hospital for an aqueous humor sample and intravenous voriconazole.resultsthe microbiological studies did not isolate any micro - organisms . however , clinical evidence of improvement was confirmed after 5 days of antimycotic intravenous therapy . complete clinical resolution was achieved at 1 month after treatment completion with oral voriconazole , as evidenced by va of 20/20 and disappearance of endothelial lesions.conclusionendothelial involvement by fungi is a rare condition . in this case , no microbes were isolated , but the characteristic morphology of the lesions , the history of onychomycosis , and the spectacular response to voriconazole turn this case into a valid presumptive diagnosis .

Purpose Methods Results Conclusion Background Case description Discussion Conclusion

the experimental animals , feeding and management : the experimental procedures complied with the guide for the care and use of agricultural animals of obihiro university . the experiment was carried out at the field center of animal science and agriculture , obihiro university of agriculture and veterinary medicine . fifty multiparous holstein cows , in which parity was from 1st to 5th at dry period , were used in this study and had calved between september 2011 and august 2012 . parity and body condition score ( bcs ) of the experimental cows at initiation of the study were 2.4 0.2 and 3.41 0.04 , respectively . the study was performed from 3 weeks before the expected parturition to 100 days pp . cows close to the dry period , about 1 month before the expected calving date , were moved to a paddock and fed a limited total mixed ration [ dry matter ( dm ) basis : 127 g of crude protein ( cp)/kg and 6.6 mj of net energy for lactation ( nel)/kg ] consisting of grass silage ( 3.5 kg , dm basis : 165 g of cp / kg and 5.5 mj of nel / kg ) , maize silage ( 5.1 kg , dm basis : 86 g of cp / kg and 6.0 mj of nel / kg ) , concentrate for dry cows ( 2.0 kg , dm basis : 170 g of cp / kg and 6.8 mj of nel / kg ) and grass hay ( dm basis : 125 g of cp / kg and 5.7 mj of nel / kg ) ad libitum until parturition . after parturition , cows were housed in a free - stall barn and fed a lactation diet , which was a mixed ration ( dm basis : 155 g of cp / kg and 6.2 mj of nel / kg ) consisting of grass ( 6.5 kg , dm basis : 165 g of cp / kg and 5.5 mj of nel / kg ) , maize silage ( 12.5 kg , dm basis : 84 g of cp / kg and 6.4 mj of nel / kg ) and concentrate for dairy cows ( 8.0 kg , dm basis : 180 g of cp / kg and 7.1 mj of nel / kg ) ad libitum . in addition , the diets were supplemented with minerals , and the dairy cow concentrate was prepared according to each cow s specific requirements for milk production . grass hay ( dm basis : 104 g of cp / kg and 5.2 mj of nel / kg ) and water were available ad libitum . cows were milked twice daily between 05:00 and 06:30 hr and between 17:00 and 18:30 hr . the experimental itt and sampling : the itt was performed 3 weeks before the expected calving date . the cows were weighed the day before the initiation of the itt , and bw was used to determine the doses of insulin for the itt . immediately before the itt , an extension catheter was inserted into the right or left jugular vein . the itt was performed by intravenously administering 0.05 iu / kg bw of insulin ( novolin r 100 iu / ml ; novo nordisk pharma , tokyo , japan ) , followed by administration of 5 ml heparinized saline ( 100 iu / ml ) . blood samples were collected via the jugular vein at 0 ( before insulin injection ) , 30 , 45 and 60 min relative to the administration of insulin via caudal venipuncture to measure glucose and insulin . bcs was assessed twice a week from 3 weeks before the expected parturition to 3 weeks after calving by the same operator by using a 1 to 5 scale with 0.25 intervals , where 1=thin and 5=very fat . blood samples were obtained by caudal venipuncture twice a week from 3 weeks before the expected parturition to 3 weeks after calving . blood samples were collected via the jugular vein from the calves immediately after birth . nonheparinized and silicone - coated 9-ml tubes ( venoject , autosep , gel + clot . act . vp - as109k ; terumo corporation , tokyo , japan ) were used for biochemical analysis , and sterile 10-ml tubes containing 200 l of stabilizer solution ( 0.3 m edta and 1% acetyl salicylic acid , ph 7.4 ) were used for hormonal analysis . serum was obtained by centrifuging the blood samples for 15 min at 38c in an incubator . all the tubes were centrifuged at 2,000 g for 20 min at 4c , and plasma samples were maintained at 30c until analysis . in addition , milk samples were collected twice a week after milking until the onset of luteal activity . the milk samples were centrifuged at 1,500 g for 15 min at 4c , and the skim milk samples were stored at 30c until analysis for progesterone concentration . daily milk yield was recorded until 100 days pp . peripartum diseases , such as milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , lameness , retained placenta , endometritis and mastitis , were recorded when that has been diagnosed from 3 weeks prepartum to 3 weeks postpartum by veterinarian in the experimental farm . the experimental measurement of hormones and metabolites : plasma and skim milk progesterone concentrations were determined using enzyme immunoassay ( eia ) after extraction with diethyl ether , as described previously ; the extraction efficiency was 90% . the standard curve ranged from 0.05 to 50 ng / ml , and the 50% effective dose ( ed50 ) of the assay was 0.66 ng / ml . the mean intra - assay and inter - assay coefficients of variation ( cvs ) were 6.0% and 9.2% , respectively . the total plasma insulin - like growth factor 1 ( igf-1 ) concentration was determined using eia by using the biotin streptavidin amplification technique after protein extraction by using acid ethanol ( 87.5% ethanol and 12.5% 2 n hydrochloric acid ) to obtain igf-1 free from binding proteins . intra- and inter - assay cvs were 5.9% and 6.1% , respectively , and the ed50 of this assay system was 7.2 ng / ml . the plasma gh concentrations were determined using eia as described previously ; the standard curve ranged from 0.78 to 100 ng / ml , and the ed50 was 21 ng / ml . intra- and inter - assay cvs were 3.1% and 8.2% , respectively . the plasma insulin concentrations were determined using an enzyme - linked immunosorbent assay ( elisa ) kit ( bovine insulin elisa 10 - 1201 - 01 ; mercodia , uppsala , sweden ) . the serum concentrations of glucose , non - esterified fatty acids ( nefa ) , -hydroxybutyrate ( bhba ) , total protein ( tp ) , albumin ( alb ) , blood urea nitrogen ( bun ) and total cholesterol ( t - cho ) and the activities of aspartate aminotransferase ( ast ) were measured using a clinical chemistry automated analyzer ( tba120fr ; toshiba medical systems co. , ltd . , tochigi , japan ) . the experimental identification of the onset of luteal activity : when the progesterone concentration in the plasma or skim milk had increased to more than 1 ng / ml , the cows were considered to show luteal activity . the experimental statistical analysis : sixteen cows were excluded from data analysis , because of the following reasons : a pregnancy period of more than 287 or less than 273 days ( n=6 ) , severe mastitis ( n=3 ) , twin calving ( n=1 ) , blood collection loss at itt ( n=3 ) and mistakes in insulin injection at itt ( n=3 ) . cows were divided into two groups based on the time required for glucose to reach the minimum levels after insulin injection . cows with a minimum glucose at 60 min after insulin injection were considered to have lower insulin sensitivity and/or lower glucose metabolism compared to cows with a minimum glucose level by 45 min after insulin injection . therefore , cows with a minimum glucose level at 60 min after insulin injection were defined as the insulin resistant group ( ir group ) , whereas those with a minimum glucose level by 45 min after insulin injection were defined as the non - insulin resistant ( nir group ) in this study . before data analysis , bcs , plasma igf-1 , gh and insulin concentrations , and serum metabolite concentrations were averaged weekly . the period of 06 days after calving was considered as the parturient week ( 0 week pp ) , and the kolmogorov smirnov test ( sas enterprise guide version 4.3 ; sas institute inc . , cary , nc , u.s.a . ) was used for statistical testing of normality . in addition , the data were analyzed separately for the prepartum and pp periods . stat view ( stat view 5.0 software ; abacus concepts inc . , berkeley , ca , u.s.a . ) was used for data analysis by using the repeated measures of the anova procedure , including time ( week ) , group ( nir or ir ) and their interaction in the model as fixed effects . diagnosis of peripartum diseases and sex of calves in the nir and ir groups were analyzed using the chi - square test , and other data , including results for calves between nir and ir , were analyzed using the student s t - test or wilcoxon s signed rank test ( sas enterprise guide version 4.3 ; sas institute inc . ) . results are presented as mean standard error of the mean ( sem ) ; differences with p<0.05 were considered significant . in 28 of the 34 experimental cows , the time required for glucose to reach the minimum level was 45 min after insulin injection with one exception ( 30 min ; n=1 , 45 min ; n=27 , nir group ) . the remaining experimental cows ( n=6 ) required 60 min after insulin injection to attain the minimum glucose levels ( ir group ) . serum glucose concentrations at 60 min after insulin injection were higher in the nir group than in the ir group , although glucose levels at the other time points did not differ between the nir and ir groups ( fig . 1.the change in serum glucose concentration after insulin injection at insulin tolerance test ( itt ) in the nir ( n=28 ) and ir ( n=6 ) groups . p<0.05 ) . the change in serum glucose concentration after insulin injection at insulin tolerance test ( itt ) in the nir ( n=28 ) and ir ( n=6 ) groups . table 1table 1.parity , calving difficulty , sex of calves , peripartum disease , luteal activity onset and milk yield in the nir and ir groupsnir groupir groupp - value(n=28)(n=6)parity at the onset of experiment2.4 0.32.2 0.70.460calving difficulty1.1 0.11.0 0.00.700sex of calves ( male / female)14/143/31.000diagnosis of peripartum disease9/28 ( 32%)1/6 ( 17%)0.645days to the onset of luteal activity ( days)38.3 3.820.3 3.60.039average of daily milk yield between days 7 and 100 pp ( kg)41.4 0.935.9 2.00.013total milk yield from days 7 to 100 pp ( kg)3,888.1 81.23,375.5 185.90.013values are the mean sem . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection . b ) 1 , unassisted birth ( natural , without human assistance ) ; 2 , easy calving with human assistance ; 3 , difficult calving with a few humans ; 4 , dystocia ( requiring considerably more force than normal ) ; and 5 , surgical treatment or death of cow . c ) milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , lameness , retained placenta , endometritis and mastitis from 3 weeks prepartum to 3 weeks postpartum . shows the parity , calving difficulty , sex of calves , peripartum disease diagnosis , luteal activity onset and milk yield until 100 days pp in the nir and ir groups . days until the onset of luteal activity in the ir group were fewer than those in the nir group ( p<0.05 ) . in addition , the average ( p<0.05 ) and total ( p<0.05 ) milk yields until 100 days pp were lower in the ir group than in the nir group . peripartum diseases were diagnosed as mastitis ( n=6 ) , hypocalcemia ( n=1 ) and milk fever ( n=2 ) in nir group , and as mastitis ( n=1 ) in ir group , and there was no significant difference in the number of cows with the peripartum diseases between nir and ir groups . no significant difference was noted in other factors between the nir and ir groups . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection . b ) 1 , unassisted birth ( natural , without human assistance ) ; 2 , easy calving with human assistance ; 3 , difficult calving with a few humans ; 4 , dystocia ( requiring considerably more force than normal ) ; and 5 , surgical treatment or death of cow . c ) milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , lameness , retained placenta , endometritis and mastitis from 3 weeks prepartum to 3 weeks postpartum . 2.serum metabolite concentrations , activities of enzymes and plasma metabolic hormones , and bcs during the experimental period [ mean sem : solid , nir ( n=28 ) ; open , ir ( n=6 ) groups ] . * indicates differences of p<0.05 , and indicates differences of p<0.1 between the nir and ir groups . shows the circulating serum metabolite concentrations , enzyme levels , plasma metabolic concentrations and bcs during the experimental period . during the prepartum period , bcs ( p<0.05 ) , and serum bun concentrations ( p<0.05 ) were lower , whereas serum glucose ( p=0.05 ) and alb concentrations ( p=0.10 ) tended to be lower in the ir group than in the nir group . during the pp period , cows of the nir group had higher serum nefa ( p<0.05 ) and bhba ( p=0.09 ) concentrations than those in the ir group . in addition , treatment and time effects were observed ( p<0.05 ) for bcs during the pp period : bcs at 0 ( p=0.08 ) and 1 ( p<0.05 ) week pp were lower in the ir group than in the nir group . no significant differences were noted in the other factors between the nir and ir groups in each period . serum metabolite concentrations , activities of enzymes and plasma metabolic hormones , and bcs during the experimental period [ mean sem : solid , nir ( n=28 ) ; open , ir ( n=6 ) groups ] . * indicates differences of p<0.05 , and indicates differences of p<0.1 between the nir and ir groups . bw , plasma metabolic hormone levels and serum glucose concentrations at birth in the calves of cows of the nir and ir groups are shown in table 2table 2.bw and plasma metabolic hormones and serum glucose concentrations at birth in the calves of the nir and ir groupscalves of nircalves of irp - value(n=28)(n=6)bw at the birth ( kg)47.2 0.942.1 1.70.020plasma gh concentration ( ng / ml)13.6 1.315.2 4.80.653plasma igf-1 concentration ( ng / ml)121.5 6.369.8 5.60.001plasma insulin concentration ( ng / ml)0.3 0.00.7 0.20.061serum glucose concentration ( mg / dl)77.4 5.272.1 14.80.684values are the mean sem . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection .. bw at birth in the calves of the ir group was lower than that in the calves of the nir group ( p<0.05 ) . furthermore , the calves of the ir group showed lower plasma igf-1 concentration ( p<0.001 ) and higher plasma insulin concentration ( p=0.06 ) . no significant differences were noted in the plasma gh and serum glucose levels at birth between the calves of the nir and ir groups . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection . in this study , the six cows that reached the minimum glucose levels at 60 min after insulin injection were considered to be ir ; the reason for ir was thought to be the slow recovery of glucose after insulin injection , which is consistent with the findings of a previous study by lee et al . . in general , bcs and blood glucose and bun concentrations are known to be associated with energy status and feed intake [ 7 , 8 , 38 ] . during the prepartum period , ir cows showed lower energy status and feed intake owing to the lower bcs and glucose and bun concentrations . although ir by itt was confirmed at 3 weeks before calving , a difference in energy status between ir and nir cows was noted . in particular , bcs can not be evaluated on the basis of the change in energy status of the real - time feed intake ; therefore , in this study , ir cows might have become insulin resistant during an earlier time . malnutrition causes imbalance in glucose homeostasis , and the decrease of insulin in circulation induces the reduction of feed intake in dairy cows . therefore , the feed intake reduction in ir cows might have inhibited the volatile fatty acid production in the rumen and thus suppressed gluconeogenesis in the liver . lower energy status , such as lower bcs , before calving of ir group in this study might be caused by long - term malnutrition from previous lactation . however , in this study , the reasons for the lower energy status in the ir cows were not clear ; thus , further studies are warranted to confirm the onset of insulin resistance in pregnant dairy cows . bcs at 0 and 1 week pp were lower in the ir group than in the nir group . nir cows showed higher serum nefa and bhba concentrations than those in ir cows , although the levels of metabolic hormones did not differ between the ir and nir cows . furthermore , the average and total milk yield until 100 days pp were lower in the ir group than in the nir group . higher nefa and bhba indicate greater mobilization of adipose tissue and failure of lipid metabolism in the liver [ 14 , 15 ] . however , cows with lower bcs had sustained reduced plasma nefa and bhba concentrations after calving compared to cows with higher bcs [ 28 , 33 ] . cows with lower bcs produce milk by protein mobilization , because of the limited body fat ; thus , fat - corrected milk yield in those cows was lower than moderate and fat cows . conversely , it was indicated that higher bcs cows have ability to mobilize fat to maintain energetic homeostasis after feed restriction . additionally , roche et al . have concluded that bcs at calving had positive effect on milk yield , and optimal bcs at calving was 3.5 in the 5-point scale . in the present study , greater bcs and better gluconeogenesis in nir group might produce greater milk yield compared with ir group , although the differences of them between nir and ir groups were not so greater . days to the onset of luteal activity in the ir group were fewer than in the nir group . in dairy cows , lowered energy status during the peripartum period is known to delay the first ovulation after parturition [ 2 , 19 ] . butler and smith showed that a negative energy balance was directly related to the pp interval to the first ovulation and that the differences in the energy balance were reflected in the milk yield . in addition , cows with a delayed first ovulation showed higher nefa and bhba concentrations after parturition [ 21 , 31 , 39 ] . therefore , in this study , higher nefa and bhba concentrations of nir cows during the pp period might have delayed the onset of luteal activity , and the lowered milk yield of ir cows might induce earlier resumption of ovarian activity . the maternal endocrine and metabolic milieu transferred through the placenta during late pregnancy affects the environment of the fetus [ 17 , 24 , 30 ] . in humans , ir of the mother is associated with low birth weight of the infant ; in cattle , maternal malnutrition during gestation is related to the lowered development of both the placenta and the fetus [ 24 , 30 ] . further , in ewes , restricted maternal feeding during gestation was related to lower bw and plasma igf-1 , insulin and glucose concentrations in the fetus , although maternal igf-1 concentrations were not affected . in the present study , calves of the ir cows showed lowered bw at birth and a lower plasma igf-1 concentration , supporting the findings of previous studies . in addition , they showed higher insulin levels than those of nir cows , despite the similar glucose levels . in the late gestation , fetal growth is mainly regulated by igf-1 , and the dominant regulator of igf-1 production in the fetus is fetal glucose and insulin . thus , the differences in blood metabolic hormones and glucose concentrations between the calves of the nir and ir groups might be attributed to the fetal nutritional condition that was affected by maternal endocrine and metabolic milieu . in humans , lower bw at birth is known to be associated with a wide range of adverse outcomes later in life , including diabetes ; further , obese children with low birth weight have higher blood insulin to glucose concentration and show higher insulin resistance as revealed by the homeostasis model assessment compared with obese children with normal birth weight . therefore , calves of ir cows might develop insulin resistance in the future . in conclusion , the findings of the present study suggest that ir at 3 weeks before parturition in dairy cows is related to the pp metabolic status , milk production and resumption of ovarian activity along with growth , as well as the metabolic status of their calves . therefore , ir evaluated on the basis of the recovery of glucose after an injection of a small dose of insulin during the dry period might be an indication of the pp performance of pregnant dairy cows , as well as the growth , fertility and milk production of their calves . in addition , the reason for ir in the present study was thought to be the slow recovery of glucose after insulin injection as well as the previous study . therefore , the enhancement of the gluconeogenesis in the liver by energy supplementation , such as glycerol , or hepatic stimulant , such as amino acids , should be confirmed in order to improve the ir .

this study aimed to investigate the effects of insulin resistance ( ir ) during the close - up dry period on the metabolic status and performance of dairy cows as well as to determine the effects on body weight ( bw ) and metabolic status of their calves . an insulin tolerance test ( itt ) was conducted by administering 0.05 iu / kg bw of insulin to 34 multiparous holstein cows at 3 weeks prepartum . blood samples were collected at 0 , 30 , 45 and 60 min after insulin injection , and cows were divided into two groups based on the time required for glucose to reach the minimum levels [ non - ir ( nir ) , 45 min ( n=28 ) ; and ir , 60 min ( n=6 ) ] . blood or milk sampling and body condition score ( bcs ) estimation were performed twice weekly during the experimental period . blood samples from calves were collected immediately after birth . cows with ir showed lower bcs ( p<0.05 ) and serum urea nitrogen ( p<0.05 ) and glucose concentration ( p=0.05 ) before calving , and lower serum non - esterified fatty acid concentration ( p<0.05 ) and milk yield ( p<0.05 ) and earlier resumption of luteal activity ( p<0.05 ) after calving ; their calves showed lower bw ( p<0.05 ) and plasma insulin - like growth factor - i concentration ( p<0.001 ) and higher plasma insulin concentration ( p<0.05 ) . in conclusion , ir at 3 weeks prepartum in dairy cows is related to postpartum metabolic status and performance along with growth and metabolic status of their calves .

MATERIALS AND METHODS RESULTS DISCUSSION

biodegradable block copolymers prepared from l - lactide , -caprolactone , 1,4-dioxan-2-one , and trimethylene carbonate have been synthesised and studied extensively during recent decades [ 1 , 2 ] . the varying physical properties of block polymers allows for the combination of soft and hard polymers giving rise to copolymers that can be tuned for specific function such as elasticity [ 3 , 4 ] . the sequence in which blocks are synthesised into block copolymers is specific and is determined by the choice of monomer and catalyst . for example , at the application of tin octoate as catalyst , the block copolymer structure polycaprolactone - polylactide ( pcl - pla ) is formed if the -caprolactone is polymerized first , followed by polymerization of l - lactide . however , a random copolymer is obtained when pla is initially synthesised followed by pcl [ 5 , 6 ] . this is a result of transesterification of the pla and segmentation of the block polymer . two competing reactions during carop ( coordinated anionic ring opening polymerisation ) occur ; ( i ) ring - opening of ester bonds in molecules of the initial cyclic monomer and ( ii ) cleavage of the ester bonds in the macromolecules of the polymer . these competing reactions depend on the choice of catalyst , the existing polymer , which acts as a macroinitiator , and the type of monomer added at the second stage of polymer synthesis . atering the catalyst utilized for example , y(cf3coo)3/al(iso - bu)3 catalyst or the complex [ y(l6)-{n(sihme2)2}(thf ) ] promotes the initial synthesis of the pla block , followed by the pcl block . showed that selectivity of al(oipr)3 catalyst could be accomplished through coordination with sb(oh)2 ( ( s)-()-2,20-[1,10-binaphtyl-2,20-diylbis(nitrylomethilidyne)]diphenol ) , allowing synthesis of the block structure pcl - pla - pcl , where the pla block was synthesised by the first route . furthermore , tin octoate has been shown to result in the transesterification of polyesters in the absence of monomer ; thus , the presence of the monomer initially prevents significant transesterification of the block polymer formed . catalysts possess an ability to promote transesterification reactions , and tin octoate is known for its strong ability to break ester bonds in a macromolecule . the ability to induce transesterification is a negative property of tin octoate ; however , it is commonly used in the synthesis of block polymers as the catalyst is safe to handle , inexpensive , and nontoxic . there is an interest in the synthesis of block copolymers with any given sequence of blocks using tin octoate as catalyst , though there are preconditions for this form of synthesis . it is known , that various tin - containing compounds possess different selectivity and activities in living polymerisation . it is has been shown that the activity is dependent on the valency and ligand size ; for example , tin ( iv ) displays higher catalytic activity than tin ( ii ) complexes . additionally , it has been found that the block structure pcl - pla - pcl can be obtained using tetrakis sn ( iv ) alkoxides , and it has been shown that additives ( triphenylphosphine and 4-picoline ( c6h7n ) ) can direct the carop reaction [ 11 , 12 ] . for example , it has been shown that addition of pyridine in the carop reaction promotes catalyst complexation , resulting in reduction of the competing transesterification . the propensity of tin octoate to form complexes with nucleophilic substances has been well established . have shown that the steric and electronic factors of catalysts and type of monomers utilised influence the formation of block copolymers [ 15 , 16 ] . preventing the competing transesterification reactions is an issue in the formation of multiblock polymers and is the focus of this study . through establishing an effective protocol that minimizes this side reaction a diverse range of polymers with a wider array of mechanical properties can be formed . by incorporation of a hard block in the centre of a rapidly tri- or starblock structure commonly , blocks possessing high degradability that terminate the macromolecule lead to a high rate of degradability for the entire copolymer and vice versa . furthermore , the hard block core surrounded by soft bloeks with low degradability can furnish tough biodegradable copolymers with slow degradation rates . these features are desirable properties in the production of biodegradable packaging that are expected to have long enough shelf life time . additionally , when crosslinked , block structures with flanking hydrophobic regions allow the formation of hydrogels with high swelling capacity , a feature required in scaffold - based tissue engineering . for ring opening polymerization to remain as a living polymerization , preventing transesterification and preserving the lengths of blocks polymers formed is important and allows for the synthesis of multiblock polymers with controlled repetition of hard and soft blocks . additionally , these improvements would allow incorporation of carop reaction into the field of biomimetics whereby the properties of naturally occurring materials could be mimicked such as the adhesive materials of sea shells or spiders silks . such polymer structures with a hard block in the middle or a highly repetitive sequence of hard and soft blocks can be useful in the production of stents and occluders such that the mechanical properties and degradation behavior can be mimicked . hence , we investigated the optimisation of carop reaction to maintain integrity of the 1st pla block in block copolymer formation . within this study , we investigate three possible variants in polymerisation utilising tin octoate to improve the carop reaction and minimise transesterification side - reactions , ( i ) identify a suitable protective additive , thereby protecting the pla formed from competing side - reactions , ( ii ) identify an additive that minimises transesterification by mimicking the characteristics of the initial monomer , and ( iii ) identify a suitable additive that is able to complex to tin octoate thereby minimising the competing transesterification reaction while promoting the cycle - opening reaction for the monomer . -caprolactone ( 99% purity ) , obtained from fluka , was dried over cah2 and distilled under nitrogen at reduced pressure . ( 3s)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione ( l - lactide ) ( 98% purity ) was obtained from sigma aldrich and purified by recrystallization with dry diethyl ether . 1,3-trimethylene carbonate ( 99% purity ) from boehringer ingelheim corporation , germany , was used without further purification . the monomer was dried for 24 h under reduced pressure at room temperature prior to polymerization . tin 2-ethylhexanoate ( 96% purity ) ( sn(oct)2 ) from sigma aldrich , hydroxy butyl vinyl ether ( 98% purity , hbve ) stabilized by 0.01% koh from basf , and 1,4-butanediol ( 99% purity ) from alfa aesar were purified by distillation under nitrogen at reduced pressure . methanol anhydrous ( 99.8% purity ) from sigma aldrich and tetrahydrofuran ( 99% purity ) from alfa aesar were used without further purification . toluene anhydrous ( 99.8% purity ) from sigma aldrich was dried over cah2 and distilled under nitrogen . synthesis was performed in a three - necked round bottom flask ( 100 ml ) equipped with a thermometer , a condenser and a magnetic stirrer . the flask was purged with dry argon and vacuumed twice , after which the reaction vessel was kept under the argon atmosphere . toluene , 1,4-butanediol or hbve ( initiators ) , and sn(oct)2 ( catalyst ) were added to the flask at 90c and stirred for 30 minutes . the quantity of the initiator and monomers used were based on the desired degree of polymerization . the quantity of the catalyst sn(oct)2 was chosen such that the ratio of initiator to catalyst was maintained at a constant of 10 for all syntheses . the necessary quantity of monomers ( -caprolactone or l - lactide or -caprolactone and l - lactide ) was added to synthesize the first block of copolymer , depending on desired polymer structure . the temperature was then increased to 110c for 24 hours for polymerization to proceed . to synthesize the 2nd block , the necessary quantity of monomers ( -caprolactone or l - lactide or trimethylencarbonate ) was added to the flask and allowed to react for a further 24 hours at 110c . mol / l , and the quantity of monomers usually taken for the synthesis was 0.08 mol . after 48 hours , the reaction mixture was poured into cold methanol and the precipitated polymer was filtered , washed several times by cold methanol , and dried in a vacuum oven for 48 hours at 40c . the molar masses of polymers were determined by size exclusion chromatography ( sec ) using an agilent 1100 series hplc . polystyrene standards with a narrow molar mass distribution in the range of 580400,000 g / mol were used for calibration . measurements were made at room temperature with a linear pl gel and 5 m mixed c column . chloroform was used as eluent with a flow rate of 1 ml / min . samples were prepared in deuterated chloroform ( 200 mg of polymer/1 ml cdcl3 ) . c - nmr and h - nmr spectra were obtained using a bruker 400 spectrometer with deuterated chloroform used as internal standard . average lengths of poly(-caprolactone ) ( lcl ) and poly(l - lactide ) blocks ( lla ) were calculated from the intensities of the carbonyl signals [ 23 , 24 ] using the equations below : ( 1)lpcl = icccilcc+1,lpla = illlilllc+1 , where iccc and illl are the intensities of -caprolactone--caprolactone and l - lactide - l - lactide triads , respectively ; ilcc and illlc represent the intensities of -caprolactone - l - lactide and l - lactide--caprolactone triads and tetrads peaks , respectively . the glass transition temperatures and melting enthalpies of polymer samples were measured using a ta instruments model q10 dsc machine equipped with a dsc refrigerated cooling system and ta instruments control software . dsc analysis was accomplished by initially heating the sample to 200c to eliminate internal stresses . samples were then equilibrated at 80c , followed by heating samples to 200c at a rate of 10c per minute . crystallinity ( c ) of the polymers was calculated using ( 2 ) : ( 2)c=hh100100 , where h is the experimental melting enthalpy of polymer in j / g and h100 is the melting enthalpy of the polymer with 100% crystallinity and h100 = 139 j / g for polycaprolactone and h100 = 93 j / g for polylactide . mass spectrometric measurements were performed using a kratos axima tof ( kratos - shimadzu biotech , manchester , uk ) time of flight instrument , equipped with a pulsed n2 laser ( 337 nm , 4 ns pulse width ) and time - delayed extraction ion source . the matrix , 2,5-dihydroxybenzoic acid ( dhb ) or dithranol , was dissolved in thf ( 20 mg / ml ) . sodium iodide was dissolved in thf ( 5 mg / ml ) and used as the ionizing agent . samples were prepared by mixing the matrix solution with the polymer solution and ionizing agent to the ratio of 10 : 1 : 1 , respectively . this mixture ( 1 l ) the average molar mass of polymers was calculated using the standard software program provided by the instrument manufacturer . gas chromatography - mass spectrometry measurement was recorded by quattro micro gc with quadrupole mass spectrometer ( waters corporation ) . the length of pla - pcl block copolymer formation can be influenced by additives that prevent segmentation due to tin octoate . linear , branched saturated or unsaturated esters , styrene and -methylstyrene were considered as suitable additives to minimise transesterification side reactions induced by tin octoate . initially , protective additives ( 10 : 1 initial monomer : protective additive ) were added to reactions upon synthesis of the 1st copolymer block . for all reactions , the targeted triblock pcl - pla - pcl was 20 - 40 - 20 kda and the physical characteristics of the obtained copolymers are outlined in table 1 . analysis of the nmr spectra showed that block copolymers where obtained in the presence of protective additives -methylstyrene , styrene , and ethyl benzoate . the presence of the protective additive esters was found to preserve the pla macromolecule , providing longer pla and pcl segments in comparison to reaction where no additive was utilised . the triblock molar mass closest to the required target was achieved when -methylstyrene was utilised . furthermore , an increase in the molar mass of the polymer was observed during generation of the 2nd copolymer block with living polymerisation sustained . 2 ) showed the observed increments of polymer molar mass by analysing the middle pla block , initially synthesised , and the final triblock copolymer by sec ( figure 1 ) . to study the influence of protective additives in the formation of copolymers , a series of reactions were performed neat with the chosen additives . upon treatment of the monomers and tin octoate , protective additives , -methylstyrene , and styrene yielded pcl - pla - pcl block copolymers with high crystallinity of pcl and pla . furthermore , introduction of the protective additive promoted the preservation of the block structure and augmentation of the pcl and pla segment lengths . from the aforementioned data ( table 1 ) , increased polymer lengths were observed when additives containing electron - rich motifs such as the saturated olefin observed in -methylstyrene were utilised . additionally , -methylstyrene is known to homopolymerize only at low temperature ( 78c ) through an anionic mechanism , due to its low ceiling temperature ( 66c ) compared to styrene ( 395c ) . since reactions of cyclic monomers was performed at 110c , -methylstyrene was further investigated in improving the polymerisation reaction as it would not interfere with the block copolymer formation . initially , a range of molar ratio of protector additive and tin octoate was investigated for carop reactions or cyclic monomers . the targeted molar mass of the triblock pcl - pla - pcl was 20 - 40 - 20 kda for all experiments . the obtained molar masses and crystallinity for each synthesized polymer are summarized in table 2 . varying the ratio of -methylstyrene compared to the catalyst from 2.5 : 1 to 2500 : 1 resulted in improved polymerisation reactions as the additive ratio increased ; however , the increase in -methylstyrene significantly reduced the rate of ring opening polymerization . when the protective additive : catalyst ratio was higher than 125 , a decrease in the resulting molar mass of the polymer obtained was observed . additionally , polymer 3 ( table 2 ) displayed the lowest pla crystallinity , while the pcl segment displayed no crystallinity . the molar ratios of monomers l - lactide and -caprolactone used and the resulting segment lengths of the copolymers are outlined in table 3 . c nmr analysis at 160180 ppm of the obtained polymers from table 3 demonstrated the formation of the desired copolymers , figure 2 . evidently , analysis of the crystallinity ( table 2 ) , block lengths ( table 3 ) , and nmr data ( figure 2 ) polymer 3 ( table 2 ) does not conform to the observed trend . the presence of dyads and triads as observed in polymer 6 ( figure 2(d ) ) suggests that polymer 3 ( figure 2(b ) ) also exists as a random copolymer due to the transesterification side reactions . to elucidate the mode of reaction for polymer 3 , we reacted tin octoate with -methylstyrene in toluene without a cyclic monomer ; however , gc - ms and maldi - tof analysis ( section 3.3 ) did not elude to the phenomena observed with polymer 3 ( table 3 ) . comparing of pcl and pla blocks length of polymers 4 and 5 ( table 3 ) with polymers 13 , we speculate that another mechanism of protection occurs when the quantity of protective additive is significantly higher . polymers 4 and 5 ( table 3 ) exhibit the largest pcl and pla segment lengths and lowest molar masses . comparing the influence of styrene and -methylstyrene , we propose that the mechanism of segment protection with -methylstyrene is a result of its higher propensity to participate in nucleophilic substitution reactions compared to styrene . it is possible that the slight difference in structure between -methylstyrene and styrene is important as it is known that the difference in one methyl group in a ligand for a given catalyst can influence the resulting product of the carop reaction . analysing the aforementioned data , a nonlinear dependence of differing quantities of -methylstyrene can be deduced and it appears that -methylstyrene interacts only with catalyst and does not react with the monomer ( -caprolactone ) or with polymer - macroinitiator ( pla ) . a series of model reactions structured on the synthesis of polymer 3 ( random structure ) were performed to determine the mode of reaction for -methylstyrene : ( i ) a twofold increases in the monomer quantity , ( ii ) twofold increase in initiator ( 1,4-butanediol ) , ( iii ) fourfold reduction in catalyst ( tin octoate ) , and ( iv ) twofold dilution ( toluene ) of the reaction mixture . it was found with increases in monomer quantity ( reaction 1 ) and initiator ( reaction 2 ) , random copolymers were obtained as observed for polymer 3 ( tables 2 and 3 ) . reduction of tin octoate ( reaction 3 ) leads to the formation of a block copolymer with pcl crystallinity of 44.4% and pla crystallinity of 45.3% , while dilution of the reaction solution gave a polymer with pcl crystallinity of 9.2% and pla crystallinity of 39.2% . the obtained pcl and pla segment lengths of copolymer ( reaction 3 ) were 22.2 and 32.9 monomer units , respectively . thus it can conclude that the protective additive ( -methylstyrene ) reacts only with the catalyst ( tin octoate ) , and the concentration of the reacting substances influences the degree of interaction between the catalyst and the protective additive used thereby dictating its effectiveness . to determine the products of the reaction between only -methylstyrene and tin octoate , three experiments with differing molar ratios of tin octoate : -methylstyrene were conducted . the molar ratios ( tin octoate : -methylstyrene ) utilised were ( i ) 1 : 2.5 , ( ii ) 1 : 125 , and ( iii ) 1 : 300 . maldi - tof analysis of the resulting products was utilised and potential products of reactions are outlined in table 4 . the most intensive signals on maldi - tof spectra were obtained for structures 3 , 4 , 6 , 7 , 8 , and 10 ( table 4 ) . interestingly the most intense signal was due to substance 6 ( table 4 ) with a mass of 413.33 da , which was observed in all three model reactions . due to the diversity of the products obtained in table 4 , we could not conclude through maldi - tof analysis the compounds responsible for random copolymer formation . hence , we performed syntheses of triblock copolymers of pcl - pla - pcl using the three molar ratios of protective additive : tin octoate . the targeted molar mass of the copolymers for maldi - tof analysis was 2000 - 2000 - 2000 da ( pcl - pla - pcl ) . analysis of the maldi - tof spectra showed the molar masses of polymers synthesised with tin octoate : -methylstyrene ratios of 1 : 2.5 ( 5130 da ) , 1 : 125 ( 4010 da ) , and 1 : 300 ( 4640 da ) . the ratios of l - lactide : -caprolactone for these copolymers , determined from h nmr , were 43 : 57 , 42 : 58 , and 44 : 56 , respectively . maldi - tof analysis of the copolymers formed ( figure 3 ) displayed a difference of 72 da , indicative of one monomer unit of lactic acid suggesting that the polymers underwent transesterification . spectrum ( b ) ( figure 3 ) displayed additional signal compared to spectra ( a ) and ( c ) indicating that an increased degree of transesterification had occurred . all significant peaks acquired from maldi - tof spectra were used in determination of the end groups . from the literature , we considered published data regarding the analysis of terminal groups of similar copolymers [ 2628 ] . analysing the monomer ratio determined through nmr and mass spectra obtained from maldi - tof analysis , copolymer synthesised with a tin octoate : -methylstyrene ratio of 1 : 2.5 contained the following terminal groups in abundance with molar masses : 188 , 385 , 397 , 413 da . the tin octoate : -methylstyrene ratio 1 : 125 formed the following terminal groups : 260 , 368 , 394 , 385 , and 397 da . the tin octoate : -methylstyrene ratio of 1 : 300 resulted in the formation of terminal groups 117 , 172 , 188 , and 260 da . it is probable that the interaction between the catalyst and -methylstyrene forms a wide spectrum of products that depend on the ratio of the reacting components . we attempted to explain the observed differences of copolymers from analysis of the maldi - tof spectra . at equal ratios of catalyst : -methylstyrene , the electrophilic attack of electrophilic sites of tin octoate ( between carbonyl carbon and tin atom ) by -methylstyrene is possible . the coordination of tin octoate by -methylstyrene occurs , as maldi - tof spectra displayed the required mass ions of 521 , 522 , and 523 da ( tin octoate + -methyl styrene h 405.12 + 118.18 1 = 522.3 da ) . studies have shown the capability of tin octoate to react with aibn , producing a complex with better catalytic working capacity . additionally , steric factors are important as tin ( iv ) does not participate in complex formation at similar conditions . we expect that complex molecules containing tin ( substances 28 from table 4 ) are formed . thus , by altering the steric structure of the catalyst used transesterification can be minimised , thus conserving the pla blocks that are formed first during polymerisation . from calculations and our experiences with these substances , compound 6 from table 4 , ms co o sn o - ms , with a mass of 413 da is most likely responsible for the protection of pla at the catalyst : -methylstyrene ratio of 1 : 2.5 . with increases of the tin octoate : -methylstyrene ratios up to 1 : 100 , the probability of interactions between the electrophilic sites of tin octoate and -methylstyrene increases . it is likely that the products from these reactions become less branched and more symmetrically linear . we speculate that the transformation of tin ( ii ) into tin ( iv ) possibly occurs . thus , the catalytic activity of the transformed catalyst increases in comparison to the initial tin octoate . a series of tin ( iv ) alkoxides have been shown to be the most active catalysts for carop . in our examples , an increase in the observed mass at 260 ( compound 13 , table 4 ) , ms o co r , suggests that at these reaction conditions , the concentration of -methylstyrene becomes sufficient to destroy two branches of tin octoate , giving different compounds . furthermore , the number of tin atoms coordinated by several molecules of -methylstyrene ( up to four ) increases among the products of reaction . from analysis of the maldi - tof and nmr spectra , and consideration of the random copolymer formation with a catalyst : -methylstyrene ratio of 1 : 125 , we can assume that this is caused by the appearance of substances 3 , 7 , and 8 ( table 4 ) in the reaction medium . finally , considering the terminal groups of the macromolecules , we suspect that the increase of transesterification activity was induced by symmetric compound 8 from table 4 with mass 385 da , ms o sn o ms . this has been shown by majerska et al . in which the formation of substances with similar structure , sn(or)2 , using tin octoate have been established and confirmed . with the further increase in the concentration of -methylstyrene , complete destruction of tin octoate is possible , and domination of tin atoms coordinated by molecules of -methylstyrene starts to prevail . catalytic activity is maintained , though it is considerably reduced in comparison with that of tin octoate owing to the massive structure and shielding of tin by ligands . it has been well proved by the incomplete reaction of monomer and low - molar - mass polymer synthesised at a catalyst : -methylstyrene ratio of 1 : 300 and above . moreover , the transesterification ability of tin vanishes for the same reason tin is shielded by aromatic rings of -methylstyrene . there is also an increase in the content of small fragments born from tin octoate that can react with 1,4-butanediol and -methylstyrene ( substances 1214 from table 4 ) . to further probe this reaction , we reacted -methylstyrene and tin ( ii ) octoate , in different ratios at reflux in toluene for 24 h and subsequently analyzed the obtained products . gc - ms analysis of the mixture revealed the presence of intermediate benzeneacetic acid-2-ethylhexyl ester ( scheme 1 ) , due to the interaction between -methylstyrene and tin ( ii ) octoate . benzeneacetic acid-2-ethylhexyl ester ( scheme 1 ) was observed as the major compound in all varying ratios . additionally , several intermediates originated from tin ( ii ) octoate were also identified , including 2-ethyl hexanoic acid , 2-ethylhexyl 2-ethylhexanoate , and hexanoic acid 2-ethyl anhydride . therefore , it was confirmed that the mechanism of the -methylstyrene protection is associated with -methylstyrene and tin ( ii ) octoate complexation . the protective properties of -methylstyrene were tested in the synthesis of diblock structures pla - pcl ( pla first route ) and triblock copolymer ptmc - pla - ptmc . we synthesised a reverse triblock ptmc - pla - ptmc in toluene using tin octoate producing a random copolymer . we obtained a ptmc - pla - ptmc triblock copolymer using -methylstyrene : tin octoate ( 125 : 1 ) , through a two - step reaction in toluene . the targeted structure of copolymer ptmc - pla - ptmc was 20 - 40 - 20 kda . the obtained properties of the block copolymers including the molar masses , crystallinity , and segment lengths of pla , and l - lactide : trimethylencarbonate ratio for two copolymers are presented in table 5 . it can be seen from figure 4 , that the use of protective additive resulted in no signals in the region of 120140 ppm ( spectrum b ) indicating that the transesterification processes did not occur . the dyad signal from the carbonyl of ptmc ( 154 ppm ) is observed on both spectra , while the dyad signal from l - lactide ( 169 ppm ) is seen only in the spectrum ( b ) , hence confirming the formation of the block structure . furthermore , the spectrum ( a ) ( reaction without protective additive ) displays smaller carbonyl peaks , indicating that reaction without the presence of the protective additive results in increased formation of random polymers . we then utilised -methylstyrene as protective additive for the synthesis of the diblock pla - pcl ( pla first rout ) . all reaction conditions were performed as previously stated with a molar ratio of tin octoate : -methylstyrene ratio of 1 : 25 ; however , hbve was used as polymerisation initiator . the targeted molar mass of diblock copolymer pla - pcl was 40 - 40 kda , which upon reaction was successfully furnished . c nmr spectra of pla - pcl displayed two carbonyl peaks in the region 160170 ppm resulting from the formation of the desired diblock , figure 5 . we note that the nmr spectra of both the tri - block ( figure 2 ) and di - block ( figure 5 ) copolymers display minimal difference in signals observed . this is a result of polymer fabrication , whereby in both examples the pla block is formed first followed by the pcl block . in both examples , we can observe in the nmr spectra the absence of carbonyl signals due to triads , tetrads , and randomised segments . as highlighted , the addition of a small quantity of -methylstyrene can preserve the pla macromolecules polymerised during the initial sequence of block copolymer synthesis ( pcl - pla - pla or pla - pcl ) . however , it is difficult to reach molar masses greater than 6070 kda for these polymers using the carop reaction due to transesterification [ 33 , 34 ] . we attempted the synthesis of pcl with a target molar mass of 100 kda using the reaction conditions previously outline with protective additive -methylstyrene ( tin octoate : -methylstyrene molar ratio ; 1 : 25 ) and without . sec analysis of the reaction products showed an mn 108.1 kda for pcl synthesised with -methylstyrene , while an mn of 72.3 kda for pcl was observed when no protector additive was used , figure 6 . we conclude that the addition of protective additives decreases the transesterification side - reactions that are especially important to the synthesis of polymers with high molar masses . it is known that after 9599% of monomer conversion , polydispersity of the polymer increases as the rate of transesterification side - reaction begins to increase at minimal quantities of monomers . we investigated this phenomena and attempted to control polydispersity through addition of the protective additive -methylstyrene . pla ( 5 kda ) was utilised as polymer ( a ) for investigation , as shown in the top spectrum ( figure 7 ) . reactions of ( i ) tin ( ii ) octoate in the presence of -methylstyrene ( b ) and ( ii ) only tin ( ii ) octoate ( c ) with starting polymer pla were investigated . model reactions were performed by dissolving 5 g of pla , 40 l of sn(oct)2 , and 40 l of -methylstyrene in 50 ml of toluene and allowing the mixture to heat to refluxing under argon atmosphere for 24 hours . analysis of the maldi - tof spectra of the initial pla and the two pla samples from the pilot studies highlighting the polydispersity are outlined in figure 7 . molecular mass ( mn ) of the obtained pla samples ( figure 7 ; ( a ) , ( b ) , ( c ) ) and the polydispersity index were determined utilising software to give an mn of 4915 da ( a ) , 4415 da ( b ) , 3414 da ( c ) and a polydispersity of 1.20 ( a ) , 1.22 ( b ) , and 1.41 ( c ) . evidently , the introduction of -methylstyrene reduced the negative effect of transesterification induced by tin octoate and decreased the polydispersity . this study has established that protective additives -methylstyrene and styrene are capable of reducing the influence of the transesterification side - reactions during the synthesis of block copolymers of polycaprolactone and polylactide or polytrimethylenecarbonate and polylactide . mechanistic studies can not elude to the mode that these protective additives suppress transesterification as there is no linear dependence of transesterification versus tin octoate : -methylstyrene molar ratio . it can be suggested that the formation of products in the carop reaction is dependent on the ratio of tin octoate : -methylstyrene . ms , with mass 413 da obtained during treatment of tin octoate with -methylstyrene is responsible for the reduction in transesterification and protection of the block polymers . the effectiveness of this model of polymer protection was tested with successful formation of triblock ( ptmc - pla - ptmc ) and diblock ( pla - pcl ) structures . the ability of -methylstyrene to decrease the transesterification reaction was also analysed by refluxing pla with an amount of tin octoate in toluene . the polymer treated with the addition of -methylstyrene remained almost unchanged while without the protective additive significant transesterification occurred . in conclusion , the application of protective additives in ring opening polymerisation will allow for the formation of block copolymers with consistent uniform structures and high molecular mass as well as the generation of previously unobtainable polymers with new physical features and properties . ultimately this new protocol could be applied to the field of biomimetics , allowing the synthesis of novel biodegradable materials that could be used in the medical industry ( as stent , occluders , scaffold tissue engineering , and suture ) and manufacturing industry ( as packaging material ) .

well - defined di- and triblock copolymers consisting of -caprolactone ( cl ) , l - lactide ( la ) , and trimethylene carbonate ( tmc ) were synthesized via pla first route in coordinated anionic ring opening polymerization / copolymerization ( carop ) with tin ( ii ) octoate as catalyst . the desired block structure was preserved by use of protective additive -methylstyrene by preventing the transesterification side - reactions . maldi - tof analysis revealed that the protection mechanism is associated with -methylstyrene and tin ( ii ) octoate complexation . additionally , it was shown that use of -methylstyrene in ring opening polymerization allowed the formation of polyesters with high molar mass .

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

the rapid improvement in quality , quantity , and cost of next generation sequencing ( ngs ) has resulted in commensurate improvements in analysis techniques . for bacteria , the availability of kits for library preparation , rapid and high content sequencing , and mature data analysis pipelines for genome resequencing and assembly had drastically reduced costs and improved reliability of these results . the commoditization of bacterial genome sequencing has led to more complex applications : clinical and agricultural diagnostics [ 14 ] , outbreak detection and monitoring [ 57 ] , human health studies [ 8 , 9 ] , biocatalysis [ 10 , 11 ] , environmental studies , and many others [ 13 , 14 ] . for ngs platforms , current sequencing technologies require that sequencing adapters be ligated to dna fragments before sequencing is possible . ligation of adapters to ( typically small ) dna fragments is an inefficient process , generating ligated hybrids from only a small fraction of targeted dna molecules . this limitation in turn increases the required dna input , with the only goal being to generate sufficient numbers of ligated fragments to allow sequencing . typical library preparation methods require large amounts ( ~1 g ) at high concentrations ( > 25 ng / ml ) of dna for successful library generation , limiting the types of samples that can be sequenced reliably . these include high variability of evenness and completeness of genome coverage as a function of % gc content , input dna quantities , and sequencing technology [ 1519 ] . these impact the amount of sequencing data required and the quality of genome assembly and analysis . several library preparation kits that require 1100 ng of input dna are now available ( new england biolabs ' nebnext , illumina 's truseq nano , bioo scientific 's nextflex , nugen 's ovation ultralow , etc . ) . this paper details the results of evaluation of the utility of the nebnext ultra library preparation kits for both resequencing and assembly of several bacterial genomes . we compare the evenness and completeness of coverage between nebnext ultra and illumina truseq kits for bacterial genomes of varying size and % gc content . our findings indicate that low dna input amounts are sufficient to generate high quality sequencing data that can be used for genome resequencing or de novo assembly ( if combined with long fragment data ) . we sequenced three different bacterial species with various genome lengths ( from 5.4 mb to 6.7 mb ) and containing various % gc contents ( from 35% to 68% ) . standard input dna amounts were 100 ng , approximately 10x lower than the required amount for the illumina truseq kit and 10x higher than the minimum dna inputs per nebnext ultra manual specifications . the most challenging ( longest genome and highest gc content ) bacterial genomes ( burkholderia a and b ) were also sequenced with minimal dna inputs ( 10 ng ) . all samples were sequenced on the illumina hiseq platform using 2 100 bp chemistry . data analyses consisted of read - mapping the short fragment data to reference genomes using bwa ( burrows - wheeler alignment ) . these data were also combined with long insert mate pair data to evaluate their utility for de novo assembly of the bacterial genomes . genomic dna from bacillus anthracis ( strain sterne 34f2 ) was isolated from a log phase culture using the mo - bio ultraclean microbial dna isolation kit . burkholderia thailandensis a ( strain e254 , accession numbers cp004381 and cp004382 ) and burkholderia thailandensis b ( strain usamru malaysia # 20 , accession numbers cp004383 and cp004384 ) are previously reported strains , and dna was provided by dr . the integrity of all genomic dna samples was evaluated using agarose gels and their quantity measured with picogreen reagents on a qubit 2.0 instrument . nebnext ultra library preparation protocol consists of several enzymatic and two purification steps , one of which is used for size selection of library fragments . genomic dna samples were sheared in 55 l of tle buffer ( 10 mm tris , 0.1 mm edta , ph 8) using covaris e220 with the following settings : duty cycle 10% , intensity 5 , cycle 200 , and time 100 sec . after shearing , two enzymatic steps ( end preparation and adapter ligation ) are performed in the same tube , followed by size selection of the library fragments using a double ampure cleanup . first ampure step used 0.4x sample volume of beads and the supernatant was transferred to a clean tube . selected library fragments were amplified with barcoded primers ( 1012 pcr cycles ) and purified one more time with ampure beads ( 0.5x bead volume ) ( see supplementary material available online at http://dx.doi.org/10.1155/2014/434575 ) . nebnext libraries were analyzed using bioanalyzer 2100 and dna 1000 or dna high sensitivity chips , to quantify the library size and assess the level of adapter - dimer and primer - dimer contamination . libraries were quantified using illumina library qpcr quantification kits from kapa biosystems and sequenced on either the illumina miseq or illumina hiseq . the illumina data from this study were trimmed to remove any ambiguous bases ; any reads shorter than 70 bp after trimming and the corresponding read pairs were discarded . the total number of reads per sample ranged from 6.2 million to 47.8 million before trimming . all data had read lengths of 151 bp with one exception which had read lengths of 101 bp . after trimming , the average read lengths were reduced by less than 3.5% for all samples . the data for each sample were normalized to 70x coverage of the genome after trimming . the average number of reads with a quality greater than q20 after trimming and normalization ranged from 61% of the total reads to 97% of the total reads . the total number of reads , the number of reads with quality greater than q20 , and the average read lengths before and after trimming for each sample can be found in table s1 . the assemblies were compared to the reference genomes to consider insertion / deletion errors and rearrangements using an in - house perl script . for read - mapping , burrows - wheeler alignment ( bwa ) mapping tool was used , combined with samtools and in - house perl scripts for coverage and insert size analysis [ 21 , 22 ] . for base coverage we used bwa global alignment option with default parameters . bwa global alignment only reports the best alignment based on score calculated by a set of parameters . if a read has several possible best alignment spots , bwa randomly assigns the read to one spot . all reads mapped to contigs were used to calculate base coverage . for insert size calculation , only properly paired reads ( read pair on the same contig and with correct orientation ) were used . we report the mean , standard deviation , the minimum , and maximum of the insert size distribution for all short fragment libraries . we utilized three thresholds for reporting coverage : 0% , 1% , and 10% of mean fold coverage . calculation of evenness of coverage was performed by calculating the average and standard deviation of coverage across nonoverlapping 10 kbp fragments of the finished genome . evenness for each fragment was calculated as 1 ( standard deviation of coverage / coverage ) . all data points ( genomic and plasmid coverage , where appropriate ) were used to generate box and whisker plots in ibm 's statistics program spss . two debruijn graph assembly tools were used to evaluate the quality of the short fragment data for the purpose of assembling high quality genomes . paired reads were randomly selected ( in silico ) from each sample to generate libraries of approximately 70-fold genome coverage for each sample , in order to normalize the data . the only exception was the e. coli sample prepared with the truseq kit , for which only 61-fold coverage was available . the 70-fold short fragment illumina data were combined with previously sequenced long insert mate pair data generated by 454 . the 454 data had an average insert size of 8 kbps and provided 7- to 8-fold base coverage , with the exception again for the e. coli samples , which had approximately 3.5-fold coverage . the library preparation protocol , as described in section 2 , yields average insert sizes of ~270 15 bps ( average library sizes of ~400 15 bps ) that are optimal for either 2 100 or 2 150 bp sequencing on illumina platforms . different insert sizes can easily be obtained by adjusting the size selection step ( ratio of dna solution to ampure beads ) as recommended by the manufacturer . it is not necessary to adjust the shearing step , as the sheared dna produced by covaris has a very broad size distribution . nebnext library process provides very consistent results in terms of library size and concentration , even when performed for the very first time . prior to normalization and sequencing , samples were analyzed using qubit ( picogreen - based method ) , bioanalyzer 2100 , and quantitative real - time pcr ( qpcr , kapa biosystems ) . unfortunately , this was not the case when molar library concentrations were obtained with qubit and bioanalyzer data only ( without qpcr ) . sequencing was performed on either illumina hiseq ( 2 100 bp ) or illumina miseq ( 2 150 bp ) . figure 1 ( b. anthracis and e. coli ) and figure 2 ( b. thailandensis a and b ) contain sliding window coverage plots that compare the coverage of each genome by different library preparation method and different dna input amount . from the figures , it can be seen that the genome coverage is remarkably similar regardless of the library preparation method ( nebnext or truseq ) . of particular interest is that even the libraries prepared from only 10 ng of genomic dna produced essentially the same evenness of genome coverage as the rest of the samples ( figures 2(a)2(d ) , top panel ) . as table 1 shows , the number of true gaps in coverage ( 0% ) is slightly higher for nebnext than for truseq libraries , while the number of gaps is lower for nebnext when using 1% or 10% average coverage thresholds . the data were not normalized among all samples prior to evenness of coverage comparisons . instead , they were normalized within each sample relative to the average coverage . figure 3 shows box and whisker plots of the evenness of coverage of 10 kbp windows for each genome . in the case of e. coli , the evenness of coverage for nebnext ultra libraries prepared with 100 ng of input dna is superior to that of truseq libraries produced with 1 g of input dna . for b. anthracis and b. thailandensis , there is more variation in coverage for the nebnext preparations at both 100 ng and 10 ng . further examination of this effect suggests that it is proportional to the amount of input dna , supporting the theory that nebnext ultra kits either do not introduce bias or introduce similar bias to truseq kits , with a lower dna requirement . figure 4 shows the results of de novo genome assemblies generated using the short fragment data either alone ( figure 4(a ) , assembled with idba ) or complemented with long insert mate pair data ( figure 4(b ) , assembled with allpaths ) . idba assemblies show very similar results for low ( b. anthracis ) to medium ( e. coli ) % gc genomes . however , data obtained from nebnext libraries show a dramatic reduction in the number of contigs for high % gc genomes from the two burkholderia strains . importantly , nebnext libraries prepared from just 10 ng of genomic dna maintain the high quality of genome assembly , producing similar numbers of contigs as with 100 ng input dna samples . however , scaffolding mostly depends on the long insert mate pair data , which are the same for all assemblies . comprehensive assembly statistics are shown in tables s2a and s2b . in conclusion , we have demonstrated that the quality of bacterial genome resequencing and de novo assembly is similar , regardless of the library preparation method and input dna amount ( from 10 to 1000 ng ) . the only significant difference was observed in the assemblies of the b. thailandensis genomes , where nebnext library data produced dramatically more contiguous assemblies ( figure 4 ) . in general , the assemblies from the truseq libraries were more prone to indels and rearrangements . the full results for the comparisons of our assemblies to the reference genomes are found in table 2 . this is likely due to the improved ability of the nebnext reagents to more effectively amplify high % gc regions that are very common in burkholderia genomes . modern library preparation methods for next generation sequencing technologies , as represented by nebnext ultra , are enabling bacterial genome sequencing from very small input amounts of genomic dna . these methods likely do not require any additional improvement , since handling and quantifying dna amounts smaller than 10 ng can become challenging . we have deposited the genomes of burkholderia thailandensis a ( strain e254 , accession numbers cp004381 and cp004382 ) and burkholderia thailandensis b ( strain usamru malaysia # 20 , accession numbers cp004383 and cp004384 ) into genbank . all experimental and computational methods used during this work are publically available or can be provided by the authors .

sequencing bacterial genomes has traditionally required large amounts of genomic dna ( ~1 g ) . there have been few studies to determine the effects of the input dna amount or library preparation method on the quality of sequencing data . several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input dna . in this study , we evaluated the nebnext ultra library preparation reagents for sequencing bacterial genomes . we have evaluated the utility of nebnext ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the truseq library preparation kit . the nebnext ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic dna . for the two most challenging genomes ( burkholderia spp . ) , which have the highest gc content and are the longest , we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic dna is used .

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

lateral epicondylitis is also known as tennis elbow or tendonitis of the extensor muscles of the forearm and refers to pain and tenderness over the lateral epicondyle of the humerus ; this pain is exaggerated by resisted dorsiflexion of the wrist or the middle finger . it has been estimated that 1 - 3% of the population suffer from this condition with equal distribution between men and women . pathophysiology of lateral epicondylitis most commonly involves osteotendinous part of the extensor muscles of the wrist at their origin ( the lateral epicondyle ) ; among these , the tendon of the extensor carpi radialis brevis is more commonly involved . it is believed that the injury involves tears ( either microscopic or macroscopic ) in the origin of the extensor muscles of the wrist , which leads to an inflammatory response and in the chronic cases granulation and fibrous tissue . studies have shown that most cases of lateral epicondylitis involve fibrotic tissue and angiogenesis as a result of which some believe that tendinosis is a more correct term for the condition than tendinitis . it has been argued that the most common causes of lateral epicondylitis are overuse injuries . tendons are relatively hypovascular and as a result are prone to injuries which are cause by hypoxia such injuries usually result from occupational and athletic activities . these activities include ( but are not limited to ) : backhand stroke in racquet sports , pitching in baseball , typing on keyboards , repetitive occupational hand movements such as hammering or driving screws and carrying heavy briefcases . symptoms of the condition include pain on the lateral aspect of the elbow , reduced grip , increase in pain with activity and reduced strength of the extensor activities of the wrist . despite the fact that this condition often lacks an inflammatory component the main sign of the lateral epicondylitis is tenderness of the lateral epicondyle coupled with pain on resisted dorsiflexion of either wrist or middle finger . the evidence shows that 95% of patients heal either spontaneously or by conservative measures alone . this has led some practitioners to believe that it is not necessary to treat lateral epicondylitis , their argument is supported by studies such as the study by smidt et al . which showed a limited benefit for treatment modalities other than expectant management . one of the treatments which has unequivocal evidence both for and against it , is corticosteroid injection . a meta - analysis by aspenberg showed that while corticosteroid injection has short term benefits , in the long term it is more likely to cause harm . however , there are others who believe that corticosteroids are a poor treatment choice for lateral epicondylitis . in order to address treatment concerns for patients with lateral epicondylitis ; we did a randomized double - blind clinical trial to test the effectiveness of steroid injection versus placebo and immobilization versus no immobilization in treating patients with lateral epicondylitis . this randomized double - blind clinical trial was conducted in the kashani university hospital in isfahan , iran during the first 6 months of the 2013 . patients with confirmed lateral epicondylitis , who had not received any treatment prior to enrolment , were entered into the study if they had none of the exclusion criteria and gave informed consent for participation [ figure 1 ] . patients were seleted from a pool of individuals who were screened for upper extremity complains , those who were suspected of suffering from lateral epicondylitis were examined by an orthopedic surgeon and if a diagnosis of lateral epicondylitis was confirmed and none of the exclusion criteria were present , then they were asked to participate in an orientation session where the trial was explained to them and information booklets were distributed between them . those patients who agreed to participate in the trial were asked to give written consents . the inclusion criteria included a history compatible with lateral epicondylitis and positive examination in palpation of the elbow over the lateral epicondyle , with resisted wrist extension , resisted middle finger extension and/or the mills test , and patients with symptoms lasting more than 6 weeks , no history of acute trauma , fracture , and/or surgery within 12 months , patients who had not received corticosteroid injection , physiotherapy , splint or casting during the past 6 months , no bilateral involvement and no history of cervical disk herniation , radiculopathy or abnormal electrophysiologic study . random numbers table was used to allocate patients between groups 1 , 2 , 3 and 4 . the trial pharmacist also prepared a series of similar vials containing either 40 mg of depomedrol ( aburaihan comp . , iran ) ( 1 cc ) or 1 cc normal saline and coded them either 1 , 2 , 3 or 4 , the group assignments were not decoded until the end of the trial when the final analysis was due to take place . patients took their envelopes to the trial pharmacist who gave them a coded vial which they took to the orthopedic surgeon who made the injection . due to the color difference of depomedrol and normal saline , both vials and syringes were covered by stickers in order to conceal the injection solution . maximum point of tenderness was identified and injection was performed in fanlike fashion in that area . after the injection the trial clerk took the patients to a technician who gave patients in groups 1 and 3 long arm splints . the trial subjects were then evaluated using oxford elbow scale ( oes ) and visual analog scale ( vas ) ( oes is the gold standard for clinical evaluation of elbow complaints ) by the trial clerk . the patients were evaluated at the baseline and before administration of treatment and they were also asked to come to the trial office at 2 weeks , 4 weeks and 24 weeks for follow - up evaluation which was also conducted by administration of oes and vas by the trial clerk . oes is a patient reported questionnaire consisting of 12 questions that has been shown to have high specificity and sensitivity in assessing the outcomes of interventions on the elbow of the patients . these questions assess three domains pertaining to the elbow : function , pain and social - psychological with four questions for each domain . the questionnaire was translated into persian by a professional translator and was then translated back into english in order to ascertain accuracy . the translated questionnaire was then validated using a group of 20 random individuals and its reliability was tested by retest of the same individuals ( which showed 95% reliability ) . after all of the enrolled patients finished the 24 weeks follow - up , the data was entered into an ibm spss database ( ver . 18 ) and analyzed . the groups were subsequently decoded so that the interventions used in each group become known . due to the number of groups and multiplicity of comparisons made , we used bonferroni correction in order to adjust the p value accordingly . this study was approved by the ethics committee of the isfahan university of medical sciences ( protocol record 387278 ) and funds were provided by the university research council . overall in the 6 months period of recruitment , 91 patients which met the inclusion and exclusion criteria were identified and from this pool of patients , 79 accepted to participate in the study . at the end of the 24 weeks follow - up , 78 patients had finished the study with one patient lost to follow - up . after decoding the groups it became known that group 1 had received 40 mg depomedrol along with long arm splint , group 2 had received 4 mg depomedrol alone , group 3 had received normal saline with splint and finally group 4 had received normal saline injection only . there were 21 , 19 , 19 and 20 test subjects in the first , second , third and fourth group , respectively . one of the patients in the second group was lost to the follow - up after the initial treatment and was subsequently excluded from the final analysis . chi - square test failed to show any significant difference between the groups with regards to gender distribution ( p = 0.866 ) . the average age of the participants was 47.39 years ( standard deviation [ sd ] : 6.53 , range : 32 - 65 ) ; repeated measure analysis of variance ( anova ) test did not show any significant difference in the age composition of the subjects of the study groups ( p = 0.622 ) . distribution of variables between the four groups the average vas score of the participants prior to the administration of the treatment was 5.93 ( out of ten , sd : 0.93 ) and no significant intergroup difference was noticed ( p = 0.577 ) . the average oxford elbow score of the patients prior to receiving treatment was 21.21 ( sd : 2.80 ) again repeated measure anova test failed to show any intergroup difference ( p = 0.658 ) [ table 1 ] . before and after tests showed that all groups reported better outcomes at different follow - up intervals and at the end of the trial . in order to understand which treatment was more effective we compared the mean differences of the groups outcomes at different stages of the trial . we measured the difference between the mean vas score at different stages of the trial [ figure 2 ] and then compared these means differences between the four groups . the analysis showed that at the 2 week follow - up , both of the corticosteroid injection groups were similar ( p = 1 ) but the patients in these two groups had a significantly larger reduction in the vas score compared with the two groups which received normal saline injection ( p < 0.001 ) . in the groups which received normal saline injection the patients in group 4 who received long arm splints in addition to normal saline injection fared better ( mean difference : 0.79 , p = 0.005 ) . at 4 weeks follow - up , again the corticosteroid injection groups were similar but significantly better than the normal saline injection groups ( p < 0.001 ) but by this time the two normal saline injection groups had become similar ( p = 0.768 ) . by the 24 week , a reversal of results was observed as the groups who received saline injection reported better results ; at 24 weeks , the two corticosteroid injection groups were similar ( p = 0.372 ) . the group who received depomedrol alone at 24 weeks reported worse scores than the normal saline only injection group ( p = 0.044 ) but they did not report significantly different outcomes compared to the normal saline and splint group ( p = 0.847 ) . the group that received both corticosteroid injection and long arm splint , however , were significantly worse than both of the groups that received normal saline injection ( p < 0.01 ) . mean visual analog scale of the four groups during the 24 weeks of study we measured the difference between the mean oes score at the beginning and the end of the trial [ figure 3 ] and then compared these means differences between the four groups . the depomedrol only group reported similar scores to that of the depomedrol and splint group ( p = 1 ) but they reported lower scores compared to the both normal saline injection groups ( p < 0.05 ) . the depomedrol and splint group reported similar scores to the normal saline and splint group ( p = 0.157 ) but reported worse scores compared to the normal saline only group ( p = 0.025 ) . the two groups that received normal saline injection reported similar scores ( p = 1 ) . mean oxford elbow scale of the four groups at beginning and at week 24 none of the patients were retreated or sought further treatment in the 6 months period of follow - up and as the results showed regardless of the intervention used the patients all reported a degree of improvement compared to their baseline status . lateral epicondylitis is believed to have three distinctive phases : the initial phase , the subacute phase and the chronic phase . in the first two stages management options include icing , rest , physical therapy and bracing with 95% of patients healing spontaneously with minimal intervention . there is , however , no consent on management of chronic epicondylitis . because of high rate of spontaneous healing , watchful waiting has been proposed as an initial approach with some even arguing that this may be the best approach overall to lateral epicondylitis . non - surgical treatment of lateral epicondylitis is a highly debated issue ; over the years many treatment modalities have been proposed but there has been no conclusive evidence to show that either of them works best for this condition . a systematic review by bisset et al . showed that while enough evidence does not exist ; there may be a short term benefit for physical joint manipulation and exercise but evidence for both of these was limited and lacked long term follow - up . ultrasound , ionization and acupuncture were also shown to be beneficial up to 3 months , but after a year of follow - up their benefit disappeared . their results also showed that laser therapy and extracorporeal shockwave therapy have no beneficial effect either in short term or long term . there are , however , more recent trials that have shown some effectiveness for radial shock wave therapy in cases where other modalities have failed . topical non - steroidal anti - inflammatory drugs may also be beneficial in reducing pain in patients . while over the years , corticosteroid injection was debated as one of the choices for treatment of lateral epicondylitis , in the recent years , the evidence has pointed that corticosteroids , at least in the long run , have limited benefit or are even harmful and cause recurrences . the evidence shows short term ( 1 month ) benefit for either corticosteroid injection or physiotherapy alone ( not both ) but this benefit fades after the early months . recent meta - analysis of the available evidence has confirmed the short term benefits of corticosteroid injection , but in the long term , the adverse effects have been shown to far outweigh the short term benefits of this treatment modality . again showed that corticosteroid injection in the short term causes pain alleviation but this effect reverses over long term . they also showed that there may be short term benefit for the patients who are treated with sodium hyaluronate injection , botulinum toxin injection or prolotherapy . compared steroid injection with injection of autologous blood for treatment of patients with lateral epicondylitis ; both treatment modalities were equally effective at 2 weeks but at 6 weeks it was shown that autologous blood injection has a significantly larger effect than steroid injection but more studies are needed for evaluation of this treatment modality . overall , more recent reviews of injection modalities have shown effectiveness for platelet rich plasma , botulinum toxin , hyaluronic acid and prolotherapy but the trials that reported clear benefits for these modalities were at high risk of bias with the exception of prolotherapy . more recent studies have also shown benefit associated with prolotherapy in treatment of lateral epicondylitis . in a more recent study by krogh et al . , it was shown that steroid is more effective in pain reduction at 1 month compared to either saline injection or platelet rich plasma injection , but in 3 months no clear advantage for either method was noticed . furthermore , it was shown that corticosteroid injection leads to reduced color doppler activity and tendon thickness compared to the other modalities which puts the patients at a higher risk for adverse effects . the high risk of recurrence with corticosteroid injection was again documented in a trial by mardani - kivi et al . where they compared corticosteroid injection with procaine injection . corticosteroid injection also has many documented adverse effects the most serious of which is tendon rupture . these adverse effects have been reported in most of the clinical trials that evaluated the effectiveness of corticosteroid injection . there seems to be adverse effects for all injection treatment modalities with the exception of sclerosant and platelet - rich plasma injections , which on the other hand have shown no clear beneficial effect either . our results have shown that when measuring the pain of the patients using the visual analog scale , they report a clear short term benefit when treated with corticosteroid injection ; namely injection of 40 mg depomedrol , but this benefit fades by the 24 week of follow - up . there is , also , no obvious pain relief advantage when splinting is added to the treatment modality . the advantage of corticosteroid injection is lost , however , when the more comprehensive oes is used ; oes combines questions about pain , with those inquiring about function and psychosocial aspects . our results show that when comparing oes scores at 24 weeks , patients who had received normal saline injection reported better scores compared to the group which received corticosteroid injection , which might be related to the rebound effect of the corticosteroid injection . our results are consistent with other similar studies in that they show a clear pain relief benefit for corticosteroid injection in the short term , we have shown that this pain relief benefit over placebo lasts through the 6 month follow - up , the advantage is , however , reduced over time and by the 24 week the difference between the groups becomes minimal . on the other hand , the superiority of saline injection at 6 months compared to corticosteroid injection when the oes scale is used shows that while patients who receive steroid injection may have greater pain relief ; it seems that in other aspects , the possible unwanted effects of corticosteroids ( such as reduced tendon thickness ) causes them to have a lower oes score . we , however , did not observe any clinically significant adverse effect in the patients . our results indicate that despite the clear pain reduction benefit associated with steroid injection in short term , this benefit in comparison with normal saline injection fades by the 24 week of follow - up . all authors have assisted in preparation of the first draft of the manuscript or revising it critically for important intellectual content . all authors have read and approved the content of the manuscript and confirmed the accuracy or integrity of any part of the work .

background : lateral epicondylitis is a common problem affecting 1 - 3% of the population . there has been much debate about the best treatment modality for this condition . there is , however , no conclusive evidence in support of any of the proposed treatment modalities . in this trial , we have studied the effect of corticosteroid injection ( with or without splinting ) with normal saline injection ( with or without splinting).materials and methods : in this double - blind , randomized clinical trial , individuals were randomly assigned to either of four treatment groups and received either 40 mg depomedrol injection alone , 40 mg depomedrol injection with splinting , normal saline injection alone , or normal saline injection with splinting . they were evaluated using the visual analog scale ( vas ) at weeks 2 , 4 and 24 and with the oxford elbow scale ( oes ) at 24 weeks.results:a total of 79 patients were participated in the study . the corticosteroid injection groups had better pain relief as measured by vas at 2 and 4 weeks compared with the two saline injection groups . mean vas difference at week 0 versus week 2 was 4.5 0.9 and 2.8 0.6 in corticosteroid injection groups and saline injection groups respectively ( p < 0.01 ) but at 24 weeks , there was only moderate benefit reported for the group which received steroid injection and splinting ( p < 0.01 ) compared to the saline injection groups . the saline injection groups reported better improvement in oes scores ( 20.1 3.7 ) at the end of the trial compared corticosteroid injection groups ( 16.1 2.9 ) ( p < 0.05).conclusion : our results indicate that despite the clear pain reduction benefit associated with steroid injection in short term , this benefit in comparison with normal saline injection fades by the 24th week of follow - up .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION AUTHORS CONTRIBUTIONS

multiple sclerosis is a chronic demyelinating disease of the central nervous system , affecting any part of the cns , but mostly white matter tracts in the cerebral hemispheres , optic nerves , brainstem , cerebellum , and spinal cord . the prevalence of the disorder ranges between 2 and 150/100,000 depending on the country or specific population , affecting approximately 1 million people worldwide . psychiatric symptoms in the context of ms were first noted by charcot , who described mania , hallucinations , and depression among other manifestations of the disease . lifetime prevalence of major depression ranges from 19 to 54% , depending on the population sample and diagnostic criteria used [ 68 ] . annual prevalence is estimated at 16% , and point prevalence of clinically significant depressive symptoms is as high as 50% . the rate of depressive disorders in a pediatric population with demyelinating disease has been estimated at 27% . evidently , prevalence is considerably higher than in the general population or among patients with general medical conditions other than ms . etiologic factors seem to be both biological and psychosocial , and risk factors have been reported to be female sex , age < 35 years , family history of major depression , and a high level of stress . disease - modifying agents , interferon beta in particular , have also been suspected to induce depressive symptomatology [ 14 , 15 ] . however , subsequent well - designed studies failed to prove a depressogenic effect , and pretreatment depressed mood was found to be the best predictor of subsequent depression [ 16 , 17 ] . the importance of this clinical issue can not be underestimated , given its high frequency , the possible adverse effects of depression on disease course , cognitive functioning , treatment adherence , deleterious consequences on quality of life , and increased risk of suicide [ 22 , 23 ] . unfortunately , mental comorbidity and depression in particular are frequently underdiagnosed and undertreated , with reported rates of missed diagnosis around 2330% [ 6 , 24 ] and rates of inadequate treatment around 20%36% of those reporting depression [ 6 , 25 ] . in a cross - sectional community study of patients with ms by cetin et al . , 59% of the patients who had significant symptoms of depression were not taking medication , possibly due to missed diagnosis , denial of symptoms , or false reporting , whereas the rest 41% received inadequate treatment . in the whole sample ( n = 542 ) , 28% of the patients were adequately treated for depression and therefore had achieved remission . in another study by mohr et al . , 65.6% of depressed patients did not receive medication , 4,7% received subthreshold treatment , 26,6% received threshold , and only 3.1% received overthreshold , indicating that in most cases , therapy is not in accordance with treatment guidelines for depression . given the fact that depression is a treatable condition , correct diagnosis and adequate treatment are issues of immense importance for the management of multiple sclerosis patients . the present paper is focused on current knowledge on diagnosis , assessment , and therapeutic interventions for depression in the context of multiple sclerosis . although the symptom of depressive mood is almost always experienced by patients suffering from disabling diseases , the syndrome of major depression , as defined by the classification systems of icd-10 and dsm - iv - tr , corresponds to a constellation of symptoms comprising depressive mood , anhedonia , fatigue , psychomotor retardation or restlessness , suicidal ideation or suicide attempt , feelings of guilt and worthlessness , and difficulty in concentrating , as well as vegetative symptoms , including altered sleep , appetite , and sexual arousal . a common challenge for correctly diagnosing depression in the context of multiple sclerosis is distinguishing whether a certain symptom emanates from a depressive disorder or can be attributed to the demyelinating disease . potential confounders are fatigue , insomnia , altered appetite , and impaired memory and concentration , and misjudgments will lead to false positives or false negatives . varied presentations of depression , for example , unexplained somatic complaints , anxiety , or hopelessness instead of sadness , could also complicate diagnosis , especially in older persons . patients with ms might exhibit pathological laughter or crying occurring with bilateral forebrain disease , which poorly correlates with underlying mood . on the other hand a way of disentangling the differential diagnosis in such cases is by paying attention to the cognitive and affective domains of the depressive symptomatology , for example , the depressive mood and depressive beliefs , which is also an approach adopted by assessment tools and rating scales for depression in the presence of a physical illness and multiple sclerosis in particular . the use of two screening questions has been proposed for detecting major depressive disorder in the context of ms and primary care setting , which correspond to depressive mood ( during the last two weeks , have you often been bothered by feeling down , depressed , or hopeless ? ) and anhedonia ( during the past two weeks , have you often been bothered by little interest or pleasure in doing things ? ) . at least one affirmative response on either question was used as a criterion of having mdd . this approach was shown to have a positive predictive value of 71.7% , with a rate of 27.3% of false positives . however , two thirds of false positives were shown to have subthreshold depression , which again should alert the clinician for the need of treatment . using the yale single question is even less time consuming , but this tool seems to lack sensitivity , as it could not identify 34.7% of patients who were depressed by beck depression inventory criteria . on the other hand , it appears to be quite specific , that is , depression can be ruled out when it does not exist . the beck depression inventory is an objective self - report assessment tool comprising 21 items , and one of the most commonly used for patients with multiple sclerosis . recommended cutoff point is set at 13 , though 30% of patients with depression will be missed in this way . concerns regarding the length of the instrument as well as the inclusion of items corresponding to neurovegetative symptoms , which might lead to overdiagnosis , have led to the implementation of shorter forms , such as the 7-item beck depression inventory - fast screen ( bdi - fs ) . validity of the instrument has been documented for the population of patients with multiple sclerosis . the hospital anxiety and depression scale is a self - assessed questionnaire consisting of 14 items , suitable for use in persons with medical conditions , if items corresponding to somatic symptoms are omitted , and recently validated for people with multiple sclerosis . the center for epidemiologic studies depression rating scale ( ces - d ) , comprising 20 items , has been used with a cutoff point of 16 ( > 15 ) , as likely to correspond to significant depression , recognizing 74.5% of depressed persons [ 9 , 45 ] . another useful scale is the chicago multiscale depression inventory that was developed to assess depression in ms and other chronic diseases . it is a 42-item self - reporting questionnaire consisting of three subscales , mood , physical malfunctioning , and self - criticism , the first of which is recommended for ms . the validity of the use of the hamilton rating scale for depression has been examined in one study . the authors conclude that the whole or part of the scale can be used , depending on study design . the depressive mood scale ( ehd , echelle d ' humeur dpressive ) is an 11-item french questionnaire specifically designed and validated for the assessment of depression in ms , focusing on mood changes rather than somatic symptoms . the zung self - rating depression scale ( zsrd ) is a 20-item tool that has been shown to have good construct validity for measuring depression in medically ill populations and has been used for patients with ms [ 53 , 54 ] , as well as the montgomery - asberg rating scale ( madrs ) , a widely known clinician - rated assessment tool for depression , consisting of 10 items . in pediatric populations , researchers most frequently use the child depression inventory for identifying depression in samples with ms [ 57 , 58 ] . the presence of depression can also be documented by applying the kiddie - sads interview . the utility of detecting depression partly depends on the efficacy and availability of treatments as well as willingness of patients to receive treatment . in a recent study , less than 30% of depressive individuals who were prompted to receive treatment did so , and possible explanations for this were distorted beliefs of depression being inevitable , practical problems , or the depression itself [ 60 , 61 ] . concluding , identifying depression , though not sufficient to ensure effective treatment , is a necessary first step towards proper management of depressed patients with multiple sclerosis . a comprehensive treatment plan for depression should include pharmacotherapy , psychotherapy , or cognitive behavioural therapy in specific or combination therapy . screening for suicidal intend is incremental due to the high cumulative lifetime risk for suicide . treating depression has been found to improve adherence , cognitive disturbances , fatigue , quality of life , and possibly disease course , by decreasing production of cytokine . there is a growing body of evidence regarding the neuroprotective effects of antidepressants such as fluoxetine and phenelzine . furthermore , escitalopram has been found to reduce stress - related relapses in a recent open - label , randomized , controlled study . treatment guidelines have been most recently reported by the canadian network for mood and anxiety disorders ( canmat ) , based on up - to - date literature . use of antidepressants is strongly recommended , as well as psychotherapies emphasizing coping strategies rather than insight . drugs with significant sedating or anticholinergic side effects , such as tricyclic antidepressants , should rather be avoided , due to issues with fatigue , orthostatic hypotension , balance , cognitive disturbances , and bladder problems . patients should also be monitored for manic or hypomanic symptoms , while on antidepressant medication , as the prevalence of bipolar disorder is quite elevated in ms , presumably two to three times higher than in the general population .should depression emerge during treatment with interferon beta , the latter needs not necessarily be discontinued , but the depression should be adequately treated instead . despite the high burden of depression in multiple sclerosis patients , few trials have been published regarding this particular population , and only two of them were double blind , randomized , placebo controlled , and meeting certain standards of quality , as already observed by koch et al . . the first one examined the efficacy of desipramine versus placebo and found a trend in favor of desipramine . the second demonstrated greater efficacy of paroxetine versus placebo , with 78.6% versus 42.1% of patients exhibiting response , respectively , but the difference did not reach significance , probably because of underpowering of the study and dosing and duration issues . another problem faced by both studies was missing data or patients who were lost at followup . in another study by mohr et al . , sertraline was found to be equally effective with cognitive behavioural therapy , and both of them were more efficacious than supportive - expressive therapy in a sample of 63 depressed patients with multiple sclerosis . open - label studies have demonstrated efficacy of sertraline , fluvoxamine , moclobemide , and fluoxetine . a few case reports deal with the usefulness of ect for severe or refractory depression in ms ; however , concerns have been raised that ect might mediate neurological deterioration by altering the number or size of the cns plaques and/or periplaque edema [ 79 , 80 ] . it has been postulated that the presence of contrast - enhanced lesions might predict deterioration , and therefore , a gadolinium - enhanced mri is suggested , but this observation is based on a report of only three patients , one of whom deteriorated after ect sessions . psychotherapy has long been considered an important treatment option for the management of depression in patients with ms , with approaches focusing on coping skills showing superiority over insight - oriented therapies . cognitive behaviour therapy ( cbt ) helps patients to correct distorted cognitive appraisal of the environment and core beliefs that lead to maladaptive behaviour , and change the connection between life events and learned reactions such as depression beliefs . regarding multiple sclerosis in particular , which imposes progressively increasing physical difficulties and challenges in everyday living , together with cognitive impairment , cbt can help maximize the development of the patients ' coping skills . in line with this observation , individual ( cbt ) has been found more beneficial than supportive expressive therapy ( set ) , administered either as usual or by telephone . telephone - administered psychotherapy or counseling is particularly appealing because of the frequent physical disability , which represents an obstacle to receiving therapy otherwise . for similar reasons , computerized forms of cbt are also available [ 86 , 87 ] . , there still may be some patients who would benefit more by insight - oriented , psychodynamic psychotherapy , which focuses on the management of psychological defenses and unconscious psychic activity [ 82 , 88 ] . the effectiveness of mindful - based intervention ( mbi ) which is based on the nonjudgmental awareness of everyday moments has also been recently demonstrated . individual preference and needs , cognitive performance , and availability of therapist should help the clinician choose among the above diverse psychotherapeutic approaches . the prevalence of depression in patients is remarkably high , yet it is still frequently underdiagnosed and undertreated . patients who are not treated are not expected to improve and are at risk for further deterioration . more and better designed studies for therapeutic interventions , particularly in the pediatric populations , are clearly needed .

multiple sclerosis is a chronic demyelinating disease affecting one million people worldwide , with a significant burden of psychiatric comorbidity . depression is the commonest psychiatric manifestation but still remains largely underdiagnosed and undertreated . the present work reviews current knowledge on diagnosis , assessment , and somatic and psychotherapeutic treatment interventions for depression in adult and pediatric populations of patients with multiple sclerosis .

1. Introduction 2. Diagnosis and Assessment 3. Treatment Approaches 4. Concluding Remarks

variability in clinical response to standard therapeutic dosage regimen was reported in the 1950s by many pioneers in the field . since then , the association between monogenic polymorphisms and variations of drugs ' metabolism , transport , or target had been identified and the vision of personalized drug therapy in health care envisioned [ 1 , 2 ] . pharmacogenomic - guided drug therapy for patient is based on the premise that a large portion of interindividual variability in drug response ( efficacy and/or toxicity ) is genetically determined . despite the widespread recognition of the scientific rationale and the clinical implementation of pharmacogenomic tests at several major academic medical institutions [ 37 ] , most clinicians and researchers engaged in the discipline would agree that the early vision of achieving personalized therapy in the form of therapeutic regimens tailored to an individual 's genetic profile remains some years away . broadly speaking , the development and implementation pathways for pharmacogenomic tests consist of several stages ( figure 1 ) : first , discovery of pharmacogenomic biomarkers and validation in well - controlled studies with independent populations ; second , replication of drug - gene(s ) association and demonstration of utility in at - risk patients ; third , development and regulatory approval of companion - diagnostic test ; fourth , assessing the clinical impact and cost - effectiveness of the pharmacogenomic biomarkers ; fifth , involvement of all stakeholders in clinical implementation . lessons learned in making pharmacogenomic - guided therapy useful to clinicians have identified multiple scientific challenges and implementation barriers existing within these stages , each of which is fueled by multitude of stakeholders with varied goals and interests . this paper will provide a perspective on these existing challenges and barriers in the complex process of implementing pharmacogenomics in clinical practice , as well as incorporating pharmacogenomics into the drug development process . many pharmacogenomic biomarkers have been identified over the last decade , but only few of them have been utilized to different extents in clinical setting ( table 1 ) . one of the major challenges for translating most discovered biomarkers to their clinical implementation as genomic tests has been the inconsistent replication result of genetic associations , whether alone or in combination . traditionally , the candidate gene approach incorporating a panel of genes that encode known drug targets , metabolizing enzymes , and membrane transporters is used in pharmacogenomic studies to test the hypothesis of an association between single nucleotide polymorphisms ( snps ) and a pharmacological or therapeutic endpoint . a good example of inconsistent replication result of genetic associations is the atypical antipsychotic clozapine with its complex pharmacological effects via the dopaminergic , serotonergic , adrenergic , and histaminergic receptors within the central nervous system . over the years , conflicting study results exist in the literature for association between clozapine response with either snps of each known pharmacological receptor subtype [ 1013 ] , combinations of polymorphisms , and metabolizing enzymes and transporters . it is also of note that the original association regarding combination of polymorphisms was not replicated in a subsequent study . the recent identification of yet another new candidate gene for clozapine treatment response illustrates the limitation of candidate gene approach in that there is always the possibility of involvement of other yet - to - be - identified genes , including those that have not been known to be linked to the pharmacology of the drug , that could account for additional variability in patient 's therapeutic response . when evaluated or used alone , most of these markers are likely of insufficient sensitivity and specificity to provide clinically useful prediction , especially of efficacy . the recognition of multiple gene variants , rather than snps , each accounting for part of the disposition and response phenotypes , has led to the increased use of whole genome approach for discovery of new biological pathways and identification of associations between pharmacogenomic biomarkers and response phenotypes . genome - wide association study ( gwas ) approach screens large number of snps ( up to 2.3 million per array ) across the whole genome in order to determine the most significant snps associated with response phenotypes . in contrast to the hypothesis - driven candidate gene approach , there is no a priori knowledge of specific gene for the discovery - driven gwas approach . rather , the large numbers of snp analyses test multiple hypotheses and necessitate large sample size , sophisticated computing and platforms ( e.g. , affymetrix genechips ) , and high cost . in addition , the level of significance associated with each test needs to be corrected for multiple hypothesis testings . refinement of the gwas approach takes a two - step design , using high - density array to discover the snp associations in a population cohort followed by replicating the initial findings above the genome - wide significance with additional patient sets in a more hypothesis - driven study of sufficient sample size . while this approach has been successfully applied in the pharmacogenomics of clopidogrel , flucloxacillin , simvastatin , and warfarin [ 1722 ] , the implications of the results are less clear for other drugs such as the psychotropics [ 2330 ] . a middle - of - the - road approach would be to limit the number of snps that warrant analysis . based on the phenomenon of linkage disequilibrium among snps , whereby two or more snps are inherited together in haplotype blocks more frequently than would be expected based on chance alone , a single representative snp within a haplotype block could serve as a tag snp ( tsnp ) for the haplotype . by genotyping a smaller number of carefully chosen tsnps to identify haplotype blocks of dna sequences that are inherited together the hapmap database created by the international hapmap project ( http://www.hapmap.org/ ) is freely available for selection of these tsnps . based on the hapmap database , many gwass of drug responses have been completed [ 18 , 19 , 32 , 33 ] . it is hoped that some of the scientific challenges for study replication related to snp genotyping may be alleviated through this approach . regardless of the choice of approach to identify the genotype - phenotype association , population variations in prevalence and relative importance of different allele variants , for example , cyp2d6 , hla - b , ugt1a1 , and slc6a4 , remind investigators of the importance of ethnicity and population stratification [ 35 , 36 ] , which could magnify the sample size requirement for statistical power in most pharmacogenomic studies . for example , although the algorithms based on the work of gage et al . and the international warfarin pharmacogenetics consortium ( iwpc ) [ 38 , 39 ] are clinically useful , they do not include detection of the cyp2c98 , an allele commonly occurring in african americans . the lower success with algorithm - based dose prediction in african americans is likely related to exclusion of this allele in most dosing algorithms . another example is hla - b1502 being a strong predictor of carbamazepine - induced severe cutaneous drug reactions in han chinese and most southeast asians but not in caucasians , who do not carry the allele variant [ 4143 ] . if not accounted for , these ethnicity - or population - related variables will confound the results of most pharmacogenomic association studies and could complicate the result interpretation . in addition , there is no universal agreement among different test platforms as to which allele variant should be tested routinely for some genetic polymorphisms , for example , cyp2d6 and ugt1a1 . in addition to the aforementioned ethnicity - related considerations , the drug disposition and response phenotypes can be affected by patient - specific variables . phenocopying with a change in metabolic phenotype secondary to concurrent enzyme inhibitor [ 44 , 45 ] could create genotype - phenotype discordance and affect the ability to predict possible drug response based on genotype - guided dosing and achievable drug concentration . inflammatory responses elicited by extrahepatic tumors have been shown to release cytokines such as interleukin-6 ( il-6 ) and resulted in transcriptional downregulation of the human cyp3a4 gene . therefore , lower docetaxel clearance reported in cancer patients could be related to tumor - associated inflammation and subsequent transcriptional repression of cyp3a4 , potentially leading to unanticipated toxicity despite normal enzymatic activity in the patient . il-6-mediated downregulation of cytochrome p-450 enzyme activities also likely contributed to a recent report of significant increase in clozapine concentration in a patient with infection and inflammation . an additional challenge for applying pharmacogenomic biomarkers in targeted cancer therapeutics is sampling of tumor tissue that carries the somatic mutations ( e.g. , testing for the epidermal growth factor receptor 1 ( her1 ) mutation in patients treated with gefitinib for nonsmall cell lung cancer and testing for overexpression of the human epidermal growth factor receptor 2 ( her2 ) protein in patients receiving trastuzumab for breast cancer ) . the presence of tumor cell heterogeneity might result in intra- and interindividual variabilities in tumor tissue content and , hence , measurable level of the biomarker . in spite of this limitation , furthermore , there is an increasing appreciation that genetic heterogeneity alone can not explain interindividual variations in drug responses . yet currently , much less is known about the influence of environmental variables and gene - environment interactions on drug disposition and response phenotypes such as mutations and polymorphisms [ 4951 ] . environmental factors , through their participation in epigenetic mechanisms , could result in many different phenotypes within a population . in the not too distant future , pharmacoepigenetic investigations focusing on studying the interaction among drugs , environment , and genes could provide additional insight of drug response variations beyond the level of genetic polymorphisms . after demonstration of a genetic association with response phenotype , there is the need of validating the biomarker , regardless of whether it is to be developed as a companion diagnostic test . for the purpose of personalized therapy , a companion diagnostic for a drug can be defined as a biomarker that is critical to the safe and effective use of the drug . the acce ( analytical validity , clinical validity , clinical utility and associated ethical , legal , and social implications ( elsi ) ) model project sponsored by the office of public health genomics , centers for disease control and prevention ( cdc ) , has been recently advocated by some investigators to be the basis for evaluation of pharmacogenomic biomarker tests . analytical validity determines how well a diagnostic test measures what it is intended to measure , regardless of whether it is an expression pattern , a mutation , or a protein . clinical validity measures the ability of the test to differentiate between responders and nonresponders , or to identify patients who are at risk for adverse drug reactions . the clinical utility measures the ability of the test result to predict outcome in a clinical environment and the additional value over nontesting , that is , standard empirical treatment . in 2004 , the cdc launched the evaluation of genomic applications in practice and prevention ( egapp ) initiative , which aims to establish an evidence - based process , including assessments of analytical validity , clinical validity , and clinical utility , for evaluating genetic tests and genomic technology that are being translated from research to clinical practice . for the pharmacogenomics discipline , one often - cited publication was the 2007 egapp working group evidence - based review of the literature on the use of cyp genotyping for clinical management of depressed patients with the selective serotonin reuptake inhibitors ( ssris ) . based on strong evidence of analytical validity , possible demonstration of clinical validity , and lack of study data to support evaluation of potential clinical utility , the working group does not recommend the application of cyp2d6 genotyping for ssri pharmacotherapy . since approval of most cyp genotyping tests by the food and drug administration ( fda ) is dependent on their technical performance in detecting cyp450 gene variants , the weak evidence of association between genotype and phenotypes ( different metabolic phenotypes , responders versus nonresponders ) is also not unexpected , since most ssris rely on multiple but not necessarily polymorphic enzymes for metabolism and have a flat dose - response relationship with wide therapeutic index . the clinical validity of the cyp genotyping tests to differentiate response phenotypes is further limited by the cyp genotype - metabolic phenotype discordance that can occur as a result of drug - drug interactions [ 44 , 45 ] or environmental influences . given these limitations as well as the lack of cost - effectiveness data , it is not surprising that the ssris are not good candidates for genotype - based pharmacogenomic therapy and , hence , the recommendation of the egapp working group . other pharmacogenomic biomarkers could be better candidates for testing association between specific genotype and clinical phenotype [ 5563 ] , as indicated by published guidelines . pharmacogenetic dosing algorithms [ 37 , 39 ] based on the patient 's cyp2c9 and vkorc1 genotypes and other nongenetic factors ( e.g. , age , body size , and concurrent interacting drug ) have been used to determine warfarin dosage regimens . as shown for clopidogrel , simvastatin , and warfarin , replication of the association in multiple cohorts or inclusion of replication data would provide further evidence of clinical validity [ 17 , 18 , 64 , 65 ] . establishing the clinical utility of pharmacogenomic biomarkers has been advocated to ensure that their use is appropriate , cost - effective , and ultimately improves clinical outcome in patients . yet within the clinical and scientific communities , there are constant debates with little agreement regarding the required levels of evidence for proof of clinical utility of diagnostic tests that are scientifically appropriate but at the same time realistically achievable [ 6671 ] . the gold standard for demonstration of clinical utility of a drug is the use of randomized controlled trials ( rcts ) . given the current evidence - based driven clinical environment , many investigators advocate that hypothesis - driven , prospective , double - blind rcts would provide the ideal approach to validate the clinical utility of pharmacogenomic biomarkers . however , within the context of personalized medicine , the biomarker as a companion diagnostic test is intended for use with a drug to produce the optimal efficacy and safety . this makes it difficult to distinguish the clinical utility of the test that is different from that of the drug or the drug - test combination . in addition , the traditional assessment of evidence of drug efficacy and safety with the use of rcts may not necessarily portray the benefit of pharmacogenomic biomarkers . complex disease etiologies , heterogeneous patient population , placebo effects , and drug response variabilities per se all contribute to statistical power issues that necessitate large patient cohort for rct . all too often , the end result is achievement of small average benefit in the entire heterogeneous patient cohort , despite the trial being costly in terms of time and sample size . in contrast to evidence - based practice , the emphasis and value of pharmacogenomics are more geared towards incremental advantages in efficacy and safety for the outliers ( the poor metabolizers , the ultra - rapid metabolizers , the nonresponders , or those susceptible to develop adverse drug reactions ) over traditional therapy or standard dosing regimen . for example , the iwpc showed that a pharmacogenetic dosing algorithm was most predictive of therapeutic anticoagulation in 46% of the patients cohort who required < 25 mg / week or > 49 mg / week . therefore , a balance between the scientific demands of rcts and the practical value of genotyping for patient care seems appropriate . given the low prevalence of genetic variants associated with drug response and the desire to generate more robust evidence , many investigators and sponsors have advocated the use of prospective enrichment design clinical trials to include patients who are more likely to respond or at least be stratified according to disease subtypes and/or exclude patients who are highly susceptible to adverse drug reactions . however , even with the assumption of ( and sometimes proven ) association between genetic variabilities and drug response , both advantages and disadvantages exist for this study design . a recent simulation study of trial designs suggested that conducting more trials with smaller sample sizes and lessened evidence - based criteria might contribute substantially to cancer survival , and assessment relying solely on the current traditional , risk - averse trial design might slow long - term progress . in this regard , it is of note that the fda recently approved crizotinib and vemurafenib with their respective pharmacogenomic biomarker tests solely on data from two single - arm studies . examples would be prescribing of abacavir in patients tested positive for hla - b5701 and 6-mercaptopurine or azathioprine in homozygous carriers of tmpt mutations . likewise , conducting a pharmacogenomic add - on as part of a head - to - head efficacy comparison of two antipsychotics in patients who are carriers of the del allele of the 141c ins / del polymorphism in the dopamine d2 receptor gene would be difficult . the del allele is associated with poor antipsychotic response ; yet , all currently marketed antipsychotics are d2 blocker , albeit with different extent of blockade . not surprisingly , pharmaceutical companies have very little financial incentive to conduct time- and cost - intensive rcts , especially for out - of - patent marketed drugs . to move the discipline forward to eventual implementation , we have to rethink the types of study design and/or the quality of study data for evidence of clinical validity and utility . the concept of conducting practical clinical trials in real - world setting had been previously proposed for regulatory decision - making [ 77 , 78 ] . the recent study by anderson et al . provided evidence of comparative effectiveness between pharmacogenetic - guided warfarin therapy in 504 patients versus standard care in 1,866 patients and a strong validation to the clinical benefit associated with the use of pharmacogenomic biomarkers in a real world setting . at the grassroot level , the concept of practical clinical trial can even be modified and adopted on a much smaller scale in clinics or physician offices . as an example , elimination of tolbutamide is known to be 50% and 84% slower in carriers of cyp2c92 and cyp2c93 variants , respectively , than in homozygous carriers of cyp2c91 . yet , to - date , there is no prospective rct to evaluate the appropriateness of 50% to 90% dose reductions for patients who are carriers of the two allelic variants . in contrast , evaluating tolbutamide efficacy can be easily done after implementation of these dosage reductions . therefore , such effort in clinical practice , instead of expensive and time - consuming rct , could constitute the first step of obtaining evidence of clinical utility of cyp2c9 genotyping in optimizing tolbutamide therapy . for patient care , a good example for the need of balance between evidence - based medicine and personalized medicine is clopidogrel . despite the extensive evidence of clopidogrel efficacy linked to cyp2c19 genetic polymorphism [ 81 , 82 ] , debates continue over the routine use of cyp2c19 genotyping to guide clopidogrel therapy [ 8385 ] . this prevents more widespread use of the biomarker in individualized therapy , despite the significantly higher rates of stent thrombosis and the associated mortality rates in carriers of the reduced - function cyp2c192 allele . based on lack of outcomes data , the joint clinical alert issued in 2010 by the american college of cardiology and the american heart association did not recommend routine genotyping and suggested the need of large , prospective , controlled trials . one such trial is the pharmacogenomics of antiplatelet intervention-2 ( papi-2 ) trial that evaluates the effect of genotype - guided antiplatelet therapy versus standard care on cardiovascular events among 7,200 patients undergoing percutaneous coronary intervention ( pci ) ( clinicaltrials.gov nct01452152 ) . the questions then become are we in the meantime sacrificing patient care on the insistence of waiting for proof of value via the evidence - based approach ? if no study results are available in the near future , should we focus on steps that can facilitate the genotyping implementation in clinical setting and examine the cost - effectiveness of genotypes - guided antiplatelet therapy with a variety of different approaches ? for many healthcare facilities and systems , it is also critical to assess whether a test offers a good return on investment . therefore , in addition to clinical validity and clinical utility , another potential barrier to test implementation is demonstration of cost - effectiveness of the companion diagnostic test . ideally , the pharmacogenomic biomarker will result in cost - effective improved clinical care in patients who will benefit from individualized therapy with the drug and avoidance of cost - ineffective treatment for patients who likely will not benefit from the drug , either as a result of lack of response or increased adverse drug reactions [ 86 , 87 ] . traditional cost - effectiveness analysis compares the relative costs and outcomes of two different approaches , typically visualized on a cost - effectiveness plane divided into four quadrants . as mentioned in the last paragraph , avoidance of cost - ineffective treatment the antipsychotic drugs offer an alternative approach to cost - effectiveness evaluation for pharmacogenomics biomarkers . with an annual cost that is at least ten times higher , the atypical antipsychotic agents are more expensive yet no more efficacious and , hence , likely to be less cost - effective , than the typical antipsychotic agents [ 89 , 90 ] . rather than focusing on using biomarkers to predict efficacy of the more expensive atypical antipsychotic agents [ 1015 ] , genotyping for the glycine9 allele of the ser9gly polymorphism in the dopamine 3 receptor gene [ 91 , 92 ] might be used to identify patients susceptible to tardive dyskinesia , a highly prevalent adverse drug reaction associated with the use of the less expensive typical antipsychotic agents . the genetic testing might enable appropriate dose reduction for the typical antipsychotic agents and lessen the incidence of adverse drug reaction . additional approaches of demonstrating cost - effectiveness of pharmacogenomic - based therapy can range from clinical trial comparing per - patient cost for specific clinical outcome between genotype - based regimen and standard regimen to decision model - based study using simulated patient cohort [ 9496 ] . alternative approach exists even within the context of cost - effectiveness comparison between genotype - based regimen and standard regimen with no genetic testing . with generic availability of clopidogrel , a cost - effectiveness study of the value of pharmacogenomic biomarker should compare clopidogrel use in cyp2c19 ems and ums versus the use of prasugrel or ticagrelor for pms . regardless of the specific approach , it should be understood that the economic impact and cost - effectiveness of screening could be affected by different variables . two separate studies utilized modeling techniques with simulated patient cohorts to evaluate the potential clinical and economic outcomes for pharmacogenomic - guided warfarin dosing . while the relatively high cost of cyp2c9 and vkorc1 bundled test ( $ 326 to $ 570 ) resulted in only modest improvements ( quality - adjusted life years , survival rates , and total adverse rates ) , the investigators also suggested that improvements in the cost - effectiveness can be achieved in several ways , specifically further cost reduction of the genotyping test and utilizing genotype - guided warfarin dosing algorithm in outliers ( patients with out - of - range inrs and/or those who are at high risk for hemorrhage [ 97 , 98 ] ) . other variables such as different population prevalence of a specific variant and cost of alternative treatment approaches would also impact the economic impact analysis . in summary , clinical utility and cost - effectiveness can not be the only measures in determining the relative value of pharmacogenomics for drug therapy optimization in individual patients . rather , they should be used to supplement the best practice strategies currently in place to achieve optimal drug therapy . over the last decade , the fda has progressively acknowledged the importance of biomarkers and provided new recommendations on pharmacogenomic diagnostic tests and data submission . these efforts included the publication of fda guidance for pharmacogenomic data submission , guidance on pharmacogenetic tests and genetic tests for heritable markers , and draft guidance for in vitro diagnostic multivariate index assays ( ivdmias ) , the introduction of the voluntary data submission program , and formation of an interdisciplinary pharmacogenomic review group ( iprg ) to evaluate the voluntary submissions , as well as the approval and classification of different biomarkers . obviously , any biomarker with fda approval will generate more confidence for clinicians , healthcare facility administrators , and payers , and could enhance test implementation and utilization in the clinical settings . additional regulatory efforts also provide an impetus of pharmacogenomic data submission for drug approval and additional research to address the debate over the utility of the information incorporated in the revised labels , for example , for clopidogrel [ 8385 ] . within the united states , there are separate regulatory oversights for a pharmacogenomic biomarker developed as an in - house test by a clinical laboratory versus that for an in vitro diagnostic device developed by a medical device manufacturer . quality standards for clinical laboratory tests are governed by the clinical laboratory improvement amendments ( clia ) . in addition , the laboratories are accredited either by the college of american pathologists , the joint commission on accreditation of healthcare organizations , or health department of each individual state , that take into consideration of clia compliance and laboratory standard practices that are in line with good laboratory practice ( glp ) regulations enforced by the fda . although there is internal validation within the laboratory , there is no external regulatory review process for the test itself . on the other hand , although currently there is no formal regulatory process for submission of companion diagnostic tests , the fda previously ruled that evaluation and approval of the amplichip cyp450 test as an in vitro diagnostic device was required . in addition , the regulatory agency had fast track approved trastuzumab with the companion diagnostic hercep test in 2001 for detecting overexpression of her2 protein in breast cancer tissue by immunohistochemistry and more recently for tests that utilize fluorescence in situ hybridization to amplify the her2 gene . further examples of fda assuming a greater role were the respective companion diagnostic tests approved for crizotinib and vemurafenib . with the formation of a personalized medicine group within the office of in vitro diagnostic device , center for device evaluation and radiological health , it is likely that more fda - approved tests would be available in the future . although no similar frameworks for premarketing regulatory review and approval of pharmacogenomic biomarkers exist in the european union and the united kingdom , there are regulations applicable for postmarketing approval . gefitinib was approved by the european medicines agency ( ema ) in june 2009 , followed by subsequent approval of a companion diagnostic test for her1 mutations . there are several challenges and practical aspects related to clinical decision support infrastructure and training of healthcare professionals ( table 2 ) that need to be addressed before pharmacogenomic biomarkers can be successfully utilized in any healthcare setting . these are further discussed in the following sections . even with a decrease in genotyping cost over time , a relatively low demand for specific biomarker test at institutional clinical laboratories may not justify the cost of equipment and technical upkeep associated with in - house testing . this not only precludes the ideal point - of - care consultation at the bedside or within the clinic , but also results in long turnaround time for obtaining test results from external clinical laboratories or research institutions . the impact of the time delay would depend on the urgency of the test , for example , her2 expression or cyp2c19 genotyping prior to scheduled pci versus on - the - spot warfarin dosing adjustment or in the setting of emergency pci . a recent commentary of pharmacogenomics in primary care reported acceptable turnaround time of 24 hours for a feasibility study of warfarin pharmacogenetic testing in a family practice clinic . in addition , a point - of - care cyp2c19 genotyping device with a turnaround time of about an hour has been developed and recently used to explore the feasibility of incorporating cyp2c192 testing into clinical protocol for antiplatelet dosing . in addition to technology advances , the concept and adoption of preemptive ( preprescription ) genotyping [ 5 , 103105 ] with result stored in electronic medical record for subsequent use would also help minimize the inconvenience of time delay in test reporting . not unexpectedly , patients expect healthcare professionals to be able to explain the pharmacogenomic diagnostic test results and answer their questions regarding treatment access and choices . while interpretation of genotype result for deciding the appropriateness of a specific drug for a patient is usually not difficult , for example , the presence of the hla - b5701 variant for excluding abacavir therapy in patients with hiv-1 infection , the contrary would be true when the genotype result is used for dosing adjustment . the challenges for genotype - based doing guidelines are related to the multitude of genetic and nongenetic variables that can affect drug disposition and response , the significant interindividual variabilities in activities of most of the metabolizing enzymes , and the possibility of phenocopying with metabolic phenotype change in the presence of drug - drug interaction [ 44 , 45 ] . this difference in interpretation complexity related to the intended use of the test is likely one of the reasons for the fda to previously separate pharmacogenomic biomarkers into three categories . despite these challenges , warfarin dosing recommendations based on cyp2c9 and vkorc1 genotypes have been incorporated by the fda into the updated product label in 2010 . the dose table provided in the product label was reported to provide better dose prediction than empiric dosing [ 99 , 107 ] . however , the inclusion of most of the pharmacogenomics biomarkers as informational pharmacogenetic tests by the fda on the revised labels of many drugs , without clear guidance on dosing recommendation and/or therapeutic alternatives , usually results in a knowledge vacuum for the clinicians . all stakeholders would agree that lack of sufficient pharmacogenomics education for health professionals remains a major barrier for practical implementation of pharmacogenomics within the healthcare system . the need of adequate training was echoed in a recent usa survey of more than 10,000 physicians . although 98% of all respondents agreed that the genetic profile of a patient could influence drug therapy decision , only 29% had received some pharmacogenomics education during their medical training , and only 10% felt they were adequately trained to apply the knowledge in clinical practice . although the international society for pharmacogenomics recommended incorporating pharmacogenomics education in medical , pharmacy , and health science curricula , pharmacogenomics courses or materials have only been included to a variable extent at most pharmacy schools [ 110 , 111 ] . the gap in knowledge can currently be addressed through clinical guidelines available from professional organizations ( clinical pharmacogenetics implementation consortium , the international aids society - usa panel , the european science foundation , the british association of dermatology , and the pharmacogenomics working group of the royal dutch association for the advancement of pharmacy ) [ 5563 , 112 ] , availability of simple dosing algorithm such as that for warfarin , and further effort to include specific dosing recommendation in product label [ 99 , 107 ] . the most logical setting for initial implementation of pharmacogenomics would be healthcare facilities affiliated with academic institutions . the concept of pharmacogenomics - guided drug therapy is similar to that of clinical pharmacokinetics consultation service ( cpcs ) or therapeutic drug monitoring ( tdm ) program . in this regard , the familiarity of the cpcs or tdm program should be emphasized to clinicians who view the adoption of pharmacogenomics with some skepticism . likewise , hospitals with established cpcs or tdm program might find the task of introducing pharmacogenetic testing less formidable simply by expanding or modifying their existing clinical services . the availability of consultation service , in any format , should be complemented by educational training of clinicians to achieve specific competences . crews et al . reported significant increase in ordering of the cyp2d6 genotyping test one year after its availability via the cpcs . in a similar manner , once more clinicians are educated about the utility of pharmacogenomic approach to drug therapy , especially how to use the information , they would over time integrate pharmacogenomic findings and technologies into their practice . the importance of healthcare informatics for implementation of pharmacogenomics in clinical practice could not be overemphasized . at the level of patient care , integration of genotyping order template and/or genotype result into a robust system of electronic medical record ( emr ) with pop - up action alert and order templates for actionable pharmacogenomic tests to be used by physicians will be necessary [ 113 , 114 ] . at the level of research , the health information technology would enable organizational management of all research data and accessibility by the emr [ 115118 ] . both the patient care- and research - level informatics should incorporate updated information when available and be linked to other health informatics such as billing , clinical laboratory , and clinical trials within the healthcare facility . although adoption of emr is not universal , health information technology is a critical area for investment by healthcare system administrators , perhaps through collaborative efforts with the technology industry and the government . successful examples incorporating a coordinated team approach ( physicians , pharmacists , information technology and laboratory personnels ) with appropriate infrastructure support ( informatics ) to facilitate clinical implementation of pharmacogenomics have been reported at several institutions [ 35 ] . to fully integrate the multifacet process of the pharmacogenomics service , other organizational aspects of clinical decision support should include fostering effective communication and collaboration between laboratory staff and clinicians , creating flexible workflow with minimal disruption to the daily activities of the practitioners , delineating policies and reward systems that allow equitable schedule to minimize the additional time burdens perceived by some healthcare providers , and standardizing procedures to incorporate up - to - date pharmacogenomics - related information into formulary review and decision by the pharmacy and therapeutics committee . all these steps would facilitate implementation with minimal effect on work efficiency and cost for the healthcare system . successful implementation of pharmacogenomic biomarkers in clinical practice not only involves multidisciplinary coordination among physicians , pharmacists , clinical laboratories , health information specialists , and healthcare system administrators , but also requires collaborative efforts and willingness from the payer , a significant stakeholder in this endeavor . with the current healthcare landscape and the high cost of providing healthcare , the reimbursability of any particular test plays a significant role in deciding its implementation status in most healthcare facilities . while the cost of testing for several oncologic biomarkers and thiopurine s - methyltransferase in the united states is reimbursed in some hospitals , that is not the case for most pharmacogenomic biomarker tests . both federal and private payers are reluctant to reimburse the cost of the tests on the basis of either ( 1 ) lack of evidence of clinical utility ( which is usually associated with endorsement by professional organizations ) , ( 2 ) tests being not medically necessary ( because it has never been classified by the fda as required test ) , or ( 3 ) lack of cost - effectiveness analysis and/or comprehensive comparative effectiveness analysis . even with the product labeling information regarding the impact of cyp variants for warfarin , the centers for medicare and medicaid services recently denied coverage for genetic testing except when the test is provided for the purpose of clinical trials . this reluctance stance is consistent with the findings by cohen et al . who reported that most payers do not consider test accuracy in identifying subpopulations of interest , test cost , medication adherence , and off - label use as relevant factors in their consideration for reimbursement . in their survey of 12 payers , the most consistent determining factor is conclusive evidence linking the use of the diagnostic test with health outcome . even though most payers understand the implications of pharmacogenomics in healthcare and the potential return on investment , their reluctance to pay for diagnostic tests costing much less ( most costing $ 500 ) than what they actually pay for the more expensive drugs ( for which the diagnostic tests could be useful ) primarily reflects their expectation of demonstration of clinical utility and comparative effectiveness [ 120 , 121 ] . accordingly , inconsistent assessment of clinical utility and benefit could only result in confusion regarding the appropriate use and interpretation of biomarker - based pharmacogenomic diagnostic tests . hopefully , more realistic clinical practice guidelines from diverse groups of organizations and expert panels that take into consideration of the issues discussed earlier , would pave the way to greater extent of implementation . to that end , it is of note that regulatory guidance has been published to support the recommendation of the clinical practice guidelines . in addition , additional clarification from regulatory agencies regarding definition of clinical utility , especially in the context of distinguishing the difference between utility of a diagnostic test versus test / drug combination versus the drug itself , would be very helpful in dealing with issues of implementation decision and test reimbursement . it should also be noted that even for trastuzumab , which is reimbursed by most insurers , there have been few cost - effectiveness analysis of her2 protein expression and treatment with trastuzumab . for most pharmacogenomic biomarkers , the ideal analyses might not be available until years after the diagnostic test is marketed . with limited comprehensive pharmacoeconomic data for cost - effectiveness evaluations [ 124 , 125 ] , other evaluation approaches ranging from comparing per - patient cost for specific clinical outcome within in - patient setting to decision model - based study that utilizes simulated patient cohort [ 9496 ] should be considered . in addition , all stakeholders should recognize that a negative cost - effectiveness conclusion based primarily on high cost of genotyping needs to be interpreted with the high likelihood of lower cost of genotyping in the foreseeable future . since revenue generation from a pharmacogenomic diagnostic companion test would likely be significantly less than that for a drug , there is not much incentive for pharmaceutical companies to include a thorough cost - effectiveness analysis as part of drug development . with much less financial resources than pharmaceutical companies , the lack of incentive for conducting similar evaluations also applies to diagnostic companies developing the biomarkers . in a way similar to the mutually beneficial codevelopment of proprietary drug and diagnostic test [ 126 , 127 ] , one possible solution is for diagnostic companies to collaborate with other stakeholders , such as pharmacy benefit manager ( pbm ) , to generate the evidence deemed necessary for reimbursement by both private payers and regulatory agencies . medco is the first pbm to use claims data in demonstrating a 28% reduction in bleeding or thromboembolic events in patients whose physicians were provided with cyp2c9 and vkorc1 genotypes results , when compared to patients without genetic testing . concurrent with the clinical effectiveness data is a $ 910 cost saving over a 6-month study period in the genotyped group . this type of economic impact data for pharmacogenomic testing could be used as evidence of cost - effectiveness to insurance payers and administrators of healthcare systems for consideration of potential implementation . given the dilemma of insistence of evidence - based data for reimbursement and the limited financial resource of most diagnostic companies in developing the biomarker , some paradigm shifts in thinking about approaches to reimbursement decision could be offered to the payers . instead of a universal reimbursement for all patients tested for a pharmacogenomic biomarker , an action - based reimbursement could be instituted . using clopidogrel as an example , the differential reimbursement could take the form of no payment for the cyp2c19 genotype test , if no pci is performed and clopidogrel is not prescribed , or even different amount of payment based on the risk of pci . this differential pay concept is currently in place for most prescription drugs in the form of copayment , as well as in coverage amount between within - network versus out - of - network physician visits . adopting such approach would lessen the financial burden for payer since the cost of the one - time test could be easily covered through cost saving associated with not using the drug when it is ineffective or harmful in specific patient populations , and it could provide a work - around to some payers ' insisting on conclusive evidence of linking diagnostic tests to health outcomes . implementation of pharmacogenomic testing could result in situations where an individual 's disease or medical condition is revealed to other parties , however unintended , as well as potential for discrimination and ineligibility for employment and insurance . therefore , even though the public is in general receptive to genetic - based prescribing [ 129 , 130 ] , effort should be directed towards alleviating their concern regarding privacy and confidentiality for the purposes of employment and insurance coverage decisions . they should be informed that there are ways to both protect patients ' privacy whilst at the same time promote the pharmacogenomic implementation in clinical practice [ 131 , 132 ] . in addition , provisions from the 2008 genetic information nondiscrimination act were designed to protect individuals from genetic discrimination . addressing these concerns also encourages informed patients to participate in necessary research [ 115 , 133 ] , for example , comparative effectiveness requested by other stakeholders , as well as facilitate healthcare professionals ' willingness to fully integrate genomic services into clinical practice . despite this , other existing concerns include ownership of genetic materials , availability and access to the information ( both locally and across different health system facilities similar to that of the veterans affairs emr ) , and patient 's awareness of the consequences of storing genetic materials and phenotypic data . these concerns would need to be addressed to the satisfaction of all stakeholders , especially the patients . most discussions and debates on the ethical , legal , and social implications of genetic tests usually make few distinctions between pharmacogenomic biomarkers designed for drug therapy individualization and genetic tests predicting disease susceptibility that usually carry a much greater potential for abuse . for the purpose of implementation , it would seem appropriate that consent for pharmacogenomic biomarker tests designed to individualize their drug therapy ( choice and/or dosage regimen ) not be treated the same extent of scrutiny and requirement as genetic testing for disease susceptibility . a lessening in regulation and consent requirements for pharmacogenomic markers might make it easier for their implementation . however , this issue of is very much open for further discussion before consensus can be made . social concerns also arise from clinical implementation of pharmacogenomic biomarkers within the healthcare systems . in the united states , patients are required to pay for some of the cost of the medical service , either in the form of copayment or coinsurance . therefore , an individual patient 's socioeconomic status could preclude any potential beneficial pharmacogenomic test information and exacerbate health - care disparities among different patients . in addition , for patients who are identified by pharmacogenomic test either as nonresponders or at high risk of adverse drug reaction to a specific drug , the use of pharmacogenomic test as a gatekeeper of accessibility to drug treatment might pose a problem if there is no suitable alternative drug available . as discussed earlier in this paper , carriers of the del allele of the 141c ind / del polymorphism of the dopamine d2 receptor gene are predicted to have poor response to antipsychotic treatment ; yet , all currently marketed antipsychotic treatments possess d2 blockade . is it ethical or appropriate if the patient and/or the physician decide to use a drug regardless of the unfavorable response and/or risk associated with a specific genotype ? these are relevant questions since the clinical validity and clinical utility of most pharmacogenomic tests have not been universally accepted in clinical practice . if a pharmacogenetic test ( e.g. , cyp2c29 ) is used to guide therapy with one drug ( e.g. , warfarin ) and the patient is later prescribed another drug that is also affected by the gene previously tested ( e.g. , phenytoin ) , should the clinician be responsible to act on the genotype results when dosing the second drug ? if the answer is affirmative , then some point - of - care mechanism must be in place , for example , in an emr with pop - up action alert containing the pharmacogenomic information , so that the clinician is aware of genetic test results relevant to the prescribed drug . the immediate implication with availability of pharmacogenomic information within the emr is that the information should not be ignored for clinical , ethical , and legal reasons . pharmacogenomic biomarker tests are a subset of the increasing universe of genetic tests advertised over the internet directly to the consumer . most of these direct - to - consumer ( dtc ) genetic tests are home brew and not subject to regulatory oversight by the fda and/or clia compliance for test quality standards and proficiency . in addition , companies selling dtc genetic tests can develop and market them without establishing clinical utility , which contrasts significantly to that demanded for pharmacogenomic biomarkers discussed earlier in this paper . the lack of regulatory oversight and concern of test validity likely contribute to the conclusion that most dtc genetic tests are not useful in predicting disease risk [ 134 , 135 ] . current knowledge suggests that genomic profiling based on a single snp , a common feature to most dtc genetic tests , is not necessarily clinically accurate or useful . in this regard , the recent report of a dtc genome - wide platform could provide a useful example of the impact of pharmacogenomic profiling on patient care . despite the increased consumer desire for health - related information and personalized medicine , most patients would need the help of clinicians to differentiate the relevance of different pharmacogenomics tests . this underscores the importance of educating clinicians and preparing them to provide the appropriate test interpretation for clinical decision - making . incorporating pharmacogenomics into the entire drug development process holds significant potentials for more efficient and effective clinical trials as well as financial implications for the industry . however , the issues of sufficient sample size , the cost and time associated with conducting a rct to address a specific study hypothesis , and the logistics of ensuring privacy concerns of institutional review board with possible delay in study approval and subject enrollment have posted a significant challenge and deterrent for the industry to fully incorporate pharmacogenomics in different phases of drug development . in addition , the blockbuster drug concept and its financial impact on revenue have historically played a major role in pharmaceutical drug development . as such , the concept of pharmacogenomics and the resultant segmented ( and smaller ) market tailored to a subpopulation with specific genotype however , trastuzumab provides a good example of the benefit of paradigm shift in thinking about market share and revenue . the manufacturer 's development of trastuzumab along with the diagnostic device results in capturing the market share associated with breast cancer drug treatment in all , albeit at a smaller number , of the women overexpressing the her2 protein . there are additional drug development advantages associated with this mental shift in business model from the traditional approach of product differentiation to the new commerce of market segmentation , sometimes even with little or no competition . identifying patients likely to respond to participate in clinical trials could enable benefits to be shown in a smaller number of patients , resulting in more efficient phases ii and iii studies conducted in shorter time frame and reducing the overall cost of drug development . it could also screen out patients likely to have unfavorable side effects that only appear in phase iv postmarketing surveillance studies , and such undesirable events sometime could lead to the inevitable and unfavorable outcomes of postmarketing product recall and litigation . the litigation and financial burden could be further minimized if the pharmaceutical company works with regulatory agencies to incorporate the pharmacogenomic information into a drug label that more accurately describes contraindications , precautions , and warnings . finally , as indicated earlier in this paper , beneficial partnership to develop and market a companion diagnostic test can also lead to additional revenue stream . with more than 50% of new chemical entities failing in expensive phase iii clinical trials , high attrition rate in drug development is a well - known fact for the pharmaceutical industry , and a much less discussed and explored role of pharmacogenomics is the potential of rescuing drugs that fail clinical trials during drug development . the prime example for this benefit is gefitinib , which originally was destined to failure because only a small number of patients with small cell lung cancer responded to the drug . however , in 2004 , published results showed that tumor response to the drug was linked to mutations in her1 . subsequently , development of pharmacogenomic biomarker tests for her1 mutations in patients enables identification of responders for gefitinib [ 139143 ] . this example showed that investigational drugs found to be ineffective or unsafe during phase ii or iii clinical trials might deserve a second look from the perspective of pharmacogenomics . another example is lumiracoxib , a selective cyclooxygenase-2 inhibitor that was withdrawn in 2005 from most global pharmaceutical markets because of hepatotoxicity . recently , singer et al . reported a strong association between patients with hla - dq variant alleles , especially hla - dqa10102 , and elevated transferase levels secondary to lumiracoxib - related liver injury [ 144 , 145 ] . as a result , the manufacturer of lumiracoxib has submitted an application to the ema for its use in targeted subpopulations . therefore , as demonstrated by gefitinib and possibly lumiracoxib , failing drugs can be further developed with a smaller target population with the genetic profile predictive of improved efficacy and/or reduced toxicity . this result can then be used for approval with appropriate product label containing the pharmacogenomic information . in reality , a go - ahead decision by the pharmaceutical company for such drug rescue with potential drug approval is dependent not only on the cost and time associated with developing a companion diagnostic test but also measurable better efficacy than competitor drugs in a smaller number of patients . to facilitate this aspect of drug development , regulatory decision incentives in the form of conditional approval with subsequent requirement of phase iv trial or approval similar to those developed and submitted under the orphan drug act could go a long way to provide sufficient incentive for the pharmaceutical industry . regulatory agencies worldwide , primarily the fda , the ema , and the japanese pharmaceuticals and medical devices agency , have recognized the opportunity to utilize pharmacogenomics in predicting drug response and incorporated pharmacogenomic information into revised labels of approved drugs as well as regulatory review , for example , by the iprg of the fda , that is independent of the drug review itself . nevertheless , relevant drug efficacy and safety data and issues that are important for regulatory decision - making were developed long before the era of pharmacogenomics , and it is unclear how traditional regulatory review would approach the inclusion of any pharmacogenomic data in a new drug application ( nda ) package . as described earlier , the fda has developed multidisciplinary workshop as well as regulatory initiatives such as the voluntary exploratory data submission in the usa , and the pharmacogenomics briefing meetings in europe and japan have attempted to encourage the use and submission of pharmacogenomic data by the pharmaceutical industry . however , concerns and questions remain regarding what type of pharmacogenomic data is necessary and when they should be incorporated in the nda process . although significant scientific and technological advances enable identification of variants in ( or haplotypes linked to ) genes that regulate the disposition and target pathways of drugs , translating the pharmacogenomic findings into clinical practice has been met with continued scientific debates , as well as commercial , economical , educational , ethical , legal , and societal barriers . despite the well - known potentials of improving drug efficacy and safety , as well as the efficiency of the drug development process , the logistical issues and challenges identified for incorporating pharmacogenomics into clinical practice and drug development could only be addressed with all stakeholders in the field working together and occasionally accepting a paradigm change in their current approach .

the mapping of the human genome and subsequent advancements in genetic technology had provided clinicians and scientists an understanding of the genetic basis of altered drug pharmacokinetics and pharmacodynamics , as well as some examples of applying genomic data in clinical practice . this has raised the public expectation that predicting patients ' responses to drug therapy is now possible in every therapeutic area , and personalized drug therapy would come sooner than later . however , debate continues among most stakeholders involved in drug development and clinical decision - making on whether pharmacogenomic biomarkers should be used in patient assessment , as well as when and in whom to use the biomarker - based diagnostic tests . currently , most would agree that achieving the goal of personalized therapy remains years , if not decades , away . realistic application of genomic findings and technologies in clinical practice and drug development require addressing multiple logistics and challenges that go beyond discovery of gene variants and/or completion of prospective controlled clinical trials . the goal of personalized medicine can only be achieved when all stakeholders in the field work together , with willingness to accept occasional paradigm change in their current approach .

1. Introduction 2. Scientific Challenges and Complexity 3. Integration of Pharmacogenomic Biomarker within the Healthcare System 4. Incorporating Pharmacogenomics into Drug Development 5. Conclusion

this study combines four coordinated protocols sharing the same dias artificial pancreas technology conducted at the universities of padova ( italy ) and montpellier ( france ) , the university of virginia ( uva ) , and the sansum diabetes research institute , santa barbara , california . to test whether a smart phone is capable of running outpatient closed - loop control , we have configured a system comprising available components , which were linked as follows : cgm idex dias ( running all closed - loop computations , user interface , and communications to peripheral devices ) idex pump . the idex is an experimental device manufactured by insulet ( bedford , ma ) , which combines a dexcom seven plus receiver and omnipod insulin pump . in addition , dias transferred data in real time to a central location allowing remote monitoring of patient state and system functions . the primary engineering end point was the percent time with all system communications working properly ; the protocol criterion for success in this early feasibility study was this time reaching > 80% of the total time of investigation . secondary end points included the estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and control algorithm performance . the clinical goal was to assess patients and clinicians subjective impressions of the system , i.e. , the feasibility of its ambulatory use , including patient usability and wearability . a total of 20 adults ( age 2165 years ) with type 1 diabetes were studied ( 5 subjects at each site ) . before the tests , a pilot subject was performed in italy , france , and in the united states . all participants were experienced insulin pump users and were required to have the following : prestudy hba1c of 69% ; predefined insulin pump parameters for basal rates , carbohydrate ratios , and insulin sensitivity factors ; and proper mental status / cognition . the exclusion criteria were directed toward safety and included recent history of diabetic ketoacidosis or severe hypoglycemia , pregnancy , breastfeeding , or intention of becoming pregnant ( females ) , uncontrolled arterial hypertension , and conditions that may increase the risk of hypoglycemia or infections . in addition , the united states based studies received food and drug administration approval ( ide # g120032 ) and the european studies received appropriate national - level certifications . all studies were registered with clinicaltrials.gov ( nct01578980 for uva / sansum , nct01447992 for padova , and nct01447979 for montpellier ) . after consent and screening , subjects were trained to use the omnipod insulin pump ( insulet ) and participated in a 3- to 7-day pump initiation if needed . two dexcom seven plus sensors ( dexcom , san diego , ca ) were inserted 2472 h before admission ; throughout the trials , the sensors were calibrated per manufacturer s instructions using commercial glucometers . a calibration was performed before dinner at 7:00 p.m. , thereby allowing for further system - required calibrations to be performed during the timeframes before dinner and before breakfast . per food and drug administration recommendation , an additional ( one - time ) calibration was entered by the study staff if there was a discrepancy in the two sensor readings of 20% or if the cgm was reading < 70 mg / dl and the hemocue value was > 85 mg / dl . participants in italy , france , and virginia stayed at hotels , and participants in california resided at a guest house like outpatient research unit of the sansum consisting of a living room , kitchen , four bedrooms , and bathrooms . the participants in the european studies were admitted individually , one subject at a time ; uva had both single and double admissions ; at sansum , all five subjects were admitted concurrently . subjects checked in by 5:00 p.m. and met with the study team , which confirmed that the subjects had brought their insulin , pump supplies , and regular medications . the subject s pump was removed and the study pump containing the subject s insulin was started . connections were established between dias and one sensor designated as primary ( via the idex ) , the insulin pump ( via the idex ) , and the remote monitoring site . the subject was then introduced to dias operation ; the orientation took 1520 min to complete . the dias user manual ( supplementary data ) and advice from the study team were available to the subjects at all times . after this introduction , the subjects were in charge of their interactions with dias , controlling the system via its graphical user interface . the protocol continued for 42 h. during the first evening / night of study , dias was used in open - loop mode with the subject s home insulin parameters . at 7:00 a.m. on day 2 , the system was switched into closed - loop mode and remained in closed - loop control for 29 h until the subject was discharged at 12:00 p.m. on day 3 . meals were delivered to the patient s room from local restaurants or consumed at local dining facilities ( e.g. , dining out at a restaurant in padova or a hotel buffet breakfast at uva ) . the carbohydrate content of the meals was estimated by the subject and proper entry of the desired carbohydrate amount into dias was confirmed by the study physician , but there were no dietary restrictions . when the subjects were outside of their room , they were accompanied by a member of the study team and dias were remotely monitored continually . figure 1 describes the timeline of the studies in europe and in the united states . protocol design in european ( a ) and united states ( b ) investigation centers . 1b ) were as follows : in padova and montpellier , the patient was moved to the hospital at 7:00 a.m. on day 2 of the study for initiation of closed - loop control and remained in the hospital for 10 h before returning to the hotel for the rest of the study ; in the united states studies the control algorithm was switched into safety - only mode for the night ( 11:00 p.m. to 7:00 a.m. ) , as requested by the food and drug administration . a study physician , a nurse , and a technician were located in nearby rooms to provide assistance if needed . reference blood glucose readings were measured simultaneously by finger stick with a hemocue ( hemocue ab , ngelholm , sweden ) and a commercial glucometer beginning at 7:00 p.m. on the evening of admission and continuing every 2 h during the day . overnight , there were no scheduled finger sticks ; reference blood glucose measurements were taken only if dias or the secondary sensor alarm indicated hypoglycemia or hyperglycemia , or if the two sensors had readings diverging by > 20% . nursing staff checked dias and secondary cgm readings hourly overnight and system alarms were monitored remotely for the dias and with a baby monitor to capture alarms from the secondary cgm . any dias hypoglycemia red - light warning triggered treatment with 15 g fast - acting carbohydrate ( e.g. , juice ) , whereas hyperglycemia red - light warnings prompted checking the insulin pump for occlusion or malfunction . mmol / l ( 250 mg / dl ) was followed by a -hydroxybutyrate test ( finger stick precision xtra -ketone measurement ) ; confirmed -hydroxybutyrate level > 0.6 mmol / l was a criterion for discontinuation of the trial . any hemocue reading < 80 mg / dl was followed - up with additional finger sticks at least every 15 min and any hemocue blood glucose < 70 mg / dl was treated with fast - acting glucose . the hub of the dias system was an off - the - shelf smart phone running the android operating system . to ensure the operation of the smart phone as a medical device , its operating system was modified to disable processes not related to closed - loop control operation and to include self - checks of system integrity . the communications between dias , the idex , and the pump and the sensor were wireless , giving the patient the freedom to be fully detached from the dias controller . the system components worn by the patient included an insulet omnipod insulin pod and a dexcom seven plus sensor / transmitter . the patient additionally wore a pouch containing a communication box ( either viliv s5 tablet or galaxy nexus phone ) attached to the idex . the idex and the communication box were only needed for automated data transfer and pump control at this early feasibility stage . these devices did not have any computing or patient interaction functions and were abandoned in subsequent studies . the subject controlled dias using graphical user interface , which allowed the following : initializing the system with the average daily insulin dose , basal rate , carbohydrate ratio , and correction factor ; displaying cgm traces and insulin delivery graphs ; and real - time interaction , such as entries of sensor calibrations , meal carbohydrate content , premeal capillary glucose level , and other information the subject wished to provide ( e.g. , exercise or hypoglycemia treatment ) . two traffic - light signals presented the degree of risks for hypoglycemia or hyperglycemia as follows : green light , no risks detected ; yellow light , the system is working actively to mitigate the risks by either attenuating insulin delivery if hypoglycemia is anticipated or administering correction insulin if hyperglycemia is predicted ; and red light , which signifies that risks can not be eliminated by adjustment of insulin alone and intervention is required to either consume carbohydrate or ensure that insulin is delivered properly . dias operated in two modes , open - loop ( first 13 h of each study ) controlling the pump per each patient s preset basal / bolus delivery instructions and displaying cgm and insulin delivery information or closed - loop ( hours 1442 of each study ) running a closed - loop control algorithm . both modes of operation included fully automated transfer of data from the sensor to dias and commands from dias to the insulin pump . user input was required only before meals and whenever the system signaled imminent risk for hypoglycemia or hyperglycemia . the closed - loop control algorithm included two modules : 1 ) safety supervision responsible for prediction of hypoglycemia , attenuation , or discontinuation of insulin delivery if hypoglycemia is anticipated and warnings if hypoglycemia is imminent and can not be prevented by insulin discontinuation alone ( 19 ) and 2 ) range - correction module responsible for injecting correction boluses . the clinical use of this algorithm is described in detail in a recent publication as standard control to range ( 12 ) ; details on its engineering architecture also have been published ( 20 ) . occasional cgm data loss ( up to 20 min ) did not stop the operation of the controller ; during loss of pump communication , insulin was not delivered . in addition , dias transmitted data in real time through either 3 g ( telephone network ) or wifi to two servers ( uva and montpellier ) , which allowed team members to log - in for remote observation from their locations . the server connections were one - directional : dias transmitted data out but could not be controlled from a remote location for safety reasons . the transmitted data contained glucose traces , insulin infusion by the pump , and technical information about the functioning of the control algorithm but did not contain any subject identifiers ; the monitoring web sites were password - protected . achieving statistical significance was not an objective of this early - feasibility investigation . the data analysis corresponded to the goals of the study and included estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and percent time of active system operation . post hoc analyses included t test and nonparametric comparisons of open versus closed - loop parameters of glucose control observed during the study ; however , the study was not powered for this outcome . before inclusion in the analyses , cgm data were sent through retrospective recalibration using reference blood glucose readings as discussed in a recent editorial ( 21 ) . a total of 20 adults ( age 2165 years ) with type 1 diabetes were studied ( 5 subjects at each site ) . before the tests , a pilot subject was performed in italy , france , and in the united states . all participants were experienced insulin pump users and were required to have the following : prestudy hba1c of 69% ; predefined insulin pump parameters for basal rates , carbohydrate ratios , and insulin sensitivity factors ; and proper mental status / cognition . the exclusion criteria were directed toward safety and included recent history of diabetic ketoacidosis or severe hypoglycemia , pregnancy , breastfeeding , or intention of becoming pregnant ( females ) , uncontrolled arterial hypertension , and conditions that may increase the risk of hypoglycemia or infections . all protocols were approved by the review boards of the participating institutions . in addition , the united states based studies received food and drug administration approval ( ide # g120032 ) and the european studies received appropriate national - level certifications . all studies were registered with clinicaltrials.gov ( nct01578980 for uva / sansum , nct01447992 for padova , and nct01447979 for montpellier ) . after consent and screening , subjects were trained to use the omnipod insulin pump ( insulet ) and participated in a 3- to 7-day pump initiation if needed . two dexcom seven plus sensors ( dexcom , san diego , ca ) were inserted 2472 h before admission ; throughout the trials , the sensors were calibrated per manufacturer s instructions using commercial glucometers . a calibration was performed before dinner at 7:00 p.m. , thereby allowing for further system - required calibrations to be performed during the timeframes before dinner and before breakfast . per food and drug administration recommendation , an additional ( one - time ) calibration was entered by the study staff if there was a discrepancy in the two sensor readings of 20% or if the cgm was reading < 70 mg / dl and the hemocue value was > 85 mg / dl . participants in italy , france , and virginia stayed at hotels , and participants in california resided at a guest house like outpatient research unit of the sansum consisting of a living room , kitchen , four bedrooms , and bathrooms . the participants in the european studies were admitted individually , one subject at a time ; uva had both single and double admissions ; at sansum , all five subjects were admitted concurrently . subjects checked in by 5:00 p.m. and met with the study team , which confirmed that the subjects had brought their insulin , pump supplies , and regular medications . the subject s pump was removed and the study pump containing the subject s insulin was started . connections were established between dias and one sensor designated as primary ( via the idex ) , the insulin pump ( via the idex ) , and the remote monitoring site . the subject was then introduced to dias operation ; the orientation took 1520 min to complete . the dias user manual ( supplementary data ) and advice from the study team were available to the subjects at all times . after this introduction , the subjects were in charge of their interactions with dias , controlling the system via its graphical user interface . the protocol continued for 42 h. during the first evening / night of study , dias was used in open - loop mode with the subject s home insulin parameters . at 7:00 a.m. on day 2 , the system was switched into closed - loop mode and remained in closed - loop control for 29 h until the subject was discharged at 12:00 p.m. on day 3 . meals were delivered to the patient s room from local restaurants or consumed at local dining facilities ( e.g. , dining out at a restaurant in padova or a hotel buffet breakfast at uva ) . the carbohydrate content of the meals was estimated by the subject and proper entry of the desired carbohydrate amount into dias was confirmed by the study physician , but there were no dietary restrictions . when the subjects were outside of their room , they were accompanied by a member of the study team and dias were remotely monitored continually . figure 1 describes the timeline of the studies in europe and in the united states . protocol design in european ( a ) and united states ( b ) investigation centers . 1b ) were as follows : in padova and montpellier , the patient was moved to the hospital at 7:00 a.m. on day 2 of the study for initiation of closed - loop control and remained in the hospital for 10 h before returning to the hotel for the rest of the study ; in the united states studies the control algorithm was switched into safety - only mode for the night ( 11:00 p.m. to 7:00 a.m. ) , as requested by the food and drug administration . a study physician , a nurse , and a technician were located in nearby rooms to provide assistance if needed . reference blood glucose readings were measured simultaneously by finger stick with a hemocue ( hemocue ab , ngelholm , sweden ) and a commercial glucometer beginning at 7:00 p.m. on the evening of admission and continuing every 2 h during the day . overnight , there were no scheduled finger sticks ; reference blood glucose measurements were taken only if dias or the secondary sensor alarm indicated hypoglycemia or hyperglycemia , or if the two sensors had readings diverging by > 20% . nursing staff checked dias and secondary cgm readings hourly overnight and system alarms were monitored remotely for the dias and with a baby monitor to capture alarms from the secondary cgm . any dias hypoglycemia red - light warning triggered treatment with 15 g fast - acting carbohydrate ( e.g. , juice ) , whereas hyperglycemia red - light warnings prompted checking the insulin pump for occlusion or malfunction . mmol / l ( 250 mg / dl ) was followed by a -hydroxybutyrate test ( finger stick precision xtra -ketone measurement ) ; confirmed -hydroxybutyrate level > 0.6 mmol / l was a criterion for discontinuation of the trial . any hemocue reading < 80 mg / dl was followed - up with additional finger sticks at least every 15 min and any hemocue blood glucose < 70 mg / dl was treated with fast - acting glucose . the hub of the dias system was an off - the - shelf smart phone running the android operating system . to ensure the operation of the smart phone as a medical device , its operating system was modified to disable processes not related to closed - loop control operation and to include self - checks of system integrity . the communications between dias , the idex , and the pump and the sensor were wireless , giving the patient the freedom to be fully detached from the dias controller . the system components worn by the patient included an insulet omnipod insulin pod and a dexcom seven plus sensor / transmitter . the patient additionally wore a pouch containing a communication box ( either viliv s5 tablet or galaxy nexus phone ) attached to the idex . the idex and the communication box were only needed for automated data transfer and pump control at this early feasibility stage . these devices did not have any computing or patient interaction functions and were abandoned in subsequent studies . the subject controlled dias using graphical user interface , which allowed the following : initializing the system with the average daily insulin dose , basal rate , carbohydrate ratio , and correction factor ; displaying cgm traces and insulin delivery graphs ; and real - time interaction , such as entries of sensor calibrations , meal carbohydrate content , premeal capillary glucose level , and other information the subject wished to provide ( e.g. , exercise or hypoglycemia treatment ) . two traffic - light signals presented the degree of risks for hypoglycemia or hyperglycemia as follows : green light , no risks detected ; yellow light , the system is working actively to mitigate the risks by either attenuating insulin delivery if hypoglycemia is anticipated or administering correction insulin if hyperglycemia is predicted ; and red light , which signifies that risks can not be eliminated by adjustment of insulin alone and intervention is required to either consume carbohydrate or ensure that insulin is delivered properly . dias operated in two modes , open - loop ( first 13 h of each study ) controlling the pump per each patient s preset basal / bolus delivery instructions and displaying cgm and insulin delivery information or closed - loop ( hours 1442 of each study ) running a closed - loop control algorithm . both modes of operation included fully automated transfer of data from the sensor to dias and commands from dias to the insulin pump . user input was required only before meals and whenever the system signaled imminent risk for hypoglycemia or hyperglycemia . the closed - loop control algorithm included two modules : 1 ) safety supervision responsible for prediction of hypoglycemia , attenuation , or discontinuation of insulin delivery if hypoglycemia is anticipated and warnings if hypoglycemia is imminent and can not be prevented by insulin discontinuation alone ( 19 ) and 2 ) range - correction module responsible for injecting correction boluses . the clinical use of this algorithm is described in detail in a recent publication as standard control to range ( 12 ) ; details on its engineering architecture also have been published ( 20 ) . occasional cgm data loss ( up to 20 min ) did not stop the operation of the controller ; during loss of pump communication , insulin was not delivered . in addition , dias transmitted data in real time through either 3 g ( telephone network ) or wifi to two servers ( uva and montpellier ) , which allowed team members to log - in for remote observation from their locations . the server connections were one - directional : dias transmitted data out but could not be controlled from a remote location for safety reasons . the transmitted data contained glucose traces , insulin infusion by the pump , and technical information about the functioning of the control algorithm but did not contain any subject identifiers ; the monitoring web sites were password - protected . the data analysis corresponded to the goals of the study and included estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and percent time of active system operation . post hoc analyses included t test and nonparametric comparisons of open versus closed - loop parameters of glucose control observed during the study ; however , the study was not powered for this outcome . before inclusion in the analyses , cgm data were sent through retrospective recalibration using reference blood glucose readings as discussed in a recent editorial ( 21 ) . the focus of this investigation was on the concept of using dias as a smart phone based control algorithm and user interface host . we assessed dias in terms of human factors and usability , system and component performance , performance of the control algorithm , utility of remote monitoring , and clinical events . before this study , a formative evaluation of the dias user interface heuristic evaluation ( expert review ) was followed by three focus groups with type 1 diabetic patients with varying exposure to diabetes technology ( n = 13 ) . feedback was gathered on various system components addressing user interaction , system features , and capabilities . change recommendations were prioritized , and users were asked to rate the system on several criteria . users indicated the importance of maintaining all existing insulin pump and cgm device functionalities ( 22 ) . the dias graphical user interface ( supplementary data ) proved to be reliable and well - understood by the subjects . all were able to easily navigate through the graphical user interface commands on their own in both open - loop and closed - loop modes of operation , view cgm and insulin information , and administer meal or correction boluses as needed . one subject used a hotel treadmill , one subject in italy rode a bike , one subject in france walked to nearby museums , and five subjects took a shower with the pouch hanging just outside of the shower . system wearability was evaluated in relative terms , comparing dias to previous laptop - based systems . with the transition to a smart phone as a system hub and to wireless data transmission , the weight of a closed - loop control system was reduced several - fold . figure 2a presents photos of dias displaying cgm and insulin delivery traces and the entire system worn by a study subject . dias communicated wirelessly with the idex / communication box ; these devices are placed in a pouch on the patient s belt . the idex communicated wirelessly to an omnipod insulin pump and to a dexcom sensor visible as attached on the subject . the communication range of the idex with the insulin pod and dexcom sensor was 5 inches , which necessitated the use of a belt pouch . with this set - up , the subjects were able to maintain activities of daily living , a necessary first step toward routine outpatient use . a : photos of the dias smart phone displaying cgm and insulin delivery traces ( left ) and the entire system worn by a study subject ( right ) . each of the five subjects participating simultaneously in these trials is represented by an icon on the computer screen . table 1 presents metrics of the technical performance of the artificial pancreas system overall and during the open - loop and closed - loop portions of the study . two subjects described developed hyperglycemia with ketones because of pump site or pod failures in the initial open - loop portion of the study and were rescheduled . only the completed second study data for the rescheduled subjects are included in this analysis . additionally , the three pilot subjects for each country were not included in the analysis ; the data of the first two from italy and france were recently published ( 18 ) . overall , the artificial pancreas system was functional 98% of the time , which exceeded the initially set primary end point goal of 80% . performance metrics for the functioning of the artificial pancreas system used in these studies and of its primary components one element of system connectivity should be noted . in the european studies and in the first united states based studies we used a viliv s5 tablet to communicate with the idex , which was then replaced by a more reliable samsung galaxy nexus smart phone . as evident from table 1 , this replacement had a substantial effect on system reliability , reducing almost five - fold the number of unplanned system restarts because of loss of signal transmission . because the communication box was dedicated solely to data transmission , its replacement did not affect the conceptual or the computing outcomes of the study . further , table 1 presents data on the performance of the principal system components : the cgm , dias , and the insulin pump . of particular importance for fully integrated closed - loop control is the stability of interdevice connections ( sensor idex smart phone idex insulin pump ) . table 1 presents the availability of cgm and insulin pump communications with dias during the study . although the study was not designed to test algorithm performance or to compare open - loop versus closed - loop , table 2 presents a set of glycemic control metrics and certain post hoc comparisons of open - loop versus closed - loop nights using retrospectively recalibrated cgm data ( 21 ) . the outpatient performance of the controller was similar to its inpatient performance of this same control algorithm observed in a previous study ( standard control to range , 12 ) ; thus , first indications are that a different platform ( e.g. , a smart phone ) under different outpatient conditions may achieve similar performance as a laptop - based system in the hospital . on open - loop versus closed - loop control , we observed 80 vs.72% time within target range ( p = 0.22 ) and 0.53 vs. 0.27 hypoglycemic episodes 3.9 mmol / l ( 70 mg / dl ) per 24 h ( p = 0.16 ) ; in other words , there were no significant differences between open - loop and closed - loop control overnight , which is an expected result for standard control to range ( 12 ) . performance of the control algorithm figure 2b presents a screenshot of the remote monitoring system operation during the study at sansum . each of the five subjects participating simultaneously in this study is represented by an icon on the computer screen . the icon summarizes real - time information , including patient identification number , current cgm reading and direction of change , the state of the hypoglycemia and hyperglycemia alerts , and a message informing the technician of possible risks or system malfunction . safety supervision module was active for three patients ( identification numbers 211 , 212 , and 214 ) as indicated by yellow hypoglycemia lights . each icon can be clicked during a monitoring session , which will display more detailed information for this subject , including detailed records of insulin delivery , glucose data , and the algorithm functions . throughout the study , observation of the participants was performed mainly through the remote monitoring system , which proved to be a useful tool . on six occasions during the study ( two during open - loop and four during closed - loop control ) , carbohydrate treatment was administered for blood glucose levels < 3.3 mmol / l ( 60 mg / dl ) , for a total of 0.17 events per 24 h of system operation . there were no instances of patient - initiated system shutdown , but the trials were discontinued by study staff on three occasions . the first two events occurred early , before initiation of closed - loop control , and the subjects were rescheduled for subsequent admissions , which concluded successfully . subject 1 experienced hyperglycemia of 260 mg / dl with -hydroxybutyrate level of 0.7 mmol / l 2 h after the insulin pump was initiated ( consistent with pod compared with insertion site failure ) . after the connection was reestablished with a new tablet , the insulin pod alarmed , prompting a pod change . the new pod occluded ( blood noted in pod tubing ) , resulting in -hydroxybutyrate of 1.3 mmol / l . 1 ) with the range controller switched off , delivering only basal rate ( 4.1 units in the previous 6 h ) . the subject s cgm glucose was noted to increase from 180 mg / dl to 295 mg / dl over the final 2 h , suggesting that a pod occlusion ( unconfirmed ) may have contributed to this event . these three patients were treated with subcutaneous insulin , resulting in prompt resolution of the mild ketosis . before this study , a formative evaluation of the dias user interface was conducted to evaluate the feasibility of the design for patient use . heuristic evaluation ( expert review ) was followed by three focus groups with type 1 diabetic patients with varying exposure to diabetes technology ( n = 13 ) . feedback was gathered on various system components addressing user interaction , system features , and capabilities . change recommendations were prioritized , and users were asked to rate the system on several criteria . users indicated the importance of maintaining all existing insulin pump and cgm device functionalities ( 22 ) . the dias graphical user interface ( supplementary data ) proved to be reliable and well - understood by the subjects . all were able to easily navigate through the graphical user interface commands on their own in both open - loop and closed - loop modes of operation , view cgm and insulin information , and administer meal or correction boluses as needed . one subject used a hotel treadmill , one subject in italy rode a bike , one subject in france walked to nearby museums , and five subjects took a shower with the pouch hanging just outside of the shower . system wearability was evaluated in relative terms , comparing dias to previous laptop - based systems . with the transition to a smart phone as a system hub and to wireless data transmission , figure 2a presents photos of dias displaying cgm and insulin delivery traces and the entire system worn by a study subject . dias communicated wirelessly with the idex / communication box ; these devices are placed in a pouch on the patient s belt . the idex communicated wirelessly to an omnipod insulin pump and to a dexcom sensor visible as attached on the subject . the communication range of the idex with the insulin pod and dexcom sensor was 5 inches , which necessitated the use of a belt pouch . with this set - up , the subjects were able to maintain activities of daily living , a necessary first step toward routine outpatient use . a : photos of the dias smart phone displaying cgm and insulin delivery traces ( left ) and the entire system worn by a study subject ( right ) . each of the five subjects participating simultaneously in these trials is represented by an icon on the computer screen . table 1 presents metrics of the technical performance of the artificial pancreas system overall and during the open - loop and closed - loop portions of the study . two subjects described developed hyperglycemia with ketones because of pump site or pod failures in the initial open - loop portion of the study and were rescheduled . only the completed second study data for the rescheduled subjects are included in this analysis . additionally , the three pilot subjects for each country were not included in the analysis ; the data of the first two from italy and france were recently published ( 18 ) . overall , the artificial pancreas system was functional 98% of the time , which exceeded the initially set primary end point goal of 80% . performance metrics for the functioning of the artificial pancreas system used in these studies and of its primary components one element of system connectivity should be noted . in the european studies and in the first united states based studies we used a viliv s5 tablet to communicate with the idex , which was then replaced by a more reliable samsung galaxy nexus smart phone . as evident from table 1 , this replacement had a substantial effect on system reliability , reducing almost five - fold the number of unplanned system restarts because of loss of signal transmission . because the communication box was dedicated solely to data transmission , its replacement did not affect the conceptual or the computing outcomes of the study . further , table 1 presents data on the performance of the principal system components : the cgm , dias , and the insulin pump . of particular importance for fully integrated closed - loop control is the stability of interdevice connections ( sensor idex smart phone idex insulin pump ) . table 1 presents the availability of cgm and insulin pump communications with dias during the study . although the study was not designed to test algorithm performance or to compare open - loop versus closed - loop , table 2 presents a set of glycemic control metrics and certain post hoc comparisons of open - loop versus closed - loop nights using retrospectively recalibrated cgm data ( 21 ) . the outpatient performance of the controller was similar to its inpatient performance of this same control algorithm observed in a previous study ( standard control to range , 12 ) ; thus , first indications are that a different platform ( e.g. , a smart phone ) under different outpatient conditions may achieve similar performance as a laptop - based system in the hospital . on open - loop versus closed - loop control , we observed 80 vs.72% time within target range ( p = 0.22 ) and 0.53 vs. 0.27 hypoglycemic episodes 3.9 mmol / l ( 70 mg / dl ) per 24 h ( p = 0.16 ) ; in other words , there were no significant differences between open - loop and closed - loop control overnight , which is an expected result for standard control to range ( 12 ) . figure 2b presents a screenshot of the remote monitoring system operation during the study at sansum . each of the five subjects participating simultaneously in this study is represented by an icon on the computer screen . the icon summarizes real - time information , including patient identification number , current cgm reading and direction of change , the state of the hypoglycemia and hyperglycemia alerts , and a message informing the technician of possible risks or system malfunction . safety supervision module was active for three patients ( identification numbers 211 , 212 , and 214 ) as indicated by yellow hypoglycemia lights . each icon can be clicked during a monitoring session , which will display more detailed information for this subject , including detailed records of insulin delivery , glucose data , and the algorithm functions . throughout the study , observation of the participants was performed mainly through the remote monitoring system , which proved to be a useful tool . on six occasions during the study ( two during open - loop and four during closed - loop control ) , carbohydrate treatment was administered for blood glucose levels < 3.3 mmol / l ( 60 mg / dl ) , for a total of 0.17 events per 24 h of system operation . there were no instances of patient - initiated system shutdown , but the trials were discontinued by study staff on three occasions . the first two events occurred early , before initiation of closed - loop control , and the subjects were rescheduled for subsequent admissions , which concluded successfully . subject 1 experienced hyperglycemia of 260 mg / dl with -hydroxybutyrate level of 0.7 mmol / l 2 h after the insulin pump was initiated ( consistent with pod compared with insertion site failure ) . after the connection was reestablished with a new tablet , the insulin pod alarmed , prompting a pod change . the new pod occluded ( blood noted in pod tubing ) , resulting in -hydroxybutyrate of 1.3 mmol / l . 1 ) with the range controller switched off , delivering only basal rate ( 4.1 units in the previous 6 h ) . the subject s cgm glucose was noted to increase from 180 mg / dl to 295 mg / dl over the final 2 h , suggesting that a pod occlusion ( unconfirmed ) may have contributed to this event . these three patients were treated with subcutaneous insulin , resulting in prompt resolution of the mild ketosis . technology advancements in the past year made possible the development of dias , wearable ambulatory artificial pancreas platforms using an off - the - shelf smart phone as a computational hub . besides more user - friendly touch - screen interface and wireless connectivity , one easily quantifiable result of the transition from a laptop - based to a phone - based closed - loop control is a significant reduction in the system weight , which brings the system one important step closer to ambulatory use . ultimately , this would lead to closing the loop with a portable minimally invasive system suitable for home use . industry is currently transitioning cgms and pumps to include wireless connectivity ; thus , dias is only the first of many portable devices that will be capable of wireless data exchange and fully integrated closed - loop control . at the time of this outpatient trial , the dexcom seven plus and the omnipod insulet pump had short - range wireless capability to communicate with an idex research platform . also , for the idex to establish wireless communication with dias , an intermediary tablet ( or a cell phone ) needed to be connected to the idex . hence , there was short - range wireless communication from the patient ( wearing a pod and sensor / transmitter ) to a pouch containing the idex and tablet ( or cell phone ) , and long - range wireless communication between the pouch and the dias artificial pancreas platform . these intermediate devices are now being phased out ; communication boxes are no longer necessary . such a technology improvement was anticipated in our study ; thus , we focused on the smart phone computing and user - interface capabilities of the dias , assuming that this would be the device that is here to stay . special emphasis should be placed on the fact that the subjects were operating the system by themselves most of the time . to the best of our knowledge , this is the first trial in which the subjects were responsible for the oversight of their closed - loop systems , a step that is critical for outpatient deployment of closed - loop control . based on this feedback , we conclude that the form factor of dias as an artificial pancreas platform is appropriate for outpatient use . however , before long - term efficacy studies comparing outpatient artificial pancreas with sensor - augmented pump therapy can proceed , system wearability during daily living and the reliability of device communications must be ensured . testing of the system at four different sites in three countries and in a variety of hotel and restaurant settings using one , two , or five systems concurrently provided an opportunity to challenge dias with multiple scenarios that are likely to be encountered in nonhospital and , ultimately , home settings . in general , the technical performance of the dias system with overall operational time of 98% exceeded the set goal of 80% . in retrospect , this goal may have been conservative , but before this study it was generally unclear whether a smart phone can run closed - loop control , and there was no experience to guide the choice of this goal . the principal system components sensor , dias , and insulin pump were reasonably reliable , with 0.03 , 0.09 , and 0.12 malfunction events necessitating device replacement per 24 h , respectively . occasional cgm data points were lost ( 8.1% ) , but this did not result in skipping control cycles or discontinuation of the study ; by design , control to range would function during transient absence of cgm data because the controller intervenes only if risks for hypoglycemia or hyperglycemia are detected ( 20 ) . finally , we must emphasize the utility of remote monitoring , which was available on site and at remote locations ( i.e. , studies in europe or in california were observed from virginia and vice versa in real time ) . this was a critical aspect for patient safety that allowed close supervision so that intervention could occur quickly if needed . our system allowed monitoring concurrently multiple patients , a feature that was tested at sansum with five patients simultaneously . this feature alone will allow acceleration of the number of subjects who could be studied at the same time , reducing staffing costs and making artificial pancreas research more efficient . in summary , a wearable inexpensive closed - loop control platform ( dias ) combined with real - time remote monitoring , this system opens the possibility for large pivotal trials that will establish the artificial pancreas as a viable mainstream treatment strategy in type 1 diabetes .

objectiveto evaluate the feasibility of a wearable artificial pancreas system , the diabetes assistant ( dias ) , which uses a smart phone as a closed - loop control platform.research design and methodstwenty patients with type 1 diabetes were enrolled at the universities of padova , montpellier , and virginia and at sansum diabetes research institute . each trial continued for 42 h. the united states studies were conducted entirely in outpatient setting ( e.g. , hotel or guest house ) ; studies in italy and france were hybrid hospital hotel admissions . a continuous glucose monitoring / pump system ( dexcom seven plus / omnipod ) was placed on the subject and was connected to dias . the patient operated the system via the dias user interface in open - loop mode ( first 14 h of study ) , switching to closed - loop for the remaining 28 h. study personnel monitored remotely via 3 g or wifi connection to dias and were available on site for assistance.resultsthe total duration of proper system communication functioning was 807.5 h ( 274 h in open - loop and 533.5 h in closed - loop ) , which represented 97.7% of the total possible time from admission to discharge . this exceeded the predetermined primary end point of 80% system functionality.conclusionsthis study demonstrated that a contemporary smart phone is capable of running outpatient closed - loop control and introduced a prototype system ( dias ) for further investigation . following this proof of concept , future steps should include equipping insulin pumps and sensors with wireless capabilities , as well as studies focusing on control efficacy and patient - oriented clinical outcomes .

RESEARCH DESIGN AND METHODS Subjects Procedure Safety Technology User interface Control strategy Remote monitoring Statistical analysis RESULTS Human factors and usability System and component performance Performance of the control algorithm Utility of remote monitoring Clinical events CONCLUSIONS

participants for this study were recruited from two urban areas ( yaound , the capital city of cameroon , and bamenda , the capital city of the north western region ) and two rural areas ( mbankomo and bafut ) of cameroon . the inhabitants of the urban sites are mainly middle - class public or private sector workers or businessmen . residential houses in the urban areas are grouped closely together and arranged in blocks with paved or graded road access . in the rural areas , the rural habitation pattern is sparse , with most of the homes and farms accessed only via foot paths . as a result of the absence of complete population registers , a sampling frame was established following enumeration of eligible adults in houses in delimited areas of the study sites . eligible participants were adults aged between 25 and 55 years without previously diagnosed diabetes or any known cardiovascular disease . all 3,854 eligible participants ( rural : n = 2,238 , mean age 35.3 7.7 years ; urban : n = 1,616 , mean age 35.4 8.3 years ) in the sampling frame were approached through door - to - door recruitment and invited to participate in the study . a total of 651 volunteers ( rural : n = 303 , mean age 38.5 8.3 years ; urban : n = 348 , mean age 37.9 9.1 years ) took part in this study . volunteers were provided detailed information about the study verbally and in writing , and invited to attend a testing session at their local hospital . volunteers were asked to refrain from eating , drinking or smoking starting at 10:00 p.m. the evening prior to testing . personal and sociodemographic data , as well as data on smoking , alcohol consumption , and fruit and vegetable intake were collected by self - report using an adapted world health organization ( who ) stepwise approach to sureveillance questionnaire . ethical approval for the study was obtained from the cameroon national ethics committee , and all participants provided signed informed consent . pregnant women and other volunteers who could not take part in exercise testing according to study protocol were excluded from the study . an exercise screening questionnaire , adapted from the who rose angina questionnaire was used to screen for preexisting cardiovascular disease which would contraindicate exercise testing . free - living physical activity was measured using a combined heart rate ( hr ) and movement sensor ( actiheart ; cambridge neurotechnology , cambridge , u.k . ) which has been validated in this population in free - living adults with doubly labeled water method as criterion ( 14 ) . the individual hr response to the mechanical work during an 8-min incremental step test was used for individual calibration of hr data ( 15 ) . during the test , hr was monitored in real time and the test was aborted if hr reached 90% of the age - predicted maximum hr ( 16 ) , if the subject could not keep up with the stepping protocol , or if they requested to stop . free - living physical activity was measured in 1-min epochs using the combined hr and movement sensor over 7 continuous days . the participants were requested to carry on with their normal habitual lifestyle and wear the monitor at all times except while showering , bathing , or swimming . participants with less than 2 full days of free - living data ( n = 51 ) were not included in these analyses . hr data were preprocessed ( 17 ) and converted to energy expenditure using the individual calibration derived from the step test , and the accelerometer data were converted to energy expenditure using group acceleration equations corresponding to level walking or running ( 15 ) . minute - by - minute hr and movement - derived physical activity intensity was combined in a branched equation model ( 18 ) to calculate free - living paee . basal metabolic rate ( ~resting energy expenditure [ ree ] ) was estimated using the oxford equations ( 19 ) , and total energy expenditure ( tee ) was calculated by adding ree and a component for the thermic effect of food ( equivalent to 10% of tee ) to the derived paee . energy expenditure variables are expressed per kilogram body weight to adjust for differences in body size . the distribution of intensity of activity was expressed as average daily time ( min / day ) spent at 1 met equal to time spent asleep or sedentary , between 1 and 3 mets equal to light physical activity , above 3 mets equal to moderate - to - vigorous physical activity . an estimate of cardiorespiratory fitness ( vo2max in ml of o2/kg / min ) was calculated as the intensity of work at the age - predicted maximum heart rate ( 15 ) by extrapolation of the individual heart rate response to mechanical work derived from the step test calibration . body weight was measured using electronic clinical scales , and body composition was measured using bio - impedance ( tanita tbf-531 scales ; tanita uk , uxbridge , middlesex , u.k . ) . bmi was calculated as the ratio of body weight ( kg ) to the square of height ( m ) and categorized as normal : < 25 ; overweight : 25 to < 30 ; and obese : 30 waist circumference was measured at the level of the midpoint between the lower costal margin and the anterior superior iliac crests . measurement was made to the nearest 0.1 cm using a nonstretch fiberglass tape measure with participants wearing light indoor clothing . central obesity was defined as waist circumference 80 cm in women or 94 cm in men , or using the cut points of the national cholesterol education program - adult treatment panel iii ( ncep - atp iii ) ( 20 ) when included to define the metabolic syndrome . measurements were made on the dominant arm after 5 min of rest with the subject seated . three measurements were taken at 1-min intervals , and the average of the three constituted the final blood pressure value . capillary blood glucose readings were measured after an overnight fast of at least 8 h , and then 2 h after ingestion of 75 g of glucose dissolved in 300 ml of water . blood glucose measurements were done on whole fresh capillary blood using a hemocue b - glucose analyzer ( hemocue ab , ngelholm , sweden ) . the metabolic syndrome was defined using the ncep - atp iii criteria ( 20 ) . to demonstrate the population distribution of global metabolic health , we computed a continuous summary metabolic risk score ( z - score ) largely on the basis of the components of the metabolic syndrome . this variable was derived by standardizing and then summing waist circumference , blood pressure ( average of systolic and diastolic ) , blood glucose ( average of fasting , 30-min , and 2-h glucose ) , triglycerides , body fat , fasting insulin , and inverse hdl cholesterol . the standardization of these continuous variables was computed by subtracting the sample mean from the individual value and then dividing by the sd of the sample mean . sex - specific calculations were made for waist circumference , body fat , fasting insulin , and hdl cholesterol . fasting blood samples were collected in the mornings between 7:30 and 9:30 a.m. plasma and serum samples were separated upon collection in a refrigerated centrifuge at 4c . samples were transported on dry ice by air to cambridge ( u.k . ) and stored at 80c until further analyses . assays were performed by the national institute for health research cambridge biomedical research centre , core biochemical assay laboratory . hdl cholesterol and triglycerides were measured using automated assays on the dade behring dimension rxl analyzer ( siemens healthcare , camberley , u.k . ) . hdl was measured using a homogenous accelerator selective detergent assay and triglycerides were measured using an enzymatic assay according to manufacturers specifications . ldl was calculated by the friedwald formula ( ldl = cholesterol hdl [ triglycerides/2.2 ] ) . fasting insulin was assayed in singleton on a 1235 autodelfia automatic immunoassay system using a two - step time resolved fluorometric assay kit manufactured by perkin elmer life sciences ( wallac oy , turku , finland ) . pmol / l , 3233 split proinsulin 1% at 2,800 pmol / l , and c - peptide < 0.1% at 5,280 analyses were carried out using stata version 10.1 ( statacorp , college station , tx ) . descriptive characteristics of the study sample are presented as means with sd , adjusted means with se , or as numbers with proportions , stratified by sex - specific paee quartiles . for dichotomous grouping variables , difference in means was assessed using the student t test and difference in proportion using the test or fisher exact test . independent associations between paee and the metabolic syndrome were examined in multivariate logistic regression models . there was no paee interaction by residential site or sex on its association with the metabolic syndrome . models were either unadjusted or adjusted for age , sex , residential site , smoking , alcohol consumption , fruit and vegetable consumption , and educational level ( as a surrogate for socioeconomic status ) . to illustrate the population impact of physical inactivity on metabolic health , we calculated the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee ( q1-paee ) . the population attributable fraction was computed as = ( prevalence of q1-paee in case subjects prevalence of q1-paee in control subjects)/(1 prevalence of q1-paee in control subjects ) . free - living physical activity was measured using a combined heart rate ( hr ) and movement sensor ( actiheart ; cambridge neurotechnology , cambridge , u.k . ) which has been validated in this population in free - living adults with doubly labeled water method as criterion ( 14 ) . the individual hr response to the mechanical work during an 8-min incremental step test was used for individual calibration of hr data ( 15 ) . during the test , hr was monitored in real time and the test was aborted if hr reached 90% of the age - predicted maximum hr ( 16 ) , if the subject could not keep up with the stepping protocol , or if they requested to stop . free - living physical activity was measured in 1-min epochs using the combined hr and movement sensor over 7 continuous days . the participants were requested to carry on with their normal habitual lifestyle and wear the monitor at all times except while showering , bathing , or swimming . participants with less than 2 full days of free - living data ( n = 51 ) were not included in these analyses . hr data were preprocessed ( 17 ) and converted to energy expenditure using the individual calibration derived from the step test , and the accelerometer data were converted to energy expenditure using group acceleration equations corresponding to level walking or running ( 15 ) . minute - by - minute hr and movement - derived physical activity intensity was combined in a branched equation model ( 18 ) to calculate free - living paee . basal metabolic rate ( ~resting energy expenditure [ ree ] ) was estimated using the oxford equations ( 19 ) , and total energy expenditure ( tee ) was calculated by adding ree and a component for the thermic effect of food ( equivalent to 10% of tee ) to the derived paee . energy expenditure variables are expressed per kilogram body weight to adjust for differences in body size . the distribution of intensity of activity was expressed as average daily time ( min / day ) spent at 1 met equal to time spent asleep or sedentary , between 1 and 3 mets equal to light physical activity , above 3 mets equal to moderate - to - vigorous physical activity . an estimate of cardiorespiratory fitness ( vo2max in ml of o2/kg / min ) was calculated as the intensity of work at the age - predicted maximum heart rate ( 15 ) by extrapolation of the individual heart rate response to mechanical work derived from the step test calibration . body weight was measured using electronic clinical scales , and body composition was measured using bio - impedance ( tanita tbf-531 scales ; tanita uk , uxbridge , middlesex , u.k . ) . bmi was calculated as the ratio of body weight ( kg ) to the square of height ( m ) and categorized as normal : < 25 ; overweight : 25 to < 30 ; and obese : 30 waist circumference was measured at the level of the midpoint between the lower costal margin and the anterior superior iliac crests . measurement was made to the nearest 0.1 cm using a nonstretch fiberglass tape measure with participants wearing light indoor clothing . central obesity was defined as waist circumference 80 cm in women or 94 cm in men , or using the cut points of the national cholesterol education program - adult treatment panel iii ( ncep - atp iii ) ( 20 ) when included to define the metabolic syndrome . measurements were made on the dominant arm after 5 min of rest with the subject seated . three measurements were taken at 1-min intervals , and the average of the three constituted the final blood pressure value . capillary blood glucose readings were measured after an overnight fast of at least 8 h , and then 2 h after ingestion of 75 g of glucose dissolved in 300 ml of water . blood glucose measurements were done on whole fresh capillary blood using a hemocue b - glucose analyzer ( hemocue ab , ngelholm , sweden ) . the metabolic syndrome was defined using the ncep - atp iii criteria ( 20 ) . to demonstrate the population distribution of global metabolic health , we computed a continuous summary metabolic risk score ( z - score ) largely on the basis of the components of the metabolic syndrome . this variable was derived by standardizing and then summing waist circumference , blood pressure ( average of systolic and diastolic ) , blood glucose ( average of fasting , 30-min , and 2-h glucose ) , triglycerides , body fat , fasting insulin , and inverse hdl cholesterol . the standardization of these continuous variables was computed by subtracting the sample mean from the individual value and then dividing by the sd of the sample mean . sex - specific calculations were made for waist circumference , body fat , fasting insulin , and hdl cholesterol . fasting blood samples were collected in the mornings between 7:30 and 9:30 a.m. plasma and serum samples were separated upon collection in a refrigerated centrifuge at 4c . samples were transported on dry ice by air to cambridge ( u.k . ) and stored at 80c until further analyses . assays were performed by the national institute for health research cambridge biomedical research centre , core biochemical assay laboratory . hdl cholesterol and triglycerides were measured using automated assays on the dade behring dimension rxl analyzer ( siemens healthcare , camberley , u.k . ) . hdl was measured using a homogenous accelerator selective detergent assay and triglycerides were measured using an enzymatic assay according to manufacturers specifications . ldl was calculated by the friedwald formula ( ldl = cholesterol hdl [ triglycerides/2.2 ] ) . fasting insulin was assayed in singleton on a 1235 autodelfia automatic immunoassay system using a two - step time resolved fluorometric assay kit manufactured by perkin elmer life sciences ( wallac oy , turku , finland ) . pmol / l , 3233 split proinsulin 1% at 2,800 pmol / l , and c - peptide < 0.1% at 5,280 analyses were carried out using stata version 10.1 ( statacorp , college station , tx ) . descriptive characteristics of the study sample are presented as means with sd , adjusted means with se , or as numbers with proportions , stratified by sex - specific paee quartiles . for dichotomous grouping variables , difference in means was assessed using the student t test and difference in proportion using the test or fisher exact test . independent associations between paee and the metabolic syndrome were examined in multivariate logistic regression models . there was no paee interaction by residential site or sex on its association with the metabolic syndrome . models were either unadjusted or adjusted for age , sex , residential site , smoking , alcohol consumption , fruit and vegetable consumption , and educational level ( as a surrogate for socioeconomic status ) . to illustrate the population impact of physical inactivity on metabolic health , we calculated the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee ( q1-paee ) . the population attributable fraction was computed as = ( prevalence of q1-paee in case subjects prevalence of q1-paee in control subjects)/(1 prevalence of q1-paee in control subjects ) . descriptive characteristics of the study sample stratified by sex and rural or urban residence are presented in supplementary table 1 . clinical and metabolic characteristics of the study sample stratified by sex - specific quartiles of paee are presented in table 1 . most of the individual metabolic and clinical parameters , as well as the clustered metabolic risk score or prevalence of the metabolic syndrome showed a significant linear trend across quartiles of paee . blood lipids were generally low in this sample with no sex or rural - urban differences . prevalence of metabolic syndrome was almost fourfold lower in the highest compared with the lowest quartile of paee . clinical and metabolic characteristics by quartiles of paee in adult cameroonians ( n = 552 ) data are age - adjusted means se or n ( % ) . bg , blood glucose ; bp , blood pressure ; ms , metabolic syndrome ; wc , waist circumference ; zms , clustered metabolic risk score . urban dwellers had a significantly lower paee than rural dwellers ( 44.2 21.0 vs. 59.6 23.7 kj / kg / day , p < 0.001 ) and a higher prevalence of the metabolic syndrome ( 17.7 vs. 3.5% , p < 0.001 ) ( supplementary tables 1 and 2 ) . there was no significant difference between the sexes in metabolic risk expressed as a continuous clustered metabolic score , but urban dwellers had a significantly higher mean score than rural dwellers ( 0.80 3.67 vs. 1.11 2.90 , p < 0.001 ) . this difference was observed to correspond to a rural - to - urban right shift in the population density distribution of the clustered metabolic z - score when stratified by residential area ( fig . a similar but reverse difference was observed with the population distribution of paee , with a rural - to - urban left shift in the distribution . population distribution of paee and clustered metabolic z - score in adults in rural and urban cameroon . table 2 presents independent associations between paee quartiles and prevalent metabolic syndrome . with the lowest quartile of paee as reference , there was a strong inverse association between paee and prevalent metabolic syndrome . compared with the lowest quartile of paee , being in the 2nd quartile of paee was associated with a reduction in the risk of prevalent metabolic syndrome , which was not statistically significant ( odds ratio 0.63 [ 95% ci 0.321.25 ] ) . the risk of prevalent metabolic syndrome was significantly lower for the 3rd and 4th quartiles of paee compared with the 1st quartile ( 0.32 [ 0.14 0.73 ] and 0.19 [ 0.08 0.49 ] , respectively ) . multivariate adjustments for age , sex , residential area , smoking , alcohol drinking , fruit and vegetable consumption , and educational level as potential confounders attenuated but did not change these associations . independent associations between paee and metabolic syndrome in adult cameroonians ( n = 552 ) data are odds ratio or odds ratio ( 95% ci ) . adjusted for age , sex , residential area , smoking , alcohol consumption , fruit and vegetable consumption , and number of years of education . defining the lowest quartile of paee to represent sedentary or inactive lifestyle , the population attributable fraction of prevalent metabolic syndrome due to a sedentary or inactive lifestyle was 26.3% ( 25.3% in women and 35.7% in men ) . expressing paee as a continuous variable , and assuming a linear relationship with the metabolic syndrome , each kj / kg / day of paee was associated with a 2.1% lower risk of prevalent metabolic syndrome ( odds ratio 0.98 , p = 0.03 ) , after adjustment for potential confounders as in table 2 , model 3 . this implies that an increase of 6.5 kj / kg / day of paee would correspond to approximately 13.7% risk reduction in prevalent metabolic syndrome . this amount of paee can be achieved by 30 min / day of brisk walking . our results suggest that free - living paee was significantly and independently associated with prevalent metabolic syndrome . this association persisted after multivariate adjustments for possible confounders and was similar in both urban and rural dwellers , even though rural dwellers were more physically active and had a more favorable metabolic profile than urban dwellers . low levels of physical activity may be contributing significantly to the metabolic syndrome in this population . theoretically , these data suggest a 26.3% population attributable fraction of prevalent metabolic syndrome as a result of being in the lowest quartile of paee . however , independent of several confounders , it is likely that small changes in paee may confer significant benefits in terms of lower risk of metabolic syndrome . because of the cross - sectional design of our study , we can not infer causality . the study sample included only adults aged 2555 years without known metabolic syndrome or cvd . therefore , these results may not be directly generalizable to younger or older individuals , as well as individuals with diagnosed prevalent metabolic or cardiovascular disease . the lack of complete population registers as well as the absence of fixed house addresses or telephone numbers limited our ability to use simple random sampling of participants . our use of volunteers from a study - established sampling frame may introduce sampling bias , which would limit the generalizability of these results even to our target population . however , our sample was similar to other larger population - based surveys with respect to bmi , blood pressure , and educational level ( 21,22 ) . separate from prevalence of risk factors , one could argue that educational level may have influenced volunteering for this study . however , the proportion of volunteers in this sample having at least a secondary education ( 44% of men and 35% of women ) was comparable to data from the 2004 demographic and health survey in cameroon ( 48% of men and 32% of women ) ( 21 ) . the observed inverse association between total volume of physical activity ( paee ) with the metabolic syndrome is comparable to results from previous studies which used objective measures of physical activity ( 12,13,23 ) ( 23 ) reported a significant inverse association between paee scaled for fat - free body mass and a clustered metabolic syndrome score . however , we scaled paee for body weight , not fat - free mass . although researchers agree that paee should be expressed taking into account differences in body size , the exact scaling factor applicable to body size is still a contentious issue . during light intensity locomotion activities , which are the main component of total activity in many people , the expression of paee per kilogram body weight may be more appropriate for the habitual free - living activity pattern , and is easy to interpret for clinical or public health usage . the importance of the distribution of free - living physical activity intensities , beyond total volume of physical activity , on metabolic health is still uncertain . many studies that have examined the effect of intensity distribution of physical activity on global metabolic risk have used self - report of physical activity ( 24 ) . studies that have used objective assessment of physical activity intensity distribution are less common ( 12,13 ) . most studies confirm the deleterious effect of increasing sedentary time or decreasing moderate - to - vigorous physical activity on the metabolic syndrome ( 12,24 ) . however , the benefits of increasing overall activity compared with shifting the intensity distribution toward more vigorous activity is still inconclusive . in this study we show that objectively measured total volume of physical activity had a significant independent association with the metabolic syndrome . our use of total volume of activity may be more applicable to health promotion initiatives in regions with low literacy . it may be more feasible to encourage overall physical activity than to recommend specific subdimensions of activity to less educated people . rapid urbanization of lifestyle in sub - saharan africa is characterized by increasing prevalence and clustering of metabolic risk factors such as diabetes , high blood pressure , and obesity ( 4,25 ) . our data show an evident rural - urban gradient of paee , which mirrors progression toward western levels of habitual physical activity . this transition is not only explained by increasing frequency of high risk individuals , but by a shift in the underlying population distribution of risk factors . our use of an aggregate variable for metabolic health ( the metabolic syndrome ) is relevant for assessing the effects of physical activity level on multiple risk factors in the whole population . further , the metabolic syndrome is associated with a higher morbidity and mortality ( 3 ) . we did not collect data on energy intake in this study because of the difficulties of collecting objective dietary data in a population - based study . however , we adjusted for self - reported fruit and vegetable intake in the analyses , which would capture part of the variance associated with healthy or unhealthy dietary habits . these data suggest a significant population burden of prevalent metabolic syndrome associated with being in the lowest quartile of paee , or a mean paee of 26 7 this level of physical activity corresponds to an average of 45 met - hours / week . typically , these individuals would be spending more than 80% of their time in sedentary activities with very little physical effort . encouraging people to increase their physical activity , even through reduction of sedentary time , may confer considerable health benefits especially in the least active individuals . in conclusion , reduction in physically active lifestyles as a result of urbanization may be critical in driving the current transition toward a higher burden of metabolic diseases in this region . public health interventions to increase physical activity may be expected to significantly curb the burden of metabolic syndrome in sub - saharan africa .

objectivewe examined the independent associations between objectively measured free - living physical activity energy expenditure ( paee ) and the metabolic syndrome in adults in rural and urban cameroon.research design and methodspaee was measured in 552 rural and urban dwellers using combined heart rate and movement sensing over 7 continuous days . the metabolic syndrome was defined using the national cholesterol education program - adult treatment panel iii criteria.resultsurban dwellers had a significantly lower paee than rural dwellers ( 44.2 21.0 vs. 59.6 23.7 kj / kg / day , p < 0.001 ) and a higher prevalence of the metabolic syndrome ( 17.7 vs. 3.5% , p < 0.001 ) . in multivariate regression models adjusted for possible confounders , each kj / kg / day of paee was associated with a 2.1% lower risk of prevalent metabolic syndrome ( odds ratio 0.98 , p = 0.03 ) . this implies a 6.5 kj / kg / day difference in paee , equivalent to 30 min / day of brisk walking , corresponds to a 13.7% lower risk of prevalent metabolic syndrome . the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee was 26.3% ( 25.3% in women and 35.7% in men).conclusionsurban compared with rural residence is associated with lower paee and higher prevalence of metabolic syndrome . paee is strongly independently associated with prevalent metabolic syndrome in adult cameroonians . modest population - wide changes in paee may have significant benefits in terms of reducing the emerging burden of metabolic diseases in sub - saharan africa .

RESEARCH DESIGN AND METHODS Physical activity measurement Anthropometry and clinical measures Metabolic syndrome and clustered metabolic risk score Biochemical assays Calculations and statistical analyses RESULTS CONCLUSIONS

cardiovascular disease ( cvd ) and diabetic nephropathy ( dn ) are the major causes of mortality in individuals with type 1 diabetes ( 13 ) , and a growing body of evidence from basic and clinical studies ( 47 ) supports the relationship between cvd and renal function in health and disease . cvd and dn have been proposed to be manifestations of the same underlying pathology and also exist as interrelated risk factors ( 8,9 ) . the increased mesangial matrix associated with dn is similar to the pathophysiology of coronary atherosclerosis ( 8) . while atherosclerosis tends to remain subclinical until adulthood in individuals with type 1 diabetes ( 10 ) , adolescents with type 1 diabetes demonstrate reduced peak exercise capacity and decreased cardiac function compared with nondiabetic adolescents ( 11 ) . although the mechanisms underlying the reduced peak exercise capacity are poorly understood , we have previously shown that insulin resistance and elevated ldl cholesterol ( ldl - c ) were associated with low peak volume of oxygen ( vo2peak ) ( 11 ) . elevated albumin - to - creatinine ratio ( acr ) and glomerular filtration rate ( gfr ) , which are early manifestations of dn , are also increasingly recognized in children and adolescents with type 1 diabetes ( 1,12 ) . increased gfr was shown to be associated with increased cardiovascular mortality in adults ( 13 ) . little is known , however , about the possible associations between early renal health and exercise capacity in adolescents with type 1 diabetes , and whether this relationship is independent of insulin sensitivity . accordingly , we sought to examine the associations between markers of renal health and peak exercise capacity in adolescents with type 1 diabetes . we hypothesized that cardiopulmonary fitness would be directly associated with acr and inversely related to estimated gfr ( egfr ) in adolescents with type 1 diabetes . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test . inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) . pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) . all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) . participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained . a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st . paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) . vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) . differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the test . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test . inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) . pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) . all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) . participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained . a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents . measurements were made during bicycle ergometer testing using a metabolic cart . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st . paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) . vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) . differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the test . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr ( low < 5.50 , middle range 5.509.13 , and high > 9.13 mg / g ) were stratified into tertiles . anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . adolescents with type 1 diabetes had reduced peak exercise capacity compared with nondiabetic control subjects ( vo2peak 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ; vo2peak / lean kg 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) , despite similar age , tanner stage , bmi , and habitual level of physical activity . when adjusting for egfr , the differences in tanner stage and sex - adjusted means for vo2peak / kg ( lsms se 32.3 1.0 vs. 34.2 1.5 , p = 0.30 ) and vo2peak / lean kg ( lsms se 49.0 2.1 vs. 45.0 1.4 , p = 0.09 ) between adolescents with and without type 1 diabetes lost significance . characteristics for adolescents with type 1 diabetes data are reported as lsms se , unless otherwise indicated . data are reported as the median ( q25q75 ) . in adolescents with type 1 diabetes , egfr correlated strongly with vo2peak ( r = 0.55 , r = 30.22% , p = 0.002 ) and vo2peak / lean kg ( r = 0.44 , r = 19.58% , p = 0.02 ) , and remained significantly associated with vo2peak and vo2peak / lean kg after adjusting for sex and tanner stage ( tables 2 and 3 ) . additional adjustments for insulin sensitivity , hba1c level , sbp , or ldl - c level did not attenuate the significance of the associations among egfr , vo2peak , and vo2peak / lean kg ; however , the association between egfr and vo2peak / lean kg became less significant ( p = 0.048 ; table 3 ) . in contrast , the associations between egfr and vo2peak / kg and vo2peak / lean kg in adolescent control subjects were not significant in fully adjusted models ( data not shown ) . in contrast to egfr , lnacr did not correlate with vo2peak ( se 1.42 1.49 , p = 0.35 ) and vo2peak / lean kg ( se 2.24 1.71 , p = 0.20 ) when adjusted for sex and tanner stage . associations between egfr and vo2peak / kg data are reported as se ; p value . age was not adjusted for because it is part of the bouvet egfr equation . * adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . associations between egfr and vo2peak / lean kg data are reported as se ; p value . adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . when stratifying egfrs into tertiles , subjects in the highest egfr tertile had significantly lower vo2peak ( lsm se 26.91 1.52 vs. 35.53 1.88 , p = 0.002 ) and vo2peak / lean kg ( lsm se 39.65 2.07 vs. 46.18 2.55 , p = 0.048 ) adjusted for sex and tanner stage , compared with those in the lowest egfr tertile ( fig . 1 ) . stratifying acr into tertiles demonstrated no differences in vo2peak or vo2peak / lean kg among the three groups ( data not shown ) . tanner stage and sex - adjusted means for vo2peak / kg and vo2peak / lean kg stratified by tertiles of egfr . our major observation in this study was that in adolescents with type 1 diabetes , a disease characterized by reduced exercise capacity , egfr was independently and negatively associated with cardiopulmonary fitness , independent of insulin sensitivity and other important risk factors . we previously demonstrated reduced peak exercise capacity , and cardiac and vascular function in otherwise healthy , nonobese adolescents with type 1 diabetes , compared with well - matched nondiabetic control subjects of similar bmi , pubertal stage , and habitual level of physical activity ( 11 ) . furthermore , we found that insulin sensitivity correlated strongly with vo2peak in youths with type 1 diabetes , as did ldl - c level to a lesser degree ( 11 ) . in this report , we present for the first time an independent relationship between renal health and cardiopulmonary fitness that is independent of insulin sensitivity , ldl - c level , and other important covariates . this association may represent a novel pathway of a cardiorenal connection in type 1 diabetes separate from atherosclerosis . type 1 diabetes is associated with shortened life span , and cardiac and vascular dysfunction , independent of coronary artery disease ( 20 ) . in fact , low fitness levels in adults with and without diabetes are associated with cvd mortality and decreased longevity ( 21,22 ) . control of conventional risk factors such as glucose level , cholesterol level , and blood pressure is important , but does not abolish the cardiovascular risk in subjects with type 1 diabetes ( 2,23 ) . although not without controversy , the finnish diabetic nephropathy study ( 24 ) and the pittsburgh epidemiology of diabetes complications study ( 3 ) demonstrated no increase in mortality in adults with type 1 diabetes compared with nondiabetic control subjects in the absence of dn . in contrast , the coronary artery calcification in type 1 diabetes study ( 9 ) demonstrated strong associations among egfr , acr , and progression of coronary artery calcification in adults with type 1 diabetes in the absence of established dn , suggesting that renal health may be associated with early cardiovascular pathophysiology that may take years to manifest clinically . the specific mechanisms underlying the association between renal and cardiovascular health , however , remain unclear , but increasing evidence supports the importance of shared risk factors and pathogenic pathways , including atherosclerosis ( 6,7,11,2528 ) . reduced exercise capacity and its association with renal health highlighted in this article , taken together with the established relationship between fitness and premature cvd mortality , may provide an alternative and novel pathway explaining the premature mortality of subjects with type 1 diabetes ( 21,22 ) . there is currently no generally accepted definition for renal hyperfiltration ( 29 ) , but it is increasingly recognized that elevated gfr is an early hemodynamic abnormality seen in diabetes that is linked with an increased risk of dn ( 1 ) . the meta - analysis from the chronic kidney disease prognosis consortium ( 30,31 ) demonstrated significantly greater all - cause mortality and cardiovascular mortality in individuals with the highest gfr values ( > 105 ml / min/1.73 m ) . similarly , the renal iohexol clearance survey in tromso 6 ( renis - t6 ) recently reported ( 13 ) significantly greater odds of both carotid atherosclerosis and left ventricular hypertrophy in participants in the highest gfr quartile ( > 101.2 ml / min/1.73 m ) . it is , however , also plausible that the mechanisms underlying the association between renal health and exercise capacity in adolescents with type 1 diabetes differ from the link seen with atherosclerosis . the mechanisms responsible for the relationship between renal health and exercise capacity are not yet clear . adults with type 1 diabetes and elevated gfr demonstrate cardiovascular dysfunction , including increases in arterial stiffness and altered flow - mediated dilatation ( 32,33 ) . we previously demonstrated reduced forearm blood flow in youths with type 1 diabetes ( 11 ) as evidence of vascular dysfunction , which correlated with reduced vo2peak . moreover , alterations in arterial stiffness have also been reported to be present in youths with type 1 diabetes ( 34 ) . elevated gfr is associated with endothelial dysfunction via cyclooxygenase-2 activity and nitric oxide synthase inhibition , and relatively higher blood pressures ( 3537 ) , both of which could directly impact exercise capacity . elevated gfr is also associated with increased carotid intimal medial thickness and left ventricular hypertrophy in nondiabetic individuals , further suggesting that increased gfr potentially reflects a generalized change in vascular function rather than simply reflecting intrarenal abnormalities ( 13 ) . endothelial dysfunction could also contribute to elevated gfr , thus the relationships could be bidirectional . we did not observe a significant association between acr and peak exercise capacity , potentially because increasing evidence supports the distinction of albuminuria as a risk factor for dn and not an early phenotype of dn ( 38 ) . furthermore , the prevalence of microalbuminuria ( n = 9 ) was low due to the young age of our population . . however , to minimize the effect of sample size , we used careful and detailed physiological measurements , including gold standard fitness testing and hyperinsulinemic - euglycemic clamp studies . we also chose groups similar in habitual physical activity , pubertal stage , sex , and bmi , and controlled for prestudy diet and physical activity . another limitation to the current study included the cross - sectional design , which prevents the determination of causality , and whether the association holds true longitudinally ; for that reason , the data should be viewed as hypothesis generating . the direction of the association between renal health and exercise capacity ( i.e. , whether renal health leads to reduced exercise capacity or vice versa ) is also difficult to determine with the available data . results from this study may also not be generalizable to older people with type 1 diabetes or to cohorts with a different ethnic distribution . finally , we used an estimate of gfr to assess renal function ; however , to estimate the gfr we used the bouvet equation , which was shown to have the best performance in estimating gfr in children and adolescents with normal to elevated gfrs ( 18 ) . the mean egfr in our lean nondiabetic control subjects is slightly lower than gfrs measured by insulin level in adolescents in some studies ( 39 ) , but is consistent with gfrs estimated by creatinine and cystatin c levels in adolescents in large - scale studies ( 40 ) . in conclusion , in adolescents with type 1 diabetes , a disease characterized by reduced peak exercise capacity compared with nondiabetic adolescents , there was a strong relationship between egfr and cardiopulmonary fitness , independent of directly measured insulin sensitivity and other important risk factors . this observation provides further evidence for the broad interactions observed between renal and cardiovascular function in type 1 diabetes . further research is needed to investigate the longitudinal relationships between renal and cardiopulmonary health in adolescents with type 1 diabetes , and to elucidate the specific mechanisms underlying this relationship as potential therapeutic targets .

objectivediabetic nephropathy and cardiovascular disease are strongly related in adults with type 1 diabetes , yet little is known about this relationship in adolescents prior to the onset of detectable clinical disease . we hypothesized that cardiopulmonary fitness would be directly associated with albumin - to - creatinine ratio ( acr ) and inversely related to estimated glomerular filtration rate ( egfr ) in adolescents with type 1 diabetes.research design and methodssixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects of similar pubertal stage and bmi had insulin sensitivity ( glucose infusion rate [ gir ] ) , measured by hyperinsulinemic - euglycemic clamp , and lean body mass , measured by dexa . cardiopulmonary fitness was measured by cycle ergometry to obtain peak volume of oxygen ( vo2peak ) , and renal function was measured by egfr using the bouvet equation ( measuring creatinine and cystatin c levels ) and acr.resultsadolescents ( 15.5 2.2 years of age ) with type 1 diabetes ( 6.3 3.8 years diabetes duration ) had reduced vo2peak ( 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ) and vo2peak / lean kg ( 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) compared with nondiabetic control subjects . egfr was inversely associated with vo2peak and vo2peak / lean kg after adjusting for sex , tanner stage , gir , hba1c level , systolic blood pressure , and ldl cholesterol level ( se , vo2peak : 0.19 0.07 , p = 0.02 ; vo2peak / lean kg : 0.19 0.09 , p = 0.048 ) . moreover , participants in the highest tertile for egfr had significantly lower sex- and tanner - adjusted vo2peak and vo2peak / lean kg compared with participants in the lowest tertile.conclusionsadolescents with type 1 diabetes had reduced exercise capacity , which was strongly associated with renal health , independent of insulin sensitivity . future studies should examine the underlying interrelated pathophysiology in order to identify probable targets for treatment to reduce cardiovascular and renal complications .

Introduction Research Design and Methods Subjects Activity Questionnaires and Body Composition Exercise Testing Insulin Sensitivity Renal Measurements Statistical Analysis Results Conclusions

plain films are likely to remain the best method of imaging gas shadows for many years to come and computed tomography scanning , isotope studies and nuclear magnetic resonance are unlikely to play any major role in the initial investigation of the acute abdomen ( field et al1 ) . shortly after the discovery of the x - ray in 1895 , the first x - rays were studied for medical purposes by wilhelm rontgen . during the decades that followed , x - rays were mainly used for detecting fractures and foreign bodies and gradually for the evaluation of various other diseases , such as acute abdominal pain . approximately 4%10% of emergency department visits are due to acute abdominal pain , making it one of the most encountered complaints.2 the wide variety in presentation of symptoms and the broad spectrum of associated diseases complicates isolation of the cause of abdominal pain , which may vary from life - threatening diseases requiring emergency surgery to mild self - limiting causes.2 an early and accurate diagnosis is essential in decision - making , and insufficient workup results in unnecessary interventions or delayed treatment.24 a study performed at the university of virginia in the us compared data from patients with acute abdominal pain at three different time points over a period of 35 years , ie , 1972 , 1992 , and 2007.5 the proportion of patients presenting with acute abdominal pain as a chief complaint remained more or less stable over the years , being 4% in 1972 , 5% in 1992 , and 6.6% in 2007 . plain abdominal radiography was the only diagnostic imaging modality available in 1972 and was ordered in 43% of all patients . in 1992 , ultrasound and computed tomography ( ct ) had come into use but were barely ordered , ie , in only 6.8% of all patients . in 2007 , the use of ultrasound and ct was widespread and the use of plain abdominal radiography decreased somewhat , but was still performed in a considerable proportion of patients ( 21% ) . ct and ultrasound were used liberally , and either one of these tests was performed in 42% of all patients . these data show increased use of ct and ultrasound and decreased use of plain abdominal radiographs ( a decrease of approximately one third ) between 1992 and 2007 . in the same time period , the average time patients spent at the emergency department had risen from 2.9 hours in 1992 to 4.26 hours in 2007 . patients who had undergone a ct scan spent an average of 6.64 hours in the emergency department compared with 3.44 hours without ct evaluation . diagnostic accuracy improved over the years due to increased use of ct and ultrasound ; in 1992 , 41.3% of all patients were diagnosed with nonspecific abdominal pain compared with 21.1% in 2007 . the number of patients admitted to the ward decreased from 27.4% in 1972 to 18.4% in 1992 . factors contributing to improved diagnostic accuracy include establishment of emergency medicine faculties and increased possibilities for laboratory testing . the widespread availability of various diagnostic modalities , such as ct , ultrasound , and nuclear imaging , probably account for the most significant change.57 diagnosing the underlying cause of acute abdominal pain remains a challenge despite the increase in diagnostic accuracy over the years . the ideal diagnostic imaging modality for evaluation of acute abdominal pain in adult patients at the emergency department should provide a balance between the highest diagnostic value and most accurate management changes on one hand , and lowest radiation exposure , discomfort , and duration of stay at the emergency department on the other hand , while ultimately resulting in the lowest cost to the health care system . several studies have demonstrated that a diagnosis based solely on a patient s medical history , physical examination , and laboratory tests is not reliable enough , despite the fact that these aspects are essential parts of the workup of a patient presenting with acute abdominal pain.2,8 further diagnostic workup such as imaging is therefore mandatory in patients suspected of an urgent medical condition . imaging workup traditionally starts with abdominal radiography.9 standard abdominal radiography consists of three views , ie , a supine abdominal view combined with an erect chest film and an upright abdominal view.10,11 uk and us guidelines advise consideration of abdominal radiography in case of hospital admission and or surgery in patients presenting with acute abdominal pain.12,13 one study compared the initial diagnosis after clinical evaluation and plain radiography with the final diagnosis , and found that these diagnoses corresponded in only 502 of a total of 1021 patients ( 49%).3 diagnosis based only on plain abdominal radiography corresponded with the final diagnosis in 514 of the 1021 patients ( 50% ) . the improvement in accuracy of plain abdominal radiography combined with clinical examination was not significant ( p = 0.14 ) . in 117 of the 1021 patients ( 11% ) , the treating physician changed the initial clinical diagnosis after plain abdominal radiography , and these changes were accurate in only 39 cases ( 22% ) . additionally , the level of confidence of the assigned diagnosis was recorded in 983 patients before and after plain abdominal radiography . for 875 patients in whom the diagnosis had not changed , the level of confidence also remained unchanged . several studies have demonstrated a high percentage of plain abdominal radiographs without abnormal or specific findings . two studies demonstrated that 77% and 78% of all requested plain abdominal radiographs showed no abnormal findings.14,15 another study demonstrated that in only 83 of 871 patients ( 10% ) , a specific diagnostic abnormality was noted on plain abdominal radiography.16 a study analyzing the value of plain abdominal radiography in addition to clinical examination showed that management changed in only 15 patients ( 8.9%).17 in 90 of 153 remaining patients ( 53.6% ) , the initial diagnosis changed due to other imaging modalities , and in 63 patients the diagnosis remained unchanged after plain abdominal radiography . whether plain abdominal radiography contributes to therapeutic decision - making or disposition remains questionable . particularly in the case of a negative result , the additional value of plain abdominal radiographs is disputed . it is for this reason that several studies suggest ordering plain abdominal radiographs for specific indications only , in order to reduce the number of unnecessary requests.1113,18,19 specific indications for ordering plain radiography include suspicion of perforated viscus , urinary tract stones , bowel obstruction , and ingested foreign body . the identification of a small amount of free intra - abdominal gas remains one of the most significant signs in medicine . the combination of abdominal pain and a pneumoperitoneum , even in the absence of other clinical signs , will usually lead to a laparotomy in search of a perforated viscus advocates of conventional radiography state that plain abdominal radiography should be the first diagnostic modality used in suspicion of a perforated viscus . it is possible , using careful radiographic technique , to demonstrate as little as 1 ml of free gas on an erect chest or left lateral decubitus abdominal film.20 the high percentage of missed cases is due to technical imperfections rather than limitations of the test ( poor quality of plain abdominal radiography , excluding the uppermost portion of the peritoneal cavity of the image).21 in that study , the radiographs demonstrated pneumoperitoneum in only 51% of patients with documented visceral perforation . left lateral decubitus radiographs demonstrated pneumoperitoneum in 96% of patients , chest radiographs in 85% , and upright and supine abdominal radiographs in 60% and 56% , respectively.22 another study described pneumoperitoneum in only 83% of all patients with documented visceral perforation.23 one study compared the diagnosis of patients suspected of perforated viscus before and after plain abdominal radiography.3 the positive predictive value was not influenced by plain abdominal radiography . the level of confidence in the diagnosis changed in six of 13 ( 46% ) patients with the clinical diagnosis of a perforated viscus . these data were obtained from a cohort of 1021 patients who presented at the emergency department with acute abdominal pain.2 this study demonstrates that plain abdominal radiographs have no added value in the diagnostic workup . several studies have demonstrated that plain abdominal radiography has a lower accuracy than other diagnostic modalities . one study compared ultrasonography with plain abdominal radiography in detection of pneumoperitoneum , and included 188 patients suspected of visceral perforation.24 all patients underwent chest and/or abdominal radiography and ultrasonography in order to detect free intraperitoneal air ; 165 patients had suspicion of pneumoperitoneum after ultrasonography , and in 157 of the suspected patients , visceral perforation was confirmed intraoperatively . after plain radiography , 126 patients were suspected of pneumoperitoneum which was confirmed intraoperatively in 120 of cases . both diagnostic modalities demonstrated high positive predictive value ( 95% in ultrasound and 94% in radiography ) and similar specificity ( 53% ) . ultrasound did have a higher sensitivity ( 92% versus 78% ) , accuracy ( 88% versus 76% ) , and negative predictive value ( 39% versus 20% ) . ct has proven to be the most accurate diagnostic modality in the evaluation of pneumoperitoneum.25 a small study compared ct with plain radiographic evaluation in 13 patients who underwent diagnostic peritoneal lavage due to abdominal trauma.25 only five of 13 patients ( 38% ) demonstrated free air on radiographs compared with 13 of 13 patients on ct . another study retrospectively reviewed ct and plain radiographs ( when available ) of 76 patients with proven perforation of the gastrointestinal tract.26 in 65 of 76 patients , ct was truly positive and in 11 patients was falsely negative . the cause of perforation was correctly predicted in 51 ( 78% ) of all patients , and the location of the perforation was correctly predicted in 55 patients ( 84.6% ) on ct . in 63 patients plain radiographs were used , of which 32 ( 52% ) were truly positive and 31 ( 48% ) were falsely negative for the presence of a perforation . the available evidence demonstrates that even though the positive predictive value of plain abdominal radiography is similar to that of other diagnostic modalities , the sensitivity and negative predictive value are far too low . the additional value of ct lies in the possibility of providing more information on the location and underlying cause of the perforation , or in providing an alternative diagnosis . in current practice , the high number of missed cases of perforation after plain abdominal radiography is unacceptable and renders the additional value of negative plain abdominal radiography very limited . moreover , a plain abdominal radiograph that is positive for free air conveys limited information on the location and underlying cause ; an additional ct scan will often need to be made to be able to adapt the operative strategy to the specific case . performing surgery without adequate information about the perforation site is a conceptual mistake and nowadays should be considered as substandard clinical practice ( not all free air is caused by a perforated gastric or duodenal ulcer ; consider , amongst others , colonic perforation , surgery - related perforation , perforated diverticular disease , perforated appendicitis , inflammatory bowel disease , or endoscopy ) . no place exists for abdominal radiography in the evaluation of patients suspected of visceral perforation or , for that matter , in any patient with acute abdominal pain . the standard imaging modality for detecting urinary tract stones should ideally provide information about size , site , and composition of the ureteral stone and presence of ureteral obstruction . most ureteral stones can be identified as a calcification causing a filling defect or ureteral obstruction on plain abdominal radiography.1 a plain film of the abdomen , including kidney , ureter , and bladder , has shown sensitivity ranging from 44% up to 77% and specificity in detection of stones from 80% to 87%.2729 in one study , the diagnosis of patients suspected of ureteral stones before and after plain abdominal radiography compared with the final diagnosis revealed a correct change in diagnosis in six of 11 patients ( 55%).3 the level of confidence in the diagnosis remained the same as well as having a positive predicting value , which reached 57% after clinical evaluation and 58% after radiographs . another study concluded that plain abdominal radiography had a sensitivity of 45% and specificity of 77% for detection of ureteral calculi.30 in patients previously diagnosed with urolithiasis presenting with similar symptoms , abdominal radiography could be useful to diagnose kidney stones . the advantage of ct over plain abdominal radiography is that an alternative diagnosis may be presented if the suspicion of urolithiasis is not confirmed . additional information concerning urolithiasis may also be obtained by ct , such as size and location , which are both crucial factors in guiding therapy . ct has replaced the use of plain abdominal radiography and intravenous urography in the detection of ureteral stones . bowel obstruction is a common cause of acute abdominal pain . about 7% of all patients with acute abdominal pain imaging in patients suspected of bowel obstruction should provide information about the site , cause , and level of the obstruction.31 plain abdominal radiography is advocated as a standard diagnostic modality for the detection of bowel obstruction.12,13 plain abdominal radiographic findings have been shown to be diagnostic in 50%60% , indifferent in 20%30% , and misleading in 10%20% of patients.32 in one study , the sensitivity of plain abdominal radiography after clinical evaluation was significantly higher than that of clinical evaluation alone ( 74% versus 57% , respectively).3 changes in diagnosis after plain abdominal radiography were correct in only 16 of 24 patients ( 66% ) and the level of confidence remained unchanged in 32 of 71 patients ( 52% ) . the sensitivity of a clinical evaluation combined with a score card for clinical signs / symptoms proved to be similar to a clinical evaluation combined with plain abdominal radiography.3 frager et al compared diagnoses after clinical evaluation combined with plain abdominal radiography or ct.33 in patients with a complete obstruction , ct demonstrated a sensitivity of 100% compared with 46% after plain abdominal radiography . for partial obstruction , ct had a sensitivity of 100% compared with 30% for plain abdominal radiography . of the 61 patients who underwent surgery , 52 patients were confirmed to be correctly diagnosed preoperatively ( 85% ) based on ct findings . the exact location of the obstruction was correctly diagnosed in 50 of 53 patients ( 94% ) on ct . in addition to its higher sensitivity , an important advantage of ct is the ability to provide information about the underlying cause of obstruction or to provide information about an alternative diagnosis if no signs of bowel obstruction are present . additional diagnostic investigations should be considered if clinically relevant , particularly if surgery is considered . most foreign bodies pass through the gastrointestinal tract harmlessly . if patients are symptomatic or if the ingested foreign body is potentially dangerous , additional diagnostic examinations should be undertaken . plain abdominal radiography demonstrates a sensitivity of 90% , specificity of 100% , and accuracy of 100% for ingested foreign bodies , but the foreign body has to be radio - opaque to be seen on plain abdominal radiography.16 there is no evidence available as to whether ct has a higher sensitivity and specificity than plain abdominal radiography in the case of ingested foreign bodies . the advantage of ct is the ability to provide information about the location of the foreign body , which is a prerequisite when surgical treatment is planned . body packers orally ingest , or rectally or vaginally insert packed drugs in order to smuggle them . plain abdominal radiography is used to establish the diagnosis of drug packing and is considered the gold standard.34 if plain abdominal radiography is negative or inconclusive but a high suspicion of body packing remains , a ct scan should be done . the sensitivity of plain abdominal radiography is 85%100% , but ct has a higher sensitivity and additionally provides more accurate information about the number and location of packages . the use of plain abdominal radiography gives rise to a high number of false negative results , which could be due to overprojection of feces or a specific packaging method.35 there is no solid evidence that ct has a higher sensitivity or diagnostic accuracy than plain abdominal radiography . the low negative predictive value of plain abdominal radiography leads to the conclusion that , if clinically relevant , a ct should be used as the diagnostic modality of choice . in spite of the recent proliferation of other imaging techniques , plain films still retain their position as one of the most useful initial investigations imaging techniques such as ct and ultrasound have been shown to increase diagnostic accuracy substantially,5 and consequently have significantly decreased the added diagnostic value of plain abdominal radiography in a clinical setting . despite recent abundant evidence of its limited value , many physicians still rely on plain abdominal radiography as a simple , cheap , and widely available first diagnostic modality with lower radiation exposure than ct . proponents of plain abdominal radiography advocate its use to prevent high radiation exposure in patients due to unnecessary ct imaging . the average plain abdominal radiograph exposes the patient to 0.7 msv and an abdominal ct exposes the patient to 10.0 msv.13,36 new techniques , such as automated dose modulation and an iterative reconstruction algorithm , reduce the ct radiation dose . in everyday clinical practice , physicians differentiate , subconsciously or consciously , between urgent and nonurgent conditions in patients . patients with indeterminant symptoms and low or no suspicion of an urgent condition can be discharged home without additional imaging . patients suspected of urgent conditions require timely identification of the underlying cause and treatment within 24 hours to prevent severe complications . a recent study designed to identify the most effective diagnostic strategy for patients with acute abdominal pain has demonstrated that the highest sensitivity for detecting urgent diagnoses is achieved when ultrasonography is performed in all patients and a ct only in the event of inconclusive or negative ultrasonography ( conditional ct strategy).31 using this strategy , ct is only needed in 49% of patients . even though ct exposes patients to a higher radiation dose , it still remains the preferred standard diagnostic modality if ultrasound fails to diagnose the cause of acute abdominal pain correctly . plain radiography demonstrates low sensitivity and accuracy and is therefore generally unhelpful ; because of its low sensitivity and negative predictive value , it can also be misleading in the workup of acute abdominal pain . although the radiation dose is lower than that of ct , the mediocre test characteristics of plain radiography may delay appropriate treatment and are therefore detrimental . numerous studies have demonstrated low sensitivity and accuracy for plain abdominal radiography in the evaluation of acute abdominal pain as well as for various specific diseases , such as perforated viscus , bowel obstruction , ingested foreign body , and ureteral stones . ct provides a better workup than that achieved with plain abdominal radiography alone , and its benefits lie in improving decision - making for management , adapting the surgical strategy , and possibly even avoiding negative laparotomies . when a new test is developed , it can have three possible roles in relation to the existing situation . the existing test can be replaced , the new test could be added on top of the existing test , or the new test could function as a triage test to distinguish between patients who need further examination and those who can be safely observed without additional examination . if we were to look at abdominal radiography as if it was a newly developed test for adult patients with acute abdominal pain in the emergency department , the evidence presented in this manuscript demonstrates that there is no added value nor could abdominal radiography replace ultrasound or ct . if abdominal radiography were to be used as triage , its main purpose would be to rule out disease in patients who genuinely do not have disease , ie , to prevent further investigations . however , for this purpose , the number of false negatives has to be relatively low and sensitivity should be high , which is not the case for abdominal radiographs.37 based on the abundant available evidence , major advances in diagnostic imaging , and changes in the management of certain diseases , we can conclude that there is no place for plain abdominal radiography in the workup of adult patients with acute abdominal pain in current practice .

several studies have demonstrated that a diagnosis based solely on a patient s medical history , physical examination , and laboratory tests is not reliable enough , despite the fact that these aspects are essential parts of the workup of a patient presenting with acute abdominal pain . traditionally , imaging workup starts with abdominal radiography . however , numerous studies have demonstrated low sensitivity and accuracy for plain abdominal radiography in the evaluation of acute abdominal pain as well as various specific diseases such as perforated viscus , bowel obstruction , ingested foreign body , and ureteral stones . computed tomography , and in particular computed tomography after negative ultrasonography , provides a better workup than plain abdominal radiography alone . the benefits of computed tomography lie in decision - making for management , planning of a surgical strategy , and possibly even avoidance of negative laparotomies . based on abundant available evidence , major advances in diagnostic imaging , and changes in the management of certain diseases , we can conclude that there is no place for plain abdominal radiography in the workup of adult patients with acute abdominal pain presenting in the emergency department in current practice .

Historical overview Place of plain abdominal x-ray in current diagnostic workup Detection of free air and perforated viscus Detection of urinary tract stones Detection of bowel obstruction Detection of ingested foreign body In diagnostic workup

1 . he had a history of bilateral brow suspension surgery 10 years ago at another institution . the patient had a 30 cm 20 cm pelvic mass located in the presacral area with boundaries between both parailiac regions displacing the rectum and bladder . it was completely excised with a pathology report of myelolipoma . in physical examination , he had hypertrichosis , finger clubbing , and radiologically cortical thickening of the bones fig . the patient presented with bilateral blepharoptosis with coarse skin folds the patient had cortical thickening of the bones and clubbing his best - corrected visual acuity was 7/10 in the right eye ( re ) and 5/10 in the left eye ( le ) . horizontal length of the upper lids of re and le was 46 mm and 41 mm , respectively . vertical fissure heights were 6 mm and 4 mm with margin - reflex distances of 0.5 mm and 0.5 mm , respectively . vertical eyelid contour of the re showed an inverse v shape for the re and a smooth curve for the le . the eyelids were floppy and easily everted . to correct these clinical findings , a surgery which combines vertical tarsal shortening accompanied with horizontal whole eyelid wedge resection and brown suspension 10 mg/0.25 ml triamcinolone acetate was injected into each supratarsal space to decrease the thickness of the tarsus . horizontal wedge resection which is not a part of routine ptosis surgery was performed in this operation with the aim of normalizing the obvious horizontal length of the lids . pathological evaluation revealed epidermal hyperplasia , severe inflammatory changes in subepidermal level , hyperplastic sebaceous glands , and collagen tissue derangement fig . reconstruction of each eyelid was achieved by vertical tarsectomy , horizontal full thickness lid resection , and supratarsal steroid injection . ptosis was assessed with silicone rod frontalis suspension , and resection of excessive skin was performed as in blepharoplasty skin biopsy showing thickening of the dermis with increased collagen content and lymphocytic infiltration ( h and e , 10 ) the postoperative course was uneventful and patient satisfaction is good within the 1 year of follow - up fig . it is characterized by skin thickening , clubbing , hyperhidrosis , and periosteal reaction in the long bones . primary hoa is predominantly a male disease that at least 90% of patients are men . although symptoms may be seen in childhood , it manifests mostly during the fifth decade of life . described the largest number of primary hoa series in the literature that the authors reported that family history was positive in all studied patients . the pathogenesis of hoa is unclear and some studies explained the role of several growth factors in the evolution of the disease . although clinical findings are similar , secondary hoa differs from primary hoa with absence of family history . secondary hoa is an acquired form that is associated with usually lung disease but also heart , liver , and intestines . the presented patient had a huge pelvic mass which was completely excised with a pathology report of myelolipoma . hence , our diagnosis was secondary hoa due to the paraneoplastic manifestations of pelvic malignancy which causes secretion of many growth factors . blepharoptosis may develop secondary to sebaceous gland hyperplasia , thickening of the dermis with increased collagen content , mucin deposition , and lymphocytic infiltration or due to additionally marked scarring . our patient was a 52-years - old man with negative family history which let us exclude the diagnosis of primary hoa . the differential diagnosis includes several diseases as acromegaly , syphilitic periostitis , and thyroid acropachy . acromegaly is characterized by enlargement of facial bones that are not present in our case . serology , radiology , and clinical findings did not support a diagnosis of syphilis or thyroid disease . hoa may develop secondary to pulmonary or congenital cyanotic cardiac diseases those were not present in our patient . the remaining possible etiological factor for secondary hoa in our case was the huge pelvic malignancy which was excised previously . the surgery for these kinds of pathologies may be planned as single or staged procedures . blepharoplasty with excessive skin excision is usually required both for better cosmesis and to reduce the tissue bulk . no complications were encountered during surgery , but bleeding was more than any other lid surgery . the profound inflammatory reaction in the tissue might be the cause of this excessive bleeding . hoa may not be always primary , particularly in patients with negative family history . in cases of findings with abnormal fibroproliferative and inflammatory changes , detailed systemic examination and investigations both using serologic and imaging modalities the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

a 52-year - old male patient presented to our hospital with a history of secondary hypertrophic osteoarthropathy ( hoa ) associated with an abdominal neoplasia and blepharoptosis . he had finger clubbing , hyperhidrosis , and hypertrichosis . he also had a recent history of extensive abdominal surgery with a pathology report of myelolipoma . routine blood work was unremarkable . upper eyelid reconstruction with blepharoplasty , upper eyelid wedge resection , and brow suspension was performed to address his eyelid concerns . by this case report , we would like to attract notice that the eyelid involvement may be a part of hoa and to emphasize the importance of systemic and pathologic evaluation in failed blepharoptosis surgery .

Case Report Discussion Declaration of patient consent Financial support and sponsorship Conflicts of interest

cyclic organic structures comprise a wide variety of interesting examples in strained , distorted , bent , and sterically hindered molecular systems in nanoscience . these structures have fascinated chemists for several decades not only because of their unique chemical bonding but also because of the potential to tailor their unique physical properties through variations in molecular size , shape , and crystallinity . cyclic nanostructures composed entirely of conjugated carbon subunits are especially fascinating because they exhibit a myriad of interesting electronic properties . in particular , because cycloparaphenylene nanostructures are composed of aromatic subunits with radially oriented p orbitals , these materials can support mobile charge carriers on the delocalized valence and conduction orbitals along the backbone chain . as a result , cycloparaphenylenes and other well - ordered macrocyles may find applications as conducting materials in molecular electronics , organic field - effect transistors ( ofets ) , and nonlinear optics ( nlos ) due to their unique optoelectronic properties . in general , the ability to control and understand the electronic response of organic nanostructures at the molecular level would have a tremendous impact in nanoscale optoelectronic technologies . carbon nanorings large enough to encapsulate fullerenes were first synthesized by kawase et al . and are composed of a conjugated carbon skeleton in a smooth belt - shaped structure . many of the nanorings synthesized by the kawase group are actually part of a larger family of molecules known as phenylacetylene macrocycles ( pams ) , which are composed of alternating phenyl and ethynyl subunits sequentially bonded to form a single ring . the phenyl subunits in pams are usually linked in the meta or para positions and can be composed of four to nine phenyleneethynylene units . in a very recent communication , jasti et al . synthesized and characterized a set of - , - , and -cycloparaphenylenes composed solely of phenyl rings sequentially connected to form a single nanohoop ( figure 1 ) . their synthesis was especially noteworthy because cycloparaphenylenes form the fundamental annular segments of armchair carbon nanotubes and may serve as templates toward the rational synthesis of carbon nanotubes with specific chiralities . [ n]-cycloparaphenylenes with n = 9 , 12 , and 18 . each cycloparaphenylene is composed solely of phenyl rings sequentially connected in the para position to form a single nanoring . a peculiar feature of these cycloparaphenylenes is their unusual optoelectronic properties as a function of molecular size . specifically , as the carbon nanoring size is increased , the lowest computed absorption energy becomes larger . this result is surprising and counterintuitive to our usual expectations ! for example , our chemical intuition from organic chemistry tells us that , as the number of repeat units in a macromolecule increases , the distance between molecular energy levels diminishes , effectively decreasing the optical absorption gap . this trend ( which actually arises from quantum confinement effects ) qualitatively describes the variation of absorbance properties in -conjugated systems as the number of monomer units increase in a polymer . however , in the case of cyclic nanorings , the excitation energies as a function of size seem to contradict this quantum - confinement - effect argument . to rationalize these trends , with a view of understanding the nature of electronic excitations in these nanorings , i carried out ab initio calculations on both cyclic and linear paraphenylenes ( figure 2 ) composed of 5 to 18 phenyl repeat units each . results obtained from time - dependent density functional theory ( tddft ) give electronic band gaps , optical absorption energies , and oscillator strengths that can be used to estimate exciton binding energies as a function of paraphenylene size . the tddft calculations also provide a two - dimensional real - space analysis of transition densities that represent coherent electronic transitions between ground and electronically excited states . these transition densities give a panoramic view of electronhole coherence and exciton delocalization in each of the paraphenylene systems . on the basis of overall trends in exciton binding energies and their spatial delocalization , i find that excitonic effects play a significant role in understanding the unique photoinduced dynamics of cycloparaphenylene nanoring systems . molecular structures and atom labels for ( a ) cyclic paraphenylenes and ( b ) linear paraphenylenes . the specific atom numbers depicted in these figures define an ordered basis for generating the density matrices discussed in section . the cyclic and linear paraphenylenes ( n = 518 ) analyzed in this work are shown in figure 2 . all quantum chemical calculations were carried out with the parameter - free pbe0 hybrid density functional that incorporates a fixed combination of 25% hartreefock exchange and perdew s gga corrections in the correlation contribution . have shown that the use of pure local and gradient - corrected functionals ( i.e. , lda and pbe ) results in unphysical unbound exciton states , whereas hybrid functionals partially overcome this problem by mixing in a fraction of nonlocal hartreefock exchange . on the basis of their studies , i have chosen the pbe0 functional for this work because the pbe0 kernel provides a balanced description of neutral excitons ( both singlets and triplets ) in conjugated polymers and carbon nanotubes . ground - state geometries of all cyclic and linear paraphenylenes were optimized at the pbe0/6 - 31g(d , p ) level of theory . geometry optimizations were calculated without symmetry constraints , and root - mean - squared forces converged to within 0.00003 au . at the optimized ground - state geometries , tddft calculations were performed with a larger , diffuse 6 - 31+g(d , p ) basis set to obtain the lowest four singlet vertical excitations . for both the ground - state and the tddft calculations , i used a high - accuracy lebedev grid consisting of 96 radial and 302 angular points . all ab initio calculations were performed with a locally modified version of gamess . within the tddft formalism , one obtains the excited - state electron density , (r ) |(r)| = i=1n|i(r)| composed of n - occupied molecular orbitals i(r ) as solutions from the time - dependent kohnsham equations . in the same way that one can calculate transition density matrices from time - dependent hartreefock orbitals , one can use orbitals from the noninteracting kohnsham system to form transition densities that include many - body correlation effects from the tddft formalism . , one can construct coordinate qv and momentum pv matrices with elements given by 12 where g and v are ground and excited states , respectively . the fermi operators ci and ci represent the creation and annihilation of an electron in the ith basis set orbital in . for the cyclic and linear paraphenylenes analyzed in this work , the qv and pv matrices each form a two - dimensional xy grid running over all the carbon sites along the x and y axes . the specific ordering of the carbon sites used in this work is shown in figure 2 . the ( pv)mn momentum matrix represents the probability amplitude of an electronhole pair oscillation between carbon sites m and n , and the ( qv)mn coordinate matrix gives a measure of exciton delocalization between sites m and n. each of these matrices provides a global view of electronhole coherence and exciton delocalization for optical transitions within the paraphenylene systems . in the ground - state optimizations for the linear paraphenylenes , the dihedral angle between adjacent benzene rings was calculated to be 37. this is close to the dihedral angle in isolated biphenyl and is consistent in all of the linear paraphenylenes regardless of length . in contrast , for the smallest and highly strained n = 5 cyclic paraphenylene , the benzene rings adopt a wide variety of smaller dihedral angles between 9 and 27. figure 3 shows the average dihedral angle between adjacent benzene rings as a function of paraphenylene size . for a given number of benzene rings , the average dihedral angle in the cyclic paraphenylenes is always smaller than their acyclic counterparts . however , as the size of the cyclic paraphenylene increases , the strain energy becomes smaller , and the dihedral angles between benzene rings increase to 36. the optimized cartesian coordinates for both the cyclic and the linear paraphenylenes can be found in the supporting information . average dihedral angle between adjacent benzene rings as a function of paraphenylene size . as the number of benzene rings increases , the average dihedral angle for the cyclic paraphenylenes asymptotically approaches the acyclic dihedral angle of 37. to investigate optoelectronic trends as a function of size and shape , optical absorption energies , eopt , were computed for all the cyclic and linear ground - state geometries . table 1 compares the lowest excitation energies and oscillator strengths between the cyclic and linear paraphenylenes , and figure 4 displays eopt as a function of size . the other higher - lying singlet excitations ( up to s4 ) can be found in the supporting information . as the number of benzene rings increases , the lowest excitation energies for both the cyclic and the linear paraphenylenes asymptotically approach a value of 3.48 ev . however , as figure 4 illustrates , the manner in which they approach this asymptotic value is considerably different : eopt for the cyclic systems increases with size , whereas eopt for the acyclic systems decreases . optical excitation energies ( eopt ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . eopt for the cyclic systems increases with size , whereas eopt for the acyclic systems decreases . all excitation energies were obtained from pbe0/6 - 31+g(d , p ) tddft calculations at pbe0/6 - 31g(d , p)-optimized geometries . another significant difference between the two systems is the variation of oscillator strengths as a function of size . for the linear paraphenylenes , the oscillator strengths increase linearly as a function of chain length , whereas the oscillator strengths for most of the even - membered cyclic systems are zero ( due to molecular symmetry ) . although the dft geometry optimizations were performed without symmetry constraints , most of the calculations for the smaller ( n 12 ) even - membered nanorings converged to a c2v - like symmetric structure . for nanorings containing an odd number of benzenes , the optimized structures have a reduced symmetry , and the oscillator strengths are nonzero . however , as the number of benzene rings increases , the strain energy becomes smaller , and several conformers with different dihedral angles can exist with similar energies . i found that these different conformers have various oscillator strengths ( due to reduced symmetry ) but nearly indentical energies . a complete analysis of the conformational landscape available to the nanorings is beyond the scope of this work and would have a negligible effect on the excitation energies in the larger systems . the small s1 s0 oscillator strengths obtained for the nanorings also have a close analogy with the very recent study by kilina et al . that calculated optoelectronic properties of finite - length carbon nanotubes . in their study , it was found that hybrid functionals determine the lowest singlet - excited state to be an optically inactive ( dark ) state with the optically active ( bright ) state lying higher in energy . similarly , for the large ( n 12 ) nanorings in this work , the second and third singlet excitations are bright states with strongly allowed transitions ( table si-1 in the supporting information ) . these trends in oscillator strengths for the nanorings can also be explained qualitatively in terms of a simple exciton model where one transition dipole moment is assigned to each benzene ring ( figure 5 ) . for the lowest s1 s0 excitation , the transition moments in both the linear and the cyclic paraphenylenes are aligned in a head - to - tail arrangement . however , because of their circular geometries , the s1 s0 transition moments in the cyclic paraphenylenes effectively cancel , while the total transition moment increases as a function of length in the linear systems ( for the larger , more flexible nanorings , some of the transition dipole moments have a small component perpendicular to the ring , and the vectorial sum is slightly nonzero ) . in contrast , for the other higher - lying singlet excitations , the transition dipoles are aligned in one direction for half of the nanoring and in the opposite direction for the other half of the ring , producing a net transition dipole and a nonzero oscillator strength . as a result , the structural difference between cyclic and linear geometries imposes additional symmetry constraints that determine the oscillator strengths in these conjugated systems . arrangements of individual transition dipole moments for ( a ) cyclic and ( b ) linear paraphenylenes during the s1 s0 excitation . the transition moments in the cyclic geometry effectively cancel , but the total transition moment increases proportionally with length in the linear systems . to provide further insight into these optoelectronic trends , i carried out a two - dimensional real - space analysis of density matrices for both the cyclic and the linear systems . figure 6 displays the absolute values of the coordinate density matrix elements , |(q1)mn| , for the lowest excitation energy ( s1 s0 ) in the n = 5 , 9 , 14 , and 18 paraphenylenes . the coordinate and momentum , |(p1)mn| , density matrices for all of the other paraphenylenes the x and y axes represent the benzene repeat units in the molecule , and the individual matrix elements are depicted by the various colors . contour plots of coordinate density matrices ( q1 ) for the lowest excitation energy ( s1 s0 ) in the cyclic and linear paraphenylenes . the x- and y - axis labels represent the number of benzene repeat units in the molecule . the elements of the coordinate matrix , qmn , give a measure of exciton delocalization between sites m ( x axis ) and n ( y axis ) . although the cyclic and linear paraphenylenes are composed of similar benzene repeat units , the density - matrix delocalization patterns in each system are considerably different . for the linear systems , the electronhole pair created upon optical excitation becomes primarily localized in the middle of the molecule and away from the edges . in contrast , the density matrices for the cyclic systems have significant off - diagonal elements that persist even for the largest n = 18 nanoring . the magnitude of the off - diagonal elements represents electronic coherence between different atoms , and figure 6 shows that the electronhole states are delocalized over the entire circumference in the cyclic systems . the coherence size , which characterizes the distance between an electron and a hole , is given by the width of the momentum density matrix along the coordinate axes in figure si-2 ( supporting information ) . in this work , i arbitrarily define the coherence width as the distance where the momentum decreases to 10% of its maximum value . these figures show that , for a given number of benzene rings , the coherence size in the linear paraphenylenes is slightly larger than their cyclic counterparts . for the smaller paraphenylenes ( n < 9 ) , the coherence size in the linear systems is larger by approximately one repeat unit in comparison with the cyclic systems . as the number of benzene units increases to 18 , the coherence size for both the cyclic and the linear systems approach the same value of 9 repeat units . both |(q1)mn| and |(p1)mn| also show that the linear systems only have strong optical coherences induced at their center , whereas the optical coherences in the cyclic geometries are nearly equally distributed throughout the entire molecule . these different density - matrix delocalization patterns and coherence sizes result in distinct excitonic properties in the cyclic and linear systems , an effect which i quantify in the next section . to understand electronhole interactions on a more quantitative level , i calculated exciton binding energies for the cyclic and linear paraphenylenes as a function of size . as illustrated in figure 7 , the exciton binding energy , eexc , is given by the difference between the quasiparticle energy gap [ ionization potential ( ip ) electron affinity ( ea ) ] and the optical absorption gap ( eopt ) . both the ip and the ea were obtained from pbe0/6 - 31+g(d , p ) electronic energy calculations on the n 1 , n , and n + 1 electron systems at the neutral pbe0/6 - 31g(d , p ) optimized geometry . in this work , the vertical ip is defined by 3 and the magnitude of the vertical ea is given by 4 with these definitions , the magnitude of the exciton binding energy is then 5 table 2 gives ionization potentials , electron affinities , and exciton binding energies for the cyclic and linear paraphenylenes , and figure 8 shows the quasiparticle energy gap ( ip ea ) as a function of size . schematic representation of energy levels defining the exciton binding energy ( eexc ) , optical absorption gap ( eopt ) , ionization potential ( ip ) , and electron affinity ( ea ) . quasiparticle energy gaps ( ip ea ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . the quasiparticle energy gap for the acyclic paraphenylenes decreases much more rapidly than the energy gap for the corresponding cyclic systems . as figure 8 shows , with the exception of some small fluctuations , the quasiparticle energy gap in both systems decreases with size , as expected from quantum - confinement effects . furthermore , the quasiparticle energy gap for the linear paraphenylenes decreases much more rapidly than the energy gap for the corresponding cyclic systems . the question therefore arises : why does the quasiparticle energy gap behave so differently in the linear and cyclic systems ? to address this question , we must take a closer look at the variations in aromatic character within the linear and cyclic geometries . first , in each of these organic systems ( regardless of molecular geometry ) , an electronic competition exists between maintaining the aromaticity of the individual benzene rings versus delocalization along the backbone chain . in the unstrained linear paraphenylenes , there is relatively little conjugation along the backbone of the system because the electrons are localized in each individual phenyl unit to maintain aromaticity . in contrast , the cyclic paraphenylenes have highly strained geometries that distort the electronic structure of the individual phenyl units . the strong deformation within the phenyl ring diminishes the overlap of orbitals , resulting in quinoidal character ( antibonding interactions within the phenyl ring and double - bond character connecting adjacent phenyl rings ) . as a result , the electronic states in the cyclic paraphenylenes are more delocalized in comparison with their acyclic counterparts , in agreement with the transition density matrix analysis discussed in section . more importantly , the quinoid form is energetically less stable than the aromatic form : the quinoid structure has a smaller quasiparticle gap because it involves destruction of aromaticity and a loss in stabilization energy . in other words , by increasing the quinoid character of the system , the highest - occupied orbital in the cyclic paraphenylenes becomes destabilized ( i.e. , raised in energy ) , and the quasiparticle energy gap is significantly reduced . to give a quantitative measure of aromaticity in these systems , i calculated the nucleus - independent chemical shift ( nics(1 ) ) at the pbe0/6 - 31g(d , p ) level of theory for each of the phenyl rings in the cyclic and linear geometries . in the nics(1 ) procedure suggested by schleyer et al . , the absolute magnetic shielding is computed at 1 above and 1 below the phenyl ring center . the resulting average nics(1 ) values give a measure of -orbital aromaticity , with more negative nics(1 ) values denoting aromaticity and more positive values corresponding to quinoidal character . as a reference point in this work , the nics(1 ) value for benzene at the pbe0/6 - 31g(d , p ) table 2 gives average nics(1 ) values of phenyl rings in the cyclic and linear paraphenylenes , and figure 9 shows the nics(1 ) values as a function of size . as anticipated from our qualitative discussion on aromaticity , the nics(1 ) values for the cyclic paraphenylenes in particular , for the smaller nanorings ( n < 8) , figure 9 shows that these structures have significant quinoidal character , resulting in unusually small quasiparticle energy gaps ( cf . however , as the size of the cyclic paraphenylene increases , the strain energy becomes smaller , and both the cyclic and the linear nics(1 ) values approach the same limit . it is also interesting to note that the fluctuations in nanoring nics(1 ) values shown in figure 9 are correlated with the deviations seen in figures 4 and 8 . specifically , the discontinuous change in nics(1 ) values at n = 8 , 10 , 13 , and 17 can also be found as discontinuities in optical excitation energies ( figure 4 ) and abrupt changes in quasiparticle energy gaps in figure 8 . even more intriguing is the direct correlation between nics(1 ) values of individual phenyl rings and the intensity of the transition density matrix elements discussed in section . returning to figure 6 , the excitonic density along the diagonal for n = 18 is not uniform ; in other words , there are four areas along the diagonal with maximal density at repeat units of 3 , 8 , 12 , and 17 . figure si-3a , b ( supporting information ) displays the nics(1 ) values of individual phenyl rings and the dihedral angle between adjacent phenyl rings for the n = 18 cyclic geometry . figure si-3a ( supporting information ) shows four distinct maxima ( i.e. , four phenyl units that have quinoidal , or delocalized , character ) at the same positions corresponding to maxima in the n = 18 transition density matrix . in contrast , figure si-3b ( supporting information ) shows that the dihedral angles are nearly identical ( all within 0.3 ) throughout the n = 18 nanoring . taken together , these figures show that the distribution of excitonic density in figure 6 is purely an electronic effect and not a result of conformational variations . average nics(1 ) values of phenyl rings in the cyclic and acyclic paraphenylenes as a function of size . all nics(1 ) calculations were obtained at the pbe0/6 - 31g(d , p ) level of theory . finally , figure 10 displays the exciton binding energies of both systems calculated from eq 5 . in contrast to figures 4 and 8 , the exciton binding energies in both systems show a smooth monotonic variation as a function of size . as expected , for a given number of benzene rings , the exciton binding energies in the cyclic paraphenylenes are always larger ( and decrease at a faster rate ) than their acyclic counterparts . this trend is in agreement with the coherence sizes of the momentum density matrices discussed in section . compared with the linear paraphenylenes , the average electronhole distance in the cyclic systems is smaller , leading to an increase in the binding energy . however , as the number of benzene units increases , the coherence sizes in both systems become nearly equal , and the binding energies asymptotically approach the same value . these results , in combination with the quasiparticle energy gaps and electronhole delocalization patterns , explain the anomalous absorption energy trends in the cyclic paraphenylenes . in the linear systems , the quasiparticle energy gap ( ip ea ) decreases much more rapidly than the exciton binding energy , eexc . as a result , the behavior of the ip ea term dominates the right - hand side of eq 5 , and the optical absorption gap ( eopt = ip ea eexc ) in the linear paraphenylenes decreases as a function of size . in contrast , ip ea in the cyclic systems decreases at a significantly slower rate than eexc ( cf . consequently , eopt in the cyclic paraphenylenes will be largely determined by the behavior of eexc . because eexc decreases faster than ip ea in the carbon nanorings , the resulting optical absorption gap in the expression eopt = ip ea eexcincreases as a function of size , in agreement with the tddft calculations . exciton binding energies ( ip ea eopt ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . for a given number of benzene rings , in this study , i investigated the optoelectronic properties in a series of cycloparaphenylenes that form the molecular structure of carbon nanorings . to understand their unusual electronic properties as a function of molecular size , i analyzed tddft excited - state energies , examined electronhole transition density matrices , and calculated quasiparticle and exciton binding energies for both the cyclic and the linear paraphenylenes systems . the tddft calculations and transition density analyses show that the cyclic and linear paraphenylenes exhibit dramatically different size - dependent effects that can be understood in the framework of a simple exciton theory . specifically , the main differences between the absorption spectra of the cyclic and linear paraphenylenes are caused by the subtle interplay of contributions to the electronhole interaction energy . in the linear systems , the exciton binding energy decreases slower than the quasiparticle energy gap , and the resulting optical absorption gap decreases with molecular size , as expected . in contrast , the effective coulomb attraction between an electronhole pair is larger in the cyclic geometry and decreases at a faster rate than the quasiparticle band gap . as a result , the overall optical absorption gap in the cyclic paraphenylenes increases as a function of size . in conclusion , i have shown that the different excitonic effects in the cyclic and linear paraphenylenes play a vital role in determining their distinct photophysical properties . the unique spatial distributions of electronhole states , as depicted by their transition density delocalization patterns , result in different electronhole interaction energies that ultimately control the electronic excitations in these systems . looking forward , it would be very interesting to see if the unusual optical trends resulting from the lowest , weakly allowed transition in these nanorings can be observed spectroscopically . the most direct way to observe these lowest transitions would be via low - temperature spectroscopic studies with an applied magnetic field to brighten the dark excitons . a magnetic flux through the open center of the nanorings would distort the circular symmetry of the electronic wave function and produce bright excitons due to the aharonovbohm effect . it is also possible that one can already observe these lowest transitions spectroscopically without the use of an applied magnetic field . because these nanorings are significantly more flexible than carbon nanotubes , the lowest dark exciton may be strongly allowed through symmetry breaking . vibronic coupling , which allows a borrowing of intensity from an energetically close bright state , may also play a significant role in observing these weakly allowed transitions . for the larger carbon nanorings in particular , the first bright state ( i.e. , the s2 state ) is energetically close to the lowest dark state . as a result , the anomalous size scaling of optical excitations should be more easily observed in the larger carbon nanorings . from a theoretical point of view , it would also be interesting to see how these effects change ( or if they change at all ) when the paraphenylenes are functionalized with both electron donor and acceptor functional groups to facilitate charge transfer . calculations of this type would require the use of recent long - range - corrected density functional methods , which are currently underway in my group . with these goals in mind , i anticipate that a critical understanding of excitonic effects in molecular nanostructures will provide a step toward their implementation in optoelectronic applications .

the electronic structure and size - scaling of optoelectronic properties in cycloparaphenylene carbon nanorings are investigated using time - dependent density functional theory ( tddft ) . the tddft calculations on these molecular nanostructures indicate that the lowest excitation energy surprisingly becomes larger as the carbon nanoring size is increased , in contradiction with typical quantum confinement effects . in order to understand their unusual electronic properties , i performed an extensive investigation of excitonic effects by analyzing electron - hole transition density matrices and exciton binding energies as a function of size in these nanoring systems . the transition density matrices allow a global view of electronic coherence during an electronic excitation , and the exciton binding energies give a quantitative measure of electron - hole interaction energies in the nanorings . based on overall trends in exciton binding energies and their spatial delocalization , i find that excitonic effects play a vital role in understanding the unique photoinduced dynamics in these carbon nanoring systems .

Introduction Theory and Methodology Results and Discussion Conclusion

osteoporosis is defined as a skeletal disorder characterized by loss of bone mass , decreased bone strength , and increased risk of bone fracture.1 the disease progresses with age , especially in postmenopausal women.2 japan is one of the most rapidly aging societies worldwide . according to the national institute of population and social security research ( tokyo , japan ) , japanese individuals over 65 years of age constitute 23.0% of the population in 2010 and 25.1% to 25.2% as of 2013 , meaning that more than one in four people in japan are elderly . according to population - based epidemiologic studies , the estimated number of people with osteoporosis in japan is 13 million , among which 130,000 suffer from hip fractures every year ; 20,000 of these individuals die and 60,000 experience functional decline . the prevalence rate of vertebral fractures in japan is reportedly comparable with or higher than in caucasian populations , and is approximately 30% in women in their 70 s and 40% in those in their 80 s.3 a study of the prevalence of vertebral fractures in hong kong , thailand , indonesia , and japan revealed that the prevalence of vertebral fractures in both men and women was highest in japan for the younger ( 6574 years ) and older ( 75 years ) age groups ( 36.6% and 37.6% for men , 18.8% and 28.7% for women , respectively).4 bisphosphonates are known to increase bone mineral density by inhibiting osteoclast - mediated bone resorption and thereby reduce the risk of fractures.5 several formulations containing bisphosphonates have been applied to the management of osteoporosis . alendronate sodium hydrate ( alendronate ) is a bisphosphonate that potently inhibits bone resorption and is used for the treatment of osteoporosis . alendronate produces a sustained reduction in the levels of biochemical markers of bone remodeling , returning them to the premenopausal range.6,7 it also increases bone mineral density612 and decreases the risk of osteoporotic fracture in postmenopausal women.812 long - term intervention studies have shown that continuous alendronate therapy is associated with a sustained therapeutic effect on bone density and remodeling.13,14 however , bisphosphonates including alendronate , suppress bone remodeling and thus may prevent the repair of microdamage . nine patients reportedly developed spontaneous nonspinal fractures after 1 to 8 years of alendronate therapy , and histomorphometric analysis of the bone biopsy samples revealed severe suppression of bone turnover.15 in japan , a patient with insufficiency fractures of the pubis and ilium related to severe suppression of bone turnover after nearly 3 years of alendronate therapy was reported.16 recent papers reported side effects of both atypical femoral fractures and osteonecrosis of the jaw after 7 years of oral alendronate therapy.17 bisphosphonates also have adverse gastrointestinal effects , such as erosions and ulcers in the stomach and small intestine , which are mediated by mitochondrial superoxide production and lipid peroxidation.18 alendronate oral tablets were produced and are used for the treatment of osteoporosis . water is required with an alendronate oral tablet to help facilitate absorption , and the drug can not be taken with other foods or drinks . taking a tablet with oral jelly is a method to prevent digestive system disease and reduce the choking hazard . in this review , this new formulation of alendronate sodium hydrate ( oral jelly ) is introduced and discussed in the treatment of osteoporosis . in addition to osteoporosis , many elderly persons suffer from cardiovascular disease , diabetes mellitus , and joint disease . to treat their diseases , they often use multiple oral drugs . ease of swallowing and handling are preferable for medications , particularly those for elderly patients . studies on the construction of new pharmaceutical preparations and new packaging containers that are optimal for administration to elderly people conducted by the ministry of health and welfare of japan in the 1980s reported that most powder , capsule , tablet , and liquid drug formulations and most packaging containers were suitable for general healthy adults , but not for elderly persons.19 development of new formulations of medicines suitable for elderly individuals with difficulty in swallowing and mastication , dry mouth , and artificial teeth was desired . in 1996 , the ministry of health and welfare of japan reported that jelly , pudding - like , biscuit - like , and wafer - like formulations were preferable for elderly patients.20 biscuit - like and wafer - like formulations were developed as orally disintegrating tablets , which were advanced and popular drug formulations.21 the oral jelly formulation was developed as an original formulation in japan.22 oral jelly formulations have many advantages : ease of swallowing , the ability to take the medication without water , lower risk of accidental ingestion , and optimal masking of drug taste.22 in addition to drug formulations , packaging containers optimal for elderly individuals were developed in japan . there are several types of packaging containers for oral jelly : a single - serving type ( figure 1 ) , a stick type ( figure 2 ) , and an air - extruded type ( table 2 ) . according to a survey among individuals aged 65 and over , the air - extruded type was better than the single - serving type and the stick type.23 elderly persons tend to suffer from a decline in physical performance . it is difficult for the elderly persons to handle the single - serving type when it involved peeling off the container s seal and taking the jelly formulation with a spoon . for the stick type , it was difficult to take all of the jelly formulation by squeezing the packaging container . an air - extruded jelly formulation was developed as a new formulation comprising the jelly formulation and clean air under increased pressure.2225 the jelly formulation is smoothly discharged by pushing the air portion . therefore , elderly patients with physical disabilities are able to easily take all of the jelly formulation from the package . the questionnaire survey indicated the applicability of the air - extruded jelly formulation in elderly patients.26 a majority of the patients ( 63.9% , 69 of 108 subjects ) intended to switch their present drug formulation to the air - extruded jelly formulation . especially among subjects who presently took a powder formulation or more than five kinds of medications daily , a high ratio intended to switch to the air - extruded jelly formulation . alendronate oral jelly 35 mg ( bonalon oral jelly 35 mg ; teijin pharma limited , tokyo , japan ) was approved in 2012 by the ministry of health , labour and welfare of japan as the world s first drug for osteoporosis in jelly formulation . alendronate oral jelly 35 mg , administered once a week , was launched in 2012 ( figure 3 ) . once - weekly alendronate oral jelly comprises a jelly portion and an air portion , which features the air - extruded jelly formulation and makes it easy to take the jelly formulation when pushed by the air . thus , the active ingredient in the once - weekly alendronate oral tablet 35 mg , which has been proven both safe and effective as an osteoporosis treatment , is offered in a jelly formulation . a dissolution test and bioequivalence study , in accordance with the japanese guideline on bioequivalence assessment for development of additional formulations and the japanese guideline for bioequivalence studies of generic products , were carried out to evaluate the bioequivalence of alendronate oral jelly 35 mg to the alendronate oral tablet 35 mg.27 in the dissolution test , the dissolution profiles of both formulations satisfied the criteria for judgment of similarities set forth in the guideline . the bioequivalence study was carried out with an open - label , randomized cross - over ( two - sequence , two - period ) study design and included 32 japanese healthy adult male volunteers . when the pharmacokinetic parameters of alendronate oral jelly 35 mg and alendronate oral tablet 35 mg were calculated from the serum alendronic acid concentration , the area under the blood concentration time curve ( auc ) showed a mean standard deviation of 21.70 10.20 ng hour / ml and 21.33 13.96 ng hour / ml , and maximum concentration ( cmax ) was 9.60 4.36 ng / ml and 9.03 4.93 ng / ml , respectively ( table 3 ) . the serum half - life ( t1/2 ) was 1.54 0.48 hours and 1.47 0.50 hours , respectively . the mean logarithmic auc and cmax were log ( 1.049 ) and log ( 1.050 ) , respectively , each within the criteria range , log ( 0.90 ) to log ( 1.11 ) , for judgment of bioequivalence with the similarities in dissolution profiles . these results indicate that alendronate oral jelly 35 mg is bioequivalent to alendronate oral tablet 35 mg and suggest no difference in clinical efficacy and safety . in european and american studies , alendronate at a dose of 5 to 20 mg / day has consistently been shown to increase bone mineral density at the spine , hip , and other skeletal sites and to reduce the risk of all types of fractures.612,2832 the fracture intervention trial ( fit ) comprised two independent but parallel studies with common recruitment conducted at 11 institutions in the united states . the alendronate dosage was 5 mg / day for 2 years , and was then increased to 10 mg / day.8,10 in the phase iii study , alendronate dosages ranged from 5 to 20 mg / day.9 in the fosamax international trial ( fosit ) and the long - term care facilities study ( ltcfs ) , the alendronate dosage was 10 mg / day . compared to caucasians , asians have a smaller body size33 and a lower dietary intake of calcium.34 because of these racial differences , the recommended drug dose established for caucasians may not be appropriate for asians . a small - scale , single - blind study of postmenopausal japanese women indicated that a dosage of 2.5 mg / day was as effective as a dosage of 10 mg / day in increasing lumbar bone mineral density with 36 weeks of treatment . however , bone turnover markers changed more markedly and rapidly in the 10 mg / day group , and the incidence of adverse effects was higher in the 10 mg / day group . these findings suggest that a 10 mg / day dosage may be an overdosage for japanese patients.35 a dose - range finding study in japan concluded that the optimal daily dose of alendronate is 5 mg in a japanese population.36 the study showed that alendronate at a dosage of 5 mg / day was effective in increasing lumbar bone mineral density , and the incidence of adverse effects was no different compared with the placebo control group after 24 weeks of treatment for japanese patients with postmenopausal osteoporosis . the study also showed that serum parathyroid hormone and serum 1,25-dihydroxyvitamin d increased only in the 5 mg / day dose group . studies to evaluate the effects of alendronate for the treatment of japanese patients have been carried out . a double - masked multicenter comparative study of japanese patients with osteoporosis indicated that alendronate treatment at a dosage of 5 mg / day for 48 weeks was more efficacious than alfacalcidol for increasing bone mineral density without serious drug - related adverse events.37 a 2 year , double - blind comparative study of japanese patients with osteoporosis indicated that alendronate at a dosage of 5 mg / day significantly lowered the incidence of one or more vertebral fractures to a greater extent than did alfacalcidol after 6 months or more of treatment.38 a consecutive 3 year follow - up study of japanese patients demonstrated that continuous alendronate therapy at 5 mg / day is associated with a sustained therapeutic effect on increasing vertebral bone mineral density and reducing the risk of new vertebral fractures.39 a prospective study to assess vertebral strength and mechanical parameters in japanese women with postmenopausal osteoporosis indicated that vertebral strength significantly increased by 26.9% from baseline at 12 months of 5 mg / day alendronate,40 and by 31.8% at 18 months,41 demonstrating a sustained therapeutic effect . once - daily treatment with alendronate has been shown to substantially increase bone mineral density and bone strength and to reduce the incidence of osteoporotic fractures . dosing convenience is a key element in the effective management of any chronic disease , and is particularly important in the long - term management of osteoporosis . alendronate must be administered after fasting with water only , at least 30 minutes before the first food of the day , which offers an additional degree of complexity . thus , it is desirable to reduce the frequency of alendronate dosing without compromising treatment efficacy . animal data support the rationale that once - weekly dosing with alendronate at 70 mg ( seven times the daily oral treatment dose for caucasians ) could provide similar efficacy to that of alendronate at a dosage of 10 mg / day because of its long duration of effect in bone.4244 in addition , animal studies have suggested that the potential for esophageal irritation , observed with daily oral alendronate , might be substantially reduced with once - weekly dosing.45,46 a randomized , double - blind , multicenter study to evaluate the efficacy and safety of oral alendronate once- and twice - weekly dosing regimens compared with daily dosing in postmenopausal women with osteoporosis showed that both the once- and twice - weekly dosing regimens fully satisfied the equivalence criteria relative to daily dosing therapy.47,48 this study also showed that there were fewer serious upper gastrointestinal adverse experiences and a trend toward a lower incidence of esophageal events in the once - weekly dosing group than in the daily dosing group . the study concluded that once - weekly treatment with alendronate would provide patients with a more convenient , therapeutically equivalent alternative to daily dosing and might enhance compliance and long - term persistence with therapy . a therapeutic equivalence study of once - weekly and once - daily alendronate was also carried out in japan . the efficacy and safety of treatment with once - weekly oral alendronate 35 mg were compared with those of once - daily oral alendronate 5 mg in a 52-week , randomized , double - blind , multicenter study of japanese patients with osteoporosis.49 the study concluded that the efficacy and safety of the alendronate 35 mg once - weekly regimen were therapeutically equivalent to those of the alendronate 5 mg once - daily regimen . for a 12 month period in the united states , a randomized and double - blind study to compare the efficacy and tolerability of once - weekly oral alendronate 70 mg and the once - daily oral selective estrogen receptor modular raloxifene 60 mg was carried out for the treatment of postmenopausal osteoporosis.50 the study concluded that the once - weekly alendronate 70 mg regimen produced significantly greater increases in spine and hip bone mineral density and greater reductions in markers of bone turnover than the once - daily raloxifene 60 mg regimen with similar safety and tolerability profiles . risedronate is a bisphosphonate that , like alendronate , binds to bone hydroxyapatite and inhibits osteoclast - mediated bone resorption . once - weekly risedronate oral tablets at a dosage of 35 mg / week ( 5 mg / day ) have been produced and used to treat osteoporosis . a study comparing the once - weekly alendronate 70 mg regimen with the once - weekly risedronate 35 mg regimen during 24 months showed that the former yielded significantly greater bone mineral density gains and larger decreases in bone turnover marker levels than the latter , with no difference in gastrointestinal tolerability.51 the us food and drug administration approved the once - monthly oral ibandronate sodium tablet as the first once - monthly oral tablet in 2005 . ibandronate sodium is a bisphosphonate that inhibits bone resorption and is commonly prescribed for the treatment and prevention of postmenopausal osteoporosis.52,53 once - weekly oral bisphosphonates have been associated with higher adherence compared with the once - daily formulations , although overall adherence has remained suboptimal.5456 a study performed to assess whether once - monthly bisphosphonates are associated with improvements in adherence showed no evidence of improved adherence with once - monthly dosing relative to once - weekly dosing , although adherence with either weekly or monthly dosing was significantly better than that with once - daily dosing.57 these study results suggests that merely reducing the dosing frequency of oral bisphosphonates is not enough to improve adherence . alendronate oral jelly , which features the air - extruded jelly formulation , is easy for elderly patients to use and take ; most of these patients have physical disabilities and swallowing difficulty . therefore , this new formulation of osteoporosis treatment may meet individual needs and improve adherence . in addition , alendronate oral jelly with its advantages of ease of swallowing and lower risk of accidental ingestion may reduce the incidence of adverse effects such as esophageal irritation first , there are no published clinical data for its use in the treatment of osteoporosis . an in vivo study to evaluate the use of alendronate sodium gel in the treatment of bone resorptive lesions in periodontitis was carried out.58 osseous defects associated with chronic adult periodontitis were filled with alendronate sodium gel , and flaps were then sutured to obtain primary closures of the interdental papilla in the alendronate gel site . in the control site the osseous defect improvement rate in the alendronate gel site after 6 months was 42.85% and showed significant improvement compared with that of the control site of ( 7.43% ) . in addition , a 6 month , randomized , controlled clinical trial showed that the local delivery of 1% alendronate gel into periodontal pockets significantly improved bone filling compared with a placebo gel.59 second , alendronate oral jelly is approved and used only in japan . third , once - weekly dosing of alendronate oral jelly is the only dosing regimen . other options , such as a once - monthly dosing regimen , will create a variety of osteoporosis treatments to meet individual needs . intravenous infusion of alendronate for the treatment of osteoporosis is one of these therapeutic options . this preparation may avoid the topical effects of oral alendronate on the gastric mucosa and may provide a useful alternative to oral dosing . a 52-week , multicenter , randomized , double - masked , active - controlled , parallel - group , non - inferiority study of different preparations of alendronate in japanese patients with osteoporosis , randomly assigned to receive intravenous alendronate 900 g every 4 weeks or oral alendronate 35 mg once weekly was conducted.60 the study confirmed that the efficacy and safety of intravenous alendronate 900 g every 4 weeks were similar to those of oral alendronate 35 mg taken once weekly , although the study did not show that intravenous administration might reduce the incidence of gastrointestinal adverse effects . an intravenous infusion of alendronate will provide a new opportunity for effective treatment of patients with osteoporosis who are unable to take oral alendronate or have difficulty complying with the procedures for oral dosing . these new formulations of alendronate oral jelly and intravenous alendronate provide treatment alternatives so that patients may select a method of dosing tailored to their preferences . management of osteoporosis involves assessing fracture risk and preventing fractures.61 higher adherence of treatment of patients with osteoporosis and prevention of fractures associated with osteoporosis remain issues to be resolved .

osteoporosis is a skeletal disorder characterized by loss of bone mass , decreased bone strength , and an increased risk of bone fracture . the disease progresses with age , especially in postmenopausal women . japan is one of the most rapidly aging societies worldwide . japanese individuals over 65 years of age constituted 23.0% of the population in 2010 and 25.1% to 25.2% as of 2013 . the estimated number of people with osteoporosis in japan is currently 13 million . bisphosphonates increase bone mineral density by inhibiting osteoclast - mediated bone resorption , thereby reducing the risk of fractures . alendronate sodium hydrate ( alendronate ) is a bisphosphonate that potently inhibits bone resorption and is used to treat osteoporosis . sufficient water is required to take an alendronate oral tablet ; insufficient water could result in digestive system diseases , such as esophageal ulceration . elderly patients with swallowing difficulty may choke on the tablet . taking a tablet with oral jelly is a method to prevent digestive system disease and reduce the choking hazard . once - weekly alendronate oral jelly was approved in 2012 by the ministry of health , labour , and welfare of japan as the world s first drug for osteoporosis in a jelly formulation . it consists of a jelly portion and an air portion . the jelly formulation is smoothly discharged by pushing the air portion . therefore , elderly patients with physical disabilities are able to easily take all of the jelly formulation from the package . in this review , this new formulation of alendronate sodium hydrate ( oral jelly ) is introduced and discussed in terms of osteoporosis treatment . this new formulation provides an alternative so that patients may select a method of dosing tailored to their preferences . management of osteoporosis involves assessing fracture risk and preventing fractures . higher adherence to the treatment of patients with osteoporosis and prevention of osteoporotic fractures are issues to be resolved .

Introduction Formulations of oral drugs and packaging containers for elderly patients Alendronate sodium hydrate (oral jelly) Discussion

however , the studies examining developing children have shown specific patterns mainly characterized by the girls earlier brain maturation compared with that of boys.14 at this time , as far as we are aware , there is no reported study that has assessed whether adhd symptoms are related to persistence of the primitive reflexes in medication - nave children with a focus on sex - specific assessment . as such , in the study reported here we examined the extent to which the persistence of the primitive atnr and stnr is related to adhd symptoms in 35 medication - nave girls of school age ( 811 years ) . data on 35 girls ( mean age 9.51 , standard deviation [ sd ] = 1.01 , age range 811 years ) with adhd diagnosed according to diagnostic and statistical manual of mental disorders : fourth edition ( dsm - iv ) criteria characterizing their primitive reflexes and psychometric measures of adhd symptoms were collected . all the patients were medication nave and their assessment was done before starting any treatment . the adhd diagnosis was combined hyperactive - inattentive subtype with occurrence of adhd - based learning difficulties and , in a few cases , episodic enuresis . exclusion criteria were other child psychiatric and neurological disorders , including conduct disorder , alcohol or drug dependency , substance abuse , and/or previous exposure to stimulant medication . recruitment was based on consecutive visits of the untreated patients to the clinic , and the control group was recruited using school advertisements . the control group of 30 girls without adhd ( mean age 9.83 , sd = 0.98 , age range 811 years ) fulfilled the same exclusion criteria . parents of all the participants gave written informed consent and the research was approved by the charles university first faculty of medicine ethical committee within the framework of collaboration with the elspac project , the project of the center for neuropsychiatric research of traumatic stress , and the msm0021620849 project at charles university . the measurement of primitive reflexes was done twice by two independent examiners and the results of the two measurements averaged . we measured atnr using the schilder test.15 atnr presents as the tonic reflex response that occurs in newborns ; it normally vanishes at around 3 months of age . in the schilder test , the subject stands with their feet together and the arms held straight out at shoulder level and height but with the hands relaxed at the wrists . in the test when i turn your head , i want you keep your arms straight out in front of you , as they are now . this means your arms remain in the same position , and only your head moves . then the tester slowly rotates the subject s head until the chin is parallel with the shoulder , pauses for 10 seconds , then returns the head to the midline and again pauses for 10 seconds . typical indicators of the atnr include movement of the extended arms in the same direction as the head turn , dropping of the arms , or swaying and loss of balance . it was scored as follows : 0 = no response ( the arms remained straight out in front ) ; 1 = slight movement of the arms up to 20 to the same side as the head is turned or slight dropping of the arms ; 2 = movement of the arms up to 45 as the head is turned or marked dropping of the arms ; 3 = arm movement greater than 45 either to the side or down , swaying or loss of balance may occur . purdue reflex test.16 this reflex emerges at 68 months of life and is inhibited at between 9 and 11 months . in the bender purdue test , the subject is instructed to maintain four point kneeling table position and to slowly move the head to look down as between the thighs . the position is held for up to 5 seconds and then the head is slowly moved upward as if looking at the ceiling . typical indicators of the reflex are any bending of the arms due to head flexion and/or raising of the feet , straightening of the arms and flexion of the knees as a result of head extension , any bending of the arms due to head flexion and/or raising of the feet , and straightening of the arms and flexion of the knees due to head extension . the test was scored as follows : 0 = no response ; 1 = tremor in one or both arms or slight hip movement ; 2 = movement of the elbow on either side and/or definite , movement in the hips , or arching of the back ; 3 = bending of the arms on head flexion or movement of the bottom back on head extension ; 4 = bending of the arms to the floor , or movement of the bottom back onto the ankles , so that the subject is sitting in the cat position . a frequently used measure of adhd symptoms is the conners parent questionnaire ( cpq).17 the cpq is 93-item scale of symptoms that are most commonly associated with behavioral disorders and related to children and adolescents ( aged 317 years ) , and can measure treatment changes and outcome assessment purposes . the questionnaire enables a total score to be calculated as well as scores for subscales based on the factor structure of the questionnaire : i conduct problems ( items 39 , 40 , 41 , 47 , 48 , 51 , and 69 ) ; ii anxiety ( items 811 , 42 , 43 , and 64 ) ; iii impulsivity - hyperactivity ( items 78 , 8084 , 89 , and 90 ) ; iv learning problems ( items 45 , 62 , 63 , 67 ) ; v psychosomatic difficulties ( items 6 and 2124 ) ; vi perfectionism ( items 3 and 7678 ) ; vii antisocial behavior ( items 7173 and 75 ) ; and viii muscular tension ( items 1214 and 36 ) . the symptoms are rated on a four - point likert scale ( from 0 to 3 ) by either one or both parents of the child . for statistical evaluation of scores of the measures of atnr , stnr , and adhd symptoms , nonparametric analyses were preferred because cpq , atnr , and stnr data do not have normal distribution . all the methods of statistical evaluation were performed using the software package statistica ( v 6 ; statsoft inc , tulsa , ok , usa ) . data on 35 girls ( mean age 9.51 , standard deviation [ sd ] = 1.01 , age range 811 years ) with adhd diagnosed according to diagnostic and statistical manual of mental disorders : fourth edition ( dsm - iv ) criteria characterizing their primitive reflexes and psychometric measures of adhd symptoms were collected . all the patients were medication nave and their assessment was done before starting any treatment . the adhd diagnosis was combined hyperactive - inattentive subtype with occurrence of adhd - based learning difficulties and , in a few cases , episodic enuresis . exclusion criteria were other child psychiatric and neurological disorders , including conduct disorder , alcohol or drug dependency , substance abuse , and/or previous exposure to stimulant medication . recruitment was based on consecutive visits of the untreated patients to the clinic , and the control group was recruited using school advertisements . the control group of 30 girls without adhd ( mean age 9.83 , sd = 0.98 , age range 811 years ) fulfilled the same exclusion criteria . parents of all the participants gave written informed consent and the research was approved by the charles university first faculty of medicine ethical committee within the framework of collaboration with the elspac project , the project of the center for neuropsychiatric research of traumatic stress , and the msm0021620849 project at charles university . the measurement of primitive reflexes was done twice by two independent examiners and the results of the two measurements averaged . we measured atnr using the schilder test.15 atnr presents as the tonic reflex response that occurs in newborns ; it normally vanishes at around 3 months of age . in the schilder test , the subject stands with their feet together and the arms held straight out at shoulder level and height but with the hands relaxed at the wrists . in the test when i turn your head , i want you keep your arms straight out in front of you , as they are now . this means your arms remain in the same position , and only your head moves . then the tester slowly rotates the subject s head until the chin is parallel with the shoulder , pauses for 10 seconds , then returns the head to the midline and again pauses for 10 seconds . typical indicators of the atnr include movement of the extended arms in the same direction as the head turn , dropping of the arms , or swaying and loss of balance . it was scored as follows : 0 = no response ( the arms remained straight out in front ) ; 1 = slight movement of the arms up to 20 to the same side as the head is turned or slight dropping of the arms ; 2 = movement of the arms up to 45 as the head is turned or marked dropping of the arms ; 3 = arm movement greater than 45 either to the side or down , swaying or loss of balance may occur . purdue reflex test.16 this reflex emerges at 68 months of life and is inhibited at between 9 and 11 months . in the bender purdue test , the subject is instructed to maintain four point kneeling table position and to slowly move the head to look down as between the thighs . the position is held for up to 5 seconds and then the head is slowly moved upward as if looking at the ceiling . typical indicators of the reflex are any bending of the arms due to head flexion and/or raising of the feet , straightening of the arms and flexion of the knees as a result of head extension , any bending of the arms due to head flexion and/or raising of the feet , and straightening of the arms and flexion of the knees due to head extension . the test was scored as follows : 0 = no response ; 1 = tremor in one or both arms or slight hip movement ; 2 = movement of the elbow on either side and/or definite , movement in the hips , or arching of the back ; 3 = bending of the arms on head flexion or movement of the bottom back on head extension ; 4 = bending of the arms to the floor , or movement of the bottom back onto the ankles , so that the subject is sitting in the cat position . we measured atnr using the schilder test.15 atnr presents as the tonic reflex response that occurs in newborns ; it normally vanishes at around 3 months of age . in the schilder test , the subject stands with their feet together and the arms held straight out at shoulder level and height but with the hands relaxed at the wrists . in the test when i turn your head , i want you keep your arms straight out in front of you , as they are now . this means your arms remain in the same position , and only your head moves . then the tester slowly rotates the subject s head until the chin is parallel with the shoulder , pauses for 10 seconds , then returns the head to the midline and again pauses for 10 seconds . typical indicators of the atnr include movement of the extended arms in the same direction as the head turn , dropping of the arms , or swaying and loss of balance . it was scored as follows : 0 = no response ( the arms remained straight out in front ) ; 1 = slight movement of the arms up to 20 to the same side as the head is turned or slight dropping of the arms ; 2 = movement of the arms up to 45 as the head is turned or marked dropping of the arms ; 3 = arm movement greater than 45 either to the side or down , swaying or loss of balance may occur . purdue reflex test.16 this reflex emerges at 68 months of life and is inhibited at between 9 and 11 months . in the bender purdue test , the subject is instructed to maintain four point kneeling table position and to slowly move the head to look down as between the thighs . the position is held for up to 5 seconds and then the head is slowly moved upward as if looking at the ceiling . typical indicators of the reflex are any bending of the arms due to head flexion and/or raising of the feet , straightening of the arms and flexion of the knees as a result of head extension , any bending of the arms due to head flexion and/or raising of the feet , and straightening of the arms and flexion of the knees due to head extension . the test was scored as follows : 0 = no response ; 1 = tremor in one or both arms or slight hip movement ; 2 = movement of the elbow on either side and/or definite , movement in the hips , or arching of the back ; 3 = bending of the arms on head flexion or movement of the bottom back on head extension ; 4 = bending of the arms to the floor , or movement of the bottom back onto the ankles , so that the subject is sitting in the cat position . a frequently used measure of adhd symptoms is the conners parent questionnaire ( cpq).17 the cpq is 93-item scale of symptoms that are most commonly associated with behavioral disorders and related to children and adolescents ( aged 317 years ) , and can measure treatment changes and outcome assessment purposes . the questionnaire enables a total score to be calculated as well as scores for subscales based on the factor structure of the questionnaire : i conduct problems ( items 39 , 40 , 41 , 47 , 48 , 51 , and 69 ) ; ii anxiety ( items 811 , 42 , 43 , and 64 ) ; iii impulsivity - hyperactivity ( items 78 , 8084 , 89 , and 90 ) ; iv learning problems ( items 45 , 62 , 63 , 67 ) ; v psychosomatic difficulties ( items 6 and 2124 ) ; vi perfectionism ( items 3 and 7678 ) ; vii antisocial behavior ( items 7173 and 75 ) ; and viii muscular tension ( items 1214 and 36 ) . the symptoms are rated on a four - point likert scale ( from 0 to 3 ) by either one or both parents of the child . for statistical evaluation of scores of the measures of atnr , stnr , and adhd symptoms , spearman correlation coefficients were determined and multiple linear regression analysis was undertaken . nonparametric analyses were preferred because cpq , atnr , and stnr data do not have normal distribution . all the methods of statistical evaluation were performed using the software package statistica ( v 6 ; statsoft inc , tulsa , ok , usa ) . adhd symptoms measured by total score of the cpq and its subscales were significantly correlated with atnr and stnr scores ; that is : cpq and atnr , r = 0.64 ; cpq and stnr , r = 0.41 ; ii anxiety and atnr , r = 0.39 ; iii impulsivity - hyperactivity and atnr , r = 0.47 ; iii impulsivity - hyperactivity and stnr , r = 0.40 ; iv learning problems and stnr , r = 0.47 ; and vi perfectionism and atnr , r = 0.44 ( table 1 , figure 1 ) . spearman rank correlation coefficients with r higher than 0.43 were significant at p < 0.01 , with fisher s z > 0.46 , and spearman rank correlation coefficients with r > 0.33 significant at p < 0.05 , with fisher s z > 0.35 . these correlations indicate that significant manifestations of adhd symptoms are associated with persisting atnr and stnr responses . we compared cpq , atnr and stnr scores for the 35 study subjects with those for the 30 control participants . the control group had no adhd symptoms , and none of this group manifested atnr ( the score was zero in all cases ) , while only one case of stnr response was found with a score of 1 . to analyze effects of atnr and stnr on cpq , we used a multiple linear regression to determine whether persisting primitive reflexes in their specific interactions are proportionally linked to increased levels of cpq . multiple r = 0.60 was found to be statistically significant ( p = 0.0008 ; f = 8.99 ; sequential consistency ( sc ) model = 2,375 ; sc residuals ( resid ) = 4,229 ; processor consistency ( pc ) model = 1187 ; pc resid = 132 ) which enables cpq to be defined as a linear function of two variables : cpq = f(atnr , stnr ) . recent findings show that a high proportion of children with adhd exhibit motor abnormalities.3,4 according to brain imaging studies , these abnormalities are probably linked to prefrontal cortex deficits that influences attention and executive functions.1820 the abnormalities may also be of cerebellar origin and adhd children in many cases exhibit atrophy in cerebellar regions associated with motor dysfunctions that are most likely linked to inhibitory deficits.3,21,22 disinhibitory processes related to disturbed motor functions are probably also linked to persisting primitive reflexes that were not sufficiently suppressed in later stages of development . according to some evidence , persisting primitive reflexes may occur in various neuropsychiatric syndromes such as schizophrenia or various forms of dementia5,6,20,23 and have also been observed in patients with dyslexia11,24 and in school - aged children with adhd12,13,25,26 in our previous study on medicated adhd children,13 we found that boys had higher levels of adhd symptoms and that there was little statistically significant difference in persisting primitive atnr between girls and boys . moreover , studies that have focused on persistent atnr in dyslexia11,24 have not reported differences between girls and boys . in this context , this study represents an important step focused on detailed analysis of adhd children in medication nave conditions . the results show that persistent atnr and other primitive reflexes that may occur in later age specifically influence adhd symptoms related to hyperactivity due to muscular tension , which also may have specific influence on attentional functions . taken together recent research predominantly does not take into account differences between adhd girls and boys.27,28 nevertheless several comparisons showed few sex effects in core clinical manifestations of adhd . in addition , girls compared with boys , have significantly more parent - rated emotional symptoms and more likely they become victims of bullying . girls and boys have similar levels of coexisting psychiatric and physical health problems , and receive the same types of treatment although fewer girls than boys are referred for adhd treatment.27,28 therefore , research focused on girls with adhd seems to be particularly important and , in the future , detailed comparison of girls and boys may provide particularly important findings and have significant therapeutic implications . the results of this study support the hypothesis that adhd symptoms are linked to the persisting primitive atnr and stnr in girls . further data are needed to find specific sex differences and to determine the relationships between adhd symptoms and various primitive reflexes that are possible to assess in school - aged children . together , our data suggest a hypothesis for future research that adhd symptoms may present a process related to primitive neural mechanisms , such as primitive reflexes , interfering with higher - level brain functions due to insufficiently developed cognitive and motor integration . in this context , hughlings jackson s concept may in principle be useful to explain and understand certain developmental changes as a consequence of disturbances between higher and lower levels of cognitive and motor functions during brain processing that might cause processes related to dissolution ( or disintegration ) of mental functions.8,12 the preliminary data reported here show that detailed analysis of persisting primitive reflexes in children with developmental disorders and comparisons of differences between girls and boys with adhd and dyslexia may offer important new findings with specific consequences for treatment .

background and objectivesrecent and historical findings suggest that later - developed functions during brain ontogenesis related to higher levels of cognitive and motor integration tend to replace the older , more primitive , ones , and the persistence of the older functions may be linked to specific neuropsychiatric disorders . currently , there is growing evidence to suggest that persisting primitive reflexes may be related to developmental and neurodegenerative disorders . preliminary data also suggest that persisting primitive reflexes may be specifically linked to attention - deficit and hyperactivity disorder ( adhd).methodsin the study reported here , we tested to what extent the persisting primitive asymmetric tonic neck reflex and symmetric tonic neck reflex are related to adhd symptoms measured by conners parent questionnaire in 35 medication - nave girls of school age ( 811 years ) with adhd . the results were compared with those of a control group of 30 girls of the same age.resultsthis study showed that persisting primitive reflexes are closely linked to adhd symptoms.conclusionthe data suggest that adhd symptoms may be linked to more primitive neural mechanisms interfering with higher brain functions due to insufficiently developed cognitive and motor integration .

Introduction Methods Participants Measurement of primitive reflexes ATNR STNR Psychometric measure of ADHD symptoms Statistical analysis Results Discussion Conclusion

community - acquired pneumonia ( cap ) is acquired during the course of everyday life by patients who do not have a known immune deficiency . despite advancements in antibiotic treatment , cap continues to negatively affect morbidity and mortality . cap is also the sixth most common infectious cause of death in hospitals.1 previous studies have shown that the mortality rate of pneumonia varies from 1% to 60% and is related to the weight of the patient.2 studies have found that the mortality rate in cap outpatient cases is 15% ; the mortality rate is 12% in inpatient cases and 40% in intensive care patients.3,4 d - dimer ( dd ) is a metabolic substance produced during the catabolization of fibrin by plasmin . dd levels have been shown to increase in patients who have disorders that trigger fibrin production and catabolization ; these disorders include pulmonary emboli ( pe ) , deep vein thrombosis ( dvt ) , solid tumors , leukemia , severe infections , trauma or a post - operative state , disseminated intravascular coagulation ( dic ) , pregnancy , acute stroke , sickle - cell anemia , congestive heart failure and chronic kidney failure.58 while the measurement of plasma dd levels is a well - known test for venous thromboemboli , the relationship between plasma dd and other diseases is still unclear . a limited number of studies have examined the relationship between cap and plasma dd levels . some of these studies suggest that an increase in dd is directly related to the intra- and extra - vascular coagulation that occurs in acute and chronic lung damage in cap cases.9 the present study investigated the relationship between the severity of cap and serum dd levels , as well as the potential correlation among plasma dd levels , the radiological extent of the disease and hospital mortality rates . the ultimate goal of this study was to determine whether plasma dd levels can be used to develop prognoses in cap cases . this study began in october 2007june 2008 at the department of chest diseases , faculty of medicine , cumhuriyet university , sivas , turkey , and included 60 consecutive cap patients and 24 healthy controls . the study was approved by the ethics committee of the faculty of medicine , cumhuriyet university , and all participants gave their informed consent . individuals were excluded from the study because of cancer , leukemia , known blood - clotting and bleeding disorders , disseminated intravascular coagulation ( dic ) , renal failure , rheumatological diseases , vasculitis or sickle - cell anemia , pregnancy , an age less than 18 years or thromboembolic diseases . the demographic characteristics , physical examination findings and laboratory findings ( leucocytes , hemoglobin , hematocrit , glucose , sodium , potassium , urea , creatinine , crp and sedimentation ) of all study participants were monitored regularly . initial lung x - rays were taken for all patients in the cap group and the control group . patients were placed into two groups according to their lung x - rays : lobar ( i.e. , more than one segment of the same lobe involved ) and multilobar ( i.e. , more than one lobe involved ) . patients were divided into five groups according to the pneumonia severity index ( psi ) ( psi groups i , ii , iii , iv and v).10,11 cap guidelines were used to determine whether patients would be treated at home or in the hospital.12 the criteria are shown in table 1 . the plasma dd levels of all participants were measured in the hospital s hematology laboratory before starting the treatments of their cap . blood samples taken from the antecubital vein with an injector were placed into 1.8 ml citrated tubes . the blood samples were centrifuged at 40,000 rpm for 15 minutes at room temperature and then evaluated using the quantitative latex method ( acltop , italy ) . kruskal - wallis ( cv ) , tukey s and mann - whitney u tests were used in the statistical analyses , and p - values of less than 0.05 were accepted as significant . the study included 60 cap patients with an overall mean age of 62.5 11.7 years ( 29 males with an average age of 63.0 11.7 years and 31 females with an average age of 62.0 11.7 years ) and 24 healthy controls with a mean age of 59.6 6.6 years ( 10 males with an average age of 59.5 4.6 years and 14 females with an average age of 59.4.0 7.9 years ) . the age and sex distributions for the cap and control groups are shown in table 2 . the relationship between plasma dd levels and psi statistically significant differences ( p<0.05 ) were found between the following groups : the control group and psi groups iii , iv and v ; psi group i and psi group iv and v ; psi group iii and psi group v as well as between psi group iv and psi group v. differences between the other groups were not found to be statistically significant ( p>0.05 ) ( table 3 ) . statistically significant ( p<0.05 ) differences were found between the following groups : the control group and psi group iii , iv and v ; psi group i and psi group iv and v ; psi group ii and psi group iv and v ; psi group iii and psi group iv as well as between psi group iv and psi group v. no statistically significant differences were found among the other classes ( p>0.05 ) . plasma dd levels were 337 195.1ng / ml in the outpatient group , 691.0 180.5ngml in the inpatient group , 1363.2 331.5ng / ml in the intensive care group and 161.3 38.1ng / ml in the control group ( see table 4 and figure 1 ) . in addition to cap , 38 patients had other accompanying diseases ( 15 patients : asthma / copd ( chronic obstructive pulmonary disease ) 9 patients : diabetes mellitus ; 8 patients : coronary artery disease ; 3 patients : chronic liver disease and 3 patients : peptic ulcus ) ; 22 patients had no accompanying disease . the mean plasma dd level for patients who had an accompanying disease was 776.15 473.58 , while the mean serum dd level for patients without an accompanying disease was 494.23 280.18 ng / ml . patients were divided into two groups ( lobar and multilobar ) , and plasma dd levels in the multilobar group were significantly higher than those in the lobar group ( table 5 ) ( p<0.001 ) five of the 36 inpatients ( 13.8% ) died ; all of the deceased patients had plasma dd levels that were higher than the normal limit ( 1429.8 511.3ng / ml ) , and all had another disease in addition to cap . an increase in plasma dd levels may result from the activation of the fibrinolytic system and from fibrin catabolization within the alveoli . in addition , plasma dd levels may be increased by the activation of the blood coagulation process ; this activation is caused by endotoxins in the gram - negative pathogens that trigger cap.1215 this study found that the plasma dd levels of cap outpatients , inpatients and intensive care patients correlated positively with the severity of their pneumonia . this study found a statistically significant relationship between patients psi scores and their plasma dd levels . shilon et al.,16 in a study performed on 68 cap patients and no control group , found that psi groups iv and v were associated with increased plasma dd levels when compared with groups i , ii and iii . ng / ml in the severe pneumonia group , 912.6 512.6 ng / ml in the non - severe pneumonia group and 387.94 99.56 ng / ml in the control group ; their results were statistically significant.17 ribelles et al.18 found that increased plasma dd levels were correlated with psi ; their results were also statistically significant . chalmers and coworkers19 also found that increased dd levels were correlated with psi . in their study , median dd levels were as follows : class i : 543 ng / ml ; class ii : 727 ng / ml ; class iii : 1192 ng / ml ; class iv : 1128 ng / ml and class v : 1801 ng / ml ; p<0.001 . the present study also found a statistically significant relationship between serum dd level and psi score ; these findings are consistent with the findings of previous studies . plasma dd levels were found to be statistically significant in the inpatient group than outpatients group ( defined according to the cap guidelines).12 guneysel et al . and ribelles et al . found increased plasma dd levels in intensive care patients,17,18 and shillon et al . found increased plasma dd levels in inpatients.16 the present study also found that plasma dd levels were significantly increased in intensive care patients . several studies have examined the relationship between plasma dd levels and the extent of disease in the lungs of cap patients.18,20 levi et al.20 reported a correlation between the extent of pulmonary disease , radiological appearance and plasma dd levels in severe pneumonia patients . ribelles et al.18 suggested that plasma dd levels were higher in patients with lobar or multilobar pneumonia than in patients with segmental pneumonia . no relationship was detected between a high plasma dd level and either the radiological extent of the disease , microorganisms present or the antibiotics previously administered no segmental involvement was determined in the present study . this could be because patients came to our hospital only in the later stages of the illness or because they had received treatment previously in other healthcare settings . patients were divided into two groups ( lobar and multilobar ) , and plasma dd levels in the multilobar group were significantly higher than those in the lobar group ( p<0.05 ) . ribelles et al . found a strong correlation between the mortality rate and serum dd levels in cap patients18 . while the mean serum dd level was 3,786 2,646 ng / ml in patients who died , it was only 1,609 1,808 ng / ml in patients who survived ( p < 0.0001).18 they also determined that cap patients in psi classes iv and v who had plasma dd levels higher than 2000 ng / ml had a higher risk of mortality.18 some researchers have suggested that increased plasma dd levels are related to the extent of pneumonia because the psi and apache ii score are closely associated with the mortality rate.21 low plasma dd levels in cap patients ( less than 500 ng / ml ) at the time of admission to the hospital has been linked to a decreased likelihood of an early death or major morbidity.19 several researchers have identified meaningful relationships between plasma dd levels and psi.16,17 however , because these studies included an insufficient number of patients , no strong relationship between plasma dd levels and mortality could be identified.16,17 the present study was able to identify a strong relationship between plasma dd levels and the rate of mortality ( 13.8% ) . cap patients show increased plasma dd levels even in the absence of an accompanying disease that can increase dd levels . increased plasma dd levels in cap patients were correlated with the severity of the disease , the radiological extent of the disease and in - hospital mortality rates . additional studies with more patients are needed to determine whether plasma dd levels can be used to develop prognoses for cases of pneumonia .

background : plasma d - dimer levels are directly related to the intra- and extra - vascular coagulation that occurs in acute and chronic lung damage in patients with community - acquired pneumonia ( cap).objectives : this study examines the relationship between the severity of community - acquired pneumonia and d - dimer levels . in addition , the study examines the correlations among community - acquired pneumonia , the radiological extent of the disease and mortality.methods:the pneumonia severity index was used to classify patients into five groups . patients were treated at home or in the hospital according to the guidelines for community - acquired pneumonia . blood samples were taken from the antecubital vein with an injector and placed into citrated tubes . after they were centrifuged , the samples were evaluated with the quantitative latex method.results:the study included 60 patients who had been diagnosed with community - acquired pneumonia ( mean age 62.5 11.7 ) and 24 healthy controls ( mean age 59.63 6.63 ) . the average plasma d - dimer levels were 337.3 195.1ng / ml in the outpatient treatment group , 691.0 180.5 in the inpatient treatment group , 1363.2 331.5 ng / mlin the intensive care treatment group and 161.3 38.1ng / ml in the control group ( p<0.001 ) . the mean d - dimer plasma level was 776.1 473.5ng / ml in patients with an accompanying disease and 494.2 280.1 ng / ml in patients without an accompanying disease ( p<0.05).conclusions : plasma d - dimer levels were increased even in community - acquired pneumonia patients who did not have an accompanying disease that would normally cause such an increase .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

bacterial multicomponent monooxygenases ( bmms ) comprise a family of enzymes capable of hydroxylating or epoxidizing a wide range of hydrocarbons , including the greenhouse gas methane and environmentally hazardous substances such as benzene and trichloroethylene . bmm enzymes can be grouped into four classes : the three - component enzymes soluble methane monooxygenases ( smmos ) , phenol hydroxylases ( phs ) , and alkene monooxygenases ( amos ) , and the four - component enzymes alkene / arene monooxygenases . all bmm enzymes contain three common components : a hydroxylase , a reductase , and a regulatory protein . the hydroxylase component is a multi - subunit dimeric ( 222 or 22 ) protein hosting a diiron center in each -subunit . the carboxylate - bridged diiron center is the locus for o2 activation and subsequent substrate hydroxylation / epoxidation . it is similar to those in the r2 subunit of ribonucleotide reductase , ferritin , stearoyl acyl carrier protein 9 desaturase , and the aging - related protein clk1 . the reductase component is an nadh oxidoreductase with an [ fe2s2 ] cluster in the ferredoxin domain ( fd ) and a flavin adenine dinucleotide ( fad ) cofactor in the fad domain , responsible for the reduction of the hydroxylase diiron center . the third component , a cofactor - less regulatory protein , couples nadh consumption to product formation . a key question regarding the catalytic mechanism of bmm enzymes is how component protein interactions achieve the timely control of electron transfer to the diiron active site , dioxygen activation , and hydrocarbon substrate oxidation . elucidating component protein binding sites on the hydroxylase is required as a foundation for answering such a question . an important clue comes from early crystallographic investigations of hydroxylases from the three - component system smmo and the four - component toluene / o - xylene monooxygenase ( tomo ) . both hydroxylases contain a structure of c2 symmetry with a shallow depression , termed the canyon the canyon region was proposed as the docking site for the other component proteins , and , indeed , later crystallographic studies revealed that the regulatory component does occupy a portion of the canyon in hydroxylase regulatory protein complexes of ph , toluene-4-monooxygenase ( t4mo , another four - component bmm ) , and , very recently , smmo ( figure 1b ) . regulatory protein complex of smmo ( pdb i d 4gam ) : ( a ) the hydroxylase mmoh showing the canyon , and ( b ) mmoh in complex with the regulatory protein mmob . there is another mmob molecule binding to the canyon on the other side of mmoh . mmoh -subunit is colored in green , -subunit in blue , -subunit in yellow , and mmob in purple . there is no crystal structure available for the hydroxylase reductase complex of any bmm enzyme . by using the zero - length cross - linker 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide ( edc ) , a chemical cross - linking study of smmo isolated from methylosinus trichosporium ob3b revealed that the reductase , mmor , cross - linked to the -subunit of the hydroxylase mmoh , and that the regulatory component , mmob , cross - linked to the -subunit . a different result was obtained , however , using smmo isolated from methylococcus capsulatus ( bath ) , where either the full - length mmor or its fd cross - linked to the -subunit using the same cross - linker , edc . further attempts to determine the binding site by identifying cross - linked residues failed . the two identified fd cross - linking sites , glu-56 and glu-91 , cross - linked to the n - terminal amino group of mmoh -subunit , which is not observed in the crystal structure of mmoh owing to disorder . because the mmor binding site on mmoh is obscure , it was unclear how the regulatory protein and the reductase might interact in the complete enzyme system . simulations of steady - state oxidase and oxygenase activities of smmo as a function of component protein concentrations favored a non - competitive model , whereby mmor and mmob bind at distinct sites on mmoh , forming a hypothetical ternary complex . the formation of such a ternary complex was also proposed in a small - angle x - ray scattering ( saxs ) study , where the species formed in the presence of large excess of mmob and mmor ( 1020 equiv of each relative to mmoh ) was modeled as a mmoh2mmob2mmor complex . later crystallographic investigations of the hydroxylase regulatory protein complexes , however , suggested that the regulatory component may block the reductase binding site , but there was no direct experimental evidence for such . the role of the regulatory protein in electron transfer from the reductase to the hydroxylase diiron center is also not well understood . a determination of the reductase binding site on the hydroxylase would clarify many of these questions . accordingly , in this study we determined the reductase binding site on the hydroxylase of smmo isolated from methylococcus capsulatus ( bath ) , by using hydrogen the results clearly reveal that the fd of mmor indeed binds to the canyon of mmoh . more importantly , the fd shares the same binding site as the core of mmob ; it therefore binds competitively with mmob to mmoh . consistent with the shared binding site , we show that mmob does not facilitate , but actually inhibits , electron transfer . overall , this work presents the first experimentally determined reductase fd binding site on the hydroxylase of a bmm enzyme , and it reveals how the regulatory component may control electron transfer in the catalytic cycle . 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid ( iaedans ) was obtained from molecular probes . other chemicals were purchased from sigma - aldrich and used without further purification . the expression system for mmor ferredoxin domain ( fd , residues 1107 ) was prepared by mutating s108 and f109 of wild - type mmor to stop codons by site - directed mutagenesis , using primers shown in table s1 . fd was expressed and purified as described previously , except that an additional step with a monoq column was employed to separate apo protein without the iron sulfur cluster from the holo protein . the expression system for the mmob 2 - 33 d36c mutant was prepared by site - directed mutagenesis using mmob d36c as the template ; the primers are shown in table s1 . the mmob 2 - 33 d36c protein was expressed and purified following the procedure described for wild - type mmob . a 60 pmol portion of mmoh was incubated with fd for a final mmoh : fd concentration ratio of 1:6 during deuterium labeling . under this condition , > 95% of the fd binding sites on mmoh were saturated , based on a kd value of 0.9 m . all mixtures were incubated for 20 min at room temperature before deuterium labeling . as a control , mmoh alone was incubated in 50 mm phosphate buffer ( ph 7.0 ) and treated exactly the same as the fd - bound protein . deuterium exchange was initiated by dilution of each sample with 15-fold 50 mm phosphate buffer ( pd 7.0 ) , 99.9% d2o at room temperature . at each deuterium exchange time point ( 10 s , 1 min , 10 min , 60 min , 4 h , 6 h , and 8 h ) , an aliquot from the exchange reaction was removed and quenched by adjusting the ph to 2.5 with an equal volume of quench buffer ( 150 mm potassium phosphate buffer , h2o ) . quenched samples were immediately frozen on dry ice and stored at 80 c until analysis . several undeuterated control samples were prepared in the same way as the deuterium - labeled samples and were used for validation of the peptic peptides of the proteins used in the deuterium labeling experiments . each flash - frozen sample was rapidly thawed and injected into a waters nanoacquity with hdx technology ( waters corp . ) . the protein samples were digested online using a 2.1 mm 30 mm poroszyme immobilized pepsin cartridge ( applied biosystems ) . the digestion temperature was set to 15 c and the digestion was performed for 30 s. the cooling chamber of the ultra - performance liquid chromatography ( uplc ) system , which housed all the chromatographic elements , was held at 0.0 0.1 c for the entire time of the measurements . the injected peptides were trapped and desalted for 3 min at 100 l / min and then separated in 14 min by a 5% to 40% acetonitrile : water gradient at 40 l / min . the separation column was a 1.0 100.0 mm acquity uplc c18 bridged ethyl hybrid particles ( beh ) column ( waters corp . ) containing 1.7 m particles , and the back pressure averaged 8800 psi at 0.1 c . the average amount of back - exchange using this experimental setup was 1825% , based on analysis of highly deuterated peptide standards . deuterium levels were not corrected for back - exchange and are therefore reported as relative ; however , all comparison experiments were done under identical experimental conditions , thus negating the need for a back exchange correction . the uplc step was performed with protonated solvents , thereby allowing deuterium to be replaced with hydrogen from side chains and the amino / carboxyl terminus that exchange much more rapidly than amide linkages . the average error in determining the deuterium levels was 0.1 da in this experimental setup , consistent with previously obtained values . in order to eliminate peptide carryover , a wash solution of 1.5 m guanidine hydrochloride , 0.8% formic acid , and 4% acetonitrile was injected after each run . mass spectra were obtained with a waters xevo g2 tof instrument equipped with standard electrospray ionization source ( waters corp . ) . the instrument configuration was the following : capillary was 3.2 kv , trap collision energy at 6 v , sampling cone at 35 v , source temperature of 80 c , and desolvation temperature of 175 c . mass accuracy was ensured by calibration with 500 fmol/l human [ glu1]-fibrinopeptide b and was less than 10 ppm throughout all experiments . the mass spectra were processed with the software dynamx 2.0 ( waters corp . ) by centroiding an isotopic distribution corresponding to the + 2 , + 3 , or + 4 charge state of each peptide . deuteration levels were calculated by subtracting the centroid of the isotopic distribution for peptide ions of undeuterated protein from the centroid of the isotopic distribution for peptide ions from the deuterium - labeled sample . the resulting relative deuterium levels were automatically plotted versus the exchange - in time . identification of the peptic fragments was accomplished through a combination of exact mass analysis and ms using identity software ( waters corp . ) . ms was performed by a series of low high collision energies ramping from 5 to 32 v , therefore ensuring proper fragmentation of all the peptic peptides eluting from the lc system . iaedans - labeled mmob d36c and 2 - 33 d36c mutants were prepared following procedures described previously . concentrations of the labeled proteins were determined by using the bradford assay ( bio - rad ) . the excitation wavelength was set to 336 nm and emission was monitored at 490 nm . samples were made in 25 mm mops , ph 7.0 buffer ; the concentration of fluorescently labeled protein was 1 m . mmoh was considered to have two non - interacting binding sites ( hsite ) . the simulation procedures are described as follows , taking the titration of fd into 1 m mmoh and 1 m iaedans - labeled mmob as an example . two equilibria were considered , eqs 1 and 2 , where [ hsite ] , [ b ] , and [ fd ] are the concentrations of free mmoh binding site , free mmob , and free fd ; [ hsite - b ] and [ hsite - fd ] are the concentrations of bound mmob and fd ; [ hsite]total , [ b]total , and [ fd]total are the total concentrations of mmoh binding site ( 2 m ) , mmob ( 1 m ) , and fd ( the total amount titrated in ) . b ) was determined previously to be 0.55 m ; several kd values for the h fd complex ( kd , h fd ) were tested to allow us to choose the one that best simulated the experimental data . the concentrations [ b ] and [ hsite b ] were first calculated for each titration point by numerically solving the simultaneous eqs 1 and 2 . these values were then used to calculate the observed fluorescence anisotropy robs ( eq 3 ) , which is the sum of fluorescence anisotropy of free and bound mmob weighted by their fractional fluorescence intensity,3 where rb and rhsite b are the fluorescence anisotropy of free and bound mmob , and fb and fhsite b are the fractional fluorescence intensity of free and bound mmob , respectively . the fb and fhsite b parameters can be expressed in terms of [ b ] , [ hsite b ] , and the molar fluorescence intensity of free and bound mmob , fb and fhsite b:45 substituting eqs 4 and 5 into eq 3 , the fluorescence anisotropy of each titration point can be calculated on the basis of [ b ] and [ hsite b ] by solving eqs 1 and 2:6 the same simulation procedures were followed for the titration of fd into 1 m mmoh and 1 m iaedans - labeled mmob 2 - 33 . the kd value of 2.67 m for the h b 2 - 33 complex determined in this study was used for the simulation ( figure s3 ) . several kd values ( 0.9 , 2 , and 6 m ) for the h fd complex ( kd , h fd ) were tested to see which one best fits the experimental data . to a mixture of 10 m mmoh , 20 m mmor , and 0120 m mmob in 50 mm mops buffer , ph 7.0 , was added 10 mm cross - linker edc . the reaction was incubated at room temperature for 10 min and then quenched by adding an equal volume of sds loading buffer . the reaction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) . the electron transfer kinetics of smmo were studied by stopped - flow optical spectroscopy at 15 c . the tubing and syringes of the hi - tech scientific sf-61 dx2 double - mixing stopped - flow instrument were made anaerobic by first flushing and then incubating with 15 mm anaerobic sodium dithionite solution for 3 h , followed by flushing with 25 mm anaerobic mops buffer , ph 7.0 right before use . the following steps were performed inside of a glovebox with an o2 level of less than 0.5 ppm . to investigate electron transfer from chemically reduced reductase , 40 m degassed fd or mmor was titrated with 3 mm sodium dithionite until the absorption at 405 nm no longer changed , and the resulting sample was then loaded into a gastight syringe . a 20 m quantity of mmoh , or 20 m mmoh premixed with 40 m mmob in the presence or absence of 10 m mmor , was degassed and sealed in another gastight syringe . to study electron transfer from nadh , a 40 m solution of anaerobic nadh was sealed in one syringe ; 20 m mmoh premixed with 40 m mmor in the presence / absence of 40 m mmob was sealed in another syringe . the syringes were then taken out from the glovebox and connected to the stopped - flow instrument . equal volumes of reagents from each syringe were rapidly mixed by the stopped - flow instrument , and the electron transfer kinetics were monitored by recording the absorbance change at 470 or 458 nm . data were fit by two ( when chemically reduced fd or mmor was used as the electron source ) or three ( when nadh was used as the electron source ) exponentials , and effective electron transfer rates were calculated as weighted averages of individual electron transfer rate constant . hdx - ms is a powerful tool for probing protein structure , dynamics , and the binding interface . the rationale behind hdx - ms relies on protein backbone amide protons that are in constant exchange with solvent protons , or deuteriums if in deuterated solvent . the number of exchangeable protons and their rates of exchange depend on factors such as ph , temperature , chemical environment , and the three - dimensional protein architecture , thus reflecting the structure and dynamics of the protein . typically , protein backbone amide protons exchange rapidly with deuterons if they are involved in weak or suboptimal hydrogen bonds , reside at / near the surface , or are readily accessible to the solvent ; the exchange rates are slower if they are involved in strong intramolecular hydrogen bonds and/or are less accessible to solvent . protein binding sites , based on the reduced solvent exposure in regions that constitute the binding interface . here mmoh is a 251.3 kda homodimer that consists of three protomer subunits in each monomer : ( 60.6 kda ) , ( 45.1 kda ) , and ( 19.8 kda ) . upon pepsin digestion , 165 overlapping mmoh peptic peptides were detected , covering 93.9% , 93.5% , and 96.4% of the sequences of the - , - , and -subunits , respectively ( figure s1 ) . the deuterium incorporation and protein dynamics were followed from 10 s up to 8 h. all the peptic peptides that were followed by hdx - ms are displayed in figure s2 . most of these peptides showed low deuterium uptake even after an 8 h incubation in deuterated buffer ( figure 2 ) . the data indicate very slow dynamics in most of the three subunits of the homodimer , suggesting that the protein is very rigid and not undergoing breathing movements indicative of a dynamic and solvent exposed structure . amide backbone hydrogens involved in hydrogen - bonding interactions in secondary structural elements such as -helices and -sheets exhibit slow exchange rates . therefore , the low deuterium uptake is consistent with the high helical content of mmoh . the and -subunits are more protected from exchange compared with the -subunit , the peptides of which seem to indicate a more accessible and dynamic structure ( figure 2 ) . summary of hdx - ms data for free mmoh in solution at four time points . the hdx - ms data are mapped onto pdb entry 1mty , with the color code indicated for deuteration times shown at the bottom of each image . the hdx - ms data are shown only on one monomer ; the second monomer is represented in sand color . we next used hdx - ms to locate regions of mmoh that show differences in exchange upon binding to the ferredoxin domain ( fd , residues 1107 , 11.8 kda ) of mmor . the two isolated domain proteins both have stable structures and retain the biochemical properties of the two domains in full - length mmor . electron transfer to the diiron(iii ) centers in the hydroxylase involves first , nadh reductions of the oxidized fad cofactor to its hydroquinone form , followed by the [ fe2s2 ] cluster in the fd sequentially shuttling two electrons from the reduced fad cofactor to the diiron center in mmoh . the fd protein is a smaller yet competent model of mmor for studying electron transfer as well as binding to mmoh . among the 165 detected peptic peptides of mmoh in the presence of fd ( figure s2 ) , six peptides derived from the -subunit and three from the -subunit exhibited lower deuterium uptake compared with samples without fd ( figures 3 and s2 ) , covering residues 7088 and 236255 of the -subunit , and residues 3748 of the -subunit . the differences in deuterium uptake were visible after 1060 min deuteration but not at earlier time points , possibly because the exchange rates in the absence of fd were already very slow due to stable h - bonds in the -helices . in the presence of fd the reduced solvent exposure at the binding site modestly reduced the exchange rates . the differences in deuterium uptake in the presence and absence of fd were subtle even at longer incubation times ( up to 8 h exchange ) , but were consistent in the set of overlapping peptides . typically , differences in deuteration greater than 0.4 da but less than 1 da are considered subtle . the experiments were performed in triplicate , and the average error of the measurements was 0.1 da . all other mmoh peptic peptides exhibited the same deuterium uptake in the presence or absence of fd ( figure s2 ) . deuterium exchange kinetics for four representative peptides that showed different deuterium uptake in the presence ( blue traces ) or absence ( red traces ) of fd : ( a ) residues 7081 , -subunit ; ( b ) residues 8288 , -subunit ; ( c ) residues 237242 , -subunit ; and ( d ) residues 4047 , -subunit . the largest number on the y - axis represents the maximum amount of deuterium that can be incorporated in each peptic peptide . we then mapped the peptides that showed decreased deuterium uptake in the presence of fd onto the crystal structure of mmoh ( pdb entry 4gam ) . these peptides cluster in the canyon region at the 22 interface , representing a possible fd binding site ( figure 4a ) . this region includes the area closest to the diiron center from the protein surface , a preferred binding site for the iron sulfur cluster of the reductase in order to facilitate fast electron transfer . this binding site determined by hdx - ms rationalizes the seemingly conflicting results of previous chemical cross - linking studies . because the binding site consists of residues from both - and -subunits , mmor can cross - link to either the -subunit ( for smmo isolated from methylococcus capsulatus ( bath ) ) or the -subunit ( for smmo isolated from methylosinus trichosporium ob3b ) . it is unlikely that smmos isolated from different species have different reductase binding sites . the different cross - linking results are most likely due to different distributions of carboxyl groups and amino groups that are required to be in close proximity for the cross - linking mediated by edc . ( a ) mmoh peptides that showed decreased deuterium uptake in the presence of fd are mapped onto the crystal structure of mmoh ( pdb i d 4gam ) . ( c ) a closer view of the binding interface in the docked model : the [ fe2s2 ] cluster of fd , mmoh residues at the binding interface , and the diiron center of mmoh , viewed from the top of the figure . ( d ) the crystal structure of mmoh-2mmob , showing that mmob covers the fd binding site . mmoh is colored in cyan ; binding site peptides of the -subunit in orange and those of the -subunit in yellow ; fd in red ; and mmob in purple . to validate the hdx - ms - determined fd binding site , we performed computational docking using the cluspro server . the structure of fd ( residues 198 , pdb i d 1j4q ) previously determined by solution - state nmr spectroscopy and the crystal structure of mmoh ( pdb i d 4gam ) were used as input structures , and the nmr - determined fd residues involved in binding to mmoh were used as docking constraints . the results show that fd covers the hdx - ms - determined binding surface ( figure 4b ) . the -subunit peptides involved in binding ( yellow region in figure 4 ) seem uncovered in the docked structure ( figure 4b ) , but upon a closer examination residues 4548 inside the canyon were found to be in close contact with fd . the docking result is therefore in full agreement with the hdx - ms data . a close examination of the docked mmoh fd complex reveals that the diiron active site of mmoh is approximately 14 away from the [ fe2s2 ] cluster of fd , a favorable distance for electron transfer . consistent with hdx - ms results , the docked model reveals that fd covers the pore region of mmoh , which we previously proposed as the proton transfer pathway from the mmoh surface to its diiron center . residue e240 in the mmoh -subunit , the gating residue of the pore , is situated midway between the [ fe2s2 ] cluster of fd and the diiron center of mmoh ( figure 4c ) . previously , this residue was found to shift its conformation toward the protein interior upon mmob binding , closing the pore and possibly bringing in a proton to the diiron active site . we propose that an identical conformational change occurs when mmor binds to mmoh during electron transfer from the fd [ fe2s2 ] cluster to the carboxylate - bridged diiron center , providing the mechanism for proton - coupled electron transfer . strikingly , the fd binding site determined here overlaps largely with that of mmob as previously determined by the crystal structure of the mmoh2mmob complex . in particular , this structure reveals that the core of mmob docks into the canyon region , covering the very same area where we now conclude fd binds , while the n - terminal tail of mmob binds to an adjacent location on mmoh , on the surface of helices h and 4 of the -subunit , adopting a ring - like conformation ( figure 4d ) . this observation requires the fd of mmor and the core of mmob compete for the same binding site in the canyon of mmoh . to test this possibility , we first investigated the binding competition between fd and the core of mmob by fluorescence anisotropy titrations . the mmob core was prepared by truncating the n - terminal tail at residue 33 ( 2 - 33 ) , and a cysteine mutation d36c was introduced in order to attach the fluorophore iaedans . the n - terminal truncated mmob ( designated mmob 2 - 33 ) is still able to bind to mmoh but with lower affinity ( kd = 2.67 m , figure s3 ; kd = 0.55 m for the full - length mmob ) . to characterize the binding competition , fd was titrated into a mixture of 1 m mmoh and 1 m iadeans - labeled mmob 2 - 33 . the fluorescence anisotropy decreased steadily as increasing amounts of fd were added ( figure 5a ) , indicating displacement of n - terminal truncated mmob by fd from mmoh ( scheme 1a ) . the titration curve could be simulated by assuming that the n - terminal truncated mmob and fd compete for the same binding sites on mmoh ( mmohsite , one on each side of mmoh ) , and an apparent kd value of 2 m was obtained for the mmoh fd complex on the basis of the simulations ( figure 5a ) . experimental evidence for mmor and mmob binding competition . ( a , b ) titrating fd into 1 m mmoh and 1 m iadeans - labeled n - terminal truncated ( 233 ) mmob ( a ) or full - length mmob ( b ) . the titration curves are simulated assuming kd = 0.9 , 2 , and 6 m for the mmoh fd complex ; kd = 2 and 6 m best fit the experimental data in ( a ) and ( b ) , respectively . edc was used as the cross - linking reagent , and 0 , 1 , 2 , 4 , and 6 equiv mmob relative to mmor were added to a mixture of 10 m mmoh and 20 m mmor . the cross - linking reaction mixtures were resolved by sds - page , and bands corresponding to the mmor-subunit cross - linking product are shown . in the binding competition between fd and full - length mmob ( b ) , fd may displace the core of mmob from the canyon , but the n - terminal tail may still bind to mmoh . mmoh complex when the titration experiment was repeated with the full - length mmob labeled with iaedans ( figure 5b ) , indicating that fd is less effective in displacing full - length mmob . this result is an expected consequence of the additional binding site of the n - terminal tail of mmob on the surface of helices h and 4 of the mmoh -subunit , a binding region not shared with fd . it is possible that the n - terminal tail serves as an anchor attached to the surface of mmoh , such that , when fd displaces the core of mmob from the canyon , mmob does not completely dissociate from mmoh ( scheme 1b ) . a simple competitive binding model would therefore no longer apply to the component interactions in this case . similar results were obtained by using the full - length mmor as the titrant . an apparent kd value of 8 m for the mmoh mmor complex is required to fit the titration curve using a competitive binding model ( figure s4 ) , considerably larger than the 0.9 m kd value previously determined for the mmoh mmor complex in the absence of mmob . these results clearly demonstrate that both fd and the full - length mmor are able to displace mmob , in particular the core of mmob , from mmoh . this property was demonstrated by studying the effect of mmob on mmor cross - linking to mmoh . as shown in figure 5c , mmor forms cross - links to the mmoh -subunit in the presence of edc , as demonstrated previously ; the yield of this cross - link decreases in a dose - dependent manner as the mmob concentration increases from 0 to 6 equiv relative to the mmor concentration , indicating that mmob blocks mmor binding , and thus its cross - linking , to mmoh . a similar finding the determination of component protein binding sites on the hydroxylase and their binding competition form a basis for understanding the role of the regulatory protein in electron transfer reactions of smmo . previously , mmob was proposed to facilitate electron transfer from mmor to mmoh , and this effect was ascribed to formation of a hypothetical mmoh2mmob2mmor ternary complex . it was hypothesized that the increased electron transfer exhibited by this ternary complex is due to a conformational change of mmoh induced by mmor . considering the substoichiometric concentration of mmor found in vivo and used during activity assays , it was further proposed that such conformational changes are retained throughout the catalytic cycle even after mmor dissociates from the ternary complex . considering our current finding that the core of mmob and the fd of mmor compete for binding to the canyon of mmoh , these proposals are deemed unlikely . because mmob can inhibit mmor binding to the canyon , mmob would inhibit electron transfer as well . to test experimentally whether mmob increases the electron transfer rate , and whether pre - equilibrating mmor with other protein components would result in such an increase , we designed two sets of single - turnover , single - mixing stopped - flow experiments . in the first set , the electron transfer reactions were initiated by mixing 2 equiv of chemically reduced fd to ( i ) 1 equiv of oxidized mmoh alone , ( ii ) 1 equiv of oxidized mmoh equilibrated with 2 equiv of mmob , and ( iii ) 1 equiv of oxidized mmoh equilibrated with 2 equiv of mmob as well as 0.5 equiv of mmor ( figure 6a ) . the reactions were monitored at 470 nm , which increases in intensity as electrons are transferred to the mmoh diiron sites from the reduced [ fe2s2 ] clusters . the previous proposal would predict that case ( iii ) , where all three components were pre - mixed and equilibrated , would exhibit the fastest electron transfer rate . the actual results ( figure 6b ) , however , showed that reaction ( i ) displays the fastest electron transfer rate . including mmob in reactions ( ii ) and ( iii ) significantly retarded the electron transfer reaction , even when mmor was added and pre - equilibrated with other components . these experiments were repeated using chemically reduced full - length mmor as the reductant , and the same trend was observed ( figure s5 ) . mmob inhibits electron transfer , using chemically reduced fd as the electron source . the electron transfer reaction was initiated by mixing 2 equiv of nadh with ( i ) 1 equiv of oxidized mmoh and 2 equiv of mmor and ( ii ) 1 equiv of oxidized mmoh , 2 equiv of mmob , and 2 equiv of mmor ( figure 7a ) . the reactions were monitored at 470 nm , where the absorption first decreased due to the reduction of the fad cofactor by nadh and the subsequent intra - mmor electron transfer to the [ fe2s2 ] cluster . the absorption at this wavelength then increased , as electrons were transferred from these cofactors to the diiron centers in mmoh . the previous proposal would predict that reaction ( ii ) , which had all three protein components pre - equilibrated , would display the faster electron transfer rate , but in fact it is slower ( figure 7b ) , again demonstrating the inhibitory effect of mmob on electron transfer ( table 1 ) . electron transfer reactions were initiated by mixing the reagent in syringe 1 with protein(s ) in syringe 2 in single - mixing stopped - flow experiments ( setups also shown in figures 6a and 7a ) . the inhibitory effect of mmob on electron transfer is in full agreement with our finding that the core of mmob shares a binding site on mmoh with the fd of mmor such that mmob inhibits mmor binding . although electron transfer is retarded , it is still much faster than the rate of substrate turnover ( kcat = 0.1 s at 15 c using propylene as substrate ) . the advantage of such competitive binding is that it provides a mechanism for modulating electron transfer during catalysis . by fine - tuning the affinity of mmor and mmob for the canyon region of mmoh , the binding of mmor can be inhibited during dioxygen activation , preventing undesired electron transfer that could quench activated dioxygen intermediate species . such quenching is suggested by oxidase activity displayed by mmoh and mmor in the absence of mmob . the modulation of electron transfer exhibited by the mmob regulatory protein is most likely a feature common to other bmm enzymes . these enzymes share conserved protein sequences as well as structures , and , like smmo , their regulatory proteins also bind to an analogous canyon region of the hydroxylase , as illustrated in the x - ray structures of the protein complexes in ph and t4mo , blocking the shortest pathway to the hydroxylase diiron center from its surface . the reductase may therefore need to compete with the regulatory protein for binding in order to deliver electrons to the diiron centers in these enzymes as well . in disagreement with this hypothesis , previous studies of ph and tomo these electron transfer rates , however , were determined in steady - state assays in the absence of substrates , by measuring nadh consumption . under these conditions , the nadh consumption rate depends on the oxidase activity of the hydroxylase , and the accelerated nadh consumption in the presence of the regulatory component is most likely due to increased oxidase activity and not an indication of accelerated electron transfer . using hdx - ms , the binding site for the ferredoxin domain of mmor was determined to be in the canyon of mmoh , the same region where the core of the mmob regulatory protein binds . this finding is consistent with previous chemical cross - linking results and the current computational docking study , as well as a series of binding competition assays . mmob inhibits mmor binding to the canyon as well as the electron transfer that leads to reduction of the hydroxylase . the previous proposals that mmob increases the electron transfer rate when all three components are pre - equilibrated has been tested experimentally and proved to be invalid . regulatory proteins of other bmm enzymes may similarly share binding sites with their reductases and inhibit electron transfer . such binding competition would provide a control mechanism for electron transfer in bmm enzymes . to initiate the catalytic cycle , the reductase displaces the regulatory protein from the canyon of the hydroxylase , reducing the diiron center from diiron(iii ) to diiron(ii ) ; the regulatory protein then re - binds to the canyon and displaces the reductase , initiating o2 activation and substrate oxidation . the binding affinities of the component proteins may be fine - tuned so that the reductase is unable to displace the regulatory protein at this step , preventing quenching of activated oxygen intermediates p * , hperoxo , and q. at the end of the catalytic cycle , the diiron center returns to the diiron(iii ) state , and the reductase can bind again to the canyon , priming the enzyme for the next cycle of catalysis .

the hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases ( bmms ) requires the interplay of three or four protein components . how component protein interactions control catalysis , however , is not well understood . in particular , the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s ) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic . here we determine the reductase binding site on the hydroxylase of a bmm enzyme , soluble methane monooxygenase ( smmo ) from methylococcus capsulatus ( bath ) . we present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase , previously determined to be the regulatory protein binding site as well . the latter thus inhibits reductase binding to the hydroxylase and , consequently , intermolecular electron transfer from the reductase to the hydroxylase diiron active site . the binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer , and other bmm enzymes are likely to adopt the same mechanism .

Introduction Experimental Section Results and Discussion Conclusions

a 46-year - old married saudi woman presented at a primary health care center with left upper abdominal pain , with no other associated symptoms . five months later , she presented to the surgery clinic because of increased abdominal pain with no response to medication ; her physical examination showed fullness of the left upper abdomen . her routine laboratory investigation as well as chest and abdominal x - rays were not remarkable . ultrasound of the abdomen showed a solid mass at the left hypochondrium separated from the spleen and just below the left hemidiaphragm . computed tomography ( ct ) scan of the abdomen ( figure 1 ) showed encapsulated hypodense mass at the left upper abdomen which was interposed but clearly separated from left liver lobe , spleen , stomach and left hemidiaphragm . magnetic resonance imaging ( mri ) of the abdomen ( figure 2 ) confirmed the location of the mass and attachment to the inner aspect of the anterior abdominal wall but separated from internal abdominal organs . the mass was isosignal intensity to muscles in t1- weighted images and heterogeneous hyperintense signal in t2 weighted images . in post ( a ) axial non - enhanced ct scan of the abdomen showing a well defined hypodense mass at the left hypchondrium , interposed between left liver lobe , left hemidiaphragm and stomach . ( a ) axial t1w1 : an oval shape mass with signal intensity parallel to the muscle . ( c ) axial post contrast t1w1 showing avid contrast enhancement of the mass . at surgery , a firm , rounded mass apparently encapsulated at the left sub - diaphragmatic area was found attached to the extra - peritoneal fascia of the anterior abdominal wall . histopathology showed a biphasic tumor composed of spindle cell and epithelial cell elements , with mild anaplasia & infrequent mitosis . the final diagnosis was low grade synovial sarcoma involving the fascial aponeurosis of the anterior abdominal wall . synovial sarcoma ( ss ) is an uncommon soft - tissue malignant tumor that is common in the extremities of middle - aged patients , close to large joints particularly the knee in the popliteal fossa.12 despite its name , the lesion does not commonly arise in an intraarticular location but usually near joints . the tumor arises from pleuripotential mesenchyme , in close association with joint capsules , tendon sheaths , bursae and fascial structures . it is generally accepted that synovial sarcoma is derived from primitive mesenchymal cells , not synovial cells.2 synovial sarcoma is the fourth most common type of soft tissue sarcoma following malignant fibrous histiocytoma , liposarcoma and rhabdomyosarcoma . synovial sarcoma accounts for 5 - 10% of all soft tissue sarcomas.3 about 85 - 90% of ss occur in the extremities.4 reported examples arising in the anterior abdominal wall are rare . synovial sarcoma in the abdominal wall tends to occur with a much greater frequency in females in contrast to such tumors in the extremities or the neck which tend to occur with a much greater frequency in males.5 radiological findings of ss are not pathognomonic . however , findings of a soft - tissue mass , particularly if calcified ( 30% ) , near but not in a joint of a young patient , are very suggestive of the diagnosis . cross - sectional imaging features are essential for staging extent of the tumor and the planning of surgical resection.6 the most common ct appearance of ss is that of a heterogeneous soft - tissue mass with attenuation similar to or slightly lower than that of muscle areas of lower attenuation representing necrosis or hemorrhage are also common with heterogeneous contrast enhancement.7 on mri , ss typically appears as a prominently heterogeneous multilobulated soft - tissue mass with signal intensity similar to or slightly higher than that of muscle on t1-weighted mr images . prominent heterogeneity with predominant high signal intensity is also a feature of ss on t2-weighted mr images . the presence of multilobulation and marked heterogeneity are highly suggestive of a diagnosis of ss . ct and mri are useful in defining the extent of the disease and in follow up response to chemotherapy.7 the presence of extensive calcification suggests a more favorable prognosis.8 similar ct and mri appearances are seen in our case . despite its name there are three main histologic subtypes of ss : biphasic , monophasic , and poorly differentiated types . poorly differentiated ss is generally epithelioid in morphology and has high mitotic activity.78 in the present case , histopathology showed a biphasic pattern of ss , the coexistence of spindle cells in a wavy pattern , and the pseudoglandular formation . both biphasic and monophasic synovial sarcomas are usually intermediate grade ( grade 2/3 ) ; however , both types can be high grade ( grade 3/3 ) . poorly differentiated synovial sarcomas are high - grade tumors.7 the specific chromosomal aberration in synovial sarcoma has recently been reported . the hallmarks for synovial sarcoma are the ( x ; 18 ) translocation and syt - ssx gene fusion products . is the molecular diagnosis in synovial sarcoma , as well as immunohistochemical study , especially in cases in which histological diagnosis is difficult.79 surgical excision is the treatment of choice , and the recurrence rate range from 28% to 36% even with adequate surgical and adjunctive therapies.4 however , the multimodality treatment approach has improved the prognosis of synovial sarcomas . tumor invasiveness , histologic grade and tumor size significantly correlate with the survival period , with a more preferable prognosis of synovial sarcomas encountered in childhood . chemotherapy has been used to treat metastatic or residual disease.710 primary synovial sarcoma of the anterior abdominal wall is a rare extra - articular tumor site . in cases of anterior abdominal wall masses ,

synovial sarcoma is a malignant mesenchymal neoplasm which commonly occurs in the extremities of adults , in close association with joint capsules , tendon sheaths , bursae and fascial structures . only a few cases of synovial sarcoma occurring in the abdominal wall have been reported . a case of a primary synovial sarcoma arising from the anterior abdominal wall fascial aponeurosis is presented .

CASE REPORT DISCUSSION

docetaxel ( dcx ) is a second generation taxane , derived from the inactive 10-deacetyl baccatin iii , extracted from the european yew tree ( taxus baccata ) . dcx has better water solubility , pharmacokinetic profile , and anticancer activity than paclitaxel . current fda approved dcx products , including taxotere , are essentially tween 80/ethanol - based solutions , which unfortunately are associated with various significant side effects . hypersensitivity reactions , which are attributed to the tween 80 in the formulations , can vary from simple skin rash to systemic anaphylaxis and necessitate premedication with corticosteroids . other problems associated with the tween 80/ethanol - based dcx formulations include the nonspecific accumulation of dcx in healthy organs , which may lead to systemic toxicity and subsequent discontinuation of therapy . nanoparticle - based , tween 80-free dcx formulations are expected to not only avoid tween 80-related side effects but also increase the concentration of dcx in tumors due to the enhanced permeation and retention ( epr ) effect . data from many previous studies demonstrate that nanoparticles of 100200 nm are most successful in tumor vasculature extravasation , although there are disagreements in the literature . the heterogeneous nature of tumor type , size , location , and metastasis may contribute to the disagreements . in order to improve the epr - related nanoparticles extravasation , nanoparticles should be designed to circulate longer in the blood , while the drug of interest is retained within the nanoparticles . pegylation is a strategy to render the surface of nanoparticles hydrophilic , thus enabling the nanoparticles to evade early opsonization and circulate longer in the blood . on the other hand , for a drug to be retained within the nanoparticles , a strong affinity between the drug and the excipient(s ) used to prepare the nanoparticles is required . solid lipid nanoparticles ( slns ) have been extensively investigated as drug carriers . advantages of such nanocarriers include high compatibility with lipophilic drugs , ease of fabrication , and controlled release . reported the development of a pegylated lipid nanocapsule formulation ( lnc ) for paclitaxel using a novel phase inversion - based method . the resultant lncs were made of an oily medium - chain triglyceride core and stabilized with soybean lecithin as a lipophilic surfactant , and peg hydroxystearate ( solutol ) as a hydrophilic surfactant . applied a high pressure homogenization technique to prepare a sln formulation of paclitaxel using triglyceryl myristate ( trimyristin ) and phospholipids . the formulation showed improved in vitro activity , but the in vivo circulation time and biodistribution profile were not improved , as compared to the market product taxol . applied a factorial design to optimize formulation parameters to prepare paclitaxel sln formulations using compritol 888 ato ( a mixture of mono- , di- , and triglycerides of behenic acid ) and precirol ato5 ( i.e. , glyceryl palmito - stearate ) , and the final optimized formulation demonstrated an improved in vitro cytotoxic activity against the murine breast cancer cell line mxt - b2 . the present study aimed at the rational selection of a triglyceride from a list of medium- and long - chain triglycerides for the development of a sln formulation to ultimately improve the antitumor activity of dcx . previously it was reported that low melting point triglycerides are excellent solubilizers for dcx , prompting us to hypothesize that high melting point triglycerides will be suitable excipients for preparing dcx - incorporated slns . triglycerides that are solid at body temperature were selected to ensure formulation stability and to avoid droplet coalescence . an oil - in - water ( o / w ) emulsion - based method was applied , where dcx and all lipid components were dissolved in the oil phase , and the aqueous phase consisted of a 0.1% ( w / v ) poloxamer 188 aqueous solution . finally , the in vitro and in vivo antitumor activities of the selected formulation were evaluated . the 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy ( polyethylene glycol)-2000 ] ( dope - peg-2000 ) and phosphatidylcholine from chicken egg ( epc ) were from avanti polar lipids , inc . sepharose 4b , mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) kit , tween 80 ( t80 ) , poloxamer 188 ( pluronic f68 ) , trimyristin ( tm ) , trilaurin ( tl ) , tristearin ( ts ) , tripalmitin ( tp ) , mannitol , sucrose , phosphate buffer saline ( pbs , ph 7.4 ) , triglyceride assay kit , and caspase 3 assay kit were all from sigma - aldrich ( st . louis , mo , usa ) . float - a - lyzer dialysis tubes ( mwco 50,000 ) were from spectrum chemicals & laboratory products ( new brunswick , nj , usa ) . tc-1 cells ( murine lung cancer cell line ) were from the american type culture collection ( atcc , rockville , md , usa ) and grown in rpmi 1640 , supplemented with 10% fetal bovine serum ( fbs ) and 1% of 100 g / ml streptomycin and 100 iu / ml penicillin ( 1% p / s ) . m - wnt cells ( murine mammary gland cell lines ) were from dr . m - wnt cells were grown in a similar medium as tc-1 , with an additional supplement of 1% glutamax . human breast adenocarcinoma cells ( mda - mb-231 ) were from atcc and grown in dmem supplemented with 5% fbs and 1% p / s . all cell culture reagents were from invitrogen ( life technologies , carlsbad , ca , usa ) . female c57bl/6 mice ( 68 weeks old ) were from charles river laboratories ( wilmington , ma , usa ) . briefly , 1 ml of dichloromethane ( dcm ) containing dcx , a triglyceride ( tm , tp , tl , or ts ) , egg pc , and dope - peg-2000 in a weight ratio of 1:20:10:2 was added to 10 ml of 0.1% poloxamer 188 aqueous solution in a glass vial , and the mixture was sonicated using a probe sonicator , with a microprobe attached , for 40 s , at a sonication intensity of 50% ( q - sonica llc , newtown , ct , usa ) . the glass vial was placed in an ice bath during sonication to prevent heat accumulation . the emulsion was stirred for 15 min at 400 rpm in a water bath ( 65 c ) to evaporate dcm , and was then stirred for an additional hour at room temperature . the resultant nanoparticle suspension was concentrated to 1 ml by ultrafiltration using an amicon device ( millipore inc . , 30,000 mwco ) ( 490 g , 25 min , 4 c ) as previously reported . finally , slns were briefly sonicated to eliminate aggregates due to the concentrating process . dcx - free slns were prepared similarly without the addition of dcx . for slns that were used in animal studies , the ultrafiltration period was extended to 60 min to further concentrate the suspension . the prolonged ultrafiltration did not result in any significant particle size change ( data not shown ) . kansas city , mo , usa ) with 9.25% ( w / v ) sucrose as a cryoprotectant . the tween 80/ethanol - based dcx formulation ( dcx in t80/e ) was prepared by dissolving dcx in tween 80 ( 20 mg / ml ) . this concentrate was then diluted with water / ethanol solution to make a final dcx solution of 4 mg / ml . the final concentrations of tween 80 and ethanol in the solution were 20% ( v / v ) and 13% ( v / v ) , respectively . particle size and zeta potential of the slns were measured using a malvern zetasizer nano zs ( malvern instruments , worcestershire , u.k . ) . briefly , 20 l of the concentrated slns in suspension were diluted to 1 ml with water , and the particle size and zeta potential were determined at room temperature . the slns were examined using an fei tecnai transmission electron microscope ( fei corporate , hillsboro , or , usa ) at the institute for cellular and molecular biology , microscopy and imaging facility at the university of texas at austin as previously reported . the content of dcx in the slns was determined using hplc after extraction as previously reported with modifications . briefly , slns in suspension were diluted 510 times with methanol in a glass vial , which was placed in a water bath ( 65 c ) for 20 min to dissolve the lipids , and placed at 20 c for 45 min . the supernatant was collected by centrifugation at 18000 g for 10 min at 4 c ( beckman coulter inc . , brea , ca , usa ) , and 5 l of the supernatant was used for hplc assay as previously described . , santa clara , ca , usa ) , with rp - c18 column ( zorbax eclipse , 5 m , 4.6 mm 150 mm ; santa clara , ca , usa ) . the mobile phase was acetonitrile and water ( 1:1 , v / v ) . the flow rate was 1 ml / min , and the detection wavelength was 230 nm . the dcx loading percentage was measured using a similar procedure , with the exception that the slns were lyophilized , and 5 ml of methanol was added to 5 mg of the lyophilized slns . the weight percentage of dcx in the slns ( % w / w ) was calculated based on the following formula : to investigate whether free dcx coexisted with dcx - slns in the nanoparticle preparation , the slns ( 100 l ) were applied to a sepharose 4b column ( 6 mm 30 cm ) equilibrated with water , and the dcx - slns were eluted with water . fractions of 0.5 ml were collected , and 0.3 ml of each fraction was lyophilized to determine the content of dcx as mentioned above . in addition , the absorbance of each fraction ( 100 l ) at 500 nm was measured using a biotek synergy ht multi - mode microplate reader ( winooski , vt , usa ) to determine their turbidity , which was used as an indication of the presence of nanoparticles in the fractions collected . finally , the concentration of triglycerides in each fraction was also measured using a sigma triglyceride assay kit following the manufacturer s instruction . dcx - slns prepared using different triglycerides were stored in parafilm - sealed vials at 4 c for eight days . particle size , zeta potential , and dcx content were measured as mentioned above shortly after the preparation and on day 8 to monitor any change of these parameters . the release of dcx from the slns made with different triglycerides was monitored using float - a - lyzer tubes ( mwco 50,000 ) . briefly , dcx - slns suspension was diluted to 1 ml with pbs and transferred to the dialysis tube , which was then placed in a 50 ml plastic tube containing 20 ml of release medium ( pbs , 0.1 mm , ph7.4 , with 1% tween 80 ) . the tubes were then placed at 37 c in an orbital shaker at 100 rpm ( max - q 5000 , thermo scientific , waltham , ma , usa ) . at predetermined time points , the whole release medium was replaced with fresh medium to maintain sink condition , and dcx concentration was analyzed using hplc as previously mentioned . the release of dcx from the dcx in t80/e formulation was evaluated similarly for comparison . for mdsc , a ta instruments model 2920 dsc ( new castle , de , usa ) was used , and the data were analyzed using ta universal analysis 2000 software . the ramp rate was 5 c / min , and the temperature range was from 10 to 200 c . samples included dcx , trimyristin , dcx - slns ( prepared with trimyristin ) , blank slns , and the physical mixture of dcx and blank slns . a philips model 1710 x - ray diffractometer ( philips electronic instruments inc . , mahwah , nj , usa ) available in the texas materials institute x - ray facility at the university of texas at austin was used to analyze the crystallinity of dcx in the slns . samples included dcx alone , dcx - slns ( prepared with trimyristin ) , dcx mixed with blank slns , and blank slns . cells were seeded in 96-well plates at a density of 3,000 cells / well and incubated at 37 c with 5% co2 overnight . they were treated with various concentrations of dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or t80/e alone for 72 h. cell viability was determined using an mtt assay as previously described . ic50 values were calculated using graphpad prism ( graphpad software , inc . , la jolla , ca , usa ) . caspase 3 activity was determined using a sigma - aldrich caspase 3 fluorimetric assay kit . in brief , tc-1 cells were seeded in 24-well plates at 25,000 cells / well and incubated overnight . the cells were treated with dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or t80/e for 72 h. the concentration of the dcx was 0.01 m . the supernatant was transferred to a clear - bottomed black plate and mixed with the assay substrate , acetyl - asp - glu - val - asp-7-amido-4-methylcourmarin ( ac - devd - amc ) . the mixture was incubated for 6 h for the hydrolysis of the ac - devd - amc by caspase 3 to release the fluorescent amc , which was quantified by measuring the fluorescence intensity at 360 nm ( excitation)/460 nm ( emission ) according to the manufacturer s instruction . the unit of the caspase 3 activity was mol amc / min / ml . a caspase 3 inhibitor ( provided in the kit ) total protein concentration in the cell lysates was determined using bio - rad dc protein assay kit following the manufacturer s instruction . all animal protocols were approved by the institutional animal care and use committee at the university of texas at austin , and the national institutes of health ( nih ) guidelines for laboratory animal use and care were followed . animals were left to acclimatize for at least 7 days upon arrival from the vendor . each mouse was injected with the murine tc-1 lung cancer cells ( 5 10 cells per mouse ) suspended in 100 l of fbs - free rpmi 1640 medium subcutaneously in the shaved left flank . six days after the implantation ( day 6 ) , mice were randomized into 4 groups , 7 mice per group , and injected intravenously via the tail vein with dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or 5% mannitol as a vehicle control . tumor sizes were measured using a digital caliper , and tumor volumes were calculated using the following formula : on day 21 , mice were euthanized to harvest tumor tissues , which were weighed , fixed in zn formalin buffer for immunohistochemistry . tissue sample preparation for immunohistochemical evaluation was carried out in the histology and tissue analysis core at dell pediatric research institute ( dpri ) at the university of texas at austin . the formalin - fixed tumor tissues were embedded in paraffin wax , sectioned , and stained with an antibody against cd-31 ( abcam , cambridge , ma , usa ) as a marker for angiogenesis ( n = 3 ) . slides were then scanned , and images were taken using the scanscope xt ( aperio technologies , vista , ca , usa ) . three weeks after tumor implantation , mice were divided into 2 groups ( n = 910 ) ; one group was injected with dcx in t80/e ( equivalent to dcx dose of 16 mg / kg ) via the tail vein , and the other group with dcx - slns prepared with trimyristin ( equivalent to dcx dose of 16 mg / kg ) . two or twelve hours later , 45 mice from each group were euthanized to collect tumor , liver , kidney , spleen , heart , lung , and blood samples . the organs and tumor tissues were weighed and then stored at 80 c the blood samples were mixed with an edta solution and allowed to stand for about 15 min and centrifuged ( 3300 g , 10 min , 4 c ) to separate the plasma , which was stored at 80 c . dcx was extracted from the samples using ethyl acetate , and dcx concentrations in the samples were determined using hplc . statistical analyses were completed by performing anova followed by fisher s protected least significant difference procedure . a p - value of 0.05 ( two - tail ) was considered significant . the nanoparticles were composed of dcx , a triglyceride , epc , dope - peg-2000 , and pluronic f68 . the average particle size of slns produced using tristearin , tripalmitin , trimyristin , and trilaurin as the triglyceride was 178.4 2.3 , 176.3 3.9 , 182.8 2.0 , and 150.7 14.5 nm , respectively ( table 1 ) . the polydispersity indices of all nanoparticle preparations were equal to , or below , 0.2 . the content of dcx in the final slns was 2.42.8% ( w / w ) ( table 1 ) . the release of dcx from the slns was slower , relative to the diffusion of dcx out of the dcx in t80/e formulation ( figure 1a ) . only about 4.59% of the dcx was released from the sln formulation within the first 6 h , whereas about 31% of dcx diffused out of the t80/e formulation within the same time period ( figure 1a ) . the rate at which the dcx was released from the dcx - slns prepared with trimyristin was the slowest ( figure 1b ) . ( a ) the release of dcx from dcx - slns ( closed circles ) prepared using tristearin ( ts ) , tripalmitin ( tp ) , trimyristin ( tm ) , or trilaurin ( tl ) . as a control , the release of dcx from dcx in t80/e ( open circles ) was also included . ( b ) a comparison of the percent of dcx released per hour from dcx - slns prepared using different triglycerides . the release rates were calculated with data in the initial 24 h period . each point represents mean ( n = 3 ) . during a short - term , 8-day stability study at 4 c , no significant change in particle size and dcx content in any of the four dcx - slns preparations was found ( figures 2a , b ) . however , the zeta potential of dcx - slns prepared with tripalmitin and trilaurin changed significantly ( figure 2c ) . based on data shown in figures 1 and 2 , the dcx - slns prepared with trimyristin were chosen for further studies , because the release of dcx from the slns prepared with trimyrstin as the triglyceride was the slowest , and the resultant dcx - slns were also relatively more stable . the particle sizes ( a ) , dcx contents ( b ) , and zeta potentials ( c ) of dcx - slns prepared with different triglycerides shortly after preparation or after 8 days of storage at 4 c . shown in figure 3a are the gpc results for the dcx - slns prepared with trimyristin as the triglyceride . about 90% of the dcx that was eluted from the column was associated with the triglyceride and with the fractions that contained the nanoparticles ( calculated based on the area under curves of the gpc profiles ) ( figure 3a ) . the slns were successfully lyophilized with 9.25% ( w / v ) sucrose as a cryoprotectant ( data not shown ) . ( a ) a representative gpc profile of dcx - slns prepared with trimyristin . collected fractions were divided into three portions : one was analyzed for dcx content ( red ) , one was used to measure turbidity ( od 500 nm ) ( blue ) , and the other one was used to measure trimyristin concentrations , and the visible absorbance of the formed colored component was measured ( od 540 ) ( black ) . ( b ) a representative tem image of dcx - slns prepared with trimyristin ( bar = 200 nm ) . dsc analysis of the dcx - slns , free dcx , blank slns , and blank slns mixed with dcx showed that dcx exhibited a characteristic melting peak at 167.4 c ( figure 4a ) . the physical mixture exhibited an endothermic melting peak at 143.4 c , which can be attributed to the presence of free dcx , as the blank slns did not show any distinct peak at that temperature . a distinct dcx endothermic melting peak was also absent in the dcx - slns ( figure 4a ) . on the other hand , the presence of the endothermic melting peak of trimyristin at approximately 5558 c confirmed the solid state of the lipid within the slns ( figure 4a ) . finally , xrd showed that a characteristic dcx peak was present in the physical mixture , but absent from the dcx - sln composition ( figure 4b ) . dsc thermograms ( a ) and x - ray diffractograms ( b ) of dcx - slns , dcx alone , trimyristin ( tm ) alone , blank slns , or the physical mixture of blank slns and dcx . mtt assay revealed that both dcx - slns and dcx in t80/e inhibited the proliferation of tumor cells , including murine mammary gland cancer cells ( m - wnt ) , murine lung cancer cells ( tc-1 ) , and human breast adenocarcinoma cells ( mda - mb-231 ) . however , the ic50 values of the dcx - slns were significantly lower than that of the dcx in t80/e in each of the three cell lines ( figure 5a ) . at the highest equivalent dcx concentrations tested ( i.e. , 0.01 , 1 , and 0.05 m in m - wnt , tc-1 , and mda - mb-231 , respectively ) , both the blank slns and the t80/e vehicle control did not show any significant toxicity in all three cell lines ( data not shown ) . ( a ) the ic50 values of dcx - slns and dcx in t80/e in mda - mb-231 , m - wnt , and tc-1 cells . cells were incubated with the dcx formulations for 72 h. ( b ) caspase 3 activity ( in nmol amc / min / ml ) in tc-1 cells following 72 h of incubation with dcx - slns or dcx in t80/e ( dcx , 0.01 m ) . caspase 3 activity was also measured in tc-1 cells treated with dcx - slns , dcx in t80/e , blank slns , and t80/e at a dcx concentration of 0.01 m . caspase 3 activity in cells treated with the dcx - slns was significantly higher than in cells treated with dcx in t80/e ( p < 0.005 , figure 5b ) . the total protein contents in the cell lysates in all four groups were not significantly different ( data not shown ) . the antitumor activity of dcx - slns was evaluated in tc-1 murine lung cancer model pre - established in c57bl/6 mice . as shown in figure 6a , both dcx - slns and dcx in t80/e significantly inhibited the growth of the tc-1 tumors in mice . however , the dcx - slns were significantly more effective than the dcx in t80/e formulation , starting on day 15 ( figure 6a ) . the average body weights of mice that were injected with blank slns or 5% mannitol ( as a vehicle control ) increased slightly ( 10% ) during the 21 days after tumor cell implantation , while the average body weight of mice that were treated with dcx - slns or dcx in t80/e did not show any significant change ( figure 6b ) . finally , the average weight of tumors in mice that were treated with the dcx - slns was also significantly lower than that in other groups at the end of the study ( figure 6c ) . anti - cd31 staining ( i.e. , angiogenesis marker ) showed that the extent of cd31 staining tended to be lower in tumors in mice that were treated with the dcx - slns , as compared to in other groups ( figure 6d ) . ( a ) the growth curves of tc-1 tumors in c57bl/6 mice ( p < 0.05 , dcx - slns or dcx in t80/e vs 5% mannitol , p < 0.05 , dcx - slns vs dcx in t80/e ) . ( b ) ( d ) representative images of tumor tissues after anti - cd31 staining ( bar = 100 m ) . mice were iv injected with dcx - slns or dcx in t80/e at a dcx dose of 15 mg / kg via the tail vein on days 6 , 9 , and 12 after tumor implantation . figure 7 shows the concentration of dcx in tumors and other organs in tc-1 tumor - bearing mice 2 and 12 h after the mice were injected intravenously with either dcx - slns ( prepared with trimyristin ) or dcx in t80/ethanol . the concentration of dcx in tumors in mice that were injected with dcx - slns was about 50% higher than in mice that were injected with the dcx in t80/ethanol formulation , 12 h after the injection ( p < 0.05 ) ( figure 7a ) . however , the concentration of dcx in the liver , spleen , kidneys , heart , and lungs of mice that were injected with the dcx - slns were lower than in mice that were injected with the dcx in t80/e formulation ( figures 7b f ) . finally , 2 h after intravenous injection , the concentration of dcx in the plasma in mice that were injected with the dcx - slns was about 5-times higher than in mice that were injected with the dcx in t80/e formulation ( figure 7 g ) . the concentrations of dcx in tumor ( a ) , liver ( b ) , lungs ( c ) , heart ( d ) , kidneys ( e ) , spleen ( f ) , and plasma ( g ) of tc-1 tumor - bearing c57bl/6 mice 2 or 12 h after iv injection of either dcx - slns or dcx in t80/e . since the discovery of taxanes in the mid-1980s , the fervent search for more efficacious and less toxic taxane formulations has led to the fda approval of three market products , namely , taxol ( bristol - myers squibb , princeton , nj , usa ) , taxotere ( sanofi - aventis u.s . llc , bridgewater , nj , usa ) , and abraxane ( celgene corporation , summit , nj , usa ) . in addition , a polymeric pegylated micelle formulation of paclitaxel ( genexol - pm ) has been marketed in south korea since 2007 , and others ( e.g. , bind-014 ) are in the pipeline . taxotere used to be the only fda - approved dcx formulation on the u.s . include formulations launched by hospira , sagent , and accord , which are all dcx in tween 80/ethanol solutions . docefrez is a lyophilized dcx powder to be reconstituted with 3.54% ethanol in tween 80 before injection . dcx has low water solubility ( 5 g / ml ) , and tween 80 and ethanol are used in the current dcx formulations to solubilize dcx . the main aim in the present study was to rationally design a tween 80-free formulation of dcx that also improves the antitumor activity of dcx . , where the authors reported that dcx solubility in low melting point triglycerides , such as tributyrin , tricaproin , and tricaprylin , is 10,00020,000 times more than in water , we postulated that triglyceride - based sln formulations may exhibit attractive drug four different medium- and long - chain saturated triglycerides that are solid at body temperature were employed in this study ; namely , trilaurin ( mp 46 c ) , trimyristin ( mp 57 c ) , tripalmitin , ( mp 66 c ) , and tristearin ( mp 69 c ) with fatty acid chain lengths of 12 ( c12:0 ) , 14 ( c14:0 ) , 16 ( c16:0 ) , and 18 ( c18:0 ) carbon atoms , respectively . it was reported that triglycerides with melting points lower than room temperature form nanodroplets that are prone to coalescence during preparation or storage . the use of high melting point triglycerides may significantly decrease the mobility of the drug molecules within the lipid core and thus reduce immature drug leakage . therefore , tricaprylin ( c8:0 , mp 9 c ) , tricaprin ( c10:0 , mp 31 c ) , and trolein ( c18:1 , mp 5 c ) were excluded . the trimyristin - based sln formulation was chosen from the four tested formulations because the rate at which the dcx was released from them was the slowest ( figure 1 ) , and the slns were also more stable ( figure 2 ) . in a mouse model with pre - established tc-1 mouse tumors , the dcx - slns were significantly more effective than dcx in t80/e in inhibiting the tumor growth ( figure 6 ) , likely because the dcx - slns significantly increased the accumulation of dcx in tumor tissues ( figure 7a ) . circulating nanocarriers usually take advantage of the leaky vasculature and/or poor lymphatic drainage in tumor tissues to accumulate in tumors . since dcx accumulation in tumor was only significantly higher at 12 h , but not at 2 h , following the administration of slns , as compared to the dcx in t80/e , it is likely that the enhanced retention of the dcx - slns in tumors due to poor lymphatic drainage was responsible for the improved accumulation . small molecules may also exhibit enhanced permeation to tumors as well due to the leaky neovasculature in the tumor tissues , however , they can easily diffuse out of tumors as well , while macromolecules and nanocarriers are entrapped and consequently accumulate . dcx not only induces apoptosis due to microtubule assembly stabilization , but also is antiangiogenic . inhibition of the formation of new blood vessels that provide rapidly growing tumors with increasing nutritional demand is expected to stunt the tumor growth . furthermore , the hastily formed tumor - associated vasculature is characterized with various fenestration and imperfections , and almost lacking any intact lymphatic drainage . nanoparticles are known to take advantage of this leaky tumor vasculature to extravasate into the tumor microenvironment , where they can accumulate . the most widely used lipid excipients are soy lecithin , glycerides , or a mixture of the two . formulated dcx into a nanostructured lipid carrier based on soy lecithin , glyceryl monostearate , and fatty acids , but the dcx in nanostructured lipid carrier was only slightly more effective than a dcx in a t80/e formulation ( duopafei ) against b16 tumors in mice . xu et al . designed a trimyristin - based sln formulation for dcx to treat hepatocellular carcinoma instead , their surface was galactosylated to target asialoglycoprotein receptors overexpressed on the surface of hepatoma cell lines . we previously reported the formulation of dcx - loaded lecithin - based pegylated slns . the resultant slns showed an improved in vitro cytotoxic activity , in addition to improved tumor accumulation in a mouse model . however , the capacity of this formulation in incorporating dcx was limited , which may be attributed to the limited affinity between dcx and the excipients . in the present study , in order to rationally select the most suitable excipient , four dcx - sln formulations were prepared using four different high melting point triglycerides . as discussed previously , a successful formulation for taxane is the one that exhibits ( a ) long plasma circulation time , ( b ) long drug retention within the delivery carrier , which requires high drug excipient affinity and slow release , ( c ) high tumor accumulation , and ( d ) favorable biodistribution profile , as less drug goes to healthy tissues . drug release behavior from the dcx - slns and short - term stability were the criteria for triglyceride selection in this study . in this regard , the absence of a burst release of dcx from the dcx - slns in the first 2 h and a subsequent slower release rate predict a limited drug leakage from the nanoparticles in the blood circulation before reaching tumors . the release of dcx from the tristearin - based and trilaurin - based dcx - slns was relatively faster ( figure 1 ) , and the tripalmitin- and trilaurin - based dcx - slns showed instability , as the zeta potential changed significantly following a short - term storage ( figure 2 ) . change in zeta potential has been used as an indicator of nanoparticle instability . several reports indicated that trilaurin does not exist in the solid state within the slns , but rather as a supercooled - liquid state that resembles o / w emulsions , even at 4 c , which was not the case with triglycerides having higher melting points . the relatively faster release of dcx from the trilaurin - based slns may also arise as a result of the same phenomenon . phenomenon was not reported , therefore , the reasons to which this sign of instability may be attributed need to be investigated . on the other hand , the absence of a burst release of dcx from the dcx - slns prepared with trimyristin may infer a strong dcx trimyristin interaction . it was reported that the solubility of dcx in tributyrin ( 4 c chain ) is about 108 mg / ml , and it gets lower with higher chain length , reaching about 56 mg / ml with tricaprylin ( 8 c chain ) . based on this , and since dsc and xrd data ( figure 4 ) implied that there is a strong interaction between dcx and the excipients , it is speculated that dcx exists in the slns in either a noncrystalline state or a dissolved state , or both , within the lipid matrix . existence of dcx in the amorphous state within lipid - based matrices was also reported previously . the disappearance of the characteristic dcx - related peaks in dsc and xrd was previously shown to be related to the loss of dcx crystallinity . in fact , we also found that the characteristic melting peak of dcx at 167 c completely disappeared upon analyzing the thermal behavior of dcx trimyristin mixtures at dcx to trimyristin ratios of 1:5 , 1:2 , and 1:1 using dsc ( data not shown ) , suggesting the prevalence of a strong interaction between the dcx and trimyristin . the relatively slower release of dcx from trimyristin - based dcx - slns may also be attributed to the strong dcx long - chain triglycerides in soybean oil and egg yolk phospholipids are commonly used in intravenous fat emulsions as components of parenteral nutrition for patients who are not able to receive nutrition via oral diets ( e.g. , intralipid , b braun medical inc . , bethlehem , pa ) . triglycerides are metabolized in the blood by lipases into corresponding fatty acids , which are cleared from the blood within about 30 min . phosphatidylcholine and pegylated phosphoethanolamine are used in products that are approved for intravenous infusion in humans ( e.g. , doxil ) . therefore , we expect that our new dcx - sln formulations will likely have a favorable safety profile . in fact , the body weights of the tumor - bearing mice that were treated with the dcx - slns did not significantly change by the end of the efficacy study ( figure 6b ) . in addition , the concentrations of dcx in vital organs such as liver , spleen , kidneys , lungs , and heart of mice that were injected with the dcx - slns were significantly lower than in mice that were injected with the dcx in t80/e formulation ( figure 7 ) , indicating that our dcx - slns may be less damaging to those vital organs than taxotere . in the present study , by taking advantage of the high solubility of dcx in triglycerides , we successfully prepared several dcx - incorporated slns using various medium- and long - chain triglycerides . the dcx - sln composition prepared with trimyristin was selected for further evaluation because the resultant dcx - slns were stable in a short - term stability study , and the rate at which the dcx was released from them was the slowest . the dcx - slns showed a stronger antitumor activity than dcx solubilized in a tween 80/ethanol mixture in cell culture and , more importantly , in a mouse model with pre - established tumors , likely because the dcx - slns significantly increased the accumulation of the dcx in tumor tissues . the decreased accumulation of dcx in vital organs after iv injection of dcx - slns , relative to after injection of dcx solubilized in a tween 80/ethanol mixture , suggests that the dcx - slns may have a favorable safety profile .

docetaxel ( dcx ) is a second generation taxane . it is approved by the u.s . food and drug administration for the treatment of various types of cancer , including breast , non - small cell lung , and head and neck cancers . however , side effects , including those related to tween 80 , an excipient in current dcx formulations , can be severe . in the present study , we developed a novel solid lipid nanoparticle ( sln ) composition of dcx . trimyristin was selected from a list of high melting point triglycerides as the core lipid component of the slns , based on the rate at which the dcx was released from the slns and the stability of the slns . the trimyristin - based , pegylated dcx - incorporated slns ( dcx - slns ) showed significantly higher cytotoxicity against various human and murine cancer cells in culture , as compared to dcx solubilized in a tween 80/ethanol solution . moreover , in a mouse model with pre - established tumors , the new dcx - slns were significantly more effective than dcx solubilized in a tween 80/ethanol solution in inhibiting tumor growth without toxicity , likely because the dcx - slns increased the concentration of dcx in tumor tissues , but decreased the levels of dcx in major organs such as liver , spleen , heart , lung , and kidney . dcx - incorporated slns prepared with one or more high - melting point triglycerides may represent an improved dcx formulation .

Introduction Materials and Methods Results Discussion Conclusion

the prevalence of diabetes mellitus ( dm ) for all age groups worldwide was estimated to be 2.8% in year 2000 and it is quite clear that male patients with dm are 3 times more likely to develop erectile dysfunction ( ed ) than those who do not have dm . in streptozotocin ( stz)-induced diabetic rats , thus , the erectile process has been shown to be strongly androgen - sensitive , at least in the rat model . testosterone substitution can reverse hypogonadism and diabetes - induced penile hyporeactivity to cavernous nerve stimulation in vivo in rats , along with increased neuronal nitric oxide synthase ( nnos ) expression . insulin - like growth factor binding protein-3 ( igfbp-3 ) is a member of the growth factor family and it is generally believed that the increased expression of igfbp-3 is associated with ed in diabetic rats , while igfbp expression is suppressed by androgens . it is well known that short hairpin rna ( shrna ) is emerging as a technology to silence gene expression by inhibiting mrna translation and/or inducing its degradation , including in the penis . in the current study , we investigated whether short hairpin ribonucleic acid constructs targeting igfbp-3 ( igfbp-3 shrna ) can rehabilitate decreased testosterone concentrations in stz - induced diabetic rats . to design igfbp-3 shrna construct , the rat igfbp-3 gene sequence ( gi : m31837 ) was analyzed for a potential sirna target using the web - based sirna target finder and design tool provided on the ambion website ( ambion , inc . double - stranded sirnas ( nucleotide position : 611 ) were transcribed in vitro using the silencer sirna construction kit ( ambion ) following the manufacturer s instructions . the inhibitory sirna ( 5-gcgctacaaagttgactatga-3 , nucleotide position 611 ) was then cloned into the pgpu6/gfp / neo plasmid vector ( 2 version , ambion ) as a short hairpin dna sequence ( 5 sense strand : 5-caccgcgctacaaagttgactatgatt caagagatcatagtcaactttgtagcgcttttttg-3 ) according to the manufacturer s instructions . the pgpu6/gfp / neo- igfbp-3 shrna plasmid was purified using the endo - free maxi kit ( qiagen , valencia , ca ) . plasmids were quantitated by spectrophotometry and prepared in 0.9% saline solution at a concentration of 1.0 g/l . twenty - seven adult spf male wistar rats ( age 3 months , weight 310330 g , certificated no . scxk[e]2008 - 0005 ) were obtained from hubei laboratory animal of research center ( wuhan , china ) . the wuhan university animal care and injections of citrate buffer ( 100 mmol / l citric acid and 200 mmol / l disodium phosphate , ph 7.0 ) . rats were considered diabetic if blood glucose levels were greater than 200 mg / dl ( table 1 ) . animals received 60 l citrate buffer ( group 1 and 2 ) and 60 ul lipofectamine - plasmid complex ( 10 g / kg ) ( group 3 ) into the corpus cavernosum 12 weeks after stz induction . a constriction band was applied at the base of the penis , and the needle was left in place for 5 min to allow the medication to diffuse throughout the cavernous space . at 12 weeks after igfbp-3 shrna administration , erectile function was evaluated by measuring intracavernous pressure ( icp ) following electrostimulation of the cavernous nerves , as previously described . the ratio of maximal icp - to - mean arterial pressure ( map ) ( icp / map ) and total icp ( area under curve ) were recorded for each rat . the penile shaft was collected for further analyses and whole blood samples from both systemic and penile shaft were also collected with serum obtained after clotting for 30 min at room temperature following centrifugation . all the serum samples were immediately snap - frozen in liquid nitrogen after collection and stored at 80c for further analyses . total rna was extracted from rat penile samples using trizol reagent ( invitrogen , merelbeke , belgium ) according to the manufacturer s protocol and re - suspended in rnase - free water . total rna was stored at 80c until analysis of igfbp-3 mrna levels by myiq single - color real - time pcr detection system ( bio - rad laboratories , hercules , ca ) with the following primers : igfbp-3 , forward 5-agccgtctcctggaaacacc-3 and reverse 5-cccgctttctgcctttgg-3 ; quantitative mrna measurements were performed in triplicate and normalized to an internal control of gapdh . after the pcr program , data were analyzed with abi 7300 sds software ( applied biosystems , foster city , ca ) . the rat penile samples were lysed in ripa buffer ( 1tbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , 0.1% sds , 0.004% sodium azide , 10 l / ml pmsf , 10 l / ml protease inhibitor cocktail , 10 l / ml sodium orthovanadate ) . protein concentration was measured using the coomassie plus protein assay reagent ( pierce biotechnology , rockford , il ) . equal quantities ( 30 g ) of lysates were separated on 10% sodium dodecyl sulfate gradient polyacrylamide gels and electro - blotted onto pvdf membranes ( bio - rad , hercules , ca ) . the membranes were blocked and then incubated with rabbit anti - igfbp-3 antibody ( sc-9028 ) at 1:1000 dilutions ( santa cruz biotechnology , santa cruz , ca ) . chemiluminescence was detected using ecl western blotting detection reagents ( amersham , buckinghamshire , uk ) . the expression of igfbp-3 protein was visualized by densitometry using the mivnt image analysis system ( shanghai institute of optical instruments , shanghai , china ) . the measurement of serum testosterone concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) testosterone detection kit ( r&d systems , minneapolis , mn ) . similarly the measurement of cgmp concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) cgmp detection kit ( r&d systems , minneapolis , mn ) . differences among normal control group , diabetic control group , and igfbp-3 shrna treatment group were considered significant at the level of p<0.05 using the 2-tailed unpaired t test . to design igfbp-3 shrna construct , the rat igfbp-3 gene sequence ( gi : m31837 ) was analyzed for a potential sirna target using the web - based sirna target finder and design tool provided on the ambion website ( ambion , inc . double - stranded sirnas ( nucleotide position : 611 ) were transcribed in vitro using the silencer sirna construction kit ( ambion ) following the manufacturer s instructions . the inhibitory sirna ( 5-gcgctacaaagttgactatga-3 , nucleotide position 611 ) was then cloned into the pgpu6/gfp / neo plasmid vector ( 2 version , ambion ) as a short hairpin dna sequence ( 5 sense strand : 5-caccgcgctacaaagttgactatgatt caagagatcatagtcaactttgtagcgcttttttg-3 ) according to the manufacturer s instructions . the pgpu6/gfp / neo- igfbp-3 shrna plasmid was purified using the endo - free maxi kit ( qiagen , valencia , ca ) . plasmids were quantitated by spectrophotometry and prepared in 0.9% saline solution at a concentration of 1.0 g/l . twenty - seven adult spf male wistar rats ( age 3 months , weight 310330 g , certificated no . scxk[e]2008 - 0005 ) were obtained from hubei laboratory animal of research center ( wuhan , china ) . the wuhan university animal care and injections of citrate buffer ( 100 mmol / l citric acid and 200 mmol / l disodium phosphate , ph 7.0 ) . rats were considered diabetic if blood glucose levels were greater than 200 mg / dl ( table 1 ) . animals received 60 l citrate buffer ( group 1 and 2 ) and 60 ul lipofectamine - plasmid complex ( 10 g / kg ) ( group 3 ) into the corpus cavernosum 12 weeks after stz induction . half the dose was administered in each crus . during intracavernosal injection , a constriction band was applied at the base of the penis , and the needle was left in place for 5 min to allow the medication to diffuse throughout the cavernous space . at 12 weeks after igfbp-3 shrna administration , erectile function was evaluated by measuring intracavernous pressure ( icp ) following electrostimulation of the cavernous nerves , as previously described . the ratio of maximal icp - to - mean arterial pressure ( map ) ( icp / map ) and total icp ( area under curve ) were recorded for each rat . the penile shaft was collected for further analyses and whole blood samples from both systemic and penile shaft were also collected with serum obtained after clotting for 30 min at room temperature following centrifugation . all the serum samples were immediately snap - frozen in liquid nitrogen after collection and stored at 80c for further analyses . total rna was extracted from rat penile samples using trizol reagent ( invitrogen , merelbeke , belgium ) according to the manufacturer s protocol and re - suspended in rnase - free water . total rna was stored at 80c until analysis of igfbp-3 mrna levels by myiq single - color real - time pcr detection system ( bio - rad laboratories , hercules , ca ) with the following primers : igfbp-3 , forward 5-agccgtctcctggaaacacc-3 and reverse 5-cccgctttctgcctttgg-3 ; quantitative mrna measurements were performed in triplicate and normalized to an internal control of gapdh . after the pcr program , data were analyzed with abi 7300 sds software ( applied biosystems , foster city , ca ) . the rat penile samples were lysed in ripa buffer ( 1tbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , 0.1% sds , 0.004% sodium azide , 10 l / ml pmsf , 10 l / ml protease inhibitor cocktail , 10 l / ml sodium orthovanadate ) . protein concentration was measured using the coomassie plus protein assay reagent ( pierce biotechnology , rockford , il ) . equal quantities ( 30 g ) of lysates were separated on 10% sodium dodecyl sulfate gradient polyacrylamide gels and electro - blotted onto pvdf membranes ( bio - rad , hercules , ca ) . the membranes were blocked and then incubated with rabbit anti - igfbp-3 antibody ( sc-9028 ) at 1:1000 dilutions ( santa cruz biotechnology , santa cruz , ca ) . chemiluminescence was detected using ecl western blotting detection reagents ( amersham , buckinghamshire , uk ) . the expression of igfbp-3 protein was visualized by densitometry using the mivnt image analysis system ( shanghai institute of optical instruments , shanghai , china ) . the measurement of serum testosterone concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) testosterone detection kit ( r&d systems , minneapolis , mn ) . similarly the measurement of cgmp concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) cgmp detection kit ( r&d systems , minneapolis , mn ) . differences among normal control group , diabetic control group , and igfbp-3 shrna treatment group were considered significant at the level of p<0.05 using the 2-tailed unpaired t test . we determined the ratio of icp / map and the total icp ( area under curve ) during electrostimulation of the cavernous nerves at 12 weeks after igfbp-3 shrna administration . the representative icp tracing in response to electrostimulation of the cavernous nerves is shown in figure 1(a c ) . electrostimulation elicited significantly increased icp / map ratio ( figure 1d ) and total icp ( figure 1e ) in the igfbp-3 shrna treatment group compared to those in the diabetic control group ( p<0.01 , respectively ) . real - time qpcr analysis using gapdh as a housekeeping gene showed that cavernous igfbp-3 mrna level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 2 ) . accordingly , western blot analysis showed that cavernous igfbp-3 protein level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 3 , lower ) . these results are consistent with the results of real - time qpcr analysis and prove that igfbp-3 shrna inhibits igfbp-3 expression in rat cavernous tissue . both serum testosterone and cavernous cgmp concentrations were measured by elisa and we found that serum testosterone and cavernous cgmp concentrations were both significantly increased in the igfbp-3 shrna treatment group compared to those in the diabetic control group at 12 weeks after igfbp-3 shrna administration ( p<0.01 , figure 4 ) . we determined the ratio of icp / map and the total icp ( area under curve ) during electrostimulation of the cavernous nerves at 12 weeks after igfbp-3 shrna administration . the representative icp tracing in response to electrostimulation of the cavernous nerves is shown in figure 1(a c ) . electrostimulation elicited significantly increased icp / map ratio ( figure 1d ) and total icp ( figure 1e ) in the igfbp-3 shrna treatment group compared to those in the diabetic control group ( p<0.01 , respectively ) . real - time qpcr analysis using gapdh as a housekeeping gene showed that cavernous igfbp-3 mrna level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 2 ) . accordingly , western blot analysis showed that cavernous igfbp-3 protein level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 3 , lower ) . these results are consistent with the results of real - time qpcr analysis and prove that igfbp-3 shrna inhibits igfbp-3 expression in rat cavernous tissue . both serum testosterone and cavernous cgmp concentrations were measured by elisa and we found that serum testosterone and cavernous cgmp concentrations were both significantly increased in the igfbp-3 shrna treatment group compared to those in the diabetic control group at 12 weeks after igfbp-3 shrna administration ( p<0.01 , figure 4 ) . in the present study , we found that concentration of serum testosterone was significantly higher in the igfbp-3 shrna treatment group compared to that in the diabetic control group ( p<0.01 ) at 12 weeks after igfbp-3 shrna administration . these results suggest that igfbp-3 shrna may rehabilitate erectile function via increases of concentrations of serum testosterone and cavernous cgmp in stz - induced diabetic rats . the possibility that hypogonadism might contribute to diabetes mellitus ( dm)-related ed has been reconsidered recently , because of a higher prevalence of low testosterone in diabetic than in nondiabetic individuals [ 1012 ] , and even in animal models of chemical diabetes , overt hypogonadism is often reported . essentially , androgens play a dual role in the erectile process by controlling both pro - erectile and anti - erectile signaling pathways . on the one hand , androgens exert a positive role in the maintenance of the general architecture of the penile contractile structure . castration induces cavernous smooth muscle atrophy and contributes to structural alteration of erectile tissue , with a relative increase in connective tissue content [ 1618 ] . furthermore , castration reduced nitric oxide ( no ) and its generating enzyme no synthase ( nos ) , as well as nos - containing nerve fibers . thus , in castrated rats , erectile response is markedly impaired , but testosterone ( t ) reverses this deficiency . a recent study reported that testosterone may prevent impaired erectile function by maintaining at the control level the expression of the key enzymes involved in the erectile process , such as nnos . on the other hand , it has been recently demonstrated that androgens up - regulate the major mechanism responsible for cgmp degradation , phosphodiesterase type 5 ( pde5 ) , and pde5 regulates the amount of cgmp available for cavernosal smooth muscle relaxation and penile erection . a clinical report demonstrated a null effect of hypogonadism on penile erection , probably because androgen reduction produces both decreased cgmp formation ( effect on no ) and degradation ( effect on pde5 ) . the major subcellular mechanism by which erection occurs involves no - induced activation of soluble guanylyl cyclase ( sgc ) and increased cgmp concentration . a significantly decreased level of cgmp in diabetes rats was associated with ed in our previous study . here , we found that serum testosterone and cavernous cgmp concentrations were both significantly increased in the igfbp-3 shrna treatment group compared to those in the diabetic control group at 12 weeks after igfbp-3 shrna administration these results suggest that igfbp-3 shrna may rehabilitate erectile function via increased concentration of serum testosterone and improvement of no - cgmp signaling activities in stz - induced diabetic rats . igfbp-3 , conventionally thought to inhibit growth through ligand sequestration , might also be antiproliferative and proapoptotic through actions independent of the insulin - like growth factor-1 ( igf-1 ) receptor . a previous study has shown that igfbp-3 is significantly correlated with erectile function in the diabetic rat model . the increased expression of igfbp-3 might lead to depletion of the smooth muscle density and elicit ed in patients with diabetes mellitus ( dm ) . since igfbp expression is suppressed by androgens , an increase of concentration of serum testosterone might decrease the expression of igfbp-3 ( the most abundant igfbp ) . thus , hyperglycemia in combination with increased igfbp-3 expression may have a role in the development of ed . the current study indicated that igfbp-3 shrna may rehabilitate erectile function via increased concentration of serum testosterone and improvement of no - cgmp signaling activities in stz - induced diabetic rats . further investigation of the mechanisms by which increased concentration of serum testosterone leads to activation of no - cgmp signaling will enrich our understanding of the pathogenesis of ed of stz - induced diabetic rats .

backgroundthe aim of this study was to determine if shrna constructs targeting insulin - like growth factor binding protein-3 can rehabilitate decreased serum testosterone concentrations in streptozotocin - induced diabetic rats.material/methodsafter 12 weeks of intracavernous administration of igfbp-3 shrna , intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated . the expression of igfbp-3 at mrna and protein levels was detected by quantitative real - time pcr analysis and western blot , respectively . the concentrations of serum testosterone and cavernous cyclic guanosine monophosphate were detected by enzyme - linked immunosorbent assay.resultsafter 12 weeks of intracavernous administration of igfbp-3 shrna , the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic control group ( p<0.01 ) . cavernous igfbp-3 expression at both mrna and protein levels was significantly inhibited . both serum testosterone and cavernous cyclic guanosine monophosphate concentrations were significantly increased in the igfbp-3 shrna treatment group compared to the diabetic control group ( p<0.01).conclusionsthese results suggest that igfbp-3 shrna may rehabilitate erectile function via increases of concentrations of serum testosterone and cavernous cyclic guanosine monophosphate in streptozotocin - induced diabetic rats .

Background Material and Methods IGFBP-3 shRNA Constructs Experimental animals Measurement of erectile responses Quantitative real-time PCR analysis Western blot analysis Enzyme-linked immunosorbent assay Statistical analysis Results Evaluation of erectile function IGFBP-3 shRNA inhibits IGFBP-3 expression in rat cavernous tissue Testosterone and cGMP concentrations in rat serum Discussion Conclusions

the plant antiaris toxicaria ( family moraceae ) is an indigenous plant common in ghanaian forests . foto or kyenkyen in akan and the bark cloth tree in english . despite considerable advancements in the treatment of neurological disorders currently available antiepileptic drugs ( aeds ) have debilitating adverse effects on cognition and behaviour . these adverse effects are commonly and consistently observed with barbiturates , benzodiazepines , and topiramate [ 4 , 5 ] . these problems are known to prevail more in developing countries due to lack of facilities for proper diagnosis and treatment along with monitoring of aed serum levels [ 6 , 7 ] . natural products and plants already used in traditional medicine can be a good place to start in the search for safer and more effective options . numerous plants used for the treatment of epilepsy traditionally have been shown to be potent in models of epilepsy and several such plants remain to be scientifically validated . leonotis leonurus , delphinium denudatum , mimosa pudica , and synedrella nodiflora are but a few examples [ 810 ] . preliminary screening of the aqueous extract of antiaris toxicaria revealed significant anticonvulsant effect in pentylenetetrazole - induced seizure test . hence , this study seeks to further explore the extract 's potential as an anticonvulsant . stem bark of antiaris toxicaria was harvested from the knust campus , kumasi , and authenticated at the pharmacognosy department of the faculty of pharmacy , knust , kumasi , ghana . a voucher specimen ( knust / hm1/011/s007 ) dried stem bark was milled into powder using a commercial grinder . the coarse powder ( 431 g ) was extracted by cold maceration with distilled water as solvent at room temperature for five days . filtrate was oven - dried to obtain a yield of 23.40% w / w of antiaris toxicaria aqueous extract ( aae ) . male icr mice and sprague - dawley rats weighing between 20 and 25 g and 120 and 145 g , respectively , were obtained from noguchi memorial institute for medical research , accra , ghana , and kept in the departmental animal house . animals were maintained under laboratory conditions of temperature , humidity , and light , housed in stainless steel cages ( 34 47 18 cm ) , and allowed access to water and food ad libitum . all animals were handled in accordance to the guide for the care and use of laboratory animals and experiments were approved by the faculty ethics committee . diazepam ( dzp ) , pentylenetetrazole ( ptz ) , picrotoxin ( pct ) , 4-aminopyridine ( 4-ap ) , and strychnine ( str ) were purchased from sigma - aldrich inc . , st . flumazenil was obtained from app pharmaceuticals , llc , schaumburg , il , usa . the method as described by vellucci and webster , 1984 , and moezi et al . , 2011 , the plant extract was administered at doses of 200 , 400 , and 800 mg kg body weight orally . intraperitoneal ( i.p ) injection of diazepam ( 0.1 , 0.3 , and 1 mg kg ) was used as reference anticonvulsant drug . animals were pretreated with the plant extract thirty minutes and diazepam fifteen minutes before administration of pentylenetetrazole ( ptz ) 85 mg kg subcutaneously . control animals were pretreated with distilled water ( 10 ml kg , p.o . ) . the onset of , total duration as well as frequency of clonic seizures were measured within a thirty minute period . animals received aae at doses of 200 , 400 , and 800 mg kg ( p.o . ) body weight . picrotoxin ( pct ) was injected intraperitoneally at a dose of 3 mg kg fifteen minutes after pretreatment with diazepam and thirty minutes after aae . diazepam ( 0.1 , 0.3 , and 1 mg kg ) served as positive control . control animals received distilled water ( 10 ml kg , p.o . ) . anticonvulsant activity was scored similarly to that stated in the ptz test [ 13 , 15 ] . tonic convulsions of hind limb extremities of mice were induced using electrical current ( 50 ma , 60 hz , and 0.2 seconds ) via ear clip electrodes . control group animals received distilled water orally ( 10 ml kg , p.o . ) . carbamazepine at doses of 3 , 10 , and 30 mg kg orally served as reference anticonvulsant . aae was tested at doses of 200 , 400 , and 800 mg kg orally . aae was administered orally at doses of 200 , 400 , and 800 mg kg body weight . strychnine was injected subcutaneously at a dose of 2 mg kg thirty minutes after aae administration . standard anticonvulsant employed was diazepam ( 0.1 , 0.3 , and 1 mg kg , i.p ) . the onset of , decrease in the total duration plus frequency of tonic convulsions were taken as indication of anticonvulsant activity . animals received aae orally at doses of 200 , 400 , and 800 mg kg body weight . 4-aminopyridine was dissolved in normal saline and injected subcutaneously at a dose of 10 mg kg body weight thirty minutes after drug treatments . control animals were pretreated with normal saline ( 10 ml kg ) and sodium valproate at 100 , 200 , and 400 mg kg served as positive control . hind limb tonic extensions and decrease in the total duration of convulsions were recorded . method as described by morales - villagrn and tapia , 1996 , was used with slight modifications . in order to investigate the mechanism of action of aae as an anticonvulsant , mice were treated with flumazenil , a benzodiazepine antagonist ( 1 mg kg , i.p ) fifteen minutes before the administration of aae ( 400 mg kg , p.o . ) . thirty ( 30 ) minutes later , convulsions were induced with pentylenetetrazole at 85 mg kg , intraperitoneally . other groups of animals received aae or flumazenil or diazepam ( 0.3 mg kg , i.p ) only . animals were observed for thirty minutes after treatment via video recording for latency and duration of convulsions . analysis of variance ( anova ) followed by newman - keuls ' post hoc test was used to determine significant differences between means . two - way anova followed by bonferroni test was used in the flumazenil experiment . in the 4-aminopyridine seizure test , the kaplan - meier method was used in estimating survival relative to time and survival differences were analyzed with the log - rank test . statistical analyses were carried out with graph pad prism version 5.0 ( graphpad software , san diego , ca , usa ) and sigmaplot version 11.0 ( systat software , inc . , pentylenetetrazole ( 85 mg kg , s.c . ) produced myoclonic jerks in all mice pretreated with the distilled water . aae ( 200800 mg kg ) produced a significant ( p = 0.0021 ; f3,16 = 7.671 ; figure 1(a ) ) dose - dependent increase in time taken to the onset of clonic seizures . in the extract - treated animals , frequency of seizures was also decreased nondose - dependently , and only the 800 mg kg dose was significant ( p = 0.0511 ; f3,16 = 3.213 ; figure 1(a ) ) . aae produced significant ( p = 0.0018 ; f3,16 = 8.005 ; figure 1(c ) ) dose - dependent decrease in the total duration of convulsions in all animals pretreated with the various doses of the extract . the reference anticonvulsant diazepam ( 0.11.0 mg kg , i.p ) also profoundly delayed the onset of myoclonic jerks , decreased frequency of jerks and significantly ( p < 0.0001 ; f3,16 = 125.8 ; figures 1(b ) and 1(d ) ) antagonized ptz - induced seizures . picrotoxin ( 3 mg kg , i.p ) produced clonic seizures in all mice pretreated with the distilled water . aae ( 200800 mg kg ) produced a dose - dependent increase in latency to seizures which was significant ( p = 0.0009 ; f3,16 = 9.108 ; figure 2(a ) ) at all doses . the frequency of seizures was also significantly ( p = 0.0081 ; f3,16 = 5.591 ; figure 2(a ) ) decreased dose - dependently . aae produced significant ( p = 0.0002 ; f3,16 = 12.53 ; figure 2(c ) ) dose - dependent decrease in the total duration of seizures in all animals . the reference anticonvulsant diazepam ( 0.11.0 mg kg , i.p . ) also profoundly delayed the onset of myoclonic jerks , decreased frequency of jerks and significantly antagonised ( p < 0.0001 ; f3,16 = 17.72 ; figures 2(b ) and 2(d ) ) pct - induced seizures . the extract did not affect the latency to onset of hind limb tonic extensions and duration of convulsions significantly ( figure 3 ) . the reference anticonvulsant carbamazepine ( 3 , 10 , and 30 mg kg , p.o . ) , however , delayed the onset of hles significantly ( p = 0.0054 ; f3,16 = 6.193 ; figure 3 ) and decreased the total duration of electroshock - induced convulsions . aae did not affect the latency to onset of hind limb tonic extensions and duration of convulsions significantly ( figure 4 ) . the reference anticonvulsant diazepam ( 1 mg kg , i.p ) , on the other hand , delayed the latency to convulsions significantly ( p < 0.0001 ; f4,20 = 36.60 ; figure 4 ) . the duration of convulsions was also significantly ( p < 0.0001 ; f4,20 = 14.31 ; 4-aminopyridine ( 10 mg kg , i.p . ) produced hind limb tonic extensions in all animals . the extract produced a significant ( p < 0.0001 ; f3,28 = 22.02 ; figure 5(a ) ) increase in time taken to the onset of convulsions . sodium valproate ( 100400 mg kg ) also significantly ( p < 0.0001 ; f3,28 = 17.61 ; figure 5(b ) ) delayed the onset of convulsions . the extract significantly ( p = 0.0004 , (df = 3 ) = 18.11 ; figure 6(a ) ) improved survival of the animals after induction of convulsions . the test for trend presented significant ( p < 0.0001 , (df = 1 ) = 15.79 ) effect of the treatment groups on median survival indicating a linear trend . sodium valproate also produced similar effects on survival ( p < 0.0001 , (df = 3 ) = 21.23 ; figure 6(b ) ) and linear trends ( p < 0.0001 , (df = 1 ) = 19.17 ) . survival curves show a decrease in probability of survival with time . however , probability of survival increased with increasing dose . flumazenil significantly ( p = 0.0009 ; f1,8 = 26.09 ; figure 7(c ) ) reversed the reduction in duration of seizures produced by the extract . a significant reversal ( p = 0.0003 ; f1,8 = 37.51 ; figure 7(a ) ) was also obtained for the latency to seizures . effects of diazepam were significantly ( p < 0.0001 ; figures 7(a ) , 7(b ) , and 7(c ) ) reversed by flumazenil . the outcome of this study provides evidence that the aqueous extract of the stem bark of antiaris toxicaria possesses anticonvulsant activity . the ability of aae to delay the onset of convulsions and/or shorten the duration of convulsions was considered an indication of anticonvulsant activity . judging from the data obtained , the plant extract exhibited anticonvulsant activity in the ptz test just like diazepam which can be due to action on gaba system [ 1921 ] . it , however , has been shown to be due to inhibition and/or attenuation of gabaergic neurotransmission [ 22 , 23 ] . it is therefore likely that aae produces its anticonvulsant effect directly or indirectly by enhancing gabaergic neurotransmission in the brain . - synaptic inhibition mediated by gaba often leads to inhibition of neurotransmitter release from the excitatory arm . pct - induced seizures which are due to the decreased gabaa - receptor - mediated inhibition in turn promote the excitatory arm of the cns mainly mediated by glutamate [ 20 , 22 ] . the extract being effective in the pct - induced seizure test points to a more specific action on gaba - mediated neurotransmission . this has been further confirmed by the use of flumazenil to reverse the anticonvulsant properties of aae in the ptz - induced seizure model . flumazenil is a benzodiazepine antagonist at the gabaa receptor possessing the ability to reverse anticonvulsant effects and the accompanying alterations in extracellular glutamate concentration . the specificity of flumazenil to this receptor has been proven as several authors have demonstrated that the antagonist has no effect on diazepam 's action on voltage - dependent na channels [ 2931 ] . retigabine is currently the only approved drug for the treatment of epilepsy which functions through activation of potassium currents [ 3234 ] and has promise in the treatment of pharmacoresistant epilepsy . it is also known that 4-aminopyridine stimulates the release of neurotransmitters , including glutamate , in numerous preparations such as brain slices [ 3538 ] and the neuromuscular junction . furthermore , it has been recently found by a microdialysis procedure in vivo that glutamate is by far the amino acid predominantly released by 4-aminopyridine in the striatum . therefore , due to the well - recognized function of excitatory amino acid receptors in convulsive and excitotoxic mechanisms , it seems feasible that the excess glutamate released may be involved in the excitatory effects . this indicates that the extract may be acting , in addition to the above , by modulating the release of glutamate . from the pattern of survival curves , it can be inferred that the dose of the extract does affect survival rate offering significant protection against death from 4-aminopyridine - induced convulsions . this was evident from the log - rank analysis which indicated significant differences between survival curves for each dose level . median survival time ranged from 30 min and death may be prevented totally at the highest dose of aae . the exact mechanism by which the extract offers this protection may be linked to its ability to reduce excitotoxic effects mediated by glutamate release . this indicates that it is not effective in partial and generalized tonic seizures and is unable to prevent seizure spread . the fact that aae produced no protective effects against strychnine - induced convulsions suggests it does not interact with the glycine - mediate inhibitory pathway . the results presented here indicate that the aqueous extract of antiaris toxicaria exhibits anticonvulsant activity , possibly through gaba - mediated inhibition and inhibition of glutamate mediated excitation via activation of potassium ion channels .

antiaris toxicaria ( moraceae ) was evaluated for anticonvulsant activity in rodents . animal models used include maximal electroshock test ( mest ) ; pentylenetetrazole - induced ( ptz ) convulsions ; picrotoxin - induced ( pct ) convulsions ; strychnine- ( str- ) and 4-aminopyridine - induced convulsions . increase in latency to seizures as well as reduction in duration and frequency of seizures indicated anticonvulsant activity . the extract was more effective in all models used except the maximal electroshock test and strychnine - induced convulsions . antiaris toxicaria aqueous extract ( 200 , 400 , and 800 mg kg1 ) significantly ( p < 0.05 0.01 ) shortened the duration of convulsions in ptz- and pct - induced seizures . delay in the onset of convulsions in the two tests was significant ( p < 0.001 ) . reduction in the frequency of seizures was also significant ( p < 0.05 0.001 ) in both tests . antiaris further delayed the onset of seizures in 4-aminopyridine model while producing 75% protection against death in mice . diazepam ( 0.1 , 0.3 , and 1 mg kg1 ) , carbamazepine ( 3 , 10 , and 30 mg kg1 ) , and sodium valproate ( 100400 mg kg1 ) were used as reference anticonvulsant drugs for various models . flumazenil blocked the effect of the extract in the ptz test significantly suggesting that antiaris toxicaria may be acting by enhancing the effects of the gabaergic system . antiaris toxicaria aqueous extract therefore possesses anticonvulsant activity .

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

a 15-year - old male , who was diagnosed with prs , was scheduled for repair of a pectus excavatum under general anesthesia . he had a history of tracheostomy at birth , a cleft palate operation at 1 month of age , and tracheostomy closure 1.5 years later . according to the consulting otolaryngologist , there was no pathological condition except sleep apnea . at the physical examination of his airway , the boy had the typical appearance of prs . he also had a mouth opening limitation and the maximum distance between incisors was 2.8 cm . his mallampati class was grade 3 , but he had no limited neck movement . because his parents disagreed with a conscious fiber - optic intubation for fear that their son would have a hard time during the procedure , we planned to induce anesthesia with propofol and succinylcholine and then intubate with the mcgrath mac video laryngoscope and a malleable stylet . we also prepared a laryngeal mask airway ( lma ) in case of difficult mask ventilation , according to the difficult airway guidelines . finally , if all of these methods failed , we planned to wake him because his parents wanted to cancel the surgery if the intubation failed . anesthesia was induced in the operating room with 2 mg / kg propofol and 1 mg / kg succinylcholine after preoxygenation with 100% oxygen . after full muscular relaxation , the mcgrath mac video laryngoscope was inserted into the oropharynx and was placed posterior to the epiglottis because the epiglottis obscured the glottis and provided a half view of the glottis ( cormack - lehane grade 2 ) . however , insertion of the 60 angled malleable styletted ett failed because the tube tip could be seen posterior to the arytenoids despite the external laryngeal maneuver . the tube tip reached the glottis , but there was considerable resistance advancing the ett through the vocal cords even though the stylet was withdrawn . finally , the frova intubating introducer with its tip modified as much as a 60 angle , similar to the mcgrath mac video laryngoscope , was inserted through the vocal cords ( figs . 1 and 2 ) . the frova intubating introducer was used as a railroad for the ett , and there was no difficulty advancing the ett under vision with the video laryngoscope ( fig . correct placement of the ett was further confirmed by auscultation and end - tidal co2 detection . we applied the mcgrath mac video laryngoscope and frova intubating introducer for endotracheal intubation in this case of a difficult airway and showed that the strategy was effective and successful . fiber - optic intubation is currently the gold standard for elective difficult airway management , but alternatives are described in the literature . the intubation techniques described include : airtraq optical laryngoscopy ( prodol meditec sa , vizcaya , spain ) , video laryngoscopy , non - fiber - optic intubation via lma , fiber - optic intubation via lma , and a paraglossal approach with tube advancement over a gum - elastic bougie . we chose a mcgrath mac video laryngoscope and malleable stylet because the mcgrath mac video laryngoscope provides a good view of the glottis , and a malleable stylet helps direct the ett to the glottis . as a precaution for a failed intubation , we also prepared a frova intubating introducer and fiber - optic bronchoscope for combined use , and an lma in case of a difficult mask ventilation according to the difficult airway guidelines . it is important to have backup airway management plans because it takes less than 10 min for patients with a difficult airway to become hypoxic . at our first attempt , insertion of the 60 angled malleable styletted ett failed because the tube tip could be seen posterior to the arytenoids . external laryngeal pressure and withdrawal of the blade to lessen tilting of the laryngeal axis and to reduce the introducing angle may have been helpful . however , these maneuvers did not work in our case . the tube tip reached the glottis , but there was considerable resistance advancing the ett through the vocal cords . this occurs from time to time , as the tube tip may strike the anterior tracheal wall because of the angle of the stylet . in this case , the operator withdraws the stylet approximately 4 cm , withdraws the video laryngoscope by 1 - 2 cm and rotates the ett slightly to facilitate passage into the trachea . we withdrew the stylet and rotated the ett to pass it into the trachea but failed . finally , the frova intubating introducer with its tip modified as much as a 60 angle like the mcgrath mac video laryngoscope was attempted , and it was inserted through the vocal cords with no difficulty . this was a remarkable result because the tip of the 60 angled malleable styletted ett , which had the same curvature as the frova intubating introducer , could be seen posterior to the arytenoids even though the external laryngeal maneuver was applied . unlike the macintosh laryngoscope , video laryngoscopes require a tilting force rather than a lifting force , and this force makes mouth opening too small to handle the ett . because the frova intubating introducer has a smaller outer diameter than that of an ett ( 4.6 mm for the frova intubating introducer vs. 8.9 mm for a # 6.5 ett ) , it is easy to handle in a narrow space , as shown in this case . thus , the frova intubating introducer with its tip modified as much as 60 reached the glottis , whereas the same 60 angled malleable styletted ett could not . although the frova intubating introducer was designed for use with a macintosh laryngoscope in the line of sight , and its tip should not be modified during macintosh laryngoscopy , modifying its tip and combined use with a video laryngoscope provides some advantages . first , the frova intubating introducer allows for easy handling in the narrow space under a video laryngoscope . second , it creates less trauma than a styletted ett . less resistance advancing the 60 angled frova intubating introducer through the vocal cords than that of a 90 angled malleable finally , preparing the frova intubating introducer is less time consuming than a fiber - optic bronchoscope , so it can be used for an unanticipated difficult intubation . the use of a video laryngoscope combined with a fiber - optic bronchoscope has been reported previously . fiber - optic bronchoscopy is a method of inserting an ett with a video laryngoscope . however , this technique has some disadvantages ; it needs more than two experienced anesthesiologists and one anesthesiologist should be familiar with the fiber - optic bronchoscope . in addition , the maintenance and preparation of a fiber - optic bronchoscope are time - consuming , limiting its use in the case of an unanticipated difficult intubation . use of a lma is another good option in patients with prs ; however , we thought it would be dangerous in this case because lung compliance might have decreased during repair of the pectus excavatum . we did not know that tracheal intubation with a macintosh laryngoscope would be a success or failure in this patient because we did not use a macintosh laryngoscope initially . sometimes tracheal intubation is attempted with a macintosh laryngoscope in anticipated difficult airway with little possibility of success , even though other methods for intubation , such as a video laryngoscope or fiber - optic bronchoscope , are available . the difficult airway management guidelines developed by the difficult airway society of the uk suggest that the initial tracheal intubation plan should not include more than four attempts . the fact that multiple attempts can lead to tissue trauma causes more difficult intubation in subsequent trials , makes mask ventilation difficult , and results in hypoxia . therefore , it is important to choose the first intubation plan carefully because fewer attempts lead to less airway trauma and increased patient safety . in conclusion , we propose that the combined use of a mcgrath mac video laryngoscope and a frova intubating introducer is a useful option for a difficult airway with an upper airway anomaly .

patients with pierre robin syndrome are characterized by micrognathia , retrognathia , glossoptosis , and respiratory obstruction and are prone to have a difficult - to - intubate airway . the mcgrath mac video laryngoscope provides a better view of the glottis than a macintosh laryngoscope , but it is not easy to insert an endotracheal tube through the vocal cords because a video laryngoscope has a much greater curvature than that of a conventional direct laryngoscope and an endotracheal tube has a different curvature . the frova intubating introducer is used as a railroad for an endotracheal tube in cases of a difficult airway . we thought that a combination of these two devices would make it easy to insert an endotracheal tube through the vocal cords , as a mcgrath mac video laryngoscope provides a better glottic view and the frova intubating introducer is a useful device for placing an endotracheal tube through the glottis . we report a successful endotracheal intubation with use of the mcgrath mac video laryngoscope and frova intubating introducer in a patient with pierre robin syndrome .

Case Report Discussion

rarely , severe cholecystitis may result in extravasation of bile through rupture of rokitansky - aschoff sinuses or mucosal ulceration causing severe fibrosis and formation of yellow intramural nodules and xanthogranulomatous reaction known as xanthogranulomatous cholecystitis ( xgc ) [ 1 - 4 ] . although there is a known association between gallbladder cancer and xgc , it is not clearly understood . computed tomography ( ct ) has recently become the gold standard investigation of choice in acute abdomen because it is quick , noninvasive , and provides a detailed map of the abdomen . furthermore , the introduction of multislice ct has increased the diagnostic accuracy of intra - abdominal conditions . however , ct may also detect incidental silent conditions requiring urgent attention such as abdominal aortic aneurysm ( aaa ) . we herein present a rare case of a patient who presented with signs of acute cholecystitis and gallbladder empyema . investigations showed evidence of chronic cholecystitis and an incidental large aaa . at surgery , a suspicious small nodule ( not seen on ct ) was excised . this rare coexistence of pathologies adds a further dimension to the management dilemma of the multidisciplinary team . a 79-year - old woman presented to the emergency department with a 5-week history of intermittent right upper quadrant and epigastric pain . her past medical history included hypertension , myocardial infarction 10 years previously , and bilateral knee replacement . on examination , she had tachycardia of 100 beats per minute with no fever . abdominal examination revealed a tender mass in the right upper quadrant with a pulsatile epigastric mass . a full blood count and serum biochemistry showed a hemoglobin level of 11.2 g / dl , leukocytosis of 15 10/l , c - reactive protein level of 141 mg / l , and normal renal and liver function tests . an urgent ct scan of the abdomen showed evidence of severe cholecystitis with a distended , thick - walled gallbladder , suspected gallbladder empyema , and an incidental 7-cm infrarenal aaa ( fig . 1a ) . immediate percutaneous decompression of the gallbladder empyema with a radiologically placed drain revealed 240 ml of pus . the patient 's condition improved , and she was discharged home . in view of the adjacent aaa , the decision of the multidisciplinary team was to carry out open cholecystectomy followed by staged endovascular aneurysm repair . at surgery , the gallbladder was adherent to the surrounding tissue with dense liver , duodenal , and bowel adhesions . a small nodule ( not clearly seen on ct ) was found adjacent to the distended gallbladder . xgc is a rare form of chronic cholecystitis , accounting for 0.7% to 13.2% of resected gallbladder specimens . it is thought that chronic inflammation leads to mucosal ulceration and rupture of rokitansky - aschoff sinuses with extravasation of lipids from bile , resulting in a xanthogranulomatous reaction within the gallbladder wall characterized by formation of multiple intramural yellow nodules ( fig . this inflammatory process is often extensive and may extend to adjacent organs , forming dense adhesions with a large mass of inflammatory tissue surrounding the gallbladder . the association between xgc and gallbladder cancer has been shown in the literature in small case series and some single case reports . it is estimated that xgc and gallbladder cancer coexist in up to 12% of cases . xgc is often mistaken for carcinoma of the gallbladder both macroscopically during surgery and on ct examinations ; thus , radiological differentiation from cancer can be extremely difficult in the presence of severe inflammation . furthermore , the fact that xgc can be associated with gallbladder cancer makes the differentiation even more difficult . in our patient , the clinical presentation suggested a diagnosis of acute cholecystitis and gallbladder empyema , as confirmed on ct ( fig . therefore , simple cholecystectomy was carried out as planned ; however , advanced gallbladder cancer was discovered during surgery . this nodule was less than 1 cm in size and was not shown on ct because of the extensive inflammation surrounding the gallbladder . diagnosis of xgc on ct can be difficult ; however , the presence of cholelithiasis , thickening of the gallbladder wall of > 3 mm , hypodense nodules within the gallbladder wall , and contrast enhancement of the mucosa with a distinctive presence of a hypodense band around the gallbladder are highly suggestive of xgc . kim et al . suggested that the hypodense nodules may represent abscesses of xanthogranulomas . in our patient following percutaneous drainage of the gallbladder empyema , a second ct scan showed that these hypodense areas remained unchanged , confirming that these areas corresponded to the yellow nodules seen macroscopically in the resected specimen ( fig . laparoscopic cholecystectomy is often associated with conversion to open surgery in 80% of cases . with the increased use of ct as a gold standard imaging modality in abdominal pain , more and more incidental pathologies are identified , some of which prompt urgent attention ( e.g. , aaa ) . the increase in the aging population has led to a rise in the prevalence of aaa coexisting with other nonvascular pathologies . it is estimated that 3.5% of patients have an aaa coexisting with intra - abdominal nonvascular disease . the management of concomitant pathology can present a real challenge to the multidisciplinary team , especially with large aneurysms . it is unclear whether performing simultaneous procedures or a staged approach results in a better outcome . a study by fry and fry concluded that the risk of simultaneous biliary and aortic procedures may subject the patient to a major risk of graft infection and death . they suggested that a staged operation can be performed safely if the time between the cholecystectomy and subsequent aortic reconstruction is less than 4 months . another report by thomas et al . supports the above findings . according to the above studies , the mortality rate due to performance of cholecystectomy in a single operation with aaa repair can reach to 9% . even when considering a staged procedure , the order of treatment of both pathologies poses a difficult surgical challenge .

there have been reports of the coexistence of abdominal aortic aneurysm ( aaa ) with intra - abdominal malignancy including gastric , colonic , pancreatic , and renal . we herein report a case of a previously undiagnosed aaa and a presenting complaint consistent with acute cholecystitis . following cholecystectomy , this was noted to be a rare form of chronic cholecystitis : xanthogranulomatous cholecystitis . there is a known possible association of this uncommon condition with gallbladder cancer . the management of concomitant pathologies can present a real challenge to the multidisciplinary team , especially with large aneurysms .

INTRODUCTION CASE REPORT DISCUSSION

neuro - cardiogenic interaction has been known for many years.1)2 ) an excessive release of catecholamines , known as a trigger of takotsubo cardiomyopathy ( tc ) , is developed when sympathetic nervous system is hyperactivated in stressful conditions including cerebral seizure . cerebral seizure has been reported as a cause of tc internationally , and cardiac complications are one of the main causes of mortality in epilepsy.3)4 ) biventricular tc is associated with more hemodynamic instability than isolated left ventricular tc . therefore , in that case , medical treatment should be more invasive and the course of hospitalization is longer.5 ) but , there has been no case report describing biventricular tc after cerebral seizure in korea . here , we report a case of a patient who presented with initial echocardiography showing biventricular apical ballooning after status epilepticus . an 83-year - old female presented to our emergency room with 2 episodes of 10 minute - duration of generalized tonic - clonic seizures at home and in the ambulance . blood pressure ( bp ) was 160/100 mm hg , pulse rate was 105/min , respiration rate was 22/min , and body temperature was 37.0. peripheral oxygen saturation was 96% with oxygen flow of 6 l / min via facial mask . she had an epilepsy 7 years prior to this attack after cerebral hemorrhage on left parietal and right temporal lobe . she was on donepezil 10 mg , methylphenidate 10 mg , choline alfoscerate 400 mg , acetyl l - carnitine 500 mg , trifrusal 300 mg because of her vascular dementia . she was independent in her activities of daily living and had never complained about chest pain or dyspnea before the admission . when she was admitted , there was another 5-minute duration of generalized tonic - clonic seizure with conjugate deviation of the eyes to the right . status epilepticus was terminated after intravenous lorazepam 4 mg injection and phenytoin 750 mg infusion . but bp was dropped from 160/100 mm hg to 70/40 mm hg and her consciousness was impaired . chest x - ray showed cardiomegaly and patchy increased opacities in right upper and lower lobes ( fig . 1 ) . arterial blood gas analysis showed a metabolic acidosis ( ph : 7.24 , pao2 : 70 mm hg , paco2 : 39 mm hg , hco3- : 16.7 mmol / l ) . a diffusion - weighted magnetic resonance brain imaging showed localized encephalomalacic lesions in left parietal lobe and right temporal pole region with peripheral old blood product deposition . division of cardiology was called for suspecting st elevation myocardial infarction ( stemi ) since an electrocardiogram ( ecg ) showed precordial v2 - 4 st segment elevation and she was found to have elevated cardiac enzymes on serial measurements ( troponin i levels 0.62 2 ) . there was no evidence of pericarditis or pheochromocytoma from her history , clinical symptoms , laboratory findings and echocardiographical features . an emergency transthoracic echocardiography ( tte ) showed an apical ballooning of the left ventricle ( lv ) with severe systolic dysfunction ( lv ejection fraction = 23% by simpsons methods ) and focal hypokinesia of right ventricular ( rv ) apex with decreased rv systolic function . fractional area change ( fac ) , [ ( rv end diastolic area - rv end systolic area ) / rv end diastolic area 100 ] was 22 % , tricuspid annular plane systolic excursion was 20 mm . despite tte finding was accordant with tc , emergency coronary angiography was done since we suspected stemi . there was no significant stenosis in left anterior descending artery , left circumflex artery and right coronary artery . however , hypokinesia of the mid to apical lv and rv from tte were discordant with coronary artery lesion . she had no further epileptic seizures during hospitalization with sodium valproate 300 mg every 12 hours and initial metabolic acidosis was resolved . after supportive care with standard heart failure therapy ( aspirin , - blocker , angiotensin converting enzyme inhibitor , 3-hydroxy-3-methylglutaryl - coenzyme a reductase inhibitor , diuretics and anti - convulsant ) , her clinical condition was getting better and moved from intensive care unit to a general ward . when patient was hospitalized for 10 days , mid to apical wall motion of lv was almost improved except for apico - anterior wall and apical rv wall motion abnormality was not observed any more ( fig . 5 ) . tc , also known as left apical ballooning syndrome or stress - induced cardiomyopathy , is named for ventricle which seems similar in appearance to a japanese octopus trap on ventriculography scan.6 ) tc is becoming well recognized as a cause of acute , reversible , and transient lv systolic dysfunction . in some cases , it would not be simple to make difference between acute coronary syndrome and tc since ecg abnormalities , elevated cardiac enzyme and symptoms mimicking stemi are common findings in tc . st - segment elevation is the most common ecg abnormality , reported in about 82% of patients , and t - wave inversion in 64%.7 ) generally in tc , there is no identifiable coronary culprit lesion explaining the wall - motion abnormality . but , as in our case , several cases of tc with coronary atherosclerotic lesion were reported.8 ) even though coronary atherosclerotic lesion was present , it was discordant with echocardiographic findings and affected regions were reversibly recovered without primary coronary intervention.9 ) in our case , there was a significant stenosis in ramus intermedius artery , however , wall motion abnormality was seen in mid to apical lv and apical rv in tte . those regional wall motion abnormalities extend beyond a single epicardial vascular distribution and discordant with coronary atherosclerotic lesion . as a result of this when seizure begins , it rises quickly within 30 min and decreases within few hours.10)11 ) a similar catecholamine release was observed in a case of tc associated with epilepsy.12 ) therefore , we can assume that seizure may trigger a condition inducing tc . in tc associated with epilepsy , complications were frequent and severe : heart failure , apical thrombus , cardiogenic shock , and left ventricular rupture were found in previous studies.13 ) although there is no hard data , it could be hypothesized that tc may be related to sudden unexpected death in epilepsy ( sudep ) . mechanisms of sudep are not clearly understood , it may include cardiac arrhythmia , myocardial ischemia , dysfunction of autonomic nervous system to the heart . cardiac abnormalities are found in up to 33% at autopsy of sudep.14 ) in our patient , apical rv hypokinesia was accompanied with lv involvement . isolated lv involvement is the most common variant , but rv involvement is becoming well recognized . it has been reported that rv involvement affects approximately 25% to 42% of patients with tc.15 ) daoko et al.5 ) said that most patients with biventricular tc are elderly women and presenting symptoms are similar to those of acute myocardial infarction . and it is known that rv involvement is associated with increasing hemodynamic instability and the risk of complications . in conclusion , tc should form a part of the differential diagnoses of cardiac event in patient with seizure . in high risk patients , such as elevated plasma troponin level and hemodynamic instability is accompanied , ecg and tte should be taken as soon as possible to detect tc and identify rv involvement . in a biventricular tc associated with epilepsy ,

we describe a case of takotsubo cardiomyopathy in an elderly woman after status epilepticus . in an emergency echocardiography , not only left ventricular apical ballooning but also right ventricular apical hypokinesia was observed . after a medical management , the patient 's condition was improved and a follow - up echocardiography showed substantial recovery of left and right ventricular apical ballooning .

Introduction Case Discussion

the main causes of snhl are advanced age , the use of ototoxic medications , noise exposure , hereditary , and autoimmune diseases . it has recently been reported that various other factors such as family history , smoking , alcohol consumption , head trauma , diseases such as diabetes , high blood pressure , and some renal pathologies also lead to changes in hearing [ 25 ] . polycystic ovary syndrome ( pcos ) is a common endocrine disease affecting at least 10% of females of reproductive age . it is described by hyperandrogenism , menstrual irregularity , anovulation , infertility , and obesity . in addition , pcos is often accompanied by hyperinsulinemia , insulin resistance , dyslipidemia , and low - grade chronic inflammation that predispose to endothelial damage . also , high - frequency hearing is often affected sooner in diseases that cause endothelial damage . high - frequency ( 40008000 hz ) hearing loss was first described in pcos patients by oghan and coksuer . kucur et al . also identified eh frequency ( 800014000 ) hearing loss in pcos patients . however , these studies did not include any information about the association between hearing thresholds and the known parameters of pcos such as ir , lh / fsh ratio , hirsutism score , and free testosterone index ( fti ) . the aim of our study was to investigate the association between pcos and hearing thresholds . in addition , the relationship among insulin resistance ( ir ) , lh / fsh ratio , hirsutism score , and fti with hearing thresholds were investigated . this prospective study enrolled 80 consecutive cases that applied to the endocrinology and gynecology polyclinics of our hospital that met the inclusion criteria . forty cases ( mean age , 24.336.38 years ; range 1646 years ) that were diagnosed with pcos composed the pcos group . diagnosis of pcos was according to rotterdam eshre / asrm - sponsored pcos consensus workshop group . forty age and body mass index ( bmi ) matched female volunteers ( mean age , 26.386.75 years ; range 1642 years ) composed the control group . the study design was confirmed by the local ethics committee ( approval number : 1126.12.2012 ) and informed consent was obtained from all of the women that participated in the study . for all participants , anamnesis was obtained , and otoscopic examination and pure - tone audiometric tests were performed . the exclusion criteria were : hyperprolactinemia , congenital adrenal hyperplasia , androgen - secreting tumors , thyroid disorders , cushing syndrome , vertigo , tinnitus , middle ear diseases , major neurological and psychiatric disease , diabetes mellitus , chronic kidney failure , chronic liver failure , hypertension or hyperlipidemia and similar chronic diseases , history of previous ear surgery , family history of hearing loss , history of acoustic trauma , conductive hearing loss , subject to ototoxic substances , autoimmune diseases , cancer , exposure to radiation in the head or neck , pregnancy , smoking , alcohol use , ongoing infection or inflammation , and history of using any medication . all patients had routine audiometric tests performed by the same expert audiometrist blinded to the study . the pure - tone audiometry ( pta ) test was performed in a soundproof environment using an interacoustic clinical audiometer ( model ac40 ; denmark ) . pta - low ( 250 , 500 , 1000 , and 2000 hz ) , pta - high ( 4000 , 6000 , and 8000 hz ) and pta - extended high ( 9000 , 10000 , 11200 , 12500 , 14000 , 16000 , 18000 , and 20000 hz ) frequencies were tested . hearing threshold levels were measured in decibels ( db ) . in each case , the threshold level was calculated for both ears and at every frequency . the threshold levels at 250 hz , 500 hz , 1000 hz , and 2000 hz were identified as low - frequency audiometry , the thresholds at 4000 hz , 6000 hz , and 8000 hz as high - frequency audiometry and the thresholds at 9000 hz , 10000 hz , 11200 hz , 12500 hz , 14000 hz , 16000 hz , 18000 hz , and 20000 hz as extended - high ( eh ) frequency audiometry and the mean value was calculated . the mean hearing thresholds of the pcos group and control group at pta - low , pta - high , and pta - eh frequencies were compared . the physical examination of each of the women was performed by the same endocrinology specialist . bmi was calculated by dividing the weight by the square of height ( kg / m ) . the degree of hirsutism was determined using the ferriman - gallwey scoring ( fgs ) system . blood samples were obtained during the follicular phase of the menstrual cycle after 12 hours fasting . the fasting plasma glucose levels ( fpg ) were evaluated using roche hitachi pp modular analyzer ( roche - hitachi , tokyo , japan ) by enzymatic colorimetric assay . follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , total testosterone ( tt ) , sex hormone - binding globulin ( shbg ) , and insulin measurements were done using the chemiluminescent immunoassay method with an immulite 2000 ( diagnostic products , los angeles , ca , usa ) analyzer . insulin resistance was measured using the homa - ir ( homeostasis model assessment for insulin resistance ) index . the free testosterone index ( fti ) was calculated according to the formula 100 serum testosterone ( nmol / l)/shbg ( nmol / l ) . the descriptive statistics of the variables studied were given as mean , standard deviation , and minimum and maximum values . the student t - test was used to compare data between the control group and pcos group . the statistical significance level was taken as 5% , and spss ( version 21 ) was utilized for all statistical calculations . this prospective study enrolled 80 consecutive cases that applied to the endocrinology and gynecology polyclinics of our hospital that met the inclusion criteria . forty cases ( mean age , 24.336.38 years ; range 1646 years ) that were diagnosed with pcos composed the pcos group . diagnosis of pcos was according to rotterdam eshre / asrm - sponsored pcos consensus workshop group . forty age and body mass index ( bmi ) matched female volunteers ( mean age , 26.386.75 years ; range 1642 years ) composed the control group . the study design was confirmed by the local ethics committee ( approval number : 1126.12.2012 ) and informed consent was obtained from all of the women that participated in the study . for all participants , anamnesis was obtained , and otoscopic examination and pure - tone audiometric tests were performed . the exclusion criteria were : hyperprolactinemia , congenital adrenal hyperplasia , androgen - secreting tumors , thyroid disorders , cushing syndrome , vertigo , tinnitus , middle ear diseases , major neurological and psychiatric disease , diabetes mellitus , chronic kidney failure , chronic liver failure , hypertension or hyperlipidemia and similar chronic diseases , history of previous ear surgery , family history of hearing loss , history of acoustic trauma , conductive hearing loss , subject to ototoxic substances , autoimmune diseases , cancer , exposure to radiation in the head or neck , pregnancy , smoking , alcohol use , ongoing infection or inflammation , and history of using any medication . all patients had routine audiometric tests performed by the same expert audiometrist blinded to the study . the pure - tone audiometry ( pta ) test was performed in a soundproof environment using an interacoustic clinical audiometer ( model ac40 ; denmark ) . pta - low ( 250 , 500 , 1000 , and 2000 hz ) , pta - high ( 4000 , 6000 , and 8000 hz ) and pta - extended high ( 9000 , 10000 , 11200 , 12500 , 14000 , 16000 , 18000 , and 20000 hz ) frequencies were tested . hearing threshold levels were measured in decibels ( db ) . in each case , the threshold level was calculated for both ears and at every frequency . the threshold levels at 250 hz , 500 hz , 1000 hz , and 2000 hz were identified as low - frequency audiometry , the thresholds at 4000 hz , 6000 hz , and 8000 hz as high - frequency audiometry and the thresholds at 9000 hz , 10000 hz , 11200 hz , 12500 hz , 14000 hz , 16000 hz , 18000 hz , and 20000 hz as extended - high ( eh ) frequency audiometry and the mean value was calculated . the mean hearing thresholds of the pcos group and control group at pta - low , pta - high , and pta - eh frequencies were compared . the physical examination of each of the women was performed by the same endocrinology specialist . bmi was calculated by dividing the weight by the square of height ( kg / m ) . the degree of hirsutism was determined using the ferriman - gallwey scoring ( fgs ) system . blood samples were obtained during the follicular phase of the menstrual cycle after 12 hours fasting . the fasting plasma glucose levels ( fpg ) were evaluated using roche hitachi pp modular analyzer ( roche - hitachi , tokyo , japan ) by enzymatic colorimetric assay . follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , total testosterone ( tt ) , sex hormone - binding globulin ( shbg ) , and insulin measurements were done using the chemiluminescent immunoassay method with an immulite 2000 ( diagnostic products , los angeles , ca , usa ) analyzer . insulin resistance was measured using the homa - ir ( homeostasis model assessment for insulin resistance ) index . the free testosterone index ( fti ) was calculated according to the formula 100 serum testosterone ( nmol / l)/shbg ( nmol / l ) . the descriptive statistics of the variables studied were given as mean , standard deviation , and minimum and maximum values . the student t - test was used to compare data between the control group and pcos group . the statistical significance level was taken as 5% , and spss ( version 21 ) was utilized for all statistical calculations . the demographic data and laboratory findings of the study groups are presented in table 1 . significantly higher fgs , fpg , insulin , homa - ir , tt , fti , and lh / fsh measurements were identified in the pcos group than in the control group ( p=0.001 , 0.009 , 0.013 , 0.005 , 0.001 , 0.001 , and 0.003 , respectively ) . the comparison of the low , high , and eh frequency hearing thresholds of the pcos and control groups is presented in table 2 . there was no statistically significant difference between the mean hearing thresholds of the groups at low or high frequencies . on the other hand , the mean hearing thresholds at eh frequencies were statistically significantly higher in the pcos group than in the control group ( p=0.001 for the right ear and p=0.015 for the left ear ) . the correlation analysis of the pcos group showed a positive correlation between the eh frequency hearing threshold levels and the fti values ( r=0.380 , p=0.016 for the right ear and r=0.360 , p=0.022 for the left ear ; figure 1a , 1b ) . also , a significant positive correlation was identified between the hearing thresholds at eh frequencies and the fgs value ( r=0.344 , p=0.030 for the right ear and r=0.432 , p=0.005 for the left ear ; figure 2a , 2b ) . but there was no significant correlation between the eh frequency and bmi , fpg , insulin , homa - ir , lh / fsh ( p>0.005 ) in the pcos group . comparison of the pcos and control groups in terms of the average hearing thresholds at each frequency in pta - eh measurements are shown in table 3 . hearing thresholds at each frequency in pta - eh were statistically significantly higher in the pcos group than in the control group except for left ear 20000 hz . there are various previous studies reporting that pcos and hearing loss are associated at high ( 40008000 hz ) and eh ( 800014000 hz ) frequencies . these studies do not provide any information about the presence of a relationship between hearing loss and known parameters of pcos such as ir , elevated lh / fsh ratio , hirsutism score , and elevated fti . to our knowledge , our study is the first study to investigate the association of hearing thresholds with parameters such as ir , lh / fsh ratio , hirsutism score , and fti in pcos cases . in histological examination , hwang et al . detected the presence of microvascular structures with leaner walls in the stria vascularis of the basal end of the cochlea in obese rats . they also reported that markers such as tumor necrosis factor alpha , hypoxia - induced factor 1 , caspase 3 , poly ( adp - ribose ) polymerase-1 , nuclear factor kappa b , and apoptosis inducing factor are present in the basal end of the cochlea at high concentrations . various methods have been used to measure hearing thresholds . in our study , the hearing threshold levels at eh frequencies ( 900020000 hz ) were statistically significantly higher in the pcos group when compared to the control group . however , at low ( 2502000 hz ) and high ( 40008000 hz ) frequencies , there was no significant difference between the hearing thresholds of the pcos group and the control group . our findings support other studies that also reported findings suggesting that eh frequencies which represent the basal end of the cochlea are often influenced sooner in disorders that cause endothelial and vascular damage . in one other study , it was reported that chronic inflammation in pcos could induce of hearing loss , particularly at eh frequencies . as exposure to the multifactorial agents of pcos continues over time , it was determined that hyperandrogenism was associated with elevated blood pressure , low hdl cholesterol , dysglycemia , insulin resistance , and abdominal obesity [ 2227 ] . endothelial damage and increased risk for cardiovascular disease in pcos patients have also been reported . some of the biochemical and hormonal changes that occur in pcos have been identified as responsible for atherosclerosis and endothelial dysfunction . in light of all this information , it can be suggested that the hyperandrogenism in pcos impairs microcirculation especially in the basal end of the cochlea through microangiopathy and leads to an elevation in hearing thresholds at eh frequencies . in our study , we detected the presence of a positive correlation between the fti and fgs values and hearing thresholds at eh frequencies in the pcos group . however , there was no significant correlation between bmi , fpg levels , insulin levels , ir , or lh / fsh ratio and hearing thresholds at eh frequencies . these results support the view that the elevation of testosterone levels ( the fundamental cause of hirsutism in pcos ) may play a significant role in the pathophysiology of elevated hearing thresholds . first , it is a cross - sectional study and therefore is incapable of assessing the changes that may occur in hearing levels over time ; new longitudinal studies are required to investigate the causal role of pcos in hearing loss . new studies conducted with larger case populations are needed to elucidate the influence of pcos and various risk factors on auditory functions . at pta - eh frequencies , we detected significantly higher hearing thresholds in pcos patients than in controls . we also determined that elevated fti and fpg levels were positively correlated with elevated hearing thresholds . these findings support the position that one of the most important factors playing a role in the elevation of hearing thresholds in pcos is hyperandrogenism , which is a fundamental component of pcos . a positive correlation was identified between elevated hearing thresholds and hirsutism scores ( fgss ) , which can easily be calculated during physical examination . we believe that pcos - related hyperandrogenism plays an important role in sensorineural hearing loss and that this effect can be avoided by managing identified risk factors . also , early hearing screening may help diagnose hl early , especially in pcos cases with significant hyperandrogenism and hirsutism .

backgroundthe purpose of our study was to investigate the association between polycystic ovary syndrome ( pcos ) and hearing thresholds.material/methodsforty women diagnosed with pcos ( mean age , 24.336.38 years ) and 40 healthy women controls ( mean age , 26.386.75 years ) were included in prospective study . each case was tested with low ( 250 , 500 , 1000 , and 2000 hz ) , high ( 4000 , 6000 , and 8000 hz ) and extended high ( eh ) ( 900020000 hz ) frequency audiometry . the fasting plasma glucose , insulin , fsh , lh , total testosterone , and sex hormone - binding globulin were measured in all patients.resultsthe mean hearing thresholds at eh frequencies were statistically significantly higher in the pcos group than in the control group ( p=0.001 right ear and p=0.015 left ear ) . there were significant positive correlations among free testosterone index ( fti ) values and hirsutism scores with eh frequency hearing thresholds.conclusionsat pure - tone audiometry ( pta ) eh frequencies , we detected significantly higher hearing thresholds in pcos patients than in controls . we also determined that elevated fti and hirsutism score were positively correlated with elevated hearing thresholds in eh frequencies . these findings support that hyperandrogenism can play a role in the elevation of hearing thresholds in pcos .

Background Material and Methods Subjects Exclusion criteria Audiometric evaluation and mean hearing levels Morphometry Laboratory analyses Statistical analysis Results Discussion Conclusions

retinal artery macroaneurysms ( rams ) are rare , acquired dilatations of the retinal arterial vasculature , usually within the first three branches of the arteriolar tree.1 the incidence is estimated to be approximately 1 in 4,500 people over the age of 40.2 hypertension and older age lead to hyaline degeneration of the vascular walls , loss of autoregulatory tone and elastic recoil , and arterial dilatation.3 approximately 8%25% of rams will undergo spontaneous involution . even with submacular hemorrhage , up to 37% of patients will have better than 20/40 visual acuity without treatment.4 however one third of patients will develop vascular leakage and retinal edema and may need treatment.5 an 84-year - old female with a past medical history of hypertension and dyslipidemia was referred for progressively decreasing visual acuity in the left eye . she had no history of diabetes . on initial examination , dilated fundus examination and fluorescein angiography of the left eye revealed a superotemporal retinal arterial macroaneurysm , with surrounding circinate exudates involving the fovea ( figures 1 and 2 ) . the patient was observed initially , but the edema increased and her vision deteriorated to 20/60 . the patient was observed for 3 months , but the vision worsened again to 20/60 , with increased edema and exudates on optical coherence tomography . she was observed for 3 months , but due to recurrent leakage , she ultimately received a total of six injections of intravitreal bevacizumab every 1 month to 3 months . argon laser at a setting of 400 mw and 200 m spot size was applied around the ram . the ram occluded , and on follow - up examination 6 months later , the patient s vision has remained at 20/30 . the first report of laser photocoagulation to treat exudative retinal artery macroaneurysm was published in 1976 by dr donald gass and colleagues.6 in a study of 27 patients with symptomatic ram , half of the patients underwent argon laser therapy and the remaining half was observed . the vision in both groups improved , but the laser group experienced a greater gain.7 approximately 16%27% of rams occlude with lasers , but there is a risk of vascular occlusion , early increase in exudates from selective reabsorption of fluid , arteriovenous shunts , macular pucker , and scotomas.7,8 other treatment options for rams include yellow dye laser and indocyanine green dye - enhanced photocoagulation.9,10 anti - vascular endothelial growth factor ( vegf ) therapy has recently been reported in the treatment of patients with exudative or hemorrhagic rams . chanana and azad11 published the first case report in 2009 , and subsequent case reports have shown encouraging results ( table 1).1219 cho et al18 described 23 patients with rams who were either observed or received intravitreal bevacizumab . both groups experienced statistically significant improvements in visual acuity and central macular thickness , but the bevacizumab group regained vision faster . a larger , prospective study of 38 eyes with hemorrhagic and exudative rams underwent three monthly injections of bevacizumab , with the vision and retinal thickness improving in both groups.19 the role of vegf and the mechanism of action of anti - vegf therapy in rams are not fully understood . in a study that compared the vegf levels in 500 l vitreous samples from patients with vitreous hemorrhage from nondiabetic etiologies ( including four patients with ram ) with those from patients with proliferative diabetic retinopathy , the vegf levels were significantly lower in the former than in the latter groups ( 2.75 pg / ml vs 821 pg / ml , respectively).20 however , these were small vitreous samples obtained up to 3 weeks after the initial hemorrhage and may not have been representative of the vitreous and microenvironment around the ram . the role of vegf in intracranial aneurysms and other biological systems has been more clearly elucidated . in intracranial aneurysms , vegf levels were found to be significantly higher than the levels in controls.20 in particular , patients with intracranial aneurysms had higher expressions of vegf receptor 2 ( vegfr2 ) and lower levels of vegf receptor 1 ( vegfr1 ) . vegfr1 is associated with angiogenesis , while vegfr2 is associated with thrombosis.21 bevacizumab has been shown to decrease vegfr1 levels and to increase vegfr2 levels to normal levels in cancer cells.22 in the circulatory system , platelets produce vegf , which stimulates nitrous oxide production , resulting in vasodilation.21 inhibition of vegf may decrease leakage of fluid and exudates through the endothelium . in mice studies , bevacizumab decreases bleeding time and increases coagulation.23 thus , vegf inhibition may block angiogenesis , decrease binding of the prothrombotic vegfr2 , and decrease vascular permeability . in conclusion , the majority of rams can be observed , but intervention may be necessary in cases of persistent or recurrent macular edema from exudative or hemorrhagic rams . our patient s ram was initially observed , but continued leakage prompted further therapy . anti - vegf injections have shown promise in improving visual acuity and hastening resolution of macular edema in ram;5 however , most of the previously published cases had relatively short follow - up . our case has one of the longest follow - ups with the most number of anti - vegf treatments , but the patient continued to have leakage into the macula . the potential benefits of anti - vegf treatment must be carefully weighed against the potential risk of infection from serial injections . laser photocoagulation remains a viable treatment option in cases of macular edema secondary to ram .

an 84-year - old female with a history of hypertension and dyslipidemia was referred for a retinal artery macroaneurysm with exudation that had extended into the macula . she underwent a total of six injections of bevacizumab , with some improvement in visual acuity and retinal thickness . due to persistent macular edema , focal laser photocoagulation was performed around the macroaneurysm . the vision remained at 20/30 during 20 months of follow up . although anti - vascular endothelial growth factor therapy may improve vision and decrease retinal thickness in retinal artery macroaneurysm , recalcitrant cases may be treated with laser photocoagulation to seal the leaking vessel .

Introduction Case report Discussion

the absorption of iron is strictly controlled through hepcidin , while there are no efficient physiologic mechanisms to excrete iron from the body . under conditions where iron metabolism is disturbed , such as in transfusion - dependent patients with thalassemia major , myelodysplastic syndromes , aplastic anemia , or sickle cell disease , excessive iron deposition occurs in the liver , heart , and endocrine organs , resulting in reactive oxygen species ( ros ) through the fenton and haber weiss reaction . ros can attack components of cells and further induce organ dysfunction , such as liver fibrosis , cirrhosis , hepatic carcinoma , myocarditis , pericarditis , and other diseases . in the liver of chronic hepatitis c ( chc ) patients , iron deposition has been found in both hepatocytes and reticuloendothelial cells although the mechanism is not fully clarified . like other viruses , hepatitis c virus ( hcv ) needs constituents of host cells to proliferate , and iron is one of the most important constituents . many studies have explored the connection between iron overload and hcv life cycle with differing results . some studies have found that iron promotes hcv replication , while others have shown that iron suppresses hcv replication . most of the studies suggest the positive role of iron on hcv translation . to summarize the interactions between hcv and iron , and understand the mechanisms of iron overload in chc , we searched articles published in the pubmed databases up to january 28 , 2017 , to gain an in - depth understanding and knowledge of relevant areas . in the human body , iron is critical for maintaining the fundamental function of many proteins . iron in the diet is absorbed through divalent metal transporter 1 ( dmt1 ) , a multi - transmembrane protein , or heme carrier protein 1 on duodenal and jejunal enterocytes . it is then exported by the ferroportin ( fpn ) to bind to transferrin in the bloodstream , and taken to the erythroblasts for erythropoiesis . iron that is not utilized can also be stored as ferritin or hemosiderin in enterocytes , macrophages , and hepatocytes . there are no efficient physiologic mechanisms to excrete iron , a little iron ( about 2 mg per day ) is lost by sloughing of intestinal epithelial cells , desquamation of skin and urinary cells , blood loss , and sweat . iron homeostasis in the human body is mainly regulated by the hepcidin / fpn and iron - regulatory protein / iron - responsive element ( irp / ire ) systems . when iron levels increase , hepcidin negatively regulates iron levels by binding to the fpn and promoting the internalization and degradation of fpn . this reduces the amount of iron absorbed by enterocytes , released by hepatocytes , and recycled from macrophages , and finally reduces transferrin levels in the bloodstream . in contrast , when iron levels decrease , hepcidin is downregulated to improve transferrin levels . transferrin receptor 1 ( tfr1 ) and bone morphogenetic protein 6 receptor ( bmp-6r ) can sense transferrin saturation ( ts ) and tissue iron content , respectively , further regulating hepcidin concentration . hepcidin can also be affected by other factors , including inflammation , erythropoietic activity of the bone marrow , and the oxygen tension within hepatocytes . while hepcidin regulates systemic iron metabolism transcriptionally and posttranslationally , cellular iron metabolism is regulated by the irp / ire system posttranscriptionally . the irp / ire system alters the expression of iron - metabolism - related proteins , such as ferritin , fpn , hypoxia - inducible factor 2 ( hif2/epas1 ) , dmt1 , tfr1 , and other proteins . many studies have found iron deposition in the liver , and in both hepatocytes and reticuloendothelial cells . were the first to find elevated serum ferritin and iron levels in patients with chronic hepatitis ( both chronic hepatitis b and chc ) . subsequently , in 1999 , a study of 209 chc patients found liver iron accumulation detected by liver biopsy in 42.1% of patients , the majority of which was mild liver iron accumulation ( 35.4% of the 209 chc patients ) . those with liver iron accumulation had significantly higher levels of serum iron ( si ) , ferritin , and ts ; liver iron accumulation was also found to have a significant relationship with the severity of histological activity based on metavir classification and cirrhosis . in a study of 100 consecutive patients with hcv infection who underwent liver biopsy , 19 patients were found to be hepatic iron stain positive , which is associated with stage iii or iv fibrosis . fifty - five patients had at least one abnormal value of si , ferritin , or ts . in multivariate analysis , the only independent predictive factor of severe hepatic fibrosis was serum ferritin . however , elevated serum ferritin can also be caused by hepatic inflammation . in 1994 , a study of 123 chronic hepatitis patients ( including 63 chc patients ) found increased si , iron saturation , and ferritin in chc patients , while no evidence of hepatic iron accumulation could be found in any of these patients . in this study , serum ferritin was elevated in the absence of liver iron overload , which indicates that elevated serum ferritin may also be caused by inflammation itself . thus , elevated ferritin reflects hepatic iron accumulation , as well as hepatic inflammation , and also predicts severe hepatic fibrosis . in other words , hepatic iron accumulation can lead to elevated ferritin , while elevated ferritin is not only induced by hepatic iron accumulation but also caused by hepatic inflammation . thus , serum ferritin levels only serve as a reference to evaluate liver iron status in chc patients , and liver biopsy is the gold standard for diagnosis of iron overload . so far , plenty of studies have been devoted to exploring the mechanism as to how hcv leads to iron overload . it is generally accepted that hcv alters iron metabolism by reducing the level of hepcidin . fujita et al . measured hepcidin messenger rna ( mrna ) levels in liver samples from 56 patients with hcv infection , and revealed that the expression of hepcidin is strongly correlated with serum ferritin and the degree of iron deposition in liver tissues . the hepcidin - to - ferritin ratio was significantly lower in hcv(+ ) patients than hbv(+ ) patients or controls . girelli et al . detected s - hepcidin ( the 25-amino acid bioactive peptide in serum ) levels in 81 untreated chc patients and 57 controls with a rigorous definition of normal iron status and found that s - hepcidin was significantly lower in chc patients than in controls . in chc patients , s - hepcidin levels examined the determinants of serum hepcidin and liver hepcidin mrna levels and their association with histological lesions in 96 patients with chc and 30 controls ; they concluded that serum hepcidin was significantly lower in patients with chc compared to healthy controls , and that liver hepcidin mrna levels did not differ between patients and controls . a study using fl - n/35 transgenic mice harboring the hcv polyprotein also found decreased hepcidin expression in the liver , accompanied by an increase in fpn expression in the duodenum , spleen , and liver . this suggests that hcv proteins may directly lead to increased duodenal iron absorption , macrophage iron release , and hepatic iron accumulation . elevated duodenal fpn levels and a significant relationship between hcv and hepcidin in chc patients were also found by other studies . all of these studies suggest that hepcidin may play a pivotal role in the pathogenesis of iron overload in patients with chc . kohjima et al . analyzed iron - metabolism - related gene expression profiles in 100 patients with chc ( genotype 1b , n = 50 ; genotype 2 , n = 50 ) and 18 living donors of liver transplantation . they found that expression of genes related to iron absorption ( transferrin , and dmt1 ) , iron export ( fpn ) , cellular iron metabolism ( irp1 and irp2 ) , and hepcidin - regulation ( bmpr1 , bmpr2 , and hemojuvelin ) were significantly higher , indicating that hcv affects the expression of iron - metabolism - related genes , leading to iron accumulation in hepatocytes . besides , patients with a sustained virological response ( svr ) had significantly lower transcription and protein expression levels of hepcidin , fpn , bmpr2 , and hemojuvelin before therapy , indicating the importance of hepatocytic iron retention for viral response during pegylated interferon plus ribavirin treatment . almost all hcv proteins , including core , e1 , e2 , ns3 , ns4a , ns4b , and ns5a , have been shown to regulate hepcidin expression through signal transducer and activator of transcription 3 , mitogen - activated protein kinase , or bmp / smad signaling pathways , and increased histone deacetylase activity , which further clarifies the mechanism of decreased levels of hepcidin caused by hcv . like other viruses , hcv needs constituents of host cells to replicate and translate , and iron is one of the most important constituents . it is indispensable for several basic metabolic processes in viruses , as well as in mammalian cells , as described in many studies . were the first to analyze the effect of iron on hcv replication in vitro . in 2000 , they cultured a nonneoplastic hcv - infected human hepatocyte line ( ph5ch8 ) treated with feso4 and proved that iron can promote hcv replication in liver cells . in 2016 , foka et al . found that iron - overloaded macrophages was infected with hcv when co - cultured with hcv - infected human hepatoma cell line ( huh7.5 ) . these iron overloaded macrophages also enhanced hcv replication in co - cultured hcv - infected huh7.5 cells through reversed ferritin flow from macrophages to huh7.5 cells . macrophages without overloaded iron was also infected with hcv and enhance hcv replication in huh7.5 cells , but the rate of infection was slower and the effect on hcv replication was weaker . on the other hand , several studies have found that iron suppresses hcv replication . a study using huh7 cells in 2005 concluded that iron could inhibit hcv replication by inactivating the rna polymerase ns5b , without significant effect on translation . fillebeen et al . concluded that iron could decrease hcv replication by reducing the activity of ns5b in huh7.5.1 cells , similar to the result of the study in 2005 . a later study , in 2011 , again found that increased iron status and down - regulated hepcidin inhibited hcv replication . . this may be due to different cell types . besides , in clinical practice , chc with primary or secondary iron overload have different prognoses . chc patients with hereditary iron overload due to hemochromatosis gene mutations have increased svrs to antiviral therapy , when excess iron deposits in parenchymal cells and macrophages become iron deficient . in contrast , iron overload secondary to chc or other chronic liver diseases significantly aggravates the disease , when excess iron deposits in macrophages . it seems that the iron content of macrophages is associated with the clinical outcome of disease , because iron overload may impair the immune function of macrophages through oxidative stress . although the role of iron in promoting or suppressing hcv replication is debatable , there are few controversies about the effect of iron on hcv translation . apart from a group in the usa that reported that iron suppresses hcv translation through increasing heme oxygenase-1 in vitro and another group that found no effect of iron on hcv translation , all other studies confirm the positive role of iron on hcv translation , but the mechanisms are not fully understood . expression of hcv is predominantly controlled at the translational level , which involves the interaction between cellular translation initiation factors and a specific mrna stem - loop structure the internal ribosome entry site ( ires ) within the 5 untranslated region . iron is reported to stimulate the expression of eukaryotic initiation factor 3 ( eif3 ) , thus enhancing hcv ires - dependent translation both in hepg2 cells and hcv - infected patients , whereas iron chelation reverses it in vitro . another group also found that hepatic iron load promotes hcv translation initiation in vitro with the mechanism involving iron - dependent increased expression of eif3 . expression of another element , la protein , is also significantly increased by hepatic iron load which can promote hcv translation . expression of both eif3 and la protein can be partially inhibited by the iron chelator deferoxamine . cho et al . also found that iron changes the affinities of common cellular factors to hcv ires which modulates hcv ires - dependent translation . in summary , iron can regulate hcv ires - dependent translation by increasing expression levels of associated factors . translation of both hcv 1b and hcv 2b is significantly increased after iron treatment , while translation of hcv 6a shows little difference . compared with subtype 1b , hcv 6a has a modified eif3 binding site within hairpin iii . it is reported that hepatic iron levels are higher in patients infected with subtype 1b than those in patients infected with subtypes 2a or 2b , which may explain the different responses of hcv genotypes to iron overload . nowadays , liver biopsy is the gold standard for diagnosis of hepatic iron overload , and there are no uniform diagnostic criteria about extracellular iron concentration . most of the studies described in the paper used cell models incubated in ferric or ferrous iron solutions with maximum concentrations ranging from 50 to 300 mol / l , whether they reach the standard of iron overload is difficult to judge . therefore , whether these results from in vitro research could be suggestive to understand the effect of iron on hcv is still debatable . each part of iron metabolism , including absorption , restoration , recycling , and utilization , is regulated by hepcidin and the ire / irp system . there is no efficient way to eliminate iron from the body , so any abnormality in the process of iron metabolism may lead to excessive iron burden . the relationship between hcv and iron overload many studies have observed iron overload in patients with chc , and most suggest that decreased hepcidin expression , as well as increased hcv protein - mediated expression of other iron metabolism - related genes , play a role . as to the detailed mechanism of hcv proteins inducing iron overload , there is still much to discover . in turn , as an essential element for all living bodies , iron can also affect the life cycle of hcv . nearly all studies so far confirm the positive role of iron on hcv translation , while the role of iron in promoting or suppressing hcv replication is still unclear . more work is needed to understand this , and the mechanisms by which iron affects hcv life cycle . iron chelation therapy can reverse the negative effect of iron overload , which offers important information for clinical application . hcv reduces the levels of hepcidin , and elevates the levels of fpn , resulting in elevated iron absorbed by enterocytes and iron deposition in hepatocytes and macrophages . although the role of iron in hcv replication is disputable , it is strongly suggested the positive effect of iron on hcv translation by increasing expression of both eif3 and la protein , and changing the affinities of common cellular factors to hcv ires . hcv : hepatitis c virus ; fpn : ferroportin ; si : serum iron ; ts : transferrin saturation ; ires : internal ribosome entry site ; eif3 : eukaryotic initiation factor 3 ; : upregulated ; : downregulated . this work was supported by grants from the 215 project of beijing municipal commission of health and family planning ( no . 303 - 01 - 005 - 0068 ) and the basic - clinic cooperative foundation of capital medical university ( no . this work was supported by grants from the 215 project of beijing municipal commission of health and family planning ( no . 303 - 01 - 005 - 0068 ) and the basic - clinic cooperative foundation of capital medical university ( no .

objective : the aim of this study was to summarize the interactions between hepatitis c virus ( hcv ) infection and iron overload , and to understand the mechanisms of iron overload in chronic hepatitis c ( chc ) and the role iron plays in hcv life cycle.data sources : this review was based on data in articles published in the pubmed databases up to january 28 , 2017 , with the keywords hepatitis c virus , iron overload , iron metabolism , hepcidin , translation , and replication.study selection : articles related to iron metabolism , iron overload in patients with chc , or the effects of iron on hcv life cycle were selected for the review.results:iron overload is common in patients with chc . the mechanisms involve decreased hepcidin levels caused by hcv through signal transducer and activator of transcription 3 , mitogen - activated protein kinase , or bone morphogenetic protein / smad signaling pathways , and the altered expression of other iron - metabolism - related genes . some studies found that iron increases hcv replication , while other studies found the opposite result . most of the studies suggest the positive role of iron on hcv translation , the mechanisms of which involve increased expression levels of factors associated with hcv internal ribosome entry site - dependent translation , such as eukaryotic initiation factor 3 and la protein.conclusion:the growing literature demonstrates that chc leads to iron overload , and iron affects the hcv life cycle in turn . further research should be conducted to clarify the mechanism involved in the complicated interaction between iron and hcv .

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colorectal cancer ( crc ) is a major cause of mortality and morbidity , and the third most common malignancy in the world ( 1 ) . the incidence of this malignancy shows considerable variation among racially or ethnically defined populations in multiracial / ethnic countries . crc is the third most common type of cancer in men and the second most common cancer in women worldwide ( 2 ) . kashmir has been reported as being a high - incidence area of gastrointestinal ( git ) cancers ( 3,4 ) . in the kashmir valley , crc represents the third most common type of git cancer , following esophageal and gastric cancer ( 5,6 ) . the tumour - suppressor gene p16ink4a codes for a cyclin - dependent kinase inhibitor p16ink4a , which acts as a negative regulator of cell growth and proliferation in the g1 phase of the cell cycle ( 7,8 ) . functionally , p16ink4a has been identified as an inhibitor of cyclin - dependent kinase 4 and 6 . as a consequence , p16ink4a plays a significant role in the proliferation process of many types of tumour cell growth and apoptosis , and correlates closely with the expression of the proliferating cell nuclear antigen ki67 , which is usually viewed as the proliferation marker in intestinal tumours ( 9,10 ) . most human colorectal carcinomas show genetic alterations in the p16ink4a - cyclin d - prb pathway ( 11 ) . the p16ink4a gene of the cyclin d / prb pathway has been found to be inactivated in human malignancies with a frequency second only to p53 ( 12,13 ) . mutation , homozygous deletions and hypermethylation of the promoter are major mechanisms of p16ink4a inactivation ( 8,14,15 ) . hypermethylation of non - mutated promoter regions is one of the common mechanisms for inactivating tumour - suppressor genes , which leads to stable allele - specific loss of transcription function ( 16 ) . such methylations tend to occur at the sites of cpg dinucleotides , which are clustered as so - called cpg islands and are frequently found in promoters of p16ink4a and other tumour - suppressor genes ( 13 ) . in fact , the major mechanism of p16ink4a gene inactivation has been found to be promoter methylation ( 11 ) . a number of studies have been carried out on p16ink4a in different populations , implicating the role of hypermethylation in the development of cancers ( 1723 ) . p16ink4a gene promoter methylation has been observed in colorectal dysplasia , adenomas , malignant tumours and normal mucosa adjacent to tumours ( 18,19,24 ) . two significant investigations were previously carried out in the kashmir valley in order to establish the role of p16ink4a mutations and promoter hypermethylation in gastric and esophageal squamous cell carcinoma , respectively ( 13,25 ) . based on the hypothesis that crc carcinogenesis is a multi - step and multi - gene event , we designed this study to elucidate the role of p16ink4a promoter hypermethylation in the development and progression of crc in the kashmiri population and to correlate it with the clinicopathological parameters of crc cases . this study included 114 crc cases recruited from the department of surgery , sher - i - kashmir institute of medical sciences ( skims ) , srinagar , india . tumour and adjacent normal tissue samples from the cases were resected in the general surgery department ( skims ) and were collected for this study . data on all crc cases were obtained from personal interviews with patients and/or guardians and medical records . all patients and/or guardians were informed of the study and their will to participate in this study was noted in a pre - designed questionnaire ( available on request ) . samples were snap - frozen immediately after collection and stored at 70c until further analysis . dna was then isolated from both tissue and blood samples using the ammonium acetate method succeeding proteinase - k digestion . the tissue for dna extraction from the tumour sample was selected by an experienced pathologist and was ascertained to comprise > 90% tumour cells . both normal and tumour dnas were subjected to sodium bisulphite modification using the ez dna methylation kit ( zymo research , usa ) . previously reported primer sets were used for the amplification of the p16 promoter ( 11,26 ) . the unmethylated primer pair p16-u1f / u1r produced an amplicon of 151 bp , while p16-u1f / u2r produced an amplicon 234 bp in length . / m1r produced an amplicon of 150 bp , while p16-m1f / m2r produced an amplicon of 234 bp in length . the presence of unmethylation and/or methylation of the p16 promoter was ascertained by the presence of both amplicons . pcr for both unmethylation as well as methylation detection was performed in a 50-l total volume reaction mixture containing 10 ng of modified genomic dna , 100 m of each dntp , 100 ng of each of the three primers ( u1f , u1r and u2r in the case of unmethylation detection ; m1f , m1r and m2r in the case of methylation detection ) , 1.5 mm mgcl2 , 5% dimethyl sulphoxide ( dmso ) , 10x taq buffer and 2 units taq dna polymerase ( fermentas , md , usa ) . the conditions of pcr were as follows : initial denaturation at 95c for 7 min , 40 cycles of denaturation at 95c for 30 sec , annealing at designated temperatures ( c ) ( see table i ) for 45 sec and extension at 72c for 45 sec , and final extension at 72c for 10 min in a bio - rad icycler . the pcr amplicons were electrophoresed on 2.5% agarose gels and visualised after staining with ethidium bromide . universal methylated human dna ( zymo research ) was used as a positive control for methylated alleles whereas dna from normal lymphocytes was used as a control for unmethylated alleles . all statistical analyses were performed using pasw software , version 18 ( ibm , ny , usa ) . pearson s chi - square two - proportion test was used to evaluate the hypothesis of equal distribution of molecular alterations with different clinicopathological variables . a fisher s two - tailed test ( p - values ) of 0.05 was considered statistically significant . this study included 114 crc cases recruited from the department of surgery , sher - i - kashmir institute of medical sciences ( skims ) , srinagar , india . tumour and adjacent normal tissue samples from the cases were resected in the general surgery department ( skims ) and were collected for this study . data on all crc cases were obtained from personal interviews with patients and/or guardians and medical records . all patients and/or guardians were informed of the study and their will to participate in this study was noted in a pre - designed questionnaire ( available on request ) . samples were snap - frozen immediately after collection and stored at 70c until further analysis . dna was then isolated from both tissue and blood samples using the ammonium acetate method succeeding proteinase - k digestion . the tissue for dna extraction from the tumour sample was selected by an experienced pathologist and was ascertained to comprise > 90% tumour cells . both normal and tumour dnas were subjected to sodium bisulphite modification using the ez dna methylation kit ( zymo research , usa ) . previously reported primer sets were used for the amplification of the p16 promoter ( 11,26 ) . the unmethylated primer pair p16-u1f / u1r produced an amplicon of 151 bp , while p16-u1f / u2r produced an amplicon 234 bp in length . / m1r produced an amplicon of 150 bp , while p16-m1f / m2r produced an amplicon of 234 bp in length . the presence of unmethylation and/or methylation of the p16 promoter was ascertained by the presence of both amplicons . pcr for both unmethylation as well as methylation detection was performed in a 50-l total volume reaction mixture containing 10 ng of modified genomic dna , 100 m of each dntp , 100 ng of each of the three primers ( u1f , u1r and u2r in the case of unmethylation detection ; m1f , m1r and m2r in the case of methylation detection ) , 1.5 mm mgcl2 , 5% dimethyl sulphoxide ( dmso ) , 10x taq buffer and 2 units taq dna polymerase ( fermentas , md , usa ) . the conditions of pcr were as follows : initial denaturation at 95c for 7 min , 40 cycles of denaturation at 95c for 30 sec , annealing at designated temperatures ( c ) ( see table i ) for 45 sec and extension at 72c for 45 sec , and final extension at 72c for 10 min in a bio - rad icycler . the pcr amplicons were electrophoresed on 2.5% agarose gels and visualised after staining with ethidium bromide . universal methylated human dna ( zymo research ) was used as a positive control for methylated alleles whereas dna from normal lymphocytes was used as a control for unmethylated alleles . all statistical analyses were performed using pasw software , version 18 ( ibm , ny , usa ) . pearson s chi - square two - proportion test was used to evaluate the hypothesis of equal distribution of molecular alterations with different clinicopathological variables . a fisher s two - tailed test ( p - values ) of 0.05 was considered statistically significant . the patients comprised 67 males and 47 females ( male / female ratio of 1.42 ) . the demographic and clinical characteristics of the crc cases are shown in table ii . out of 114 confirmed cases of crc , 109 cases were sporadic , 4 were familial adenomatous polyposis ( fap ) and 1 was lynch syndrome . all but 1 case were adenocarcinoma and only 1 was squamous cell carcinoma ( scc ) of basal cell type ; 79 came from a rural and 35 from an urban environment ; 49 cases had carcinoma in the colon and 65 in the rectum ; 74 were smokers and 40 non - smokers ; and 89 cases had well - differentiated adenocarcinoma . ms - pcr analysis revealed a high methylation status of the p16ink4a promoter in crc cases . forty - eight ( 42.1% ) of the tumours were found to be hypermethylated at the promoter region of p16ink4a , while 8 ( 7% ) of the tumours were partially hypermethylated and the remaining 58 ( 50.9% ) of the tumours were not methylated at all ( table ii ) . among the 48 patients with tumours which were methylated for the p16ink4a gene promoter , 26 were female , 36 had a higher tumour stage ( c+d ) , 38 were smokers and 31 were of a well - differentiated ( wd ) grade ( table ii ) . statistical analysis between the p16ink4a hypermethylation status and clinicopathological parameters of the crc cases revealed a significant association ( p<0.05 ) with gender ( females ) , lymph node status ( present ) , tumour stage ( c+d ) , smoking status ( smokers ) and tumour grade ( wd ) of the crc patients ( table ii ) . this is the first study to report on the association of p16 gene promoter hypermethylation with the risk of development of crc in the kashmiri population . the kashmir valley , located in the northern part of india and walled by the himalayas has a unique ethnic population living in discrete temperate environmental conditions and having unique eating habits , which , along with genetic factors , play a large role in the development of git cancers ( 46,13 ) . as previously reported , the etiology and incidence of various git cancers in this population has been attributed to a probable exposure to nitroso compounds , amines and nitrates reported to be present in local foodstuffs , such as hoakhe suen ( sun - dried vegetables ) , pharei and hoggade ( sundried and/or smoked fish and meat ) , hakh ( a leafy vegetable of the brassica family ) , hot noon chai ( salted tea ) , dried and pickled vegetables and red chilli , and also through smoking hukka ( a water pipe ) ( 3,4,27 ) . cancer cell lines have in general demonstrated an increased frequency of hypermethylation in comparison to primary tumours ( 28 ) . the inactivation of tumour - suppressor genes by promoter hypermethylation has been recognised to be as common as gene disruption by mutation in tumourigenesis ( 2931 ) . a number of studies on crc around the globe have demonstrated the role of promoter hypermethylation of a number of different genes in the development and progression of crc ( 3234 ) . promoter hypermethylation of p16ink4a , similarly to that of other genes , plays a pivotal role in the inactivation of p16ink4a , which in turn enhances tumour development ( 11,18,23 ) . in the present study , we observed hypermethylation of the p16ink4a gene promoter in 42.1% ( 48/114 ) of crc cases , which is higher than reported by other major studies in the world ( 11,20,21 ) . however , our observation is consistent with other studies ( 22,23 ) which have reported a similar frequency of p16ink4a hypermethylation in crc tumours in the japanese population . this may be due to the fact that this population is exposed to a special set of environmental challenges , including extreme temperature , high altitude and special food habits , as well as exposure to agricultural by - products , such as pesticides and nitrosamines ( 6,13 ) . liang et al , urosevic et al and krtolica et al have already proposed that geographical differences or other unknown factors supplementary to p16 methylation may increase tumour aggressiveness ( 3537 ) . we found a significant association of the p16ink4a methylation status with higher dukes stage ( c+d ) . these results are in concordance with those of other studies ( 20,21,23 ) , which have reported the same observations of a higher methylation status of p16ink4a gene promoter with higher dukes stage ( c+d ) . duke s staging has been considered a most significant prognostic determinant in cancers ( 20 ) . dukes staging , proposed by dr cuthbert e. duke in 1932 , is a clinical classification for crc based on the tumour size , local extent and metastatic status , i.e. , lymph node involvement . hence , the present study supports the observation of yi et al ( 20 ) in suggesting that p16ink4a methylation may be involved in the malignant transformation of crc . furthermore , a significant association was found between p16ink4a methylation status and lymph node metastasis in our population , as was also reported by goto et al in their study ( 23 ) . we also found that females were more likely than males to have p16ink4a methylated crc tumours in our population ; this supports the study of wiencke et al ( 38 ) . however , contrary to this observation , we found the methylation status of the p16ink4a gene to be associated with a well - differentiated tumour grade . jie et al previously demonstrated the association of p16ink4a methylation status with the proliferative activity of crc ( 39 ) . they reported the loss of proliferation and invasion in crc in tumours with re - expression of p16ink4a protein . thus , epigenetic silencing of p16ink4a may have a role in driving crc tumours to higher differentiation and consequently to invasiveness . another unique observation in our study was the association of the methylation status with smoking status . tumours of the crc patients who had a history of smoking ( cigarettes and/or hookah ) were found to have methylated p16ink4a gene promoter ( p<0.05 ) . in conclusion , in the kashmir valley population , the high level of epigenetic silencing of p16ink4a plays a pivotal role in the initial tumourigenesis and also enhances the chances of tumour development and progression to advanced stages .

hypermethylation of the promoter region of the p16ink4a ( p16 ) gene plays a significant role in the development and progression of colorectal cancer ( crc ) . the aim of the present study was to establish the role of the methylation status of the p16 gene in 114 crc cases and to correlate it with the various clinicopathological parameters . analysis of p16 promoter methylation was performed by methylation - specific pcr . forty - eight ( 42.1% ) of the crc cases were found to be methylated for the p16 gene in our population . the methylation status was found to be associated with the gender , lymph node status , tumour stage , smoking status and tumour grade of the crc patients . p16 plays a pivotal role in tumour development and progression to advanced stages .

Introduction Materials and methods Colorectal cancer cases and controls DNA extraction Methylation-specific polymerase chain reaction (MS-PCR) of the p16INK4a promoter Statistical analysis Results Discussion

anabolic androgenic steroids ( aass ) represent a large group of synthetic derivatives of testosterone , produced to maximize anabolic effects and minimize the androgenic ones [ 1 , 2 ] . the most common oral and injectable aass with their chemical structures are reported in table 1 . androgens act by binding to the nuclear androgen receptor ( ar ) in the cytoplasm and then translocate into the nucleus . this binding results in sequential conformational changes of the receptor affecting the interaction between receptor and protein , and receptor and dna . the basic structure of all steroids is a perhydro - cyclopentano phenanthrene ring system that can be modified in order to obtain several designed chemical modifications . the most important chemical modification , in which the basic structure can undergo , is reported in fig . 1 . anabolism is defined by kuhn as any state in which nitrogen is differentially retained in lean body mass , either through stimulation of protein synthesis and/or decreased breakdown of protein anywhere in the body . skeletal muscle can be considered as the main target tissue for the anabolic effects of aas , which are mediated by ars which after exposure to aass are up - regulated and their number increases with body building . therefore , aass determine an increase in muscle size as a consequence of a dose - dependent hypertrophy resulting in an increase of the cross - sectional areas of both type i and type ii muscle fibers and myonucleardomains . moreover , it has been reported that aass can increase tolerance to exercise by making the muscles more capable to overload therefore shielding them from muscle fiber damage and improving the level of protein synthesis during recovery . despite some therapeutic use of aass ( severe burns , primary or secondary hypogonadism , short stature , hiv wasting syndrome etc . ) there is also wide abuse among athletes especially bodybuilders in order to improve their performances and to increase muscle growth and lean body mass , taking into account the significant anabolic effects above reported . not by chance , these substances fall within the vast group of performance - enhancing drugs , which also include : stimulants , painkillers , sedatives and anxiolytics , diuretics , blood boosters and masking drugs . a high - dose regimen is stacked by combining numerous oral and injectable aass , which are self - administered in drug cycles which last from 4 to 12 weeks [ 7 - 9 ] . furthermore , aas users frequently associate other substances to aass , the so called steroid - accessory drugs , such as ephedrine , growth hormone , insulin , diuretics , ghb etc [ 10 - 15 ] for several reasons . the prolonged misuse and abuse of aass can determine several adverse effects , some of which may be even fatal especially on the cardiovascular system because they may increase the risk of sudden cardiac death ( scd ) , myocardial infarction , altered serum lipoproteins , and cardiac hypertrophy . 2 . the aim of this review is to focus on deaths related to aas abuse , trying to evaluate the autoptic , histopathological and toxicological findings in order to investigate the pathophysiological mechanism that underlines this type of death , which is still obscure in several aspects . some databases , from 1975 to june 2014 , were searched : medline , cochrane central , scopus , web of science , science direct , embase and google scholar , using the following keywords : anabolic androgenic steroid , death , cardiovascular effects , toxicity , side / adverse effects . the main key word anabolic androgenic steroid was individually searched in association to each of the others . the 189 sources found after the initial screening in order to exclude duplicate sources and retrospective studies , were selected according to the inclusion criteria , which allowed the identification of 10 sources . a comprehensive flow diagram with inclusion criteria the review of the literature using the flow diagram reported in fig . 3 allowed us to identify 19 fatal cases between 1990 and 2012 . the most important autoptic , histopathological findings and circumstantial data are reported in table 2 , while the toxicological findings are reported in table 3 . of the 19 cases , 17 ( 89.5% ) were males whereas only 2 ( 10.5% ) were females . the age ranged from 18 to 37 years ( mean age : 28 4.4 ) . among the 19 fatal cases , in 14 bodies ( 12 males and 2 females ) the data available allowed to calculate the body mass index ( bmi ) and the results are reported in table 4 . in none of the cases the bmi was lower than 24.9 , which is considered the upper limit of the normal healthy weight . for all cases the autopsy excluded extracardiac causes of death , only in one case a bilateral pulmonary embolism from deep venous thrombus of lower extremities was found ( table 2 ) . toxicological investigation performed mainly on urine samples but also in blood and hair samples , by using several screening tests and analytical methods revealed in 12 cases [ 16 , 18 , 20 - 25 ] the presence of aass and/or their metabolites in urine specimens ; in one case nandrolone was detected in blood , whereas in another case stanozolol was found in hair . in the remaining 6 cases in which the toxicological analysis was negative , circumstantial data and evidences reported by relatives and friends of the deceased highlighted a previous prolonged use of aass . the chronic use of aass can cause various pathological alterations , which are related to dose , frequency and patterns of use . taking into account that numerous organs and apparatus are the target of aass , several adverse effects can involve the liver , cardiovascular , reproductive , musculoskeletal , endocrine , renal , immunologic and hematological systems as well as some psychological effects ; a schematic representation is reported in fig . 19 fatal cases are reported ; although only single case report or small series of cases were included , whereas retrospective studies and other papers that did not fulfill the inclusion criteria were not taken into account , some consideration can be formulated ; in all cases the autopsy findings together with the histological examination have highlighted cardiac causes of death . only in one case a mechanical cardiovascular cause of death was found ( a bilateral pulmonary embolism from deep venous thrombus of lower extremities ) . in numerous cases [ 16 , 18 , 19 , 22 , 25 ] , a common finding was a left ventricular hypertrophy , frequently associated with fibrosis and myocytolisis . a myocardial hypertrophy was not found in the 4 cases reported by fineschi et al in two different reports [ 21 , 23 ] . what is the significance that could be attributed to the myocardial hypertrophy ? a vigorous training in young athletes can determine a left ventricularhypertrophy , independently of the use of aass ( the so called athlete 's heart ) [ 25 - 28 ] . melchert and welder categorized the effects of aas on the cardiovascular system into four groups of activities : vasospastic , atherogenic , thrombotic and direct myocardial injuries . aas can induce adverse cardiovascular effects such as left ventricular hypertrophy ( lvh ) , hypertension , impaired diastolic filling , arrhythmia , erythrocytosis , thrombosis and altered lipoprotein profiles . abnormalities in cardio - vascular reflex control of the cardiovascular system [ 31 - 35 ] and in vascular reactivity [ 36 - 40 ] have also been reported . studies on isolated hearts from rats treated chronically with nandrolonedecanoate ( nd ) have also shown a raise in myocardial susceptibility to ischemia / reperfusion injuries [ 41 , 42 ] . nandrolone abuse combined with vigorous exercise training may lead to impaired diastolic function and concentric hypertrophy of the left ventricular ( lv ) wall . vigorous weight lifting itself would also cause lv wall mass and thickness increase but cardiac function would not be affected . however , when combined with aas abuse pathological cardiac hypertrophy could be caused . in another study , rats were treated with nd for 6 weeks ( total dose 30 mg /kg ) . rocha et al . studied the effects on cardiac function in rats undergoing swimming training and those not undergoing it . they investigated that swimming training combined with high doses of nandrolone ( 5 mg / kg per injection , equal to 10 mg / kg per week ) sharpens cardiac hypertrophy with interstitial fibrosis . without a doubt . an explanation might be the change of the sympathetic autonomic activity modulated by the renin - angiotensin - system ( ras ) . experiments have shown that ras plays a significant role in the development of lvh and myocardial fibrosis . angiotensin ii type 1 receptor s ( at1r s ) stimulation is associated with the regulation of cell growth and proliferation of vascular smooth muscle cells , cardiomyocytes and endothelial cells involved in endothelial dysfunction , atherosclerotic vascular phenomena , congestive heart failure and myocardial infarction . marques neto et al . pointed out that the treatment with supraphysiological , chronic doses of aass induce cardiac parasympathetic disturbances in ventricular depolarization in both exercised and sedentary rats . unambiguously , it has been shown that the blockage of the ras , and in particular of at1r by losartan , obstructs qt prolongation . down - regulation of ion channel subunits , kchip2 , kv1.4 and kv4.3 , could explain the autonomic dysfunction and cardiac repolarization disturbances caused by chronic treatment with supraphysiological doses of nd . moreover , prolonged qt intervals and ventricular action potential could be explained by the reduced density of the transient outward potassium . no augmentation in tissue collagen content or in the mrna expression of types i and iii collagens have been shown by histological analysis the previously mentioned unconformity could be attributed to the duration of treatment with nd and the age of rats used . participation of the potassium ( k ) current in the generation of prolonged qt and potential action duration has been noticed . ito is the transient outward k+ current which is one of the main repolarizing currents in the mammalian myocardium and is generally believed to flow through kv1.4 , kv4.2 and kv4.3 channels in rats [ 49 , 50 ] . in heart hypertrophic cases ito is down - regulated [ 51 - 53 ] . low ito density , kv1.4 and kv4.3 down - regulation in the left ventricle and prolonged action was noticed in the group treated with nandrolone compared to the control group . homogenous distribution of kv4.3 channel in the rat s ventricular wall , higher kv4.2 in the epicardial and lower in the endocardial ventricular wall have been observed [ 49 , 50 ] . these differences may partially explain the up - regulation of kv4.2 and prolonged qtc interval and action potential . the expression of kchip2 is considerably decreased in heart failure and hypertrophy [ 54 , 55 ] . kchip2 was found to be significant for ito expression in the human heart and the correlation between kchip2 absence and a total loss of ito together with an increased susceptibility to ventricular arrhythmias in mice has been shown . according to riezzo the following effects were produced in physically trained mice intramuscularly treated with nd : moderate increase of heart weight , morphologically extensive cardiac hypertrophy and a wide colliquative myocytolysis which together could result in a significant heart failure . medei et al . found approximately 25% less nuclei and higher cardiomyocyte nuclei diameter in the ventricles of the group treated with nd . lower nuclei suggests a toxic effect of nd which may involve a pro - apoptotic mechanism . tanno et al found that nd treatment whether combined with resistance training or not induced pathological concentric hypertrophy , re - expression of fetal genes , systolic and diastolic function impairment and an incremented myocardial collagen content leading to lvh . increased relative left ventricle wall thickness ( rwt ) was observed as a consequence of intensive physical training in rats treated with nd compared to the respective non - trained ones . in addition , the non - trained nandrolone treated group also produced higher rwt compared to the non - trained treated group . increased interventricular septum thickness in the end - diastole ( ivsdia ) was noticed in both the non - trained nandrolone treated and trained vehicle - treated groups , compared to the non - trained vehicle treated rats . a considerable lower ratio of maximum early to late transmitral flow velocity ( e / a ratio ) was observed in the trained groups , in comparison with non - trained groups . nandrolone - treated groups ( both trained and non - trained ) showed lower e / a ratio in comparison with the respective vehicle - treated groups . moreover , significant decrease in the expression of alpha - myosin heavy chain ( -mhc ) mrna and beta - myosin heavy chain ( -mhc ) mrna in the left ventricle was induced by nandrolone and resistance training respectively . . found that short - term nd treatment induces an overexpression of 2-adrenoceptors without cardiac hypertrophy . increment in cardiovascular mortality has been associated to an imbalance of ( ans ) activity . aass can acutely inhibit the reuptake of catecholamines into extraneuronal tissue and consequently the increment of catecholamine concentrations at receptor sites occurs . although , the neuronal catecholamine transporter is normally responsible for the reuptake of noradrenaline , it has also been proved responsible for nonexocytotic release of noradrenaline from sympathetic nerve terminals during ischemia . an increased release of noradrenaline has been implicated in ischemia - induced arrhythmia [ 63 , 64 ] . an increased activity of 6-phosphogluconate dehydrogenases and glucose-6-phosphate was observed in rat hearts , also nd activated isocitrate dehydrogenase and malic enzyme , which are other nadp - linked dehydrogenases . during the same study a significantly increased heart weight it was shown that treatment with nd causes small qrs complex extension that might slightly reduce the spreading rate of the action potential through the heart ventricles , possibly because of the greater heart mass . it is known that administration of doses higher than normal ( supraphysiological ) of nd impair exercise - induced cardioprotection in treadmill - exercised rats . chaves et al . were the first to say that enhanced levels of antioxidant enzyme levels produced after exercise are impaired with nd treatment ( 10mg / kg for 8 weeks ) , a fact which is well correlated to the cardiac injurious effects of the drug . it was observed that the hearts of animals treated with nandrolone and having undergone training ( dt group ) showed lower glutathione peroxidase ( gpx ) , superoxide dismutase ( sod ) and glutathione reductase ( gr ) activities compared with controls and trained groups of animals ( ct ) . the latter observation indicates that nandrolone could act through blocking or down regulating the mechanisms implicated in the improvement of antioxidant defenses in dt animals , which might explain the lower percentage of left ventricular developed pressure and augmented infarct size in dt group . it has been shown , in other researches on rats that exercise training improves myocardial resistance to reperfusion injury / ischemia [ 67 - 69 ] since physiological cardiac hypertrophy amends the sensitiveness of that heart making it more resistant to the previously mentioned disorders in vivo rat hearts . notwithstanding the strenuous research efforts , the molecular mechanism(s ) involved in exercise - induced cardio protection is still debatable . the numerous studies above reported in animal models , especially in rats , have called into question several pathophysiological mechanisms , which may explain some of the macroscopic and microscopic finding regarding the 19 cases here reported ; however , we have to underline that these cases are single case report or small series of cases and not experimental studies . moreover , the users of these substances frequently associate numerous steroids , in different forms , singularly and in several temporal combinations and cycles , and commonly , various steroid - accessory drugs are also used . therefore , the interpretation of the postmortem findings is particularly difficult and no comprehensive conclusions can be done . finally , a brief remark must be placed regarding the bmi which was in all cases ( 12 males and 2 females ) higher than 25 . although according to the bmi ranges from an overweight ( 9 cases ) to a very severely obese ( 2 cases ) were found , however , the bmi is not a direct measure of body fatness and bmi is calculated from an individual 's weight which includes both muscle and fat . as a result , some subjects such as highly trained athletes may have a high bmi because of increased muscularity rather than increased body fatness ; therefore , these results should be evaluated with caution . the relationship between aas abuse , vigorous exercise training , and cardiac death can be evaluated only by the application of an investigative protocol , which must include a rigorous methodology covering : a complete autopsy with a special regard to aas target organs and apparatus ( the cardiovascular system in primis ) . a broad toxicological investigation , preceded by a careful evaluation of clinico - anamnestic data , in order to confirm an aas consumption ( including type of aass , concentration and interval of exposure ) and the possible detection of other substances which could have contributed to the fatal outcome . for this purpose , different matrices can be used ; urine is the most common , because it provides a prolonged detection time window , but also several other matrices such as : blood , serum , plasma , hair , oral fluid and nails can be used . the comparison of the cases reported here , allows us to support the hypothesis that the combined effects of strong workout , the prolonged / chronic or previous abuse of aass in different forms and combinations , have predisposed these subjects to develop different patterns of myocardial injuries and consequent sudden cardiac death [ 21 , 23 ] . therefore , the authors would strengthen the warning already expressed in previous reports [ 21 , 23 ] against the use / abuse of these substances among professional and non - professional athletes . moreover , only through a careful examination of all suspicious cases of aas related deaths with the application of a rigorous investigative protocol , these cases can be identified and they could provide further information and data that may increase the knowledge of this type of deaths .

anabolic androgenic steroids ( aass ) represent a large group of synthetic derivatives of testosterone , produced to maximize anabolic effects and minimize the androgenic ones . aas can be administered orally , parenterally by intramuscular injection and transdermally . androgens act by binding to the nuclear androgen receptor ( ar ) in the cytoplasm and then translocate into the nucleus . this binding results in sequential conformational changes of the receptor affecting the interaction between receptor and protein , and receptor and dna.skeletal muscle can be considered as the main target tissue for the anabolic effects of aas , which are mediated by ars which after exposure to aass are up - regulated and their number increases with body building . therefore , aass determine an increase in muscle size as a consequence of a dose - dependent hypertrophy resulting in an increase of the cross - sectional areas of both type i and type ii muscle fibers and myonuclear domains . moreover , it has been reported that aass can increase tolerance to exercise by making the muscles more capable to overload therefore shielding them from muscle fiber damage and improving the level of protein synthesis during recovery.despite some therapeutic use of aass , there is also wide abuse among athletes especially bodybuilders in order to improve their performances and to increase muscle growth and lean body mass , taking into account the significant anabolic effects of these drugs.the prolonged misuse and abuse of aass can determine several adverse effects , some of which may be even fatal especially on the cardiovascular system because they may increase the risk of sudden cardiac death ( scd ) , myocardial infarction , altered serum lipoproteins , and cardiac hypertrophy . the aim of this review is to focus on deaths related to aas abuse , trying to evaluate the autoptic , histopathological and toxicological findings in order to investigate the pathophysiological mechanism that underlines this type of death , which is still obscure in several aspects . the review of the literature allowed us to identify 19 fatal cases between 1990 and 2012 , in which the autopsy excluded in all cases , extracardiac causes of death .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS CONFLICT OF INTEREST

in general relativity , kinetic theory has been used relatively sparsely to model phenomenological matter in comparison to fluid models , although interest has increased in recent years . from a mathematical point of view there are fundamental advantages to using a kinetic description . in non - curved spacetimes kinetic theory has been studied intensively as a mathematical subject during several decades , and it has also played an important role from an engineering point of view . the main purpose of this review paper is to discuss mathematical results for the einstein - vlasov system . however , in the first part of this introduction , we review kinetic theory in non - curved spacetimes and focus on the special - relativistic case , although some results in the non - relativistic case will also be mentioned . the reason that we focus on the relativistic case is not only that it is more closely related to the main theme in this review , but also that the literature on relativistic kinetic theory is very sparse in comparison to the non - relativistic case , in particular concerning the relativistic and non - relativistic boltzmann equation . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to good comprehension of kinetic theory in general relativity . moreover , it is often the case that mathematical methods used to treat the einstein - vlasov system are carried over from methods developed in the special relativistic or non - relativistic case . the purpose of kinetic theory is to model the time evolution of a collection of particles . for instance , the particles are atoms and molecules in a neutral gas or electrons and ions in a plasma . in astrophysics the particles are stars , galaxies or even clusters of galaxies . a characteristic feature of kinetic theory is that its models are statistical and the particle systems are described by density functions f = f(t , x , p ) , which represent the density of particles with given spacetime position ( t , x ) and momentum p. a density function contains a wealth of information , and macroscopic quantities are easily calculated from this function . in a fluid model the quantities that describe the system do not depend on the momentum p but only on the spacetime point ( t , x ) . a choice of model is usually made with regard to the physical properties of interest for the system or with regard to numerical considerations . it should be mentioned that a too naive fluid model may give rise to shell - crossing singularities , which are unphysical . in a kinetic description the time evolution of the system is determined by the interactions between the particles , which depend on the physical situation . for instance , the driving mechanism for the time evolution of a neutral gas is the collision between particles ( the boltzmann equation ) . for a plasma the interaction is through the electromagnetic field produced by the charges ( the vlasov - maxwell system ) , and in astrophysics the interaction is gravitational ( the vlasov - poisson system and the einstein - vlasov system ) . of course , combinations of interaction processes are also considered but in many situations one of them is strongly dominating and the weaker processes are neglected . consider a collection of neutral particles in minkowski spacetime . let the signature of the metric be ( , + , + , + ) . in this section we assume that all the particles have rest mass m = 1 , and we normalize the speed of light c to one . we point out that in section 2 on the einstein - vlasov system , the dependence on the rest mass and the speed of light will be included in the formulation of the system . the four - momentum of a particle is denoted by p , a = 0 , 1 , 2 , 3 . since all particles have equal rest mass , the four - momentum for each particle is restricted to the mass shell , ppa = m = 1 . thus , by denoting the three - momentum by p , p may be written p = ( p , p ) , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p^0 } = \sqrt { 1 + \vert p{\vert ^2}}$\end{document } is the energy of a particle with three - momentum p , and p is the usual euclidean length of p. the relativistic velocity of a particle with momentum p is denoted by p and is given by 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat p = { p \over { \sqrt { 1 + \vert p\vert ^{2}}}}.$$\end{document } note that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vert \hat p\vert < 1 = c$\end{document}. the relativistic boltzmann equation models the spacetime behavior of the one - particle distribution function f = f(t , x , p ) , and it has the form 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({{\partial _ t } + { p \over { { p^0 } } } \cdot { \nabla _ x } } \right)f = q(f , f),$$\end{document } where the relativistic collision operator q(f , g ) is defined by 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , g ) = \int\nolimits_{{{\mathbb r}^2 } } { \int\nolimits_{{{\mathbb s}^2 } } { k(p , q,\omega)[f(p + a(p , q,\omega)\omega)g(q - a(p , q,\omega)\omega ) - f(p)g(q)]d\omega \;dp.}}$$\end{document } note that g = f in equation ( 2 ) . here d is the element of surface area on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{{\mathbb s}}}^2}$\end{document } and k(p , q , ) is the scattering kernel , which depends on the differential cross - section in the interaction process . we refer to , and for examples of differential cross - sections in the relativistic case . if two particles , with momentum p and q respectively , collide elastically with scattering angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega \in { { \rm{{\mathbb s}}}^2}$\end{document } , their momenta will change , i.e. , p p and q q. the relation between p , q and p , q is given by 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^{\prime } = p + a(p , q,\omega)\omega , \quad q^{\prime } = q - a(p , q,\omega)\omega,$$\end{document } where 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(p , q,\omega ) = { { 2({p^0 } + { q^0}){p^0}{q^0}(\omega \cdot ( \hat q - \hat p ) ) } \over { { { ( { p^0 } + { q^0})}^2 } - { { ( \omega \cdot ( p + q))}^2}}}.$$\end{document } this relation is a consequence of four - momentum conservation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a } + { q^a } = { p^{a^{\prime } } } + { q^{a{\prime}}},$$\end{document } or equivalently 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^0 } + { q^0 } = { p^{0{\prime } } } + { q^{0{\prime}}},$$\end{document } 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + q = { p^{\prime } } + { q^{\prime}}.$$\end{document } these are the conservation equations for relativistic particle dynamics . in the classical case the corresponding conservation equations read 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert p\vert ^{2 } + \vert q\vert ^{2 } = \vert p^{\prime}\vert ^{2 } + \vert q^{\prime}\vert ^{2},$$\end{document } 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + q = { p^{\prime } } + { q^{\prime}}.$$\end{document } the function ( p , q , ) gives the distance between p and p ( q and q ) in momentum space , and the analogue function in the non - relativistic , newtonian , classical case has the form 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_{{\rm{cl}}}}(p , q,\omega ) = \omega \cdot ( q - p).$$\end{document } by inserting ac1 in place of a in equation ( 3 ) we obtain the classical boltzmann collision operator ( disregarding the scattering kernel , which is also different ) . we point out that there are other representations of the collision operator ( 3 ) , cf . . in and classical solutions to the relativistic boltzmann equations are studied as c , and it is proven that the limit as c of these solutions satisfies the classical boltzmann equation . the former work is more general since general initial data is considered , whereas the latter is concerned with data near vacuum . the latter result is stronger in the sense that the limit , as c , is shown to be uniform in time . the main result concerning the existence of solutions to the classical boltzmann equation is a theorem by diperna and lions that proves existence , but not uniqueness , of renormalized solutions . an analogous result holds in the relativistic case , as was shown by dudyski and ekiel - jeewska , cf . also . regarding classical solutions , illner and shinbrot have shown global existence of solutions to the non - relativistic boltzmann equation for initial data close to vacuum . glassey showed global existence for data near vacuum in the relativistic case in a technical work . he only requires decay and integrability conditions on the differential cross - section , although these are not fully satisfactory from a physics point of view . by imposing more restrictive cut - off assumptions on the differential cross - section , strain gives a different proof , which is more related to the proof in the non - relativistic case than is . for the homogeneous relativistic boltzmann equation , global existence for small initial data has been shown in under the assumption of a bounded differential cross - section . for initial data close to equilibrium , global existence of classical solutions has been proven by glassey and strauss using assumptions on the differential cross - section , which fall into the regime hard potentials , whereas strain has shown existence in the case of soft potentials . in the non - relativistic case , we refer to [ 189 , 172 , 119 ] for analogous results . the collision operator q(f , g ) may be written in an obvious way as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , g ) = { q^ + } ( f , g ) - { q^ -}(f , g),$$\end{document } where q and q are called the gain and loss term , respectively . if the loss term is deleted the gain - term - only boltzmann equation is obtained . it is interesting to note that the methods of proof for the small data results mentioned above concentrate on gain - term - only equations , and once that is solved it is easy to include the loss term . in it is shown that the gain - term - only classical and relativistic boltzmann equations blow up for initial data not restricted to a small neighborhood of trivial data . thus , if a global existence proof of classical solutions for unrestricted data will be given , it will necessarily use the full collision operator . the gain term has a nice regularizing property in the momentum variable . in it is proven that given f l( ) and g l( ) with f , g 0 , then 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert \vert { q^ + } ( f , g)\vert \vert _ { { { h^1}({\mathbb r}_p^3 ) } } \leq c\vert \vert f\vert \vert _ { { { l^2}({\mathbb r}_p^3)}}\vert \vert g\vert \vert _ { { { l^1}({\mathbb r}_p^3)}},$$\end{document } under some technical requirements on the scattering kernel . here h is the usual sobolev space . the proof relies on the theory of fourier integral operators and on the method of stationary phase , and requires a careful analysis of the collision geometry , which is very different in the relativistic case . simplified proofs in the classical and relativistic case are given in [ 193 , 194 ] . more precisely , lions used the regularizing theorem to prove that solutions to the classical boltzmann equation , with periodic boundary conditions , converge in l to a global maxwellian , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = { e^{- \alpha \vert p\vert ^{2 } + \beta \cdot p + \gamma}}\quad { \rm{with}}\;\alpha , \gamma \in r,\quad \alpha > 0,\quad \beta \in { { \mathbb r}^3},$$\end{document } as time goes to infinity . it should be pointed out that the convergence takes place through a sequence of times tending to infinity and it is not known whether the limit is unique or depends on the sequence . in the relativistic situation , the analogous question of convergence to a relativistic maxwellian , or a jttner equilibrium solution , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { e^{- \alpha \sqrt { 1 + \vert p\vert ^{2 } } + \beta \cdot p + \gamma}},\quad \alpha , \beta , \;{\rm{and}}\;\gamma \;{\rm{as}}\;{\rm{above}},\;{\rm{with}}\;\alpha > \vert \beta \vert,$$\end{document } was studied by glassey and strauss [ 87 , 88 ] . in the periodic case , they proved convergence in a variety of function spaces for initial data close to a jttner solution . having obtained the regularizing theorem for the relativistic gain term , it is a straightforward task to follow the method of lions and prove convergence to a global jttner solution for arbitrary initial data ( satisfying the natural bounds of finite energy and entropy ) , which are periodic in the space variables , cf . . we also mention that in the non - relativistic case desvillettes and villani have studied the convergence rate to equilibrium in detail . a similar study in the relativistic case has not yet been achieved . for more information on the relativistic boltzmann equation on minkowski space we refer to [ 54 , 68 , 181 , 79 ] and in the non - relativistic case we refer to [ 190 , 79 , 53 ] . let us consider a collision - less plasma , which is a collection of particles for which collisions are relatively rare and the interaction is through their charges . for simplicity we assume that the plasma consists of one type of particle , although the results below hold for plasmas with several particle species . the particle rest mass and the particle charge are normalized to one . in the kinetic framework , the most general set of equations for modeling a collision - less plasma is the relativistic vlasov - maxwell system : 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + \hat v \cdot { \nabla _ x}f + ( e(t , x ) + \hat v \times b(t , x ) ) \cdot { \nabla _ v}f = 0$$\end{document } 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ \quad \nabla \cdot e = \rho,$$\end{document } 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}b = - c\nabla \times e,\quad \quad \nabla \cdot b = 0.$$\end{document } the notation follows the one already introduced with the exception that the momenta are now denoted by v instead of p. this has become a standard notation in this field . e and b are the electric and magnetic fields , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \upsilon}$\end{document } is the relativistic velocity , 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat v = { v \over { \sqrt { 1 + \vert v\vert ^{2}/{c^2}}}},$$\end{document } where is the speed of light . the charge density and current j are given by 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho = \int\nolimits_{{{\mathbb r}^3 } } { fdv,\quad j = } \int\nolimits_{{{\mathbb r}^3 } } { \hat vfdv.}$$\end{document } equation ( 12 ) is the relativistic vlasov equation and equations ( 13 , 14 ) are the maxwell equations . a special case in three dimensions is obtained by considering spherically - symmetric initial data . for such data it can be shown that the solution will also be spherically symmetric , and that the magnetic field has to be constant . the maxwell equation e = tb then implies that the electric field is the gradient of a potential . hence , in the spherically - symmetric case the relativistic vlasov - maxwell system takes the form 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + \hat v \cdot { \nabla _ x}f + \beta e(t , x ) \cdot { \nabla _ v}f = 0,$$\end{document } 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = \nabla \phi , \quad \nabla \phi = \rho.$$\end{document } here = 1 , and the constant magnetic field has been set to zero , since a constant field has no significance in this discussion . this system makes sense for any initial data , without symmetry constraints , and is called the relativistic vlasov - poisson system . another special case of interest is the classical limit , obtained by letting c in equations ( 12 , 13 , 14 ) , yielding : 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + v \cdot { \nabla _ x}f + \beta e(t , x ) \cdot { \nabla _ v}f = 0,$$\end{document } 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = \nabla \phi , \quad \nabla \phi = \rho,$$\end{document } where = 1 . this is the vlasov - poisson system , and = 1 corresponds to repulsive forces ( the plasma case ) . taking = 1 means attractive forces and the vlasov - poisson system is then a model for a newtonian self - gravitating system . one of the fundamental problems in kinetic theory is to find out whether or not spontaneous shock formations will develop in a collision - less gas , i.e. , whether solutions to any of the equations above will remain smooth for all time , given smooth initial data . if the initial data are small this problem has an affirmative solution in all cases considered above [ 81 , 86 , 32 , 33 ] . for initial data unrestricted in size the picture is more involved . in order to obtain smooth solutions globally in time , the main issue is to control the support of the momenta 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) : = \sup \{\vert v\vert:\exists ( s , x ) \in [ 0,t ] \times { { \mathbb r}^3}\;{\rm{such}}\;{\rm{that}}\;f(s , x , v ) \neq 0\},$$\end{document } i.e. , to bound q(t ) by a continuous function so that q(t ) will not blow up in finite time . that such a control is sufficient for obtaining global existence of smooth solutions follows from well - known results in the different cases , cf . [ 85 , 104 , 39 , 96 , 34 , 81 ] . for the full three - dimensional relativistic vlasov - maxwell system , the problem of establishing whether or not solutions will remain smooth for all time is open . a different sufficient criterion for global existence in this case is given by pallard in , and he also shows a new bound for the electromagnetic field in terms of q(t ) in . in two space and three momentum dimensions , glassey and schaeffer [ 82 , 83 ] have shown that q(t ) can be controlled for the relativistic vlasov - maxwell system , which thus yields global existence of smooth solutions in that case . the relativistic and non - relativistic vlasov - poisson equations are very similar in form . however , the mathematical results concerning the two systems are very different . in the non - relativistic case , batt gave an affirmative solution in 1977 in the case of spherically - symmetric data . pfaffelmoser was the first one to give a proof for general smooth data . a simplified version of the proof is given by schaeffer in . pfaffelmoser obtained the bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c{(1 + t)^{(51 + \delta)/11}},$$\end{document } where > 0 can be taken as arbitrarily small . the sharpest bound valid for = 1 and = 1 has been given by horst and reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c(1 + t)\log ( 2 + t).$$\end{document } in the case of repulsive forces ( = 1 ) rein has found a better estimate by using a new identity for the vlasov - poisson system , discovered independently by illner and rein and by perthame . rein s estimate reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c{(1 + t)^{2/3}}.$$\end{document } independently , and at about the same time as pfaffelmoser gave his proof , lions and perthame used a different method for proving global existence . their method is more generally applicable , and the two studies and are examples of problems in related systems , where their method has been successful . on the other hand , their method does not give such strong growth estimates on q(t ) as described above . for the relativistic vlasov - poisson equation , glassey and schaeffer showed in the case = 1 that if the data are spherically symmetric , q(t ) can be controlled , which is analogous to the result by batt mentioned above . also in the case of cylindrical symmetry they are able to control q(t ) ; see . if = 1 it was shown in that blow - up occurs in finite time for spherically - symmetric data with negative total energy . more recently , lemou et al . have investigated the structure of the blow - up solution . they show that the blow - up is determined by the self - similar solution of the ultra - relativistic gravitational vlasov - poisson system . it should be pointed out that the relativistic vlasov - poisson system is unphysical since it lacks the lorentz invariance ; it is a hybrid of a classical galilei invariant field equation and a relativistic transport equation ( 17 ) , cf . . in particular , in the case = 1 , it is not a special case of the einstein - vlasov system . only for spherically - symmetric data , in the case the situation for weak solutions is different , in particular the existence of weak solutions to the relativistic vlasov - maxwell system is known [ 70 , 139 ] . we also mention that models , which take into account both collisions and the electric and magnetic fields generated by the particles have been investigated . classical solutions near a maxwellian for the vlasov - maxwell - boltzmann system are constructed by guo in . a similar result for the vlasov - maxwell - landau system near a jttner solution is shown by guo and strain in . we refer to the book by glassey and the review article by rein for more information on the relativistic vlasov - maxwell system and the vlasov - poisson system . before turning to the main theme of this review , i.e. , the einstein - vlasov system nordstrm gravity is an alternative theory of gravity introduced in 1913 . by coupling this model to a kinetic description of matter the nordstrm - vlasov system results . in nordstrm gravity the nordstrm - vlasov system reads 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\partial _ t^2\phi - { \delta _ x}\phi = - { e^{4\phi}}\int\nolimits_{{{\mathbb r}^3 } } { { { { \mathfrak f}dp } \over { \sqrt { 1 + \vert p\vert ^{2}}}},}$$\end{document } 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}{\mathfrak f } + \hat{p } \cdot { \nabla _ x}{\mathfrak f } - \left [ { ( { \partial _ t}\phi + \hat{p } \cdot { \nabla _ x}\phi)p + { { ( 1 + \vert p\vert ^{2})}^{- 1/2}}{\nabla _ x}\phi } \right ] \cdot { \nabla _ p}{\mathfrak f } = 0.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p } = { p \over { \sqrt { 1 + \vert p\vert ^{2}}}},$$\end{document } denotes the relativistic velocity of a particle with momentum p. the mass of each particle , the gravitational constant , and the speed of light are all normalized to one . a solution ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathfrak f},\phi)$\end{document } , ) of this system is interpreted as follows . the spacetime is a lorentzian manifold with a conformally - flat metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\mu \nu } = e^{2\phi}{\rm diag}(- 1,1,1,1).$$\end{document}. the particle distribution f defined on the mass shell in this metric is given by 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(t , x , p ) = { \mathfrak f}(t , x,{e^\phi}p).$$\end{document } the first mathematical study of this system was initiated by calogero in , where the existence of static solutions is established . although the nordstrom - vlasov model of gravity does not describe physics correctly , the system approaches the vlasov - poisson system in the classical limit . indeed , it is shown in that solutions of the nordstrm - vlasov system tend to solutions of the vlasov - poisson system as the speed of light goes to infinity . the cauchy problem was studied by several authors [ 51 , 50 , 15 , 108 , 131 ] and the question of global existence of classical solutions for general initial data was open for some time . another interesting result for the nordstrm - vlasov system is given in , where a radiation formula , similar to the dipole formula in electrodynamics , is rigorously derived . in this section we consider a self - gravitating collision - less gas in the framework of general relativity and we present the einstein - vlasov system . it is most often the case in the mathematics literature that the speed of light c and the gravitational constant g are normalized to one , but we keep these constants in the formulas in this section since in some problems they do play an important role . however , in most of the problems discussed in the forthcoming sections these constants will be normalized to one . let m be a four - dimensional manifold and let gab be a metric with lorentz signature ( , + , + , + ) so that ( m , gab ) is a spacetime . the metric is assumed to be time - orientable so that there is a distinction between future and past directed vectors . the possible values of the four - momentum p of a particle with rest mass m belong to the mass shell pm tm , defined by 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_m } : = \{({x^a},{p^a } ) \in tm:{g_{ab}}({x^a}){p^a}{p^b } = - { m^2}{c^2},{p^a}\;{\rm{is}}\;{\rm{future}}\;{\rm{directed}}\}.$$\end{document } hence , if m > 0 , pm(x ) is the set of all future - directed time - like vectors with length cm , and if m = 0 it is the set of all future - directed null vectors . on pm we take ( x , p ) , a = 0 , 1 , 2 , 3 and j = 1 , 2 , 3 ( letters in the beginning of the alphabet always take values 0 , 1 , 2 , 3 and letters in the middle take 1 , 2 , 3 ) as local coordinates , and p is expressed in terms of p and the metric in view of equation ( 25 ) . below we drop the index m on fm and simply write f. since we are considering a collisionless gas , the particles follow the geodesics in spacetime . the geodesics are projections onto spacetime of the curves in pm defined in local coordinates by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { d{x^a } } \over { ds } } = { p^a}},\\ { { { d{p^j } } \over { ds } } = - \gamma _ { bc}^j{p^b}{p^c}}.\\ \end{array}$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { bc}^a$\end{document } are the christoffel symbols . along a geodesic the density function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = f({x^a},{p^j})$\end{document } is invariant so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${d \over { ds}}f({x^a}(s),{p^j}(s ) ) = 0,$$\end{document } which implies that 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a}{{\partial f } \over { \partial { x^a } } } - \gamma _ { ab}^j{p^a}{p^b}{{\partial f } \over { \partial { p^j } } } = 0.$$\end{document } this is accordingly the vlasov equation . we point out that sometimes the density function is considered as a function on the entire tangent bundle tm rather than on the mass shell pm tm . the vlasov equation for f = f(x , p ) then takes the form 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a}{{\partial f } \over { \partial { x^a } } } - \gamma _ { bc}^a{p^b}{p^c}{{\partial f } \over { \partial { p^a } } } = 0.$$\end{document } this equation follows from ( 26 ) if we take the mass shell condition gabpp = mc into account . indeed , by abuse of notation , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \partial f } \over { \partial { x^a } } } = { { \partial f } \over { \partial { x^a } } } + { { \partial f } \over { \partial { p^0}}}{{\partial { p^0 } } \over { \partial { x^a}}},}\\ { { { \partial f } \over { \partial { p^j } } } = { { \partial f } \over { \partial { p^j } } } + { { \partial f } \over { \partial { p^0}}}{{\partial { p^0 } } \over { \partial { p^j}}}.}\\ \end{array}$$\end{document } here f is considered as a function on pm in the left - hand side , and on tm in the right - hand side . from the mass shell condition gabpp = mc we derive \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \partial { p^0 } } \over { \partial { x^a } } } = - { 1 \over { { p_0}}}{p^b}{p_c}\gamma _ { ab}^c,}\\ { { { \partial { p^0 } } \over { \partial { p^j } } } = - { { { p_j } } \over { { p_0}}}.}\\ \end{array}$$\end{document } inserting these relations into ( 26 ) we obtain ( 27 ) . if we let t = x , and divide the vlasov equation ( 26 ) by p we obtain the most common form in the literature of the vlasov equation 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial f } \over { \partial t } } + { { { p^j } } \over { { p^0}}}{{\partial f } \over { \partial { x^j } } } - { 1 \over { { p^0}}}\gamma _ { ab}^j{p^a}{p^b}{{\partial f } \over { \partial { p^j } } } = 0.$$\end{document } in a fixed spacetime the vlasov equation ( 28 ) is a linear hyperbolic equation for f and we can solve it by solving the characteristic system , 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{x^i } } \over { ds } } = { { { p^i } } \over { { p^0}}},$$\end{document } 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{p^i } } \over { ds } } = - \gamma _ { ab}^i{{{p^a}{p^b } } \over { { p^0}}}.$$\end{document } in terms of initial data f0 the solution of the vlasov equation can be written as 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f({x^a},{p^i } ) = { f_0}({x^i}(0,{x^a},{p^i}),{p^i}(0,{x^a},{p^i})),$$\end{document } where x(s , x , p ) and p(s , x , p ) solve equations ( 29 , 30 ) , and where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x^i}(t,{x^a},{p^i } ) = { x^i}\;{\rm{and}}\;{p^i}(t,{x^a},{p^i } ) = { p^i}.$$\end{document } in order to write down the einstein - vlasov system we need to know the energy - momentum tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{ab}^m$\end{document } in terms of f and gab . we define 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{ab}^m = c\sqrt { \vert { g_{ab}}\vert } \int\nolimits_{{{\mathbb r}^3 } } { f{p_a}{p_b}{{d{p^1}d{p^2}d{p^3 } } \over { - { p_0}}}},$$\end{document } where , as usual , pa = gabp , and gab denotes the absolute value of the determinant of gab . we remark that the measure \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu : = { { \sqrt { \vert { g_{ab}}\vert } } \over { - { p_0}}}d{p^1}d{p^2}d{p^3},$$\end{document } is the induced metric of the submanifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_m}({x^a } ) \subset { t_{{x^a}}}m$\end{document } , and that is invariant under lorentz transformations of the tangent space , and it is often the case in the literature that is written as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{ab}^m = c\int\nolimits_{{p_m}({x^a } ) } { f{p_a}{p_b}\mu.}$$\end{document } let us now consider a collisionless gas consisting of particles with different rest masses m1 , m2 , , mn , described by n density functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_{{m_j}}}$\end{document } , j = 1 , , n. then the vlasov equations for the different density functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_{{m_j}}}$\end{document } , together with the einstein equations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab } } - { 1 \over 2}r{g_{ab } } + \lambda { g_{ab } } = { { 8\pi g } \over { { c^4}}}\sum\limits_{k = 1}^n { t_{ab}^{{m_k}},}$$\end{document } form the einstein - vlasov system for the collision - less gas . here rab is the ricci tensor , r is the scalar curvature and is the cosmological constant . henceforth , we always assume that there is only one species of particles in the gas and we write tab for its energy momentum tensor . moreover , in what follows , we normalize the rest mass m of the particles , the speed of light c , and the gravitational constant g , to one , if not otherwise explicitly stated that this is not the case . we define the particle current density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n^a } = - \int\nolimits_{{{\mathbb r}^3 } } { f{p^a } } \sqrt { \vert { g_{ab}}\vert } { { d{p^1}d{p^2}d{p^3 } } \over { { p_0}}}.$$\end{document } using normal coordinates based at a given point and assuming that f is compactly supported , it is not hard to see that tab is divergence - free , which is a necessary compatibility condition since the left - hand side of ( 2 ) is divergence - free by the bianchi identities . a computation in normal coordinates also shows that n is divergence - free , which expresses the fact that the number of particles is conserved . if v is a future - directed time - like or null vector then we have nav 0 with equality if and only if f = 0 at the given point . hence , n is always future - directed time - like , if there are particles at that point . moreover , if v and w are future - directed time - like vectors then tabvw 0 , which is the dominant energy condition . this is called the non - negative pressure condition , and it implies that the strong energy condition holds as well . that the energy conditions hold for vlasov matter is one reason that the vlasov equation defines a well - behaved matter model in general relativity . another reason is the well - posedness theorem by choquet - bruhat for the einstein - vlasov system that we state below . before stating that theorem we first discuss the conditions imposed on the initial data . the initial data in the cauchy problem for the einstein - vlasov system consist of a 3-dimensional manifold s , a riemannian metric gij on s , a symmetric tensor kij on s , and a non - negative scalar function f0 on the tangent bundle ts of s. the relationship between a given initial data set ( gij , kij ) on s and the metric gab on the spacetime manifold is , that there exists an embedding of s into the spacetime such that the induced metric and second fundamental form of (s ) coincide with the result of transporting ( gij , kij ) with . for the relation of the distribution functions f and f0 we have to note that f is defined on the mass shell . the initial condition imposed is that the restriction of f to the part of the mass shell over (s ) should be equal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_0 } \circ ( { \psi ^{- 1}},d{(\psi)^{- 1 } } ) \circ \phi$\end{document } , where sends each point of the mass shell over (s ) to its orthogonal projection onto the tangent space to (s ) . an initial data set for the einstein - vlasov system must satisfy the constraint equations , which read 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r - { k_{ij}}{k^{ij } } + { ( { \rm{tr}}\;k)^2 } = 16\pi \rho,$$\end{document } 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ i}k_l^i - { \nabla _ l}({\rm{tr}}\;k ) = 8\pi j_{l}.$$\end{document } here = tabnn j = htbcn , where n is the future directed unit normal vector to the initial hypersurface , and h = g + nn is the orthogonal projection onto the tangent space to the initial hypersurface . in terms of f0 we can express and j by ( j satisfies naj = 0 , so it can naturally be identified with a vector intrinsic to s ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \rho = - \int\nolimits_{{{\mathbb r}^3 } } { { f_0}{p^a}{p_a } } \sqrt { \vert { g_{ij}}\vert } { { d{p^1}d{p^2}d{p^3 } } \over { 1 + { p_j}{p^j}}},}\\ { { j_l } = \int\nolimits_{{{\mathbb r}^3 } } { { f_0}{p_l } } \sqrt { \vert { g_{ij}}\vert } d{p^1}d{p^2}d{p^3}.}\\ \end{array}$$\end{document } we can now state the local existence theorem by choquet - bruhat , for the einstein - vlasov system . theorem 1 let s be a 3-dimensional manifold , gij a smooth riemannian metric on s , kij a smooth symmetric tensor on s and f0 a smooth non - negative function of compact support on the tangent bundle ts of s. suppose that these objects satisfy the constraint equations ( 33 , 34 ) . then there exists a smooth spacetime ( m , gab ) , a smooth distribution function f on the mass shell of this spacetime , and a smooth embedding into m , which induces the given initial data on s such that gab and f satisfy the einstein - vlasov system and (s ) is a cauchy surface . moreover , given any other spacetime ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({m^\prime},g_{ab}^\prime)$\end{document } ) , distribution function f and embedding satisfying these conditions , there exists a diffeomorphism from an open neighborhood of if (s ) in m to an open neighborhood of (s ) in m , which satisfies and carries gab and f to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g_{ab}^\prime$\end{document } and f , respectively . the above formulation is in the case of smooth initial data ; for information on the regularity needed on the initial data we refer to and . in this context we also mention that local existence has been proven for the yang - mills - vlasov system in , and that this problem for the einstein - maxwell - boltzmann system is treated in . however , this result is not complete , as the non - negativity of f is left unanswered . also , the hypotheses on the scattering kernel in this work leave some room for further investigation . the local existence problem for physically reasonable assumptions on the scattering kernel does not seem well understood in the context of the einstein - boltzmann system , and a careful study of this problem would be desirable . the mathematical study of the einstein - boltzmann system has been very sparse in the last few decades , although there has been some activity in recent years . since most questions on the global properties are completely open let us only very briefly mention some of these works . global existence for the homogeneous einstein - boltzmann system in robertson - walker spacetimes is proven in , and a generalization to bianchi type i symmetry is established in . in the following sections we present results on the global properties of solutions of the einstein - vlasov system , which have been obtained during the last two decades . before ending this section we mention a few other sources for more background on the einstein - vlasov system , cf . in this section , we discuss results on global existence and on the asymptotic structure of solutions of the cauchy problem in the asymptotically - flat case . in general relativity two classes of initial data are distinguished in the study of the cauchy problem : asymptotically - flat initial data and cosmological initial data . the initial hypersurface is topologically and appropriate fall - off conditions are imposed to ensure that far away from the body spacetime is approximately flat . spacetimes , which possess a compact cauchy hypersurface , are called cosmological spacetimes , and data are accordingly given on a compact 3-manifold . in this case , the whole universe is modeled rather than an isolated body . one can also consider a case , which is un - physical , in which spacetime is asymptotically flat except in one direction , namely cylindrically - symmetric spacetimes , cf . the majority of the work so far has been devoted to the spherically - symmetric case but recently a result on static axisymmetric solutions has been obtained . in contrast to the asymptotically - flat case , cosmological spacetimes admit a large number of symmetry classes . this provides the possibility to study many special cases for which the difficulties of the full einstein equations are reduced . the main goal of these studies is to provide an answer to the weak and strong cosmic censorship conjectures , cf . the study of the global properties of solutions to the spherically - symmetric einstein - vlasov system was initiated two decades ago by rein and rendall , cf . also [ 135 , 156 ] . they chose to work in coordinates where the metric takes the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\mu ( t , r)}}d{t^2 } + { e^{2\lambda ( t , r)}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\varphi ^2}),$$\end{document } where t , r 0 , [ 0, ] , [ 0 , 2 ] . these are called schwarzschild coordinates . asymptotic flatness is expressed by the boundary conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r \rightarrow \infty}{\lim } \lambda ( t , r ) = \underset{r \rightarrow \infty}{\lim } \mu ( t , r ) = 0,\quad \forall t \geq 0.$$\end{document } a regular center is also required and is guaranteed by the boundary condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( t,0 ) = 0\quad \forall t \geq 0.$$\end{document } the coordinates ( r , , ) give rise to difficulties at r = 0 and it is advantageous to use cartesian coordinates . with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x = ( r\sin \phi \cos \theta , r\sin \phi \sin \theta , r\cos \phi)$$\end{document } as spatial coordinates and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v^j } = { p^j } + ( { e^\lambda } - 1){{x \cdot p } \over r}{{{x^j } } \over r}$$\end{document } as momentum coordinates , the einstein - vlasov system reads 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + { e^{\mu - \lambda}}{v \over { \sqrt { 1 + \vert v\vert ^{2 } } } } \cdot { \nabla _ x}f - \left({{\lambda _ t}{{x \cdot v } \over r } + { e^{\mu - \lambda}}{\mu _ r}\sqrt { 1 + \vert v\vert ^{2 } } } \right){x \over r } \cdot { \nabla _ v}f = 0,$$\end{document } 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}\rho,$$\end{document } 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}p,$$\end{document } 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ t } = - 4\pi r{e^{\lambda + \mu}}j,$$\end{document } 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}({\mu _ { rr } } + ( { \mu _ r } - { \lambda _ r})({\mu _ r } + { 1 \over r } ) ) - { e^{- 2\mu}}({\lambda _ { tt } } + { \lambda _ t}({\lambda _ t } - { \mu _ t } ) ) = 8\pi { p_t}.$$\end{document } the matter quantities are defined by 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v\vert ^{2 } } f(t , x , v)dv,}$$\end{document } 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { \left({{{x \cdot v } \over r } } \right)}^2}f(t , x , v){{dv } \over { \sqrt { 1 + \vert v\vert ^{2}}}},}$$\end{document } 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j(t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { x\cdot v } \over r}f(t , x , v)dv,}$$\end{document } 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_t}(t , x ) = { 1 \over 2}\int\nolimits_{{{\mathbb r}^3 } } { \vert { { x \times v } \over r}\vert ^{2}f(t , x , v)dv.}$$\end{document } here is the energy density , j the current , p the radial pressure , and pt the tangential pressure . let us point out that these equations are not independent , e.g. , equations ( 38 ) and ( 39 ) follow from ( 35)(37 ) . as initial data we take a spherically - symmetric , non - negative , and continuously diferentiable function f0 with compact support that satisfies 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{\vert y\vert < r } { \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v{\vert ^2 } } { f_0}(y , v)dv\;dy\ ; < { r \over 2}.}}$$\end{document } this condition guarantees that no trapped surfaces are present initially . the schwarzschild coordinates have the advantage that the resulting system of equations can be written in a quite condensed form . moreover , for most initial data , solutions are expected to exist globally in schwarzschild time , which sometimes is called the polar time gauge . let us point out here that there are initial data leading to spacetime singularities , cf . hence , the question of global existence for general initial data is only relevant if the time slicing of the spacetime is expected to be singularity avoiding , which is the case for schwarzschild time . however , one disadvantage is that these coordinates only cover a relatively small part of the spacetime , in particular trapped surfaces are not admitted . hence , to analyze the black - hole region of a solution these coordinates are not appropriate . here we only mention the other coordinates and time gauges that have been considered in the study of the spherically symmetric einstein - vlasov system . the einstein - vlasov system is investigated in double null coordinates in [ 64 , 63 ] . maximal - areal coordinates and eddington - finkelstein coordinates are used in [ 21 , 17 ] , and in respectively . in it is shown that for initial data satisfying ( 44 ) there exists a unique , continuously - differentiable solution f with f(0 ) = f0 on some right maximal interval [ 0 , t ) . if the solution blows up in finite time , i.e. , if t < , then (t ) becomes unbounded as t t. moreover , a continuation criterion is shown that says that a local solution can be extended to a global one , provided q(t ) can be bounded on [ 0 , t ) , where 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t):\sup \{\vert v\vert : \exists ( s , x ) \in [ 0,t ] \times { { \mathbb r}^3}\;{\rm{such}}\;{\rm{that}}\;f(s , x , v ) \neq 0\}.$$\end{document } this is analogous to the situation for the vlasov - maxwell system . a control of the v - support immediately implies that and p are bounded in view of equations ( 40 , 41 ) . in the vlasov - maxwell case the field equations have a regularizing effect in the sense that derivatives can be expressed through spatial integrals , and it follows that the derivatives of f can also be bounded if the v - support is bounded . for the einstein - vlasov system such a regularization is less clear , since , e.g. , r depends on p in a point - wise manner . however , in view of equation ( 39 ) certain combinations of second and first order derivatives of the metric components can be expressed in terms of the matter component pt , which is a consequence of the geodesic deviation equation . this fact turns out to also be sufficient for obtaining bounds on the derivatives of f , cf . [ 142 , 135 , 156 ] for details . the local existence result discussed above holds for compactly - supported initial data . the compact support condition in the momentum variables is in replaced by the fall - off condition 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{(x , v ) \in { { \mathbb r}^6}}{\sup}{(1 + \vert v\vert)^5}\vert \overset{\circ}{f}(x , v)\vert < \infty.$$\end{document } we also refer to where a subclass of non - compactly - supported data is treated . local existence of solutions in double null coordinates and in eddington - finkelstein coordinates is established in , and respectively . in the authors also consider the problem of global existence in schwarzschild coordinates for small initial data for massive particles . they show that for such data the v - support is bounded on [ 0 , t ) . hence , the continuation criterion implies that t = . the resulting spacetime in is geodesically complete , and the components of the energy - momentum tensor as well as the metric quantities decay with certain algebraic rates in t. the mathematical method used by rein and rendall is inspired by the analogous small data result for the vlasov - poisson equation by bardos and degond . this should not be too surprising since for small data the gravitational fields are expected to be small and a newtonian spacetime should be a fair approximation . in this context we point out that in it is proven that the vlasov - poisson system is indeed the non - relativistic limit of the spherically - symmetric einstein - vlasov system , i.e. , the limit when the speed of light c . in this result is shown without symmetry assumptions . as mentioned above the local and global existence problem has been studied using other time gauges , in particular rendall has shown global existence for small initial data in maximal - isotropic coordinates in . the previous results refer to massive particles but they do not immediately carry over to massless particles . this case is treated by dafermos in where global existence for small initial data is shown in double null coordinates . the spacetimes obtained in the studies [ 142 , 156 , 63 ] are all causally geodesically complete and appropriate decay rates of the metric and the matter quantities are given . in the case of small initial data the resulting spacetime is geodesically complete and no singularities form . a different scenario , which leads to a future geodesically complete spacetime , is to consider initial data where the particles are moving rapidly outwards . if the particles move sufficiently fast the matter disperses and the gravitational attraction is not strong enough to reverse the velocities of the particles to create a collapsing system . it is shown that the scenario described above can be realized , and that global existence holds . in section 3.7 we discuss results on the formation of black holes and trapped surfaces ; in particular , the results in will be presented . a corollary of the main result in concerns the issue of global existence and thus we mention it here . it is shown that a particular class of initial data , which lead to formation of black holes , have the property that the solutions exist for all schwarzschild time . the initial data consist of two parts : an inner part , which is a static solution of the einstein - vlasov system , and an outer part with matter moving inwards . the set - up is shown to preserve the direction of the momenta of the outer part of the matter , and it is also shown that in schwarzschild time the inner part and the outer part of the matter never interact in schwarzschild time . as was mentioned at the end of section 3.1 , the issue of global existence for general initial data is only relevant in certain time gauges since there are initial data leading to singular spacetimes . however , it is reasonable to believe that global existence for general data may hold in a polar time gauge or a maximal time gauge , cf . , and it is often conjectured in the literature that these time slicings are singularity avoiding . however , there is no proof of this statement for any matter model and it would be very satisfying to provide an answer to this conjecture for the einstein - vlasov system . a proof of global existence in these time coordinates would also be of great importance due to its relation to the weak cosmic censorship conjecture , cf . [ 61 , 62 , 65 ] . the methods of proofs in the cases described in sections 3.3 and 3.4 , where global existence has been shown , are all tailored to treat special classes of initial data and they will likely not apply in more general situations . in this section these results are all conditional in the sense that assumptions are made on the solutions , and not only on the initial data . the first study on global existence for general initial data is , which is carried out in schwarzschild coordinates . the authors introduce the following variables in the momentum space adapted to spherical symmetry , 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l : = \vert x{\vert ^2}\vert v{\vert ^2 } - { ( x \cdot v)^2},\;w = { { x \cdot v } \over r},$$\end{document } where l is the square of the angular momentum and w is the radial component of the momenta . a consequence of spherical symmetry is that angular momentum is conserved along the characteristics . in these variables the vlasov equation for f = f ( t , r , w , l ) becomes 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}w + { e^{\mu - \lambda}}{\mu _ r}e - { e^{\mu - \lambda}}{l \over { { r^3}e } } } \right){\partial _ w}f = 0,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = e(r , w , l ) = \sqrt { 1 + { w^2 } + l/{r^2}}.$$\end{document } the main result in shows that as long as there is no matter in the ball \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\{x \in { { \mathbb r}^3}:\vert x\vert \leq \epsilon \},$$\end{document } the estimate 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq { e^{\log q(0){e^{c(\epsilon)t}}}},$$\end{document } holds . here c( ) is a constant , which depends on . thus , in view of the continuation criterion this can be viewed as a global existence result outside the center of symmetry for initial data with compact support . this result rules out shell - crossing singularities , which are present when , e.g. , dust is used as a matter model . the bound of q is obtained by estimating each term individually in the characteristic equation associated with the vlasov equation ( 48 ) for the radial momentum . the einstein equations imply that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mu _ r } = { m \over { { r^2}}}{e^{2\lambda } } + 4\pi rp{e^{2\lambda}}$$\end{document } where 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m(t,\,r ) = 4\pi \int\nolimits_0^r { { \eta ^2}\rho ( t,\eta ) d\eta,}$$\end{document } is the quasi local mass . thus , using ( 38 ) the characteristic equation consists of the two terms t1 = 4re , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_2 } = { e^{\mu + \lambda}}{m \over { { r^2}}}$\end{document } , together with a term , which is independent of the matter quantities . there is a distinct difference between the terms t1 and t2 due to the fact that m can be regarded as an average , since it is given as a space integral of the energy density , whereas j and p are point - wise terms . the method in makes use of a cancellation property of the radial momenta in t1 so that outside the center this term is manageable but in general it seems very unpleasant to have to treat point - wise terms of this kind . in rendall the bound on q(t ) is again obtained by estimating each term in the characteristic equation . in this case . however , the terms are , in analogy with the schwarzschild case , strongly singular at the center . in particular , the method avoids the point - wise terms by using the fact that the characteristic system can be written in a form such that green s formula in the plane can be applied . this results in a combination of terms involving second - order derivatives , which can be substituted for by one of the einstein equations . this method was first introduced in but the set - up is different in and the application of green s formula becomes very natural . in addition , the bound of q is improved compared to ( 49 ) and reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq q(0){e^{c(1 + t)/\epsilon t}}.$$\end{document } this bound is sufficient to conclude that global existence outside the center also holds for non - compact initial data . in addition to the global existence result outside the centre , it is shown in that as long as 3m(t , r ) r and j 0 , singularities can not form . note that in schwarzschild coordinates 2m(t , r ) r always , and that there are closed null geodesics if 3 m = r in the schwarzschild static spacetime . the method in also applies to the case of maximal - isotropic coordinates studied in . there is an improvement concerning the regularity of the terms that need to be estimated to obtain global existence in the general case . a consequence of is accordingly that the quite different proofs in and in are put on the same footing . we point out that the method can also be applied to the case of maximal - areal coordinates . the results discussed above concern time gauges , which are expected to be singularity avoiding so that the issue of global existence makes sense . an interpretation of these results is that first singularities ( where the notion of first is tied to the causal structure ) , in the non - trapped region , must emanate from the center and that this case has also been shown in double null - coordinates by dafermos and rendall in . the main motivation for studying the system in these coordinates has its origin from the method of proof of the cosmic - censorship conjecture for the einstein - scalar field system by christodoulou . an essential part of his method is based on the understanding of the formation of trapped surfaces . in it is shown that a single trapped surface or marginally - trapped surface in the maximal development implies that weak cosmic censorship holds the theorem holds true for any spherically - symmetric matter spacetime if the matter model is such that first singularities necessarily emanate from the center . the results in and in are not sufficient for concluding that the hypothesis of the matter needed in the theorem in is satisfied , since they concern a portion of the maximal development covered by particular coordinates . therefore , dafermos and rendall choose double - null coordinates , which cover the maximal development , and they show that the mentioned hypothesis is satisfied for vlasov matter . the main reason that the question of global existence in certain time coordinates discussed in the previous section 3.5 is of great importance is its relation to the cosmic censorship conjectures . now there is , in fact , no theorem in the literature , which guarantees that weak cosmic censorship follows from such a global existence result , but there are strong reasons to believe that this is the case , cf . and . hence , if initial data can be constructed , which lead to naked singularities , then either the conjecture that global existence holds generally is false or the viewpoint that global existence implies the absence of naked singularities is wrong . in view of a recent result by rendall and velazquez on self similar dust - like solutions for the massless einstein - vlasov system , this issue has much current interest . let us mention here that there is a previous study on self - similar solutions in the massless case by martn - garca and gundlach . however , this result is based on a scaling of the density function itself and therefore makes the result less related to the cauchy problem . also , their proof is , in part , based on numerics , which makes it harder to judge the relevance of the result . the main aim of the work is to establish self - similar solutions of the massive einstein - vlasov system and the present result can be viewed as a first step to achieving this . in the set - up , two simplifications are made . first , the authors study the massless case in order to find a scaling group , which leaves the system invariant . more precisely , the massless system is invariant under the scaling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r \rightarrow \theta r,\;\;t \rightarrow \theta t,\;\;w \rightarrow { 1 \over { \sqrt \theta}}w,\;\;l \rightarrow \theta l.$$\end{document } the massless assumption seems not very restrictive since , if a singularity forms , the momenta will be large and therefore the influence of the rest mass of the particles will be negligible , so that asymptotically the solution can be self - similar also in the massive case , cf . the second simplification is that the possible radial momenta are restricted to two values , which means that the density function is a distribution in this variable . thus , the solutions can be thought of as intermediate between smooth solutions of the einstein - vlasov system and dust . for this simplified system it turns out that the existence question of self - similar solutions can be reduced to that of the existence of a certain type of solution of a four - dimensional system of ordinary differential equations depending on two parameters . the proof is based on a shooting argument and involves relating the dynamics of solutions of the four - dimensional system to that of solutions of certain two- and three - dimensional systems obtained from it by limiting processes . the reason that an ode system is obtained is due to the assumption on the radial momenta , and if regular initial data is considered , an ode system is not sufficient and a system of partial differential equations results . the solution is not asymptotically flat but there are ideas outlined in of how this can be overcome . it should be pointed out here that a similar problem occurs in the work by christodoulou for a scalar field , where the naked singularity solutions are obtained by truncating self - similar data . the singularity of the self - similar solution by rendall and velazquez is real in the sense that the kretschmann scalar curvature blows up . the asymptotic structure of the solution is striking in view of the conditional global existence result in . indeed , the self similar solution is such that j 0 , and 3m(t , r ) r asymptotically , but for any t , 3m(t , r ) > r for some t > t. in global existence follows if j 0 and if 3m(t , r ) r for all t. it is also the case that if m / r is close to 1/2 , then global existence holds in certain situations , cf . . hence , the asymptotic structure of the self - similar solution has properties , which have been shown to be difficult to treat in the search for a proof of global existence . we have previously mentioned that there exist initial data for the spherically - symmetric einstein - vlasov system , which lead to formation of black holes . the first result in this direction he shows that there exist initial data for the spherically - symmetric einstein - vlasov system such that a trapped surface forms in the evolution . the occurrence of a trapped surface signals the formation of an event horizon . as mentioned above , dafermos has proven that , if a spherically - symmetric spacetime contains a trapped surface and the matter model satisfies certain hypotheses , then weak cosmic censorship holds true . in it hence , by combining these results it follows that initial data exist , which lead to gravitational collapse and for which weak cosmic censorship holds . however , the proof in rests on a continuity argument , and it is not possible to tell whether or not a given initial data set will give rise to a black hole . this is in contrast to the result in , where explicit conditions on the initial data are given , which guarantee the formation of trapped surfaces in the evolution . the analysis is carried out in eddington - finkelstein coordinates and a central result in is to control the life span of the solution to ensure that there is sufficient time to form a trapped surface before the solution may break down . in particular , weak cosmic censorship holds for these initial data . in the formation of the event horizon in gravitational collapse one inner part and one outer part , in which all particles move inward initially . the reason for the inner part is that it is possible to choose the parameters for the data such that the particles of the outer matter part continue to move inward for all schwarzschild time as long as the particles do not interact with the inner part . this fact simplifies the analysis since the dynamics is much restricted when the particles keep the direction of their radial momenta . the main result is that explicit conditions on the initial data with adm mass m are given such that there is a family of outgoing null geodesics for which the area radius r along each geodesic is bounded by 2 m . it is furthermore shown that if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t \geq 0,\;{\rm{and}}\;r \geq 2 m + \alpha { e^{- \beta t}},$$\end{document } where and are positive constants , then f(t , r , , ) = 0 , and the metric equals the schwarzschild metric 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - ( 1 - { { 2 m } \over r})d{t^2 } + { ( 1 - { { 2 m } \over r})^{- 1}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\phi ^2}),$$\end{document } representing a black hole with mass m. hence , spacetime converges asymptotically to the schwarzschild metric . the latter result does not reveal whether or not all the matter crosses r = 2 m or simply piles up at the event horizon . in it is shown that for initial data , which are closely related to those in , but such that the radial momenta are unbounded , all the matter do cross the event horizon asymptotically in schwarzschild time . this is in contrast to what happens to freely - falling observers in a static schwarzschild spacetime , since they will never reach the event horizon . the result in is reconsidered in , where an additional argument is given to match the definition of weak cosmic censorship given in . it is natural to relate the results of [ 20 , 24 ] to those of christodoulou on the spherically - symmetric einstein - scalar field system and . in it is shown that if the final bondi mass m is different from zero , the region exterior to the sphere r = 2 m tends to the schwarzschild metric with mass m similar to the result in . in explicit conditions on the initial data this paper played a crucial role in christodoulou s proof of the weak and strong cosmic censorship conjectures . the conditions on the initial data in allow the ratio of the hawking mass and the area radius to cover the full range , i.e. , 2m / r ( 0 , 1 ) , whereas the conditions in require 2m / r to be close to one . hence , it would be desirable to improve the conditions on the initial data in , although the conditions by christodoulou for a scalar field are not expected to be sufficient in the case of vlasov matter . in a numerical study on critical collapse for the einstein - vlasov system it has been shown by rein and rodewis that the numerical scheme has desirable convergence properties . ( in the vlasov - poisson case , convergence was proven in , see also ) . the speculation discussed above that there may be no naked singularities formed for any regular initial data is in part based on the fact that the naked singularities that occur in scalar field collapse appear to be associated with the existence of type ii critical collapse , while vlasov matter is of type i. the primary goal in was indeed to decide whether vlasov matter is type i or type ii . given small initial data , no black holes form and matter will disperse . for large data , black holes will form and consequently there is a transition regime separating dispersion of matter and formation of black holes . if we introduce a parameter a on the initial data such that for small a dispersion occurs and for large a a black hole is formed , we get a critical value ac separating these regions . if we take a > ac and denote by mb(a ) the mass of the black hole , then if mb(a ) 0 as a ac we have type ii matter , whereas for type i matter this limit is positive and there is a mass gap . for more information on critical collapse the conclusion of is that vlasov matter is of type i. there are two other independent numerical simulations on critical collapse for vlasov matter [ 128 , 21 ] . in these simulations , we end this section with a discussion of the spherically - symmetric einstein - vlasov - maxwell system , i.e. , the case considered above with charged particles . whereas the constraint equations in the uncharged case , written in schwarzschild coordinates , do not involve solving any difficulties once the distribution function is given , the charged case is more challenging . however , in it is shown that solutions to the constraint equations do exist for the einstein - vlasov - maxwell system . in local existence is shown together with a continuation criterion , and global existence for small initial data is shown in . in this section we discuss the einstein - vlasov system for cosmological spacetimes , i.e. , spacetimes that possess a compact cauchy surface . the particles in the kinetic description are in this case galaxies or even clusters of galaxies . the main goal is to determine the global properties of the solutions to the einstein - vlasov system for initial data given on a compact 3-manifold . in order to do so , a global time coordinate t must be found and the asymptotic behavior of the solutions when t tends to its limiting values has to be analyzed . this might correspond to approaching a singularity , e.g. , the big bang singularity , or to a phase of unending expansion . presently , the aim of most of the studies of the cosmological cauchy problem has been to show existence for unrestricted initial data and the results that have been obtained are in cases with symmetry ( see , however , , where to some extent global properties are shown in the case without symmetry ) . a recent and very extensive work by ringstrm has , on the other hand , a different aim , i.e. , to show stability of homogeneous cosmological models , and concerns the general case without symmetry . the size of the cauchy data is in this case very restricted but , since ringstrm allows general perturbations , there are no symmetries available to reduce the complexity of the einstein - vlasov system . the only spatially - homogeneous spacetimes admitting a compact cauchy surface are the bianchi types i , ix and the kantowski - sachs model ; to allow for cosmological solutions with more general symmetry types , it is enough to replace the condition that the spacetime is spatially homogeneous , with the condition that the universal covering of spacetime is spatially homogeneous . spacetimes with this property are called locally spatially homogeneous and these include , in addition , the bianchi types ii , iii , v , vi0 , vii0 , and viii . one of the first studies on the einstein - vlasov system for spatially - homogeneous spacetimes is the work by rendall . he chooses a gaussian time coordinate and investigates the maximal range of this time coordinate for solutions evolving from homogeneous data . for bianchi ix and for kantowski - sachs spacetimes he finds that the range is finite and that there is a curvature singularity in both the past and the future time directions . for the other bianchi types there is a curvature singularity in the past , and to the future spacetime is causally geodesically complete . although the questions on curvature singularities and geodesic completeness are very important , it is also desirable to have more detailed information on the asymptotic behavior of the solutions , and , in particular , to understand in which situations the choice of matter model is essential for the asymptotics . in recent years several studies on the einstein - vlasov system for spatially locally homogeneous spacetimes have been carried out with the goal to obtain a deeper understanding of the asymptotic structure of the solutions . roughly , these investigations can be divided into two cases : ( i ) studies on non - locally rotationally symmetric ( non - lrs ) bianchi i models and ( ii ) studies of lrs bianchi models . in case ( i ) rendall shows in that solutions converge to dust solutions for late times . under the additional assumption of small initial data this result is extended by nungesser , who gives the rate of convergence of the involved quantities . in rendall also raises the question of the existance of solutions with complicated oscillatory behavior towards the initial singularity may exist for vlasov matter , in contrast to perfect fluid matter . note that for a perfect fluid the pressure is isotropic , whereas for vlasov matter the pressure may be anisotropic , and this fact could be sufficient to drastically change the dynamics . this question is answered in , where the existence of a heteroclinic network is established as a possible asymptotic state . this implies a complicated oscillating behavior , which differs from the dynamics of perfect fluid solutions . the results in were then put in a more general context by calogero and heinzle , where quite general anisotropic matter models are considered . in case ( ii ) the asymptotic behaviour of solutions has been analyzed in [ 159 , 160 , 48 , 47 ] . in , the case of massless particles is considered , whereas the massive case is studied in . both the nature of the initial singularity and the phase of unlimited expansion are analyzed . the main concern in these two works is the behavior of bianchi models i , ii , and iii . a general conclusion is that the choice of matter model is very important since , for all symmetry classes studied , there are differences between the collision - less model and a perfect fluid model , both regarding the initial singularity and the expanding phase . the most striking example is for the bianchi ii models , where they find persistent oscillatory behavior near the singularity , which is quite different from the known behavior of bianchi type ii perfect fluid models . in it is also shown that solutions for massive particles are asymptotic to solutions with massless particles near the initial singularity . for bianchi i and ii , it is also proven that solutions with massive particles are asymptotic to dust solutions at late times . the investigation concerns a large class of anisotropic matter models , and , in particular , it is shown that solutions of the einstein - vlasov system with massless particles oscillate in the limit towards the past singularity for bianchi ix models . considers the homogeneous spacetimes with a cosmological constant for all bianchi models except bianchi type ix . she also obtains the time decay of the components of the energy momentum tensor as t , and she shows that spacetime is asymptotically dust - like . anguige studies the conformal einstein - vlasov system for massless particles , which admit an isotropic singularity . he shows that the cauchy problem is well posed with data consisting of the limiting density function at the singularity . in the spatially homogeneous case the metric can be written in a form that is independent of the spatial variables and this leads to an enormous simplification . another class of spacetimes that are highly symmetric but require the metric to be spatially dependent are those that admit a group of isometries acting on two - dimensional spacelike orbits , at least after passing to a covering manifold . the group may be two - dimensional ( local u(1 ) u(1 ) or t symmetry ) or three - dimensional ( spherical , plane , or hyperbolic symmetry ) . in all these cases , the quotient of spacetime by the symmetry group has the structure of a two - dimensional lorentzian manifold q. the orbits of the group action ( or appropriate quotients in the case of a local symmetry ) are called surfaces of symmetry . thus , there is a one - to - one correspondence between surfaces of symmetry and points of q. there is a major difference between the cases where the symmetry group is two- or three - dimensional . in the three - dimensional case no gravitational waves are admitted , in contrast to the two - dimensional case where the evolution part of the einstein equations are non - linear wave equations . three types of time coordinates that have been studied in the inhomogeneous case are cmc , areal , and conformal coordinates . a cmc time coordinate t is one where each hypersurface of constant time has constant mean curvature and on each hypersurface of this kind the value of t is the mean curvature of that slice . in the case of areal coordinates , the time coordinate is a function of the area of the surfaces of symmetry , e.g. , proportional to the area or proportional to the square root of the area . in the case of conformal coordinates , the advantage with a cmc approach is that the definition of a cmc hypersurface does not depend on any symmetry assumptions and it is possible that cmc foliations will exist for general spacetimes . the areal coordinate foliation , on the other hand , is adapted to the symmetry of spacetime but it has analytical advantages and detailed information about the asymptotics can be derived . the conformal coordinates have mainly served as a useful framework for the analysis to obtain geometrically - based time foliations . let us now consider spacetimes ( m , g ) admitting a three - dimensional group of isometries . the topology of m is assumed to be s f , with f a compact two - dimensional manifold . the universal covering \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f}$\end{document } of f induces a spacetime \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat g)$\end{document } by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat m = { \mathbb r } \times { s^1 } \times \hat f$\end{document } and = p*g , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } a three - dimensional group g of isometries is assumed to act on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat g)$\end{document}. if f = s and g = so(3 ) , then ( m , g ) is called spherically symmetric ; if f = t and g = e2 ( euclidean group ) , then ( m , g ) is called plane symmetric ; and if f has genus greater than one and the connected component of the symmetry group g of the hyperbolic plane h acts isometrically on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f = { h^2}$\end{document } , then ( m , g ) is said to have hyperbolic symmetry . in the case of spherical symmetry the existence of one compact cmc hypersurface implies that the whole spacetime can be covered by a cmc time coordinate that takes all real values [ 151 , 42 ] . the existence of one compact cmc hypersurface in this case was proven by henkel using the concept of prescribed mean curvature ( pmc ) foliation . accordingly , this gives a complete picture in the spherically symmetric case regarding cmc foliations . in the case of areal coordinates , rein has shown , under a size restriction on the initial data , that the past of an initial hyper - surface can be covered , and that the kretschmann scalar blows up . hence , the initial singularity for the restricted data is both a crushing and a curvature singularity . in the future direction it is shown that areal coordinates break down in finite time . in the case of plane and hyperbolic symmetry , global existence to the past was shown by rendall in cmc time . this implies that the past singularity is a crushing singularity since the mean curvature blows up at the singularity . also in these cases rein showed under a size restriction on the initial data , that global existence to the past in areal time and blow up of the kretschmann scalar curvature as the singularity is approached . hence , the singularity is both a crushing and a curvature singularity in these cases too . in both of these works the question of global existence to the future was left open . this gap was closed in , and global existence to the future was established in both cmc and areal time coordinates . the global existence result for cmc time is a consequence of the global existence theorem in areal coordinates , together with a theorem by henkel which shows that there exists at least one hypersurface with ( negative ) constant mean curvature . in addition , the past direction is analyzed in using areal coordinates , and global existence is shown without a size restriction on the data . it is not concluded if the past singularity , without the smallness condition on the data , is a curvature singularity as well . the issues discussed above have also been studied in the presence of a cosmological constant , cf . [ 182 , 184 ] . in particular it is shown that in the spherically - symmetric case , if < 0 , global existence to the future holds in areal time for some special classes of initial data , which is in contrast to the case with = 0 . in this context we also mention that surface symmetric spacetimes with vlasov matter and with a maxwell field have been investigated in . an interesting question , which essentially was left open in the studies mentioned above , is whether the areal time coordinate , which is positive by definition , takes all values in the range ( 0 , ) or only in ( r0 , ) for some positive r0 . it should here be pointed out that there is an example for vacuum spacetimes with t symmetry ( which includes the plane symmetric case ) where r0 > 0 . this question was first resolved by weaver for t symmetric spacetimes with vlasov matter . her result shows that if spacetime contains vlasov matter ( f 0 ) then r0 = 0 . smulevici has recently shown , under the same assumption , that r0 = 0 also in the hyperbolic case . smulevici also includes a cosmological constant and shows that both the results , for plane ( or t ) symmetry and hyperbolic symmetry , are valid for 0 . the important question of strong cosmic censorship for surface - symmetric spacetimes has recently been investigated by neat methods by dafermos and rendall [ 67 , 65 ] . the standard strategy to show cosmic censorship is to either show causal geodesic completeness in case there are no singularities , or to show that some curvature invariant blows up along any incomplete causal geodesic . in both cases no causal geodesic can leave the maximal cauchy development in any extension if we assume that the extension is c. in [ 67 , 65 ] two alternative approaches are investigated . the first method is independent of the matter model and exploits a rigidity property of cauchy horizons inherited from the killing fields . the areal time described above is defined in terms of the killing fields and a consequence of the method by dafermos and rendall is that the killing fields extend continuously to a cauchy horizon , if one exists . now , since global existence has been shown in areal time it follows that there can not be an extension of the maximal hyperbolic development to the future . the second method is dependent on vlasov matter and the idea is to follow the trajectory of a particle , which crosses the cauchy horizon and shows that the conservation laws for the particle motion associated with the symmetries of the spacetime , such as the angular momentum , lead to a contradiction . in most of the cases considered in there is an assumption on the initial data for the vlasov equation , which implies that the data have non - compact support in the momentum space the results of the studies [ 67 , 65 ] can be summarized as follows . for plane and hyperbolic symmetry strong cosmic censorship the restriction that matter has non - compact support in the momentum space is here imposed except in the plane case with = 0 . in the spherically - symmetric case cosmic censorship the difficulties to show cosmic censorship in this case are related to possible formation of extremal schwarzschild - de - sitter - type black holes . cosmic censorship in the past direction is also shown for all symmetry classes , and for all values of , for a special class of anti - trapped initial data . although the methods developed in [ 67 , 65 ] provide a lot of information on the asymptotic structure of the solutions , questions on geodesic completeness and curvature blow up are not answered . in a few cases , information on these issues has been obtained . as mentioned above , blow up of the kretschmann scalar curvature has been shown for restricted initial data . in the case of hyperbolic symmetry causal future geodesic completeness has been established by rein when the initial data are small . the authors show global existence to the future in areal time , and in particular they show that the spacetimes are future geodesically complete . it is shown that the de sitter solution acts as a model for the dynamics of the solutions by proving that the generalized kasner exponents tend to 1/3 as t , which in the plane case is the de sitter solution . the first study of spacetimes admitting a two - dimensional isometry group was carried out by rendall in the case of local t symmetry . for a discussion of the possible topologies of these spacetimes we refer to the original paper . in the model case the spacetime is topologically of the form t , and to simplify our discussion later on we write down the metric in areal coordinates for this type of spacetime : 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = { e^{2(\eta - u)}}(- \alpha d{t^2 } + d{\theta ^2 } ) + { e^{- 2u}}{t^2}{{[dy + h\;d\theta + m\;dt]}^2}}\\ { \quad \quad + { e^{2u}}{{[dx + a\;dy + ( g + ah)\;d\theta + ( l + am)dt]}^2}.}\\ \end{array}$$\end{document } here the metric coefficients , u , , a , h , l , and m depend on t and and , x , y s. in cmc coordinates are in fact considered rather than areal coordinates . under the hypothesis that there exists at least one cmc hypersurface , rendall proves for general initial data that the past of the given cmc hypersurface can be globally foliated by cmc hypersurfaces and that the mean curvature of these hypersurfaces blows up at the past singularity . the result in holds for vlasov matter and for matter described by a wave map . that the choice of matter model is important was shown in , where a non - global existence result for dust is given , which leads to examples of spacetimes that are not covered by a cmc foliation the plane case , where the symmetry group is three - dimensional , is one subcase and the form of the metric in areal coordinates is obtained by letting a = g = h = l = m = 0 and u = logt/2 in equation ( 52 ) . another subcase , which still admits only two killing fields ( and which includes plane symmetry as a special case ) , is gowdy symmetry . it is obtained by letting g = h = l = m = 0 in equation ( 52 ) . in gowdy symmetric spacetimes with vlasov matter are considered , and it is proven that the entire maximal globally hyperbolic spacetime can be foliated by constant areal time slices for general initial data . the areal coordinates are used in a direct way for showing global existence to the future , whereas the analysis for the past direction is carried out in conformal coordinates . these coordinates are not fixed to the geometry of spacetime and it is not clear that the entire past has been covered . a chain of geometrical arguments then shows that areal coordinates indeed cover the entire spacetime . the method in was in turn inspired by the work for vacuum spacetimes , where the idea of using conformal coordinates in the past direction was introduced . as pointed out in , the result by henkel guarantees the existence of one cmc hypersurface in the gowdy case and , together with the global areal foliation in , it follows that gowdy spacetimes with vlasov matter can be globally covered by cmc hypersurfaces as well . the more general case of t symmetry was considered in , where global cmc and areal time foliations were established for general initial data . in these results , the question whether or not the areal time coordinate takes values in ( 0 , ) or in ( r0 , ) , r0 > 0 , was left open . as we pointed out in section 4.2.1 , this issue was solved by weaver for t symmetric spacetimes with the conclusion that r0 = 0 , if the density function f is not identically zero initially . in the case of t symmetric spacetimes , with a positive cosmological constant , smulevici has shown global existence in areal time with the property that t ( 0 , ) . the issue of strong cosmic censorship for t symmetric spacetimes has been studied by dafermos and rendall using the methods , which were developed in the surface symmetric case described above . in strong cosmic censorship is shown under the same restriction on the initial data that was imposed in the surface symmetric case , which implies that the data have non - compact support in the momentum variable . their result has been extended to the case with a positive cosmological constant by smulevici . the present cosmological observations indicate that the expansion of the universe is accelerating , and this has influenced theoretical studies in the field during the last decade . one way to produce models with accelerated expansion is to choose a positive cosmological constant . another way is to include a non - linear scalar field among the matter fields , and in this section we review the results for the einstein - vlasov system , where a linear or non - linear scalar field have been included into the model . lee considers in the case where a non - linear scalar field is coupled to vlasov matter . the form of the energy momentum tensor then reads 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\alpha \beta } } = t_{\alpha \beta}^{{\rm{vlasov } } } + { \nabla _ \alpha}\phi { \nabla _ \beta}\phi - \left({{1 \over 2}{\nabla ^\gamma}\phi { \nabla _ \gamma}\phi + v(\phi ) } \right){g_{\alpha \beta}}.$$\end{document } here is the scalar field and v is a potential , and the bianchi identities lead to the following equation for the scalar field : 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^\gamma}{\nabla _ \gamma}\phi = v^{\prime}(\phi){.}$$\end{document } under the assumption that v is a non - negative c function , global existence to the future is obtained , and if the potential is restricted to the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v(\phi ) = { v_0}{e^{- c\phi}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0 < c < 4\sqrt \pi$\end{document } , then future geodesic completeness is proven . in the einstein - vlasov system with a linear scalar field is analyzed in the case of plane , spherical , and hyperbolic symmetry . here . a local existence theorem and a continuation criterion , involving bounds on derivatives of the scalar field in addition to a bound on the support of one of the moment variables , is proven . for the einstein scalar field system , i.e. , when f = 0 , the continuation criterion is shown to be satisfied in the future direction , and global existence follows in that case . the work extends the result in the plane and hyperbolic case to a global result in the future direction . in the plane case it is also shown that the singularity is crushing and that the kretschmann scalar diverges uniformly as the singularity is approached . in standard cosmology this is a strong assumption leading to severe restrictions of the possible geometries as well as of the topologies of the universe . thus , it is natural to ask if small perturbations of an initial data set , which corresponds to an expanding model of the standard type , give rise to solutions that are similar globally to the future ? in a recent work , ringstrm considers the einstein - vlasov system and he gives an affirmative answer to the stability question for some of the standard cosmologies . the standard model of the universe is spatially homogeneous and isotropic , has flat spatial hypersurfaces of homogeneity , a positive cosmological constant and the matter content consists of a radiation fluid and dust . hence , to investigate the question on stability it is natural to consider cosmological solutions with perfect fluid matter and a positive cosmological constant . however , as is shown by ringstrm , the standard model can be well approximated by a solution of the einstein - vlasov system with a positive cosmological constant . approximating dust with vlasov matter is straightforward , whereas approximating a radiation fluid is not . by choosing the initial support of the distribution function suitably , ringstrm shows that vlasov matter can be made to mimic a radiation fluid for a prescribed amount of time ; sooner or later the matter will behave like dust , but the time at which the approximation breaks down can be chosen to be large enough that the radiation is irrelevant to the future of that time in the standard picture . the main results in are stability of expanding , spatially compact , spatially locally homogeneous solutions to the einstein - vlasov system with a positive cosmological constant as well as a construction of solutions with arbitrary compact spatial topology . in other words , the assumption of almost spatial homogeneity and isotropy does not seem to impose a restriction on the allowed spatial topologies . let us mention here some related works although these do not concern the einstein - vlasov system . ringstrm considers the case where the matter model is a non - linear scalar field in and . the background solutions , which ringstrm perturb and which are shown to be stable , have accelerated expansion . in the expansion the corresponding problem for a fluid has been treated in and , and the newtonian case is investigated in and for vlasov and fluid matter respectively . equilibrium states in galactic dynamics can be described as static or stationary solutions of the einstein - vlasov system , or of the vlasov - poisson system in the newtonian case . here we consider the relativistic case and we refer to the excellent review paper for the newtonian case . first , we discuss spherically - symmetric solutions for which the structure is quite well understood . on the other hand , almost nothing is known about the stability of the spherically - symmetric static solutions of the einstein - vlasov system , which is in sharp contrast to the situation for the vlasov - poisson system . at the end of this section let the metric have the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\mu ( r)}}d{t^2 } + { e^{2\lambda ( r)}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\varphi ^2}),$$\end{document } where r 0 , [ 0 , ] , [ 0 , 2 ] . as before , asymptotic flatness is expressed by the boundary conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r \rightarrow \infty}{\lim } \lambda ( r ) = \underset{r \rightarrow \infty}{\lim } \mu ( r ) = 0,$$\end{document } and a regular center requires \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( 0 ) = 0.$$\end{document } following the notation in section 3.1 , the time - independent einstein - vlasov system reads 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{\mu - \lambda}}{v \over { \sqrt { 1 + \vert v{\vert ^2 } } } } \cdot { \nabla _ x}f - \sqrt { 1 + \vert v{\vert ^2 } } { e^{\mu - \lambda}}{\mu _ r}{x \over r } \cdot { \nabla _ v}f = 0,$$\end{document } 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}\rho,$$\end{document } 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}p.$$\end{document } recall that there is an additional equation ( 39 ) of second order , which contains the tangential pressure pt , but we leave it out since it follows from the equations above . the matter quantities are defined as before : 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( x ) = \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v{\vert ^2 } } f(x , v)dv,}$$\end{document } 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { \left({{{x \cdot v } \over r } } \right)}^2}f(x , v){{dv } \over { \sqrt { 1 + \vert v{\vert ^2}}}}.}$$\end{document } the quantities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e : = { e^{\mu ( r)}}\sqrt { 1 + \vert v{\vert ^2 } } , \quad l = \vert x{\vert ^2}\vert v{\vert ^2 } - { ( x \cdot v)^2 } = \vert x \times v{\vert ^2},$$\end{document } are conserved along characteristics . if we let 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = \phi ( e , l),$$\end{document } for some function , the vlasov equation is automatically satisfied . a common assumption in the literature is to restrict the form of to 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e , l ) = \phi ( e)(l - { l_0 } ) _ + ^l,$$\end{document } where l > 1/2 , l0 0 and x+ = max{x , 0}. if we furthermore assume that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e ) = ( e - { e_0 } ) _ + ^k,\;k > - 1,$$\end{document } for some positive constant e0 , then we obtain the polytropic ansatz . the case of isotropic pressure is obtained by letting l = 0 so that only depends on e. in passing , we mention that for the vlasov - poisson system it has been shown that every static spherically - symmetric solution must have the form f = (e , l ) . this is referred to as jeans theorem . it was an open question for some time whether or not this was also true for the einstein - vlasov system . this was settled in 1999 by schaeffer , who found solutions that do not have this particular form globally on phase space , and consequently , jeans theorem is not valid in the relativistic case . however , almost all results on static solutions are based on this ansatz . by inserting the ansatz f(x , v ) = (e , l ) in the matter quantities and p , a non - linear system for and is obtained , in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}{g_\phi}(r,\mu),}\\ { { e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}{h_\phi}(r,\mu),}\\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_\phi}(r,\mu ) = { { 2\pi } \over { { r^2}}}\int\nolimits_1^\infty { \int\nolimits_0^{{r^2}({\epsilon ^2 } - 1 ) } { \phi ( { e^{\mu ( r)}}\epsilon , l){{{\epsilon ^2 } } \over { \sqrt { { \epsilon ^2 } - 1 - l/{r^2}}}}dld\epsilon}}}\\ { { h_\phi}(r,\mu ) = { { 2\pi } \over { { r^2}}}\int\nolimits_1^\infty { \int\nolimits_0^{{r^2}({\epsilon ^2 } - 1 ) } { \phi ( { e^{\mu ( r)}}\epsilon , l)\sqrt { { \epsilon ^2 } - 1 - l/{r^2 } } dld\epsilon.}}}\\ \end{array}$$\end{document } existence of solutions to this system was first proven in the case of isotropic pressure in , and extended to anisotropic pressure in . the main difficulty is to show that the solutions have finite adm mass and compact support . the argument in these works to obtain a solution of compact support is to perturb a steady state of the vlasov - poisson system , which is known to have compact support . two different types of solutions are constructed , those with a regular centre [ 144 , 134 ] , and those with a schwarzschild singularity in the centre . in , rein and rendall go beyond the polytropic ansatz and obtain steady states with compact support and finite mass under the assumption that satisfies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e , l ) = c(e - { e_0 } ) _ + ^{\delta + k}){l^l}\;{\rm{as}}\;e \rightarrow { e_0},$$\end{document } where k > 1 , l > 1/2 , k + l + 1/2 > 0 , k < l + 3/2 . this result is obtained in a more direct way and is not based on the perturbation argument used in [ 144 , 134 ] . their method is inspired by a work on stellar models by makino , in which he considers steady states of the euler - einstein system . in there is also a discussion about steady states that appear in the astrophysics literature , and it is shown that their result applies to most of these steady states . an alternative method to obtain steady states with finite radius and finite mass , which is based on a dynamical system analysis , all solutions described so far have the property that the support of contains a ball about the centre . in rein showed that steady states also exist whose support is a finite , spherically - symmetric shell with a vacuum region in the center . in it a systematic study of the structure of spherically - symmetric static solutions was carried out mainly by numerical means in and we now present the conclusions of this investigation . by prescribing the value (0 ) , the equations can be solved , but the resulting solution will in general not satisfy the boundary condition ( ) = 0 , but it will have some finite limit . it is then possible to shift both the cut - off energy e0 and the solution by this limit to obtain a solution , which satisfies ( ) = 0 . a convenient way to handle the problem that e0 and (0 ) can not both be treated as free parameters is to use the ansatz \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = ( 1 - e/{e_0 } ) _ + ^l , k \geq 0,\;l > - 1/2,k < 3l + 7/2,$$\end{document } as in . this gives an equation for , which can be rewritten in terms of the function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y(r ) = { { { e^{\mu ( r ) } } } \over { { e_0}}}.$$\end{document } in this way the cut - of energy disappears as a free parameter of the problem and we thus have the four free parameters k , l , l0 and y(0 ) . the structure of the static solutions obtained in is as follows : if l0 = 0 the energy density can be strictly positive or vanish at r = 0 ( depending on l ) but it is always strictly positive sufficiently close to r = 0 . hence , the support of the matter is an interval [ 0 , r1 ] with r1 > 0 , and we call such states ball configurations . if l0 > 0 the support is in an interval [ r0 , r1 ] , r0 > 0 , and we call such steady states for shells . the value y(0 ) determines how compact or relativistic the steady state is , and the smaller values the more relativistic . for large values , recall y(0 ) 1/e0 , a pure shell or a pure ball configuration is obtained , cf . note that we depict the behavior of but we remark that the pressure terms behave similarly but the amplitudes of p and pt can be very different , i.e. , the steady states can be highly anisotropic . figure 1a pure shell for moderate values of y(0 ) the solutions have a distinct inner peak and a tail - like outer peak , and by making y(0 ) smaller more peaks appear , cf . figure 2multi - peaks of ball configurations , l0 = 0 multi - peaks of ball configurations , l0 = 0 in the case of shells there is a similar structure but in this case the peaks can either be separated by vacuum regions or by thin atmospheric regions as in the case of ball configurations . an example with multi - peaks , where some of the peaks are separated by vacuum regions , is given in figure 3 . figure 3multi - peaks of a shell multi - peaks of a shell a different feature of the structure of static solutions is the issue of spirals . for a fixed ansatz of the density function f , there is a one - parameter family of static solutions , which are parameterized by y(0 ) . a natural question to ask is how the adm mass m and the radius of the support r change along such a family . by plotting for each y(0 ) the resulting values for r and m a curve is obtained , which reflects how radius and mass are related along such a one - parameter family of steady states . figure 4 . it is shown in that in the isotropic case , where l = l0 = 0 the radius - mass curves always have a spiral form . figure 4(r , m ) spiral for k = 0 , l = 10.5 , lo = 0 , 0.01 y(0 ) 0.99 ( r , m ) spiral for k = 0 , l = 10.5 , lo = 0 , 0.01 y(0 ) 0.99 another aspect of the structure of steady states investigated numerically in concerns the buchdahl inequality . if a steady state has support in [ r0 , r1 ] , then the adm mass m of the configuration is m = m(r1 ) , where the quasi local mass m(r ) is given by equation ( 50 ) in schwarzschild coordinates . in view of the schwarzschild metric ( 51 ) , schwarzschild asked already in 1916 the question : how large can 2m / r possibly be ? he gave the answer 2m / r 8/9 in the special case of the schwarzschild interior solution , which has constant energy density and isotropic pressure . in 1959 buchdahl extended his result to isotropic solutions for which the energy density is non - increasing outwards and he showed that also in this case 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{2 m } \over r } \leq { 8 \over 9}.$$\end{document } this is sometimes called the buchdahl inequality . let us remark that the buchdahl inequality can obviously be written as m / r 4/9 , but since it is the quantity 2m / r , which appears in the schwarzschild metric ( 51 ) , it is common to keep the form of equation ( 62 ) . a bound on 2m / r has an immediate observational consequence since it limits the possible gravitational red shift of a spherically - symmetric static object . in particular , the overwhelming number of the steady states of the einstein - vlasov system have neither an isotropic pressure nor a non - increasing energy density , but nevertheless 2m / r is always found to be less than 8/9 in the numerical study . also for other matter models as pointed out by guven and murchadha , neither of the buchdahl assumptions hold in a simple soap bubble and they do not approximate any known topologically stable field configuration . in addition , there are also several astrophysical models of stars , which are anisotropic . lemaitre proposed a model of an anisotropic star already in 1933 , and binney and tremaine explicitly allow for an anisotropy coefficient . hence , it is an important question to investigate bounds on 2m / r under less restrictive assumptions . in it is shown that for any static solution of the spherically - symmetric einstein equation , not necessarily of the einstein - vlasov system , for which p 0 , and 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + 2{p_t } \leq \omega \rho,$$\end{document } the following inequality holds 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{2m(r ) } \over r } \leq { { { { ( 1 + 2\omega)}^2 } - 1 } \over { { { ( 1 + 2\omega)}^2}}}.$$\end{document } moreover , the inequality is sharp and sharpness is obtained uniquely by an infinitely thin shell solution . note in particular that for vlasov matter = 1 and that the right - hand side then equals 8/9 as in the buchdahl inequality . an alternative proof was given in and their method applies to a larger class of conditions on , p and pt than the one given in equation ( 63 ) . on the other hand , the result in is weaker than the result in in the sense that the latter method implies that the steady state that saturates the inequality is unique ; it is an infinitely thin shell . the studies [ 10 , 103 ] are of general character and in particular it is not shown that solutions exist to the coupled einstein - matter system , which can saturate the inequality . for instance , it is natural to ask if there are solutions of the einstein - vlasov system , which have 2m / r arbitrarily close to 8/9 . this question is given an affirmative answer in , where in particular it is shown that arbitrarily thin shells exist , which are regular solutions of the spherically - symmetric einstein - vlasov system . using the strategy in it follows that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r}{\sup}{{2m(r ) } \over r } \rightarrow { 8 \over 9},$$\end{document } in the limit when the shells become infinitely thin . the question of finding an upper bound on 2m / r can be extended to charged objects and to the case with a positive cosmological constant . the spacetime outside a spherically - symmetric charged object is given by the reissner - nordstrm metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - ( 1 - { { 2 m } \over r } + { q \over { { r^2}}})d{t^2 } + { ( 1 - { { 2 m } \over r } + { q \over { { r^2}}})^{- 1}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\phi ^2}),$$\end{document } where q is the total charge of the object . the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1 - { { 2 m } \over r } + { q \over { { r^2}}}$\end{document } is zero when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r _ \pm } = m \pm \sqrt { { m^2 } - { q^2}}$\end{document } , and r is called the inner and outer horizon respectively of a reissner - nordstrm black hole . a buchdahl type inequality gives a lower bound of the area radius of a static object and this radius is thus often called the critical stability radius . it is shown in that a spherically - symmetric static solution of the einstein - maxwell system for which p 0 , p + 2pt , and q < m satisfy 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt m \leq { { \sqrt r } \over 3 } + \sqrt { { r \over 9 } + { { { q^2 } } \over { 3r}}}.$$\end{document } note , in particular , that the inequality holds for solutions of the einstein - vlasov - maxwell system , since the conditions above are always satisfied in this case . this inequality ( 65 ) implies that the stability radius is outside the outer horizon of a reissner - nordstrm black hole . in the relevance of an inequality of this kind on aspects in black - hole physics an infinitely thin shell solution does saturate the inequality ( 65 ) , but numerical evidence is given in that there is also another type of solution , which saturates the inequality for which the inner and outer horizon coincide . the study in is concerned with the non - charged situation when a positive cosmological constant is included . the following inequality is derived \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m \over r } \leq { 2 \over 9 } - { { \lambda { r^2 } } \over 3 } + { 2 \over 9}\sqrt { 1 + 3\lambda { r^2}},$$\end{document } for solutions for which p 0 , p + 2pt , and 0 r 1 . in this situation , an infinitely thin shell solution does not generally saturate the inequality but does so in the two degenerate situations r = 0 and r = 1 . in the latter case there is a constant density solution , and the exterior spacetime is the nariai solution , which saturates the inequality and the saturating solution is thus non - unique . in this case , the cosmological horizon and the black hole horizon coincide , which is in analogy with the charged situation described above where the inner and outer horizons coincide when uniqueness is likely lost . an important problem is the question of the stability of spherically - symmetric steady states . at present , there are almost no theoretical results on the stability of the steady states of the einstein - vlasov system . wolansky has applied the energy - casimir method and obtained some insights , but the theory is much less developed than in the vlasov - poisson case and the stability problem is essentially open . the situation is very different for the vlasov - poisson system , and we refer to for a review on the results in this case . however , there are numerical studies [ 21 , 100 , 171 ] on the stability of spherically - symmetric steady states for the einstein - vlasov system . the latter two studies concern isotropic steady states , whereas the first , in addition , treats anisotropic steady states . here we present the conclusions of , emphasizing that these agree with the conclusions in [ 171 , 100 ] for isotropic states . to allow for trapped surfaces , maximal - areal coordinates are used , i.e. , the metric is written in the following form in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - ( { \alpha ^2 } - { a^2}{\beta ^2})d{t^2 } + 2{a^2}\beta dtdr + { a^2}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta \,d{\phi ^2}).$$\end{document } here the metric coefficients , , and a depend on t and r 0 , and a are positive , and the polar angles [ 0 , ] and [ 0 , 2 ] parameterize the unit sphere . a maximal gauge condition is then imposed , which means that each hypersurface of constant t has vanishing mean curvature . the boundary conditions , which guarantee asymptotic flatness and a regular center , are given by 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(t,0 ) = a(t,\infty ) = \alpha ( t,\infty ) = 1.$$\end{document } steady states are numerically constructed , and these are then perturbed in order to investigate the stability . more precisely , to construct the steady states the polytropic ansatz is used , cf . section 5.1 , 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(r , w , l ) = \phi ( e , l ) = ( { e_0 } - e ) _ + ^l.$$\end{document } by specifying values on e0 , l0 and (0 ) steady states are numerically constructed . the distribution function fs of the steady state is then multiplied by an amplitude a , so that a new , perturbed distribution function is obtained . we remark that also other types of perturbations are analyzed in . for k and l fixed each steady state is characterized by its central red shift zc and its fractional binding energy eb , which are defined by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${z_c } = { 1 \over { \alpha ( 0 ) } } - 1,\;{e_b } = { { { e_b } } \over { { m_0}}},\;{\rm{where}}\;{e_b } = { m_0 } - m.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_0 } = 4{\pi ^2}\int\nolimits_0^\infty { \int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty { a(t , r)f(t , r , w , l ) } dl\;dw\;dr}}$$\end{document } is the total number of particles , which , since all particles have rest mass one , equals the rest mass of the system . m is the adm mass given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = \int\nolimits_0^\infty { \left({4\pi \rho ( t , r ) + { 3 \over 2}{\kappa ^2}(t , r ) } \right){r^2}dr,}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = \beta / r\alpha$\end{document } both m0 and m are conserved quantities . the central redshift is the redshift of a photon emitted from the center and received at infinity , and the binding energy eb is the difference of the rest mass and the adm mass . in figure 5 and figure 6 the relation between the fractional binding energy and the central redshift is given for two different cases . figure 5k = 0 , l = 0 , l0 = 0.1 figure 6k = 0 , l = 3/2 , l0 = 0.1 k = 0 , l = 0 , l0 = 0.1 k = 0 , l = 3/2 , l0 = 0.1 the relevance of these concepts for the stability properties of steady states was first discussed by zeldovich and podurets , who argued that it should be possible to diagnose the stability from binding energy considerations . zeldovich and novikov then conjectured that the binding energy maximum along a steady state sequence signals the onset of instability . varying the parameters k , l and l0 give rise to essentially the same tables , cf . . table 1k = 0 and l = 1/2 . z c e b a < 1a > 10.210.032stablestable0.340.040stablestable0.390.040stablestable0.420.041stableunstable0.460.040stableunstable0.560.036stableunstable0.650.029stableunstable0.820.008stableunstable0.950.015unstableunstable1.200.078unstableunstable if we first consider perturbations with a > 1 , it is found that steady states with small values on zc ( less than approximately 0.40 in this case ) are stable , i.e. , the perturbed solutions stay in a neighbourhood of the static solution . a careful investigation of the perturbed solutions indicates that they oscillate in a periodic way . for larger values of zc hence , for perturbations with a > 1 the value of zc alone seems to determine the stability features of the steady states . plotting eb versus zc with higher resolution , cf . , gives support to the conjecture by novikov and zeldovich mentioned above that the maximum of eb along a sequence of steady states signals the onset of instability . consider a steady state with eb > 0 and a perturbation with a < 1 but close to 1 so that the fractional binding energy remains positive . the perturbed solution then drifts outwards , turns back and reimplodes , and comes close to its initial state , and then continues to expand and reimplode and thus oscillates , cf . figure 7zc = 0.47 , eb = 0.04 , a = 0.99 , t = 90.0 zc = 0.47 , eb = 0.04 , a = 0.99 , t = 90.0 in it is stated ( without proof ) that if eb > 0 the solution must ultimately reimplode and the simulations in support that it is true . for negative values of eb , the solutions with a < 1 disperse to infinity . a simple analytic argument is given in , which relates the question , whether a solution disperses or not . it is shown that if a shell solution has an expanding vacuum region of radius r(t ) at the center with r(t ) for t , i.e. , the solution disperses in a strong sense , then necessarily m0 m , i.e. , eb 0 . as we have seen above , a broad variety of static solutions of the einstein - vlasov system has been established , all of which share the restriction that they are spherically symmetric . the recent investigation removes this restriction and proves the existence of static solutions of the einstein - vlasov system , which are axially symmetric but not spherically symmetric . from the applications point of view this symmetry assumption is more realistic than spherical symmetry , and from the mathematics point of view the complexity of the einstein field equations increases drastically if one gives up spherical symmetry . before discussing this result , let us mention that similar results have been obtained for two other matter models . in the case of a perfect fluid , heilig showed the existence of axisymmetric stationary solutions in . these solutions have non - zero angular momentum since static solutions are necessarily spherically symmetric . in this respect the existence of static axisymmetric solutions of elastic matter , which are not spherically symmetric , was proven in . let us now briefly discuss the method of proof in , which relies on an application of the implicit function theorem . also , the proofs in [ 92 , 1 , 2 ] make use of the implicit function theorem , but apart from this fact the methods are quite different . the set - up of the problem in follows the work of bardeen , where the metric is written in the form 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { c^2}{e^{2\nu/{c^2}}}d{t^2 } + { e^{2\mu}}d{\rho ^2 } + { e^{2\mu}}d{z^2 } + { \rho ^2}{b^2}{e^{- 2\nu/{c^2}}}d{\varphi ^2}$$\end{document } for functions , b , depending on and z , where t , [ 0 , [ , z and [ 0 , 2 ] . the killing vector fields t and correspond to the stationarity and axial symmetry of the spacetime . solutions are obtained by perturbing off spherically symmetric steady states of the vlasov - poisson system via the implicit function theorem and the reason for writing /c in the metric , instead of , is that converges to the newtonian potential un of the steady state in the limit c . asymptotic flatness is expressed by the boundary conditions 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{\vert ( \rho , z)\vert \rightarrow \infty}{\lim}\nu ( \rho , z ) = \underset{\vert ( \rho , z)\vert \rightarrow \infty}{\lim } \mu ( \rho , z ) = 0,\;\;\underset{\vert ( \rho , z)\vert \rightarrow \infty}{\lim } b(\rho , z ) = 1.$$\end{document } in addition the solutions are required to be locally flat at the axis of symmetry , which implies the condition 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu ( 0,z)/{c^2 } + \mu ( 0,z ) = \ln b(0,z),\;z \in { \mathbb r}.$$\end{document } let us now recall from section 5.1 the strategy to construct static solutions by using an ansatz of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = \phi ( e , l),$$\end{document } where e and l are conserved quantities along characteristics . due to the symmetries of the metric ( 68 ) the following quantities are constant along geodesics : 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { e : = - g(\partial/\partial t,{p^a } ) = { c^2}{e^{2\nu/{c^2}}}{p^0}}\\ { \quad \ ; = { c^2}{e^{\nu/{c^2}}}\sqrt { 1 + { c^{- 2}}({e^{2\mu}}{{({p^1})}^2 } + { e^{2\mu}}{{({p^2})}^2 } + { \rho ^2}{b^2}{e^{- 2\nu/{c^2}}}{{({p^3})}^2})},}\\ \end{array}$$\end{document } 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l : = - g(\partial/\partial \varphi , { p^a } ) = { \rho ^2}{b^2}{e^{- 2\nu/{c^2}}}{p^3}.$$\end{document } here p are the canonical momenta . e can be thought of as a local or particle energy and l is the angular momentum of a particle with respect to the axis of symmetry . for a sufficiently regular the ansatz function f satisfies the vlasov equation and upon insertion of this ansatz into the definition of the energy momentum tensor ( 32 ) the latter becomes a functional ta = t( , b , ) of the unknown metric functions , b , . it then remains to solve the einstein equations with this energy momentum tensor as right - hand side . the newtonian limit of the einstein - vlasov system is the vlasov - poisson system and the strategy in is to perturb off spherically symmetric steady states of the vlasov - poisson system via the implicit function theorem to obtain axisymmetric solutions . indeed , the main result of specifies conditions on the ansatz function such that a two parameter ( and ) family of axially - symmetric solutions of the einstein - vlasov system passes through the corresponding spherically symmetric , newtonian steady state , whose ansatz function we denote by . the parameter = 1/c turns on general relativity and the parameter turns on the dependence on l. since l is not invariant under arbitrary rotations about the origin the solution is not spherically symmetric if f depends on l. it should also be mentioned that although is a priori small , which means that c is large , the scaling symmetry of the einstein - vlasov system can be used to obtain solutions corresponding to the physically correct value of c. the most striking condition on the ansatz function , or rather on the ansatz function of the corresponding vlasov - poisson system , needed to carry out the proof is that it must satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$6 + 4\pi { r^2}{a_n}(r ) > 0,\quad r \in [ 0,\infty [ , $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_n}(r ) : = \int\nolimits_{{{\mathbb r}^3 } } { \phi ^{\prime } } \left({{1 \over 2}\vert v{\vert ^2 } + { u_n}(r ) } \right)dv.$$\end{document } an important argument in the proof is indeed to justify that there are steady states of the vlasov - poisson system satisfying this condition . it is of course desirable to extend the result in to stationary solutions with rotation . moreover , the deviation from spherically symmetry of the solutions in is small and an interesting open question is the existence of disk - like models of galaxies . in the vlasov - poisson case this has been shown in .

the main purpose of this article is to provide a guide to theorems on global properties of solutions to the einstein - vlasov system . this system couples einstein s equations to a kinetic matter model . kinetic theory has been an important field of research during several decades in which the main focus has been on non - relativistic and special relativistic physics , i.e. , to model the dynamics of neutral gases , plasmas , and newtonian self - gravitating systems . in 1990 , rendall and rein initiated a mathematical study of the einstein - vlasov system . since then many theorems on global properties of solutions to this system have been established . this paper gives introductions to kinetic theory in non - curved spacetimes and then the einstein - vlasov system is introduced . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to a good comprehension of kinetic theory in general relativity .

Introduction to Kinetic Theory The Einstein-Vlasov System The Asymptotically-Flat Cauchy Problem: Spherically-Symmetric Solutions The Cosmological Cauchy Problem Stationary Asymptotically-Flat Solutions

there are at least 40 types of legionella bacteria , half of which are capable of producing disease in humans . the legionella pneumophila bacterium we describe the case of a 60-year - old woman with a history of diabetes mellitus and arterial hypertension who was admitted to our hospital with fever and symptoms of respiratory infection , diarrhea , and acute renal failure . we used real - time polymerase chain reaction ( pcr ) to detect l. pneumophila dna in peripheral blood and serum samples and urine antigen from a patient with pneumonia . since legionella is a cause of 2% to 15% of all community - acquired pneumonias that require hospitalization , legionellosis should be taken into account in an atypical pulmonary infection and not be forgotten . moreover , real - time pcr should be considered a useful diagnostic method . legionella species are gram - negative bacteria that are ubiquitous in both natural aquatic and moist soil and muddy environments and in artificial aquatic habitats.1,2 human infection with legionella spp . has two distinct forms : legionnaires disease , a more severe form of infection which includes pneumonia , and pontiac fever , a milder febrile flu - like illness without pneumonia.3 legionella stands as the cause of community- acquired pneumonia ( cap ) in 2%15% of all caps that require hospitalization . the clinical and radiological features of legionella pneumonia are nonspecific , and the diagnosis depends on laboratory tests.4 this paper reports a case of pneumonia caused by legionella pneumophila that was admitted to the general hospital of komotini in greece . a 60-year - old female with a history of diabetes mellitus , chronic obstructive pulmonary disease , and arterial hypertension with a 4-day history of watery diarrhea and temperature was admitted to our hospital . the findings from physical examinations on admission were as follows : temperature 38.6c , blood pressure 130/95 mm hg , heart rate 117 beats / min , and respiratory rate 28 breaths / min . laboratory findings on admission were as follows : white blood cell ( wbc ) count was 14,280/l , creatinine was 3.4 mg / dl , the erythrocyte sedimentation rate ( esr ) was 110/h , procacitonin ( pct ) 2 ng / ml , c - reactive protein ( crp ) 14 mg / dl ( table 1 ) . we considered the elevated creatinine levels as acute , because at her previous laboratory tests her creatinine values never surpassed 1.3 mg / dl . arterial blood gas showed hypoxemia , as partial pressure of oxygen on air was 56 mm hg.5 the chest x - ray showed infiltration of the left lung ( figure 1 ) . with a clinical diagnosis of pneumonia , urine samples for l. pneumophila and streptococcus pneumoniae antigen , gram stain sputum , and blood specimens upon admission were collected from the patient for culture testing.6 the results were reported negative . the pneumonia severity index ( psi ) was evaluated as class 3 with a mortality rate of 0.9%.7 the patient was treated empirically with 1 g amoxycillin / clavulanic acid three times daily and 500 mg clarithromycin two times daily for 2 days . empiric antibiotic treatment was added upon admission based on elevated values of crp , wbc , esr , and chest x - ray findings , since early antibiotic treatment prevents progression of the disease and these markers are known to be elevated in infectious diseases.8,9 the clinical status of the patient was deteriorating , and there was a marked progression of the infiltrates on the chest x - ray ; the left infiltrate progressed to bilateral shadows . the laboratory values were as follows on the 2nd hospital day : wbc count 17,780/l , crp 18 mg / dl , pct 2 ng / ml , and esr 120/h . the patient s hypoxemia increased : partial pressure of oxygen in arterial blood ( pao2 ) 47 mmhg , partial pressure of carbon dioxide in arterial blood ( paco2 ) 25.4 mm hg , and ph 7.5 ( table 1 ) . one day after her admission to hospital ( day 2 ) ( figure 2 ) , two blood samples , one serum sample and a second urine sample were taken and tested for l. pneumophila with a real - time polymerase chain reaction ( pcr ) kit ( aqua screen l. pneumophila - detection kit for real - time pcr ; minerva biolabs , minerva , oh ) and with culture method . a portion of 200 l of blood and an equal volume of serum were plated on buffered charcoal yeast extract ( bcye ) ( oxoid , reading , uk ) with l - cysteine , on bcye without l - cysteine , and on gvpc ( gas vesicle protein c ) . the antibiotics were immediately changed to levofloxacin after the positive real - time pcr and legionella urinary antigen detection test . the patient responded after therapy with levofloxacin , on day four ( figure 3 ) . her general condition , as well as radiographic and laboratory findings , gradually improved and the patient was discharged on the 21st day of hospitalization ( figure 4 ) . to identify the source of the infection , the patient s relatives were interviewed . exposure histories revealed that the patient had travelled abroad and visited an operating spa pool 2 weeks before the day of onset . after she returned , she remained in her village until her admission to hospital . as there were no cooling towers or any aerosol - generating systems in an area of 2 km from the house , and there was no access to the spa pool abroad , environmental samples were taken only from the home water supplies , and standard methods were processed . the incidence of legionnaires disease has increased in the last decade since the introduction of urinary antigen immunoassays.10,11 this test accounts for most of the diagnostics due to its high sensitivity and ease of use.12 l. pneumophila has become one of the leading causes of cap in adults , accounting for 6%14% of cases requiring hospitalization in recent studies.13,14 legionnaires disease occurs sporadically and in outbreaks , with the sporadic form representing 65%82% of the cases.10,11,15 nevertheless , the number of confirmed community outbreaks including more than 100 cases has increased in recent years due to the use of legionella antigenuria.11,15 routine testing for legionella urinary antigen has increased the number of diagnostics of legionnaires disease and has allowed earlier diagnosis and treatment , greatly improving the prognosis.16 this has been particularly true for milder cases , mainly in the outbreak setting.17 however , most of the knowledge on risk factors , clinical presentation , and outcome of community - acquired legionnaires disease is based on studies performed before routine urinary antigen testing was adopted.18,19 moreover , recent community outbreaks have contributed to the better understanding of legionnaires disease in this setting.2022 legionella pneumophila has been recognized as an important cause of both cap and nosocomial pneumonia.1,10,11,23 environmental systems , such as air conditioning cooling towers , evaporative condensers , whirlpools , and hot spring baths have hosted and transmitted the organism . cases of legionella pneumonia presumably transmitted from contaminated hot spring spa water have been reported from greece.23 the early recognition of infection due to legionella plays a major role in its treatment and preventing mortality in patients with any underlying disease.24,25 in this case , the pcr method was lifesaving for the patient because it confirmed the infection , leading to the administration of the right treatment , whereas the first urine antigen test was misleading . this way of diagnosing legionella has been well established in previous published studies , and it should be applied in such cases where a differential diagnostic problem exists.26,27 legionnaires disease is an acute bacterial infection generally caused by l. pneumophila , primarily involving the lower respiratory tract . outbreaks have been described related to a common source of contamination.1 erythromycin has been the treatment of choice ever since a retrospective study of the original outbreak in philadelphia indicated a lower mortality rate with this antibiotic . because legionella is an intracellular pathogen , antibiotics that penetrate intracellularly are likely to be active against this pathogen . both fluoroquinolones and macrolides penetrate cells well , and fluoroquimolones achieve high intracellular levels and have a lower minimum inhibitory concentration against legionella than erythromycin.28 they are more active than erythromycin in inhibiting l. pneumophila in different intracellular models . three observational studies with a total of 458 patients have indicated that fluoroquinolones ( mainly levofloxacin ) are associated with a superior clinical response when compared with that of macrolides ( erythromycin and clarithromycin ) , as evidenced by a shorter time to apyrexia , shorter hospital stay , and fewer drug - related complications.2931 however , no randomized trials have been performed yet . although it has been traditional to add rifampin to erythromycin to treat severe legionellosis , an observational cohort study revealed that the addition of rifampin to clarithromycin was associated with more side effects and a longer hospitalization stay than erythromycin alone.30,32 in all cases of severe , life - threatening pneumonia , prompt administration of appropriate antibiotics is associated with improved outcomes . this case should urge every clinical doctor to study carefully the medical history of a patient and consider alternative diagnosis when the patient is not responding to the initial treatment . flouoroquinolones have established an equal if not superior therapeutic profile for legionellosis based upon published studies.2830 it should be mentioned at this point that a very important limitation of the study was that we were unable to take water samples from the spa in order to have a solid confirmation of the source of legionellosis . pcr is a useful tool in the hands of the clinical doctor and should be used where possible in suspicious cases , such as the above . also , early antibiotic treatment prevents the progression of the infection and should be administered upon admission . finally , flouoroquinolones , and in this case levofloxacin , should be considered an effective and efficacious treatment for legionellosis . written informed consent was obtained from the patient s next - of - kin for publication of this case report and any accompanying images .

introductionthere are at least 40 types of legionella bacteria , half of which are capable of producing disease in humans . the legionella pneumophila bacterium , the root cause of legionnaires disease , causes 90% of legionellosis cases.case presentationwe describe the case of a 60-year - old woman with a history of diabetes mellitus and arterial hypertension who was admitted to our hospital with fever and symptoms of respiratory infection , diarrhea , and acute renal failure . we used real - time polymerase chain reaction ( pcr ) to detect l. pneumophila dna in peripheral blood and serum samples and urine antigen from a patient with pneumonia . legionella dna was detected in all two sample species when first collected.conclusionsince legionella is a cause of 2% to 15% of all community - acquired pneumonias that require hospitalization , legionellosis should be taken into account in an atypical pulmonary infection and not be forgotten . moreover , real - time pcr should be considered a useful diagnostic method .

Introduction Case presentation Conclusion Introduction Case report Discussion Conclusion Consent

bat guano , an excrement of the cave - dwelling bats forms the basis of the ecology inside the cave by acting as a food source for detritivorous microbes . it contains high content of organic carbon , nitrogen , phosphate , and potassium , . bacteria present in bat guano were reported to be involved in nitrification process and were also known as potential chitinase producer . a clone library based study in bat guano samples has revealed the presence of group 1.1a and 1.1b crenarchaeota , an efficient ammonia oxidizer , . analyzing bat guano is also important since they often harbor various pathogens which can be thread for speleologists , and tourists . although the microbial communities in diverse cave ecosystems have been studied , little is known about the microbial communities of bat guano heaps , and there has been no studies using high throughput sequencing technology . meghalaya is known to possess the largest and most diverse karst caves in the world . pnahkyndeng cave located in ri - bhoi district of meghalaya , india is a home of various bats and offering an ideal environment for studying the bat guano microbiota without any anthropological influence . samples were collected on february 2014 from the bat guano of pnahkyndeng cave ( 255722.70n , 915543.10e ) , nongpoh , ri - bhoi district , india . ten composite guano samples were collected from different places of the cave floor and the soil community dna was extracted separately using the fast dna spin kit for soils ( mp biomedical , solon , oh , usa ) . the freshly extracted dna was purified twice using 0.5% low melting point agarose gel and mixed to prepare a composite sample . final dna concentrations were quantified by the using a microplate reader ( bmg labtech , jena , germany ) . the v4 region of the 16s rrna gene was amplified using f515/r806 primer combination ( 5-gtgccagcmgccgcggtaa-3 ; 5-ggactachvgggtwtctaat-3 ) . amplicon was extracted from 2% agarose gels and purified using the qia quick gel extraction kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's instructions . quality filtering on raw sequences was performed according to base quality score distributions , average base content per read and gc distribution in the reads . singletons , the unique otu that did not cluster with other sequences , were removed as it might be a result of sequencing errors and can result in spurious otus . chimeras were also removed using uchime and pre - processed consensus v4 sequences were grouped into operational taxonomic units ( otus ) using the clustering program uclust at a similarity threshold of 0.97 , . all the pre - processed reads were used to identify the otus using qiime program for constructing a representative sequence for each otus . the representative sequence was finally aligned to the greengenes core set reference databases using pynast program , . , 403,529 reads were classified at the phylum , 282,350 at the order , 188,406 at the family and 2926 sequences were classified at the species levels . classified otus belonged to 18 different phyla dominated by chloroflexi , crenarchaeota , actinobacteria , bacteroidetes , proteobacteria , and planctomycetes ( fig . 1 ) . analysis of bacterial communities revealed the two most dominant bacteria 's chloroflexi ( 29.97% ) and actinobacteria ( 22.55% ) , which are known to be a common inhabitant of cave microflora . other identified phyla include crenarchaeota ( 16.96% ) , planctomycetes ( 12.41% ) and proteobacteria ( 12.03% ) . chloroflexi was divided into 11 classes thermomicrobia , planctomycetia , gitt - gs-136 , ktedonobacteria , anaerolineae , tk10 , tk17 , s085 , chloroflexi , ellin6529 , and gitt - gs-136 . the most dominant otu within this phyla was denovo 317 , classified under the class thermomicrobia ( 40.52% ) followed by denovo 710 under the thermomicrobia ( 7.61% ) , denovo 235 under the genus thermogemmatisporaceae ( 7.18% ) and denovo 3 under the genus gemmataceae ( 2.98% ) . the dominant otu within this phyla was denovo 74 under the genus mycobacterium ( 29.39% ) followed by denovo 993 ( 8.27% ) and denovo 372 ( 5.27% ) classified under the genus acidimicrobiales and actinomycetales , respectively . only five otus were classified up to the species level ( mycobacterium llatzerense and mycobacterium celatum ) . a third dominant phylum in this sample was identified as planctomycetes comprising of 91 otus and 46,063 reads . seventeen archeal otus were classified under the order nrp - j , methanomicrobiales and methanosarcinales . four of them were identified at the genus level ( methanosarcina , haloquadratum , methanosaeta and methanocorpusculum ) . the phylogenetic tree based on the genus level relationships is provided as supplementary fig . 1 . previous study on archaeal communities present in bat guano identified many ammonia oxidizing bacteria but it was limited with a few number of clones . our analysis provides in - depth and high throughput identification of the bacterial communities present in bat guano . in the present study , we identified 18 bacterial phyla and most of the bacterial genus identified was known to be involved in nitrogen cycling as seen in previous study . a significant portion of otus still remains unclassified which indicates the possibility for the presence of novel species in pnahkyndeng cave . further studies like whole metagenome sequencing or functional metagenomics can illustrate the detailed information of this bacterial community .

v4 hypervariable region of 16s rdna was analyzed for identifying the bacterial communities present in bat guano from the unexplored cave pnahkyndeng , meghalaya , northeast india . metagenome comprised of 585,434 raw illumina sequences with a 59.59% g+c content . a total of 416,490 preprocessed reads were clustered into 1282 otus ( operational taxonomical units ) comprising of 18 bacterial phyla . the taxonomic profile showed that the guano bacterial community is dominated by chloroflexi , actinobacteria and crenarchaeota which account for 70.73% of all sequence reads and 43.83% of all otus . metagenome sequence data are available at ncbi under the accession no . srp051094 . this study is the first to characterize bat guano bacterial community using next - generation sequencing approach .

Direct link to deposited data Experimental design, materials and methods Nucleotide sequence accession number Competing interests

differential lipid composition between the apical and basolateral membrane domains of epithelial cell plasma membranes made it clear that membrane lipids are not laterally distributed in a homogeneous fashion . the lipid raft hypothesis was developed to explain lateral separation of bilayer lipids , and this idea quickly found applications in viral budding , endocytosis , and signal transduction ( reviewed in ) . in model membranes , lipids can separate into microscopically resolvable raft - like domains . plasma membrane surrogates formed by chemical membrane blebbing or cell swelling procedures also show phase behaviour [ 5 - 8 ] . similar domains are not evident upon direct observation of unperturbed plasma membranes in living cells , but the non - equilibrium nature of cell membranes , including endocytosis , exocytosis , and other motile processes , may prevent overt phase separation . likewise , quantitative analysis of lipid - anchored protein and lipid diffusion in cell membranes by fluorescence recovery after photobleaching ( frap ) , frster resonance energy transfer ( fret ) , and fluorescence correlation spectroscopy ( fcs ) [ 9 - 11 ] indicated that rafts in the plasma membrane of resting cells must be very small or ephemeral ( or both ) , forcing an evolution of the lipid raft hypothesis . these tiny clusters do not represent lipid phase separations but are probably short - range ordering imposed upon lipids by transmembrane proteins and cortical actin structures . thus , the current challenge for the field is to understand the interplay between protein and lipid that converts the exceedingly small , unstable clusters of components into larger , more stable membrane microdomains required for function . the recent development of sensitive quantitative microscopy methods has advanced our knowledge of lipid dynamics in resting cells . the diffusion of raft lipids ( e.g. , sphingomyelin ) and non - raft lipids ( e.g. , phosphatidylethanolamine ) was measured by an elegant fcs technique within regions as small as 30 nm in diameter using stimulation emission depletion fluorescence microscopy . the results indicate that raft lipids , but not non - raft lipids , are indeed preferentially trapped , albeit for short distances ( < 20 nm ) and for short periods ( 10 - 20 ms ) . homofret measurements , combining frap , emission anisotropy , and theoretical model fitting to test models of lateral organization in the membrane , were used determine the degree of clustering of glycosylphosphatidylinositol ( gpi)-anchored proteins in the plasma membrane . the formation of gpi - anchored protein nanoclusters ( of ~4 molecules or even less ) is an active process involving both actin and myosin , and these nanoclusters are nonrandomly distributed into larger domains of < 450 nm . additionally , high - speed single - particle tracking ( 50 khz ) revealed that gpi - anchored proteins , along with other membrane proteins , undergo rapid hop diffusion between 40 nm actin - regulated compartments , with a compartment dwell time of 1 - 3 ms on average . however , when gpi - anchored proteins were deliberately cross - linked by gold or quantum dot particles , they underwent transient confinement or stall ( stimulation - induced temporary arrest of lateral diffusion ) from a cholesterol - dependent nanodomain in a src family kinase mediated manner [ 17 - 19 ] . a recent study identified a transmembrane protein ( carboxyl - terminal src kinase [ csk]-binding protein ) involved in the linkage between the particle - cross - linked gpi - anchored protein , thy1 , and the cytoskeleton ( figure 1 ) . thy-1 crosslinking by streptavidin - coated quantum dots aggregates gpi lipid tails in the outer leaflet of the plasma membrane in a cholesterol - dependent manner . carboxyl - terminal src kinase ( csk)-binding protein ( cbp ) , a transmembrane protein , is recruited to or captured by thy-1 clusters along with src - family kinase substrates ( ks ) . cbp or ks ( or both ) are phosphorylated by src - family kinases ( sfk ) , enabling cbp to bind to actin filaments via an ebp50-erm ( ezrin / radixin / moesin - binding phosphoprotein 50-ezrin / radixin / moesin ) adaptor linkage resulting in a transient anchorage . when either cbp or the adaptors are dephosphorylated by an unspecified protein tyrosine phosphatase ( ptp ) the anchorage is terminated . the lipid envelope of influenza and hiv virions , but not those of the vesicular stomatitis virus ( vsv ) or semliki forest virus ( sfv ) , is enriched in raft - like lipids , leading to the notion that these viruses bud from lipid microdomains in the plasma membrane [ 21 - 25 ] . by contrast , the lipidomes of vsv and sfv are very similar to each other and to that of the plasma membrane suggesting that these viruses do not select or generate lipid raft domains for budding . the protein and lipid environment of the budding domains of hemagglutinin ( ha ) and hiv has been the source of several recent papers examining the process of viral budding using quantitative live - cell imaging techniques . influenza buds from ha clusters ( ranging up to micrometers in diameter ) regulated by ha transmembrane region length and palmitoylation . recent fluorescence lifetime imaging microscopy ( flim)-fret experiments in living cells indicate that ha colocalizes with lipid microdomain markers , further supporting the role of lipid - protein interactions in influenza virus budding . proton magic angle spinning nuclear magnetic resonance was used to detect a minor fraction ( ~10 - 15% ) of liquid - ordered membrane phospholipids in ha virions and virion lipid extracts at 37c . while lipid ordering increased at lower temperatures , it was not required for virion fusion with target membranes . progressive recruitment of cytoplasmic hiv-1 gag to the membrane , via post - translational acyl lipid modification and pip2/basic residue interactions , forms membrane domains that culminate in virion budding [ 31 - 33 ] . while the hiv-1 lipid envelope composition indicates enrichment in lipids and proteins associated with rafts , paradoxically , one report has failed to observe an enrichment of enhanced green fluorescent protein ( egfp)-gpi at gag domains in living cells , suggesting that the local lipid microenvironment may not exactly parallel the classic raft lipid composition . recent work has implicated the tetraspanin family of proteins in gag domain formation and function . tetraspanins , a widely expressed and highly conserved class of transmembrane proteins ( reviewed in ) , form tetraspanin - enriched microdomains ( tems ) through lateral tetraspanin - tetraspanin interactions and binding to non - tetraspanin membrane proteins . tetraspanins can be palmitoylated and the lipid environment within tems contains cholesterol , but gpi - anchored proteins and caveolin are not enriched in tems ( reviewed in ) . recently , cholesterol and tetraspanin palmitoylation were implicated in the confined diffusion and co - diffusion ( of two tetraspanin molecules ) of the tetraspanin cd9 . tetraspanins appear to induce order in the plasma membrane by virtue of protein clustering , but they likely also stabilize lipid microenvironments in the plasma membrane allowing for lateral organization of hiv-1 gag and virion budding . some lectin - based membrane domains form in the absence of post - translational lipid modifications or known lipid binding activity . dendritic cell - specific intracellular adhesion molecule-3-grabbing non - integrin ( dc - sign ) , a tetrameric c - type lectin with affinity for high - mannose glycans , forms microdomains on the plasma membrane [ 40 - 42 ] that serve as high - avidity binding sites for numerous pathogens . a previous report suggested that dc - sign interacts with lipid rafts , but this was based on detergent insolubility and cholera toxin colocalization assays , which generally do not faithfully report on intrinsic membrane lateral heterogeneity . also , dc - sign domains do not depend on cholesterol ( unpublished data ) . this result implies that dc - sign within domains does not exchange with the surrounding membrane . the source of this stability remains a mystery , and its cause may not reside in the membrane - apposed cytoskeleton but in extracellular cross - linking factors such as galectins ( reviewed in ) . dc - sign membrane domains that are multiplexed with another c - type lectin , cd206 ( unpublished data ) , appear to mediate the formation of fungipods , novel cellular protrusive structures involved in fungal recognition by dendritic cells ( figure 2 ) . thus , the lateral heterogeneity in membranes provided by rafts and other microdomains continues to provide surprising functional consequences . c - type lectins ( clrs ) form a type of plasma membrane domain that is not dependent on cholesterol . ( a ) plasma membrane domains containing mixtures ( yellow ) of dendritic cell - specific intracellular adhesion molecule-3-grabbing non - integrin ( dc - sign ) ( green ) and cd206 ( red ) are observed on a monocyte - derived dendritic cell ( dc ) by immunofluorescence . dc - sign domains are known sites of binding and entry for a range of pathogens including hiv-1 . ( b ) yeast cell wall material is sensed by these clr membrane domains , triggering a unique protrusive response , the fungipod . the image shows an example of a dc fungipod formed via cd206 ligation by a fixed saccharomyces cerevisiae particle ( zymosan ) , visualized by scanning electron microscopy ( 9500 ) . a variety of membrane domain forming systems have a wide gamut of lipid and protein constituents and possess a correspondingly broad range of functions . recent advances have shown that preferential lipid trapping or confinement in the resting plasma membrane occurs only on very small spatiotemporal scales . critical attention must be paid when determining if and when such confinement becomes biologically meaningful for processes such as endocytosis and signal transduction . while lipid ordering can be stabilized by oligomerization of membrane - associated proteins ( i.e. , gm1 crosslinking , influenza ha clustering ) , the lipids in these domains may still exchange between domain and surrounding membranes , making even these stabilized raft - like domains dynamic environments . at what point does a membrane domain become stable enough to be biologically relevant ? what is the range of protein and lipid turnover rates seen in membrane domains and are there different turnover rates for each constituent ? it is likely that a spectrum of membrane microdomains exists with different compositions and physical characteristics suited to their diverse purposes . the lipid species and their ordering within raft - like complexes appear to be key factors in determining intradomain cohesiveness and resultant domain size and lifetime .

evidence in support of the classical lipid raft hypothesis has remained elusive . data suggests that transmembrane proteins and the actin - containing cortical cytoskeleton can organize lipids into short - lived nanoscale assemblies that can be assembled into larger domains under certain conditions . this supports an evolving view in which interactions between lipids , cholesterol , and proteins create and maintain lateral heterogeneity in the cell membrane .

Introduction and context Major recent advances Future directions Competing interests

repetitive effortless vomiting is termed as rumination syndrome and is common in infants and mentally retarded individuals . it is associated with considerable morbidity in children who frequently miss the school , have history of multiple hospitalizations , and also suffer significant psychiatric morbidity . unfortunately , before reaching a correct diagnosis , a number of patients undergo unnecessary invasive testing and surgical treatments . symptoms are often confused with that of gastric motility disorders and diagnosis is often delayed due to poor awareness regarding adult rumination syndrome among physicians . here we are presenting a case that posed diagnostic confusion as already mentioned and showed improvement soon after the correct diagnosis was made . the main factors that helped in remission of symptoms were supportive psychotherapy and the firm attitude of the physician . a 26-year - old lady presented with her husband and mother with the complaints of recurrent and intractable vomiting since past 2 years . the vomiting was regular , occurring throughout the day , and it used to increase after meals . for the past 6 months , she was not able to hold even the liquid diet to the extent that few sips of water were sufficient to induce vomiting . along with vomiting , she also complained of episodic loss of consciousness , which she used to regain after 1 - 2 h. these episodes of unconsciousness specifically occurred when she was under some stress . besides this , she was not having any other complaint , e.g. , abdomen pain , fullness in the stomach after having meals , diarrhea or constipation , or swallowing difficulties . her history did not suggest any cardiac problem , focal neurological deficits , or convulsions . at the time of presentation , she was pregnant ( second trimester ) with normal fetal growth . her family history was unremarkable except that she had strained relations with her parents - in - law . though her husband was very supportive but for almost 1 year she was staying at her mother 's house due to this illness . since the onset of illness she underwent extensive laboratory investigations including upper gastro - intestinal endoscopy , barium meal , and mri brain but any of the tests did not reveal any finding that could explain the symptoms . results of her liver and kidney function tests were within normal limits since the onset of illness . in the past , she was treated with a number of drugs including antidepressants , antipsychotics , promethazine , proton - pump inhibitors , and prokinetics for optimal periods without any relief . her husband also took her for a vacation when suggested by a physician , but it also did not improve the situation . looking at such a long history , multiple consultations , and extensive laboratory work - up , she was interviewed in the presence of her husband . then it became clear that her vomiting was effortless ; in other words , she used to stand in front of a wash basin and without putting any finger or anything else in the mouth , used to expel all the food ( liquid or solid ) that she had . the vomiting act was not associated with lacrimation , salivation , cramping of the abdomen , cessation of respiration followed by hyperventilation , and the gag sound that is otherwise common during true vomiting . it always took her about half an hour to expel the food . to make the information clearer , she was made to drink a glassful of water and her vomiting was observed which confirmed the information provided by her husband . her general physical examination showed pale conjunctiva but we did not find any signs of induced vomiting . however , she was not meeting all criteria for the major depressive disorder according to dsm - iv tr . looking at the concern of her family members and her pregnancy , she was hospitalized and reassured . when she did not improve even after 2 days , in view of her pregnancy , oral feeding from a naso - gastric tube was started . to our surprise , she improved within few hours and started taking liquids orally without vomiting them . she also insisted on the removal of the naso - gastric tube , but due to our concerns for the maintenance of feeding , it was kept for 2 days . she kept on taking liquid diet orally as well as through the tube and the symptoms did not recur . now her tube was removed and she was insisted to have solid food . for the next 2 days , she even had solid food without vomiting anything ; at this juncture , supportive psychotherapy was started . she was discharged after 3 days with the advice to attend regular supportive psychotherapy sessions . the diagnosis often poses a challenge to the physicians , especially when encountered in adults with normal intelligence and is often confused with gastroparesis and gastroesophageal reflux . in these adults , it is commonly associated with psychiatric morbidity in themselves or in their family members . one common psychiatric problem is personality disturbances with high scores on hypochondriasis and depression sub - scales . previous literature suggests that these patients often show multiple physician consultations and diagnosis is often delayed by years , and the food is usually expelled within few minutes of having it . could find manometric abnormality only in one - third of patients while levine et al . the present case , gastroscopy , barium meal and mr scan of head were done before presenting to us . however , none of them showed any abnormality . in view of the available literature and her pregnancy the treatment of adult rumination syndrome consists of reassurance , behavior therapy , psychotherapy , and relaxation therapies . these may improve a number of patients while others suffer persistence of symptoms.[36 ] nissen fundoplication has been reported to provide complete relief in the symptoms in one report . this patient was also shown to a number of physicians and was given all kinds of medical treatment including gastrokinetics , antacids , antidepressants , and benzodiazepines without any improvement . the relief was obtained with reassurance , supportive psychotherapy , and our firm attitude to take care of the patient 's health . in conclusion , adult rumination syndrome may be more prevalent than it appears and adequate knowledge of this syndrome among the physicians is necessary to reach to a diagnosis and early intervention . in addition , further studies are required to assess therapeutic benefits of various modalities in this entity .

rumination syndrome is known to exist in infants and mentally retarded adults since long time . in past few years , some reports appeared that showed its existence in adult patients also . it is frequently confused with the intractable vomiting in adults and misdiagnosis leads to delay in appropriate management . we are here describing the case of a female patient with rumination syndrome where specific points in the history delineated the presence of this illness and helped in appropriate management . the patient became symptom free soon after the diagnosis was reached .

INTRODUCTION CASE REPORT DISCUSSION

an atlanto - axial synovial cyst is very rare . since the first report of this lesion by onofrio and mih1 ) in 1988 , to our knowledge . however , the current report described a patient with a large hemorrhagic cystic mass which was seen around prevertebral space of the atlantoaxial joint on the left side and the obstruction of the nasopharyngeal cavity on cervical magnetic resonance image ( mri ) . we report a symptomatic case associated with rheumatoid atlanto - axial subluxation , which regressed after the surgical management . a 72-year - old woman presented with sudden severe headache in her left occipital area with dyspnea . a laboratory examination of the patient 's blood revealed no remarkable abnormality indicative of inflammation , but high - titer of rheumatoid factor ( 95.9 iu / ml ) . seven months ago before visiting to our department , she checked brain mri due to sudden attack of severe headache on left occipital area , which revealed non - specific lesion except rheumatoid pannus with small prevertebral cyst of c1 - 2 junction ( fig . , there was a large hemorrhagic cystic mass around prevertebral space of the atlanto - axial joint on the left side , obstructing the nasopharyngeal cavity on cervical mri ( fig . the cystic mass was connected to atlanto - axial joint capsule on axial view of computed tomography ( ct ) . multiple bony erosion , rheumatoid atlanto - axial instability including left tilted c1 - 2 subluxation and cranial settling were demonstrated . and there were loss of lordosis and retrolisthesis in c 3 - 4 , 4 - 5 ( fig . 3 ) . in the first operation stage , considering patient 's dyspnea , aspiration of the cystic lesion was performed via transoral approach with otolaryngology surgeon ( fig . after 0.5 cm - sized mucosal incision on left side oropharyngeal wall , residual material of the cyst was squeezed by forceps and removed by suction tools . in the second operation stage , there were rheumatoid atlanto - axial instability and retrolisthesis in c 3 - 4 , 4 - 5 , so it was followed by posterior occipito - cervical fusion that connected from occiput to c5 , using vertex screw & rod system and iliac bone graft ( fig . 5 ) . the patient was tolerable on her postoperative course and showed good respiration and relieved headache . cervical synovial cysts are rare and located at the c1 - 2 junction or lower cervical spine . only 24 cases of synovial cysts of the c1 - 2 junction have been reported in the literature5 ) . moreover , the pathogenesis of spinal synovial cysts remains unclear but is thought to be attributable to degenerative changes of the facet joints or excessive joint motion2,3,5,7 ) . it revealed multiple bony erosions and a large pannus formation of c1 - 2 junction on this case . this patient also showed atlanto - axial instability caused by rheumatoid arthritis , such as left tilted antlanto - axial subluxation and basilar impression . the atlantoaxial articulation is a true synovial joint and is responsible for a large proportion of normal cervical mobility . the etiology of articular cysts is unclear , but they are assumed to be degenerative because minor chronic damage to articular surfaces produces a reactive proliferation of synovium or fibrocartilage that includes loculated collections of mucinous fluid . so , we hypothesized that hemorrhagic event was developed as a result of microtrauma caused by rheumatoid atlantoaxial instability . it is known that the higher titer of rheumatoid factor , the more destructive manifestations of joint occur1 ) . the patient was in the controlled state in serologic inflammatory marker , such as esr(erythrocyte sedimentation rate ) and crp ( c - reactive protein ) . however , the high titer of rheumatoid factor , in spite of long standing medication of rheumatology , was related factor of progressive destruction of joints . we report a rare case of large hemorrhagic cyst on prevertebral space of left side c1 - 2 area associated with rheumatoid arthritis causing airway obstruction and left occipital pain , successfully managed anterior and posterior approach . we suggest repeated microtrauma due to atlanto - axial subluxation associated with rheumatoid arthritis as a main cause of hemorrhagic event on the cyst .

synovial cyst on prevertebral space of c1 - 2 joint is rare but may be associated hemorrhagic event . we describe a case of a 72-year - old woman who presented with sudden severe headache in her left occipital area with dyspnea . she had rheumatoid arthritis for 14-years . large hemorrhagic cystic mass was seen around prevertebral space of the atlantoaxial joint on the left side on cervical mri ( magnetic resonance image ) and it obstructed the nasopharyngeal cavity . aspiration of the cystic lesion was performed via transoral approach , followed by posterior occipito - cervical fusion . the specimen was xanthochromic , suggesting old hemorrhage . the patient was tolerable on her postoperative course and showed good respiration and relieved headache . we suggest that repeated microtrauma due to atalantoaxial subluxation associated with rheumatoid arthritis as a main cause of hemorrhagic event on the cyst .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

methanol has the potential to be an efficient fuel for direct methanol fuel cells ( dmfcs ) with many applications ranging from small portable devices to large stationary power plants . however , one major challenge that dmfc is facing is the fragility of the proton exchange membrane which is very thin in a dmfc . the thickness is typically less than 100 m . a tiny crack or defect could fail the whole fuel cell . ionic liquids ( ils ) can be a replacement of the conventional proton exchange membrane to overcome the risk of fragility . used as the electrolyte , ionic liquids possess many advantages such as high ionic conductivity , chemical stability , and resistance to high temperature . the current generation ionic liquids are stable even in the environment of moisture and air , making them ideal for general use as electrochemical media [ 3 , 4 ] . in developing il - based dmfc , the concentration of methanol must be controlled and monitored at several locations : the fuel feed , the electrolyte , the electrode assembly , and so forth . it is critical to develop a fast sensing technique for methanol in the environment of ionic liquids . in this paper the technique is based on potential step analysis and is reliable even when water is present in the ionic liquids . chemicals and electrodes were purchased directly from the manufacturers or from venders such as fisher scientific and vwr : 1-butyl-3-methylimidazolium tetrafluoroborate ( basf quality , 98% ) from aldrich , methanol ( reagent grade acs ) from pharmco - aaper , phosphate buffer saline ( 25x ) from thermo scientific , l(+)-ascorbic acid ( 99% ) from acros , potassium tetrachloroplatinate ( ii ) 98% from aldrich , sulfuric acid 2.0 n from labchem , and gold disk electrode ( 2 mm dia . ) from ch instruments . 1-butyl-3-methylimidazolium tetrafluoroborate ( bmimbf4 ) is one of a few ionic liquids with relatively small viscosity , but it is still very viscous when compared to aqueous solutions . the presence of trace amount of less viscous methanol may reduce the viscosity or increase the conductivity significantly . therefore , our most straightforward thinking was to sense methanol by the change in conductivity . our thought was confirmed as the conductivity of bmimbf4 showed a linear relationship with the concentration of methanol in it . as shown in figure 1 , the conductivity would be a good indicator of methanol content if there was no interference of water . secondly , water is produced on the cathode and it could cross over to the electrolyte and to the anode ( 2 ) . thirdly , ionic liquids including bmimbf4 absorb water from the environment and the water content in bmimbf4 could reach as high as 0.29 wt% : ( 1)anode : ch3oh+h2oco2 + 6h++6e ( 2)cathode : 32o2 + 6h++6e3h2o water will also affect the conductivity of ionic liquids as reported before . for this reason , the conductivity sensing method has little use in a dmfc . a practical method must be water - resistant . we found a potential step method with pt - nanoparticle - coated au - nanoporous film ( pgnf ) was a reliable sensing method for methanol even when water was present . methanol was oxidized on pgnf electrode when the potential step was applied ( 1.9 v versus ag / agcl with saturated kcl solution ) . the current became smooth after 1 second and it was still measurable with amplitude of 175 a after 2 seconds . our previous study has found that the adsorption / desorption and double layer charging were dominant within the first 2 seconds of potential step . in the following report , the potential was fixed at 1.9 v versus ag / agcl ( saturated kcl ) and the sampling time at 2 seconds . under the small a / v ( electrode area to electrolyte volume ) condition and with a planar electrode , the current in a single potential step chronoamperometry can be predicted according to the cottrell equation : ( 3)it = nfad01/2c01/2t1/2 . in ( 3 ) , n is the number of electrons involved in redox reaction , f is the faraday constant , a is the electrode area , d 0 is the diffusion constant , c 0 is the bulk concentration , and t is the sampling time . if t is fixed in the potential step analysis , the sampling current i(t ) should be directly proportional to the concentration c 0 or the concentration of methanol in bmimbf4 . the current at the sampling time ( 2 seconds ) at various concentrations of methanol in bmimbf4 is shown in figure 3 . the sampling current did not change too much when the methanol concentration was smaller than 1 m. desorption / adsorption and double layer charging were predominant at the sampling time when the concentration was low . when the concentration was above 1 m , the electrooxidation of methanol became the predominant process and the sampling current had a linear relationship with the methanol concentration . the potential step method described above not only had a good linearity but also was waterproof . when water was added to the methanol solution in bmimbf4 , it did not interfere with the oxidation of methanol . the only effect of water was that it changed the molarity of methanol in bmimbf4 . as shown in figure 4 , the linearity was not destroyed by the presence of water , even when the water content was as high as 2.4 m. conductivity monitoring was a reliable method in sensing methanol in ionic liquids , but its use was limited to the situation without the presence of water . a potential step method with properly selected potential step and sampling time could be a more practical method to analyze methanol concentration in ionic liquids especially when the methanol concentration was high so that the sampling current was predominantly contributed by the methanol oxidation .

the development of direct methanol fuel cells required the attention to the electrolyte . a good electrolyte should not only be ionic conductive but also be crossover resistant . ionic liquids could be a promising electrolyte for fuel cells . monitoring methanol was critical in several locations in a direct methanol fuel cell . conductivity could be used to monitor the methanol content in ionic liquids . the conductivity of 1-butyl-3-methylimidazolium tetrafluoroborate had a linear relationship with the methanol concentration . however , the conductivity was significantly affected by the moisture or water content in the ionic liquid . on the contrary , potential step could be used in sensing methanol in ionic liquids . this method was not affected by the water content . the sampling current at a properly selected sampling time was proportional to the concentration of methanol in 1-butyl-3-methylimidazolium tetrafluoroborate . the linearity still stood even when there was 2.4 m water present in the ionic liquid .

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

epithelial monolayer ( em ) is the largest body tissue lining many organs in the human body . in the intestine , em provides protection to the internal body from toxic and infectious agents while at the same time it facilitates absorption of digested food and water from the gut . epithelial monolayer integrity and paracellular transport are the important features that can be protected and maintained with the help of epithelial barrier function . epithelial cells are connected with each other by four types of junctions , that is , desmosomes , gap junctions , adherens junctions , and tjs [ 24 ] . tight junctions are impermeable and control the movement of molecules and ions via a paracellular pathway . until recently , tight junction functions were categorized as fence as they separate the apical and basolateral cell surface domain defining cell polarity or a barrier due to their control over solutes and liquid flow through the paracellular space between the epithelial cells [ 58 ] . however tjs are not restricted to the fence and barrier function but have been defined to participate in signal transduction processes , gene expression , cell proliferation , and differentiation [ 911 ] . various unidentified external and internal regulators impair the normal function of tjs causing loss of water and solute in the passive manner that leads to leaky - flux watery diarrhea . tight junction ( tj ) is a complex structure constituting of growing numbers of components , including integral membrane proteins ( claudins , occludin , and junctional adhesion molecules jams ) and peripheral membrane proteins . the peripheral membrane proteins include ( 1 ) scaffold pdz ( postsynaptic density protein ( psd95 ) , drosophila discs large tumor suppressor ( dlg1 ) , and zonula occludens-1 protein ( zo-1 ) ) , multi - pdz domain protein-1 ( mupp-1 ) , and membrane - associated guanylate kinase ( magi-1 ) ; ( 2 ) no - pdz expressing proteins such as cingulin , symplekin , atypical protein kinase c , ras - related protein rab-3b ( rab-3b ) , ras - related protein rab-13 ( rab-13 ) , phosphatase and tensin homolog ( pten ) , and 7h6 antigen ; ( 3 ) cell polarity molecules asip / par-3 , partitioning defective 6 homolog alpha ( par-6 ) , and pals1-associated tj protein ( patj ) [ 13 , 14 ] . besides these proteins , tricellulin protein has recently been identified at the epithelial cell junctions with involvement in the barrier function . claudin family so far includes 24 reported members in different types of mammalian cells ; among them 21 are known components of tj in em in the kidneys , liver , brain , and intestine . these are involved in various physiological processes such as regulation of paracellular permeability and conductance . claudins are found in homo and heterotypic manner in single tj [ 13 , 26 ] . they can be divided into two main categories , pore - sealing and pore - forming claudins . claudin-1 , -3 , -4 , -5 , -7 , and -19 are known as pore - sealing claudins and an increased expression of these claudin proteins leads to increased tightness of em and increased transepithelial electrical resistance ( teer ) and decreases solute permeability across the monolayer [ 2731 ] . on the other hand , claudin-2 and -15 are considered as the pore - forming claudins , because of their ability to form paracellular anion / cation pores as well as water channels and therefore they decrease epithelial tightness and increase solute permeability [ 13 , 32 ] . epithelial barrier dysfunctions occur in inflammatory bowel diseases ( ibds ) like crohn 's disease ( cd ) or ulcerative colitis ( uc ) that contribute to leaky - flux diarrhea , that is , loss of solutes and water in increased amount dependent upon the components of tj proteins . downregulation of pore - sealing claudins ( e.g. , 4 , 5 , and 8) while upregulation of pore - forming claudin-2 is observed in active crohn 's disease patients [ 33 , 34 ] . numerous studies have reported leaky diarrhea in patients undergoing immunosuppressive therapy after organ transplantation [ 3538 ] . recently our group has reported mycophenolic acid- ( mpa- ) mediated increased expression of myosin light chain kinase ( mlck ) , myosin light chain-2 ( mlc-2 ) , and mlc-2 phosphorylation and redistribution of zo-1 and occludin in caco-2 and in hek-293 cells [ 39 , 40 ] as a possible mechanism of diarrhea in patients undergoing immunosuppressive therapy . transcription factors ( tfs ) play an important role in the gene regulation at the promoter level working either as an activator or as a repressor of a specific gene . the current review will focus major claudins family members ( table 1 ) and their regulators , which alter claudins gene activity at promoter level and therefore modulate tjs structure and function . claudin-1 protein is a key constituent of tjs and its altered expression is reported in a variety of cancers , most prominently colorectal cancers [ 13 , 17 , 41 , 42 ] . promoter region ( 1160 bps to 850 bps ) of claudin-1 consists of putative binding sites for caudal - related homeobox ( cdx-1 , -2 ) , gata4 , and t - cell factor / lymphoid enhancing factor-1 ( tcf / lef-1 ) transcription factors . there is a direct correlation between claudin-1 and cdx-2 expression in human colon cancer patient . cdx-2 is a homeobox domain - containing nuclear transcription factor that plays an important role in intestinal development by regulating the proliferation and differentiation of intestinal cells [ 4345 ] , and it is expressed in all cells along the crypt villus axis . cdx-2 transcriptional activity is controlled through mitogen - activated protein kinase / extra cellular signal regulated kinase pathway ( mapk / erk pathway ) which phosphorylates it at ser-60 position and resultantly reduces cdx-2 transcription activity in crypt and lowers villus cells . on the other side , cyclin - dependent kinase 2 ( cdk2 ) phosphorylates cdx-2 at ser-281 which coordinates cdx-2 polyubiquitination and degradation by the proteasome [ 43 , 4649 ] . specificity protein-1 ( sp-1 ) is the first identified transcription factor of specificity protein / krppel - like factor ( sp / xklf ) family , consisting of 785 amino acids ( aa ) with molecular weight of 100 to 110 kda . sp-1 's dna binding domain is the most conserved among other domains of sp family members which consisted of cy2his2 zinc ( zn ) fingers . mutational analysis has revealed that zn fingers 2 and 3 are essential for dna binding activity . sp-1 binds to the gc - rich elements that are common regulatory elements in the promoters of numerous genes . sp-1 binds its individual binding sites as a multimer and is capable of synergic activation of promoters containing multiple binding sites and regulates transcription by dynamically recruiting and forming complexes with many factors associated with transcription . normally sp-1 has been described as a transcriptional activator but it can also act as a repressor . claudin-1 promoter region ( 138 to 76 bp ) contains sp-1 binding site and a mutation in this region results in a significant loss of claudin-1 transcription . claudin-2 , also known as leaky protein , forms paracellular water channels in tjs and mediates paracellular transport of water molecules across the em . it is also involved in many signaling pathways , including vitamin d receptor , epidermal growth factor receptor ( egfr ) , and c - jun n - terminal kinases ( jnk ) signaling pathways , and contributes to inflammatory bowel disease and colon cancer [ 33 , 5558 ] . salmonella infection facilitates bacterial invasion across the em by inducing claudin-2 expression and altering its localization in tjs which is reversible by specific inhibitors ( egfr ( gefitinib ) and jnk ( sp600125 ) ) , making claudin-2 as a potential therapeutic target to prevent bacterial invasion and inflammation . interleukin-6 ( il-6 ) increases tj permeability of caco-2 monolayer from the basal side by inducing caludin-2 expression . il-6 activates the mitogen - activated protein kinases / extracellular signal - regulated kinases ( mek / erk ) pathway by inducing phosphorylation of erk and phosphatidylinositol 3-kinase ( pi3k / akt ) by phosphorylating akt , which in turn enhances cdx-2 expression . in the claudin-2 promoter region ( 1067 to 1 ) , four cdx-2 ( cdx - a , -b , -c , and -d ) , stat , and nuclear factor - kappa - light - chain - enhancer of activated b cells ( nf-b ) putative binding sites are identified . il-6 induced expression of claudin-2 can be reversed by using either specific inhibitors of mek / erk and pi3k / akt pathways ( u0126 ( a mek inhibitor ) and ly294002 ( a pi3k inhibitor ) ) or site directed mutagenesis in the putative cdx-2 binding sites in the promoter region of claudin-2 gene . a sp-1 binding site ( 112 and 74 bps ) in the promoter region of claudin-3 is crucial for its activation . claudin-3 expression is significantly decreased at mrna and protein levels , by knocking down the sp-1 with sirna , indicating an essential role of sp-1 in claudin-3 activation . claudin-4 is mainly expressed in the em of colon , renal tubules , mammary gland , and thyroid gland and is considerably raised in their cancers . there are two known sp-1 binding sites ( between 105 and 49 bps ) in the promoter region of claudin-4 . caludin-5 is mostly expressed in the tjs of em of pancreatic acinar cells , alveolar lung cells , colon , and endothelial cells forming the blood - brain barrier and endoneurial blood - nerve barrier . in colonic regions , its expression is mainly involved in the paracellular sealing of tjs [ 33 , 6163 ] . both downregulation and redistribution of claudin-5 can alter tjs structure leading to barrier dysfunction in active crohn 's disease . forkhead box ( foxo ) gene family members are potent transcriptional activators with four known members ; foxo1 ( also known as foxo1a ) , foxo3 ( also known as foxo3a ) , foxo4 , and foxo6 which bind to conserved consensus core recognition motif ttgtttac [ 6466 ] . four pairs of putative binding sites for foxo and tcf--catenin ( tcf--catenin act as a stabilizer ) are identified in the three regions of claudin-5 promoter ( region 1 , position 2,906/2,871 ; region 2 , position 2,317/2,287 ; region 3 , position 1,103/1,008 ) . both foxo1 and e74-like factor 3 ( elf3 ) belongs to e26 transformation - specific sequence ( ets ) family of transcription factors and binds to the ets binding site in the promoter region ( 150 bps ) of claudin-7 . members of ets family are mainly involved in cell differentiation , proliferation , and cell transformation . regulation of the target genes by ets factors depends upon their activation by mapk and their association with other cofactors [ 68 , 69 ] . an essential role of elf3 is reported in epithelial cell differentiation [ 7072 ] and small interference rna ( sirna ) treatment downregulates the claudin-7 expression validating the central role of elf3 in claudin-7 activation . claudin-15 is a pore - forming protein expressed in the em of intestine , liver , and kidney tissues . four putative binding sites ( bs1 - 4 ) of transcription factor hepatocyte nuclear factor 4 alpha ( hnf4 ) are present in the ( 693 to 47 bps ) region of claudin-15 promoter . hnf4 is considered as an important regulator of em barrier integrity and is involved in the regulation of metabolism , cell junction , differentiation , and proliferation of liver and intestine epithelial cells . both animal model and ibd patients ' biopsy studies have shown that an altered expression of hnf4 directly influences the expression and distribution of claudin-15 . 5060% of filtered mg , and 3035% of filtered ca are reabsorbed into the body by thick ascending limb , the loop of henle . claudin-16 and -19 play a main role in the regulation of mg reabsorption and loss of either claudin-16 or -19 leads to excessive renal waste of mg . four putative transcription factor ( not characterized , ap2 , nf - e , and sp-1 ) binding sites are located between 139 and 75 in the promoter region of mouse claudin-19 . however , only sp-1 is described for having an important role in the expression of claudin-19 and a mutation in sp-1 binding site significantly reduces the claudin-19 expression . tight junctions play an important role in the regulation of paracellular movement of molecules across the em , impart mechanical strength , maintain the polarity of cells , and prevent the passage of unwanted molecules and pathogens through the space between the plasma membranes of adjacent cells . the efficiency of the junction in preventing ion passage increases exponentially with the number of strands of claudins family proteins which are having important role in the structure as well as controlling paracellular movement across the tight junctions . altered expression of claudins family proteins in tjs plays a key role in numerous abnormalities like cancers , ibds , and leaky diarrhea and a better understanding of their regulatory mechanism could help in designing innovative therapeutic strategies .

human gastrointestinal tract is covered by a monolayer of specialized epithelial cells that constitute a protective barrier surface to external toxic and infectious agents along with metabolic and digestive functions . intercellular junctions , among epithelial cells , such as desmosomes , adherens , gap , and tight junctions ( tjs ) , not only provide mechanical integrity but also limit movement of molecules across the monolayer . tj is a complex structure composed of approximately 35 different proteins that interact with each other at the apical side of two adjacent epithelial cells . claudin family proteins are important members of tj with so far 24 known isoforms in different species . claudins are structural proteins of tj that help to control the paracellular movement by forming fence and barrier across the epithelial monolayer . altered function of claudins is implicated in different form of cancers , inflammatory bowel diseases ( ibds ) , and leaky diarrhea . based on their significant role in the molecular architecture of tj , diversity , and disease association , further understanding about claudin family proteins and their genetic / epigenetic regulators is indispensable .

1. Introduction 2. Claudin-1 3. Claudin-2 4. Claudin-3, Claudin-4, and Claudin-5 5. Claudin-7 6. Claudin-15 7. Claudin-19 8. Conclusion

drug induced liver injury ( dili ) is a major issue worldwide , both for patients and health providers . it is one of the primary causes for attrition during clinical and preclinical studies and the main reason for drug withdrawal from the market . dili is divided into types , ( i ) hepatocellular , ( ii ) cholestatic , or ( iii ) mixed ( hepatocellular and cholestatic ) , according to the type of liver damage and the clinical chemistry biomarker alterations . the cholestatic and mixed hepatocellular and cholestatic type are the two most severe manifestations of dili and yield almost half of the recorded cases of dili . cholestatic liver injury , or more simply cholestasis , is the disruption of the bile flow , which might be either due to biliary tract obstruction or to complications in bile acid uptake . while the mechanistic basis for hepatocellular dili is still a mystery for the majority of the cases , more knowledge exists for cholestatic dili . there is growing evidence for a vast amount of cholestasis cases pinpointing the important role of hepatic transporters . basolateral transporters are responsible for the uptake of drugs and other endobiotics and xenobiotics from the blood , influencing the exposure of the hepatocyte to potential damage . canalicular transporters regulate the hepatic clearance , as well as the secretion of bile salts and bile conjugates into bile . any disturbance of the transporters physiological function may result in the accumulation of potentially harmful bile products that can finally cause cholestasis.figure 1 provides an overview on the respective location of hepatocyte transporters . blue symbols represent mainly the canalicular transporters , and red symbols , the basolateral ones . mrp16 multidrug resistance - associated proteins 16 , ost/ost organic solute transporter , bsep bile salt export pump , bcrp breast cancer resistance protein , mate1 multidrug and toxin extrusion transporter 1 , abcg5/g8 atp - binding cassette subfamily g member 5/8 , mdr3 multidrug resistance protein 3 , p - gp p - glycoprotein , atp8b1 atpase - aminophospholipid transporter , oatp organic anion transporting polypeptide , ntcp sodium ( na ) taurocolate cotransporting polypeptide , oct organic cation transporter 1 , oat organic anion transporter . the most important one , due to its pivotal role in bile salts clearance , is the bile salt export pump ( bsep ) . apart from bsep , there is evidence for the implication of other canalicular efflux transporters such as the multidrug resistance - associated protein 2 ( mrp2 ) , breast cancer resistance protein ( bcrp ) , multidrug resistance protein 3 ( mdr3 ) , and p - glycoprotein ( p - gp ) . mdr3 functions as an atp - dependent phospholipid flippase , translocating phosphatidylcholine from the inner to the outer canalicular membrane . canalicular phospholipids are then solubilized by canalicular bile salts to form mixed micelles , protecting cholangiocytes from the detergent properties of bile salts . while p - gp is also not transporting bile salts , it is implicated in cholestasis because of its large amount of substrates and inhibitors which cause drug drug interactions that disrupt the smooth function of the hepatocyte . the basolateral transporters play also an important role , both the uptake transporters , such as organic anion transporting polypeptides 1b1 , 1b3 , and 2b1 ( oatp1b1 , 1b3 , and 2b1 ) and sodium ( na ) taurocolate cotransporter ( ntcp ) , and the efflux transporters , like multidrug resistance - associated protein 3 and 4 ( mrp3 and mrp4 ) . in particular , in cases of cholestasis , the basolateral uptake transporters ntcp and oatp1b1 have been found down - regulated . however , in this case , oatp1b3 is up - regulated as a compensatory mechanism for the elimination of xenobiotics from sinusoidal blood . on the contrary , in cases of cholestasis , mrp3 and mrp4 are up - regulated to facilitate the efflux of the toxic bile salts out of the hepatocyte . thus , simultaneous inhibition of several of these transporters could induce drug toxicity due to inadequate elimination from the blood or increase the cholestatic effect due to accumulation of bile salts in the hepatocyte . consequently , drug - induced liver injury and cholestasis are important toxicity alerts to be considered in drug development . interestingly , there are only a few computational studies for the prediction of cholestasis reported in literature . with respect to the involvement of hepatic transporter , there are some in vitro studies correlating cholestasis with transporter inhibition , such as bsep , mrp3 , mrp4 , and ntcp . also several in silico studies for the identification of potentially cholestatic compounds via modeling of transporters and then associating them with the cholestatic effect of their inhibitors have been conducted . a characteristic example is the study by greupink et al . in 2012 , who developed a pharmacophore approach for ntcp in order to identify potentially ntcp inhibitors . under the same principles , in 2014 ritschel and colleagues performed a 3d ligand - based pharmacophore model for bsep inhibition . however , in most of these cases the amount of validated drugs is small and what is basically described is the association between transporter inhibition and cholestasis . thus , as the respective is associated with cholestasis , it is assumed that an inhibitor is causing cholestasis . most recently , muller et al . , in order to model dili , also modeled some more hepatotoxicity end points , including cholestasis . moreover , mulliner et al . presented a multilevel modeling approach for dili , where cholestasis was also included as a morphological hepatobiliary finding . however , examining the liver transporters contribution was not within the scope of their work . dilisym ( www.dilisym.com ) is a mechanistic mathematical model of dili , that has been used to investigate the effects of bsep inhibition on drug - induced liver injury , as well as on bile acid - mediated dili . developed a multiscale , liver - centric in silico modeling framework for acetaminophen pharmacology and metabolism that can be extended in predicting hepatotoxicity due to acetaminophen overdosing . in this study we present a classification scheme in order to predict cholestasis from a public data set , using physicochemical descriptors as well as predicted transporter inhibition profiles . for the latter we used our in house classification models for bsep , bcrp , p - gp , oatp1b1 , and oatp1b3 . for compiling the dili training data set we searched in pubmed ( http://www.ncbi.nlm.nih.gov/pubmed ) , google , scopus ( https://www.scopus.com/ ) , and the sider database v2 using the search terms : drug - induced cholestasis or the retrieved publications were then investigated manually for human data , i.e. compounds that are positive or negative for drug - induced cholestasis in humans . those compounds in principle drugs were added to those obtained from the sider v2 database . unfortunately , cholestasis is an end point that is not widely examined in terms of experimental or in silico studies that would potentially guide us to big data sets . thus , even though we were able to compile several drugs positive for cholestasis , there was almost no information in terms of the negatives . on the other hand , dili in general is studied quite extensively and there are several respective data sets . since choleastasis is a possible manifestation for dili , we can consider safely that any compound negative for dili will definitely be also negative for cholestasis . thus , the negative compounds for dili that we had compiled and curated in a previous work were also used as negatives for this study . the data set was carefully curated according to the following rules : all inorganic compounds were removed based on their chemical formula in moe 2014.09.salt parts and compounds containing metals and/or rare or special atoms were removed and the chemical structures were standardized using the standardiser tool created by francis atkinson.duplicates and permanently charged compounds were removed using moe 2014.09 . here we have to note that stereoisomers , even if biologically can be considered as different compounds , were considered as duplicates , since they give the exactly same vector of descriptors . if two ( or more ) stereoisomers are of the same class , only one was kept . if they were of different classes , they were all removed.3d structures were generated using corina ( version 3.4 ) , and their energy was minimized with moe 2014.09 , using default settings , but changing the gradient to 0.05 rms kcal/(mol a ) . all inorganic compounds were removed based on their chemical formula in moe 2014.09 . salt parts and compounds containing metals and/or rare or special atoms were removed and the chemical structures were standardized using the standardiser tool created by francis atkinson . here we have to note that stereoisomers , even if biologically can be considered as different compounds , were considered as duplicates , since they give the exactly same vector of descriptors . if two ( or more ) stereoisomers are of the same class , only one was kept . 3d structures were generated using corina ( version 3.4 ) , and their energy was minimized with moe 2014.09 , using default settings , but changing the gradient to 0.05 rms kcal/(mol a ) . in addition , the existing chirality was preserved . after these curation steps 152 compounds remained as positives for cholestasis . the negatives for dili , and subsequently for cholestasis , were 466 compounds . however , when uniting the data , there were compounds with contradictory class assignments . these compounds were removed from the data set , yielding a data set of in total 578 compounds ( 131 positives and 447 negatives ) . the compiled data set is provided in the supporting information . recently and after having already compiled our training set for cholestasis and developed the respective model a data set covering multiple levels of hepatotoxicity was published by mulliner and co - workers . the data are hierarchically clustered by the authors into three levels of hepatotoxicity : level 0 corresponds to general hepatotoxicity , level 1 corresponds to clinical chemistry findings and morphological finding as distinguished parts of general hepatotoxicity , and level 2 discriminates both clinical chemistry and morphological findings into hepatocellular and hepatobiliary injury . we use only clinical data , i.e. the human data , of morphological findings for hepatobiliary injury as an external test set for validating the developed cholestasis model . once more , we performed chemical data curation and removed the compounds overlapping with the training set , which led to 1347 compounds ( 230 positives and 1117 negatives ) as an external test set . we also merged the training with the test set and tried to generate a model based on the united data . the merged data set comprises 1904 compounds : 355 positives and 1549 negatives for cholestasis . here we must note that the total number of compounds in the merged data set is not the same as the sum of the compounds of training set and test set . the reason is that when removing the overlapping compounds between training set and test set , all of the compounds were removed from the test set , since we had already selected the final model for cholestasis . from those overlapping compounds , this did not matter so much , since they were totally removed from the test set . however , when merging the two data sets for modeling , we did not want to decide regarding the class of those compounds . for both data sets , several types of molecular descriptors have been calculated , such as all 192 2d moe descriptors , the 3d volsurf series of descriptors , as well as ecfps ( extended connectivity fingerprints ; ecfp6 ) , using rdkit ( http://www.rdkit.org/ ) . the list of the final descriptors used for the proposed model is given in the supporting information ( table s1 ) . in addition to this , predicted hepatic transporter inhibition profiles were also included in the list of descriptors . the transporters investigated comprise bsep , p - gp , bcrp , oatp1b1 , and oatp1b3 . in particular , for basolateral transporters we calculated the predictions for four in silico classification models built upon padel descriptors for oatp1b1 and oatp1b3 inhibition . for obtaining the predictions we use the models version implemented in etoxlab , an open source modeling framework for implementing predictive models . out of each model we use the sum of these binary scores , denoted sum binary score . the sum binary score can take values between 0 ( if all models predict the compound as negative ) and 4 ( if all models predict the compound as positive ) . for basolateral transporters a more thorough description of the transporters inhibition models is provided in the supporting information ( table s2 ) , where the size of the training set , the inhibition threshold of the training set , and the algorithm and performance of each individual model based on auc values are provided . the two - class classification models were built using the software package weka ( version 3.7.12 ) . we investigated the performance of several base classifiers , such as logistic regression , tree methods ( random forest and j48 tree ) , support vector machines ( smo in weka with polynomial , rbf , and puk kernels ) , nave bayes , and k - nearest neighbors . moreover , because the data set is slightly imbalanced , in order to equilibrate the effect of the majority class on model performance , we also applied the cost - sensitive meta - classifier metacost . the cost matrix applied corresponds to the imbalance ratio of the data ( 3:1 ) . additionally , several meta - classifiers were explored for attribute selection ( attributeselectedclassifier ) , as well as for improving the statistical performance , such as bagging and boosting . the models were originally validated via 10-fold cross validation , which is considered a quite trustworthy method of validation . the best models according to 10-fold cross - validation evaluation were further validated via using the data set by mulliner . subsequently , for the best obtained models , 50 iterations were performed by changing the cross - validation seed ( for splitting the data within cross validation ) and the respective performance parameters were calculated . in order to compare whether the inclusion of the transporters predictions in the descriptors set improves significantly the model s performance , a two - sample t test was performed in r. the statistics metrics taken into consideration were accuracy , sensitivity , specificity , matthews correlation coefficient ( mcc ) , area under the curve ( auc ) , precision and weighted average precision . apart from the conventional metrics of accuracy , sensitivity , and specificity , we also use auc , since it is a measure of the models capability to rank the predictions , while it decouples classifiers from class imbalance and error costs . moreover receiver operating characteristic ( roc ) graphs are very useful for visualization of the models results . the mcc value , considering its formula , takes into account all values of the confusion matrix : where , tp is true positives , fp is false positives , tn is true negatives , and fn is false negatives . thus , it is considered more balanced and informative than the column- or linewise metrics . weighted average precision is the average precision obtained for the two classes but weighted from the total number of instances of the classes . it is a quite helpful parameter in multiclass classification problems , as well as for imbalanced data sets where the number of negatives is greater than the number of positives . especially for the latter case , due to the definition of precision [ ppv = tp/(tp + fp ) ] , its value for the positive class would be low , which not necessarily means that the total performance of the model is bad . of course , since we are dealing with a toxicity classification problem , like cholestasis , the metrics that is of particular interest and that should by no means drop below 0.5 is sensitivity or true positive rate . in order to be confident regarding the validity of the models we used , we investigated the coverage of the transporters models for the cholestasis data . additionally , we checked how reliable the predictions of the cholestasis model for the cholestasis test set are . the applicability domain was checked on knime with the enalos nodes that compute the applicability domain on the basis of the euclidean distances . the number of compounds within the model s applicability domain for each model and for each cholestasis data set is provided in the supporting information ( table s3 ) . several combinations of descriptors and classifiers were investigated and the optimal classification model was selected on the basis of the results of 10-fold cross validation . with respect to the classifier , the best results were obtained using as base classifier ibk the k - nearest neighbors implementation in weka with k = 5 . the meta - classifier metacost was also applied , with the application of the cost matrix [ 0.0 , 1.0 ; 3.0 , 0.0 ] , i.e. weighting the minority class 3 times more than the majority class , in order to cope with the slightly imbalanced training set . 2d moe descriptors were performing better than fingerprints and/or volsurf descriptors , especially for sensitivity , mcc and auc . combining the volsurf descriptors with 2d moe descriptors also did not provide any significant improvement of the results . from the whole set of 2d moe descriptors we decided to use a subset of 93 interpretable descriptors that give almost the same performance compared to using all 2d moe descriptors . apart from the 93 2d descriptors , we also included the predicted transporter inhibition profiles . in order to assess the importance and significance of this additional information individually , we used them in different combinations : all transporters , only bsep , all transporters excluding either bsep , or p - gp , or bcrp , or the oatps . this led to in total seven models ( table 1 ) . it is interesting to mention that we also exchanged the training - test set roles between the two data sets and tried to generate a model for cholestasis based on the bigger data set from the work of mulliner et al . however , even though the results for 10-fold cross validation were equivalent to the model we propose ( generated on the compiled training set of 578 compounds ) , the results for the external validation of the 578 compounds were rather poor ( results not shown ) . furthermore , we tried to generate a cholestasis model on the merged data for the subset of 93 2d moe descriptors , with or without the transporters interaction profiles . interestingly , the k - nearest neighbor settings ( k = 5 ) , which gave quite satisfactory results for 10-fold cross validation while modeling either the training or the test set standalone , did not have the same effect for the united data . for the merged data set svm ( smo implementation in weka ) using a polynomial kernel , with exponent equal to 2 , performs better . the use of metacost with a cost matrix of [ 0.0 , 1.0 ; 5.0 , 0.0 ] , due to the new imbalance ratio of the data , is also necessary . additionally , under these settings , the performance of the model is significantly better when using the transporters predictions as additional descriptors . the obtained performance of this model , as well as the respective p - values of the performed two - sample paired t test out of 50 iterations , is presented in the supporting information ( table s4 ) . inspecting the obtained results in table 1 , it becomes obvious that the best settings for the model for 10-fold cross validation are achieved with the inclusion of all transporter inhibition predictions in the list of descriptors . nevertheless , this is not the case for the external validation , where including predicted inhibitor profiles for all transporters yields lower accuracy and specificity values , while sensitivity remains almost the same . interestingly , the use of bsep inhibition prediction stand - alone does not seem to be sufficient . there is a drop in the statistics especially for sensitivity in comparison to the use of the whole set of transporter predictions , both for 10-fold cross - validation and for the external test set . in order to assess if the predicted transporter inhibition profiles indeed statistically significantly improve the models , we performed 50 iterations of 10-fold cross validation followed by a two sample t test on the performance parameters . for this we used the models with 2d moe descriptors , 2d moe plus all transporters , 2d moe plus bsep , and 2d moe plus all transporters without bsep ( table 2 ) . analyzing the p - values for the pairwise comparisons ( supporting information table s5 ) the main conclusion is that indeed the use of liver transporter inhibition predictions contributes significantly to the models performance when compared to the use of only 2d physicochemical descriptors . interestingly , it is not only the bsep inhibition contribution , which matters . on the contrary , when only bsep predictions are used , there is only a slight increase in sensitivity and specificity of the model , while sensitivity decreases . the other way round , if all transporters predictions are used , sensitivity increases , but accuracy and specificity reach their peak only after the inclusion of bsep predictions . nevertheless , it does not contain all the important information , despite the fact that it is the most widely discussed transporter in literature with respect to cholestasis . a possible explanation for this interesting result could be the different thresholds required for bsep inhibition to cause cholestasis versus the threshold of the bsep inhibition model . for the bsep model , every compounds with an ic50 > 50 m was classified as noninhibitors , while ic50 < 10 m was the threshold for characterizing a compound as inhibitor . however , for the development of cholestasis , an ic50 < 300 m has been reported as sufficient . the other transporters included in our study are in general not well described in literature via experimental procedures , but they are rather pinpointed due to the fact that they are transporting bile salts or bile conjugates ( with the exception of p - gp , whose role is attributed mainly to drug thus , our study gives extra weight to literature indications concerning bcrp , p - gp , oatp1b1 , and 1b3 . it was quite curious that even though the transporters predictions significantly increase the performance both for the model built on the training set of 578 compounds and for the model trained with the merged data set of 1904 compounds ( but with a different base classifier ) ; this was not the case for the prediction on the test set . the performance of all statistics metrics decrease , when transporters predictions are used as descriptors . we can only speculate a different way of class assignment between the two data sets , since they are coming from different sources . another plausible explanation could be the fact that the external validation set had a contradiction rate of almost 20% regarding the class labels of those compounds shared with the training set ( 71 out of 419 shared compounds had contradictory class labels ) . we assume that this is due to the drawbacks of the toxicity reporting system : under - reporting , voluntarily basis , difficulties to obtain the data , which are often proprietary , as well as the lack of the prerequisite of a causal relationship between drug and adverse event . in any case , despite these contradictions between the training and the test set , the model retained its satisfactory performance . furthermore , we would like to mention that there is also experimental evidence for the implication of the basolateral efflux transporters mrp3 and mrp4 , as well as for the canalicular efflux transporter mrp2 . we are aware of this fact , but for these transporters there are currently not sufficient data available to develop high quality models that can be further used for contributing to our cholestasis model . additionally , we should pinpoint the fact that we are using predictions for the inhibition of transporters rather than real in vitro data . this could potentially accumulate some additional error in the final predictions of the cholestasis model . in any case , despite any deteriorating factors , our final in silico models for cholestasis were extensively validated with 10-fold cross validation and statistical tests . furthermore , the external validation set was of a significant size being even bigger than the training set . for both cases moreover , for both training sets ( regular and merged one ) there is a trend indicating the importance of transporter predictions in the development of cholestasis . the performance of the classification models from which we obtained the predictions provides us with enough confidence to use them in our input matrix . moreover , the percentage of the cholestasis data that are within the applicability domain of the transporters models ranges between 93.1% and 99.5% ( table s3 , supporting information ) , which is quite satisfactory and further enhances our confidence in using predicted transporter interaction profiles as descriptors . in this study we present a two - class classification model for the prediction of cholestasis ( or cholestatic dili ) based on a public data set of 578 compounds . the model is incorporating information both from 2d physicochemical descriptors , as well as predictions of inhibition of the hepatic transporters bsep , bcrp , p - gp , oatp1b1 , and oatp1b3 . the performance of the resulting model is rather satisfactory and is validated both via 50 iterations of different 10-fold cross validations , as well as an external test set of over 1300 compounds . our results demonstrate that adding transporter predictions as additional descriptors to the list of 2d physicochemical descriptors is improving model performance . this is in alignment with evidence from literature which shows that inhibition of selected hepatic transporters contributes to cholestasis . interestingly , the increase in model performance can not be attributed solely to bsep inhibition , which is the transporter that is most correlated to cholestasis in literature . the performance increases only when the whole list of transporter inhibition predictions is included . this result points toward a rather synergistic effect of several transporters , including the less elucidated role of oatps , bcrp , and p - gp in cholestasis . our study is the first of its kind regarding combining physicochemical descriptors and predicted transporter information in order to predict cholestasis .

cholestasis represents one out of three types of drug induced liver injury ( dili ) , which comprises a major challenge in drug development . in this study we applied a two - class classification scheme based on k - nearest neighbors in order to predict cholestasis , using a set of 93 two - dimensional ( 2d ) physicochemical descriptors and predictions of selected hepatic transporters inhibition ( bsep , bcrp , p - gp , oatp1b1 , and oatp1b3 ) . in order to assess the potential contribution of transporter inhibition , we compared whether the inclusion of the transporters inhibition predictions contributes to a significant increase in model performance in comparison to the plain use of the 93 2d physicochemical descriptors . our findings were in agreement with literature findings , indicating a contribution not only from bsep inhibition but a rather synergistic effect deriving from the whole set of transporters . the final optimal model was validated via both 10-fold cross validation and external validation . it performs quite satisfactorily resulting in 0.686 0.013 for accuracy and 0.722 0.014 for area under the receiver operating characteristic curve ( auc ) for 10-fold cross - validation ( mean standard deviation from 50 iterations ) .

Introduction Methods Results and Discussion Conclusions

this was a double - blind randomized paralleled trial conducted at 74 sites in the u.s . and six sites in puerto rico ( nct00698230 ) . the study consisted of five periods : screening , metformin dose stabilization , 14-day placebo single - blind run - in , 12-week double - blind treatment , and 3-week off - treatment follow - up . the study was conducted pursuant to the declaration of helsinki and was approved by institutional review boards at participating sites . patients provided informed consent before screening . patients ( 1875 years ) with type 2 diabetes , a bmi between 25 and 45 kg / m , and a1c between 711% while taking metformin monotherapy at a stable dose for 10 weeks were eligible . exclusion criteria included a medical history of disorders involving glucocorticoid , mineralocorticoid , or androgen excess ; a history of type 1 diabetes or secondary forms of diabetes ; previous insulin therapy ; triglycerides > 500 mg / dl ; and treatment with any oral , systemic , topical , or inhaled glucocorticoids , thiazolidinediones , or exenatide within 3 months of screening . no inclusion criteria were specified for cholesterol or blood pressure and patients could enter the study on ( and maintain ) any hypolipidemic or antihypertensive regimen . patients were randomized equally to once - daily incb13739 ( 5 , 15 , 50 , 100 , or 200 mg ) or placebo . dose selection was based on phase 1 pharmacokinetic and pharmacodynamic data , with the goal of evaluating regimens that achieve different degrees of inhibition , from < 50 to > 90% , with the duration of inhibition varying across the five dose levels . patients with a fasting plasma glucose ( fpg ) > 270 mg / dl through week 8 or > 240 mg / dl subsequently were discontinued and offered rescue therapy . the primary end points were the change from baseline to week 12 compared with placebo in a1c , safety , and tolerability . secondary end points included the change from baseline to week 12 compared with placebo in fpg and lipid profiles and the proportion of patients achieving an a1c 7% at week 12 . tertiary end points included the change from baseline in homeostasis model assessment insulin resistance ( homa - ir ) , weight , blood pressure , and the proportion of patients meeting rescue therapy criteria . on - treatment study visits occurred at weeks 2 , 4 , 8 , and 12 and a follow - up visit at week 15 off treatment . monitoring for adverse events ( aes ) ( intensity , duration , outcome , and causality ) , physical examinations , vital signs , body weight and morphometrics , 12-lead electrocardiograms , and safety laboratory assessments including hematology , serum chemistry , and urinalysis were also performed . there were 40 patients per group completing week 12 who provided 90% power to detect a mean 0.6% difference in a1c between the 200-mg group and placebo assuming an emax dose - response model ( 13 ) with a half - maximal stimulation ( ed50 ) of 30 mg and an sd in a1c of 1.2% . this emax model is commonly used for phase 2 dose - ranging studies and was prespecified with the following optimal linear contrast : 0.45666 ( placebo ) , 0.31381 ( 5 mg ) , 0.12333 ( 15 mg ) , 0.168336 ( 50 mg ) , 0.312566 ( 100 mg ) , and 0.412901 ( 200 mg ) based on the half - maximal concentration ( ed50 ) = 30 mg assumption . two populations were prespecified : the evaluable analysis set was defined as all patients randomized who have completed the 12 weeks of study treatment with 80% compliance ; and the full analysis set was defined as all patients randomized who have taken at least one dose of study drug with any missing week 12 data imputed by last observation carried forward . the a1c and fpg end points were prespecified to be analyzed using the evaluable analysis set ; all other efficacy end points were prespecified to be analyzed using the full analysis set . for all end points , treatment effect was assessed using a linear model with treatment as the model factor and baseline as a covariate . on - treatment study visits occurred at weeks 2 , 4 , 8 , and 12 and a follow - up visit at week 15 off treatment . monitoring for adverse events ( aes ) ( intensity , duration , outcome , and causality ) , physical examinations , vital signs , body weight and morphometrics , 12-lead electrocardiograms , and safety laboratory assessments including hematology , serum chemistry , and urinalysis were also performed . there were 40 patients per group completing week 12 who provided 90% power to detect a mean 0.6% difference in a1c between the 200-mg group and placebo assuming an emax dose - response model ( 13 ) with a half - maximal stimulation ( ed50 ) of 30 mg and an sd in a1c of 1.2% . this emax model is commonly used for phase 2 dose - ranging studies and was prespecified with the following optimal linear contrast : 0.45666 ( placebo ) , 0.31381 ( 5 mg ) , 0.12333 ( 15 mg ) , 0.168336 ( 50 mg ) , 0.312566 ( 100 mg ) , and 0.412901 ( 200 mg ) based on the half - maximal concentration ( ed50 ) = 30 mg assumption . two populations were prespecified : the evaluable analysis set was defined as all patients randomized who have completed the 12 weeks of study treatment with 80% compliance ; and the full analysis set was defined as all patients randomized who have taken at least one dose of study drug with any missing week 12 data imputed by last observation carried forward . the a1c and fpg end points were prespecified to be analyzed using the evaluable analysis set ; all other efficacy end points were prespecified to be analyzed using the full analysis set . for all end points , treatment effect was assessed using a linear model with treatment as the model factor and baseline as a covariate . baseline assessments were performed in 302 patients who entered the treatment phase of the study , and 228 patients ( 75% ) completed the 12-week treatment period . the most common reasons for discontinuation were loss to follow - up ( 5% ) , withdrawal of consent ( 5% ) , lack of efficacy ( 4% ) , noncompliance with study procedures / medication ( 4% ) , and adverse events ( 4% ) , none of which related to the dose level of study medication . the clinical characteristics of the population at baseline were similar between treatment groups ( supplementary table 1 ) : the mean duration of diabetes was 6.2 years , bmi 32.4 kg / m , a1c 8.3% , and fpg 173 mg / dl . at week 12 , treatment with incb13739 resulted in a dose - dependent reduction in a1c ( pemax = 0.016 ; table 1 , fig . the placebo - adjusted least - squares ( ls ) mean difference from baseline in a1c reached statistical significance for the 100-mg ( 0.47% ; p < 0.05 ) and 200-mg ( 0.56% ; p a1c decreased compared with placebo in a time - dependent manner , reaching its maximum at week 12 ( fig . . a greater proportion of patients ( 25% ) randomized to 100 or 200 mg incb13739 achieved an a1c < 7% when compared with placebo ( 9.5% ) at week 12 . in a predefined subgroup analysis in patients with a baseline a1c 8% , the response to incb13739 was more pronounced , with the 50- , 100- , and 200-mg groups achieving a significant ( p < 0.05 ) change in a1c from baseline of 0.65 to 0.72% . the placebo - adjusted change in a1c for the 100- and 200-mg groups was greater in subjects with a baseline bmi > 30 kg / m ( 0.53% and 0.93% , respectively ) than in subjects with a baseline bmi 30 kg / m ( 0.35% and 0.17% , respectively ) . the number of patients requiring rescue therapy ( 12 ) did not differ significantly between treatment groups . fpg decreased in a dose- and time - dependent manner in the 100- and 200-mg treatment groups ( fig . 1c ) and reached statistical significance ( p < 0.01 ) from placebo in the 200-mg group with an ls mean difference of 24.1 mg / dl . a dose - dependent reduction in homa - ir was observed , reaching significance ( p < 0.05 ) in the 200-mg group with an ls mean difference of 1.32 ( 24% ) , suggesting an insulin - sensitizing mechanism of action . p < 0.01 , active vs. pbo . # p < 0.01 , week 12 vs. baseline . a : ls mean ( se ) change from baseline in a1c at week 12 . b : ls mean difference ( se ) from placebo in a1c from baseline to week 12 in the 100-mg ( ) and 200-mg ( ) treatment groups . c : ls mean difference ( se ) from placebo in fpg from baseline to week 12 in the 100- and 200-mg treatment groups . * p < 0.1 , p < 0.05 , p < 0.01 , active vs. placebo ( pbo ) . body weight decreased with incb13739 treatment , with statistical significance from baseline ( p < 0.05 ) achieved in the 15 ( 0.6 kg ) , 100 ( 1.1 kg ) , and 200 mg ( 0.9 kg ) treatment groups ( table 1 ) . plasma lipids and blood pressure were generally well controlled at baseline ( supplementary table 1 ) . treatment with incb13739 resulted in a modest dose - dependent ( ptrend = 0.026 ) decrease in total cholesterol , reaching a maximum of 7 mg / dl ( 3% ) from baseline in the 200-mg group ( table 1 ) . in a prespecified analysis , patients with adult treatment panel ( atp ) iii defined hyper - lipidemia ( total cholesterol > 200 mg / dl ; ldl cholesterol > 130 mg / dl ) or hyper - triglyceridemia ( > 200 mg / dl ) at baseline exhibited a greater improvement , reaching statistical significance ( p < 0.05 ) in the 100-mg group for all three lipid categories ( cholesterol 16 mg / dl , 6% ; ldl 17 mg / dl , 10% ; triglycerides 74 mg / dl , 16% ) . similar responses were observed in the 200-mg group , but these did not reach significance , possibly because of the smaller size of the subgroups . changes in hdl and free fatty acids were not significantly different between the treatment groups . treatment with incb13739 was well tolerated and aes were reported at similar frequencies across all treatment groups ( table 2 ) . one death occurred in the 200-mg group because of complications after a serious ae of acute ischemia of the lower extremities . this ae occurred 2 weeks after the last dose of study medication in a subject with preexisting congestive heart failure and aortic valvular disease . the death was due to cardiac arrest immediately after induction of anesthesia before bilateral iliofemoral embolectomy . the most frequent aes reported were typical for this population and did not exhibit dose dependence . there were four reports of nausea in the 200-mg group ( compared with one in the placebo group ) ; however , all of these resolved during continued dosing and three were categorized by the investigator as unrelated to study medication . there were no clinically relevant differences between treatment groups in electrocardiograms , hematology , serum chemistry , or urinalysis . end point endocrine assessments and safety summary endocrine data are week 12 ls mean sem unless otherwise noted . treatment emergent ae data are n ( % ) for all aes or for those occurring in at least 3% of patients . a4 , androstenedione ; d / c , discontinuation ; fai , free androgen index ; sae , serious adverse event ; t , testosterone . * determined by the investigator to be possibly , probably , or definitely drug related . the anticipated compensatory activation of the hypothalamic - pituitary - adrenal axis to overcome reduced cortisol regeneration on 11hsd1 inhibition was evaluated . incb13739 caused a dose - related increase in morning plasma acth and the acth - sensitive adrenocorticosteroid dehydroepiandrosterone sulfate ( dheas ) levels , although mean concentrations of both hormones remained within laboratory reference ranges ( table 2 ) . acth and dheas rises after incb13739 reached a plateau at week 4 ( + 102 and + 54% , respectively , versus + 19 and + 6% in the placebo group ) ; did not exhibit a further increase at week 12 , even in the 200-mg treatment group ( + 114% and + 55% , respectively ) ; and returned to baseline levels by the 3-week follow - up visit ( fig . morning plasma cortisol and evening salivary cortisol levels were unaltered by incb13739 at any dose ( fig . 2c ) , suggesting that the rise in acth was a compensatory response . change in acth , dheas , and cortisol . a : ls mean percent ( % ) change ( se ) from baseline in acth by treatment group and time on therapy . b : ls mean percent ( % ) change ( se ) from baseline in dheas by treatment group and time on therapy . c : ls mean ( se ) cortisol concentrations at week 12 in the morning ( plasma concentrations , , left axis ) or at night ( salivary concentrations , , right axis ) . incb13739 treatment resulted in a dose - related increase in morning fasting serum androstenedione ( a4 ) , although mean concentrations remained within the laboratory reference range ( table 2 ) . in males , there were no differences between treatment groups in total testosterone , sex hormone binding globulin ( shbg ) , or free androgen index ( fai ) . in females , total testosterone ( available at baseline and week 8) increased in a dose - dependent manner with mean concentrations within the laboratory reference range . maximal concentrations were observed in the 200-mg group ( 1.8 vs. 1.3 nmol / l in the placebo group ; p < 0.05 ) . these changes occurred alongside modest increases in shbg ( assessed at week 12 ) , apparent in the 50- and 100-mg groups ( p < 0.05 ) , but not the 200-mg group . importantly , there were no significant differences between treatment groups in calculated fai in females ( placebo = 6.9 ; incb13739 range = 5.78.2 ) . at week 12 , treatment with incb13739 resulted in a dose - dependent reduction in a1c ( pemax = 0.016 ; table 1 , fig . 1a ) . the placebo - adjusted least - squares ( ls ) mean difference from baseline in a1c reached statistical significance for the 100-mg ( 0.47% ; p < 0.05 ) and 200-mg ( 0.56% ; p < 0.01 ) groups . a1c decreased compared with placebo in a time - dependent manner , reaching its maximum at week 12 ( fig . 1b ) . a greater proportion of patients ( 25% ) randomized to 100 or 200 mg incb13739 achieved an a1c < 7% when compared with placebo ( 9.5% ) at week 12 . in a predefined subgroup analysis in patients with a baseline a1c 8% , the response to incb13739 was more pronounced , with the 50- , 100- , and 200-mg groups achieving a significant ( p < 0.05 ) change in a1c from baseline of 0.65 to 0.72% . the placebo - adjusted change in a1c for the 100- and 200-mg groups was greater in subjects with a baseline bmi > 30 kg / m ( 0.53% and 0.93% , respectively ) than in subjects with a baseline bmi 30 kg / m ( 0.35% and 0.17% , respectively ) . the number of patients requiring rescue therapy ( 12 ) did not differ significantly between treatment groups . fpg decreased in a dose- and time - dependent manner in the 100- and 200-mg treatment groups ( fig . 1c ) and reached statistical significance ( p < 0.01 ) from placebo in the 200-mg group with an ls mean difference of 24.1 mg / dl . a dose - dependent reduction in homa - ir was observed , reaching significance ( p < 0.05 ) in the 200-mg group with an ls mean difference of 1.32 ( 24% ) , suggesting an insulin - sensitizing mechanism of action . p < 0.01 , active vs. pbo . # p < 0.01 , week 12 vs. baseline . a : ls mean ( se ) change from baseline in a1c at week 12 . b : ls mean difference ( se ) from placebo in a1c from baseline to week 12 in the 100-mg ( ) and 200-mg ( ) treatment groups . c : ls mean difference ( se ) from placebo in fpg from baseline to week 12 in the 100- and 200-mg treatment groups . * p < 0.1 , p < 0.05 , p < 0.01 , active vs. placebo ( pbo ) . body weight decreased with incb13739 treatment , with statistical significance from baseline ( p < 0.05 ) achieved in the 15 ( 0.6 kg ) , 100 ( 1.1 kg ) , and 200 mg ( 0.9 kg ) treatment groups ( table 1 ) . plasma lipids and blood pressure were generally well controlled at baseline ( supplementary table 1 ) . treatment with incb13739 resulted in a modest dose - dependent ( ptrend = 0.026 ) decrease in total cholesterol , reaching a maximum of 7 mg / dl ( 3% ) from baseline in the 200-mg group ( table 1 ) . in a prespecified analysis , patients with adult treatment panel ( atp ) iii defined hyper - lipidemia ( total cholesterol > 200 mg / dl ; ldl cholesterol > 130 mg / dl ) or hyper - triglyceridemia ( > 200 mg / dl ) at baseline exhibited a greater improvement , reaching statistical significance ( p < 0.05 ) in the 100-mg group for all three lipid categories ( cholesterol 16 mg / dl , 6% ; ldl 17 mg / dl , 10% ; triglycerides 74 mg / dl , 16% ) . similar responses were observed in the 200-mg group , but these did not reach significance , possibly because of the smaller size of the subgroups . changes in hdl and free fatty acids were not significantly different between the treatment groups . treatment with incb13739 was well tolerated and aes were reported at similar frequencies across all treatment groups ( table 2 ) . no drug - related serious aes occurred in the trial . one death occurred in the 200-mg group because of complications after a serious ae of acute ischemia of the lower extremities . this ae occurred 2 weeks after the last dose of study medication in a subject with preexisting congestive heart failure and aortic valvular disease . the death was due to cardiac arrest immediately after induction of anesthesia before bilateral iliofemoral embolectomy . the most frequent aes reported were typical for this population and did not exhibit dose dependence . there were four reports of nausea in the 200-mg group ( compared with one in the placebo group ) ; however , all of these resolved during continued dosing and three were categorized by the investigator as unrelated to study medication . there were no clinically relevant differences between treatment groups in electrocardiograms , hematology , serum chemistry , or urinalysis . end point endocrine assessments and safety summary endocrine data are week 12 ls mean sem unless otherwise noted . treatment emergent ae data are n ( % ) for all aes or for those occurring in at least 3% of patients . a4 , androstenedione ; d / c , discontinuation ; fai , free androgen index ; sae , serious adverse event ; t , testosterone . * determined by the investigator to be possibly , probably , or definitely drug related . the anticipated compensatory activation of the hypothalamic - pituitary - adrenal axis to overcome reduced cortisol regeneration on 11hsd1 inhibition was evaluated . incb13739 caused a dose - related increase in morning plasma acth and the acth - sensitive adrenocorticosteroid dehydroepiandrosterone sulfate ( dheas ) levels , although mean concentrations of both hormones remained within laboratory reference ranges ( table 2 ) . acth and dheas rises after incb13739 reached a plateau at week 4 ( + 102 and + 54% , respectively , versus + 19 and + 6% in the placebo group ) ; did not exhibit a further increase at week 12 , even in the 200-mg treatment group ( + 114% and + 55% , respectively ) ; and returned to baseline levels by the 3-week follow - up visit ( fig . morning plasma cortisol and evening salivary cortisol levels were unaltered by incb13739 at any dose ( fig . 2c ) , suggesting that the rise in acth was a compensatory response . change in acth , dheas , and cortisol . a : ls mean percent ( % ) change ( se ) from baseline in acth by treatment group and time on therapy . b : ls mean percent ( % ) change ( se ) from baseline in dheas by treatment group and time on therapy . c : ls mean ( se ) cortisol concentrations at week 12 in the morning ( plasma concentrations , , left axis ) or at night ( salivary concentrations , , right axis ) . incb13739 treatment resulted in a dose - related increase in morning fasting serum androstenedione ( a4 ) , although mean concentrations remained within the laboratory reference range ( table 2 ) . in males , there were no differences between treatment groups in total testosterone , sex hormone binding globulin ( shbg ) , or free androgen index ( fai ) . in females , total testosterone ( available at baseline and week 8) increased in a dose - dependent manner with mean concentrations within the laboratory reference range . maximal concentrations were observed in the 200-mg group ( 1.8 vs. 1.3 nmol / l in the placebo group ; p < 0.05 ) . these changes occurred alongside modest increases in shbg ( assessed at week 12 ) , apparent in the 50- and 100-mg groups ( p < 0.05 ) , but not the 200-mg group . importantly , there were no significant differences between treatment groups in calculated fai in females ( placebo = 6.9 ; incb13739 range = 5.78.2 ) . the results from this study indicate , for the first time , that decreasing local cortisol exposure through 11hsd1 inhibition improves hyperglycemia over 12 weeks in patients with type 2 diabetes . the addition of once - daily incb13739 in patients inadequately controlled with metformin significantly reduced a1c , fpg , and homa - ir . these effects were dose dependent , and the greatest improvements were achieved at the highest dose administered ( 200 mg ) , with evidence for a more profound a1c reduction in subjects with a bmi > 30 kg / m , compatible with elevated 11hsd1 in adipose tissue in obesity . preliminary data from pharmacokinetic analyses ( data not shown ) indicate that the 100- and 200-mg groups achieved , 4 h after administration , mean free drug exposures that reached 100 mg or exceeded 200 mg , the concentrations required to inhibit 90% of the enzyme activity in cellular assays ; however , only the 200-mg group retained such a mean exposure at the end of the dosing interval . thus , glycemic efficacy may be associated with a high degree of enzyme inhibition , and it is possible that greater glycemic improvement might be achieved with increased dose levels or frequency of administration . plasma lipids were generally well controlled in this population , and 30% of patients were receiving lipid - lowering medications . incb13739 treatment resulted in a dose - dependent reduction in total cholesterol , and while of modest magnitude , these changes also associated with directional beneficial trends in ldl cholesterol and triglycerides . of interest , patients who met atp iii criteria for borderline high ldl cholesterol ( > 130 mg / dl ) , total cholesterol ( > 200 mg / dl ) , or hyper - triglyceridemia ( > 200 mg / dl ) exhibited a larger improvement in all three lipid parameters . the magnitude of effect was equivalent in the 100- and 200-mg groups , reaching statistical significance for the 100-mg group , which had the largest subgroup size . incb13739 treatment resulted in a dose - dependent modest decrease in body weight of 1 kg at the highest dose studied . this change was time dependent and did not plateau over the 12-week treatment period ( data not shown ) . the thiazolidinedione insulin sensitizers increase body weight through adipocyte differentiation ( 14,15 ) . as cortisol can drive adipocyte differentiation and expansion ( 16 ) , it is possible that attenuating cortisol signaling in adipose may decrease adipocyte size . this has been reported in preclinical models with an 11hsd1 inhibitor ( 17 ) and suggests the potential for positive effects of incb13739 on total body weight and/or regional adiposity with longer exposure . incb13739 was well tolerated at all dose levels , and there were no differences in ae frequency relative to placebo nor were there any apparent dose - dependent changes in aes . while 11hsd1 is not involved in adrenal cortisol biosynthesis , 11hsd1 activity within the splanchnic bed does contribute 25% of total cortisol production ( 18 ) . an expected consequence of 11hsd1 inhibition is increased clearance of cortisol and compensatory hypothalamic - pituitary - adrenal axis activation to maintain blood cortisol concentrations . incb13739 treatment did result in a dose - related increase in acth levels that was generally within the normal reference range . the acth response reached a plateau with the 50-mg dose at week 4 , suggesting that the maximal response to incb13739 had been realized . this plateau in acth and its rapid return to baseline levels after cessation of therapy are consistent with an adaptive endocrine process driven by reversible 11hsd1 inhibition . these data indicate normal hypothalamic - pituitary - adrenal axis function after 12 weeks of incb13739 therapy that adjusted appropriately to the inhibition of 11hsd1 activity to maintain basal cortisol homeostasis . the leftward shift in the acth dose relationship relative to efficacy might reflect a greater contribution of hepatic 11hsd1 inhibition to splanchnic cortisol reactivation . aldosterone and renin were unaltered by incb13739 treatment ( table 2 ) , and serum electrolytes were unchanged ( supplementary table 2 ) . modest elevations in the androgenic precursors dheas and a4 paralleled changes in acth . like acth , these changes were generally within the reference range , plateaued with respect to both dose and time , and were reversed at follow - up . the highest concentration of dheas observed in this study ( 13.2 mol / l in males and females ) is equivalent to levels observed after 50 mg / day dehydroepiandrosterone supplement use ( 19 ) . in men , there was no change in plasma testosterone , shbg , or fai after incb13739 treatment , consistent with the testes being the main source of androgens . in females , a modest rise in total testosterone at week 8 was observed that was paralleled by a rise in shbg such that the resulting fai calculation was not significantly different in any incb13739 group compared with placebo or baseline levels . shbg is known to increase in response to improved insulin sensitivity ( 20 ) , and whether the changes observed in this study reflect this or result from more complex endocrine adaptation to small changes in total testosterone are unknown . importantly , fai is an accepted surrogate in clinical practice for free testosterone and a marker of biologic androgen activity in women ( 21 ) . no signs or symptoms of androgen excess were observed , and longer - term studies will be required to ascertain the clinical relevance of the small androgen changes observed . in summary , in patients with type 2 diabetes who had inadequate glycemic control with metformin alone , the addition of once - daily incb13739 was well tolerated and resulted in significant improvements in a1c , fpg , and homa - ir . incb13739 treatment decreased body weight and improved cholesterol and triglycerides in patients with hyper - lipidemia at baseline . 11hsd1 inhibition offers a new potential approach to control glucose and cardiovascular risk factors in type 2 diabetes .

objective11--hydroxysteroid dehydrogenase type 1 ( 11hsd1 ) converts inactive cortisone into active cortisol , thereby amplifying intracellular glucocorticoid action . the efficacy and safety of the 11hsd1 inhibitor incb13739 were assessed when added to ongoing metformin monotherapy in patients with type 2 diabetes exhibiting inadequate glycemic control ( a1c 711%).research design and methodsthis double - blind placebo - controlled paralleled study randomized 302 patients with type 2 diabetes ( mean a1c 8.3% ) on metformin monotherapy ( mean 1.5 g / day ) to receive one of five incb13739 doses or placebo once daily for 12 weeks . the primary end point was the change in a1c at study end . other end points included changes in fasting glucose , lipids , weight , adverse events , and safety.resultsafter 12 weeks , 200 mg of incb13739 resulted in significant reductions in a1c ( 0.6% ) , fasting plasma glucose ( 24 mg / dl ) , and homeostasis model assessment insulin resistance ( homa - ir ) ( 24% ) compared with placebo . total cholesterol , ldl cholesterol , and triglycerides were all significantly decreased in hyperlipidemic patients . body weight decreased relative to placebo after incb13739 therapy . a reversible dose - dependent elevation in adrenocorticotrophic hormone , generally within the normal reference range , was observed . basal cortisol homeostasis , testosterone in men , and free androgen index in women were unchanged by incb13739 . adverse events were similar across all treatment groups.conclusionsincb13739 added to ongoing metformin therapy was efficacious and well tolerated in patients with type 2 diabetes who had inadequate glycemic control with metformin alone . 11hsd1 inhibition offers a new potential approach to control glucose and cardiovascular risk factors in type 2 diabetes .

RESEARCH DESIGN AND METHODS Study assessments Statistical analysis RESULTS Efficacy Safety CONCLUSIONS Supplementary Material

however , it is unclear whether to perform reoperation in asymptomatic patients with low transprosthetic gradient . especially , in patients with depressed left ventricular function , it is important to clarify which the main cause of left ventricular dysfunction is structural valve deterioration or other factors such as cardiomyopathy . this case is a 78-year - old male who underwent aortic valve replacement with a 23 mm carpentier - edwards perimount pericardial bioprosthesis ( edwards lifesciences , irvine , ca , usa ) for aortic valve stenosis 7 years ago at another hospital . pulmonary sarcoidosis was diagnosed in his 50s , and cardiac sarcoidosis was diagnosed by a myocardial biopsy at the time of the previous cardiac surgery . he had a past history of thyroid cancer , and his thyroids and parathyroids were resected at the age of 45 , and then after that , he had been prescribed with levothyroxine and alfacalcidol . the echocardiography 6 years after the previous operation showed a normal opening of his prosthesis with an effective orifice area index of 0.62 cm / m , a mean pressure gradient of 15 mmhg , and mildly reduced left ventricular function with an ejection fraction of 43% . although he was asymptomatic , his transthoracic echocardiography 1 year later revealed the restricted motion of the prosthetic valve leaflets , with an effective orifice area index of 0.28 cm / m and a mean pressure gradient of 28 mmhg . it also revealed depressed left ventricular function with an ejection fraction of 37% , moderate mitral insufficiency , and severe tricuspid insufficiency . considering comparably early deterioration of his bioprosthesis valve and left ventricular function , we decided to perform a dobutamine stress echocardiography to differentiate structural valve deterioration from pseudo - aortic stenosis due to cardiac sarcoidosis . at the baseline , the aortic valve peak velocity and the stroke volume index were 3.8 m / s and 31 ml / m . at a dose of 10 g / kg / min of dobutamine infusion , they increased to 4.4 m / s and 41 ml / m , respectively , and the mean pressure gradient across the bioprosthesis increased to 47 mmhg , as the effective orifice area index remained small at 0.29 cm / m . therefore , he was diagnosed as having structural valve deterioration of bioprosthesis at aortic position . he underwent redo aortic valve replacement with a 20 mm ats standard mechanical valve ( ats medical , minneapolis , minnesota , usa ) , mitral annuloplasty with a 26 mm carpentier - edwards physio ii annuloplasty ring ( edwards lifesciences , irvine , ca , usa ) , and tricuspid annuloplasty with a 28 mm edwards mc3 tricuspid annuloplasty ring ( edwards lifesciences , irvine , ca , usa ) . a transthoracic echocardiography at 6 months after the reoperation showed a recovery of left ventricular function with ejection fraction of 49% . pathologic examination of the explanted prosthesis showed calcified deposit and desmoplasia in all the leaflets without an evidence of infiltration of monocyte [ figure 1 ] . we reported a rare case of low transprosthetic gradient structural valve deterioration at aortic position in a patient with cardiac sarcoidosis . the principal manifestations of cardiac sarcoidosis are conduction abnormalities , ventricular arrhythmias , and heart failure . considering early bioprosthetic deterioration in a patient with cardiac sarcoidosis , there were three challenging points to be discussed in this case . first , it was difficult to diagnose which the main cause of depressed left ventricular function was structural valve deterioration or cardiac sarcoidosis . third , the optimal strategy for this case including the type of surgery and prosthesis was worth debating . this case was low - gradient aortic stenosis , which was defined as an aortic valve area < 1.0 cm and a mean transvalvular gradient < 40 mmhg . one of the diagnostic challenges in low - gradient aortic stenosis with depressed left ventricular function is to differentiate true severe aortic stenosis from pseudo - aortic stenosis . in this case , if the cause of depressed left ventricular function was structural valve deterioration , redo aortic valve replacement would be strongly recommended . on the other hand , if his depressed left ventricular function was the clinical manifestation of the cardiac sarcoidosis , the benefit of reoperation on the ventricular reverse remodeling was uncertain . a dobutamine stress echocardiography is useful to differentiate true severe aortic stenosis from pseudo - aortic stenosis in case of native aortic valve . it remains unclear whether it is also useful in case of bioprosthetic valve deterioration , but it could lead us to the diagnosis of true structural valve deterioration . only 7 years had passed until the reoperation for structural valve deterioration in this case . reported several risk factors of early bioprosthetic deterioration , but this case did not have any of these factors . skolnick et al . reported the association between osteoporosis and decreased progression of aortic stenosis , and speculated inhibition of valvular calcification might be due to alterations in levels of vitamin d and parathyroid hormone . on the other hand , it is reported chronic secondary hyperparathyroidism in renal failure is associated with aortic calcification . in patients with sarcoidosis , therefore , the possible cause of early bioprosthesis deterioration in this case might be alfacalcidol , an analog of vitamin d or parathyroid hormone - related protein due to sarcoidosis . the patient was asymptomatic , but it was reasonable to perform reoperation because the structural valve deterioration caused the left ventricular dysfunction . in this case , we decided to perform surgical aortic valve replacement with mechanical valve considering his early bioprosthetic valve deterioration and the possibility of the need for another redo aortic valve replacement . however , it might be another option to perform surgical aortic valve replacement with bioprosthetic valve and transcatheter valve - in - valve procedure if necessary in the future . future studies are warranted to determine what strategies about the optimal type of surgery and valve selection are reasonable .

we report a case of structural valve deterioration , which occurred 7 years after aortic valve replacement in a 78-year - old male with cardiac sarcoidosis . his echocardiography showed low transprosthetic valve gradient and depressed left ventricular function . a dobutamine stress echocardiography was performed to identify his pathophysiology , and it revealed that his depressed left ventricular function was not due to cardiac sarcoidosis but to structural valve deterioration . reoperation for structural valve deterioration was performed , and his left ventricular function recovered .

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periodontitis is a chronic inflammatory disease of supporting tissues of the teeth in response to noxious stimuli whether mechanical , chemical or infectious , resulting in progressive destruction of periodontal apparatus , hence leading to pocket formation , recession or both . the tissue destruction in periodontitis has been attributed to the production of enzymes and reactive oxygen species ( ros ) by polymorphonuclear cells and other cells . it has been demonstrated that patients with periodontitis have higher oxidative dna and lipid damage biomarkers and lower antioxidant ( ao ) enzymatic activities in saliva than healthy subjects . there are a variety of defense mechanisms in the body to combat excessive ros production , and aos are one among these . aos are those substances when present at a low concentration compared to those of an oxidizable substrate significantly delays or prevents oxidation of the substrate . superoxide and hydrogen peroxide are the main oxidative species produced , which are either enzymatically eliminated by preventive aos , or metal ions are sequestrated , hence preventing fenton reactions and subsequent hydroxyl radical formation e.g. catalase , glutathione ( gsh ) peroxidase , 7 s - transferase . interception involves scavenging / chain breaking aos which inhibit chain initiation , chain propagation and also lipid peroxidation e.g. lipophilic substances such as ubiquinol , vitamin a , vitamin e , carotenoids and hydrophilic substances like uric acid , ascorbic acid , albumin , and bilirubin . at repair level various aos function by repairing the damaged and reconstituting membranes . when the level of ros increases intracellularly , the cellular ao defenses are insufficient to maintain these harmful molecules , this condition is generally referred to as oxidative stress . the concept of oxidative stress dates back to 1986 and was elaborated as the relation between free radicals and disease . gsh is a ubiquitous tripeptide made from the combination of three amino acids , that is , cysteine , glutamate , and glycine . it is a low molecular weight thiol ( up to 510 mm ) present in the cell and existing in two forms which are oxidized glutathione ( gssg ) and reduced gsh forms . out of the total gsh present in the body , the reduced gsh constitutes about 90% , and gssg is 10% . for survival of cell , oxidative damage results when the there is a deficiency of reduced gsh in the cell , which puts it at risk for oxidative damage . anti - oxidant function [ figure 1]detoxification functionimmune function . anti - oxidant function [ figure 1 ] detoxification function schematic representation of the biochemical interactions between neutrophil superoxide and glutathione the purpose of this study is to compare the levels of gsh , both oxidized and reduced forms in patients with and without chronic periodontitis in gingival crevicular fluid ( gcf ) . this study was a 6 months randomized case control study , which was conducted at the department of periodontology . an approval for the study was obtained from the ethical committee , and written informed consent was obtained before enrolling the subjects for the study . the study enrolled 40 patients , which included patients with chronic periodontitis n = 20 ( test group ) and patients without chronic periodontitis n = 20 ( control group ) . subjects in the test group were selected on the basis of clinical criteria of having at least two nonadjacent sites per quadrant with probing pocket depths 5 mm , along with bleeding on probing and demonstrable radiographic bone loss . control patients did not show evidence of attachment loss or probing pocket depths 3 mm and bleeding scores were below 10% . the subjects with a history of taking vitamin supplements , anti - inflammatory or antibiotic medication in the preceding 3 months and pregnant ladies and patient with special dietary needs were not included in the study . after enrollment , baseline gcf samples were collected , before recording clinical measures which included silness and loe plaque index , loe and silness gingival index , muhlemann and son sulcus bleeding index and probing pocket depth . after data collection , chronic periodontitis patients ( test group ) underwent scaling and root planing and oral hygiene instruction which included brushing technique , dental flossing were reinforced at baseline . subjects were then recalled at 1-month , 3 months and 6 months posttherapy for recording clinical and biochemical parameters . gcf samples were collected from mesiobuccal / distolingual sites on any teeth in the maxillary quadrant ( test and control group ) using microcapillary pipettes . gcf samples were then immediately transferred to top sealed cuvettes to prevent oxidation of aos present in it . all the results of this study were obtained by comparing intragroup and intergroup parameters , at various designated phases at baseline , 1 month , 3 month and 6 month for test over control groups using paired student 's t - test using software computer software spss version 20 ( ibm spss inc , chicago , il , usa ) . this study was a 6 months randomized case control study , which was conducted at the department of periodontology . an approval for the study was obtained from the ethical committee , and written informed consent was obtained before enrolling the subjects for the study . the study enrolled 40 patients , which included patients with chronic periodontitis n = 20 ( test group ) and patients without chronic periodontitis n = 20 ( control group ) . subjects in the test group were selected on the basis of clinical criteria of having at least two nonadjacent sites per quadrant with probing pocket depths 5 mm , along with bleeding on probing and demonstrable radiographic bone loss . control patients did not show evidence of attachment loss or probing pocket depths 3 mm and bleeding scores were below 10% . the subjects with a history of taking vitamin supplements , anti - inflammatory or antibiotic medication in the preceding 3 months and pregnant ladies and patient with special dietary needs were not included in the study . after enrollment , baseline gcf samples were collected , before recording clinical measures which included silness and loe plaque index , loe and silness gingival index , muhlemann and son sulcus bleeding index and probing pocket depth . after data collection , chronic periodontitis patients ( test group ) underwent scaling and root planing and oral hygiene instruction which included brushing technique , dental flossing were reinforced at baseline . subjects were then recalled at 1-month , 3 months and 6 months posttherapy for recording clinical and biochemical parameters . gcf samples were collected from mesiobuccal / distolingual sites on any teeth in the maxillary quadrant ( test and control group ) using microcapillary pipettes . gcf samples were then immediately transferred to top sealed cuvettes to prevent oxidation of aos present in it . all the results of this study were obtained by comparing intragroup and intergroup parameters , at various designated phases at baseline , 1 month , 3 month and 6 month for test over control groups using paired student 's t - test using software computer software spss version 20 ( ibm spss inc , chicago , il , usa ) . the nonsurgical periodontal therapy provided to the test group resulted in observed reductions of whole mouth mean plaque scores ( p < 0.001 ) , mean gingival scores , and mean sulcus bleeding index scores in the group ( p < 0.001 ) which in turn showed reduced inflammation as compared to control group [ tables 16 ] . intra - group comparison of mean plaque scores and sd in test group intra - group comparison of mean gingival index scores and sd in test group intragroup comparison of mean sbi scores and sd in test group intragroup comparison of mean plaque scores and sd in control group intragroup comparison of mean gingival index scores and sd in control group intragroup comparison of mean sulcus bleeding index score and sd in control group in all groups , mean reduced gsh and gssg levels were detected in the millimolar range ( range : 0.305.14 mm [ table 7 ] ) . lower levels of gsh and gssg concentrations were reported in the gcf from chronic periodontitis patients before and after treatment , compared with those detected in nonchronic periodontitis subjects ( control group ) [ table 8 ] . mean ratio of reduced glutathione and oxidized glutathione and sd in test group intergroup comparison between various parameters used in study between test and control group comparative analysis of difference in mean gsh and gssg value for test group was done at different time intervals , namely , baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months and 16 months . the differences were observed to be statistically significant ( p < 0.001 ) at a confidence interval of 95% , but statistically nonsignificant at the 36 months interval [ tables 9 and 10 ] . comparison of mean gsh and gssg score with a standard deviation between test and control group is depicted [ figures 2 and 3 ] gsh and gssg value for control group were depicted at different time intervals , namely , baseline 1 month , baseline - 3 months , baseline - 6 months , 13 months and 16 months in tables 11 and 12 . intragroup comparison of mean reduced glutathione level and sd in test group intragroup comparison of mean oxidized glutathione level and sd in test group reduced glutathione levels at different time intervals for test and control groups oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean reduced glutathione level and sd in control group intragroup comparison of mean oxidized glutathione level and sd in control group ratio of reduced gsh and gssg for test group at different time interval during the study was reported to be statistically significant ( p < 0.001 ) at confidence interval of 95% at baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months , 16 months [ table 7 ] . comparison of the mean ratio of reduced and oxidized gsh ( gsh : gssg ) score with a standard deviation between test and control group is depicted in figure 4 . this improvement in the gsh : gssg ratio after treatment was as a result of an increase in levels of reduced gsh and a reduction in levels of oxidized gsh . ratio of reduced to oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean ratio of reduced glutathione and oxidized glutathione and sd in control group the nonsurgical periodontal therapy provided to the test group resulted in observed reductions of whole mouth mean plaque scores ( p < 0.001 ) , mean gingival scores , and mean sulcus bleeding index scores in the group ( p < 0.001 ) which in turn showed reduced inflammation as compared to control group [ tables 16 ] . intra - group comparison of mean plaque scores and sd in test group intra - group comparison of mean gingival index scores and sd in test group intragroup comparison of mean sbi scores and sd in test group intragroup comparison of mean plaque scores and sd in control group intragroup comparison of mean gingival index scores and sd in control group intragroup comparison of mean sulcus bleeding index score and sd in control group in all groups , mean reduced gsh and gssg levels were detected in the millimolar range ( range : 0.305.14 mm [ table 7 ] ) . lower levels of gsh and gssg concentrations were reported in the gcf from chronic periodontitis patients before and after treatment , compared with those detected in nonchronic periodontitis subjects ( control group ) [ table 8 ] . mean ratio of reduced glutathione and oxidized glutathione and sd in test group intergroup comparison between various parameters used in study between test and control group comparative analysis of difference in mean gsh and gssg value for test group was done at different time intervals , namely , baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months and 16 months . the differences were observed to be statistically significant ( p < 0.001 ) at a confidence interval of 95% , but statistically nonsignificant at the 36 months interval [ tables 9 and 10 ] . comparison of mean gsh and gssg score with a standard deviation between test and control group is depicted [ figures 2 and 3 ] gsh and gssg value for control group were depicted at different time intervals , namely , baseline 1 month , baseline - 3 months , baseline - 6 months , 13 months and 16 months in tables 11 and 12 . intragroup comparison of mean reduced glutathione level and sd in test group intragroup comparison of mean oxidized glutathione level and sd in test group reduced glutathione levels at different time intervals for test and control groups oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean reduced glutathione level and sd in control group intragroup comparison of mean oxidized glutathione level and sd in control group ratio of reduced gsh and gssg for test group at different time interval during the study was reported to be statistically significant ( p < 0.001 ) at confidence interval of 95% at baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months , 16 months [ table 7 ] . comparison of the mean ratio of reduced and oxidized gsh ( gsh : gssg ) score with a standard deviation between test and control group is depicted in figure 4 . this improvement in the gsh : gssg ratio after treatment was as a result of an increase in levels of reduced gsh and a reduction in levels of oxidized gsh . ratio of reduced to oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean ratio of reduced glutathione and oxidized glutathione and sd in control group the present data confirm the study conducted by chapple et al . , 2002 suggesting that a millimolar concentration of gsh was present in gcf , which decreases during periodontitis . the reduction in gsh levels is suggestive of its protective role for vital cells and tissue structures from host - derived free radicals . due to the conversion of gsh to gssg , there is a reduction in gsh levels at inflamed tissue sites as also seen in this study . similarly , the reduced gsh : gssg ratio at baseline was due to reduced levels of gsh as well as increased accumulation of gssg . these results were consistent with findings reported in earlier studies demonstrating a reduction in total antioxidant capacity of saliva in patients with chronic periodontitis along with reduced levels of gsh . in this study , a significant increase in gsh levels along with improvement in gsh : gssg ratio at 1-month was observed following nonsurgical periodontal therapy viz . , scaling and root planing , which is consistent with the observations made by several others studies . these results were suggestive of an improvement in ao status and reduction in oxidative stress following nonsurgical periodontal therapy . other possibilities for the improvement seen following nonsurgical therapy is the reduction in proteolytic activity of microorganisms and also reductions in inflammation posttherapy , thereby lowering levels of oxidants in gcf , both exogenously and endogenously . the improvement was seen in gsh : gssg ratio at 3 months is due to relatively long duration taken by subgingival microflora to reestablish , hence their reduced activity . the preliminary findings of studies stated that millimolar concentrations of gsh is present in gcf gets significantly reduced in chronic periodontitis patients . in addition , this study demonstrated that successful , nonsurgical periodontal therapy does restore the redox balance ( gsh : gssg ) till 3 months , but at 6 months no significant improvement in either gsh or gsh : gssg ratio was noted . thus after 3 months thorough oral hygiene practice is not self - sufficient for restoring the gsh levels and gsh : gssg ratio to health , this implies that there is a reduced potential of periodontal tissues against ros activity in chronic periodontitis patients , even after successful nonsurgical therapy . such findings have opened up the potential of using pharmacological agents to elevate buffering capacity within tissues by elevating gsh levels , e.g. use of the gsh promoting drug n - acetyl cysteine . anti - inflammatory tissue redox state can be created by supplementing the individuals with micronutrients for elevating gsh concentrations . for various chronic inflammatory diseases that are associated with redox imbalance , ao supplementation duration of the study , the reliability of the spectroscopic method used for gsh estimation and method of gcf collection . this study opens up scope for various future studies dealing with the role of gsh as biological diagnostic disease marker , ao , detoxifier , and immune modulator . long - term studies can be undertaken to evaluate the efficacy of novel therapeutic approaches for improving the buffering capacity within periodontal tissues by elevating the gsh levels . duration of the study , the reliability of the spectroscopic method used for gsh estimation and method of gcf collection . this study opens up scope for various future studies dealing with the role of gsh as biological diagnostic disease marker , ao , detoxifier , and immune modulator . long - term studies can be undertaken to evaluate the efficacy of novel therapeutic approaches for improving the buffering capacity within periodontal tissues by elevating the gsh levels . the study concluded that at baseline due to prevailing oxidative stress the levels of reduced gsh were reduced in patients with chronic periodontitis . following nonsurgical therapy the levels of gsh were improved at 1-month and 3 months . at 6 months recall following nonsurgical therapy , an adjunctive use of micronutritional supplements to boost ao concentration in tissues by preserving gsh or by elevating its level at the inflamed sites is recommended .

objective : to compare the levels of glutathione ( gsh ) , both oxidized and reduced forms in patients with and without chronic periodontitis in gingival crevicular fluid ( gcf).materials and methods : twenty gcf samples from maxillary quadrants were collected using capillary micropipettes from the chronic periodontitis patients ( test group ) at baseline before treatment , at 1-month , 3 months , and 6 months after scaling and root planing and samples from 20 patients without chronic periodontitis ( control group ) from maxillary quadrants were also collected . gsh , oxidized glutathione ( gssg ) levels and gsh : gssg ratios were determined using the spectrophotometric method.statistical analysis : results were concluded for the test over control groups using paired student 's t-test.results:lower concentrations of gsh ( p < 0.001 ) and gssg ( p < 0.001 ) were detected in gcf in patients with chronic periodontitis ( test group ) than patients without chronic periodontitis ( control group ) at baseline . treatment had a significant effect in improving the gsh and reducing gssg levels postscaling and root planing at 1-month and 3 months but not significant effect at 6 months . scaling and root planing increased the gsh : gssg ratio ( p < 0.001 ) in the test group as compared to the control group ( p < 0.001).conclusions : the concentrations of gsh within gcf are reduced in chronic periodontitis patients . scaling and root planing ( nonsurgical therapy ) restores gsh concentration in gcf post 1-month and 3 months along with redox balance ( gsh : gssg ratio ) , but at 6 months the balance is not maintained . adjunctive use of micronutritional supplements to boost antioxidant concentration in tissues by preserving gsh or by elevating its level at the inflamed sites is recommended , as nonsurgical periodontal therapy alone is not able to maintain redox balance for longer duration .

INTRODUCTION MATERIALS AND METHODS Study groups and design Gingival crevicular fluid collection and glutathione estimation Data analysis RESULTS Clinical data Glutathione levels in gingival crevicular fluid DISCUSSION Limitations CONCLUSIONS Financial support and sponsorship Conflicts of interest

breast carcinoma is the commonest malignant tumor and the leading cause of cancer death in women , with more than one million new cases occurring worldwide annually . it is well known that proto - oncogenes and tumor suppressor genes are two types of genes that play a key role in the regulation of cell growth and differentiation . hence , any alteration in one or more of these genes appears to play an important role in the pathogenesis of most human malignancies . her-2/neu proto - oncogene amplification and/or overexpression is one of the most important alterations seen in breast cancer . the her-2/neu proto - oncogene ( also called c - erbb2 ) is located on chromosome 17q11[35 ] which encodes for p185 , a transmembrane glycoprotein with tyrosine kinase activity that belongs to the family of epidermal growth factor receptors . her2/neu proto - oncogene is amplified and/or overexpressed in approximately 2530% of invasive breast cancers . an association has been found to exist between amplification and/or overexpression of her-2/neu and a wide variety of different clinical and pathological features of breast carcinoma . from a clinical point of view , her-2/neu receptor has become an important target for antibody - based therapy with trastuzumab ( herceptin ) . in iraq , where the population was exposed to high levels of depleted uranium following the first and second gulf wars , breast cancer is the most common tumor type in females . over the last ten years , there has been a three - fold increase in the incidence of breast cancer , with most of this increase being attributed to a particularly aggressive type of the cancer . this finding inspired an investigation of her2/neu expression in a series of iraqi women with breast cancer to see whether any differences in her2/neu expression , in correlation with tumor characteristics , could be found in a population exposed to depleted uranium . ninety specimens of formalin - fixed , paraffin embedded breast cancer tissue , collected from breast cancer patients over a period from september 2007 to june 2008 were included in this study . all cases were collected from major hospitals and private laboratories in kufa district area ( located in the middle of iraq ) . the age range of patients was 21 to 70 years , with a mean age of 48.8 years . a group of 25 patients with benign breast lesions ( fibroadenoma ) was included as a comparative group and 20 normal breast tissue sections were included as controls . confirmation of histopathological diagnosis , grade and stage of tumor was carried out after reviewing all slides before proceeding to the immunohistochemical approach . tissue sections with a thickness of 5- were taken from the formalin - fixed , paraffin embedded blocks for immunohistochemistry . labeled streptavidin - biotin ( lsab ) method was employed for immunohistochemical detection of her-2/neu using hercep test kit ( k5204 dako co. ) . the intensity of her-2/neu cell membrane stain was classified into score 0 ( negative ; no stain is observed or faint membrane staining presents in less than 10% of tumor cells ) , score 1 + ( negative ; a faint / barely perceptible membrane staining was detected in more than 10% of tumor cells ; these cells exhibit incomplete membrane staining ) , score 2 + ( weakly positive ; a weak to moderate complete membrane staining was observed in more than 10% of tumor cells ) and score 3 + ( strongly positive ; a strong complete membrane staining was observed in more than 10% of tumor cells ) . all biopsies were classified according to the modified bloom richardson grading system into three grades : grade i , grade ii and grade iii . the results were statistically evaluated with a chi - squared test ( at a significant level of < 0.05 ) and correlation - regression analysis ( at significant level of r = 0.3 ) using ssps software . the study received ethical approval from the research ethics committee of the middle euphrates centre for cancer research and followed the tenets of the declaration of helsinki . her-2/neu immuno - expression was positive in 67.8% of breast cancer cases and negative in all sections of the normal breast tissue and benign breast lesions ( fibroadenomas ) with significant differences among these groups ( p < 0.05 ) [ table 1 ] . her-2/neu overexpression was detected in 73.2% of invasive ductal carcinoma ( idc ) cases and in 12.5% of invasive lobular carcinoma . her-2/neu(c - erbb-2 ) overexpression and histological characteristics figures in parentheses are in percentage overexpression of her-2/neu was detected in 65.6% of those with pure invasive ductal carcinomas ; all of them were of a nonspecific type . a 94.4% of invasive ductal carcinoma with an in situ comedo component ( dcis ) and 100% of invasive ductal carcinoma with overlying paget 's disease were her-2/neu positive with a significant difference ( p < 0.05 ) in comparison with pure invasive ductal carcinomas [ table 1 ] . there was a significant difference between the intensity of her-2/neu overexpression and the histological type of breast cancer ( p < 0.05 ) [ table 2 ] . her-2/neu overexpression was detected in 70.6% of grade ii and 69.0% of grade iii breast cancer samples , while none of grade i showed her-2/neu overexpression [ table 1 ] . there was no significant difference in her-2/neu overexpression between grade ii and grade iii of breast cancer ( p > 0.05 ) , but in comparison with grade i a significant difference was noticed ( p < 0.05 ) . her-2/neu overexpression was highly correlated with grade of tumor ( r = 0.96 ) , indicating that her-2/neu - positive breast cancers are biologically aggressive . no statistically significant difference in the correlation between the intensity of her-2/neu immunostaining and histological grade was found ( p > 0.05 ) [ table 2 ] . the intensity of her-2/neu(c - erbb-2 ) overexpression and histological characteristics in addition , a higher detection rate of her-2/neu overexpression was noticed in recurrent breast cancer than primary lesions ( 86.2% and 59.1% , respectively ) with a significant difference ( p < 0.05 ) [ table 1 ] . no statistically significant differences between her-2/neu overexpression and tumor size , tumor stage , lymph node involvement , and age of breast cancer patients were found ( p > 0.05 ) [ table 1 ] . depleted uranium levels were estimated to be around 320800 tons in the aftermath of the first gulf war in 1991 with further comparable levels occurring in 2003 . since the targets were always in heavily populated areas in the middle and south of iraq , the extent of exposure on individuals was extensive but has been very hard to document accurately . this study showed that her-2/neu overexpression was completely absent in both normal breast tissue and benign lesion ( fibroadenomas ) sections . the percentage of her-2/neu overexpression in malignant breast lesions is disputed in our study as it ranges from 12.5% to 100% , but the finding is that her-2/neu overexpression appears to be a biomarker for malignant breast tissue and does not play any role in the benign ( fibroadenomas ) breast lesions . this observation confirms the results of many previous studies that reached the same conclusion.[1112 ] indeed , our study reported her-2/neu overexpression in 67.8% out of 90 breast cancer cases [ figure 1a ] . furthermore , most of the ductal carcinoma cases were purely invasive ductal carcinomas of nonspecific type ( 74.4% ) that showed significant her-2/neu overexpression ( 65.6% ) . this provides more evidence of the hypothesis that aggressive tumors seem to show significant her-2/neu overexpression and demonstrates the association between the nature of the biological expression of her-2/neu by the tumor and its degree of malignancy since it has been argued that nonspecific type ductal carcinomas are the most aggressive variants of breast cancer . the current study also demonstrated that 12.5% of lobular carcinoma cases exhibited her-2/neu expression [ figure 1d ] . ( a ) invasive ductal carcinoma , ( ) showing strong complete cytoplasmic membrane staining of score 3 + ( 40 ) . ( b ) insitu ductal carcinoma of comedo type , ( ) showing strong complete cytoplasmic membrane staining of score 3+positive ( 10 ) . ( c ) paget 's disease , ( ) showing strong complete cytoplasmic membrane staining of score 3 + ( 40 ) . ( d ) invasive lobular carcinoma , pleomorphic type , ( ) showing moderate complete membrane staining of score 2 + ( 40 ) from the above results , a significant difference in her-2/neu overexpression was observed between invasive ductal and lobular carcinoma ( p < 0.05 ) . this high discrepancy in her-2/neu overexpression between invasive ductal and lobular carcinoma suggests that invasive lobular carcinomas are both morphologically and biologically different from invasive ductal carcinomas . however , further studies are required to provide a more definitive answer . all 18 cases of idc with comedo - type in situ component showed her-2/neu overexpression [ figure 1b ] . the finding that her-2/neu expression is seen more frequently in dcis than in invasive carcinoma implicates that her-2/neu signaling pathway is playing a critical role in the early stages of breast tumorogenesis . overexpression was seen in all cases of both the invasive ductal carcinoma and paget 's disease components , when both components were seen simultaneously [ figure 1c ] . her-2/neu overexpression of low intensity ( score 1 + , considered as negative ) was observed more frequently in ilc ( 50% ) , while weak to moderate intensity of her-2/neu overexpression ( score 2 + ) occurred more frequently in invasive ductal carcinoma ( idc ) + ductal carcinoma with an in situ comedo component ( dcis ) ( 55.5% ) . high intensity of her-2/neu overexpression ( score 3 + , considered as strong positive ) was reported more frequently in paget 's disease ( 100% ) . these results suggest that the intensity of her-2/neu overexpression is well correlated with histopathological type . previous studies have shown faint or absent her-2/neu overexpression in lobular carcinoma and a strong positive score ( score + 3 ) in all cases of paget 's disease . the findings of this study show that a high incidence of samples of idc with in - situ component ( 55.5% ) were moderately stained ( score + 2 ) . this compares with the findings of lan et al . , who examined the prevalence of c - erbb-2 gene amplification in moderately stained ( score + 2 ) taiwanese specimens using ihc and found a higher prevalence of her-2 overexpression ( 44.4% ) than that found in similar studies from western countries ( 13%-23% ) . this study found no significant association between her-2/neu overexpression and age of the patients [ table 1 ] , which concurs with a previous study . a high proportion of her-2/neu overexpression was seen in moderately and poorly differentiated cases of breast cancer , while a lower proportion of her-2/neu expression was reported in cases of well differentiated breast cancer . her-2/neu expression appears to be well correlated with tumor grade ( r = 0.96 ) . immunohistochemical analysis showed her-2/neu overexpression to be higher in recurrent than in primary cases of tumor . these results reflect the significant role of her-2/neu overexpression in increasing the risk of local recurrence . it has been well documented that positive her/2 breast cancer patients have good prognosis as they respond well to the blocking effect of these receptors by anti - her/2 receptor antibody using monoclonal ( herceptin ) drug that plays a role in the regression of the tumor size and prevention of recurrence . the driving factor for the increased expression of her-2/neu to the level of 68% compared to general expression of 30% might be due to some underlying genetic factors , although other environmental factors can not be excluded . further investigations are needed to ascertain the extent of the effect of depleted uranium and the types of mutations noted in the overexpressed oncogene before any final conclusions can be reached despite the three - fold increase of all types of breast cancer in iraq due to long - term exposure to depleted uranium , this heightened exposure does not appear to be correlated with her-2/neu overexpression . further longitudinal studies and the use of techniques such as microarrays are required to investigate whether any effect of depleted uranium does manifest in the pathology of breast cancer .

backgroundthis study aimed to estimate the rate of her-2/neu ( c - erbb2 ) immunohistochemical overexpression in different histological types of breast cancer found in the middle euphrates region of iraq , a region that was exposed to high levels of depleted uranium . her-2/neu ( c - erbb2 ) overexpression was correlated with common clinicopathological parameters such as age , grade , stage , tumor size and lymph node involvement to determine if any particular biomarker for exposure to depleted uranium could be found in the tumor samples from this region.materials and methodsthe present investigation was performed over a period starting from september 2007 to june 2008 . formalin - fixed , paraffin - embedded blocks from 90 patients with breast cancer were included in this study . a group of 25 patients with benign breast lesions ( fibroadenoma ) was included as a comparative group , and 20 breast tissue sections were used as controls . labeled streptavidin - biotin ( lsab ) complex method was employed for immunohistochemical detection of her-2/neu.resultsher-2/neu immuno - expression was positive in 67.8% of breast cancer , while it was negative in all benign breast lesions ( fibroadenoma ) ( p < 0.05 ) . her-2/neu immunostaining was significantly associated with histological type and recurrence of breast cancer ( p < 0.05 ) . it was positively correlated with tumor grade , but this finding was not significant ( p > 0.05).conclusionbased upon the findings of this study , it can be concluded that her-2/neu overexpression plays an important role in the pathogenesis of breast cancer and is associated with a worse prognosis . the findings indicate that in regions exposed to high levels of depleted uranium , her-2/neu overexpression is high , but its correlation with age , grade , stage , tumor size , and lymph node involvement is similar to studies that have been conducted on populations not exposed to depleted uranium .

Background Materials and Methods Results Discussion Conclusion

infantis resulted from a general increase in different biotypes or a successful spread of 1 clone . infantis identified in israel during 20072009 ( 21 human sources , 28 poultry sources , and 22 food sources ) and 16 historical strains isolated during 19702005 ( 12 human sources , 2 poultry sources , and 2 food sources ) were subjected to pulsed - field gel electrophoresis ( pfge ) . macrorestriction with the xbai enzyme discriminated the isolates into 23 distinct profiles ( pulsotypes ) , designated i1i23 . although the historical isolates showed high diversity in their pfge patterns , most ( 58/71 , 82% ) recent ( 20072009 ) isolates were homogeneous and showed an indistinguishable pfge profile ( pulsotype i1 ) , which was not found among the historical isolates ( figure 2 ; table a1 ) . these results indicate that most of the emerging isolates belong to 1 genetic clone that probably started to spread in israel sometime during 20052007 . furthermore , comparison of the i1 pulsotype with other pfge profiles through pulsenet ( www.cdc.gov/pulsenet/ ) and pulsenet europe ( www.pulsenetinternational.org/networks/europe.asp ) indicated a pattern not reported elsewhere , suggesting the emerging clone is endemic to israel . pulsed - field gel electrophoresis ( pfge ) patterns of salmonella enterica serovar infantis isolates from clinical , food , and poultry sources isolated in israel , 19702009 , showing a high degree of clonality . macrodigestion performed using xbai restriction enzyme and genetic similarity ( in % ) was based on dice coefficients . pfge was conducted according to the standardized salmonella protocol centers for disease prevention and control pulsenet as described ( 4 ) by using s. enterica ser . a complete list is provided in table a1 . to further characterize the isolates , we performed susceptibility tests to 16 antimicrobial compounds . overall , resistance to 11 antimicrobial agents was detected ( table ; table a1 ) . first , although 6/16 ( 38% ) of the historical strains were sensitive to all tested antimicrobial agents and 5/16 ( 31% ) were resistant to only 1 ( nitrofurantoin ) , none of the 20072009 isolates were sensitive to all of the tested antimicrobial agents . most ( 68/71 , 96% ) of the recent isolates were resistant to > 3 antimicrobial agents , which suggests a process of resistance acquisition over time . second , whereas isolates from 19702005 did not share any obvious resistance pattern , most ( 66/71 , 93% ) of the 20072009 strains showed a combined resistance pattern to nalidixic acid , nitrofurantoin , and tetracycline with or without additional resistance to trimethoprim / sulfamethoxazole ( table ) . the convergence of the recent serovar infantis clones to a dominant resistance pattern is consistent with their common pfge profile and shows that they share high similarity on phenotypic and genotypic levels . * pfge , pulsed - field gel electrophoresis ; i1 , emerging pfge pattern ; d , different from the emerging pattern . next , we characterized the molecular mechanisms responsible for the common antimicrobial drug resistance phenotype . in bacteria , an efficient means of acquisition and dissemination of resistance genes is through mobile genetic elements such as plasmids , transposons , or integrons ( 5 ) . plasmid analysis for 15 emerging ( 20072009 ) and 7 historical ( 19702005 ) randomly selected isolates demonstrated that all possessed 1 large plasmid of 100 kb . to identify antimicrobial drug resistance genes that are possibly encoded on this plasmid , mating experiments were conducted with a plasmid - free , rifampin - resistant escherichia coli j53 strain and recent s. enterica ser . conjugation experiments showed the obtained e. coli transconjugants received the large ( 100-kb ) plasmid and acquired the tetracycline resistance phenotype but remained susceptible to nalidixic acid and nitrofurantoin . molecular analysis by pcr showed the teta gene encoded within the tn1721 transposon in 6 of 6 randomly selected emerging isolates but in only 1 of 5 older historical strains . we examined class 1 integrons using pcr primers designed to amplify the variable region of class 1 integrons . sulfamethoxazole followed by the orfc gene of unknown function . in contrast , 3/5 historical isolates did not possess any integron , and 2/5 contained a disparate integron with a variable region of 1.3 kb . sequencing analysis indicated a different cassette encoded by the aminoglycoside adenyltransferase aada1 gene conferring resistance to spectinomycin and streptomycin . resistance to quinolones is often associated with point mutations in the quinolone - resistance determining region of the gyra gene ( 6 ) . to examine this possibility , we determined the gyra sequence from 6 recent naladixic acid resistant and 4 naladixic acid sensitive isolates . all resistant clones showed the same nucleotide substitution from guanine to thymine at position 259 ( g259 t ) in the gyra gene , resulting in the exchange of asparagine in position 87 to tyrosine ( asp87tyr ) in the quinolone resistance determining region domain . no mutations were found in the gyra sequence of the naladixic acid sensitive isolates , suggesting that the asp87tyr point mutation is responsible for the observed naladixic acid resistance phenotype . it is likely that environmental selective pressure caused by use of antimicrobial drugs has led to the distribution of appropriate resistant genes . nitrofurans and sulfonamides , for example , have been widely used to treat infections and promote growth of livestock ( 7 ) . because the emerging clone was dominant in all levels of the food chain , including broiler chickens , it is possible that the emerging clone was originally introduced from a poultry source . recent studies from other countries identified healthy poultry as a potential reservoir of s. enterica ser . infantis isolates from different sources and regions in israel showed high homogeneity of emerging isolates that differ genetically and phenotypically from previously isolated strains . we showed that the emerging clone is multidrug resistant and is characterized by a large conjugative plasmid harboring the tn1721 transposone and teta gene , which provides reduced susceptibility to tetracyclines . additional characteristics include a class 1 integron containing the dfra1 cassette , a gyra mutation that mediates nalidixic acid resistance and furthers resistance to nitrofurantoin . our results suggest the recent emergence of serovar infantis is an outcome of a clonal expansion and establishment of a specific biotype that took place during a relatively short period .

to determine whether rapid emergence of salmonella enterica serovar infantis in israel resulted from an increase in different biotypes or spread of 1 clone , we characterized 87 serovar infantis isolates on the genotypic and phenotypic levels . the emerging strain comprised 1 genetic clone with a distinct pulsed - field gel electrophoresis profile and a common antimicrobial drug resistance pattern .

The Study Conclusions