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anxiety affects quality of life in those living with parkinson 's disease ( pd ) more so than overall cognitive status , motor deficits , apathy , and depression [ 13 ] . although anxiety and depression are often related and coexist in pd patients , recent research suggests that anxiety rather than depression is the most prominent and prevalent mood disorder in pd [ 5 , 6 ] . yet , our current understanding of anxiety and its impact on cognition in pd , as well as its neural basis and best treatment practices , remains meager and lags far behind that of depression . overall , neuropsychiatric symptoms in pd have been shown to be negatively associated with cognitive performance . for example , higher depression scores have been correlated with lower scores on the mini - mental state exam ( mmse ) [ 8 , 9 ] as well as tests of memory and executive functions ( e.g. , attention ) [ 1014 ] . likewise , apathy and anhedonia in pd patients have been associated with executive dysfunction [ 10 , 1523 ] . however , few studies have specifically investigated the relationship between anxiety and cognition in pd . one study showed a strong negative relationship between anxiety ( both state and trait ) and overall cognitive performance ( measured by the total of the repeatable battery for the assessment of neuropsychological status index ) within a sample of 27 pd patients . furthermore , trait anxiety was negatively associated with each of the cognitive domains assessed by the rbans ( i.e. , immediate memory , visuospatial construction , language , attention , and delayed memory ) . two further studies have examined whether anxiety differentially affects cognition in patients with left - sided dominant pd ( lpd ) versus right - sided dominant pd ( rpd ) ; however , their findings were inconsistent . the first study found that working memory performance was worse in lpd patients with anxiety compared to rpd patients with anxiety , whereas the second study reported that , in lpd , apathy but not anxiety was associated with performance on nonverbally mediated executive functions and visuospatial tasks ( e.g. , tmt - b , wms - iii spatial span ) , while in rpd , anxiety but not apathy significantly correlated with performance on verbally mediated tasks ( e.g. , clock reading test and boston naming test ) . furthermore , anxiety was significantly correlated with neuropsychological measures of attention and executive and visuospatial functions . taken together , it is evident that there are limited and inconsistent findings describing the relationship between anxiety and cognition in pd and more specifically how anxiety might influence particular domains of cognition such as attention and memory and executive functioning . it is also striking that , to date , no study has examined the influence of anxiety on cognition in pd by directly comparing groups of pd patients with and without anxiety while excluding depression . given that research on healthy young adults suggests that anxiety reduces processing capacity and impairs processing efficiency , especially in the central executive and attentional systems of working memory [ 26 , 27 ] , we hypothesized that pd patients with anxiety would show impairments in attentional set - shifting and working memory compared to pd patients without anxiety . furthermore , since previous work , albeit limited , has focused on the influence of symptom laterality on anxiety and cognition , we also explored this relationship . seventeen pd patients with anxiety and thirty - three pd patients without anxiety were included in this study ( see table 1 ) . the cross - sectional data from these participants was taken from a patient database that has been compiled over the past 8 years ( since 2008 ) at the parkinson 's disease research clinic at the brain and mind centre , university of sydney . inclusion criteria involved a diagnosis of idiopathic pd according to the united kingdom parkinson 's disease society brain bank criteria and were confirmed by a neurologist ( sjgl ) . patients also had to have an adequate proficiency in english and have completed a full neuropsychological assessment . ten patients in this study ( 5 pd with anxiety ; 5 pd without anxiety ) were taking psychotropic drugs ( i.e. , benzodiazepine or selective serotonin reuptake inhibitor ) . patients were also excluded if they had other neurological disorders , psychiatric disorders other than affective disorders ( such as anxiety ) , or if they reported a score greater than six on the depression subscale of the hospital anxiety and depression scale ( hads ) . thus , all participants who scored within a depressed ( hads - d > 6 ) range were excluded from this study , in attempt to examine a refined sample of pd patients with and without anxiety in order to determine the independent effect of anxiety on cognition . this research was approved by the human research ethics committee of the university of sydney , and written informed consent was obtained from all participants . self - reported hads was used to assess anxiety in pd and has been previously shown to be a useful measure of clinical anxiety in pd . a cut - off score of > 8 on the anxiety subscale of the hads ( hads - a ) was used to identify pd cases with anxiety ( pda+ ) , while a cut - off score of < 6 on the hads - a was used to identify pd cases without anxiety ( pda ) . this criterion was more stringent than usual ( > 7 cut - off score ) , in effort to create distinct patient groups . the neurological evaluation rated participants according to hoehn and yahr ( h&y ) stages and assessed their motor symptoms using part iii of the revised mds task force unified parkinson 's disease rating scale ( updrs ) . in a similar way this was determined by calculating a total left and right score from rigidity items 3035 , voluntary movement items 3643 , and tremor items 5057 from the mds - updrs part iii ( see table 1 ) . processing speed was assessed using the trail making test , part a ( tmt - a , z - score ) . attentional set - shifting was measured using the trail making test , part b ( tmt - b , z - score ) . working memory was assessed using the digit span forward and backward subtest of the wechsler memory scale - iii ( raw scores ) . language was assessed with semantic and phonemic verbal fluency via the controlled oral word associated test ( cowat animals and letters , z - score ) . the ability to retain learned verbal memory was assessed using the logical memory subtest from the wechsler memory scale - iii ( lm - i z - score , lm - ii z - score , % lm retention z - score ) . the mini - mental state examination ( mmse ) demographic , clinical , and neuropsychological variables were compared between the two groups with the independent t - test or mann whitney u test , depending on whether the variable met parametric assumptions . chi - square tests were used to examine gender and symptom laterality differences between groups . all analyses employed an alpha level of p < 0.05 and were two - tailed . spearman correlations were performed separately in each group to examine associations between anxiety and/or depression ratings and cognitive functions . as expected , the pda+ group reported significant greater levels of anxiety on the hads - a ( u = 0 , p < 0.001 ) and higher total score on the hads ( u = 1 , p < 0.001 ) compared to the pda group ( table 1 ) . groups were matched in age ( t(48 ) = 1.31 , p = 0.20 ) , disease duration ( u = 259 , p = 0.66 ) , updrs - iii score ( u = 250.5 , p = 0.65 ) , h&y ( u = 245 , p = 0.43 ) , ledd ( u = 159.5 , p = 0.80 ) , and depression ( hads - d ) ( u = 190.5 , p = 0.06 ) . additionally , all groups were matched in the distribution of gender ( = 0.098 , p = 0.75 ) and side - affected ( = 0.765 , p = 0.38 ) . there were no group differences for tmt - a performance ( u = 256 , p = 0.62 ) ( table 2 ) ; however , the pda+ group had worse performance on the trail making test part b ( t(46 ) = 2.03 , p = 0.048 ) compared to the pda group ( figure 1 ) . the pda+ group also demonstrated significantly worse performance on the digit span forward subtest ( t(48 ) = 2.22 , p = 0.031 ) and backward subtest ( u = 190.5 , p = 0.016 ) compared to the pda group ( figures 2(a ) and 2(b ) ) . neither semantic verbal fluency ( t(47 ) = 0.70 , p = 0.49 ) nor phonemic verbal fluency ( t(47 ) = 0.39 , p = 0.70 ) differed between groups . logical memory i immediate recall test ( u = 176 , p = 0.059 ) showed a trend that the pda+ group had worse new verbal learning and immediate recall abilities than the pda group . however , logical memory ii test performance ( u = 219 , p = 0.204 ) and logical memory % retention ( u = 242.5 , p = 0.434 ) did not differ between groups . there were also no differences between groups in global cognition ( mmse ) ( u = 222.5 , p = 0.23 ) . participants were split into lpd and rpd , and then further group differences were examined between pda+ and pda. importantly , the groups remained matched in age , disease duration , updrs - iii , dde , h&y stage , and depression but remained significantly different on self - reported anxiety . lpda+ demonstrated worse performance on the digit span forward test ( t(19 ) = 2.29 , p = 0.033 ) compared to lpda , whereas rpda+ demonstrated worse performance on the digit span backward test ( u = 36.5 , p = 0.006 ) , lm - i immediate recall ( u = 37.5 , p = 0.008 ) , and lm - ii ( u = 45.0 , p = 0.021 ) but not lm % retention ( u = 75.5 , p = 0.39 ) compared to rpda. this study is the first to directly compare cognition between pd patients with and without anxiety . the findings confirmed our hypothesis that anxiety negatively influences attentional set - shifting and working memory in pd . more specifically , we found that pd patients with anxiety were more impaired on the trail making test part b which assessed attentional set - shifting , on both digit span tests which assessed working memory and attention , and to a lesser extent on the logical memory test which assessed memory and new verbal learning compared to pd patients without anxiety . taken together , these findings suggest that anxiety in pd may reduce processing capacity and impair processing efficiency , especially in the central executive and attentional systems of working memory in a similar way as seen in young healthy adults [ 26 , 27 ] . although the neurobiology of anxiety in pd remains unknown , many researchers have postulated that anxiety disorders are related to neurochemical changes that occur during the early , premotor stages of pd - related degeneration [ 37 , 38 ] such as nigrostriatal dopamine depletion , as well as cell loss within serotonergic and noradrenergic brainstem nuclei ( i.e. , raphe nuclei and locus coeruleus , resp . , which provide massive inputs to corticolimbic regions ) . over time , chronic dysregulation of adrenocortical and catecholamine functions can lead to hippocampal damage as well as dysfunctional prefrontal neural circuitries [ 39 , 40 ] , which play a key role in memory and attention . recent functional neuroimaging work has suggested that enhanced hippocampal activation during executive functioning and working memory tasks may represent compensatory processes for impaired frontostriatal functions in pd patients compared to controls . therefore , chronic stress from anxiety , for example , may disrupt compensatory processes in pd patients and explain the cognitive impairments specifically in working memory and attention seen in pd patients with anxiety . it has also been suggested that hyperactivation within the putamen may reflect a compensatory striatal mechanism to maintain normal working memory performance in pd patients ; however , losing this compensatory activation has been shown to contribute to poor working memory performance . anxiety in mild pd has been linked to reduced putamen dopamine uptake which becomes more extensive as the disease progresses . this further supports the notion that anxiety may disrupt compensatory striatal mechanisms as well , providing another possible explanation for the cognitive impairments observed in pd patients with anxiety in this study . noradrenergic and serotonergic systems should also be considered when trying to explain the mechanisms by which anxiety may influence cognition in pd . although these neurotransmitter systems are relatively understudied in pd cognition , treating the noradrenergic and serotonergic systems has shown beneficial effects on cognition in pd . selective serotonin reuptake inhibitor , citalopram , was shown to improve response inhibition deficits in pd , while noradrenaline reuptake blocker , atomoxetine , has been recently reported to have promising effects on cognition in pd [ 45 , 46 ] . overall , very few neuroimaging studies have been conducted in pd in order to understand the neural correlates of pd anxiety and its underlying neural pathology . future research should focus on relating anatomical changes and neurochemical changes to neural activation in order to gain a clearer understanding on how these pathologies affect anxiety in pd . to further understand how anxiety and cognitive dysfunction are related , future research should focus on using advanced structural and function imaging techniques to explain both cognitive and neural breakdowns that are associated with anxiety in pd patients . research has indicated that those with amnestic mild cognitive impairment who have more neuropsychiatric symptoms have a greater risk of developing dementia compared to those with fewer neuropsychiatric symptoms . future studies should also examine whether treating neuropsychiatric symptoms might impact the progression of cognitive decline and improve cognitive impairments in pd patients . previous studies have used pd symptom laterality as a window to infer asymmetrical dysfunction of neural circuits . for example , lpd patients have greater inferred right hemisphere pathology , whereas rpd patients have greater inferred left hemisphere pathology . thus , cognitive domains predominantly subserved by the left hemisphere ( e.g. , verbally mediated tasks of executive function and verbal memory ) might be hypothesized to be more affected in rpd than lpd ; however , this remains controversial . it has also been suggested that since anxiety is a common feature of left hemisphere involvement [ 48 , 49 ] , cognitive domains subserved by the left hemisphere may also be more strongly related to anxiety . results from this study showed selective verbal memory deficits in rpd patients with anxiety compared to rpd without anxiety , whereas lpd patients with anxiety had greater attentional / working memory deficits compared to lpd without anxiety . although these results align with previous research , interpretations of these findings should be made with caution due to the small sample size in the lpd comparison specifically . recent work has suggested that the hads questionnaire may underestimate the burden of anxiety related symptomology and therefore be a less sensitive measure of anxiety in pd [ 30 , 50 ] . in addition , our small sample size also limited the statistical power for detecting significant findings . based on these limitations , our findings are likely conservative and underrepresent the true impact anxiety has on cognition in pd . additionally , the current study employed a very brief neuropsychological assessment including one or two tests for each cognitive domain . future studies are encouraged to collect a more complex and comprehensive battery from a larger sample of pd participants in order to better understand the role anxiety plays on cognition in pd . another limitation of this study was the absence of diagnostic interviews to characterize participants ' psychiatric symptoms and specify the type of anxiety disorders included in this study . future studies should perform diagnostic interviews with participants ( e.g. , using dsm - v criteria ) rather than relying on self - reported measures to group participants , in order to better understand whether the type of anxiety disorder ( e.g. , social anxiety , phobias , panic disorders , and generalized anxiety ) influences cognitive performance differently in pd . one advantage the hads questionnaire provided over other anxiety scales was that it assessed both anxiety and depression simultaneously and allowed us to control for coexisting depression . although there was a trend that the pda+ group self - reported higher levels of depression than the pda group , all participants included in the study scored < 6 on the depression subscale of the hads . controlling for depression while assessing anxiety has been identified as a key shortcoming in the majority of recent work . considering many previous studies have investigated the influence of depression on cognition in pd without accounting for the presence of anxiety and the inconsistent findings reported to date , we recommend that future research should try to disentangle the influence of anxiety versus depression on cognitive impairments in pd . considering the growing number of clinical trials for treating depression , there are few if any for the treatment of anxiety in pd . anxiety is a key contributor to decreased quality of life in pd and greatly requires better treatment options . moreover , anxiety has been suggested to play a key role in freezing of gait ( fog ) , which is also related to attentional set - shifting [ 52 , 53 ] . future research should examine the link between anxiety , set - shifting , and fog , in order to determine whether treating anxiety might be a potential therapy for improving fog .

research on the implications of anxiety in parkinson 's disease ( pd ) has been neglected despite its prevalence in nearly 50% of patients and its negative impact on quality of life . previous reports have noted that neuropsychiatric symptoms impair cognitive performance in pd patients ; however , to date , no study has directly compared pd patients with and without anxiety to examine the impact of anxiety on cognitive impairments in pd . this study compared cognitive performance across 50 pd participants with and without anxiety ( 17 pda+ ; 33 pda ) , who underwent neurological and neuropsychological assessment . group performance was compared across the following cognitive domains : simple attention / visuomotor processing speed , executive function ( e.g. , set - shifting ) , working memory , language , and memory / new verbal learning . results showed that pda+ performed significantly worse on the digit span forward and backward test and part b of the trail making task ( tmt - b ) compared to the pda group . there were no group differences in verbal fluency , logical memory , or tmt - a performance . in conclusion , anxiety in pd has a measurable impact on working memory and attentional set - shifting .

small non - coding rnas are transcribed into mrna but remain untranslated in eukaryotic cells . they include sirna ( small interfering rna ) , mirna ( microrna ) , pirna ( piwi - interacting rna ) and snorna ( small nucleolar rna ) . mirnas are a class of multifunctional singled - stranded small rna which are ~20 nt in length and regulate the stability or translational efficiency of targeted messenger rna depending on the base - pairing complementarity between the mirna and its target mrna [ 1 , 2 ] . although over 1,000 mirna sequences have been identified from the tissues or cells of human origin and other species , as many as 1,000 to 10,000 mirnas per genome have been predicted [ 3 , 4 ] . mirnas regulate a broad range of biological processes including timing of development , cell cycle progression , stem cell self - renewal , differentiation , cancer initiation , cancer cell proliferation , metastasis and apoptosis [ 511 ] . cancer is caused by multiple processes including uncontrolled cellular proliferation and inappropriate survival of apoptotic cells . many regulatory factors switch on or off genes that govern cell division and direct cellular proliferation . mirnas regulate gene expression and play important roles in the onset and progression of tumorigenesis . emerging evidence demonstrates the involvement of mirna in mammary gland tumorigenesis , functioning either as tumor suppressors or oncogenes . although the current treatment of radiation therapy , chemotherapy and hormone therapy slow mammary gland tumor growth , prolong survival and improve the quality of patients life , metastatic breast cancer still remains incurable due to our limited understanding of the molecular mechanisms through which tumorigenesis and metastasis occur . as small non - coding rnas regulate gene expression and tumorigenesis , they may represent a novel cancer therapy . unlike mrna , mirnas are transcribed but never translated . some mirnas are transcribed from non - coding regions between genes , deriving from independent transcription unit . other mirnas are transcribed together with coding mrnas from the coding region of the genome , deriving from the introns of gene transcripts [ 13 , 14 ] . mirna gene copy number gain / loss and mirna gene mutation have been observed in breast cancer resulting in the aberrant expression of mirna . the first study about the altered expression of mirnas in human breast cancer patients and human breast cancer cell lines was reported in 2005 by lorio et al . , in which 29 mirnas were identified with aberrant expression based on microarray and northern blot analysis of 76 breast tumor samples and 14 human breast cell lines . zhang and colleagues analyzed 283 human mirna genes on 55 human breast primary tumors and 18 human breast cancer cell lines using array - based comparative genomic hybridization . the results demonstrated a high frequency ( ~72.8% ) of gene copy number abnormality in mirna - containing regions in human breast cancer . wang et al . collected 68 patients with newly diagnosed breast cancer and examined the expression of selected mirnas in tumor and adjacent non - tumor tissues . mir-21 , mir-106a and mir-155 were significantly over - expressed in the tumor specimens compared with normal controls , whereas mir-126 , mir-199a and mir-335 were significantly decreased in expression in the tumor samples . our studies of the mir-17 - 92 cluster demonstrated decreased expression of mir-17/20 in human breast cancer specimens compared with matching normal breast tissue from the same patient . subsequent analysis identified reduced mir-17/20 expression in node - positive compared with node - negative breast cancers and demonstrated that mir-17/20 inhibited breast cancer cell migration and invasion via a heterotypic signaling . although the tendency for a global decrease of mirna expression in human cancers originally suggested a general tumor suppressor function of mirnas , subsequent studies showing the aberrant expression of specific mirnas in breast cancer suggest mirna - specific roles in breast cancer onset and progression . many distinct mirnas have been shown to regulate breast cancer cell proliferation , apoptosis , cancer stem cell expansion , and tumorigenesis . mirna may function as either tumor suppressors or oncogenes depending on the cell type , culture conditions , target genes and pathway . the involvement of mirna in mammary gland tumorigenesis has been reviewed recently [ 21 , 22 ] . le et al . described the expression pattern and regulatory network of key mirnas in breast cancer , including let-7 , mir-34 , mir-125 , mir-200 family , mir-205 , mir-21 , mir-10 and the mir-17 - 92 cluster . adams et al . reviewed the mirna regulation of estrogen signaling pathway and erbb2/her signaling pathway in breast cancer . the understanding of how mirnas are involved in breast cancer through regulating the cell cycle remains rudimentary . herein we summarize the recent literature and research progress on the mechanism by which mirnas regulate the breast cancer cell cycle and cellular proliferation ( fig . 1mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint cyclin d1 is either overexpressed or amplified in ~50% of breast cancer . the abundance of cyclin d1 is rate - limiting in breast cancer cellular proliferation and g1-s phase transition [ 23 , 24 ] . in addition , cyclin d1 is a critical downstream target of erbb2- , ras- and -catenin- induced breast cancers , and is sufficient for the induction of mammary tumors when targeted to the mammary gland of mice . antisense inhibition of cyclin d1 expression in vivo suppressed the growth of neut - transformed mammary adenocarcinoma cells in nude mice . conserved sequences of the cyclin d1 3utr contain potential binding sites for multiple mirnas including mir-17/20/106 , mir-15/16 , mir-23 and let-7 . mir-17/20 binds the cyclin d1 3utr , inhibiting the expression of cyclin d1 , resulting in cell cycle arrest at the g1 phase and suppression of mcf-7 cell proliferation [ 18 , 26 ] . the regulation of cyclin d1 expression by mir-17 - 92 , as well as mir-15/16 , was confirmed by deshpande et al . . the let-7 family functions as a tumor suppressor in a variety of cancers including lung , colon , ovarian and breast cancer . schultz et al . demonstrated the downregulation of cyclin d1 by mirna let-7 in control of cancer cell growth . the regulation of cyclin d1 by mirna is likely of broad importance as cyclin d1 encodes the regulatory subunit of a kinase that phosphorylates and inactivates the prb family proteins to inhibit dna synthesis , and phosphorylates nuclear respiratory factor 1 ( nrf-1 ) to inhibit mitochondria biogenesis [ 32 , 33 ] . furthermore , cyclin d1 promotes breast epithelial cell angiogenesis and migration , and promotes chromosomal instability which in turn contributes to tumorigenesis . the mir-221/222 cluster regulates the cell cycle , cell growth and epithelial - to - mesenchymal transition ( emt ) in breast cancer . mir-221/222 inhibited p27 and p57 abundance , facilitating g1-s phase transition , thereby promoting cancer cell proliferation [ 36 , 37 ] . moreover , mir-221/222 may contribute to the aggressive clinical behavior of basal - like breast cancers . the breast cancer basal - like subtype - specific mirnas , mir-221 and mir-222 , promote emt in breast cancer by targeting trps1 ( trichorhinophalangeal syndrome type 1 ) which inhibits emt by repressing zeb2 expression . mir-221 and/or mir-222 expression in mcf-7 and t47d breast cancer cells decreased er expression associated with tamoxifen resistance . the onco - mirna mir-21 is overexpressed in a wide variety of cancers including breast cancer [ 40 , 41 ] . mir-21 induced cellular proliferation , migration , invasion , emt , cancer stem cell characteristics and chemotherapy resistance in human breast cancer [ 42 , 43 ] . high mir-21 level is associated with poor prognosis , advanced stage , positive lymph node status and reduced survival time in breast cancer . mir-21 promotes mcf-7 cellular proliferation in part through inhibiting the expression of a tumor suppressor gene programmed cell death 4 ( pdcd4 ) . in colon cancer , mir-21 participates in a dna damage - induced g2-m checkpoint through suppressing the cell cycle regulator cdc25a . a recent report demonstrated the mir-21 regulates the cell cycle through targeting cdc25a in mcf-7 breast cancer cells . with a potential anti - cancer chemical 3,3-diindolylmethane treatment , mir-27a expression is upregulated in human breast cancer cell lines . in mda - mb-231 cells , mir-27a negatively regulated the zinc finger zbtb10 gene and myt-1 , thereby promoting breast cancer cell proliferation . mir-27a suppressed myt-1 , increased cdc2/cyclin b activity and promoted the g2-m checkpoint in mda - mb-231 cells . thus , distinct mirnas affect key genetic targets that govern distinct cell - cycle checkpoints including cyclins , cdks , cdk inhibitors and the g2-m regulation apparatus . in addition to cell - cycle control , breast tumor onset and progression and breast tumor stem cells are also regulated by distinct mirnas . cancer stem cells ( cscs ) are characterized by their self - renewal capacity , an ability to differentiate into non - tumorigenic cell progeny , and their ability to seed tumors when transplanted into animal hosts . cell surface markers such as cd44 , cd24 , cd133 , epithelial - specific antigen and aldehyde dehydrogenase-1 are frequently used to isolate and enrich cscs . the involvement of mirnas in regulating tumor formation by cscs or tumor - initiating cells ( t - ic ) has been widely investigated . let-7 expression is very low to undetectable level in embryonic stem cells ( es cells ) and increases with differentiation . a comparison of mirna expression between breast t - ic and non - t - ic demonstrated reduced let-7 expression in t - ic and increased abundance with differentiation . transduction of breast cscs with let-7 reduced the proportion of undifferentiated cells , inhibited cell proliferation , mammosphere formation , and tumor formation in vivo . clarke and colleagues identified 37 mirnas which were differentially expressed between human breast cscs ( cd44cd24lineage ) and lineage nontumorigenic breast cancer cells . the mir-200 family members were downregulated in human breast cscs and normal mammary stem / progenitor cells . expression of mir-200 inhibited breast cancer stem cell expansion in vitro , and suppressed the tumor formation ability of human breast cancer stem cell in vivo . ectopic mir-34c expression reduced breast t - ics self - renewal , inhibited emt and suppressed tumor cell invasiveness via silencing notch4 . zhu et al . found the reduced mir-128 expression in human breast t - ic was accompanied by bmi-1 and abcc5 overexpression , and associated with chemotherapeutic resistance and poor survival . enforced mir-128 expression increased the sensitivity of breast cancer cells to doxorubicin - induced apoptosis and dna damage . emerging evidence has demonstrated the importance of cscs in cancer initiation , cancer metastasis and drug resistance . cscs are believed to be one of the most promising targets for cure of cancer . the discovery that non - coding rnas regulate cscs widens our understanding of cscs , and may provide potential novel strategies for breast cancer therapy . deletion of chromosome 6q , including region 6q14-q16 , is frequently observed in breast cancer . the small non - coding snorna u50 is a candidate tumor suppressor gene in the 6q14 - 16 region , playing a role in the development and/or progression of breast cancer . genomic deletion and transcriptional downregulation of snorna u50 was detected in breast cancer cell lines . re - expression of snorna u50 inhibited colony formation of the human breast cancer cells hs578 t and mda - mb-231 . pirnas are small non - coding rna that form rna - protein complexes through interactions with piwi proteins . pirna was initially discovered in germ line cells , and considered as germ line - specific small rnas . emerging evidence indicates that pirna expression occurs in somatic cells [ 57 , 58 ] and piwil2 expression has been identified in human breast cancer cells . high - throughput deep sequencing identified a group of small rnas matching pirna sequences in human breast cancer tissues and breast cancer cell lines . the study of these non - coding small rnas in human cancer is just starting . the identification of the expression signature of these non - coding small rnas in breast cancer subtypes , and an understanding of their functional significance to oncogene expression , tumor initiation and tumor cell metastasis may shed important new perspectives on the role of these non - coding small rnas in breast cancer . small non - coding rna - based diagnostic and therapeutic applications for human cancer are expected in the near future . although tumor - targeted delivery and local administration are still major challenges to the practical application of gene therapy for cancer , mirna - based cancer therapeutic approaches are being established and tested in animal models . synthetic mirna mimics or mirna expression vectors have been successfully applied to restore or overexpress mirna in vitro . chemically modified lna anti - sense mirna inhibitor and other approaches have been used to block mirna function in cells . kim et al . recently reported significant anti - tumor effect of virus - mediated delivery of mir-145 combined with 5-fu to treat breast cancer . intranasal delivery of let-7 and intravenous delivery of mir-34a mimics for non - small - cell lung cancer treatment and a virus - mediated delivery of mir-26a for liver cancer treatment in mouse model demonstrate the promise of mirna for treatment of cancer . dysregulated expression of mirnas has implicated components of the non - coding genome as either oncogenes or tumor suppressors of breast cancer . experimental evidence has shown specific mirnas regulating the initiation , progression , metastasis and drug resistance of breast cancer via control of the cell cycle , altering cellular proliferation , altering cellular apoptosis and/or controlling the population of tumor stem cells . dysregulated mirna expression has also been observed in cancer associated fibroblasts ( caf ) and in the systemic circulation [ 65 , 66 ] . the circulating mirnas have the potential to serve as novel diagnostic and prognostic biomarkers for breast cancer . a specific subset of dysregulated mirnas in breast cancer cells may serve as targets for gene therapy either alone or as an adjuvant treatment to current clinical protocols for breast cancer patients .

small non - coding rnas include sirna , mirna , pirna and snorna . the involvement of mirnas in the regulation of mammary gland tumorigenesis has been widely studied while the role for other small non - coding rnas remains unclear . here we summarize the involvement of mirna in breast cancer onset and progression through regulating the cell cycle and cellular proliferation . the regulation of breast cancer stem cells and tumor regeneration by mirna is reviewed . in addition , the emerging evidence demonstrating the involvement of pirna and snorna in breast cancer is briefly described .

ohss is a serious complication of ovulation induction , occurring in 1 - 10% of in vitro fertilization patients ( rizk and aboulghar , 1991 ; brinsden et al . , 1995 ) . this iatrogenic condition has a spectrum of clinical and laboratory manifestations ranging from mild to severe , even life - threatening conditions ( rizk et al . , 1990 ) . among the serious manifestations of ohss are ascites and pleural effusion ( rizk and smitz , 1992 ) . vascular endothelial growth factor has emerged as one of the factors most likely involved in the pathophysiology of ohss ( geva and jaffe , 2000 ; rizk and aboulghar , 2010 ) . vascular endothelial growth factor is an angiogenic cytokine that is a potent stimulator of the vascular endothelium and appears to play an integral role in follicular growth , corpus luteum function and ovarian angiogenesis ( rizk et al . , 1997 ) . vascular endothelial growth factor causes increased capillary permeability with its sequelae of ascites and possible pleural effusion ( rizk et al . , 1997 ; gomez et al . , 2002 ) . human chorionic gonadotrophin is thought to play a crucial role in the development of ohss ( rizk et al . two distinct forms of severe ohss have been described : early and late forms ( papanikolaou et al . , 2005 ; mathur et al . , 2000 ) . early ohss is caused by the administration of exogenous human chorionic gonadotrophin ( hcg ) and it starts before the 10th day after oocyte retrieval , while the late form is the result of endogenous human chorionic gonadotrophin release in the event of pregnancy , and it starts after the 10th day following oocyte aspiration ( papanikolaou et al . , 2005 ; such strategies included the use of low dose gonadotropins and frequent monitoring during controlled ovarian stimulation , cycle cancellation , coasting , freeze all embryos and the use of gonadotropin releasing hormone ( gnrh ) agonist as an oocyte trigger in gnrh antagonist cycles ( rizk et al . the use of antagonist in the luteal phase with total freeze of embryos to prevent severe ohss has also been suggested ( lainas et al . , 2012 ) . such strategies should be used especially in patients at high risk for developing severe ohss , including those who are young , have low body weight , have polycystic ovary syndrome , those who experience high estradiol levels , rapidly increasing estradiol levels , high number of follicles during controlled ovarian stimulation and those with high number of oocytes retrieved ( rizk and aboulghar , 1999 ; delvigne , 2009 ) . dopamine agonists have been proposed for the prevention of ohss in women at high risk . however , the study by lvarez et al . ( 2007 ) suggested that it was only effective in reducing the incidence of moderate ohss , but not in its severe form ( alvarez et al . , 2007 ) . low - dose aspirin therapy and doxycycline have also been suggested to inhibit angiogenesis ( fainaru et al . , 2009 ) . ( 2011 ) believes that the concept of an ohss - free clinic has become a reality . the authors suggest that this approach should include pituitary down - regulation using a gnrh antagonist , ovulation triggering with a gnrh agonist and vitrification of oocytes or embryos ( devroey et al . , 2011 ) . severe and critical forms of ohss are associated with significant ascites ( golan et al . , 1989 ; rizk et al . , ascites results in an increased abdominal pressure compromising venous return , cardiac output and renal perfusion ( navot et al . paracentesis is frequently needed , as prompt drainage of fluid produces a significant clinical and biochemical improvement , including spontaneous diuresis and hastening the resolution process ( rizk et al . , multiple paracenteses may be required to relieve symptoms and avoid serious sequelae of hemoconcentration , hypotension , decreased renal perfusion and severe respiratory compromise . instead of multiple interventions , the placement of a catheter temporarily for a few days will permit complete drainage through one procedure ( rizk and aboulghar , 1999 ) . pigtail catheters have been used for drainage in various clinical situations . in a previous study we described the use of percutaneous placement of a pigtail catheter for drainage of ascites caused by severe / critical ohss ( abuzeid et al . , 2003 ) . in this study we established the efficacy and safety of pigtail catheter drainage in the management of severe / critical ohss in patients who underwent in vitro fertilization and embryo transfer at our centre between 1999 and 2001 on both inpatient and outpatient basis . the aim of the current study was to evaluate the role of the pigtail catheter drainage in outpatient management of patients with early onset severe ohss . this is a retrospective study that included all patients who underwent in vitro fertilization and developed severe or critical ohss by the golan classification ( golan et al . , 1989 ) in the period between 2004 and 2009 . all patients underwent an ultrasound evaluation which documented the presence of severe ascites and bilateral ovarian enlargement . all patients presented with shortness of breath , marked abdominal distension , nausea , weight gain and lower abdominal pain . thirteen patients complained of vomiting , and 3 patients reported decreased urine output ( less than 720 cc per 24 hours ) and had haemoglobin 15 g / dl . all 33 patients were offered treatment using pigtail catheter insertion as an alternative to trans - vaginal paracentesis . blood studies including a complete blood count , creatinine , electrolytes , serum albumin and total protein , and coagulation panel were ordered prior to the procedure . all 33 patients were rehydrated with intravenous crystalloid solutions ( 0.9% normal saline ) . care was taken to give the patient the same amount of fluid as the amount that was drained from the peritoneal cavity . all pigtail catheter placements were performed in the operating room at our surgical centre . the catheter ( 6 - 0 french , 2.0 mm , boston scientific , quincy , ma , usa ) was introduced utilizing a non - disposable metal gamete intra - fallopian treatment ( gift ) trocar and cannula ( embryon gift catheter , ivf online , gilford , ct , usa ) which was introduced into the largest accessible ascitic fluid pocket in the upper outer quadrant of the abdominal wall under local anaesthesia and trans - abdominal ultrasound guidance . the trocar was then removed and a glide wire ( boston scientific corp . , watertown , ma , usa ) was threaded through the gift canula . the canula was removed with care so as to keep the glide wire in the peritoneal cavity . the pigtail catheter was guided into the peritoneal cavity by the wire after removal of the stylet of the pigtail catheter . the catheter was anchored to the skin using 2 - 0 silk suture and it was supported by gauze , which was covered by a tegaderm . the catheter was connected to a three - way stop cock which was connected to a drainage bag . initial amount of drained fluid was calculated and subsequently the patients were trained to drain 1000 cc per day ( 500 cc in the morning and 500 cc in the evening ) . one liter normal saline and 500 cc of 6% hydroxyethyle starch solution ( hespan ) was administered to the patient on the same day of pigtail catheter placement . any patient with critical ohss according to the golan classification , or those in whom the presenting symptoms and signs did not completely subside after drainage of ascitic fluid and iv fluid replacement , were admitted to the hospital for observation and further management . all other patients were sent home . once there was minimal or no drainage from the catheter , the patient returned to the office for removal of the catheter . the patients were encouraged to drink plenty of fluids and to increase their protein intake . all patients kept a diary for body weight , abdominal girth , and urine output before and after the procedure . patients were seen in the office as needed for evaluation and hydration with normal saline . demographic data including mean age ( years ) , duration of infertility ( years ) , bmi ( kg / m2 ) and aetiology of infertility are illustrated in table i. ovarian stimulation data including number of treatment days , total dose of recombinant follicle stimulating hormone , hormone serum levels , endometrial thickness , number of follicles and number of patients receiving only 5000 iu of human chorionic gonadotrophin is illustrated in table ii . our data include the number of patients requiring early versus late placement of pigtail catheter , mean amount of ascitic fluid drained immediately after pigtail placement , number of days of pigtail catheter drainage and the number of days between oocyte retrieval and the onset of severe ohss symptoms requiring pigtail placement . table iii also illustrates the number of patients who underwent coasting , number of patients who received prophylactic hespan on day of egg retrieval . values are expressed as mean + standard deviation values are expressed as mean + standard deviation : rfsh = recombinant fsh ; hmg = human menopausal gonadotropins ; hcg = human chorionic gonadotropin ; e2 = estradiol values are expressed as mean + standard deviation three patients ( 9% ) required an early placement of a pigtail catheter while 30 patients ( 91% ) had late placement of the catheter ( table iii ) . twenty - nine patients ( 88% ) were managed on an outpatient basis without any complications . four patients ( 12% ) required hospital admission for 1 - 7 days ( 3 + 2.7 ) , three for associated severe pleural effusion requiring thoracentesis . these three patients were admitted to the hospital immediately after insertion of the pigtail catheter for management of severe pleural effusion and careful observation . one patient was admitted 48 hours after insertion of the pigtail catheter for work up for chest pain , all tests were negative . navot et al . , 1992 ; rizk and aboulghar , 1999 ) , but perhaps the most accepted classification of ohss is the one described by golan et al as mild , moderate and severe ( golan et al . , 1989 ) . however , clinicians should be aware that ohss is a dynamic condition and moderate ohss may progress to severe ohss within hours ( rizk and aboulghar , 1999 ; rizk , 2009 ) . therefore , the practice committee of the american society for reproductive medicine ( asrm ) proposed a classification for ohss of mild , worsening and serious ( the practice committee of the american society for reproductive medicine , 2008 ) . recognizing the patients at high - risk , prevention and early recognition are the most important steps in the management of ohss ( rizk , 2006 ) . however , severe forms of ohss , especially late onset type that occurs when patients are pregnant , most likely will continue to happen , albeit at much lower rate , unless one reverts to a total freeze of all embryos after the use of gnrh agonist as a trigger shot in a gnrh antagonist cycle ( donoso and devroey , 2013 ; devroey et al . , 2011 ) . traditional treatment consists mainly of supportive management until spontaneous resolution occurs ( rizk , 1994 ) . although moderate ohss does not usually require intervention , outpatient monitoring is essential to identify progression to a severe form ( rizk , 2006 ) . patients with moderate ohss can be managed on an outpatient basis by adequate hydration and close daily monitoring of weight gain and abdominal girth to detect progression to severe ohss , and to provide intervention , if required ( rizk , 2010 ) . patients presenting with severe forms of ohss are usually managed on inpatient basis and most patients with critical ohss are admitted to intensive care units for aggressive supportive measures to avoid maternal morbidity and mortality ( the practice committee of the american society for reproductive medicine , 2008 ) . correction of hypovolemia , electrolyte imbalance and hypoalbuminemia are the basic principles during the treatment of severe ohss ( rizk , 1994 , 2006 ; myrianthefs et al . , 2000 ) . ( 1992 ) suggested that paracentesis is the single most important treatment modality in life - threatening ohss not controlled by medical therapy . paracentesis and removal of ascites result in relieving the pressure on the inferior vena cava and improvement in venous return , cardiac output and renal perfusion ( padilla et al . , 1990 ) . therefore , prompt drainage of fluid produces significant clinical and biochemical improvement , including spontaneous diuresis and hastening the resolution process ( abuzeid et al . , 2003 ; the practice committee of the american society for reproductive medicine , 2008 ) . it is also possible that removal of ascites decreases the amount of vegf and its effects on vascular permeability . therefore ultrasound guided drainage of the ascitic fluid should be performed in severe cases of ohss with respiratory distress , oliguria or severe abdominal pain ( padilla et al . , 1990 ) . this can be done either trans - vaginally ( aboulghar et al . , 1990 ) or trans - abdominally ( padilla et al . , 1990 ) . however , recurrence of ascites is common in severe cases of ohss . placement of a pigtail catheter has been proposed by some investigators to allow drainage of ascitic fluid whenever it accumulates without the need for further surgical procedures ( abuzeid et al . , 2003 ) . in a previous study we reported the use of percutaneous pigtail catheter for drainage of ascites in severe ohss as an alternative to repeated paracentesis ( abuzeid et al . , 2003 ) . this procedure is relatively simple and requires a short period of time as compared to the time needed to gradually aspirate any fluid accumulation transvaginally . immediate relief of symptoms and signs of severe ohss after pigtail catheter insertion was an important finding . in addition , the ease by which any accumulated ascitic fluid was drained resulted in a faster resolution of ohss ( abuzeid et al . , no infection was reported ( abuzeid et al . , 2003 ) . in that study the first 13 patients they were discharged home with the pigtail catheter in place and managed on an outpatient basis until ohss subsided ; thereafter the catheter was removed . the last 13 patients were managed on an outpatient basis without any problem ( abuzeid mi , et al . , 2003 ) . our previous study confirmed the safety and effectiveness of the pigtail catheter for the management of severe ohss . therefore , we embarked on the current study to evaluate the role of pigtail catheter drainage in outpatient management of patients with severe ohss syndrome . in the current study we demonstrated that patients who were diagnosed very early with severe ohss can be managed safely and effectively on outpatient basis using pigtail catheter drainage , iv fluid replacement and careful monitoring . although all 33 patients presented with shortness of breath , marked abdominal distension , nausea , weight gain and lower abdominal pain , and 13 of them complained of vomiting , ( all are criteria for hospital admission according to the practice committee of the american society for reproductive medicine on ohss ) , 30 patients were sent home as their presenting symptoms were relieved . early diagnosis and intervention resulted in rapid elimination of the symptoms of severe ohss and normalization of vital signs , which allow us to manage the patients on outpatient basis . the pigtail catheter placements were performed without complications and patients were discharged home after a few hours of observation . the patients who needed hospital admission were those with associated severe pleural effusion requiring thoracentesis . another patient was admitted to the hospital for 1 day for work up for chest pain and possible diagnosis of pulmonary embolism . once pulmonary embolism was ruled out , she was discharged home with a pigtail catheter in place and she was managed on outpatient basis . outpatient management of ohss has no place in those who fail to have complete resolutions of their symptoms , or those with severe hyperproteinuria or electrolyte imbalance . certainly it has no place in patients with critical ohss such as those with severe pleural effusion , diminished urine output , severe hemoconcentration and those at increased risk of thromboembolic risks ( rizk , 2010 ) . it is imperative to stress that such a protocol can only be used in patients who are motivated , reliable and have the necessary support at home to allow them to return to the clinic or go to the emergency room if the need arises . it also requires dedicated in vitro fertilization nurses to educate and follow up the patient on a daily basis . contrary to other reports that state that access to intravenous albumin is a critical part of outpatient management for severe ohss , we did not use any albumin replacement ( lincoln et al . , 2002 ) . the reason behind the use of intravenous albumin is to increase intravascular oncotic pressure , thereby curtailing the exodus of free water from the intravascular space . in our study we used hespan instead of albumin , as some investigators have shown that it is beneficial in reducing the incidence of severe ohss ( youssef et al . , 2011 ) . by increasing the intravascular oncotic pressure in addition , the majority of our patients had late onset ohss as a result of pregnancy . hespan should be used with caution in patients with late onset ohss only when benefits outweigh risks.it is a fact that outpatient management of early severe ohss is a common practice among infertility specialists after trans - vaginal aspiration of ascitic fluid and iv fluid hydration . outpatient management by repeated abdominal or trans - vaginal paracentesis has been reported to be safe , effective and cost effective ( lincoln et al . , 2002 ; shrivastav et al . , 1994 ; fluker et al . , 2000 ( 1994 ) were the first to suggest that day care management in patients with severe ohss with abdominal paracentesis and iv hydration was simple , safe and effective compared with traditional treatment with in - patient hospitalization and supportive measures . ( 2002 ) concluded that the need for hospitalization for hyperstimulated patients can be minimized by outpatient management of severe ohss with trans - vaginal culdocentesis and rehydration with crystalloids and albumin . ( 2000 ) advocated in a pilot study an active management strategy for moderate ohss , which involved early paracentesis designed to minimize the progression from moderate to severe ohss , instead of late paracentesis aimed at speeding up recovery in severely ill patients . ( 2010 ) concluded that early outpatient paracentesis for moderate to severe ohss is the most cost effective management plan when compared with traditional conservative inpatient therapy . ( 2009 ) reported a large series of patients with severe ohss in whom the vast majority were successfully managed as outpatients with use of aggressive trans - vaginal paracentesis ( smith et al . , 2009 ) . despite the findings in our study it is important to emphasize that ohss remains a serious disorder with the potential for rapid deterioration , requiring hospitalization and intensive treatment of a critically ill patient . the life - threatening complications of ohss are secondary to thromboembolism or compromised pulmonary or cardiovascular function and multi - organ failure . there have been isolated reports of women dying from the complications associated with ohss . the mortality rate related to ohss is very low and was estimated by brinsden et al . more recently , reports regarding maternal mortality rates due to ohss in the netherlands and united kingdom demonstrate an incidence of approximately 3 deaths per 100,000 in ivf cycles ( braat et al . , 2006 ; lewis , 2007 ) . eleven lethal complications of assisted reproductive technology have been documented and summarized by braat et al . several investigators reported sporadic fatality as a result of ohss due to adult respiratory distress syndrome , massive pulmonary oedema , myocardial infarction , and carotid artery occlusion resulting in cerebral infarction . in this study we describe a modified technique of what interventional radiologists usually use for pigtail catheter placement . we observed that in the obese patients the metal introducer provided in the pigtail catheter kit is difficult to use ; instead we used the non disposable gamete intra - fallopian treatment trocar and cannula to facilitate the introduction of the catheter through the abdominal wall of such patients . this technique helps to manage the patients in the office without the need for sending her to the hospital for an interventional radiologist to place the catheter . however , the patients in this study were identified from our in vitro fertilization database ; the study included every patient who developed severe / critical ohss . in addition , the lack of a control group that was managed without pigtail catheter placement limits the conclusions that can be drawn from our study . in summary , drainage of ascitic fluid in patients with severe / critical ohss is an important step in the management of this condition ( rizk , 2010 ) . the placement of a pigtail catheter resulted in a safe and effective outpatient management of the majority of patients with severe ohss . however , outpatient management of this potentially fatal condition requires clear understanding that rapid deterioration may occur , which may necessitate hospitalization and intensive treatment of a critically ill patient ( rizk , 2006 , 2010 ) . patients with critical ohss and those with persistence of symptoms of severe ohss despite insertion of a pigtail catheter and drainage of ascitic fluid should be admitted to the hospital for inpatient management .

objective : to evaluate the efficacy and safety of outpatient management of severe ovarian hyperstimulation syndrome ( ohss ) requiring placement of a pigtail catheter.methods : retrospective analysis of thirty - three consecutive patients who underwent in - vitro fertilization ( 2003 - 2009 ) and developed severe / critical ohss requiring placement of a pigtail catheter . patients who were managed on outpatient basis were monitored by frequent office visits , daily phone calls , and received iv normal saline for hydration when required.results : in 3 patients ( 9.1% ) ohss started early , requiring placement of a pigtail catheter 4.3 + 0.6 days after retrieval . in 30 patients ( 90.9% ) ohss started late ( 14 4 days after retrieval ) . the mean amount of ascitic fluid drained immediately after placement of the catheter was 2085 1018 cc . the pigtail catheter was removed after 7.8 5.3 days . of the 31 patients who had embryo transfer ( two had total freeze ) , 84% conceived . twenty - nine patients ( 88% ) were managed on outpatient basis without any complications . four patients required hospital admission for 1 - 7 days ( 3.0 2.7 ) . one patient with severe ohss was admitted for work up for chest pain . three patients with critical ohss with severe pleural effusion requiring thoracentesis were admitted for supportive measures.conclusion : the placement of a pigtail catheter resulted in safe and effective outpatient management for the majority of patients with severe ohss .

congenital adrenal hyperplasia ( cah ) refers to a group of autosomal recessive disorders caused by an enzyme deficiency which leads to defects in biosynthesis of steroid precursors . depending on the severity and degree of 21 hydroxylase deficiency , the clinical spectrum may vary from mild form of non classical cah to classic cah . however , the non classical cah variant is more common with a prevalence rate of 1 in 1000 . it also helps in maintaining normal levels of precursors by suppressing adreno cortico trophic hormone ( acth ) . during childhood , the management is largely focused on achieving normal growth and attaining appropriate final adult height . johns medical college hospital , bangalore by the department of endocrinology on patients diagnosed to have cah and seen in the outpatient clinic between january 2012 and october 2012 . during this period data regarding demography , clinical presentation at time of diagnosis , treatment details , height sds and bmi were collected . all patients underwent biochemical testing for 17 hydroxy progesterone ( 17 ohp ) levels for assessment of adequacy of therapy . bone age assessment with left hand and wrist bmi was calculated for all patients and obesity was defined using who charts as values above 95th percentile . 17 ohp levels between 1 ng / ml and 12 ng / ml were considered appropriate ; values below 1 ng / ml suggested suppression and over treatment and values above 12 ng / ml suggested under treatment . 29 patients were included in the study of which 22 were females ( 76% ) and 7 were males ( 24% ) . based on the cross sectional data collected , 11 patients were adults ( age > 18 ) and 18 patients were children ( 62% ) . among the males , one child was identified at birth via a neonatal screening program , one child presented with early pubarche , the other 5 infants presented between their 3rd and 6th week of life with features suggestive of adrenal crisis - poor feeding , vomiting and failure to gain weight . one of the male patients incidentally also had a penoscrotal hypospadias which was surgically corrected . among the females , 9 infants were identified at birth due to presence of genital ambiguity ( 40% ) , 1 presented with symptoms of adrenal crisis at 4 weeks of life , 4 patients presented in the pre pubertal period due to early onset adrenarche ( 18% ) , 5 patients presented in the late adolescent period with marked virilization ( 23% ) and 3 patients presented with features of poly cystic ovarian disease ( pcos ) . all the five patients who presented in the late adolescent period had obvious genital ambiguity from birth , however , they sought medical attention only much later due to marked virilization and failure to attain menarche . one among them had actually been evaluated and even underwent a clitoroplasty at a young age , but unfortunately , the diagnosis of cah was missed as she was mistakenly categorized as probable ovo testicular dsd due to presence of mullerian structures with a phallic length of around 6 cm . all these 5 women had a masculine built at presentation with poor breast development , severe hirsutism , muscular body habitus , temporal balding and varying degrees of deepening of voice . the non classical cah ( nccah ) patients presented in adulthood with complaints of irregular cycles and hirsutism ; they had no features of virilisation . simple virilizing was the most common sub class seen among the women ( 81% ) whereas the salt wasting type was predominant among boys ( 85% ) . around 35% of patients had short stature as defined by height sds < -2.0 ; 2 patients who presented with adrenarche had increase in height sds , however , their bone age was also correspondingly advanced . all the children were treated with hydrocortisone ; almost all adult patients were on dexamethasone . none of the nccah patients were treated with glucocorticoids ; they were on anti - androgen therapy along with oral contraceptive pills . most adult classic cah patients with complaints of hirsutism were treated with either spironolactone or finasteride . pubertal induction with ethinyl estradiol was initiated for three of the adult patients who sought medical care late . analysis of 17 ohp levels revealed that 32% were suppressed with levels less than 1ng / ml ; appropriate in 47% and inadequate in 21% of patients . we report the phenotypic features of a cohort of patients with cah being evaluated and managed in a tertiary centre in southern india . in this series , the typical presentation of male babies were with adrenal crisis between the 3 and 6 week of life . around 20% of female patients with classic cah were identified and appropriately treated only after late adolescence even when genital ambiguity was present since birth . short stature was seen in one third of patients on therapy and the average final adult height among patients with classic cah was 142.37 , which was significantly lower than the mean adult height for females in our population . the mean final adult height of 157.5 cm for those with nccah was better than their classic cah counterpart . obesity and hypertension were not found to be significantly higher in this cohort of cah patients . one third of patients had suppressed 17 ohp levels suggestive of over replacement therapy which may also contribute to the reduction in the final adult stature .

congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by enzyme deficiency which leads to defects in biosynthesis of steroid precursors . most common is 21 hydroxylase deficiency . clinical spectrum varies from non - classical cah to classic cah , and it may be simple virilising form or salt - wastinfg type . 29 patients were included in our study from january 2012 to october 2012 . 76% were females . male babies typically presented with adrenal crisis between 3rd to 6th week of life . around 20% of females were identified and appropriately treated only after late adolescence . short stature was seen in 1/3rd of patients . 1/3rd of patients had suppressed 17 ohp levels suggestive of over - replacement therapy which may contribute to final reduction in adult height .

type 1 diabetes ( t1d ) results from the destruction of insulin producing pancreatic cells by a cell specific autoimmune process ( 1 ) . chronic pancreatic inflammation ( insulitis ) and destruction of islet -cells in type 1 diabetes is mediated by the immune cells , particularly autoreactive cd4 and cd8 t lymphocytes , b cells , macrophages and dendritic cells ( 2 ) . in order to obtain insight into type 1 diabetes pathogenic mechanisms in humans and to test novel therapeutic approaches for its treatment , different preclinical models of the disease such as spontaneous and accelerated diabetes in the non - obese diabetic ( nod ) mice , biobreeding rats , or diabetes induced in susceptible rodent strains by multiple low doses of streptozotocin ( mlds ) are now available ( 3 ) . in the pathogenesis of t1d , several proinflamma - tory cytokines including ifn - , tnf- , il-1 , as well as il-17 ( 4 - 6 ) , have been implicated . it is also thought that the production of anti - inflammatory cytokines such as il-4 , il-10 and tgf- correlates with protection from t1d ( 6 ) . it has been shown that some drugs such as pentoxifylline ( ptx ) have immunomodulatory and anti - inflammatory activity , which might represent a potential preventive therapy for autoimmune diseases . ptx is a methyl xanthine - derived general phosphodiesterase ( pde ) inhibitor that has been available for many years to treat vascular disorder ( 7 ) . phosphodiesterases ( pdes ) are a family of enzymes that regulate intracellular levels of the cyclic nucleotides cyclic adenosine monophosphate ( camp ) and cyclic guanosine monophosphate ( cgmp ) by catalyzing their breakdown to inactive metabolites . drugs that increase the levels of camp , tend to reduce the production of proinflammatory mediators and increase the production of anti - inflammatory mediators by immune cells ( 8) . in recent years , the potential of ptx as an immunomodulatory and anti - inflammatory agent gained interest as it has been shown to effectively suppress the synthesis of tnf- and other pro inflammatory cytokines such as ifn - and il-12 in vitro and in vivo ( 9 - 10 ) . ptx has been successfully used for the treatment of experimental autoimmune diseases including experimental autoimmune myocarditis ( eam ) ( 11 ) , experimental autoimmune encephalomyelitis ( 12 ) , and adjuvant arthritis ( 13 ) . in the present study , we hypothesized that pentoxifylline , due to its anti - inflammatory and immunosuppressive activity , may affect autoimmune diabetes . consequently , we decided to investigate whether pentoxifylline treatment could prevent the development of mlds - induced diabetes in mice . streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) . male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle . diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice . stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days . the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) . mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) . subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip ) plasma , separated from blood by centrifugation , was stored at -80c until insulin assay . mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test . non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) . before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes . mice were euthanized and their spleens were removed on day 21 for cytokine production assay . after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle . a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris hcl and 1% nh4cl ( ph 7.2 ) . subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes . remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2 the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn - were measured by elisa kits according to the manufacturers instructions . mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr . after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a. one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups . streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) . male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle . diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice . stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days . the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) . mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) . subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip ) was initiated in treatment group when they were considered diabetic . at the same time , the control groups received saline vehicle alone with the same schedule . plasma , separated from blood by centrifugation , was stored at -80c until insulin assay . mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test . non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) . before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes . mice were euthanized and their spleens were removed on day 21 for cytokine production assay . after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle . a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris hcl and 1% nh4cl ( ph 7.2 ) . subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes . remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2 mmol / l l - glutamine . the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn - were measured by elisa kits according to the manufacturers instructions . mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr . the plate was then centrifuged and followed by removal of medium . after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a. one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups . all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group . however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) . effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) . ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage . effect of pentoxifylline treatment on plasma levels of insulin ( * p<0.05 ) treatment of mice with ptx significantly decreased mlds - induced production of ifn- and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) . effect of pentoxifylline on productions of ifn il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group ) the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) . effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 ) all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group . however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) . effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) . ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage . treatment of mice with ptx significantly decreased mlds - induced production of ifn- and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) . effect of pentoxifylline on productions of ifn il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group ) the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) . effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 ) in rodents , experimental insulin - dependent diabetes can be induced by multiple low doses of streptozotocin ( mlds ) ( 15 ) . this model is a commonly used animal model that has many histological and clinical features similar to those of human type 1 diabetes and involves the participation of macrophages and t cells . streptozotocin ( stz ) is a pancreatic cell toxin that induces inflammation of the islets by immune cells when it is given in multiple low doses ( 16 ) . this model of diabetes offers some advantages , such as simultaneous appearance of diabetes mellitus in all animals and not having immune abnormalities that may complicate studies in spontaneous diabetes in non - obese diabetic ( nod ) mice or bio breeding ( bb ) rats . therefore , this model as a good model has been applied extensively to investigate the type 1 diabetes mellitus , especially study the immune pathways and modulations of autoimmune insulitis and cell death ( 17 - 18 ) . c57bl/6 mice , also called c57 black 6 or simply black 6 has the advantage of strain stability and easy breeding . the most common application of c57bl/6 mice is to serve as physiological or pathological models for in vivo experiments ( 19 ) . as such , mlds induced c57bl/6j mice were chosen to evaluate the anti - hyperglycemic activity of ptx and investigate the immunotherapeutic molecular mechanisms of how ptx attenuated the development of t1d . ptx is commonly used for the treatment of microcirculatory disorders and yields only minimal side effects in patients . in addition , due to its varied immunomodulatory effects , ptx has been used in several autoimmune diseases including rheumatoid arthritis ( 20 ) , multiple sclerosis ( 21 ) , and systemic lupus erythematosus ( 22 ) . drugs that increase the level of camp , including pde inhibitors , are known to prevent or attenuate inflammatory cell responses ( 20 ) . previous studies have also shown that ptx lowers blood glucose levels in diabetic animals ( 23 ) , and the results from the present studies also indicated that ptx possessed an anti - hyperglycemic property . more importantly , the present data demonstrated that ptx could increase serum insulin concentrations in type 1 diabetic mice . it has been shown that ptx could reduce ( but not abrogate ) the insulin requirements or lengthen the honeymoon ( non - insulin - requiring ) period in a study of 21 children with new - onset type 1diabetes ( 24 ) . t helper 1 cells produce proinflammatory cytokines ( tnf- , ifn - , il-1 , il-6 , and il-12 ) , which activate macrophages and cytotoxic t cells to destroy -cells , whereas anti - inflammatory cytokines ( il-4 and il-10 ) that are produced by activated t helper 2 cells , prevent -cell destructive insulitis ( 25 ) . il-10 has been shown to prevent the onset of diabetes in mice ( 26 ) . in response to cytokine stimulation , -cells generate reactive oxygen species ( ross ) and reactive nitrogen species such as nitric oxide ( no ) , which facilitate their destruction ( 27 ) . no is also synthesized within cytokine - activated macrophages by inducible nitric oxide synthase ( inos ) ( 28 ) . more recently , the role of il-17-producing t cells have been demonstrated in the development of several autoimmune diseases , including multiple sclerosis , rheumatoid arthritis and type 1 diabetes . il-17 is a proinflammatory cytokine that is detrimental to pancreatic islet cells ( 29 ) . il-17 promotes infiltration of neutrophils and macrophages , stimulates the production of other proinflammatory cytokines , including tnf- , il-1 , il-6 and il-12 , by activated macrophages and induces nitric oxide release from cells that interferes with their function and cause -cell destruction ( 30 , 5 ) . it has been assumed that ptx similar to camp elevating agents , selectively suppresses the production of t helper type 1(il-2 , ifn - ) , but not t helper type 2(il-4 , il-6 , il-10 ) cytokines ( 31 ) . however , it has also been demonstrated that the effect of ptx on cytokine production is cell - specific ( 32 ) . the present study shows that ptx is not only able to inhibit the release of proinflammatory cytokines ( ifn - and il-17 ) , but can also increase the production of anti - inflammatory cytokines ( il-10 ) . since ptx has been found to be able to inhibit the release of proinflammatory cytokines ( 33 ) , and favor th2 response ( 34 ) , it may indirectly interfere with the induction of inos . it has been shown that pde inhibitors elevate camp , prevent or attenuate inflammatory cell responses ( 35 ) and suppress proliferation of lymphocytes ( 36 ) . our findings are consistent with previous studies , showing that ptx treatment prevented lymphocyte proliferation . in summary , results of the present work demonstrated that the suppressive effect of the ptx treatment on t1d was accompanied by decreased blood glucose level , increased plasma insulin level , suppression of t cell proliferation , down - regulation of th1 and th17 cytokines ( ifn - and il-17 ) and increase in the production of il-10 in supernatant of splenic culture of the treated mice . it seems that pentoxifylline treatment has a therapeutic effect against mlds - induced diabetes in mice .

objective(s):pentoxifylline is an immunomodulatory and anti - inflammatory agent and is used in vascular disorders . it has been shown that pentoxifylline inhibits proinflammatory cytokines production . the purpose of this study was to investigate the therapeutic effects of pentoxifylline on the treatment of autoimmune diabetes in mice.materials and methods : diabetes was induced by multiple low dose of streptozotocin ( mlds ) injection ( 40 mg / kg / day for 5 consecutive days ) in male c57bl/6 mice . after induction of diabetes , mice were treated with pentoxifylline ( 100 mg / kg / day ip ) for 21 days . blood glucose levels and plasma levels of insulin were measured . splenocytes were tested for proliferation by mtt test and cytokine production by elisa.results:pentoxifylline treatment prevented hyperglycemia and increased plasma insulin levels in the diabetic mice . aside from reducing lymphocyte proliferation , pentoxifylline significantly inhibited the production of proinflammatory interleukin 17 ( il-17 ) as well as interferon gamma ( ifn- ) , while increased anti - inflammatory cytokine il-10 as compared with those in mlds group ( diabetic control group).conclusion : these findings indicate that pentoxifylline may have therapeutic effect against the autoimmune destruction of the pancreatic beta - cells during the development of mlds - induced type 1 diabetes in mice .

determinar a presena de anticorpos ige especficos para superantgenos estafiloccicos e o grau de sensibilizao mediada por esses , assim como se esses esto associados gravidade da asma em pacientes adultos . estudo transversal incluindo asmticos adultos em acompanhamento ambulatorial em um hospital universitrio tercirio no rio de janeiro ( rj ) . os pacientes foram alocados consecutivamente em dois grupos de gravidade da asma segundo critrios da global initiative for asthma : asma leve ( al ) , com asmticos leves intermitentes ou persistentes , e asma moderada ou grave ( amg ) . foram determinados os nveis sricos de anticorpos ige antitoxinas estafiloccicas , e os resultados foram comparados por anlise estatstica . foram includos 142 pacientes no estudo : 72 no grupo al ( mediana de idade = 46 anos ; 59 do sexo feminino ) e 70 do grupo amg ( mediana de idade = 56 anos ; 60 do sexo feminino ) . na amostra geral , 62 pacientes ( 43,7% ) apresentaram resultados positivos para dosagens de anticorpos ige antitoxinas estafiloccicas : enterotoxina ( tx ) a , em 29 ( 20,4% ) ; txb , em 35 ( 24,6% ) ; txc , em 33 ( 23,2% ) ; e toxic shock syndrome toxin ( tsst ) , em 45 ( 31,7% ) . as mdias das dosagens sricas de anticorpos ige especficos anti - txa , txb , txc e tsst foram , respectivamente , de 0,96 u / l , 1,09 u / l , 1,21 u / l , e 1,18 u / l . a presena de anticorpos ige sricos anti - txa , txb , txc e tsst , foi detectada em 43,7% nessa amostra de pacientes , mas no houve associao estatisticamente significativa entre seus resultados qualitativos ou quantitativos e gravidade clnica da asma . staphylococcus aureus is a gram - positive bacterium that can colonize the human skin and respiratory tract . the most important are toxic shock syndrome toxin ( tsst ) , staphylococcal enterotoxin a ( sea ) , seb , sec , sed , see , seg , seh , and sei , the activities of which include superantigen activity , pyrogenicity , and potentiation of lethality of other toxins . the superantigen activity of staphylococcal toxins consists of direct stimulation of class ii mhc receptors and t cells , independently of antigen presentation by antigen - presenting cells , stimulating the proliferation and activity of cd4 and cd8 t lymphocytes . this mechanism is related to the worsening of allergic diseases by the production of staphylococcal toxin - specific ige antibodies , as well as by a direct effect on tissue mast cells , leading to their degranulation . in asthma patients , staphylococcal toxins also act as superantigens , stimulating cd4 t lymphocyte proliferation and activity and leading to an increased production of staphylococcal toxin - specific ige antibodies , causing an allergic - type reaction by biding to mast cells in the respiratory tract . this reaction results in the release of mediators such as histamine , kinins , platelet - activating factor , and arachidonic acid metabolites ( prostaglandins and leukotrienes ) , as well as of chemokines , eliciting immediate and late inflammatory responses ( by the recruitment and activation of neutrophils and eosinophils ) and culminating in asthma worsening . staphylococcal superantigens have been shown to play roles in atopic dermatitis , rhinosinusitis , and asthma , being correlated with their severity . with regard to asthma , kowalski et al . found ige antibodies to sea , sec , and tsst in 89.7% of 237 asthma patients ( mean levels of 1.096 3.25 ku / l ) ; although there was no significant difference between those with severe asthma and those with non - severe asthma in terms of the prevalence of staphylococcal toxin - specific ige antibodies ( 81.4% vs. 69.2% ) , mean levels were higher in the former than in the latter ( 1.65 3.25 ku / l vs. 0.54 0.72 ku / l ) . in another study ( n = 210 ) , the same authors obtained similar results , the prevalence of staphylococcal toxin - specific ige antibodies being 76.1% in patients with severe asthma and 71.1% in those with non - severe asthma , mean levels being three times higher in the former than in the latter . bachert et al . found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe asthma when compared with those with mild asthma and controls ( n = 70 ) . in a more recent study ( n = 387 ) , the same group of authors found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe uncontrolled asthma ( 59.6% ) when compared with those with controlled asthma ( 40.8% ) and controls ( 13.0% ) . high levels of staphylococcal toxin - specific ige antibodies have been found to be a risk factor for asthma ( or = 7.6 ) and severe asthma ( or = 11.09 ) . in latin america , there have been no studies correlating staphylococcal superantigens with the severity of asthma . therefore , we investigated a population of asthma patients treated at a university hospital in the city of rio de janeiro , brazil , and having no risk factors for increased staphylococcal colonization or infection in order to correlate the clinical severity of asthma with the presence of staphylococcal toxin - specific ige antibodies and degree of ige - mediated sensitization . this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 , consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria : the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group . according to the global initiative for asthma , asthma severity can be evaluated on the basis of the treatment required in order to control the disease . patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two . patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses . patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control . the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma , regardless of the presence of rhinitis and positive skin test results to aeroallergens . the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent . the sample size calculation was based on a study by kowalski et al . and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% , the required sample size was calculated to be 140 ( 70 per group ) . procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) . spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines and the reference values proposed by knudson et al . a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma . for pef measurement , a peak flow meter ( mini - wright afs ; clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg . skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens . for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used . , we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables , we used the chi - square test or fisher 's exact test , as appropriate . the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant . the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital . all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision . this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 , consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria : the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group . according to the global initiative for asthma , asthma severity can be evaluated on the basis of the treatment required in order to control the disease . patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two . patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses . patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control . the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma , regardless of the presence of rhinitis and positive skin test results to aeroallergens . the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent . the sample size calculation was based on a study by kowalski et al . and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% , procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) . spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines and the reference values proposed by knudson et al . a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma . for pef measurement , a peak flow meter ( mini - wright afs ; clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg . skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens . for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used . in order to compare numerical variables , we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables , we used the chi - square test or fisher 's exact test , as appropriate . the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant . the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital . all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision . a total of 142 patients were studied . of those , 72 ( 17 with mild intermittent asthma and 55 with mild persistent asthma ) were allocated to the ma group and 70 ( 53 with moderate asthma and 17 with severe asthma ) were allocated to the msa group . the median age was 52.5 years ( 46 years in the ma group and 56 years in the msa group ) , females and white individuals having predominated . in the sample as a whole , the mean body mass index ( bmi ) was 27.09 kg / m , 128 patients had rhinitis , 131 had positive skin test results to aeroallergens , and 99 had a family history of atopy . only 37 ( 26.1% ) had a history of smoking . mean percent predicted pef was 72.59% , mean percent predicted pre - bronchodilator fev1 was 71.55% , and mean percent predicted post - bronchodilator fev1 was 81.48% . mean eosinophil count was 4.4% , and mean total ige levels were 574.92 iu / ml . table 1 shows the distribution of sociodemographic and clinical variables , and table 2 shows lung function parameters and laboratory findings in the ma and msa groups . student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) . * student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) . table 2lung function parameters and laboratory findings in the study population , by asthma severity . student 's t - test or mann - whitney test . of the sample as a whole , 62 patients ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies : sea , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and tsst , in 45 ( 31.7% ) . the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively . as can be seen in tables 3 and , there were no statistically significant differences between the two groups regarding the frequency of ige - mediated sensitization and serum levels of staphylococcal toxin - specific ige antibodies . table 3frequency of ige - mediated sensitization to staphylococcal toxins in the study population , by asthma severity . sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . table 4serum levels of staphylococcal toxin - specific ige antibodies in the study population , by asthma severity . sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test . sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test . there were statistically significant differences between the two groups of patients in the present study regarding their clinical and sociodemographic characteristics ( including age , bmi , and prevalence of rhinitis ) . the fact that the patients in the msa were significantly older than were those in the ma group might be due to the fact that asthma tends to be more severe in older individuals , especially those in whom the onset of asthma occurred at an older age . our finding of a significantly higher bmi in the msa group is consistent with the literature , obesity having been reported to be a risk factor for and an aggravator of asthma . recent studies have established a relationship between obesity - induced changes in the gastrointestinal and respiratory microbiome and the etiopathogenesis of obesity - related asthma . our finding of a higher prevalence of rhinitis in the ma group suggests that atopy was more common in the ma group than in the msa group , supporting the concept that atopic manifestations tend to be less common in asthma patients with disease that is more severe . with regard to the lung function parameters assessed in the present study , absolute and percent predicted pef were significantly lower in the msa group , as were percent predicted pre - bronchodilator fev1 and percent predicted post - bronchodilator fev1 . these findings were expected and are consistent with the literature , showing the usual correlation between clinical severity and lung function parameters . of the 142 patients studied , 62 ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies , ige antibodies to tsst being the most prevalent . our findings are different from those of two studies in the literature and similar to those of two other studies . in one study , kowalski et al . found an 89.7% prevalence of positivity for staphylococcal toxin - specific ige antibodies in severe and non - severe asthma patients ; in another study , they found a 76.1% prevalence in patients with severe refractory asthma and a 71.1% prevalence in patients with non - severe asthma . in one study , bachert et al . found a 38.1% prevalence of positivity for staphylococcal toxin - specific ige antibodies in patients with asthma ( independently of disease severity ) and a 62% prevalence in patients with severe asthma ; in another study , they found a 59.6% prevalence in patients with severe uncontrolled asthma and a 40.8% prevalence in patients with controlled asthma , the latter prevalence being closer to that found in the present study . aureus , such as sinonasal polyposis , bronchiectasis , chronic bronchitis , and atopic dermatitis , were not included in the present study . by facilitating colonization or infection with s . aureus , the aforementioned conditions can lead to increased quantities of staphylococcal toxins in the body , resulting in increased ige - mediated sensitization and , consequently , a heterogeneous population of asthma patients . the studies conducted by kowalski et al . and bachert et al . had no such exclusion criteria and included patients with chronic sinus disease and sinonasal polyposis , as was the case with the most recent study by kowalski et al . , who nevertheless found no statistically significant differences between asthma patients with polyposis and those without regarding their levels of staphylococcal toxin - specific ige antibodies . this might explain the differences between our results and those obtained by the two aforementioned groups of authors regarding the degree of ige - mediated sensitization . aureus in the brazilian population ; therefore , it is currently impossible to determine whether or not it is lower than that in the european population . it is also impossible to determine whether the allergic immune response to staphylococcal toxins is lower in asthma patients in brazil than in those in other countries . in the present study , there were no statistically significant differences between the two groups regarding the frequency of staphylococcal toxin - specific ige antibodies . our results are qualitatively similar to but quantitatively different from those obtained by kowalski et al . , who found significantly higher levels of staphylococcal toxin - specific ige antibodies in patients with severe asthma . in addition , our results are qualitatively and quantitatively different from those obtained by bachert et al . these differences are also due to the exclusion criteria used in the present study , which were not used in any of the aforementioned studies , and to specific characteristics of our study population , as previously mentioned . in the present study , ige antibodies to sea , seb , sec , and tsst were found in 62 ( 43.7% ) of the 142 asthma patients analyzed , and neither the frequency of staphylococcal toxin - specific ige antibodies nor the serum levels of those antibodies were associated with the clinical severity of asthma . these results are extremely relevant because this is the first study on this topic in latin america , the results of which differed from those of previous studies conducted in europe , indicating a " negative " association . the limitations of the present study lie in the fact that it was a single - center study conducted at a tertiary care university hospital , in a single demographic area of brazil . our primary objective was to evaluate the influence of serum levels of staphylococcal toxin - specific ige antibodies and their association with asthma severity , meaning that this was not a population prevalence study of ige sensitization in asthma patients and healthy individuals in our region . if that had been the case , a much larger sample size would have been required , and it would have been impossible to obtain that in a single - center study . therefore , in the present study , each group served as the control group for the other , without the use of a third group , comprising healthy individuals . multicenter studies in brazil and other latin american countries are needed in order to determine more accurately the role of ige - mediated sensitization to staphylococcal toxins as an aggravator of asthma , as well as to determine its prevalence in asthma patients and healthy individuals . such studies should include asthma patients with and without diseases that can lead to increased colonization or infection with s .

abstractobjective : to determine the presence of staphylococcal superantigen - specific ige antibodies and degree of ige - mediated sensitization , as well as whether or not those are associated with the severity of asthma in adult patients . methods : this was a cross - sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of rio de janeiro , brazil . consecutive patients were divided into two groups according to the severity of asthma based on the global initiative for asthma criteria : mild asthma ( ma ) , comprising patients with mild intermittent or persistent asthma ; and moderate or severe asthma ( msa ) . we determined the serum levels of staphylococcal toxin - specific ige antibodies , comparing the results and performing a statistical analysis . results : the study included 142 patients : 72 in the ma group ( median age = 46 years ; 59 females ) and 70 in the msa group ( median age = 56 years ; 60 females ) . in the sample as a whole , 62 patients ( 43.7% ) presented positive results for staphylococcal toxin - specific ige antibodies : staphylococcal enterotoxin a ( sea ) , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and toxic shock syndrome toxin ( tsst ) , in 45 ( 31.7% ) . the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively . there were no statistically significant differences between the two groups in terms of the qualitative or quantitative results . conclusions : serum ige antibodies to sea , seb , sec , and tsst were detected in 43.7% of the patients in our sample . however , neither the qualitative nor quantitative results showed a statistically significant association with the clinical severity of asthma .

the family is the cornerstone of human social support network and its presence is essential in everyone s life . changes inevitably occur in families with illness and hospitalization of a family member . in other words , among the sources of stress for families are accidents leading to hospitalization particularly intensive care unit ( icu ) . statistics show that 8% of hospital beds in the united states are occupied by the intensive care units . stress in the family while the patient is in the icu can disrupt the harmony power of the family members and finally , it may causes disturbances in the support of the patient . in addition to the various sources of stress in intensive care units such as the patient s fear of death , financial problems , lack of awareness about the environment and etc . , their satisfaction level is another important source of stress for the patient s family . today , the family needs of hospitalized patients in the icu are summarized in five sections . these factors may include receiving assurance , staying close to the patient , receiving information , feeling comfortable and receiving support . however , in many cases , the patient s family needs and their expectations in the icu will not be fulfilled which will cause dissatisfaction . since , how families deal with mental stress is the most important components of comprehensive care , it should be known that attention to the family needs of the patients hospitalized in intensive care units could increase their satisfaction and thus a reduction in stress disorders . studies showed that the nurses in particular sections focus primarily on the patient and the disease . therefore , attention to the families should be considered as an important part in patient management . effective interventions targeted at the needs and expectations of family members should help to reduce the stress and improve their satisfaction . fox et al . stated that the needs of the family members were not often overlooked by the nurses . however , the nurses spend a lot of time with the patients and their families and are in a good position to assess their needs and plan for meeting these needs with appropriate interventions . the reason of investigating the satisfaction of the families of the patients in the icu is that the patients are facing life - threatening illness experiences and complex treatments , different technologies and different equipment are used for them . these cases could potentially lead to the dissatisfaction of their families . in the meantime , the families of trauma and neurosurgical patients are more vulnerable than the other groups of patients . they need supports that are more emotional and should be accepted by the icu staff . today , there are many theories expressed that in intensive care units , the patient and his family should be considered as a single unit . by providing care to this unit , the aim of this study was to analyze the satisfaction of the families of icu patients . the tool of society of critical care medicine s family needs assessment ( sccmfna ) ( which has been validated in 1998 by johnson ) was used in that study . the results showed that most of the family satisfaction was related to the communication and patient care and the lowest level of satisfaction was about the ability of staff to patients family comfort . bailey et al . have conducted a study in 2009 with the purpose of investigating the relationship between informational support to families of special care patients and their anxiety and satisfaction . they showed that there is a direct link between the informational support and their satisfaction . the study of fumis et al . in 2008 also showed that the increase in the availability of physicians for giving the information to the patient s family and as well as nurses efforts to provide understandable explanations about the patient s condition to them will increase the patient s family satisfaction . this clinical trial was performed with respect to the existing gap for a study carried out for investigating the interventions for the satisfaction of the patients in the special care units and their families in iran . the purpose of the study was to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery intensive care unit of al - zahra hospital in 2010 . the statistical research community was the families of hospitalized patients in neurosurgery intensive care unit of al - zahra ( sa ) hospital , isfahan , iran from may to september 2010 . with respect to inclusion criteria , the families of 64 patients were selected with simple sampling . after obtaining the informed consents from the hospitalized patients , by using the envelopes , which were prepaid by using a table of random numbers , they were divided into two groups ( intervention and control ) . the inclusion criteria was included as the following cases : to be a first - degree member of the family , to be older than 18 years , willing to participate in the study , having the power to speak and write in farsi , to be hospitalized one day before the study , lack of physical or mental problem in the family member , no responsibility for the care of another patient , introduced as the first person responsible for the patient care . obtaining the written informed consent , completion of the questionnaires about demographic information and the families satisfaction were performed in the second day of hospitalization of the patients in icu in both groups . johnson questionnaire for the satisfaction of the family of icu patients was used to measure the family satisfaction . the rating of the questionnaire was performed by using the likert scale with four options of 0 to 4 as follows : almost all the time ( 3 scores ) , often ( 2 scores ) , only occasionally ( 1 score ) and never ( zero score ) . johnson s questionnaire was the modified type of multer patient s family needs , which was evaluated in various scientific researches and there are extensive evidences for its validity . the interventions were performed in the second and the third day of hospitalization of the patient in the neurosurgery icu by the researcher in the morning shift for the intervention group . thus , the researcher provided understandable explanations about the disease to the patient s family and answered honestly to the questions about the patient and the disease . the researcher ensured them for providing the best care in the section and spoke about the prognosis of the disease . the researcher took the family member to the patient s room and gave the necessary information about the room space , equipment , personnel departments and the actions that were done for the patient . the patient family member was contacted if there was a change in the patient condition . they let the family help the patient in some cases of the public health ( such as hand and foot massage and giving food ) . locations of rest , nutrition , prayer room , bathroom and buffet of the hospital were introduced to them . the telephone numbers of hospitals and wards ( and if necessary , the social worker and supervisor ) were given . various departments and personnel of the hospital were taught to accept the families and communicate with them . the hospital social worker was introduced and if necessary , the patient s family was allowed to express their feelings . these interventions were performed in the evening shift of the second and the third day of the patient reception . the actions were carried out by the fellow researcher . while , for the control group , only the routine work was done . on the fourth day , the satisfaction survey was completed by the selected family member in both of the intervention and control groups . statistical methods used in this study were 2 test , man - whitney , independent student s t - test and paired t - tests . the findings did not show statistically significant difference in the demographic characteristics of the intervention and control groups , such as : age ( p < 0.99 ) , gender ( p < 0.79 ) , marital status ( p < 0.41 ) , educational level ( p < 0.12 ) and relation with patient ( p < 0.54 ) . as shown in table 1 , there was no significant difference in the mean satisfaction score before the intervention in the intervention and control groups ( p > 0.05 ) . the mean satisfaction score after the intervention in the intervention group was significantly more than the control group ( p < 0.001 ) . the mean satisfaction score in the intervention group after the intervention was significantly higher than before the intervention ( p < 0.001 ) . there was no significant difference in the mean satisfaction score in the control group before and after the intervention ( p > 0.05 ) ( table 2 ) . comparison of mean satisfaction score ( 100 * ) of participants in the intervention and control groups the mean of satisfaction score changes of the studied subjects in the intervention and control groups after intervention the results of the present study showed that the nursing interventions based on the family needs increased the patient s family satisfaction in the neurosurgery intensive care unit of al - zahra hospital . due to the differences in the study design , study population , the number of samples and methods of intervention , it would be difficult to compare the results . in chien et al . study , the results showed that performing the training based on the patients family needs in the icu , decreased the anxiety and increased their satisfaction . in the study of myhren et al . indicated that effective communication of the staff with the families of icu patients would increase their satisfaction . in the study by rosher and robinson , it showed that involving the families in patient care led to increase their satisfaction than before the intervention . the mentioned results were consistent with the results of this study , because all of these interventions were some parts of the interventions performed in this study . however , the study of steele et al . indicated that clinical training had no impact on the family satisfaction of the patient hospitalized in the icu . therefore , there was no statistically significant difference in the satisfaction level in the control and intervention group . in this study , it was shown that the use of nursing interventions based on family needs ( confidence , support , information , proximity and convenience ) had significant impact on the family satisfaction of the patient hospitalized in intensive care unit . they should be based on the family investigation and recognition and their priority needs should be determined in order to cause the greatest satisfaction . the icu nurses must also accept their role in the care of patient s family members . the findings of this study could be a basis for performing further studies about family needs of the patients in the icu especially with the different forms of culture and economy . consequently , the increase in family satisfaction of patients can reduce the stress disorders and improve their mental state and ultimately better support of the patient by the family . this study only examined the effect of nursing interventions in the first days of hospitalization on the satisfaction of the families but the effects on the patient s entire hospitalization period has not been assessed . by performing other researches in this field , it would be possible to compare the differences in the effects of nursing interventions based on the needs of families in the early days and the whole period of the patient s hospitalization .

background : since the family is a social system , the impairment in each of its component members may disrupt the entire family system . one of the stress sources for families is accidents leading to hospitalization particularly in the intensive care unit ( icu ) . in many cases , the families needs in patient care are not met that cause dissatisfaction . since the nurses spend a lot of time with patients and their families , they are in a good position to assess their needs and perform appropriate interventions . therefore , this study was conducted to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery icu.materials and methods : this clinical trial was conducted in the neurosurgery icu of al - zahra hospital , isfahan , iran in 2010 . sixty four families were selected by simple sampling method and were randomly placed in two groups ( test and control ) using envelopes . in the test group , some interventions were performed to meet their needs . in the control group , the routine actions were only carried out . the satisfaction questionnaire was completed by both groups two days after admission and again on the fourth day.findings:both of the intervention and control groups were compared in terms of the mean satisfaction scores before and after intervention . there was no significant difference in mean satisfaction scores between test and control groups before the intervention . the mean satisfaction score significantly increased after the intervention compared to the control group.conclusions:nursing interventions based on family needs of hospitalized patients in the icu increase their satisfaction . attention to family nursing should be planned especially in the icus .

development of human societies and industrialization as well as changes in stress sources has changed disease pattern in civilized societies . as a result , the disease pattern has changed from traditional diseases such as infectious diseases and malnutrition to diseases such as heart disease , diabetes , accidents and so forth . among them coronary artery disease is the most cardiovascular disorder as a health problem in developing and developed countries . in fact , this is not only a chronic disease associated with high mortality , but it causes limitations in life and disability in a large part of the productive forces of the country . it is also associated with reduced production and increased medical costs [ 3 - 5 ] . nowadays , cardiovascular diseases are among the most widespread chronic diseases in most countries . according to forecasts , the mortality rate in eastern mediterranean countries including iran will be 30 - 35% [ 6 , 7 ] . despite the emphasis on prevention and development of newtreatments , surgery is the only choice for many patients with cardiovascular disease . one of the surgical procedures for the treatment of cardiovascular patients is coronary artery bypass surgery . in fact , heart surgery significantly influences on the quality of life of patients with cardiovascular diseases . hence , heart surgery is an important event in the lives of patients causing collapse of economic , professional and personal life . on the other hand , each surgical procedure is associated with several psychological complications for patients . of the most important complications are anxiety and depression so that approximately 65% of cardiovascular patients experience them after surgical and medical interventions . in fact , anxiety and depression complicate the treatment process [ 7 , 8 ] . some scientists believe that anxiety is one of the essential elements of human life . however , acute and long - term anxiety is integral part of all psychological diseases . according to nemati and colleagues , in fact , anxiety is a common psychological response of cardiovascular patients which is associated with reduced quality of life and psychological morbidity [ 11 , 12 ] . on the other hand , anxiety causes increased heart rate and breathing as well as high blood pressure and even mortality . it is associated with increased risk of mortality , disability , increased medical care and functional impairment in daily activities [ 12 , 13 ] . clinical depression has been reported in 54% of patients after bypass surgery . according to world health organization ( who ) , depression is the fourth chronic disease where its disability is comparable with 8 major chronic disorders . the outcome of depression in patients with cardiovascular disease is deterioration of physical and emotional state . given the incidence of such complications in patients with cardiovascular disease the best practice in this area is appropriate communication with patient [ 13 , 15 ] . good and effective communication is very valuable from the perspective of patients . in general , communication is a set of learning skills . in fact , poor communication is the cause of most problems in treatment of patients . in fact , communication at the bedside is a therapeutic and professional communication . in health professionals , communication and communication skills play a very important role in satisfaction of patients and solving their problems . this is more important , especially in patients with chronic diseases or those requiring long - term care . the therapeutic communication is formed from the first encounter of the patient with the medical team [ 21 , 22 ] . however , studies have shown that the communication between patient and medical team is not efficient . the health personnel don not have adequate communication skills . in fact , nursing and medical team spend very little time to communicate with patients . accordingly , patients are not often satisfied with received information as well as the level and method of communication . hence , to achieve best therapeutic results , special attention should be paid to communication [ 16 , 19 , 22 ] . since nursing is a practical discipline based on professional knowledge , it is necessary to use knowledge infrastructure to develop new approaches in clinical practice . theories proposed by nursing scientists can be useful in this area [ 16 , 19 , 23 ] . for this purpose the nurse - patient communication is an essential element of this theory [ 21 , 24 ] . according to peplau theory , the purpose of nursing care is to achieve a common good nurse - patient communication . according to peplau , peplau refers to the importance of therapeutic communication with patients and its important role in reducing anxiety [ 21 , 23 , 24 ] . in fact , this theory provides a framework for nurse - patient communication . following this framework , the nurse will be able to respond communication needs of patients through establishing a good therapeutic communication with the patient [ 16 , 19 , 23 ] . hospitalization and a complex surgery such as heart surgery is a stressful process leading to depression in patients . furthermore , the need for security and psychological comfort is one of the basic needs of patients achieved through a proper structured therapeutic communication with low cost . accordingly , the objective of the present study is to examine the impact of peplau therapeutic communication model on anxiety and depression in patients who were candidate for coronary bypass surgery . this is a clinical trial with pre / post testing scheme examined the impact of independent variable ( peplau therapeutic communication ) on the dependent variables ( anxiety and depression ) . the population consisted of all patients who were candidates for coronary artery bypass referred to the research environment on non - emergency basis . the research environment was al - zahra heart hospital in shiraz due to the easy access to subjects and sufficient number of patients . data were collected using the hospital anxiety and depression scale ( kanter et al . ) . the reliability and validity of the scale have been studied by montazeri et al in 2003 . an internal consistency of 78% and 86% was respectively calculated for anxiety and depression using cronbach 's alpha . there are 14 four - option questions , each with a score of zero to 3 . odd and even questions are used to measure anxiety and depression , respectively ( seven questions to assess anxiety and seven questions to assess depression ) . a score of zero to 21 is given to each patient based on responses in each area ( a score of zero - seven : normal , eight-10 : moderate and > 10 : disease ) . due to the lack of access to error rate to calculate the sample size , 10 patients were selected as pilot ( with a depression and anxiety score of 10 or more based on the hospital anxiety and depression scale ) . after performing tests , the subjects were randomly divided into test and control groups ( using a coin ( toss ) ) . z12+z12s12+s22x1x22 the inclusion criteria were : being in a bypass list , having moderate to severe depression and anxiety scores , no history of mental illness , interesting in participating in the study ( i.e. the tendency of the patient and his family to participate in the intervention ) , lack of previous bypass surgery , aged between 35 and 70 years , ability to communicate verbally and ability to speak persian . the exclusion criteria included : lack of cooperation of patients and families during the intervention , failure to perform coronary artery bypass surgery for various reasons , mortality during the study , failure to attend therapeutic communication sessions ( at least the absence in two sessions ) . therapeutic communication sessions were held for the intervention group based on peplau 's model at four stages including : orientation , identification , exploitation and resolution . in total , seven sessions were held individually with the consent of the patient and his family at the hospital and patient 's home . it should be notedthat during therapeutic communication , duration of each session was variable given the location and patient 's needs . the place of each meeting was determined with the consent of the patient . at all meetings , the researcher used verbal and nonverbal communication skills to communicate with patients . to ensure the correct application of therapeutic communication skills , a self - control researcher - made tool approved by experts was used . the depression and anxiety of patients in both groups were assessed using the hospital anxiety and depression scale ( hads ) at baseline and then two and four months after surgery . the obtained data were analyzed using descriptive statistics as well as covariance analysis with the help of spss 16 . this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code . the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details . furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) . this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code . the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details . furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) . the percentage of male and female patients in the intervention group was 70.3% and 29.7% , respectively . the percentage of males and females in the control group was 51.4% and 48.6% , respectively . the status of the underlying disease was depicted in both intervention and control groups . according to results , the majority of patients were suffering from high blood pressure in both groups with a high frequency of 35.1% ( table 1 ) . as shown in table 2 , anxiety was reduced in the intervention group , while postoperative anxiety in the control group was increased . the hospital anxiety in the intervention group this indicates the role of peplau 's therapeutic communication in reducing hospital anxiety of patients . analysis of covariance was used to determine the effect of peplau 's therapeutic communication on anxiety level of the subjects . since hospital anxiety scale was used to measure anxiety , analysis of variance was performed using hospital anxiety scores . the results showed significant differences between experimental and control groups in terms of anxiety level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test . adjusted mean anxiety scores suggest that anxiety in the intervention group was lower than the control group . in fact , the mean anxiety scores of patients in the intervention group in pre - test and post - test stages represent the independent role of peplau 's therapeutic communication model in reducing anxiety in the intervention group ( f=174.02 , p=0.000 ) . moreover , the anxiety level in the intervention group two and four months after the intervention was decreased compared to the control group ( f=38.37 , p=0.000 ; f=11.58 , p=0.000 ) ( table 3 ) . as shown in table 4 , the mean depression in the intervention group was reduced after the surgery . the mean postoperative depression in the control group was increased . in fact , the hospital depression in the intervention group was reduced after the therapeutic communication . this indicates that the role of peplau 's therapeutic communication model in reducing hospital depression . analysis of covariance was used to determine the effect of peplau 's therapeutic communication on depression level of the subjects . since hospital depression scale was used to measure depression , analysis of variance was performed using hospital depression scores . the results showed significant differences between experimental and control groups in terms of depression level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test . adjusted mean depression scores suggest that anxiety in the intervention group was lower than the control group . in fact , the mean depression scores of patients in the intervention group in pre - test and post - test indicate the independent role of peplau 's therapeutic communication model in reducing depression in the intervention group ( f=163.27 , p=0.000 ) . such a difference is not observed in the control group . moreover , the depression level in the intervention group four months after the intervention was decreased compared to the control group ( f=20.58 , p=0.000 ) . however , no significant difference was found between two groups in terms of hospital depression two months after intervention ( p=0.61 ) ( table 5 ) . according to the results , peplau 's therapeutic communication is effective in reducing anxiety and depression in patients who were candidate for coronary artery bypass . it resulted in a dramatic decrease in anxiety and depression scores in the intervention group . depression and anxiety are the most important preventive factors in cardiovascular diseases causing disease rejection by the patient and reduce the incentive to treat and increase the likelihood of disease recurrence [ 25 - 27 ] . most patients with coronary heart disease who survive after surgery resume their normal life after several weeks or several months . previous studies show that patients suffer from anxiety and depression several months and sometimes several years after the surgery [ 26 , 27 , 29 ] . therefore , follow - up and strategies to deal with postoperative psychological complications are of utmost importance . it is not possible except through establishing a coherent and targeted therapeutic communication . indeed , effective communication enables the medical team to identify patients needs and take steps to solve problems and meet their needs . according to dadashi , hosseini and moghaddam ( 2009 ) , staff had close encounters with patients in 85.5% of cases . according to mastaneh and mouseli ( 2013 ) , the rate of respecting patient rights in terms of communication was 60% indicating a moderate level of communication skills . on the other hand , duration and how to communicate with patients , particularly in dealing with patients with chronic diseases are essential in the hope and positive attitude to treatment and follow - up after discharge [ 29 - 31 ] . in fact , the patient gains motivation to cope with illness and trust in nurses through proper therapeutic communication , because the care of patients with either physical or mental illness requires correct and consistent communication [ 30 , 31 ] . therefore , implementing a communication pattern will help medical team to establish a proper communication . peplau 's model is one of these communication patterns . to communicate with patients , especially patients with chronic diseases , peplau 's described four stages and defined roles for the nurse at every step as someone who is in close relationship with the patient [ 16 , 23 ] . peplau 's communication model helps nurses and patient to identify the disease , concerns and questions of the patient . through defining different roles for the nurse this model refers to nursing skills and abilities to establish a simple convenient therapeutic communication . the model enables nurses help the patient after discharge and to spend more time with patient [ 16 , 23 , 24 ] . for example , it has been used in communicating with cancer patients and solving systematic problems of families [ 32 , 33 ] . in this study , peplau 's communication models were employed as communication sessions for patients who were candidates for coronary artery bypass through applying nursing roles . the results showed that holding communication meetings with patients immediately after surgery dramatically reduced hospital anxiety and depression . accordingly , to improve the nurse - patient communication and establish more effective therapeutic communication processes which improves clinical basis and can have a positive impact on the treatment and discharge processes and rehabilitation of patients , especially in patients with chronic diseases , training courses should be included in in - service courses to train and introduce medical team with simple and inexpensive communication skills . furthermore , there is evidence that workshops on communication skills for health team can improve health outcomes and satisfaction of patients . another limitation of this study is the lower percentage of females ( 29.7% ) in the intervention group with respect to the control group ( 51.4% ) . the lower anxiety score in the intervention group could be due to therapeutic communication but also to gender differences . to write this article not contributed any financial resources and costs are the responsibility of the authors . all rights of this article reserved is owned by the ahvaz jundishapur university of medical sciences .

background and objective : anxiety and depression are among the psychological disorders in heart surgeries . establishing a simple communication is essential to reduce anxiety and depression . hence , the objective of the present studywas to examine the impact of peplau therapeutic communication model on anxiety and depression in patients , who were candidate for coronary artery bypass in al - zahra heart hospital , shiraz during 2012 - 2013 . methods : this is a clinical trial in which 74 patients were randomly divided into intervention and control groups , each consisted of 37 patients . anxiety and depression levels were assessed before , and two and four months after intervention using the hospital anxiety and depression scale ( hads ) . seven therapeutic communication sessions were held in four stages . data were analyzed with the spss ( version 16 ) using analysis of covariance . results : the mean anxiety and depression levels decreased in the intervention group after the therapeutic communication ( p<0.01 ) . anxiety scores in the intervention group before and after intervention were 10.23 and 9.38 , respectively . while the corresponding scores in the control group were 10.26 and 11.62 , respectively . depression scores in the intervention group before and after intervention were 11 and 9.13 , respectively . the corresponding scores in the control group were 11.30 and 12.08 , respectively . conclusion : the results demonstrated the positive role of therapeutic communication in reducing anxiety and depression of the patients . therefore , the therapeutic communication is recommended as a simple , cost effective and efficient method in this area .

sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp . throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) . antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) . rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification , 5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3 and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3 ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) . pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min . all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination . the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3 ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3 ) . unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations . nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications . multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) . ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos . sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp . throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) . antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) . rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification , 5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3 and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3 ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) . pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min . all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination . the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3 ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3 ) . unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations . nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications . multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , madison , wi , usa ) software package . three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) . ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos . clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) . of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals . most of the patients were < 10 years of age ; 36 were male and 28 were female . none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture . the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 . ( map courtesy of http://www.maps.com ) hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera . six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) . * test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference . a rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype . twenty - six of the 42 influenza a positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene . the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) . alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) . all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . * n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) . this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position . prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 . additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season . two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively . both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) . the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) . the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions . unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains . the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow ) was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season . nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no . the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 . three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively . the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution . this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features . this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) . interestingly , this proline residue is located within a barrel ( a protein motif consisting of an antiparallel sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 . three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 . most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) . magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 . hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) . clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) . of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals . most of the patients were < 10 years of age ; 36 were male and 28 were female . none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture . the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 . hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera . six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) . * test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference . a rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype . twenty - six of the 42 influenza a positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene . the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) . alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) . all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . * n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) . this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position . prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 . additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season . two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively . both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) . the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) . the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions . unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains . the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow ) was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season . nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no . the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 . three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively . the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution . this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features . this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) . interestingly , this proline residue is located within a barrel ( a protein motif consisting of an antiparallel sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 . three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 . most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) . magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 . hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) . the 4 substitutions described represent a growing lineage of influenza a ( h3n2 ) viruses characterized since july 2004 . three amino acid changes are confined within known antibody - binding sites , i.e. , the s189n change within antibody - binding site b ( 4,5 ) and the v226i and s227p changes residing in antibody - binding site d ( 4,5 ) . because of rotational restrictions , a proline substitution at position 227 ( s227p ) would typically give rise to considerable conformation change ; however , this particular substitution is located within a barrel motif and therefore has little effect on regional protein conformation . cumulatively , field isolates characterized subsequent to this outbreak continue to exhibit these 4 changes , and they appear to constitute a distinct branch in the phylogeny of ha sequences when compared with h3n2 isolates from the 20032004 season . . this change may affect protein - protein interactions since it is immediately adjacent to antibody - binding site a , where neutralizing antibodies have been shown to bind ( 4,5 ) . furthermore , since the k145n substitution is located within a glycosylation site , the charge alteration may affect glycosyl transferase activity , which results in altered glycosylation . differences in glycosylation have been shown to contribute to antigenic variation by preventing antibody binding to antigenic sites ( 15 ) . additionally , 3-dimensional analysis suggests this amino acid substitution may also promote enhanced receptor binding since the asparagine r group is shorter , which may make binding requirements less stringent and the receptor cleft more accessible . the 3-dimensional depiction provides a unique regional residue perspective , demonstrating how the rapidly evolving ha surface antigens in the vaccine strain differ at the molecular level . collectively , the clinical isolates obtained from this outbreak in nepal can not be considered antigenically distinct from the a / wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by hi ( table 1 ) . furthermore , the varying reactivity noted in several isolates from this outbreak having identical ha1 sequences is suggestive that other viral antigens aside from the ha1 protein may have contributed to the antigenic variability observed in the hi panel . with the exception of a / nepal/1670/2004 and a / nepal/1672/2004 , all isolates evaluated by hi ( table 1 ) exhibited identical ha1 amino acid sequences and varying antigenicity profiles to a / wyoming/03/2003 reference antisera . one explanation for this observation is that genetic differences in other influenza surface proteins contribute to the observed immunoreactivity . alternative viral surface protein candidates include the neuraminidase , ha2 , and m2 glycoproteins , which have been shown to exhibit antigenic properties ( 1619 ) . in this report , we describe the genetic analysis of the ha proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains . three amino acids changes ( s189n , i226v , and s227p ) were noted in known ( 4,5 ) antibody - binding sites ( table 2 ) . the fourth change ( k145n ) , which was located within a glycosylation site , may enhance viral binding since the smaller asparagine r group is located close to the ha receptor - binding cleft ( figure 3 ) . phylogenetic analyses show that the nepal isolates make up a distinct branch in the evolution of h3n2 viruses when they are compared with vaccine and reference strains ( figure 2 ) . however , antigenic data appear more ambiguous , suggesting a multigenic effect that can not solely be attributed to properties of the influenza ha ( table 1 ) . studies are in progress to characterize the neuraminidase , m2 , and ha2 proteins to determine the molecular basis responsible for antigenicity differences observed within isolates from this outbreak . the k145n substitution change has become a marker for an increasingly large subset of the fujian - like viruses . cdc and the us department of defense have recently characterized viruses with the k145n change in singapore , taiwan , china , australia , canada , and the united states . in february 2005 , who reported the emergence of a new influenza h3n2 strain in the united states . the a / california/7/2004 strain , which was first identified in the united states in september 2004 , contains all 4 changes observed in isolates from this nepalese outbreak . the a / california/7/2004 strain differs by only 1 amino acid in ha1 ( which is of no immunologic importance ) from most isolates from the outbreak in nepal . all viruses characterized ( 150 globally isolated strains ) subsequent to the preparation of this report ( march 2005 ) by the us department of defense are genetically similar in amino acid sequence to these nepalese strains ( and the a / california strain ) . most of the isolates ( 80% ) analyzed by cdc since october 2004 are antigenically related to a / california ( 20 ) , which indicates that this strain has emerged as the dominant influenza a h3n2 strain . these data indicate that these viruses may persist as the dominant strain at the onset of the 20052006 influenza season . in february 2005 , who recommended inclusion of an a / california/7/2004-like strain in the 20052006 trivalent influenza vaccine to afford immunologic protection from this variant h3n2 virus . our findings emphasize the importance of continued molecular surveillance for characterizing emerging influenza drift variants .

worldwide emergence of variant viruses has prompted a change in the 20052006 h3n2 influenza a vaccine strain .

rising levels of obesity are becoming a worldwide phenomenon and are increasingly identified as a health problem across the globe [ 14 ] . higher weight has been associated with adverse health indicators and outcomes , including cardiovascular disease [ 512 ] , stroke [ 5 , 13 ] , cognitive and functional decline [ 1418 ] , metabolic syndrome [ 19 , 20 ] , inflammation [ 21 , 22 ] , and mortality [ 20 , 2325 ] . obesity among aging populations is relatively recent and aging among people who have been obese for much of their lives is also a new phenomenon . from 1980 to 2004 , the prevalence of obesity in the us has continued to rise from about 17% to 25% for men aged 5059 . while obesity in england has also increased during this period , from approximately 9% in 1980 to 15% in 2004 for men aged 5564 , the level of obesity remains much lower in england . additionally , the difference in obesity between the us and england is more pronounced for women . the level of obesity in us women was about 24% in 1980 and rose to 37% in 2004 ( age 5059 ) ; in england , levels for women were 9% in 1980 and 14% in 2004 ( age 5564 ) . the aim of this paper is to investigate differences in how obesity relates to indicators of physiological dysregulation in men and women of diverse populations . this comparison will lead to an improved understanding of how obesity might be differentially related to health and mortality across cultures and lifestyles . obesity was relatively rare in most populations until the second part of the last century , but it has now become common in many countries . the us population is recognized as among the most obese in the world , although many other countries are now approaching the us level and most countries are experiencing increasing obesity . this worldwide obesity epidemic began with the epidemiological revolution and the virtual elimination of infectious disease ; the decline in manual labor needed to provide sustenance with the industrial revolution ; and the increasing availability and decreasing cost of food [ 2729 ] . obesity reflects some combination of calorie intake , diet content , and amount of physical activity . in some cultures , lack of physical activity can be a more important determinant of obesity ; in other cultures , overeating or food composition may be the more important determinant of obesity . it is also true that within countries , individuals could differ in the causes of obesity . for instance , changes in activity might be more characteristic of women or men resulting in different reasons for obesity by gender . these differences may affect how obesity is related to other risk factors for poor health , and it may determine the overall health risk associated with obesity . if physical activity is maintained , the overall effect of obesity may be less than if the activity is not . one indication of the cause of obesity may be the relationship between waist size and weight [ 30 , 31 ] . in societies where obese people are more physically active , waist size of the obese waist circumference has also been linked to late life mortality , where high waist circumference has been associated with increased mortality among men and women in the netherlands , while high bmi was not associated with mortality . this paper builds upon current obesity research by using both bmi and waist circumference to quantify obesity in order to determine how a combined indicator of weight and adiposity is related to physiological dysregulation in populations with different cultures , diets , behaviors , and epidemiological histories . obesity has been related to many indicators of physiological dysregulation including cardiovascular risk factors such as hypertension and metabolic dysregulation in lipid levels or insulin resistance . most studies that investigate the differences in biological risk associated with excess weight have examined western populations [ 33 , 36 ] . comparative studies on the health risks associated with obesity that examined the us and england reported that obese americans had an increased risk of diabetes and a higher waist circumference [ 37 , 38 ] . these studies suggested that differences in physical activity , diet , and social environments may explain these national differences . while these differences have been observed between the us and england , these two western countries have roughly similar life expectancy , levels of living , history , and culture even while the us has poorer health by a number of indicators of disease prevalence and biological risk [ 36 , 39 ] . comparative studies of the links between obesity and health outcomes and risk factors for obesity comparing western and non - western countries indicate important differences in the causes and consequences of obesity . a comparison of the association of disease with overweight and obesity in japan and the us indicated that the associations were stronger in the us than in japanese women and that there was no association in japanese men . links between social , demographic , and behavioral risk factors for obesity also differ markedly in japan , korea , and the us . the availability of biomarker data from taiwan a middle - income country undergoing rapid economic growth , increasing obesity , and with life expectancy recently increasing to levels similar to that of the us and england allows for investigation of the biological risk associated with obesity in a population characterized by very different cultural , behavioral , socioeconomic , and dietary parameters . we examine how elevated weight and obesity ( using an indicator that considers both bmi and waist circumference ) relate to having levels defined as clinical risk for cardiovascular , metabolic , and inflammatory markers in three aging societies that are now relatively similar in life expectancy but that differ in the timing of the epidemiological transition and obesity epidemic , history of economic development , socioeconomic levels , general lifestyle habits , health behaviors , and health care systems : the us , england , and taiwan . finding differences in the relationship between obesity and indicators of physiological dysregulation in these three aging societies will clarify whether increases in weight gain are equally problematic across all countries . we use data from three nationally representative samples : the us national health and nutrition examination survey ( nhanes , 20032006 ; n = 3855 ) , the english longitudinal study of ageing ( elsa , 2004 - 2005 ; n = 9139 ) , and the social environment and biomarkers of aging study ( sebas ) in taiwan ( 2000 ; n = 1023 ) . these surveys collect information on demographics , as well as anthropometric , physical , and biological measures . every year , approximately 5,000 individuals undergo detailed interviews and medical examinations , which include collection of several physiological measures . nhanes utilizes a complex sampling design , and when weights are applied , the sample is representative of the noninstitutionalized american population . we use the 20032006 data since nhanes data is released in two - year intervals , and this sample is centered on 2004 - 2005 , which matches the period in which elsa was collected . for nhanes , we use individual - level data based on a sample of 1,513 fasting individuals aged 54 and older . elsa includes participants drawn from households responding to the health survey for england ( hse ) in 1998 , 1999 , and 2001 and is representative of the english population aged 50 and older . the core elsa sample ( wave 1 : 2002 - 2003 ) includes people living in an hse responding household who were born prior to march 1 , 1952 , and their partners who could be under age 50 . wave 4 of elsa ( 2008 - 2009 ) , which includes a nurse visit , includes wave 1 core members , if they are still alive and do not refuse further contact after the first interview at wave 1 . it also includes a refresher sample to maintain the age structure of the sample ( in waves 3 and 4 ) , and their partners . for elsa , we use individual - level data based on a sample of 7,384 individuals aged 54 and older . sebas is drawn from a follow - up survey of the survey of health and living status of the near elderly and elderly in taiwan ( also known as the taiwan longitudinal study of aging ( tlsa ) ) , a nationally representative survey of taiwanese adults ( including institutionalized individuals ) collected in 1989 , 1993 , 1996 , 1999 , and 2000 . in 2000 , a subsample of individuals was randomly selected for inclusion in sebas . sebas consists of adults aged 54 and older in 2000 , with in - home interviews and medical exams taken in a hospital . for sebas , the sample averages 66.8 years of age in england , and the us and taiwan mean age is about the same ( table 1 ) . there are more men in taiwan ( 56% ) and england ( 53% ) and fewer in the us ( 44% ) . we examine the following indicators of physiological dysregulation often associated with obesity and also associated with increased risk for multiple adverse health outcomes and obesity [ 43 , 44 ] : ( 1 ) cardiovascular markers : high systolic ( sbp ) and diastolic blood pressure ( dbp ) ; ( 2 ) metabolic markers : high levels of blood lipids ( total and low - density lipoprotein ( ldl ) cholesterol , and fasting triglycerides ) , low levels of high - density lipoprotein ( hdl ) cholesterol , and high fasting glucose and glycated hemoglobin ; ( 3 ) high levels of inflammatory markers c - reactive protein ( crp ; available in nhanes and elsa ) and interleukin-6 ( il-6 ; available in sebas ) , as crp and il-6 have been positively associated with bmi . for each indicator we use clinical cutpoints or widely used research - based cutpoints to indicate high levels of risk which are shown in table 1 [ 39 , 43 , 45 ] . there has been debate as to the best indicator of obesity : body mass index ( bmi ) or waist circumference . waist circumference is thought to be a better measure of abdominal adiposity than bmi and a better indicator of risk of poor health outcomes , including all - cause and cardiovascular mortality [ 46 , 47 ] . for this reason we investigate the association between bmi and biomarkers across categories of bmi ( underweight < 18.5 kg / m , normal and overweight 18.529.9 kg / m , obese 3034.9 kg / m , and very obese 35 kg / m ) and waist circumference categorized as normal or high waist ( high waist : men 120 cm , women 88 cm ) . we create a composite measure of obesity and adiposity by categorizing individuals into five groups : ( 1 ) underweight and normal waist ( all underweight individuals had a normal waist circumference ) , ( 2 ) normal / overweight bmi ( termed normal bmi ) and normal waist circumference ( reference group ) , ( 3 ) normal / overweight bmi ( termed normal bmi ) and high waist circumference ( termed high waist ) , ( 4 ) obese and high waist ( all obese individuals had a high waist circumference ) , and ( 5 ) very obese . we also evaluate an alternate definition for obesity in taiwan based on bmi 27 , as it has been suggested by some that obesity levels should be differentially defined for asians [ 48 , 49 ] . a similar composite measure of obesity and adiposity was calculated using this alternate definition of bmi in taiwan . because these risk factors are all assumed to be associated with obesity and because dysregulation in multiple physiological systems has been shown to predict many of the poor health risk outcomes associated with aging , we also create two summary measures of risk based on the total number of at - risk levels of biomarkers , either 9 or 8 . because crp values for sebas are not available , this measure is not included in the 9-item summary measure for taiwan , but a summary measure ( range 09 ) was calculated for taiwan using il-6 , another indicator of inflammation , instead of the crp values included for the us and england . a second alternate summary measure of biological risk , excluding the inflammatory marker ( range 08 ) , biological risk summary scores were computed for individuals who had missing values on no more than 3 biological markers . we examine multiple covariates in our investigation of the relationship between obesity and biological risk . self - reported use of antihypertensives was determined in all three countries , and use of lipid - lowering statins was only asked in the us sample . dichotomous variables were created to indicate whether the respondent reported being a current smoker and participating in at least moderate physical activity for exercise ( e.g. , brisk walking , running , or swimming ) in the past 30 days ( for the us and england ) or generally exercising once a week ( for taiwan ) . we use logistic regression models to determine the odds of having at - risk levels of a specific biomarker for obese men and women among the three populations . for all countries , the comparison group for bmi and waist circumference is the normal bmi and normal waist group . the regressions included indicators of age , use of antihypertensives , current smoking status , and having exercised in the past 30 days . ordinary least squares ( ols ) regression models were used to determine the relationship between the summary measures of biological risk and the composite measure of obesity and adiposity . we begin by examining national differences in the high risk levels of individual biomarkers ( table 1 ) . low levels or high risk levels of hdl cholesterol are also more common in taiwan . high total and ldl cholesterol is more common among the english ; lower levels of plasma glucose , crp , and glycated hemoglobin are also characteristic of the english . few adults in england have elevated levels of fasting glucose ( 2.2% ) , while this is observed in 17.3% and 13.2% of american and taiwanese adults . most people in this age range are in the normal to overweight category ( 64.7% , 67.2% , and 89.4% in the us , england , and taiwan , respectively ) ( table 2(a ) ) . americans are more likely to be obese ( 33.7% ) compared to the english ( 32.1% ) and taiwanese ( 7.2% ) . among the obese , americans are much more likely to be very obese : 13.4% of the total us sample , 10.1% in england , and about 1% in taiwan . both among the obese and very obese , the average bmi is higher in the us and england compared to taiwan . few are underweight in any country ( 1.7% , 0.8% , and 3.4% in the us , england , and taiwan , resp . ) . when we examine waist circumference , the us has the highest average waist circumference , with 65.5% of americans , 55.9% of english , and 15.8% of taiwanese having a high waist size ( table 2(a ) ) . this means that high waist characterizes a substantial number of those who would be categorized as normal weight in the us and england . among those in the normal and overweight group about half ( 49.5% ) of americans and a third ( 36.4% ) of the english almost all obese individuals have a high waist in the us and england ( 98.3% in the us and 96.5% in england ) , but only 78.4% of the obese in taiwan also have a high waist . when we use the alternate obese cutpoint of 27 kg / m in taiwan , less than half of the obese individuals have a high waist ( not shown here ) . americans exhibit the highest proportion of the older population taking antihypertensive medication ( 47.1% ) ( table 1 ) . the percentage who reports taking antihypertensives is lower in england ( 32.0% ) and taiwan ( 28.6% ) . americans are more likely to be current smokers ( 24.5% ) than persons in taiwan ( 22.5% ) and england ( 13.9% ) . more than half of the population in all countries report having exercised in the past 30 days , with more english exercising ( 82.2% ) compared to taiwan ( 61.4% ) and the us ( 58.5% ) . men with normal bmi and high waist have a greater likelihood than men with normal bmi and normal waist size of having high - risk levels of triglycerides in all three countries . in the us and england , men with high waist are more likely to have high levels of glycosylated hemoglobin and higher crp ; fasting glucose is also elevated among this group in the us ( table 3(a ) ) . men who are obese in the us have fewer elevated risk factors than those with high waist who are not obese ; in the us , obese men are only more likely than normal weight men without high waist to have elevated glycated hemoglobin , fasting glucose and crp . english men who are obese have more elevated risk : both blood pressure indicators , hdl cholesterol , triglycerides , glycated hemoglobin , and crp . very obese men in england have the same elevated risk factors with the exception of dbp . very obese men in the us are more likely to have elevated fasting glucose in addition to crp and glycated hemoglobin . english and taiwanese women with normal weight and high waist are more likely to have elevated sbp , dbp , and glycosylated hemoglobin ; only british women with higher waist have significantly elevated triglycerides and only the taiwanese women had more hdl risk . high risk crp is more common among both american and english women with normal bmi and high waist , and this risk of elevated crp is also higher in the obese and very obese ( table 3(b ) ) . obese women in britain had elevations in the same markers as normal bmi and high waist english women , while obese women in the us only have elevated fasting glucose , glycated hemoglobin , and crp ; obese women in taiwan only had high dbp . with the exception of taiwan , levels of the inflammatory markers ( crp in the us and england ; il-6 in taiwan ) are more likely to be elevated among persons with a high waist and normal bmi , obese or very obese , compared to their normal bmi and normal waist counterparts . among men , an increase in the biological risk summary score ( range 09 ) is associated with having a high waist relative to being of normal bmi and normal waist in all three countries ( table 4(a ) ) . being obese or very obese is also related to a higher biological risk summary score ( 09 ) for men in the us and england . these equations explain 6 to 11 percent of the variance in the summary indicator of biological risk . these relationships are similar for women ( table 4(b ) ) , with one exception : obese taiwanese women do not have a significantly increased biological risk compared to their normal weight and normal waist counterparts . when we consider the alternate summary score that excludes our indicators of inflammation ( range 08 ) , being obese or very obese is no longer associated with a higher biological risk summary score in us men compared to men with a normal bmi and normal waist when controls for health behaviors and medication use are included ( table 5(a ) ) . the size of the effects of the obesity categories is reduced on the 8-indicator summary measure in both england and the us , indicating the strong link between crp and obesity . the r is also reduced in these equations for england and the us . for taiwan , women in england and taiwan , but not women in the us , with a higher waist but who are not obese have significantly higher physiological dysregulation compared to their normal bmi and normal waist counterparts . obese women in all three countries have elevated risk and the very obese have even higher risk ( table 5(b ) ) . the alternate biological risk summary measure ( 08 ) yields different relationships between weight and physiological dysregulation in the us and taiwan . in the us , only very obese women have a higher alternate biological risk summary score ( 08 ) . in taiwan , obese and normal bmi and high waist women exhibit higher alternate biological risk summary scores compared to their normal bmi and normal waist counterparts , except when smoking status , physical activity , and use of hypertensives are included . figures 1(a ) and 1(b ) illustrate the predicted alternate biological risk score ( 08 ) for each weight category for men and women ( respectively ) aged 65 who are nonsmokers , do not engage in physical activity , and are currently taking antihypertensive medication . this figure allows for country comparisons of individuals with these characteristics within each weight category and across weight categories . the predicted values indicate that the us has the highest biological risk score within each respective weight category among men and women of the same age and lifestyle behaviors . with the exception of women in the normal bmi and high waist group , england has the second highest biological risk score within each weight category , followed by taiwan . among 65-year - old women with the noted lifestyle behaviors and with a normal bmi and high waist when we consider the alternate bmi cutpoint for obesity in taiwan ( bmi 27 kg / m ) , our findings for the individual biomarkers and summary measures of biological risk are similar to using the bmi 30 kg / m cutoff for taiwan except that the category normal weight with high waist no longer differs from the omitted category ( results not shown ) . this study observes three general findings about how biological risk is associated with obesity in three countries that differ in lifestyle and culture . first , obesity is associated with physiological dysregulation in all countries with differences in the links between specific indicators of biological risk and obesity . generally , obesity in england is associated with hypertension , dyslipidemia , and elevated glycated hemoglobin ; americans who are obese are not more likely to have hypertension . in taiwan , obese women are more likely to have elevated dbp and obese men have an increased risk of elevated triglycerides and glycated hemoglobin compared to their nonobese , normal waist counterparts . our biological risk summary scores indicate that at all levels of weight physiological dysregulation was highest in the us , followed by england ( with one exception ) , with taiwanese exhibiting the lowest biological risk in all groups among the three countries . second , these relationships remain after controlling for demographic factors , participation in physical activity , and other behavioral factors . third , similar to obese older adults , high waist individuals with normal bmi also exhibit greater physiological dysregulation in all countries compared to their normal bmi and normal waist counterparts . our finding of a higher physiological dysregulation , as shown by the alternate biological risk summary score , in taiwan compared to the us and england could be due to a couple of potential explanations . the prevalence of obesity in the us and england is much higher than in taiwan , indicating an earlier initial rise in obesity relative to taiwan . from 1978 to 2002 , the proportion of obese americans and britons exhibited stark increases ( 1332% and 623% for men and women , resp . ) . the estimates for obesity prevalence in taiwan indicate a recent increase for men but not women . from 19931996 to 2000 - 2001 , the age - adjusted prevalence of obesity rose from 10.5% to 15.9% for men and declined from 13.2% to 10.7% in women . it may be that the lower levels of risk among older adults who have lived longer years with obesity could be a reflection of better pharmacologic control of physiological dysregulation ( e.g. , through statin use ) , which may in turn confer less biological risk in these populations compared to populations of currently obese taiwanese adults who may have more recently begun living with obesity . a second reason for the observed country differences in obesity may be due to differences in dietary habits and lifestyle . the us and england are two modern , western populations whose diets have been influenced by increased industrialization and have over time come to be characterized by high glycemic loads and high fatty acid composition . taiwan , on the other hand , represents a country that has experienced the effects of the industrial and scientific revolutions later than that of the us and england but is currently rapidly undergoing economic development and demographic change . the recent economic changes in taiwan may indicate that obese older adults in taiwan have more recently begun to consume high - fat diets , which could result in greater initial physiological dysregulation associated with access to western - influenced dietary habits . despite controlling for lifestyle behaviors thought to be linked with health , the country differences in obesity and physiological regulation remain . moreover , the consideration of antihypertensives does not alter our substantive conclusions on these associations . this suggests that despite the greater use of medications to treat hypertension in the us , obesity among americans is associated with greater overall biological risk than the other two countries . this is supported by findings from the general population of americans relative to england , which report that the us is faced with greater health disadvantages than england in adulthood and across the life span . we also note differences in biological profiles of obese individuals between the two westernized countries : the us and england . the excess risk of hypertension associated with obesity in england was not found in the us . these differences may be due to the higher use of medications among americans compared to the english , with about 16% more men and 18% more women in the us aged 65 + taking antihypertensive medication compared to their british counterparts . the greater use of hypertensive medications in the us is also noted when compared to japan and countries across europe . two notable differences in country patterns of the relationship between obesity and physiological dysregulation by sex are found . among men in england and taiwan , the order of magnitude of physiological dysregulation increases with higher weight categories ; however , this is not observed for us men . this difference may be due to our inability to consider statin use in england and taiwan , which may be particularly important in the relationship between obesity and physiological dysregulation for men . conversely , the importance of considering statin use may be less vital to understanding the country differences in the association between obesity and physiological dysregulation among women , given that the relationship for women is more consistent across countries , namely in the us and england . women , underweight corresponds with higher biological risk ( though nonsignificant ) compared to women with normal bmi and normal waist . underweight among men in taiwan is significantly associated with much lower biological risk than their normal bmi and normal weight counterparts . further studies will be required to explore possible explanations for these differences in physiological dysregulation . the higher biological risk observed among normal bmi and high waist individuals relative to normal bmi and normal waist older adults builds upon previous studies that report on alternate indicators of body shape , which vary across countries . the importance of waist circumference is underscored by our current study , as well as a growing body of literature on the predictive value of waist circumference on indicators of health . higher rates of diabetes among older americans compared to britons have been accounted for by high waist circumference as opposed to bmi differences . additionally , increasing waist circumference is more predictive of greater risk of incident diabetes than bmi in middle - aged british men ( and the european prospective investigation into cancer and nutrition ( epic)-potsdam study ) . waist circumference , as an indicator of central fat mass , is thought to be more strongly associated with disease risk , and in our case with physiological dysregulation , compared to bmi , which is considered a cruder index of adiposity . banks and colleagues cite differences in physical activity , diet and greater psychosocial environmental challenges in america compared to england as potential mechanisms linking central adiposity and type 2 diabetes . our study considers some of these possible mechanisms ( e.g. , physical activity and antihypertensive use ) but finds that they explain little of the relationship between biological risk and adiposity among the three countries . together , these results highlight the importance of considering waist circumference in investigating the links between indicators of health and adiposity . our finding of the biological risks associated with obesity among older taiwanese adults underscores the growing concern for risks associated with obesity in countries rapidly undergoing modernization . in comparing the biological risk of obese individuals among the three countries , we are able to use these international comparisons to our advantage to examine how differences in modernization influence the health of older adults in different populations this may have potential health policy implications that underscore the importance of addressing and controlling the rising obesity epidemic that has become most widespread in countries , like the us and england , that have long experienced high economic growth and in countries currently undergoing rapid economic development . the increasing use of biological information to inform our understanding of health represents an innovative method in biodemography that will further contribute to the testing of current comparative theory and the potential creation of new paradigms surrounding the influence of modernization on health . first is the use of a broad range of biological markers across three large - scale population surveys . the inclusion of biological information as objective precursors of health allows , to some extent , a fairly comparable comparison of indicators of health across the different populations . an exception to this uniform comparison of biomarkers across the three surveys is our use of inflammatory marker crp in the us and england and our inclusion of a different marker of inflammation ( il-6 ) in taiwan . of note , crp seems to be more strongly associated with obesity than il-6 . a growing body of literature has made distinctions between bmi and waist circumference , namely , suggesting that waist circumference is a better indicator of abdominal obesity , which in turn has been associated with obesity - related health risks . our findings generally report a similar association between increased biological risk and ( 1 ) normal bmi and high waist and ( 2 ) obese and high waist . using the us nhanes , reported that when both waist circumference and bmi were included in their analyses , only waist circumference was a significant predictor of comorbidity . although this and other studies have suggested that waist circumference may be a better indicator of obesity and risk for adverse health outcomes , our study finds the two indicators to be similarly associated with biological risk across the three countries . first , we examine population - based data from three countries at a single time point . future studies of longitudinal data will allow for further investigations of the potential role of obesity on biological risk observed in the current associations . second , we do not have measures of some lifestyle and medical behaviors for some of the datasets ( e.g. , statin use ) , which likely influence the relationship between obesity and biological risk . as such , we are unable to include such factors in our analyses of all three countries . it is possible that these lifestyle behaviors are key explanatory factors to the noted cross - country differences in obesity - related biological risk . the cross - country differences in the relationship between increased biological risk for individuals who are obese and have a high waist underscore potential differences in health and lifestyle behaviors . these behaviors may be a result of country differences in economic development that we are not able to observe in this study . the country differences in the links between obesity and physiological dysregulation are particularly marked when comparing obesity among taiwanese older adults relative to westernized populations , such as the us and england . further examination of these relationships over time and across other countries will contribute to our understanding of the potential factors responsible for these country - specific variations in biological risk , as obesity becomes increasingly more prevalent and older adults in various countries live more years with obesity and increased adiposity .

excess weight has generally been associated with adverse health outcomes ; however , the link between overweight and health outcomes may vary with socioeconomic , cultural , and epidemiological conditions . we examine associations of weight with indicators of biological risk in three nationally representative populations : the us national health and nutrition examination survey , the english longitudinal study of ageing , and the social environment and biomarkers of aging study in taiwan . indicators of biological risk were compared for obese ( defined using body mass index ( bmi ) and waist circumference ) and normal weight individuals aged 54 + . generally , obesity in england was associated with elevated risk for more markers examined ; obese americans also had elevated risks except that they did not have elevated blood pressure ( bp ) . including waist circumference in our consideration of bmi indicated different links between obesity and waist size across countries ; we found higher physiological dysregulation among those with high waist but normal bmi compared to those with normal waist and normal bmi . americans had the highest levels of biological risk in all weight / waist groups . cross - country variation in biological risk associated with obesity may reflect differences in health behaviors , lifestyle , medication use , and culture .

male macroprolactinomas ( mprl ) are usually revealed by headaches , visual troubles and gonadal insufficiency . suppurative meningitis ( sm ) , a life - threatening condition , is scarcely observed in subjects with macro tumors secreting prolactin ( prl ) and in other pituitary tumors ( pt ) . however , in some very rare cases it can be a primary presentation or appear after radiotherapy or medical treatment used for tumors destroying the sellar floor and/or the skull base . this destruction leads to cerebral spinal fluid ( csf ) leak , which can act as an entry portal for organisms predisposing to meningitis . our aim was to analyze sm frequency among male mprl deemed to be very invasive tumors , to report our cases and analyze the circumstances under which the dangerous neurological complication appeared . in this retrospective study , we analyzed 82 subjects with mprl to look for symptoms , clinical signs and biological proof of sm . , we took into account medical history , clinical examination , routine exploration , csf analysis and hormonal assessment . that one was based on prl , growth hormone ( gh ) , insulin growth hormone ( igf1 ) , cortisol , adrenocorticotropic hormone , testosterone , follicle stimulating hormone , luteinizing hormone , thyroid stimulating hormone and free thyroxin . radiological assessment was based on cerebral computed tomography scan and/or magnetic resonance imaging ( mri ) . three were first hospitalized in the department of infectious diseases for typical symptoms and signs of sm ; that one was proved by csf biochemical analyses . the fourth one was hospitalized in our department for the fourth episode of sm which was proved by csf analysis after lumbar puncture . cerebral mri showed a pt invading cavernous sinuses , the sphenoid sinus and the brain [ figure 1 ] . hormonal assessment [ table 1 ] demonstrated high prl with gonadotroph deficit . then , the pt was treated with dopamine agonists that were successful on prl ( 25 ng / ml ) and on tumor size [ figure 1 ] . after a follow - up of 7 years , invasive and aggressive pituitary tumor destroying the sella floor , responsible for suppurative meningitis ( sm ) in a 22-year - old man . ( a ) before bromocriptine ; ( b ) after treatment , bromocriptine intake and sm disappearance tumor size , hormonal assessment of our 4 patients , and bromocriptine dose used to reduce tumor volume and normalize prolactin . cerebral mri showed an invasive and multidirectional pt measuring 47 mm 40 mm 30 mm destroying the sella floor and filling the sphenoid sinus [ figure 2 ] . the pt was secreting prl [ table 1 ] . that one was normalized by bromocriptine and the tumor size was significantly reduced . large invasive tumor destroying the sella floor , responsible for suppurative meningitis in a 49-year - old man . ( a ) before medical treatment ; ( b ) liquification of the tumor after bromocriptine a man aged 25 , with a history of chronic otitis and arrested puberty , was diagnosed as a multidirectional prolactinoma destroying the sella floor and measuring 30 mm 30 mm 30 mm [ figure 3 ] . sm was sensitive to antibiotics . under dopamine agonists prl was normalized and the tumor volume decreased . the pituitary tumor filling the sphenoid sinus before bromocriptine ( a ) and after bromocriptine ( b ) a 29-year - old man was sent to our unit for a prolactinoma revealed by epilepsy crises . the pt was very large ( 68 mm 50 mm 50 mm ) and multidirectional with an extension to the chiasm , cavernous sinuses , posterior and nasal areas [ figure 4 ] . after 3 months he had rhinorrhea , then one episode of sm sterilized by antibiotics . sm never relapsed after a follow - up of over 2 years . mri showing a large tumor invading cavernous sinuses , the brain and sphenoid sinus ( a ) . after dopamine agonists ( b ) the tumor size was reduced the table 1 shows tumor sizes , hormonal parameters and bromocriptine dose which was used to treat the 4 prolactinomas . cerebral mri showed a pt invading cavernous sinuses , the sphenoid sinus and the brain [ figure 1 ] . hormonal assessment [ table 1 ] demonstrated high prl with gonadotroph deficit . then , the pt was treated with dopamine agonists that were successful on prl ( 25 ng / ml ) and on tumor size [ figure 1 ] . after a follow - up of 7 years , invasive and aggressive pituitary tumor destroying the sella floor , responsible for suppurative meningitis ( sm ) in a 22-year - old man . ( a ) before bromocriptine ; ( b ) after treatment , bromocriptine intake and sm disappearance tumor size , hormonal assessment of our 4 patients , and bromocriptine dose used to reduce tumor volume and normalize prolactin . the previous ones were proved in 1984 , 1988 and 1990 . clinical examination confirmed the meningeal syndrome and noticed rhinorrhea . cerebral mri showed an invasive and multidirectional pt measuring 47 mm 40 mm 30 mm destroying the sella floor and filling the sphenoid sinus [ figure 2 ] . the pt was secreting prl [ table 1 ] . that one was normalized by bromocriptine and the tumor size was significantly reduced . large invasive tumor destroying the sella floor , responsible for suppurative meningitis in a 49-year - old man . ( a ) before medical treatment ; ( b ) liquification of the tumor after bromocriptine a man aged 25 , with a history of chronic otitis and arrested puberty , was diagnosed as a multidirectional prolactinoma destroying the sella floor and measuring 30 mm 30 mm 30 mm [ figure 3 ] . sm was sensitive to antibiotics . under dopamine agonists prl was normalized and the tumor volume decreased . the pituitary tumor filling the sphenoid sinus before bromocriptine ( a ) and after bromocriptine ( b ) a 29-year - old man was sent to our unit for a prolactinoma revealed by epilepsy crises . the pt was very large ( 68 mm 50 mm 50 mm ) and multidirectional with an extension to the chiasm , cavernous sinuses , posterior and nasal areas [ figure 4 ] . after 3 months he had rhinorrhea , then one episode of sm sterilized by antibiotics . mri showing a large tumor invading cavernous sinuses , the brain and sphenoid sinus ( a ) . after dopamine agonists ( b ) the tumor size was reduced the table 1 shows tumor sizes , hormonal parameters and bromocriptine dose which was used to treat the 4 prolactinomas . when it happens , it is usually observed in post - operative period or after tumor reduction by radiotherapy , apoplexy or medicine intake . the situation is observed in people in whom meninges and sella floor are destroyed by an aggressive pt or an ectopic one arising from the skull base . our 4 cases can be explained by the fact that prolactinomas are the largest and the most invasive pt , especially in males . male prolactinomas often destroy the sellar floor and invade the sphenoid sinus , then the nasopharynx . the meningeal and bony breaches lead to intra and extradural spaces communication , which is responsible for bacterial infection of meninges and encephalic structures . the most difficult differential diagnosis with meningitis occurring in people with pt is pituitary apoplexy , which mimics perfectly bacterial meningitis symptoms . however , a real microbial meningitis with or without rhinorrhea can be observed in apoplexy . the true microbial meningitis secondary to pt is an exceptional phenomenon compared to pituitary apoplexy , which is relatively more frequent . to our best knowledge only 15 sm , revealing pituitary adenomas or appearing under medical treatment have been reported so far . csf leak may proceed or not sm , and can be totally unnoticed . according to lam et al . who analyzed 29 articles published between 1980 and 2011 there were only 52 pt with csf leak . the seven cases were divided in two groups : the primitive meningitis or meningitis occurring prior to any treatment of the pt , and secondary meningitis occurring after surgery and/or dopamine agonists intake as in our 4 observation . our personal research found 15 cases meningitis secondary to pt . if we add our 4 cases , one can totalize 19 cases . in this group most pt were prolactinomas , especially males ones . among this group 14 were primitive . secondary ones ( n = 5 ) appeared under medical treatment , preceded or not by surgery or radiotherapy . sellar floor destruction was obvious on radiographies in all except one case [ table 2 ] . their age ( varying between 22 and 69 years ) did not seem to be a determining factor , but the nature of the adenoma and sphenoid sinus invasion seem fundamental . according to ciatto and to lascelles et al . we find only one case concerning gh adenoma treated by somatostatin 's analogs . a case of recurrent meningitis which appeared many years after neurosurgery was observed in nelson syndrome too . curiously sm was not reported in mixed pt and gonadotroph adenomas which are usually very large and very invasive . contrary to many authors and in agreement with ciatto , we think the association of pituitary adenoma and meningitis is more common than it is generally supposed as we observed it in nearly 5% of our male mprl . so , one should think about it more often even if a cause of sm seems obvious from the first sight as in our 4 case who suffered from chronic otitis . radiological research for pt should be systematic , especially if headaches , visual troubles , and gonadal abnormalities with or without anterior or posterior pituitary deficiencies are present . physicians who deal with large pt should be aware of high risk spontaneous meningitis , but also of secondary sm which can appear 1 month to 4 years or even more after medical treatment initiation by dopamine agonists or somatostatin 's analogs or radiotherapy . on another hand , medical treatment initiation should be gradual , especially for huge tumors as rapid shrinkage may lead to csf leak apparition . if this situation appears , a medical solution may be tried using temporary lumbar puncture or a lumboperitoneal shunt until the bony defect is clogged by fibrosis . if this attitude fails , surgical repair is recommended although the reparation may represent a great challenge because of the many potential sites of the csf around the tumor . if surgical repair in not feasible some authors recommend a vaccination against pathogens of the upper respiratory tract such as streptococcus pneumonia , haemophilus influenza and neisseria meningitidis to prevent sm . three revealed the pt , and one was observed after medical treatment . in 2 cases the described cases emphasize the necessity of an early diagnosis and treatment of large and invasive pt , especially male mprl . medical treatment , which is now the gold standard for prolactinomas , should be gradual and well monitored to avoid rhinorrhea or otorrhea which can lead to potentially fatal bacterial meningitis .

background : suppurative meningitis ( sm ) or bacterial meningitis is a life - threatening condition , which is exceptionally due to pituitary tumors ( pt ) . our aim was to analyze its frequency among male macroprolactinomas ( mprl ) deemed to be aggressive , to report the cases we observed in our practice and describe the circumstances under which sm appeared.materials and methods : we retrospectively analyzed 82 male mprl in order to look for a history of well proved sm and the circumstances under which sm appeared . we also took into account the possibility of sm relapsing.results:four out of 82 male mprl had sm = 4.87% . three consulted for sm symptoms . sm was confirmed in infectious diseases department , but only one had rhinorrhea . hormonal assessment and cerebral magnetic resonance imaging pleaded for aggressive prolactinomas . after antibiotics , sm was sterilized . then , mprl were treated with bromocriptine , which normalized prolactin and reduced pt . sm never relapsed . the 4th case was hospitalized for a large multidirectional prolactinoma invading and/or arising from the skull base . he was operated on 3 times and then he was given bromocriptine . after 3 months , he had rhinorrhea and then sm which was successfully treated by antibiotics . sm never relapsed after tumor reduction.conclusion:sm was demonstrated in 4.87% . sm has revealed mprl in 3 cases and appeared after bromocriptine intake in the 4th one . endocrinologists should be aware of this severe condition , which can be avoided by repairing as soon as possible the bony defect secondary to aggressive tumors , unless it is clogged by fibrosis : what probably happened in our cases .

many studies have not been reported in literature for lumbar discectomy by destandau endospine system . we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system . a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008 . technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision . endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route . the results were evaluated by macnab 's criteria after a minimum followup of 12 months and maximum up to 24 months . based on modified macnab 's criteria , 90% patients had excellent to good , 8% had fair , and 2% had poor results . the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients . 90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months . the advantages of use of minimal invasive spinal surgical techniques in treatment of lumbar disc herniation is small incision , limited tissue disruption , enhanced visualization due to better magnification and illumination , shorter hospital stay , and faster recovery time.13 among many posterior spinal endoscopic systems used for disc surgery , destandau endospine system and foley and smith 's metrx system are seen as viable alternatives to open disc surgery.46 the aim of this study was to present results in 300 patients operated by edoscopic discectomy and to discuss technical points to shorten the learning curve . a total of 475 patients suffering from different type and level of lumbar disc herniation with radiculopathy and degenerative lumbar canal stenosis were operated between january 2002 and december 2008 . the inclusion criteria were patients having lumbar disc prolapse with unilateral radiculopathy , on clinical evaluation , positive straight leg raise or femoral stretch test , and identification of a single nerve root lesion on mri . patients with bilateral symptoms , double root involvement , cauda equina syndrome and whose clinical symptoms did not match mri picture were excluded from present study . all these patients had fair trial of conservative treatment in the form of rest , medication ( nsaid ) , activity modification , and physiotherapy ( minimum 6 weeks ) before they were advised to undergo surgery . however , in present study , none of the patients opted for surgery at 6 weeks after completion of conservative treatment . there were 206 males and 94 females aged between 18 to 72 years ( mean , 38.4 years ) . levels operated upon included l1-l2 ( n=3 ) , l2-l3 ( n=2 ) , l3-l4 ( n=6 ) , l4-l5 ( n=205 ) , and l5-s1 ( n=84 ) . there were 235 extruded , 20 contained , 15 foraminal , and 30 sequestrated herniations . results were evaluated as poor , fair , and good or excellent using modified macnab 's criteria . modified macnab 's grading was as follows : excellent - no pain / restriction of activity and being able to do all activities ; good - occasional pain with relief of presenting symptoms and returning to work with some modification ; fair - some improved functional capacity but still handicapped or unemployed ; and poor results - having objective symptoms of root involvement or repeat surgery at the index level . the clinical material included preoperative history , physical examination , plain x - rays and mri studies of lumbosacral spine , laboratory tests , and intraoperative video documentation . postoperative follow - up was carried out on third day , 2 weeks , 6 weeks , 3 , 6 , 12 , and 24 months . it consists of endospine tube , trocar , and working insert [ figure 1a and b ] . one port for 0 degree endoscope , second for suction cannula , third port ( biggest ) for working instrument , and fourth port for dural and nerve root retractor . the procedure of discectomy can be carried out under general , spinal , epidural , or local anesthesia . the operative technique consists of knee chest positioning after administration of anesthesia followed by level localization by localization devise [ figure 1c ] . at marked point , 15 mm skin incision is made aponeurosis is incised using mayo 's scissors ; 1.5 cm wide periosteal elevator is used to elevate paravertebral muscles subperiostealy , thus exposing the interlaminar window and part of the affected side facet . ( a ) clinical photograph showing patient positioning and level marking ( b ) destandau endospine system ( c ) iitv picture of marked level ( d ) position of endospine tube ( e ) endoscopic view of decompressed nerve root the endospine tube with trocar is pushed through the incision in the direction of posterior arch over interlaminar window followed by withdrawal of trocar . the working insert any soft tissue bulging in the mouth of tube is removed till boundaries of interlaminar window such as superior and inferior lamina , facet joint are clearly visualized [ figure 1e ] . this follows part resection of inferior margin of the superior lamina followed by excision of ligamentum flavum leading to exposure of the dural sac and nerve root under endoscopic vision . once the nerve root has been accurately identified , it is retracted using a nerve root retractor . . it also helps to keep the field dry . depending on local findings , discectomy involving the extraction of the nucleus pulposus once satisfactory nerve root decompression is achieved , endospine tube along with working insert is withdrawn . aponeurosis is sutured using vicryl fine suture followed by closure of the skin in a subcuticular fashion . a water - impermeable dressing ( a ) preoperative sagittal t2wi mri of prolapsed l5-s1 disc ( b ) postoperative sagittal t2wi mri of l5-s1 disc after endoscopic discectomy at 2 years follow up ( c ) axial t2wi mri section at l5-s1 disc after endoscopic discectomy at 2 years follow up shows no compression these patients were followed up on third day , 2 weeks , 6 weeks , 3 , 6 , 12 , and 24 months . patients were followed up for minimum of one year and maximum of 2-year duration . on second visit on third day , wound was inspected for any drainage or evidence of infection . complains about fever , backache , and leg discomfort at final follow - up , 90% patients were relieved of sciatica and were satisfied with procedure . 285 patients were operated as day care cases and were mobilized and discharged same evening from day care facility . based on modified macnab 's criteria 90% patients had excellent to good , 8% had fair , and 2% had poor results . five patients who had interaoperative minor dural tears were hospitalized and were observed for any dural leak . causative factor for dural tears in present study were as follows : three patients had dural rent due to forceful retraction of dura and nerve root by dural and nerve root retractor . this was observed in patients in whom there was significant posterolateral herniation resulting in tenting of dura and nerve root at recess . in this situation , authors have found gentle mobilization of nerve root and dura by nerve root hook or approaching and debulking the offending disc through axilla before proceeding with retraction of nerve root and discectomy . this happened when kerrison rongeur was used to open the tight recess resulting in dural tear and nerve root injury . these dural tears were managed by water tight closure of muscle , fascia , and skin and bed rest for duration of one week . superficial delayed wound healing was observed in 20 patients , which healed in 21 days by regular dressings rest and administration of antibiotics . the diagnosis of postsurgical discitis was based on mainly clinical grounds and laboratory evidence of raised counts , esr , and c - reactive proteins . clinical criteria included recurrence of severe and unrelenting back pain within first week of surgery , keeping patient awake at night after initial recovery . no biopsy of disc was resorted to ; however , mri of lumbosacral spine was ordered in all these patients which did not contribute much to the diagnosis . lizolid 600 mg / bd ) for first week followed by oral antibiotics for 5 weeks . all these patients responded well to antibiotics and no further intervention of any kind was carried out . after initial back pain for 6 weeks , these patients had occasional residual backache which was treated by analgesics , activity modification , lumbar support , and rest during subsequent follow - up visits . nerve root injuries ( n=2 ) were encountered while trying to do a medial facetectomy to open the recess by a kerrison rongeur causing severe laceration of nerve root . however , nerve root was in continuity . , patties were used for gentle retraction and procedure of discectomy was successfully completed endoscopically without resorting to open discectomy . 276 patients were able to return to sedentary work with an average delay of 2 weeks , except 24 patients who had complains of backache , occasional leg discomfort , discitis , and nerve root injuries . medial facetectomy was resorted to in 59 patients to open the recess to decompress the nerve root in addition to discectomy . mixter and barr7 discovered the pathophysiology of discogenic sciatica and suggested laminectomy and discectomy as operative treatment . the overall results of standard discectomy range from 68 to 95% in different series.8 the operative microscope and microsurgical techniques were developed in mid-1960 's by yasargil and krayenbuhl910 and these techniques revolutionized spine surgery leading to smaller incisions , less blood loss , increased visualization of site of pathology , decreased hospitalization , shorter postoperative recovery , and earlier return to activities compared with previous operative interventional techniques . the results of microdiscectomy also range from 85 to 98%.1113 katayama et al.14 compared the results of open vs gold standard microdiscectomy and observed no difference between the surgical outcomes in both the groups but microdiscectomy gave better lighting , magnification , and therefore decreased the length of incision and tissue invasion . microdiscectomy also allowed the patients to return to early work with lesser use of narcotic medication . microendoscopic dissectomy ( med ) combines standard microsurgical technique with an endoscope , enabling the surgeons to address all types of disc herniations including decompression of nerve root and lateral recess . chemonucleolysis was reported by smith.15 nonetheless , based upon various randomized clinical trials , the efficacy of chemonucleolysis compared with more traditional and open procedures for the operative treatment of lumbar disc herniations remained speculative.16 the use of percutaneous nucleotomy , laser discectomy , and intradiscal electrothermal annuloplasty ( idet ) compared with microdiscectomy remains unclear , and it is attributed to the lack of high - quality studies.17 conclusions about the efficacy of some of the aforementioned minimally invasive procedures ( e.g. , chemonucleolysis , apd , idet ) were questionable with regard to disc - related pathology.18 therefore , lumbar microdiscectomy remained the gold standard for addressing a herniated or sequestrated intervertebral disc ; however , a movement toward more minimally invasive approaches that would yield superior outcomes , while minimizing excessive soft and bony tissue removal and minimizing soft tissue trauma , were sought . as such , an evolution in procedures toward smaller incisions , less tissue trauma , and quicker return to daily activities took center stage in spine surgery . the use of muscular retractor system was reported initially by faubert and caspar.1920 perez - curet and fessler,21 described for the first time a myriad of spine pathologies that could be addressed using tubeology . though from our initial experience , endospine technique is minimal invasive , but limitation of study has been lack of comparison with gold standard microscopic discectomy technique . however , in a study by shin et al.,22 15 cases each were compared of med and microscopic group ( md ) . the mean cpk - mm levels were lower for the med group than for the md group at both 3 ( 576.1286.3 iu / l compared with 968.1377.8 iu / l ) and 5 days ( 348.1231.0 iu / l compared with 721.7463.2 ) postoperatively ( p<0.05 ) . the mean vas scores for postoperative back pain were lower in the med group than in the md group , both at 1 ( 3.32.3 compared with 5.81.5 ) and 5 days ( 1.91.1 compared with 3.61.1 ) postoperatively ( p<0.01 ) . aforementioned authors concluded that the med procedure is less invasive than md , and causes less muscle damage and backache . the 90% excellent results in present study is comparable with other surgical procedures for herniated lumbar discs such as those of destandau , perez - cruet et al . , and their average surgical time was 66 minutes , average blood loss was 22 ml , average hospital stay was 7.7 hours , complication rate was 5% , reoperation rate was 4% , and average return to work was 17 days with excellent result in 94% patients . average operative time was 50 minutes average blood loss was 45 ml ( range , 30 - 70 ml ) . return to work ( 21 days ) and overall results ( 90% ) which are comparable . in another prospective and randomized evaluation of surgical treatment for lumbar disc herniation by hermantin et al.,24 satisfactory results of 97% in endoscopic group ( n=30 ) and 93% in open laminectomy group ( n=30 ) were reported . however , in endoscopic group , these authors had excluded large central herniations and extra ligamentous herniations between l5 and first sacral vertebra . however , present endoscopic technique could be used for all levels and all type of herniations . in our current series , there was 5% discitis and 5% incidence of dural injury . caspar and ebling,2526 authors have reported reoperation rate of 5.5 , 5.7 , and 3% , respectively . another measure of success is reflected by the patient 's ability to return to previous employment . our patients returned to previous employment on an average at 15 days with restriction to avoid heavy manual work for 2 months . discectomy ( med ) by endospine system has claimed even lesser tissue invasion than microdiscectomy with even smaller skin incision , lesser use of analgesics , and early return to work . least tissue invasion is established by many reports comparing the postoperative mri signal of paraspinal muscles,27 intraoperative electromyographic findings establishing less invasion to nerve roots,28 and by measuring serum levels of biochemical parameters reflective of a postoperative inflammatory reaction and damage to the paravertebral muscles.29 our personal opinion is similar , though this was not the parameter studied in our series . minimal invasive microendoscopic decompression technique has been used not only for paracentral disc herniations , but also for all types including far lateral , cephalad , caudal migrated , and central and recurrent disc herniations.3032 one of the driving forces behind the minimal invasive spine surgery is economics , shorter hospital stay , reduced postoperative morbidity , and quicker recovery times . in our series , 90% patients were operated as day care cases . posterior paraspinous process endoscopic access to lumbar disc herniation requires creation of working space where no or little space existed before . internal view of operating site is magnified and well illuminated . with advent of this system , discectomy can be done as day care procedure ensuring reduced postoperative morbidity , minimal or no hospitalization , less pain , and faster recovery . with proper patient selection , discectomy and adequate nerve root decompression by doing foraminotomy or opening a lateral recess stenosis by minimally invasive technique can be achieved with this system . however , the endospine system has been excellent modality to address discogenic radiculopathy and to decompress lumbar canal stenosis . many surgeons are convinced of advantages of the system and have included this system as part of their inventry . however , due to difficulty in orientation with scope and two - dimensional vision , availability of less space , frustrating and steep learning curve , and inability to master hand eye coordination , majority of surgeons are not able to continue with the technique . the patience and persvrance to work through narrow confines and work closely with a surgeon who has mastered the technique is the key to learn . second step would be to become comfortable with 2 dimensional vision of endoscopic camera and to master orientation , triangulation . depth perception in these techniques comes from experience rather than observation ; hence , surgeon keen to learn these techniques must combine these procedures during early phase of learning with standard procedures he is doing in his clinical practice . gradually , as surgeons master the learning curve , he will be able to use this as treatment method for his patients . there is also a need to establish cadaveric labs and dummy models on line of arthroscopic learning centers where surgeons can practice hands - on cadavers and models to improve triangulation , depth perception , and hand eye coordination .

background : posterior endoscopic discectomy is an established method for treatment of lumbar disc herniation . many studies have not been reported in literature for lumbar discectomy by destandau endospine system . we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system.materials and methods : a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008 . all patients were operated as day care procedure . technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision . endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route . the results were evaluated by macnab 's criteria after a minimum followup of 12 months and maximum up to 24 months.results:based on modified macnab 's criteria , 90% patients had excellent to good , 8% had fair , and 2% had poor results . the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients . 90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months.conclusion:edoscopic discectomy provides a safe and minimal access corridor for lumbar discectomy . the technique also allows early postoperative mobilization and faster return to work .

medical tourism is illustrated as occurrence in which individuals travel abroad to receive healthcare services ( 1 ) . it is a multi - billion dollar industry and countries like india , thailand , singapore , malaysia , belgium , costa rica , cuba , dubai , hungary , israel , jordan , south africa and many others are being benefited in their economy by this recent phenomenon ( 2 , 3 ) . the prime driving factors in medical tourism are increased medical costs , increased insurance premiums , increasing number of uninsured or partially insured individuals in developed countries , long waiting lists for procedures in countries having public healthcare system , availability of high quality services at affordable price , and cheaper airfare . increased communication and internet access in developing countries are other supporting factors , which help patients to develop awareness about international travel for medical care ( 47 ) . estimations can vary but still some of reliable sources claim ; gross medical tourism revenue worldwide was more than us$ 40 billion in year 2004 and reached up to us$ 100 billion by year 2012 ( 8) . in year 2007 , it was estimated that around 750,000 us residents traveled abroad . accordingly , the base case estimated form 2007 to 2010 for the annual growth rate for outbound patients was 100 per cent in us ( 9 ) . forbes business estimated around 1.25 million americans were expected to travel outside for medical treatment in year 2014 ( 10 ) . after thorough review it was found that there is huge gap in literature available in medical tourism . accounts detailing size and market of the industry are large in number yet there is scarcity of literature detailing about role and significance of different variables and linking them to form conceptual and theoretical framework which reflects decision making of international patients for taking part in medical tourism . available literature explore various factors related to patients travel such as source of information , perceived risks , benefits of medical tourism and attributes of medical tourism destinations in sufficient amount but almost all of these related accounts are exploratory in nature and do not provide conceptual frameworks for testing . many researchers ( 2 , 6 , 11 , 12 ) made calls to explore more about the decision making process based on conceptual models and test them empirically , so managers in medical tourism industry will be more acquainted about the needs and requirements of patients and design their future strategies accordingly and at the same time patients will also be benefited by receiving more quality services from the providers . this article will draw a conceptual model based on patients source of information , perceived benefits , perceived risks , and medical tourism destination image with available literature that will be helpful to mangers to draw their future course of action for competitive advantage and at the same time fulfill the gap in available literature . image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place . various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) . consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) . amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) . source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) . there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation . medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) . judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) . these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own . many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) . literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) . word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors . studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) . websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) . printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) . major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations . some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) . print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel . los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients . fox , cbs s 60 minutes and cnn aired their segments on patients traveling to medical services . magazines like forbes and wall street journal analyzed the business aspect of medical tourism as brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) . literature revealed that not every physical attribute of a destination has influence on image formation process . there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) . it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one . this is due to dimensions of liking may not be agree with the dimensions of similarity . literature revealed that many researchers made distinction between physical and beneficial aspect of a product . few researchers explained it as characteristic and benefits to the physical and beneficial aspect of product respectively ( 41 ) . the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery . there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) . the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds . benefits offered by a product or service are considered as consumption value of the same ( 44 ) . sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) . conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values . consumption value model define the characteristics of functional value as price , credibility , and durability . medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) . quality of medical services is one of the aspects which drive medical tourism high towards success . most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) . high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) . although availability of literature explain social value in medical tourism is less , little available literature clearly define the social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) . senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) . most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) . however , the concept of social value and epistemic value may not be associated with patients from developed regions . many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) . such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) . conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value . perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision , making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) . researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) . however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) . in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) . researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) . hence , perceived risks and cognitive image should consider individual variables for image related studies in travel . credence goods as the quality of these good can not be assessed accurately even after consumption . patients may vulnerable to many risks if consider to take medical services at medical tourism destinations . researchers described six dimension of risks associated with health travelers visited israel as human - induced risks , financial risks , service quality risks , socio - psychological risk , natural disasters and car accident risk , and after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as physical - health related risks , service related risks , and destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) . in medical tourism , international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism . health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) . literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) . researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel . apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) . according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) . destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel , researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) . attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination . later on , a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) . the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) . researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) . thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) . it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination . the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) . after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ; medical amenities , general infra - structure , tourism attractions , and social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency . majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff . command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) . based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services . physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians . prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk . the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) . collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules . most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) . most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients . many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world . language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) . facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients . essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination . thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) . potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place . various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) . consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) . amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) . source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) . there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation . medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) . judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) . these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own . many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) . literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) . word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors . studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) . websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) . printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) . major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations . some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) . print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel . los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients . fox , cbs s 60 minutes and cnn aired their segments on patients traveling to medical services . magazines like forbes and wall street journal analyzed the business aspect of medical tourism as brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) . literature revealed that not every physical attribute of a destination has influence on image formation process . there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) . it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one . this is due to dimensions of liking may not be agree with the dimensions of similarity . literature revealed that many researchers made distinction between physical and beneficial aspect of a product . few researchers explained it as characteristic and benefits to the physical and beneficial aspect of product respectively ( 41 ) . the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery . there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) . the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds . benefits offered by a product or service are considered as consumption value of the same ( 44 ) . sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) . conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values . consumption value model define the characteristics of functional value as price , credibility , and durability . medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) . quality of medical services is one of the aspects which drive medical tourism high towards success . most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) . high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) . although availability of literature explain social value in medical tourism is less , little available literature clearly define the social value factor with - in the patient groups especially from less developed regions . social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) . senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) . most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) . however , the concept of social value and epistemic value may not be associated with patients from developed regions . many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) . such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) . conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value . perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision , making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) . researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) . however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) . in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) . researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) . hence , perceived risks and cognitive image should consider individual variables for image related studies in travel . credence goods as the quality of these good can not be assessed accurately even after consumption . patients may vulnerable to many risks if consider to take medical services at medical tourism destinations . researchers described six dimension of risks associated with health travelers visited israel as human - induced risks , financial risks , service quality risks , socio - psychological risk , natural disasters and car accident risk , and after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as physical - health related risks , service related risks , and destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) . in medical tourism , international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism . health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) . literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) . researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel . apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) . according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) . destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel , researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) . attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination . later on , a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) . the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) . researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing sleepy , pleasant unpleasant , exciting gloomy , and relaxing distressing ( 70 ) . furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) . thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) . it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination . the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) . after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ; medical amenities , general infra - structure , social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency . majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff . command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) . based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services . physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians . prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk . the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) . collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules . most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) . most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients . many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world . language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) . facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients . essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination . thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) . potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability prominent databases ( 2013 , 2014 , 2015 ) have been searched to obtain desire literature related to medical tourism including science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral and so on and the keywords used are medical tourism , information sources , medical travel , health travel and so on . google search was performed to get latest data related to medical tourism . to know in detail about the variables used in the framework scientific research databases the key words used to find out research variables as well as relationship between researches variables were destination image , destination information sources , beneficial image , perceived travel risks , cognitive image , affective image , consumption values and so on . to obtain data about medical tourism various types of available literature were reviewed such as industry reports , market research reports , media reports and internet sources , however for identification and explanation of variables scientific literature published in reputed journals only were included . the theory of planned ( tpb ) is an established and comprehensively tested model details the relationship between beliefs , attitude , intention , and actual behavior of consumers ( 85 ) . the model is perfectly applied in a variety of studies including leisure and tourism travel , and hospitality ( 36 , 8688 ) . these research studies emphases on the factors such as motivations , information sources , attitudes , and visit intentions , thus authors argue that attitudinal theory provides a sound foundation to understand travel intentions of international patients and underpin the conceptual model discussed in this article . studies explained that consumers access more information in case of high association of risks or benefits or both in a particular act ( 89 , 90 ) and perform rigorous information search in case of planning first time trip ( 91 ) because first time trip is associated with unknown risks as well as unknown leisure activities . in general consumer behaviour practices risk - handling activity increases in case of high level of perceived risks . importance of risk handling activity also determines by associated benefits gained after engaging with risk taking activity ( 92 ) . the benefits of information search includes possibility of finding superior alternative to those already considered and the reduction in risk achieved from eliminating inferior , but a priori uncertain , alternatives ( 93 ) . with the discussion of above literature related to tourism it could be assumed that medical tourism information sources will have positive influence on perceived travel benefits of international patients . whereas , information sources of medical tourism will negatively influence perceived travel risks of international patients . destination image is considered as perceptions or impressions of destination held by tourists with respect to the expected benefit or consumption values . the benefits sought by the travelers are highly associated with the image of destination and the affective image of destination is largely highly influenced by the motivations / benefits sought by individuals ( 13 , 44 ) . the affective image of a destination is more positive in individual s mind when emotions evoked by the place coincide with the benefits sought ( 95 ) . risks and constraints associated with travel have significant impact on destination image formation at the time of early decision - making process ( 96 ) . according to researchers , two dimensions significantly influence cognitive and affective image of destination ( 58 ) . on the basis of previous studies in tourism literature it can be assumed that perceived travel benefits of international patients will positively influence the destination image , whereas perceived travel risks of international patients will negatively influence the destination image . intention of visit related to travel is considered as tourists perceived likelihood to visit particular destination within a specific time period ( 97 ) . thus , to be closely correlated to the travel behaviour intention of visit believes as an important outcome variable in tourism research ( 16 , 97 ) . intention to visit a destination is highly influenced by cognitive / perceptual and affective evaluation ( 98 ) . destination image is a direct antecedent of perceived quality , satisfaction and revisit intention and recommendation ( 99 ) . according to researchers , there is significant theoretical and empirical link between cognitive destination image and behavioral intention ( 100 ) . based on above discussion it could be assumed that image of medical tourism destination will positively influence visit intention of international patients . conceptual framework international patients travel decision making the article is a sincere effort by authors to establish conceptual framework which explains decision - making process of international patients . though , there has been advance in explaining different factors play important roles in international patients travel , however , there is scarcity of literature conceptualizing these factors into a framework and test it empirically . literature reveals about associated benefits and risks related to travel abroad for medical purposes but do not conceptualize it in the process of decision - making . sources of information in medical tourism play significant role in development of attitude as well as intention , has already been established in consumer behavior . however , need is to test its significance in medical tourism decision - making process . the proposed framework will encourage future researchers to test different variables empirically and establish the model of international patients travel behavior . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : role of information source , perceived benefits and risks , and destination image has significantly been examined in travel and tourism literature ; however , in medical tourism it is yet to be examined thoroughly . the concept discussed in this article is drawn form well established models in tourism literature.methods:the purpose of this research was to identify the source of information , travel benefits and perceived risks related to movement of international patients and develop a conceptual model based on well - established theory . thorough database search ( science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral ) was performed to fulfill the objectives of the study.results:international patients always concern about benefits and risks related to travel . these benefits and risks form images of destination in the minds of international patients . different sources of information make international patients acquaint about the associated benefits and risks , which later leads to development of intention to visit . this conceptual paper helps in establishing model for decision - making process of international patients in developing visit intention.conclusion:ample amount of literature is available detailing different factors involved in travel decision making of international patients ; however literature explaining relationship between these factors is scarce .

squamous cell carcinoma of the head and neck ( hnscc ) is a heterogeneous disease that includes tumors arising from the mucosal epithelial surface of the oral cavity , oropharynx , hypopharynx , and larynx . although these tumors originate within different anatomic sites within the upper aerodigestive tract , they are histologically identical ( 95% of hnscc are squamous cell carcinomas ) , share common etiologic risk factors and overlapping metastatic target site profiles ( reviewed in [ 13 ] ) . recent genetic analysis of human head and neck tumors has revealed common molecular alterations including p53 mutation , p14arf , and p16 methylation , as well as cyclin d and egfr amplification [ 36 ] . despite these similarities , the distinct anatomic subsites are associated with differing rates of regional metastasis for example , vocal cord lesions tend to metastasize less frequently than oropharyngeal or hypopharyngeal lesions . this variation may be attributed to differing densities of lymph draining vessels within each of the relevant subsites . patients who exhibit metastases into the regional nodal basin exhibit a 50% decrease in survival irrespective of treatment [ 715 ] . currently , it is the 5th leading cause of cancer by incidence and the 6th leading cause of cancer mortality in the world [ 16 , 17 ] . recurrent and/or metastatic hnscc patients have a poor prognosis , with a median survival of less than 1 - 2 years [ 18 , 19 ] . several lines of evidence indicate that cancer is a disease resulting from dynamic changes in the genome that promote the progressive transformation of normal human cells into highly malignant derivatives [ 20 , 21 ] . during this process , cancer cells acquire several unique capabilities including self - sufficiency in response to growth signals , insensitivity to antigrowth signals , evasion of programmed death ( apoptosis ) , limitless replicative potential , sustained angiogenesis as well as invasion and metastasis , reprogramming of energy metabolism , and avoiding immune destruction [ 21 , 22 ] . detailed global genomic analyses of several human tumors has revealed that certain classes of signaling proteins appear to be targeted more frequently by oncogenic mutations . receptor tyrosine kinases ( rtks ) are a good example . of the 59 transmembrane rtks identified to date , dysregulation of ~30 rtks are associated with neoplastic transformation and cancer progression [ 2325 ] . interestingly , ninety percent of primary head and neck squamous cell cancers , irrespective of subsite , have alterations in members of the epidermal growth factor ( egf ) family of receptor tyrosine kinases ( erbbs ) , in particular erbb1/egfr . ten to fifteen percent of tumors will also have an alteration in another egfr family member , the erbb2/her2/neu receptor [ 27 , 28 ] . these findings suggest a strong etiologic role for rtk dysregulation in this type of tumors . given this association , patients with head and neck squamous cell cancers are well positioned to benefit from existing and future molecular targeted agents directed against oncogenic rtks such as egfr ( reviewed in ) . rtks are a family of transmembrane proteins that mediate many important physiological processes in both normal and cancerous cells . ligand binding to the extracellular domain of rtks induces receptor dimerization and activation of rtk activity . subsequent autophosphorylation of the receptor at specific tyrosine residues within the cytoplasmic domain generates binding sites for proteins that relay downstream biological signals to regulate protein function , protein - protein interactions , and gene expression . under physiological conditions , rtk dysregulation can occur through several mechanisms including gene amplification or rtk overexpression , chromosomal translocation to produce constitutively active rtks , gain of function mutations or deletions that promote ligand - independent rtk activity , escape from negative regulatory mechanisms or local environmental changes , all of which lead to potent oncogenic signaling and hence neoplastic growth . these complex signaling networks use multiple factors to drive the outcome of rtk signaling . although often depicted as linear pathways , they actually represent an integrated network with various modes of cross - talk , overlapping and distinct functions . known signaling pathways involved in head and neck tumorigenesis include the phosphatidylinositol-3-kinase ( pi3k)-akt - mammalian target of rapamycin ( mtor ) , signal transducer and activator of transcription ( stats ) and raf kinase - mitogen - activated protein kinase kinase ( mek)-p42/p44 mitogen activated protein kinase ( mapk ) signaling pathways [ 1 , 30 ] . this review highlights three rtk signaling pathways involved in head and neck squamous cell carcinoma ; egfr , the type 1 insulin - like growth factor receptor ( igf-1r ) and the hepatocyte growth factor ( hgf ) receptor ( met ) . this short review will explore the relative contribution of each signaling axis to disease progression , potential modes of cross - talk , and targeted clinical approaches under investigation for disease management . the egfr family of rtks is comprised of four different receptors known as erbb1 ( also referred to as egfr ) , erbb2 ( her2/neu in rodents ) , erbb3 ( her3 ) , and erbb4 ( her4 ) ( reviewed in [ 3133 ] ) . each receptor , with the exception of erbb3 , contain an intracellular tyrosine kinase domain that is activated by binding to extracellular egf - like ligands , which result in receptor dimerization and hence activation of downstream signaling cascades including mapk , pi3k / akt and stat signaling . eleven egf - like ligands have been identified to date that can be categorized into four groups those that bind egfr only ( egf , transforming growth factor alpha ( tgf ) , and amphiregulin ) , those that bind to egfr and her4 ( heparin binding - egf , betacellulin and epiregulin ) , those binding directly to either her3 and her4 ( neuregulin 1 and neuregulin 2 ) and her4 binding only ( neuregulin 3 and neuregulin 4 ) ( reviewed in ) . epigen , the most recently discovered member of the egf - like ligand family appears to be a low affinity and broad specificity ligand that effectively activates egfr . erbb2 is considered a ligand - less coreceptor as it does not have any known ligands that bind directly with high affinity , despite its established role as a potent oncogene in several cancer types including breast , colorectal , nonsmall cell lung carcinoma ( nsclc ) and hnscc [ 36 , 37 ] . aberrant egfr activity has been strongly linked to the etiology of 5890% of hnscc [ 26 , 38 ] . these rates can vary due to the inclusion of cancers from different subsites within the head and neck , methods used to assess gene amplification and tumor scoring methods . in contrast to lung adenocarcinomas in which activating egfr mutations result in ligand - independent signaling [ 3943 ] , such activating egfr mutations are infrequent in hnscc [ 44 , 45 ] . egfr gene amplification resulting in upwards of 12 copies per cell has been reported in hnscc patients compared to copy numbers detected in normal mucosa from noncancer patients . this and other pathways of ligand - independent receptor activation that do not require egfr overexpression have been characterized as the likely drivers of egfr activity in hnscc . egfr gene amplification remains a strong indicator for poor patient survival , radioresistance , and locoregional failure [ 4749 ] . egfr overexpression is detected in healthy mucosa in cancer patients ( field cancerization ) that will increase in proportion to observed histological abnormalities such as hyperplasia , carcinoma in situ and invasive carcinoma , indicating that it is an early event in hnscc . accordingly , significant effort has focused on egfr signaling as a therapeutic target for treating hnscc patients . cetuximab , matuzumab and nimotuzumab represent humanized antiegfr antibodies , whereas gefitinib and erlotinib are small tyrosine kinase inhibitors ( tkis ) ( figure 1 ) . cetuximab ( erbitux ) competitively inhibits endogenous ligand - binding to egfr and thereby inhibits subsequent receptor activation [ 5053 ] . cetuximab is a valuable treatment option in head and neck patients as it synergizes with current treatment modalities . cetuximab enhances the effects of many standard cytotoxic agents , including cisplatin ( the conventional platinum - fluorouracil chemotherapeutic ) , and in combination with chemotherapy it can elicit antitumor responses in tumors that previously failed to respond to that chemotherapy . notably , cetuximab did not dramatically exacerbate the common toxic effects associated with radiotherapy of the head and neck , including mucositis , xerostomia , dysphagia , pain , weight loss , and performance status deterioration . cetuximab has been approved for use in combination with radiation for treating patients with locally advanced hnscc and as monotherapy for patients with recurrent hnscc . matuzumab ( formerly emd 72000 ) binds to egfr with high specificity and affinity to block receptor signaling , and also modulates antibody - dependent cellular cytotoxicity ( adcc ) when combined with cetuximab [ 5860 ] . phase i clinical trials report excellent antitumor activity of matuzumab against several human tumor types including head and neck cancers . a randomized phase iib , four - arm , open - label study recently assessed the safety and efficacy of nimotuzumab in combination with radiation therapy ( rt ) or chemoradiation therapy ( crt ) in patients with advanced ( stage iii or iva ) hnscc . the addition of nimotuzumab to both the radiation and chemoradiation regimens was reported to improve the overall response rate , survival rate at 30 months , median progression - free survival and median overall survival . a combined group analysis of the nimotuzumab arms versus the non - nimotuzumab arms demonstrated a significant difference in overall survival favoring nimotuzumab . this study is compelling as patient response rates compare favorably with studies combining cetuximab with radiotherapy , but with fewer side effects . gefitinib ( iressa ) is a small molecule tki - targeted to the intracellular active site for phosphorylation that has been tested in clinical trials involving hnscc patients , as a single agent or in combination with radiation treatment . unfortunately , gefitinib has shown limited clinical efficacy with response rates of 1015% [ 63 , 64 ] . erlotinib is a selective inhibitor of the egfr that also shows antitumor activity in hnscc comparable to standard combination chemotherapy . another promising rtk under preclinical and clinical evaluation for head and neck cancers includes the igf-1r ( reviewed in [ 66 , 67 ] ) . two ligands , insulin - like growth factor 1 ( igf1 ) and igf2 bind to igf-1r . ligand binding to the igf-1r stimulates its intrinsic tyrosine kinase activity , activating downstream signaling networks including ras - raf , mapk and erk , and pi3k ( figure 1 ) to drive cellular functions such as cell growth , survival and differentiation . it is widely accepted that the igf - axis activates antiapoptotic signaling , which in turn upregulates the pi3k - akt and mapk pathways in cancer cells . additionally , igf - ir also regulates vascular endothelial growth factor ( vegf ) production , suggesting a role in tumor angiogenesis . several studies indicate that igf-1r is overexpressed and functional in 94% of hnscc patient samples [ 70 , 71 ] . consistent with this , igf - ir signaling significantly enhances the proliferation , motility and tumorigenicity of human head and neck cancer cell lines . igf-1r down regulation in a hnscc cell line using antisense oligonucleotides resulted in a dose - dependent decrease in cellular proliferation , induction of apoptosis , caspase activation and reduced expression of proangiogenic cytokines such as vegf . interest in targeting the igf-1r in hnscc was bolstered by the observation that treatment of head and neck cancer cells with either igf or egf resulted in igf - ir and egfr heterodimerization [ 71 , 72 ] . however , only igf resulted in the phosphorylation of both receptors . using a mouse xenograft model for hnscc , treatment with antibodies against igf-1r , egfr or it remains to be determined whether cellular cross - talk between igf-1r and egfr has an important role in determining the biological aggressiveness of hnscc or resistance to egfr - targeted therapies . several monoclonal antibodies and tkis for igf-1r have been tested in preclinical studies and early phase clinical studies . however , the efficacy of igf-1r - targeted therapy for treating patients with hnscc , particularly cross - talk with egfr , warrants further investigation . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer cell lines [ 7375 ] . treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] . targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) . similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) . these studies will be critical for evaluating whether the use of anti - igf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc . the met receptor is a single pass transmembrane protein that upon binding its ligand hgf also known as scatter factor - promotes increased cell proliferation , survival and motility ( reviewed in [ 78 , 79 ] ) . hgf is the only physiological ligand for met and is secreted as an inactive precursor polypeptide chain by mesenchymal cells . hgf is proteolytically cleaved to form an active / heterodimer by a number of serine proteases including urokinase plasminogen activator ( upa ) , tissue - type plasminogen activator ( tpa ) , coagulation factors x. xi and xii . met is a disulphide - linked / heterodimer derived from the proteolytic cleavage of a 170 kda precursor . the chain and n - terminal region of the -chain form sema domain , a seven -propeller structure in which blades 2 and 3 bind to hgf . the sema domain is flanked by a cysteine - rich region followed by four immunoglobulin repeats . it is proposed that the cysteine - rich region and immunoglobulin repeat domains undergo a conformational change following hgf binding allowing for met dimerization [ 80 , 81 ] . binding of hgf to met results in receptor autophosphorylation at key catalytic residues and subsequent recruitment of several cytosolic signaling molecules that are shared with the egfr and igf-1r signaling pathways , including the grb2/sos complex , the p85 regulatory subunit of pi3k , gab1 and jak / stat3 ( figure 1 ) . subsequent activation of the mapk and jun - n - terminal kinase ( jnk ) pathways is responsible for the mitogenic and motogenic properties of met / hgf signaling resulting in invasive growth , depending on the physiological setting . increased met signaling in human cancers can be the result of enhanced ligand - binding ( autocrine and paracrine ) , met overexpression or missense mutations that often induce constitutive kinase activity , failure of met down regulation and interactions with other cell surface receptors such as egfr ( reviewed in [ 8284 ] ) . met is overexpressed in 84% of hnscc patient samples . interestingly , amplification of the met gene ( > 10 copies per cell ) is present only in 3 of 23 ( 13% ) tumor tissues . hgf overexpression is detected in 45% of hnsccs , suggesting that hgf functions predominantly in a paracrine manner to drive met signaling in these cancers . moreover , high levels of hgf are detected in hnscc patient plasma samples supporting the idea that ligand availability is not a limiting factor for met activation . mutations in the met ligand - binding domain ( t230m / e168d ) , transmembrane or jm domain ( r988c , t1010i ) and the tyrosine kinase domain ( t1275i , v14333i ) have also been identified in hnscc tumor samples , although their relative contribution to hnscc progression remains to be determined . two somatic met mutations have been detected in hnscc that result in constitutively active receptor signaling that confers an invasive phenotype when ectopically expressed in cell lines . the y1230c mutation confers anchorage - independent growth and an invasive phenotype in transfected cells , whereas the y1235d met mutation stimulates epithelial cells to invade reconstituted basement membrane in the absence of hgf . in the case of the mety1235d mutation , genomic analyses of hnscc patient samples detected the presence of this mutant allele in 50% of metastatic tumors versus 26% in primary tumors , raising the possibility that this could be a critical genetic lesion for the acquisition of a metastatic phenotype . alternatively , increased met signaling could afford hnscc a selective advantage for growth and/or survival in metastatic sites , such as the lymph node and lung . indeed several studies indicate that met overexpression correlates highly with lymph node metastasis , pathologic stage , and disease reoccurrence [ 8891 ] . moreover , patient survival was significantly reduced in biopsy samples with positive met expression relative to negative met expression , suggesting the association of met with hnscc disease progression . consistent with these findings , treatment with the tki pf-2341066 caused a significant reduction in tumor growth , a high level of apoptosis and cellular debris within the tumor using a xenograft animal model for hnscc . selective inhibitors of met / hgf signaling include humanized monoclonal antibodies for hgf and met , and small - molecule tyrosine kinase inhibitors directed against met ( figure 1 ) . although their efficacy for treating a variety of solid tumors is increasingly recognized , we await results of preclinical and clinical trials for head and neck cancer that are ongoing . the humanized antibody amg 102 shows high potency towards the mature and processed form of hgf with no detected effects on proteolytic activation of prohgf . amg 102 interferes with met signaling , by competing with hgf for binding to the chain of the met receptor . in phase i clinical studies in patients with advanced solid tumors , 70% of patients had a best response in terms of achieving stable disease [ 93 , 94 ] . importantly , no antiamg 102 antibodies were detected and circulating hgf levels were dose dependent . another promising clinical therapeutic is the one - armed 5d5 humanized antibody ( oa5d5/metmab ) directed against met . metmab binds met with high affinity , preventing hgf binding , met phosphorylation , receptor internalization and downstream signaling events and has been shown to inhibit tumor growth in animal models by more than 95% [ 95 , 96 ] . metmab is currently in phase i / ii human clinical trials in comparison with erlotinib in patients with nsclc ( http://www.clinicaltrials.gov/ , identifier nct00854308 ) . future clinical trials will be required to determine the suitability of amg102 and metmab as either single agents or combinatorial therapeutics for treating hnscc patients . foretinib ( formerly xl880 ) is a tki whose primary targets include met and vegf , and to a lesser extent the platelet - derived growth factor ( pdgf ) receptor , ron , kit and tie2 rtks . foretinib recently completed phase ii clinical trials in head and neck patients ( http://www.clinicaltrials.gov/ , identifier nct00725764 ) . interim results suggest that after 12 months , 12 of 18 patients had stable disease . a phase i dose - escalation study of the safety and pharmacokinetics of xl184 administered orally to patients with advanced malignancies ( showed that , on average , patients survived for more than 3 months with several up to 6 months while on treatment ) ( reviewed in ) . due to encouraging data from this study , a randomized phase iii trial of xl184 in hnscc patients was initiated to investigate xl184 as a first - line treatment ( compared with placebo ) for survival benefit to patients with hnscc ( http://www.clinicaltrials.gov/ , identifier nct00704730 ) . arq197 ( arqule ) is a nonatp - site competitive , selective small molecule inhibitor of the met intracellular region . although the mechanism of arq197 is presently unknown , the results of phase i trials suggest potential antiinvasive activity for this compound . overall , met , and hgf - targeted therapies have been well tolerated in clinical trials with negligible toxicities . however , it remains to be determined whether met is a better therapeutic target than hgf . clearly , in patients where met is activated by autocrine hgf secretion , both hgf and met targeted therapies may prove to be more efficacious treatment options . acquired resistance is likely the result of several mechanisms including ( 1 ) egfr mutations initially present as well as those acquired during therapy , ( 2 ) receptor independent activation of downstream signaling cascades , ( 3 ) cross - talk with other rtks and converging signaling pathways and ( 4 ) environmental factors including inflammatory agents and viral infection . resistance to cetuximab has been associated with the coexpression of the truncated egfr mutant , egfrviii with wild - type egfr . egfrviii is the result of an in frame deletion of exons 27 spanning the extracellular ligand - binding domain . the deletion results in a truncated egfr receptor that signals in a ligand - independent manner . egfrviii expression has been detected in 42% of hnscc patient samples , and closely correlates with increased hnscc cell proliferation in vitro and increased tumor growth using in vivo xenograft models . egfrviii preferentially activates the pi3k pathway instead of the ras / raf / mek pathway , which is activated by wild - type egfr . of particular interest to the therapeutic treatment of hnscc , egfrviii expression decreases the proliferative response of egfr expressing tumor cells to cetuximab treatment relative to vector control cells . in a recent study , egfrviii cells were shown to be resistant to the antiinvasive effects of cetuximab due to an increase in phosphorylation of stat3 rather than increased pi3k signaling . egf - induced expression of the stat3 target gene hif1 was abolished by cetuximab in hnscc cells expressing wild - type egfr under hypoxic conditions , but not in egfrviii - expressing hnscc cells [ 102 , 103 ] . these data suggest a role for egfrviii in mediating hnscc resistance to cetuximab . despite egfrs critical role in the development of hnscc , clinical data indicate modest clinical benefits for locoregional control and survival of head and neck cancer patients treated with egfr - targeted therapies . hnscc patients resistant to cetuximab , often succumb to local tumor recurrence as well as regional and distant metastasis . the addition of cetuximab to radiation therapy was reported to show improved locoregional disease control , progression - free survival , and overall survival in patients with locally advanced hnscc . however the data revealed a disproportionate benefit of cetuximab with radiotherapy to oropharyngeal cancer patients when compared to patients treated with hyperfractionated radiotherapy . accumulating evidence suggests that human papilloma virus ( hpv ) 16 status ( hpv+ ) is an important prognostic factor associated with a favorable outcome in a subset of head and neck cancers , including oropharyngeal and tonsilar cancers . hpv+ tumors tend to have unique genetic aberrations including decreased egfr expression , whereas increased igf-1r levels characteristic of hnscc appear to be independent of hpv status . clinically , hpv+ tumors are characterized by more favorable patient prognosis regarding disease - free survival as well as overall survival [ 104 , 105 ] , possibly as a result of increased genomic stability associated with global gene hypermethylation in hpv+ tumors . thus it will be interesting to determine whether hpv+ status explains some of the benefits derived from the addition of cetuximab to radiotherapy in this subset of hnscc patients . at present , there are few clinical indicators of which hnscc patients will most likely respond to egfr - targeted therapies . accordingly , strategies to optimize egfr - targeted therapy remain an active area of research . additional mechanisms that result in egfr activation include activating mutations in downstream signaling components or cross - talk between different rtk pathways . activating mutations in the pi3ka oncogene occurs in 10% of hnscc tumors whereas elevated levels of phosphorylated stat3 correlates with lymph node metastasis and poor patient prognosis [ 108110 ] . conversely , h - ras mutations are infrequent in hnscc cases ( less than 5% ) , although a higher incidence has been detected in asian populations and correlates with areca nut chewing [ 111 , 112 ] . met signaling has been shown to contribute to resistance in cell lines derived from multiple tumor types including breast , gastric and lung . in one key study , nsclc with activating mutations in the egfr acquire resistance to the tki gefitinib and erlotinib , by amplification of the met gene to maintain akt and her3 signaling . these studies underscore the role of cross - talk between rtks to preferentially signal through the pi3k - akt survival pathway as a mechanism for acquired drug resistance . the relevance of met as a mechanism for escape from egfr - targeted therapy in head and neck cancers remains to be determined . hypoxia results in the transcriptional upregulation of met gene expression via hif1 in a number of tumors including head and neck , often downstream of egfr signaling . in normoxia , hydroxylation of 2 prolines in hif1 enables its binding to the von hippel - lindau tumor suppressor protein ( pvhl ) linking hif1 to a ubiquitin ligase complex . during hypoxia , minimal or no hydroxylation occurs enabling hif1 to avoid proteasomal degradation and dimerize to other hif family members such as hif1 and coactivators , to form an active transcriptional hif complex on the hypoxia response element ( hre ) of target genes such as met . the ubiquitin ligase catalyzes polyubiquitination of hif1 targeting it for proteasomal degradation . under hypoxic conditions , increased met signaling directs the invasive growth program , enabling cells to invade more oxygenated tissues . since met has been reported to promote invasive and angiogenic effects in the tumor microenvironment , the use of hgf / met inhibitors may afford a means of impairing tissue colonization as well as tumor vascularization in head and neck cancer patients . studies on other solid tumor types , most notably glioblastoma , indicate a role for igf-1r upregulation in resistance to egfr - targeted therapies . igf-1r mediates resistance to anti - egfr therapy in primary glioblastoma through the continued activation of the pi3k / akt survival pathway . the apparent cooperation between igf-1r and egfr in promoting hnscc pathogenesis as well as resistance to egfr - targeted therapy , suggests an advantage to cotargeting these signaling axes for the treatment of head and neck cancers . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer lines . treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] . targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) . similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) . these studies will be critical for evaluating whether the use of antiigf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc . targeted therapies that block egfr , met , and igf-1r signaling in head and neck cancers continue to show promising results in preclinical studies and clinical trials . however , it is difficult to predict which patients are most likely to benefit from these therapeutics and potential side effects during long - term in vivo use . given the interplay between these rtk signaling pathways and the mediocre results obtained with monotherapy regimens thus far , clinical trials will be required to determine how egfr- , met- , and igf-1r - targeted therapies can be used in combination in order to definitively abrogate their common downstream oncogenic signaling networks . although gaps in our knowledge concerning the role of met and igf-1r in head and neck tumorigenesis , as well as acquired resistance to antiegfr therapies remain to be addressed , efforts to translate current information towards clinical applications continue to be impressive .

molecular therapeutics for treating epidermal growth factor receptor-(egfr- ) expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics . however , the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment . yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross - talk between receptor pathways with a key finding of common downstream signaling pathways . such cross talk may represent a key mechanism for resistance to egfr - directed therapy . here we review the interplay between egfr and met and the type 1 insulin - like growth factor receptor ( igf-1r ) tyrosine kinases , as well as their contribution to anti - egfr therapeutic resistance in the context of squamous cell cancer of the head and neck , a tumor known to be primarily driven by egfr - related oncogenic signals .

this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals . the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation . primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) . then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated . the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium . dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) . the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div , half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem . serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls . the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons . the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) . the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10 ) , and 10 m of mpp on the tenth div for 48 h. dopaminergic neurons were identified immunocytochemically by staining tyrosine hydroxylase . cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature . cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures , cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages . the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification . cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium . the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes . the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored . the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period . the ldh release is proportional to the number of damaged or destroyed cells.11,12 lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes . cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div , culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs , cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m ) the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells . the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) . after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software . cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) , the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) . nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software . data was obtained from 12 wells ( from 2 repeats ) for each treatment condition . comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . , this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals . the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation . primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) . then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated . the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium . dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) . the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div , half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem . serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) was prepared in dimethyl sulfoxide ( dmso ) . four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls . the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons . the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) . the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10 ) , and 10 m of mpp on the tenth div for 48 h. cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature . cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures , cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages . the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification . cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium . the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes . the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored . the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period . lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes . cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div , culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs , cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m ) the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells . the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) . after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software . cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) , the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) . nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software . data was obtained from 12 wells ( from 2 repeats ) for each treatment condition . comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . , treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) . treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) . surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) . co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) . anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures . 100% corresponds to the total number of thir neurons after 12 div in untreated controls . values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) . the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) . treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures . th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture . the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) . the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) . div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture . lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) . lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures . 100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div . fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures . div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m . cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) . as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures . 100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div . red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) . against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) . 4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures . 100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture . treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) . treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) . surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) . co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) . anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures . 100% corresponds to the total number of thir neurons after 12 div in untreated controls . values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) . the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) . treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures . th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture . the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) . the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) . div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture . lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) . lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures . 100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div . fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures . div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m . cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) . as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures . 100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div . red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) . against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) . 4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures . 100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture . in the present study , tq was investigated to ascertain whether it protected mesencephalic dopaminergic neurons against mpp - induced cell death through activation of enzymatic degradation , preservation of mitochondrial function , and inhibition of apoptotic cell death . clearly , mpp was found to significantly decrease the survival of dopaminergic neurons and increase the release of ldh into the culture medium . the mpp toxicity involves its selective uptake by dopaminergic neurons through the dopamine transporter and inhibition of mitochondrial complex i activity with subsequent mitochondrial depolarization.14 in parallel , the use of jc-1 fluorescence dye in our current study showed that mpp significantly decreased the m of cultured cells as indicated by the decreasing red : green fluorescence ratio of jc-1 . similar mpp - induced reduction of m was reported in other in vitro disease models.15,16 mitochondrial damage has long been implicated in the death of nigrostriatal dopaminergic neurons in both pd patients and experimental models.17,18 staining of primary dopaminergic cultures with blue - fluorescent dapi nucleic acid stain showed that a significant number of the cells displayed features of apoptosis , most notably chromatin condensation , and fragmentation . previously , tang et al19 and xu et al16 demonstrated that mpp caused apoptotic cell death in pc12 and sh - sy5y cells . the mpp - induced apoptosis was reported to occur as the result of disruption of mitochondrial transmembrane potential and opening of the permeability transition pore.20 similar to our previous report,9 co - treatment of primary mesencephalic cell cultures with tq and mpp was found to protect dopaminergic neurons and decreased the release of ldh into the culture medium . since that time , no evidence in the literature has shown how tq protected dopaminergic neurons in the primary mesencephalic cell culture . staining of cultures with lysotracker deep red showed that tq significantly increased the red fluorescence of the dye compared with mpp - treated cultures , indicating enhancement of the formation of many autophagolysosomes , the sites of lysosomal degradation , by tq . this is supported by the findings of he and klionsky21 who correlated the fluorescent signals of lysotracker deep red to the upregulation of autophagy in zebrafish . increased red fluorescence of lysotracker deep red is attributed to the formation of many autophagosomes and autophagolysosomes that retain much dye as the result of increasing their acidification . using jc-1 fluorescent dye showed that tq significantly enhanced m as it increased the red : green fluorescence ratio of jc-1 compared with mpp - treated cultures . the tq was similarly found to protect rat cortical neurons against ethanol- and a1 - 42-induced neurotoxicity through inhibition of mitochondrial membrane depolarization.22,23 counting of apoptotic nuclei using blue - fluorescent dapi nucleic acid stain indicated that tq decreased mpp - induced apoptotic cell death in primary mesencephalic cell cultures . in accordance , ullah et al22 reported that tq inhibited apoptotic cell death in ethanol - treated rat cortical neurons and attributed this effect of tq to the preservation of mitochondrial integrity . zhang et al24 reported that mitochondrial clearance protected cultured cortical neurons against ischemia - reperfusion - induced cell damage . in conclusion , correlating such results would therefore suggest that tq might activate a lysosomal degradative process in dopaminergic neurons , where clearance of damaged mitochondria results in reduced mitochondria - mediated apoptotic cell death . this might raise the possibility of using tq as a potentially therapeutic intervention in pd patients . whenever a manuscript contains material ( tables , figures , etc . ) which is protected by copyright ( previously published ) , it is the obligation of the author to obtain written permission from the holder of the copyright ( usually the publisher ) to reproduce the material in neurosciences . please submit copies of the material from the source in which it was first published .

objectives : to investigate potential mechanisms mediating the neuroprotective effect of thymoquinone ( tq ) on dopaminergic neurons.methods:this study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 . primary cultures were prepared from embryonic mouse mesencephala ( ofi / spf ) at gestation day 14 . four sets of cultures were kept untreated , treated with tq on the eighth day in vitro ( div ) for 4 days , treated with 1-methyl-4-phenylpyridinium ( mpp+ ) on the tenth div for 48 hours and co - treated with thymoquinone and mpp+ . on the twelfth div , cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using lysotracker deep red , 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazolylcarbocyanine ( jc-1 ) and 4,6-diamidino-2-phenylindole stains.results:the mpp+ decreased the number of dopaminergic neurons by 40% , and increased the release of lactate dehydrogenase ( ldh ) into the culture medium . the tq significantly rescued dopaminergic neurons and decreased the release of ldh at the concentrations of 0.1 and 1 m . the tq significantly shifted the red fluorescent intensity of the lysotracker deep red , increased the mitochondrial membrane potential as it increased the red : green florescent ratio of jc-1 , and decreased mpp+-induced apoptotic cell death.conclusion:the tq protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria - mediated apoptotic cell death .

the femoral head often leads to healing complications , while the more predictable prosthetic replacements are associated with poorer function and significant complications . the treatment of these fractures depends on the age of the patient , fracture displacement , bone quality , timing of surgery and activity level of the patient . displaced fractures in healthy , active patients are best treated by reduction and internal fixation . there are however , complications unique to femoral neck fractures which are almost impossible to predict . non - union and avascular necrosis non - union usually can definitely diagnosed within a year of fracture fixation with the same being achieved within 3 months at times . after non - union has been established , intervention is inevitable . the decision to proceed in the management of failed fixation is based on the careful consideration of various factors . in young patients revision internal fixation with cancellous or muscle pedicle bone grafting ( vascularised bone graft ) or an osteotomy results in useful outcome . we report a case of non - union of the fracture of the neck of the femur , who refused additional procedures after his non - union had been established . the study conforms to the declaration of helsinki and was approved by the institutional ethical board . a 38-year old male businessman reported to the out door department of our hospital with a history of a fall from height . clinical and radiological examination revealed a displaced fracture of the neck of the femur which was graded as garden type 4 ( fig . the patient was operated within 24 h. intraoperatively the garden alignment index was used as a yardstick of acceptable reduction . the fracture was fixed with 3 ao 6.5 mm cannulated screws in an inverted triangle . onward the patient complained of persistent but mild groin pain on ambulation which was assisted . the patients conservative line was continued till 1 year when his symptoms continued and radiologically there was no progress . at this point twenty - six months into the post operative followup the patients pain subsided and radiographs started showing signs of union . a radiograph taken 32 months after the fixation showed full union . non - union after femoral neck fracture can be defined as a lack of radiographic evidence of union 6 months after the fracture . delayed or non - union often manifests as continued pain with weight bearing beyond 3 months post fixation . incidence of non - union of femoral neck fractures has been reported to be between 2 and 22% and generally becomes apparent within 1 year [ 69 ] . 3radiograph at 32 months depicting union garden 4 type fracture of the neck of the femur showing persistent non - union at 2 years radiograph at 32 months depicting union anatomical reduction and rigid internal fixation are the two important surgeon controlled factors that may contribute to outcome . the preferred treatment for symptomatic non - unions in the elderly is prosthetic replacement . in the young active patients , if failure is due to technical errors , revision open reduction and internal fixation may be adequate . our case demonstrates that as long as the implant is holding and the patient is regularly followed up good results might be expected in cases as far as 32 months into the post fixation period .

fracture of the femoral neck continues to be a vexing clinical and therapeutic challenge for the orthopedic surgeon . the fracture has a propensity for non - union and avascular necrosis . it is a challenge for the orthopedic surgeon to decide when to intervene in a case with non - union where the implant continues to be in place . we present a case with persistent clinical and radiological non - union signs where the fracture eventually united after 32 months . the case bolsters the view that a continued conservative regime might entail good results in such situations .

granuloma faciale ( gf ) is an uncommon , benign , inflammatory skin disorder of unknown etiology . it is characterized by single or multiple , grey - brown or violaceous nodules or plaques primarily occurring on the face and occasionally at extra - facial sites . the disease is notoriously resistant to therapies and often tends to relapse when treatment is discontinued . we present a patient with multiple lesions of gf and its response to topical tacrolimus . a 35-year - old female presented to our department with a 5 year history of single , asymptomatic , grey - brown pigmented , nodule over the left cheek [ figure 1 ] . it started as a pin head sized papule which gradually increased to 2.5 cm 1.5 cm in size . two years later similar lesions appeared on the forehead , both arms and upper back . past and personal history was unremarkable . before treatment single , grey - brown nodule with prominent follicular orifices over left cheek . after treatment residual lesion after three months of tacrolimus application general physical and systemic examination was normal . cutaneous examination revealed multiple , well - defined , grey - brown , indurated , non - tender plaques , varying in size from 0.5 cm 0.5 cm to 1.5 cm 2.5 cm , present on the left cheek , left forehead , both arms and upper back . multiple grey - brown plaques over upper back routine hematological and biochemical investigations were normal . skin biopsy ( 4 mm ) from plaque revealed normal epidermis with clear sub epidermal grenz zone and pan dermal dense infiltrate comprising of neutrophils , lymphocytes , histiocytes and plasma cells . small dermal vessels showed infiltration of neutrophils in the vessel wall along with peri - appendageal and peri - neural infiltrate in subcutaneous fat [ figure 3 ] . ( h and e , 100 ) skin biopsy with normal epidermis and dense , mixed inflammatoey infiltrate beneath a narrow grenz zone in the dermis . infiltrate is composed of mononuclear cells with neutrophils and eosinophils she was started on intralesional triamcilone acetonide 10 mg / ml injection monthly with tab . six sessions of cryotherapy were performed once monthly after which she developed erythema and itching over the plaques and discontinued treatment . the lesions showed 40 - 50% improvement after 3 months of therapy [ figure 1 ] . classically , red - brown or violaceous nodules or plaques with associated telangiectasia and follicular accentuation are seen on the face over sun - exposed sites . differential diagnosis includes lupus pernio , lupus vulgaris , lymphoma , discoid lupus erythematosus and deep mycotic infection . skin biopsy is characterized by a mixed inflammatory infiltrate with a predominance of neutrophils and eosinophils in the dermis , in conjunction with small vessel vasculitis . there is a grenz zone that separates the infiltrate from the epidermis and pilosebaceous units . the disease is notoriously resistant to therapies and often tends to relapse once the treatment is discontinued . several medical and surgical modalities like topical and intralesional corticosteroids , cryotherapy , pulsed dye laser , puva , systemic corticosteroids , dapsone and antimalarials have been tried with variable success rates . carbon dioxide laser has also been used in a case of recurrent gf . surgical excision has been performed with often unsatisfactory results . ablative procedures may leave residual pigmentation and scarring , whereas long - term application of corticosteroids is associated with skin atrophy , telangiectasia and other possible adverse effects . in recent years successes with topical calcineurin inhibitors has been reported . several authors have reported complete or near - complete resolution of lesions after application of topical tacrolimus 0.1% ointment.[48 ] treatment regimens , duration and time to resolution of lesions have varied in these case reports [ table 1 ] . others have found time to resolution to be between 4 and 6 months . in our patient , treatment with tacrolimus 0.1% ointment twice daily for 3 months has resulted in improvement . reported cases of successful treatment of granuloma faciale ( gf ) with topical tacrolimus tacrolimus inhibits t - cell proliferation , production and release of several pro - inflammatory cytokines like interleukin-2 ( il-2 ) , il-4 , tumor necrosis factor - alpha , and interferon - gamma ( ifn - gamma ) . although the pathogenesis is still unknown , it has been suggested that gf may be an ifn - gamma mediated disease . in addition , an increased production of il-5 , probably induced by the clonal expansion of a locally recruited t - cell population may enhance the attraction of eosinophils into the lesions of gf . therefore , a possible mechanism of action of topical tacrolimus in this condition may be the inhibition of ifn - gamma and il-5 production and release , induced by the down - regulation of the t - cell activity , primarily involving the calcineurin binding and inactivation . however , we did observe eosinophils in skin biopsy in our case , probably since the biopsy was taken after one year of oral dapsone . our patient experienced a relevant improvement within 3 months of treatment with tacrolimus ointment after no response with intra lesional steroids , dapsone and cryo therapy . in conclusion , the previous reports and our observation suggest that topical tacrolimus may be a well - tolerated , efficacious therapy for gf .

granuloma faciale ( gf ) is a chronic condition characterized by red - brown plaques with follicular accentuation present usually on the face . we present a case of 35-year - old female with 5 year history of plaques over cheek and extra facial sites consistent with gf and its response to topical tacrolimus . this case supports previous reports of successful treatment of gf with topical tacrolimus .

chronic myelomonocytic leukaemia ( cmmol ) is a rare disease with an incidence of 0.370.72 per 100 000 population . the median age at diagnosis varies between 65 years and 75 years [ 14 ] . the radical treatment is allogeneic stem cell transplantation , although this treatment is unsuitable for most patients because of their advanced age [ 6 , 7 ] . the median survival of patients with cmmol is 1520 months [ 5 , 6 , 8 ] . several case reports have indicated that excess surgical stress causes an acute blastic crisis , which contributes to a poor outcome [ 9 , 10 ] . a 68-year - old man complained of having a reduced appetite for approximately 5 months and weight loss during the previous 4 months . on 12 may 2012 , he visited a nearby general internal medicine clinic for the evaluation of left lower quadrant abdominal pain . he was referred to the department of internal medicine at asahi general hospital ( asahi , japan ) for investigation and treatment . he began taking oral hydroxyurea ( hydrea ; bristol - myers squibb , princeton , nj , usa ) . he was unable to stop the gingival bleeding that had been ongoing for 5 hours . he then visited the emergency department of asahi general hospital and was referred to the department of dentistry and oral surgery . at the initial visit the right molar teeth ( 47 and 48 ) showed grade iii mobility , and their gingivae were painful with continuous bleeding . 1a ) . the right molar teeth ( 46 and 47 ) showed class iii alveolar ridge deficiency on panoramic radiographs and were mobile teeth ( fig . we packed an absorbable haemostat ( surgicel ; ethicon , somerville , nj , usa ) into the gingivae and sutured 30 silk threads above it . finally , we used surgical dressing packs ( coe - pak ; g.c . figure 1:(a ) the oral photograph at the initial visit , immediately after the gauze with the astringent has been removed . ( b ) the oral photograph after the extraction of teeth 47 and 48 . ( a ) the oral photograph at the initial visit , immediately after the gauze with the astringent has been removed . the internal medicine department assigned a clinical diagnosis of cmmol , based on the blood and bone marrow examinations conducted on 13 june 2012 . computed tomography on june 25 indicated that bone absorption had nearly reached the right inferior alveolar canal , and a ring of sequestrum was present under tooth 47 ( fig . we diagnosed right mandibular osteomyelitis , based on these clinical features . on 11 july 2012 , the patient was hospitalized in the department of haematology ward because of significant anaemia and decreased platelet numbers . on the same day , the internal medicine specialist placed him on intravenous piperacillin tazobactam ( zosyn ; pfizer , new york , ny , usa ) . he afterwards complained of severe pain in the socket ( 47 and 48 ) , despite receiving the potent opioid analgesic dihydrohydroxycodeinone ( fentanyl ; daiichi - sankyo , chiyoda - ku , tokyo , japan ) . hence , we decided to apply a surgical anti - inflammatory treatment in consideration of an acute blastic crisis . a bone marrow examination on july 13 showed no signs of acute blastic crisis of his cmmol . after we consulted an internal medicine specialist , we planned a surgical procedure to clean the inflamed region in his mouth . on the day before the surgery , he was administered a transfusion of platelet concentrate ( 20 units ) and piperacillin tazobactam to prevent bleeding and further infection . on july 20 , with the patient under local anaesthesia , we surgically resected the right lower sequestrum and extracted molar 37 , which displayed severe caries . the blood examination indicated improvement in the platelet count after the transfusion . after performing an inferior alveolar block , we removed the necrotic bone with ultrasonic bone surgery ( variosurg ; nsk , ashikaga , tochigi , japan ) until there was bleeding from the bone surface ( fig . we placed gauze with tetracycline ointment in the socket and sutured 30 silk thread to compress it ( fig . ( b ) we removed necrotic bone with ultrasonic bone surgery until there was bleeding from the bone surface . ( b ) we removed necrotic bone with ultrasonic bone surgery until there was bleeding from the bone surface . ( d ) we used 30 silk thread sutures to compress the gauze . on august 11 the formation of granulation tissue had nearly covered the surface of the bone , and all oral discomfort had disappeared ( fig . we later placed a bridge in the upper anterior teeth and added a partial denture ( fig . we placed a bridge for upper anterior teeth 12 , 11 , 22 and 23 , and a partial denture . we placed a bridge for upper anterior teeth 12 , 11 , 22 and 23 , and a partial denture . on 3 january 2013 , he admitted himself to the hospital in the haematology ward because of exertional dyspnoea . the internal medicine specialists diagnosed cmmol that was undergoing acute leukaemic transformation by bone marrow examination . he died on 10 february 2013 because of cardiac arrest , following haemorrhagic shock and cardiovascular failure due to cmmol . in all patients with cmmol , every effort must be made to use the least invasive procedure possible . in the present patient , our deepest concerns were to have a proper balance between invasiveness and healing , and to avoid evoking an acute blastic crisis [ 9 , 10 ] . our most important goal was to alleviate the patient 's discomfort by using a minimally invasive procedure to eliminate infection . based on haematology results , the oral surgery did not trigger the acute blastic crisis of cmmol in this patient . the surgery was necessary to improve his oral environment and to prevent further infection and more pain . because cmmol is associated with a risk of acute blastic crisis and a poor prognosis , any surgical procedure in these patients must be considered on a case - by - case basis .

we report a case of chronic myelomonocytic leukaemia ( cmmol ) in a 68-year - old man who developed osteomyelitis of the mandible . at the initial visit , he reported uncontrolled gingival bleeding , despite self - administered haemostasis . he complained of severe pain in the socket , despite potent opioid analgesia . after consultation with the internal medicine specialists , we undertook a surgical anti - inflammatory approach that included sequestrectomy with massive blood transfusion . his physical condition was ameliorated after the surgical procedure , and he was discharged from the hospital . however , 3 months later , he died because of cardiac arrest after haemorrhagic shock and cardiovascular failure because his cmmol had developed to an acute blastic crisis . this experience demonstrates that the most important goal in such cases is to alleviate a patient 's discomfort by applying minimally invasive actions to eliminate infection and improve the quality of life without causing deterioration in the cmmol status .

in a recent issue of critical care , we presented a series of 13 patients undergoing post - cardiotomy extracorporeal membrane oxygenation ( ecmo ) with bivalirudin - based anticoagulation . after this series , we satisfactorily continued this experience , reaching about 20 patients treated . however , we think that a word of caution should be added to our recently published experience . owing to its pharmacological profile , bivalirudin is rapidly cleaved by proteolytic enzymes , and its half - life is about 25 minutes when the renal function is normal . the use of bivalirudin for cardiopulmonary bypass ( cpb ) and ecmo is a feasible option . however , it is recommended that , during cpb , blood stagnation in the circuit be avoided because the rapid cleavage of bivalirudin may result in thrombosis . the ecmo circuit is devoid of a reservoir , and therefore blood stagnation is usually not a circuit- related problem . conversely , under some circumstances , the cardiac chambers may act as a ' natural reservoir ' , which entails blood stagnation and the risk for spontaneous intracardiac thrombosis . this is particularly true in the case of a very large right or left atrium with insufficient venous drainage or the case of very poor ventricular systolic function with intraventricular blood stagnation . to avoid this condition , we think that maintaining a partial ecmo support , leaving a minimal degree of intracardiac blood flow , may be a reasonable choice . this may be checked by echocardiographic view of heart valve movement or simply by the observation of some degree of pulsatile arterial blood pressure . we think that , in the event of echocardiographic evidence of a ' smoke effect ' within one or more cardiac chambers , bivalirudin should be replaced by standard heparin anticoagulation . intracardiac thrombus formation during ecmo with heparin anticoagulation has been described as well , but the pharmacokinetic properties of heparin may limit the risk of thrombus formation due to blood stagnation .

bivalirudin has been proposed as the sole anticoagulant in patients under extracorporeal membrane oxygenation ( ecmo ) or cardiopulmonary bypass . owing to the pharmacodynamic properties of bivalirudin , areas of blood stagnation should be carefully avoided in order to limit the risk of thrombosis . the ecmo circuit has no reservoir and is usually devoid of blood stagnation areas . conversely , under some circumstances , intracardiac blood stagnation areas may exist . in this case , there is a potential risk for the spontaneous formation of an intracardiac thrombus . we suggest that , under bivalirudin anticoagulation , a minimal degree of intracardiac blood flow with left heart valve movement is allowed .

global and regional left ventricular ( lv ) functions are well - known indicators of cardiac disease . quantitative values of ventricular volumes and of myocardial mass are independent predictors of morbidity and mortality in patients with coronary artery disease . classically , echo has been used to evaluate lv volume and function because it is relatively inexpensive and noninvasive . however , a component of operator dependence and poor contrast between blood and myocardium are considerable limitations of this technique . cardiac magnetic resonance imaging ( mri ) is considered the clinical gold standard for lv function assessment , but it is expensive , of limited availability , and can not be performed in patients with implanted pacemakers or defibrillators . in recent years , multidetector ct ( mdct ) has gained acceptance as a promising imaging method for coronary arteries . mdct acquired in a single breath - hold with retrospective electrocardiogram ( ecg ) gating can cover the entire heart with 1-mm slice thickness with a temporal resolution of 125 - 250 ms . when performed for coronary imaging , this method provides excellent opportunity to create , image reformation in any desired plane , including anatomically optimized long axis , short axis , or four - chamber views . diastolic and systolic images can easily be produced from the same data set with a retrospective ecg - gating technique , thus obtaining lv end - diastolic and end - systolic volumes ( edvs and esvs ) . mdct has a potential of being utilized as tool for the combined assessment of the coronary anatomy and lv function . in addition , ventricular wall motion can be assessed visually by the use of cine loop displays of multiple cardiac phases . recently we observed increasing tendency for utilizing low radiation dose , prospective gating for coronary angiography , thus limiting possibility of volumetric assessment of ventricular function . however , a small number of patients may need a retrospective gating , thus providing possibility of reconstructions in various phases of cardiac activity . according to published reports , measurements for various lv functional parameters with mdct were well - correlated and agree with measurements obtained with mri , two - dimensional transthoracic echocardiography ( 2d - tte ) , and ecg - gated single photon emission ct ( spect ) . experience with 64-slice mdct for cardiac function assessment remains limited by small patient numbers and the inclusion of homogeneous patient populations . the purpose of this study was to assess lv ejection fraction ( lvef ) using 64-slice as a byproduct of mdct coronary examination and to compare efficacy of technique with 2d - tte in a heterogeneous patient population . also , review the role of mdct lv function with a relation to evolution in the technology of coronary mdct imaging . study included 113 patients referred for 64-slice mdct coronary angiography for evaluation of coronary artery disease . all patients were scanned on 64 slice ge - helical ct ( ge high speed advantage ) scanner and had an echo done within 1 week of the ct scan . this prospective study was approved by the institutional review board and written informed consent was obtained from all patients . patients with absolute contraindication to contrast or radiation were excluded from the study . in patients with relative contraindications such as atopy , asthma , and renal failure scan was performed if the benefit of examination outweighed the risk in such patients . patients with arrhythmias and ectopic heart beats were excluded as stable heart rate is required for ct coronary angiogram . apart from the routine contraindications , patients with pacemaker and ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool is not possible in these patients due to artifacts and incorrect segmentation by software . all patients undergoing ct coronary angiogram , who had heart rate of more than 60 bpm , were premedicated with 50 - 200 mg oral b adrenergic blocking agent : metoprolol , 1-h prior to the study . a 60 - 120 mg calcium channel blocker : diltiazem , cta was performed with contrast volume of 1.2 ml / kg body weight of iohexol 350 . the intravenous contrast agent was followed by 30 ml of saline chaser bolus at the same injection rate . scan parameters were 0.35 s rotation time , 120 kv tube voltage , 600 - 800 effective ma , 0.6 mm collimation , and a helical pitch of 0.22:1 . the image acquisition was caudocranial for post - coronary artery bypass grafting ( cabg ) patients and craniocaudal for the rest of the patients . no complications encountered in any of the patients . retrospectively , ecg - correlated image reconstruction was performed . the reconstruction was performed with the reconstruction window starting at 10% of r - r interval and up to 90% r - r interval with increment of 10% . this included data sets reconstructed in systole , if diastolic data sets showed motion artifact . diastolic and systolic axial image sets were then transferred to the scanner 's workstation - ge advanced workstation advantage windows 4.4 p. image data were evaluated with a prototype version of a commercially available program ( auto ejection fraction , circulation ; ge medical solutions ) that performs a fully automatic segmentation of the blood volume in the lv by defining the mitral valve plane and the lv . the software uses this mitral valve plane as an upper boundary for the segmentation of the lv . the software identifies the hinges of the mitral and aortic valve leaflets closest to the ventricle wall and selects these as defining points for the plane [ figure 1a ] . all ct scans were analyzed according to this method , which allowed for optimal segmentation of the lv . papillary muscles were automatically excluded from the blood pool , which allows for precise determination of blood volume in the lv . multiplanar reformats are then performed by the software in long and short axes of left ventricle . the long axis image is obtained parallel to the interventricular septum connecting the lv apex and the middle level of mitral valve . the short axis images are obtained parallel to the plane of mitral valve [ figure 1b ] . lv = left ventricular ( b ) line perpendicular to long axis of left ventricle ( parallel to mitral valve ) plane for short axis images once the region of interest is finalized , the edv and esv are measured by this software using simpson 's method by summing the endocardial area of all lv ed and es short - axis slices multiplied by the slice thickness [ figure 2a and b ] . the stroke volume ( sv ) and ef were automatically calculated from these values and displayed by the software [ figure 3a d ] . ( a ) lv short axis image in diastole ( b ) short axis image in systole 3d display of volumetric data in different patients . ( a ) patient 1 : left ventricular ejection fraction by ct 58.4% ( echo 58% ) . ( b ) patient 2 : left ventricular ejection fraction by ct 55.7% ( echo 53% ) . ( c ) patient 3 : left ventricular ejection fraction by ct 60.0% ( echo 60% ) . ( d ) patient 4 : left ventricular ejection fraction by ct 38.3% ( echo 35% ) . 3d = three - dimensional , echo = echocardiography , ct = computed tomography two - dimensional echo examination was performed either 1 week before or after coronary cta in either of two ultrasound units , an acuson sequoia ( siemens medical systems usa , mountain view , ca ) or a ge vivid 3 ( ge healthcare , milwaukee , wi ) . images were obtained using a 3.5 mhz transducer and images were acquired in standard apical and parasternal two- and four - chamber views . the chamber and wall dimensions were measured using standard recommendations for chamber quantification in consensus . results on continuous measurements are presented on mean sd ( min max ) and results on categorical measurements are presented in number ( % ) . the mean standard deviation ( sd ) lvef calculated by clinical echo similarly mean sd of lvef calculated by fully automated software based on cta data was obtained [ table 2 ] . agreement for lvef was determined by the use of pearson 's regression analysis [ figure 4 ] and calculating correlation coefficient ( r ) . bland - altman analysis [ figure 5 ] was used to compare the lvef measured with mdct and that with 2d - tte . mountain plot [ figure 6 ] was used to see the relationship between two groups . echo - ejection fraction ( ef ) mean standard deviation ( sd ) : 58.67 4.53 , echo = echocardiography ct - ejection fraction ( ef ) mean standard deviation ( sd ) : 58.93 5.43 , ct = computed tomography pearson regression analysis between echo - ef and ct - ef . r = pearson correlation coefficient , p = p - value , ef = ejection fraction bland and altman showing the correlation of echo - ef and ct - ef sd = standard deviation mountain plot showing the correlation of echo - ef and ct - ef . in our study , 86 ( 76.1% ) were males and 27 ( 23.9% ) were females . the mean age of the patients was 51.19 10.10 years and majority of subjects belonged to age group between 51 and 60 years . 28.3% were between 41 and 50 years , 15.9% subjects were between 61 and 70 years , and 12.4% subjects between 31 and 40 years . most of the patients had at least one symptom , the commonest being chest pain in 88 ( 77.9% ) cases . the most common coronary risk factor association was hypertension , accounting for 77.9% of the cases . the mean heart rate of the patients at the time of scan was 61.5 8.6 bpm with maximum patients having a heart rate range of 61 - 70 bpm . the mean lvef calculated by clinical echo was 58.67 4.53% with maximum number of patients having an ef range of 56 - 60% . the mean lvef calculated by fully automated software based on cta data was 58.93 5.43% with maximum number of patients having an ef range of 61 - 65% . in our study using the fully automated software , the pearson 's regression analysis showed a good interstudy correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) . bland - altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo ; however , this observation was not statistically significant . the mountain plot analysis reinforced that ef measured by ct correlated well with that measured by echo . mdct coronary angiography has emerged as a valuable technique for the evaluation of coronary artery disease in patients with low to intermediate pretest probability of ischemic heart disease . utilizing analytic software , gated volumetric ct data can be processed to provide quantitative functional analysis of the lv in patients with coronary artery disease . we were able to obtain satisfactory artifact free datasets from 113 consecutive subjects who underwent coronary cta . all of our patients were either known cases of coronary artery disease or had suggestive clinical symptoms . patients with pacemaker , ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool was not possible . achieving a stable heart rate for the examination was variable component of the examination . however , no examination had to be postponed or cancelled due to this limitation . our study using automated software showed a good interstudy correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) . the bland - altman plot revealed a slight mean difference between ef measurements on ct and echo with most differences falling within two sds of the mean . hence , we found that software is user - friendly and capable of providing good reproducibility for ef measurements in comparison with echo . in a previous study by krishnam et al . , similar results were recorded , though the number of subjects was small . in a study by cury et al . , a trend of mdct slightly underestimating lvef compared with tte was observed . we observed a trend towards mdct resulting in slightly higher values for lvef when compared with echo , contrary to expected mild reduction in beta blocked patients . trend however was not statistically significant , could be due to recognized limitation of evaluation technique leading to over or underestimation . mean difference in ef measurements between cta and echo is small ; although standard deviation of the mean difference is quite high , leading to wide limits of agreement . possibly observation results from the fact that the ef measurements from echo were obtained in a clinical setting , on visual estimation and calculation of ef using simpson 's method based on geometrical assumptions . our observations are in accordance with the previous studies of 64-slice coronary ct , confirming that lvef estimation is feasible with the mdct data and may be regarded as a useful clinical index , correlating with results of echo . there are studies with semiautomated software for quantitative functional analysis of lv with user defined mitral valve plane and an arbitrary point within the lv , with the option to expand or reduce the area of segmentation . many earlier studies have compared the use of 4- , 8- , and 16-slice ct scanners for evaluation of lv volumes . larger detector configuration in 64-slice ct scanner , has the advantage of being faster , capable of smaller slice thickness and higher temporal resolution . relatively higher radiation dose results from the protocol optimized for thin slice high - resolution imaging of the coronary arteries . the ecg - dependent tube current modulation is currently the most effective tool for dose reduction and may reduce patient dose by up to 50% . it is important to note that two points of the cardiac cycle ( end - systole and end - diastole ) with modulation of tube current were not used in our study because ecg - gated dose modulation was only applicable to 50 - 90% of the rr interval on ecg . if the aim is to evaluate coronary arteries only , it is recommended to use an ecg - dependent dose modulation technique or newer prospective gated techniques . new developments in mdct technology is allowing examination of patients with higher heart rates and reducing the dose of beta - blockers . presently , mdct examination is possible which contains all the phases with a considerably lower dose of the order of 2 - 3.3 msv . the software identifies the lv blood pool based on hounsfield unit values and continuity of adjacent voxels . in patients with a ventricular septal defect , there is contrast opacification in both ventricles and a bridge of contrast through the septal defect . therefore , the software identifies the right and lv blood pools as a single chamber , resulting in incorrect segmentation and thus inaccurate assessment of lv functional parameters . in patient with pacemakers , software identifies a pacemaker wire as high - density contrast and segments the pacing wire as part of the ventricle , resulting in failed segmentation . the version of the software used for this study it was not able to segment the myocardium in order to quantify the lv myocardial mass , which may be important in certain cardiac diseases such as hypertrophic cardiomyopathy . it is important to note that cardiac mri is considered as a gold standard for lv function assessment . cardiac mri ( cmri ) provides excellent temporal and spatial resolution , image acquisition in any desired plane , and a high degree of accuracy and reproducibility . concerning quantitative measurements cine mri technique is potentially the most comprehensive cardiac imaging modality available because of its excellent contrast between blood - filled ventricles and the surrounding myocardium . yamamuro et al . , have shown high linear correlation between ef measurements on ct and on mri . the temporal resolution of many available mdct is still considerably lower than that of echo and cine mri . this lower temporal resolution can make evaluation of isovolumetric ed and es phases of the cardiac cycle , and thus ef , less precise . there is considerable improvement in temporal resolution of mdct with improvement in gantry rotation , multiple and partial segment processing techniques . tr of 80 - 250 ms has been achieved in state of the art units . though it is short of fluoroscopic resolution , currently available options are more than sufficient for motion free systolic and diastolic cardiac imaging with heart rate below 100/min . additionally , the use of a b - blocker to reduce the heart rate to less than 65 bpm can influence the functional parameters that are to be measured . beta blocker do influence the lvef , lead to underestimation . overestimation or underestimation of the lv volume has been reported because of the different criteria for selecting the endocardial boundary or the inclusion / exclusion of papillary muscle . ideally estimation of the real - ef from all 20 phases is more precise ; however , this significantly increases effort and processing time . effect of beta - blocker has to be factored in interpretation of ef by mdct . introduction of new ct imaging methods , including dual source ct in clinical practice will overcome these two problems significantly owing to its two - fold increase in temporal resolution . also , newer options in mdct technology may partially obviate need for use of beta blockers . functional parameters derived from 2d - tte are compared with ct derived 3d volumetric data , which are not strictly comparable . our study design did not allow realistic comparison of mdct and echo lv volume data . it would be interesting to compare the same for assessment of accuracy of respective data . with evolution of speckle - tracking 3d echography and new low radiation , high tr scanning such studies are possible . the delay time between ct and echo and premedication with -blockers could have changed myocardial contraction and lv volumes as measured with the two methods . in the present set up , the number of patients referred to coronary angiography who will have volumetric data will be significantly smaller , limiting the application of this utility to a smaller offset of patients . the radiation issue will certainly be an important consideration to use this technique in the larger group of patients . emerging new applications of echography in the form of 2d and 3d speckle - tracking echo certainly will have a greater role to play in the future noninvasive assessment of the myocardial function . in conclusion our study confirms useful complementary functional information in coronary cta datasets , using fully automated analysis software for rapid assessment of lvef . it is irrational to utilize mdct alone to assess lv function in clinical patients , given the radiation exposure involved . however , additional clinically useful information from a clinically indicated coronary ct examination with a lowest possible radiation dose is invaluable in patients known or suspected of ischemic heart disease . validating consistency of results with mri will further lend support to the use of mdct derived results . going forward with changing trends in ccta imaging , it is conceivable that number of patients undergoing cta with retrospective gating will substantially be reduced , thus limiting the functionality to a small group of patients .

background : coronary computed tomography angiography ( ccta ) is a frequently performed examination for coronary artery disease . when performed with retrospective gating , there is an opportunity to derive functional parameters of left ventricle utilizing automated software . complementary information , if validated with established standards , will enhance the total value of study.objective:study evaluates the usefulness of fully automated software for the assessment of left ventricular ejection fraction ( lvef ) using 64-slice ccta data and to correlate ct results with echocardiography ( echo ) . role of ct derived lv function is reviewed in the light of emerging technologies and recent developments in multidetector ct ( mdct).materials and methods : a total of 113 patients referred for mdct ccta for evaluation of coronary artery disease . all patients were scanned on 64 slice ge - helical ct scanner and had an echo done within 1 week of the ct scan . retrospectively electrocardiogram ( ecg)-correlated image reconstruction was performed with the reconstruction at 10% r - r interval increment . axial image sets were analyzed with advanced workstation using a program - auto ejection fraction , circulation : ge medical solutions.results:the mean lvef calculated by clinical echo was 58.6 4.5% and by fully automated software based on cta data was 58.9 5.4% . the pearson 's regression analysis showed a large correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) . bland - altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo.conclusion:the fully automated software is simple , reliable , and user - friendly , and can provide rapid assessment of lv functional parameters with good reproducibility . despite of good correlation , fewer patients are likely to benefit , in future , from this function due to smaller number of patients undergoing ccta with retrospective gating .

moraxella catarrhalis is a gram - negative , aerobic diplococcus human mucosal pathogen which causes middle ear infections in infants and children [ 13 ] , and it is one of the three major causes of otitis media along with streptococcus pneumonia and haemophilus influenzae . although moraxella catarrhalis is frequently found as a commensal of the upper respiratory tract , recently it has emerged as a genuine pathogen and is now considered an important cause of upper respiratory tract infections in healthy children and elderly people , lower respiratory tract infections in adults with chronic obstructive pulmonary disease [ 1 , 5 ] , and hospital - acquired pneumonia . amikacin , cefixime , fosfomycin , cefuroxime , cotrimoxazole , doxycycline , and erythromycin resistant strains of moraxella catarrhalis were isolated and the widespread production of a -1actamase enzyme renders the bacterium resistant to the penicillin [ 79 ] . this has led to the search for new and effective therapeutic alternatives among natural compounds . plants remain an important source of diverse chemical entities which have been used as drugs or provide scaffolds from which new drugs have been derived . the selection of a suitable candidate species for investigations can be done on the basis of long - term use by humans . this approach is based on an assumption that the active compounds isolated from such plants are likely to be safer than those derived from plant species with no history of human use . aristolochia is an important genus in the family of aristolochiaceae and is widespread across tropical asia , africa , and south america . it is used in traditional medicine as a gastric stimulant and in the treatment of cancer , lung inflammation , dysentery , and snakebites . it is also used in the treatment of tumors and malaria and for fevers , but its usage as an antimalarial is not recommended in its crude form . aristolochia bracteolata showed a definite positive effect on wound healing , with significant increase in the level of powerful antioxidant enzymes . the whole plant was used as a purgative , antipyretic , and anti - inflammatory . organic solvent extracts of the plant showed antibacterial activities while the water extract showed antifungal activity . although aristolochia has been used for thousands of years in many cultures for many indications due to its various pharmacological activities , it was later discovered that consuming these plants can certainly be dangerous . the genus of aristolochia contains a naturally carcinogenic compound aa which has been shown to be the cause of so - called chinese herb nephropathy or aa nephropathy [ 19 , 20 ] , and mutations in the cells of people who consume it , causes more mutations than two of the best - known environmental carcinogens : tobacco smoke and uv light [ 21 , 22 ] . there are many cases of nephropathy reported in the literature caused by the systemic and long term application of chinese snakeroot ( aristolochia fangchi ) ; this highlighted the risk of using preparations which contain aristolochic acids . although aristolochia is being known in many countries that is containing a toxic compound aa , but this has not stopped it from being a popular herbal remedy for thousands of years . it is still extensively used in india and in traditional chinese medicine for slimming , menstrual symptoms , and rheumatism . it is also widespread used in sudan and other african countries as one of the most effective herbal remedies for infectious diseases . therefore , it was our objective to assess the potential antimicrobial activity of aristolochia bracteolata using a bioassay - guided fractionation , in order to produce pure compound that can act as the lead compound in developing new , safe , and effective drug to replace the use of the harmful crude plant material . sephadex lh-20 ( pharmacia fine chemical co. ltd ) was used for column chromatography . precoated silica gel plates ( merck , kieselgel 60 f254 , 0.25 mm ) and precoated rp-18 f254s plates ( merck ) were used for thin - layer chromatography ( tlc ) analysis . high resolution fab - ms and esi - ms were recorded on jeol jms700n and jms-100td , respectively . h- and c - nmr , h - h cosy , noesy , hsqc , and hmbc spectra were recorded with a unity plus 500 spectrometer ( varian inc . , u.s.a . ) operating at 500 mhz for h and 125 mhz for c , respectively . h - nmr chemicals shifts are expressed in values referring to the solvent peak h 2.49 for dmso and coupling constants are expressed in hz . c - nmr chemical shifts are expressed in values referring to the solvent peak c 39.5 for dmso . piperonylic acid was purchased from commercial sources ( tci ) and used without further purification . the plant material ( whole ) was collected in the period from ( october to december 2012 ) from khartoum state in sudan . standard strains : moraxella catarrhalis ( gtc 01544 ) , klebsiella pneumoniae ( atcc 13883 ) , escherichia coli ( k12 ) , salmonella typhimurium ( atcc 14028 ) , streptococcus pyogenes ( atcc 19615 ) , streptococcus agalactiae ( atcc 13813 ) , staphylococcus epidermidis ( atcc 12228 ) , neisseria lactamicus ( atcc 23970 ) , enterobacter cloacae , ( atcc 23355 ) , bacillus subtilis ( atcc 6633 ) , staphylococcus aureus ( 209p ) , and pseudomonas aeruginosa ( ifo 3445).clinical strains : moraxella catarrhalis , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica . standard strains : moraxella catarrhalis ( gtc 01544 ) , klebsiella pneumoniae ( atcc 13883 ) , escherichia coli ( k12 ) , salmonella typhimurium ( atcc 14028 ) , streptococcus pyogenes ( atcc 19615 ) , streptococcus agalactiae ( atcc 13813 ) , staphylococcus epidermidis ( atcc 12228 ) , neisseria lactamicus ( atcc 23970 ) , enterobacter cloacae , ( atcc 23355 ) , bacillus subtilis ( atcc 6633 ) , staphylococcus aureus ( 209p ) , and pseudomonas aeruginosa ( ifo 3445 ) . clinical strains : moraxella catarrhalis , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica . in addition to a sea urchin ( anthocidaris crassispina ) derived bacillus sp . which obtained from the laboratory in medical plants garden , nagasaki university.(c)fungal strains : the fungal strains used were aspergillus niger ( nbrc 33023 ) , penicillium crustosum ( nbrc 33015 ) , schizophyllum commune ( nbrc 30749 ) , trichophyton concentricum ( nbrc 31068 ) , and candida albicans ( nbrc 10108 ) . fungal strains : the fungal strains used were aspergillus niger ( nbrc 33023 ) , penicillium crustosum ( nbrc 33015 ) , schizophyllum commune ( nbrc 30749 ) , trichophyton concentricum ( nbrc 31068 ) , and candida albicans ( nbrc 10108 ) . the air - dried powdered whole plant ( 200 g ) was exhaustively extracted with cold maceration method with sufficient quantity of 70% methanol for 7 days at room temperature . the methanolic extract was passed through whatman number 1 filter paper ( whatman england ) and the concentrated extract ( 40 g ) was digested with 100 ml distilled water and successively partitioned with n - hexane ( 4 400 ml ) , chloroform ( 3 400 ml ) , ethyl acetate ( 5 400 ml ) , and n - butanol ( 2 400 ml ) . each fraction was concentrated under reduced pressure to a constant weight to give the corresponding n - hexane fraction ( 0.4 g ) , chloroform fraction ( 2 g ) , ethyl acetate fraction ( 0.7 g ) , n - butanol fraction ( 6 g ) , and aqueous fraction ( 30 g ) . the most active fraction against bacillus sp . and m. catarrhalis ( chloroform fraction ) was subjected to sephadex lh20 column chromatography to give three subfractions ( a - c ) . fraction ( b ) was resubjected again to sephadex lh20 to afford very active and pure compound aa-1 ( 150 mg ) . to the solution of aa-1 ( 50 mg ; 0.23 mmol ) in dimethylformamide ( dmf ) , 1 ml potassium carbonate was added ( 95 mg ; 0.69 mmol ) . to the resulting suspension iodomethane was added ( 72 l ; 1.15 mmol ) and stirred for 8 hours . the reaction mixture was poured onto water ( 10 ml ) and ethyl acetate ( 20 ml ) . the organic layer was washed with 1 m hcl ( 10 ml ) three times and then with brine ( 10 ml ) once . after removal of solvent under reduced pressure , the residue was purified by silica - gel chromatography ( chloroform - methanol ) to afford the titled ester ( 88% ) . h - nmr ( 400 mhz , cdcl3 , tms , r.t . ) ( ppm ) : 3.88 ( 3h , s ) , 4.06 ( 3hs ) , 6.38 ( 2h , s ) , 7.11 ( 1h , d , j = 7.8 hz ) , 7.72 ( 1h , dd , j = 7.8 hz , 8.0 hz ) , 7.77 ( 1h , s ) , 8.70 ( 1h , d , j = 8.0 hz ) , and 8.83 ( 1h , s ) . suspension of the tested bacteria ( 100 l of 10 cfu / ml ) was spread onto solid media plates . the sterile paper discs ( 6 mm in diameter ) which were impregnated with the plant extract ( 14 mg ) and pure compound ( 10100 g ) were placed aseptically over the bacterial culture on nutrient agar plates . after incubation at 37c for 24 hours , the zone of inhibition around the discs was measured by millimeter scale . sterile , blank paper discs impregnated with only sterile solvents served as negative control each time . the sterile paper discs ( 6 mm in diameter ) which were impregnated with individual extract were placed on the inoculated plates . these plates were incubated for 2472 h at 2528c and the growth was evaluated visually by comparing a particular plate with the negative control plates ( having only test fungi ) . the antifungal activity was evaluated by measuring the inhibition zone diameter ( in millimeter ) observed . the mic and mbc values were determined by broth dilution method in accordance with clsi methodology . bacterial strains were cultured for 24 h at 37c on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5 1.0 10 cfu / ml . dilutions ranging from 1.56 to 400 g / ml of the compound were prepared in tubes including broth and dmso 10% ( v / v ) , in addition to one negative control ( broth + dmso 10% v / v + test microorganism ) to ensure that the final concentration of dmso in the assays did not interfere with the bacterial growth and one sterility control ( broth + dmso 10% v / v + test compound ) . a 100 l suspension of test microorganism was added to individual tubes and incubated at 37c for 24 h. the mic of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the mbc was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic - free plates . initial steps in new drug discovery involve identification of new chemical entities , which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation . bioassay - guided fractionation of the identified plant may lead to standardized extract or isolated bioactive lead compounds as the new drug . the whole plant of aristolochia bracteolata was extracted successively with meoh and subjected to liquid - liquid fractionation with n - hexane , chloroform , ethyl acetate , and n - butanol . the resulting fractions were tested for antibacterial and antifungal activities . the crude extract and chloroform fraction were significantly active against sea urchin - derived bacillus sp . and both standard strain and clinical isolates of moraxella catarrhalis and were moderately active against s. aureus , b. subtilis , and ps . the n - hexane fraction had moderate activity against s. aureus and b. subtilis while ethyl acetate fraction showed moderate activity against ps . the crude extract failed to inhibit the growth of all test fungi in addition to the following bacterial strains : klebsiella pneumoniae , escherichia coli , salmonella typhimurium , streptococcus pyogenes , streptococcus agalactiae , staphylococcus epidermidis , neisseria lactamicus , enterobacter cloacae , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica . the chloroform soluble fraction was therefore selected for further chromatographic separations and resulted in the isolation of known compound aa-1 ( figure 1 ) . aa-1 showed strong activity against moraxella catarrhalis ( standard strain and clinical isolates ) and sea urchin - derived bacillus sp . ( table 2 ) , with equal mic and mbc values of 25 and 50 g / ml . both the piperonylic acid moiety of aa-1 ( figure 1 ) and aa-1-methyl ester showed no activity against bacteria ( table 2 ) , which suggests that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity . bioguided fractionation of methanolic extract of aristolochia bracteolata led to isolation of aa-1 and its structure was elucidated by interpretation of its nmr and ms data and by comparison with those reported in the literature . electrospray ionization mass spectrometry ( esims ) showed pseudomolecular ion signal at m / z 364.03 [ m + na ] and high resolution fast atom bombardment mass spectrometry ( hr - fabms ) afforded m+1 ion signal at m / z 342.0620 which was corresponding to the molecular formula c17h12no7 ( calculated for 342.06138 ) . h nmr and c - nmr spectra were matched with those of aa-1 which were previously reported . two weak signals which did not show any correlation with proton in hsqc and hmbc spectroscopy were considered as quaternary carbons at positions 5 ( 124.3 ppm ) and 6 ( 143.5 ppm ) , respectively . laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganisms in vitro . more of these compounds should be subjected to animal and human studies to determine their effectiveness in whole - organism systems , including in particular toxicity studies and an examination of their effects on beneficial normal microbiota . in spite of the fact that herbal remedy is a mixture of many chemicals in unknown doses and might result in unpleasant side effects , many people believe that treatments that are natural are somehow magically safe and effective . aristolochia is used in traditional medicine for the treatment of various diseases [ 13 , 15 ] , including those associated with bacteria . this study showed clearly that the excellent effect of aristolochia in treating such diseases is due to the toxic compound aa-1 . although aa-1 is highly effective in killing m. catarrhalis , it is ineffective against the other microorganisms tested . this highlights the importance of m. catarrhalis in discovering the cellular target of aa-1 and the mechanism of aa-1 toxicity . the widespread use of aristolochia is not sufficient to ensure that it is effective or even that it is safe . therefore , hit - to - lead exploration is necessary to identify related compounds with low toxicity , low cost , and improved potency that can replace the use of the harmful crude plant material . it is impossible to ban the use of these remedies , especially in the rural areas in sudan and other african countries ; therefore , we strongly recommend educating the public of the risks versus the benefits of aristolochia and gradually replacing them with either economical new drugs or standardized extracts and homogenous batches of other plant material with known levels of safe active constituents . using bioassay - guided fractionation technique , the present study directly linked the antibacterial activity of aristolochia bracteolata to the aa-1 . although aa-1 had strong activity against m. catarrhalis , it had a narrow spectrum of activity than expected based on the activity of the crude extract from which it was isolated or from its traditional usage . this may be the result of synergism between different compounds in the complex extracts or may be due to placebo effect .

a bioassay - guided fractionation of methanol extract of aristolochia bracteolata whole plant was carried out in order to evaluate its antimicrobial activity and to identify the active compounds in this extract . antibacterial and antifungal activities of methanol extract against gram - positive , gram - negative , and fungal strains were investigated by the agar disk diffusion method . among the strains tested , moraxella catarrhalis and sea urchin - derived bacillus sp . showed the highest sensitivity towards the methanol extract and hence they are used as test organisms for the bioassay - guided fractionation . from this extract , aristolochic acid 1 ( aa-1 ) has been isolated and has showed the greatest antibacterial activity against both standard strain and clinical isolates of moraxella catarrhalis with equal minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) values of 25 and 50 g / ml . modification of the aa-1 to aa-1 methyl ester completely abolished the antibacterial activity of the compound and the piperonylic acid moiety of aa-1 which suggested that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity .

anti - neutrophil cytoplasmic antibodies ( ancas ) correlate well with a wide spectrum of vasculitis manifestations , including wegener 's granulomatosis , microscopic polyangiitis , and churg - strauss syndrome , all of which are commonly referred to as anca - associated vasculitis ( 1 ) . several infections , particularly infective endocarditis , have been reported to show positive findings on anca tests ; furthermore , their clinical features have been acknowledged to mimic anca - associated vasculitis , which may lead to a misdiagnosis and inappropriate treatment ( 2,3 ) . however , to our knowledge , infected aortic aneurysms have not been reported to show positive findings on anca tests . we herein report a patient with an infected thoracic aortic aneurysm mimicking anti - proteinase 3-antineutrophil cytoplasmic antibody ( pr3-anca)-associated vasculitis . vegetations in the descending aorta , which were detected using transesophageal echocardiography , contributed to the diagnosis in this case . a woman in her 60s was admitted to the nagoya city university hospital complaining of a fever , which had persisted for three weeks , along with a loss of appetite , proteinuria , hematuria , and renal dysfunction . her renal function test values had been in the normal range three months prior to the admission . she also had a history of orbital mucosa - associated lymphoid tissue lymphoma , which was in remission after treatment with only radiation therapy . a physical examination at the time of admission revealed a body temperature of 38.1 , pulse rate 109 beats / min , and blood pressure 104/53 mmhg . the laboratory tests revealed a white blood cell count of 18,900/mm , hemoglobin 6.6 g / dl , blood urea nitrogen 30.8 mg / dl , serum creatinine 2.24 mg / dl , and c - reactive protein ( crp ) 9.44 mg / dl ( table ) . the pr3-anca titer was 47.4 iu / ml ( normal range < 10 iu / ml ) , although her myeloperoxidase - antineutrophil cytoplasmic antibody ( mpo - anca ) titer was not elevated . a urinalysis gave results of 2 + for protein and 3 + for occult blood in a dipstick examination , and a microscopic examination detected more than 100 red blood cells per high - power field . alt : alanine aminotransferase , ana : antinuclear antibodies , ast : aspartate transaminase , c3 : compliment 3 , c4 : compliment 4 , ch50 : 50% hemolytic unit of complement , crp : c - reactive protein , dna : deoxyribonucleic acid , ldh : lactate dehydrogenase , mpo - anca : myeloperoxidase - antineutrophil cytoplasmic antibody , rf : rheumatic factor , pr3-anca : anti - proteinase 3-antineutrophil cytoplasmic antibody she was initially diagnosed with pr3-anca - associated vasculitis and was thereafter treated with methylprednisolone pulse therapy ( 500 mg / day ) for three days ( fig . however , streptococcus sanguis was serially detected in blood cultures ; therefore , the administration of steroids was stopped . transesophageal echocardiography also revealed no vegetation in the heart ; however , it showed many oscillating masses attached to the intima in the descending aorta ( fig . 2 ) . contrast - enhanced computed tomography ( ct ) revealed a new descending aortic aneurysm ( fig . effects of antibiotics on fever and c - reactive protein during hospitalization . the patient finally became afebrile and has c - reactive protein levels within the normal range after receiving antibiotics . crp : c - reactive protein , dap : daptomycin , pcg : penicillin g , taz / pipc : tazobactam / piperacillin , tx : therapy transesophageal echocardiographic images of the descending aorta , with sectioning planes at 90 ( a ) and 0 ( b ) . many oscillating masses attached to the intima were seen in the lumen of the descending aorta . contrast - enhanced computed tomographic images in the axial ( a ) and frontal ( b ) sections . these images show a descending aortic pseudoaneurysm and a contrast defect ( arrow ) beside the aneurysm that is compatible with echo - documented vegetation . based on the results of microbiological testing , antibiotic treatment with intravenous penicillin g ( 24,000,000 unit / day ) was started , after which she became afebrile . we added intravenous daptomycin ( 300 mg every 24 hours ) and tazobactam / piperacillin ( 2.25 g every 6 hours ) to the penicillin g because recurrent fever was observed two weeks later . this additional antibiotic treatment made her afebrile again , and her renal function was recovered ( fig . , she developed a janeway lesion , which was confirmed by the pathological finding of a skin biopsy specimen ( fig . 4 ) . transesophageal echocardiography finally demonstrated that the vegetation had disappeared after the antibiotic treatment ( fig . 5 ) , but contrast - enhanced ct revealed that the aneurysm remained unchanged in form . a nontender hemorrhagic macular on the sole of the foot ( a ) . photomicrograph of the macular lesion shows microembolization with fibrin and infiltration of neutrophilic cells hematoxylin and eosin staining ( b , c ) . a transesophageal echocardiographic image of the descending aorta after the antibiotic treatment , with the sectioning plane at 90. many oscillating masses have disappeared . on the 54th hospital day , she underwent endovascular aortic repair ( evar ) of the aneurysm ( fig . 6 ) after confirmation that her crp level was in the normal range and once blood cultures were consistently negative . one year after the procedure , she continues to take oral antibiotics ; no complications related to stent - graft deployment or recurrent infections have been encountered . her pr3-anca titer has normalized . a contrast - enhanced computed tomographic image after endovascular stent graft repair . in this case , an infected aortic aneurysm exhibited elevated the patient 's pr3-anca level , and its clinical course mimicked pr3-anca - associated vasculitis . the detection of ancas is highly specific for a diagnosis of anca - associated vasculitis ( 4 ) ; however , a number of infections can result in a positive anca test . there are several reports of infective endocarditis with the presence of ancas that mimic the clinical manifestations of an anca - associated vasculitis such as glomerulonephritis ( 2,3 ) . in the present case , based on the finding of positive blood cultures , we suspected that the patient was experiencing not anca - associated vasculitis but infective endocarditis . however , no vegetations were detected in the cardiac chambers using transthoracic echocardiography or transesophageal echocardiography . instead , contrast - enhanced ct revealed a pseudoaneurysm in the descending aorta , which suggested that the patient had an infected aneurysm . her renal dysfunction and elevated pr3-anca levels normalized with clinical resolution of the infected aneurysm after the administration of appropriate antibiotic therapy and evar . the patient has remained free of any evidence of systemic vasculitis during follow - up . the presence of pr3-anca may have been falsely positive in the present patient . to our knowledge , this is the first report of an infected aortic aneurysm with a positive pr3-anca test and clinical features mimicking anca - associated vasculitis . the presence of vegetations in the heart is one of the most characteristic findings of infective endocarditis . these vegetations are composed of bacteria , platelets , and inflammatory cells interspersed with fibrin mesh , either on damaged endocardium , including native cardiac valves , or prosthetic valves ( 5 ) . in the present case , because these masses disappeared after antibiotic treatment , they were considered to be vegetations . a pathological examination of the macular hemorrhages on the patient 's sole indicated not a cholesterol embolization but rather a janeway lesion , which was the consequence of septic embolic events . we therefore suspected that these masses were not mobile plaques but bacterial vegetations . to our knowledge , few cases of infected native aortic aneurysm manifesting vegetations in the aneurysm have been reported ( 6 - 8 ) . several mechanisms have been proposed regarding how ancas are formed during the course of infection , such as autoantigen complementarity , molecular mimicry , epigenetics , neutrophil extracellular traps , and microbial - sensing proteins called toll - like receptors ( 9 ) . there may be a great degree of overlap in the clinical and laboratory manifestations between anca - associated vasculitis and anca - positive infected aortic aneurysm , potentially leading to a misdiagnosis . it is important to correctly diagnose these cases in order to avoid administering inappropriate therapy , such as immunosuppressive treatment , which might have life - threatening consequences in a patient with an infected aortic aneurysm . positive blood cultures and imaging findings , such as a pseudoaneurysm and/or vegetations in the aorta , suggest the presence of an infected aortic aneurysm . a combination of conventional surgical treatment with resection of the aneurysm , extensive local debridement , and revascularization by in situ reconstruction or extra - anatomic bypass may be the gold standard , but such a treatment strategy is accompanied by a high mortality and morbidity . in the present case , it was recently reported that evar of an infected aortic aneurysm is feasible and , for most patients , a durable treatment option ( 10 ) . given that the continuous administration of antibiotics for the infected aneurysm should prevent a recurrence of local infection , we continued using antibiotics until the infection was controlled and carefully monitored the shape of the pseudoaneurysm on contrast - enhanced ct . we were ready to perform an emergency surgical correction at any point during this patient 's treatment course . in conclusion , we herein reported an unusual case of infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation and with serological findings and clinical features mimicking pr3-anca - associated vasculitis .

we herein report an unusual case of an infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation with serological findings and clinical features mimicking anti - proteinase 3-antineutrophil cytoplasmic antibody - associated vasculitis . the positive blood cultures and imaging findings , including a pseudoaneurysm and vegetations in the aorta , suggested the presence of an infected aortic aneurysm . the patient was successfully treated with antibiotics and endovascular aortic repair . a precise diagnosis is crucial in order to avoid inappropriate therapy such as immunosuppressive treatment , which could result in life - threatening consequences in a patient with an infected aortic aneurysm .

endometriosis is a prevalent cause of infertility , pelvic pain , dysmenorrhea , and dyspareunia in reproductive - age women . its diagnosis is by inspection of the pelvis during laparoscopy . in women with pelvic pain and infertility , endometriosis mimics some autoimmune and malignant diseases , including familial occurrence and immunological abnormalities in b and t cells , increased angiogenesis , invasion of endometrial cells to adjacent organs ( e.g. , bladder and bowels ) , and need for repeat surgeries due to recurrence [ 1,57 ] . multiple mechanisms for etiology and improvement of endometriosis are suggested and the treatments are based on these unclear mechanisms . therefore , progestin , gnrh agonists and antagonists , and drugs related to lipid metabolism ( e.g. , simvastatin ) are used for treatment . it has been shown that inflammation is important in the pathogenesis of endometriosis , so endometriosis treatment should not be different from that of other inflammatory disorders . there may be a correlation between vitamin d levels and the risk of polycystic ovarian disease , endometriosis , breast and ovarian cancer , increased arterial stiffness in older patients , and myasthenia gravis [ 1218 ] . it has been found that vitamin d has a role in normal cellular growth regulation . vitamin d increases anti - inflammatory cytokines production and decreases pro - inflammatory cytokines [ 1922 ] . vitamin d induces apoptosis and suppression of angiogenesis in vitro and in vivo [ 2528 ] . although an indirect relationship between vitamin d and endometriosis has been reported in multiple studies , here has been no published randomized clinical trial on endometriosis and vitamin d treatment in women . we explored the relationship between vitamin d and endometriosis in a double - blind , randomized clinical trial looking at the effect of vitamin d supplementation on cessation of pain in proven endometriosis after laparoscopic diagnosis and treatment . this randomized , double - blind clinical trial was performed in a single tertiary university hospital from nov 2014 to feb 2016 . to find patients with endometriosis , we did laparoscopy for various indications , including ovarian cyst , infertility , pelvic pain , and dysmenorrhea . using laparoscopy , the surgeons diagnosed patients with endometriosis and tried to excise or ablate all diseased tissue . the day before laparoscopy , a data collection form was completed by a physician , including the reason for laparoscopy , and the severity of pelvic pain and dysmenorrhea were estimated using a visual analogue scale test ( vas test ) , with a score of 0 being no pain and a score 10 being the worst pain ever experienced . the laparoscopies were done by 2 gynecologic laparoscopic surgeons ; both were involved in each operation and they recorded the severity of endometriosis according to the revised american society for reproductive medicine ( asrm ) classification . in the patients with endometriosis in the second menses after laparoscopic diagnosis and treatment , the vas test was repeated . patients with vas scores of at least 3 for dysmenorrhea and/or pelvic pain were invited to participate in this clinical trial , and after consultation we asked them to sign the informed consent . the dyspareunia score was not a criterion for entering the study because some of the patients were not married and had not had intercourse . women aged 1540 years with proven endometriosis by laparoscopy and a vas test score of 3 or more for dysmenorrhea and/or pelvic pain at second menses after operative laparoscopy . patients with vitamin d treatment in the last 6 months prior to surgery ; patients with known systemic diseases ( e.g. , hypertension , diabetes , coronary , renal , and hepatic diseases ) ; patients with known malignancy ; patients with hormonal treatment , including oral contraceptive pills , in the last 6 months . after authorization by the university ethics committee , eligible patients were assigned by simple randomization to receive either vitamin d or placebo . in the vitamin d group ( d group ) , we prescribed oral vitamin d 50 000 iu / weekly for 12 weeks ( capsule d - vigel , vitamin d3 50 000 iu , daana pharma co. tabriz - iran ) and in the placebo group ( p group ) we prescribed 1 capsule of placebo ( daana pharma co. tabriz - iran ) weekly for 12 weeks . four weeks after the end of the intervention ( 24 weeks after surgical treatment ) , the vas test was repeated for the 2 groups . we used the ks test ( one - sample kolmogorov - smirnov test ) for normality of data distribution , levine s test for equality of variances and independent samples , and the t test for equality of means for comparing quantitative normal data between the 2 groups . we used the paired - samples t test for comparing quantitative normal data between before and after treatment in each group and the pearson chi - square test for matching and comparing categorical variables between the 2 groups . due to the small sample size , we conducted mann - whitney non - parametric analysis between the 2 groups and wilcoxon signed ranks test for comparing before and after treatment data in each group . this study was funded and supported by iran university of medical sciences ( iums ) , grant no . women aged 1540 years with proven endometriosis by laparoscopy and a vas test score of 3 or more for dysmenorrhea and/or pelvic pain at second menses after operative laparoscopy . patients with vitamin d treatment in the last 6 months prior to surgery ; patients with known systemic diseases ( e.g. , hypertension , diabetes , coronary , renal , and hepatic diseases ) ; patients with known malignancy ; patients with hormonal treatment , including oral contraceptive pills , in the last 6 months . after authorization by the university ethics committee , eligible patients were assigned by simple randomization to receive either vitamin d or placebo . in the vitamin d group ( d group ) , we prescribed oral vitamin d 50 000 iu / weekly for 12 weeks ( capsule d - vigel , vitamin d3 50 000 iu , daana pharma co. tabriz - iran ) and in the placebo group ( p group ) we prescribed 1 capsule of placebo ( daana pharma co. tabriz - iran ) weekly for 12 weeks . four weeks after the end of the intervention ( 24 weeks after surgical treatment ) , the vas test was repeated for the 2 groups . we used the ks test ( one - sample kolmogorov - smirnov test ) for normality of data distribution , levine s test for equality of variances and independent samples , and the t test for equality of means for comparing quantitative normal data between the 2 groups . we used the paired - samples t test for comparing quantitative normal data between before and after treatment in each group and the pearson chi - square test for matching and comparing categorical variables between the 2 groups . due to the small sample size , we conducted mann - whitney non - parametric analysis between the 2 groups and wilcoxon signed ranks test for comparing before and after treatment data in each group . this study was funded and supported by iran university of medical sciences ( iums ) , grant no . we did 146 laparoscopies for different indications in gynecologic patients , and endometriosis was diagnosed in 75 . at the second menses after diagnostic and therapeutic laparoscopy , 40 cases met the inclusion criteria for our study . the remaining 39 cases were randomly assigned in vitamin d ( n=19 ) or placebo treatment ( n=20 ) groups . one patient in the placebo group became pregnant at the third month after the operation and discontinued placebo treatment . at the end of the study , we had 19 patients in each group ( figure 1 ) . table 1 shows a baseline comparison between the 2 study groups for general characteristics , reason for laparoscopy , severity of endometriosis , and severity of dysmenorrhea and pelvic pain . causes of laparoscopy in women with endometriosis diagnosed by laparoscopy were dysmenorrhea ( n=28 ) , ovarian cyst ( n=23 ) , chronic pelvic pain ( n=19 ) , and infertility ( n=9 ) . in some patients , there was more than 1 reason for laparoscopy . severity of endometriosis in 45% was moderate ( n=17 ) and 47% had severe endometriosis ( n=18 ) . before laparoscopy , the mean pelvic pain score in the vitamin d group was 4.053.45 and 4.824.1(p=0.513 ) in the placebo group . before laparoscopy , the mean dysmenorrhea pain score in the vitamin d group was 7.372.61 and in placebo group it was 6.423.04 ( p=0.325 ) . table 2 shows a comparison between the 2 groups for severity of pelvic pain and/or dysmenorrhea at different time points ( before laparoscopy , in second menses after laparoscopy , and at 24 weeks after laparoscopy ) . at the second menses after laparoscopy , there was no significant difference between the 2 groups for pelvic pain ( p=0.583 ) and dysmenorrhea ( p=0.365 ) , and at 24 weeks after laparoscopy there was no significant difference between mean pain scores in the 2 groups . mean pelvic pain at 24 weeks after laparoscopy in the vitamin d group was 0.841.74 and in placebo group it was 0.681.70 ( p=0.513 ) . mean dysmenorrhea was 2.102.33 in the vitamin d group and 2.732.84 in the placebo group ( p=0.45 ) . all of the patients had good cooperation for follow - up and continued their treatment until the end of the study . in this double - blind , randomized clinical trial , at 24 weeks after laparoscopic treatment of endometriosis there was no significant difference between effect of vitamin d3 ( cholecalciferol ) and placebo on severity of dysmenorrhea and/or pelvic pain . this is the first clinical trial on women with endometriosis to explore the possible relationship between vitamin d treatment and relief of endometriosis - related pain . our literature search found only studies that were indirectly related to vitamin d and endometriosis [ 2743 ] . the etiology of endometriosis is poorly understood , and many etiologic factors have been suggested , including the serum level of 1 , 25-dihydroxy vitamin d3 . 1 , 25-dihydroxy vitamin d3 is a fat - soluble vitamin with an unclear role in endometriosis . it is suggested that nuclear vitamin d receptors can have a regulatory role in inflammation and can be used as an index of cellular metabolic health . a study on vitamin d receptor gene polymorphism in endometriosis compared 132 infertile women with endometriosis with 132 fertile women , reporting no significant difference and suggesting that vitamin d receptor gene polymorphism does not play an important role in the pathogenesis of endometriosis . in some studies , higher plasma levels of 1 , 25-dihydroxy vitamin d3 and higher intake of dairy foods was associated with lower risk of endometriosis . conversely , another study compared serum vitamin d levels of 87 women with endometriosis with 53 women without endometriosis ; the mean serum levels of 1 , 25-dihydroxy vitamin d3 in women with and without endometriosis were 24.914.8 ng / ml and 20.411.8 , respectively ( p=0.05 ) and the study concluded that endometriosis is associated with higher serum levels of vitamin d . a systematic review of 10 case - control studies and 1 cohort study on women s diet found that women with endometriosis had lower consumption of vegetables and omega-3 , and reported a significant association between diet and endometriosis . vitamin d binding protein ( dbp ) is a plasma glycoprotein that modulates immune and inflammatory responses and also controls transport of vitamin d metabolites and bone development . in a study comparing 13 ectopic endometrial tissues and 6 normal endometrial tissues , vitamin d binding protein was significantly higher in the ectopic endometrial tissues ( p<0.05 ) . a systematic review of research from 1946 to 2013 on vitamin d and endometriosis reported that women with endometriosis had higher serum levels of vitamin d binding protein . another study compared serum and peritoneal levels of dbp in 26 women with endometriosis and 17 women with other benign gynecological conditions and reported that women with endometriosis had higher serum levels of dbp than in the control group . a study comparing urinary levels of dbp in 57 women with endometriosis with levels in 38 controls found that the urinary level of dbp was significantly higher in patients with endometriosis . a study using a rat model of endometriosis reported that treatment with vitamin d3 produced fibrosis and apoptosis in the stroma of tissues with endometriosis . a study on induced endometriosis in adult balb female mice reported that administration of 100 g / kg / day elocalcitol ( a vitamin d receptor agonist ) for 3 weeks reduced total lesion weight . because a relationship between vitamin d and endometriosis has been suggested by multiple studies , and since there has been no randomized clinical trial on endometriosis and vitamin d treatment in women , we decided to explore this relationship . in the present study on endometriosis - related pain , we found no significant difference in results of vitamin d treatment vs. placebo at 24 weeks after surgical diagnosis and treatment . vitamin d is a fat - soluble vitamin mainly produced in the body from food and supplements and cutaneous sun exposure . vitamin d deficiency is defined as serum 1.25-dihydroxy vitamin d3 levels under 20 ng / ml . it is reported in 52% of black and hispanic adolescents in boston and in 48% of girls in maine , and it is seen in 40100% of elderly men and women in the usa and europe . a study in germany found that 57% of people 1879 years old were vitamin d deficient . the prevalence of vitamin d deficiency was reported to be 90% in healthy subjects in delhi , india . in a systematic review of 195 studies in 44 countries found that 37.3% of studies found that the mean serum vitamin d levels were less than 20 ng / ml . the prevalence in pregnant turkish women was 81.4% . in a study of high school students in iran , the serum mean vitamin d level was 14.79.4 ng / ml . in another study in university students in shiraz , iran , 51.2% of female students had low serum levels of vitamin d . because the incidence of vitamin d deficiency in iran is high and we did not check the serum vitamin d levels in samples before intervention , it is possible that this dose and duration of vitamin d prescription was beneficial only for treatment of vitamin d deficiency and not endometriosis . in 1072 women attending an infertility center , an in vitro study compared the effect of vitamin d3 on 25 human endometriosis stromal cell cultures ( ovarian endometrioma ) with the effect of vitamin d3 on culture of 20 endometrial samples of non - endometriosis women ; vitamin d3 inhibited proliferation , invasion , and pro - inflammatory cytokine production in endometriosis and reduced production of interleukin 6 and other inflammatory cytokines that stimulate adhesion of endometrial cells to the peritoneal cavity . a study on in vitro effects of vitamin d3 on human endometriosis stromal cells found that vitamin d3 significantly reduced interleukin 1 and tumor necrotizing factor- inflammatory responses , and also reported fewer endometrial stromal cells and reduced dna synthesis . the study found significantly lower serum vitamin d3 levels in severe endometriosis compared to normal controls and patients with mild endometriosis . a study in italy investigated the effect of vitamin d on primary dysmenorrhea . the samples were 40 women aged 1840 years old with 4 consecutive painful periods in the past 6 months . they measured the serum levels of 25 hydroxy vitamin d with high - performance liquid chromatography . then the women were randomized into a group of 20 women who received a single oral dose of 300 000 iu vitamin d ( cholecalciferol ) at 5 days before their next menstrual cycle and another group of 20 women received placebo . there was a negative correlation between the baseline dysmenorrhea pain score and the level of 25 hydroxy vitamin d3 ( r=0.36 , p=0.2 ) . the researchers found a significant reduction of dysmenorrhea pain in the vitamin d group in comparison with the placebo group in the next 2 menstruations ( p<0.01 ) . they suggested this significant reduction of dysmenorrhea pain in vitamin d prescription group was due to decreased levels of pro - inflammatory cytokines and decreased the biological activity of prostaglandins . the samples were not assessed for existing endometriosis . because low levels of serum vitamin d are common in healthy italian pre - menopausal women , these results only show that vitamin d was effective in relieving dysmenorrhea at least for 2 months after treatment in women with or without endometriosis and also in women with or without vitamin d deficiency . there may be a relationship between vitamin d and pathogenesis of endometriosis , but in our study vitamin d was not effective in treatment of endometriosis - related pain . larger clinical trials are needed to determine the possible effects of vitamin d supplementation in endometriosis treatment . the first limitation of this study is the high prevalence of vitamin d deficiency in our country and worldwide , meaning that a high percentage of our samples may have had vitamin d deficiency , which may have affected the results of our research . further clinical trials are needed on the role of vitamin d treatment for endometriosis - related pain . future studies should assess the serum levels of vitamin d before enrolling study subjects , and those with vitamin d deficiency should be excluded . the first limitation of this study is the high prevalence of vitamin d deficiency in our country and worldwide , meaning that a high percentage of our samples may have had vitamin d deficiency , which may have affected the results of our research . further clinical trials are needed on the role of vitamin d treatment for endometriosis - related pain . future studies should assess the serum levels of vitamin d before enrolling study subjects , and those with vitamin d deficiency should be excluded .

backgroundendometriosis is a disabling disease of reproductive - age women . dysmenorrhea , dyspareunia , and pelvic pain are the main symptoms of endometriosis . its etiology is not clear . endometriosis may have various causes , including vitamin d deficiency , but its effect is controversial.material/methodsin this double - blind clinical trial , we enrolled patients with endometriosis diagnosed and treated by laparoscopy , with scores of at least 3 for of dysmenorrhea and/or pelvic pain at 8 weeks after surgical treatment . they were randomly prescribed vitamin d ( 50 000 iu weekly for 12 weeks ) or placebo . severity of pain in the 2 groups ( placebo and treatment ) was compared by vas test at 24 weeks after surgical treatment.resultsthere were 19 patients in the vitamin d group and 20 in the placebo group . baseline characteristics in the 2 groups were similar . following the treatment with vitamin d or placebo , we did not find significant differences in severity of pelvic pain ( p=0.24 ) and dysmenorrhea ( p=0.45 ) between the 2 groups . mean pelvic pain at 24 weeks after laparoscopy in the vitamin d group was 0.841.74 and in placebo group it was 0.681.70 ( p=0.513 ) . mean dysmenorrhea was 2.102.33 in the vitamin d group and 2.732.84 in the placebo group ( p=0.45).conclusionsafter ablative surgery for endometriosis , vitamin d treatment did not have a significant effect in reducing dysmenorrhea and/or pelvic pain .

it is generally accepted that microleakage between the filling materials and root canal walls might adversely affect the outcome of root canal treatment . therefore , it is critical the complete sealing of the root canal system after cleaning and shaping in order to avoid the bacterial penetration and re - infection of the root and periapical tissues . the association of gutta - percha cones and root canal sealer has been traditionally used for this purpose . however , in the last decade , the dentin adhesive technology has been incorporated into the root canal filling techniques to reduce apical and coronal leakage by bonding to root canal walls . etch - and - rinse adhesives have been tested with resin cements and the combination of a dentin - bonding agent and an epoxy resin - based root canal sealer significantly reduced apical leakage . in restorative dentistry , the self - etch adhesive systems have shown less technique sensitivity , with reliable long - term performance of a two - step mild self - etch adhesive . following this trend , pentron clinical technologies ( wallingford , ct , usa ) has developed the epiphany system , which contains a self - etch primer , a dual - cured composite resin sealer and a polyester - based thermoplastic root - filling material ( resilon ; resilon research llc , madison , ct , usa ) . according to shipper , et al . ( 2004 ) this material has been shown to be more resistant to bacterial leakage than epoxy resin - based sealers for filling root canals . in endodontics , the controversy about the performance of adhesive systems inside the root canal remains . despite that , other possibility is combining epiphany primer with ah plus in an attempt to add the hybridization capacity to the gold standard endodontic sealer . the reason for this is that the removal of the smear layer with ethylenediamine tetraacetic acid ( edta ) does not provide the same etching pattern usually associated with the hybrid layer , which is considered an important factor for dentin bonding . the majority of studies have evaluated apical or coronal microleakage , and few have focused gap formation at the dentin / sealer interface . so far , no correlation between microleakage and gap formation has been established in the literature . thus , it is reasonable to believe that the association of both methods would provide a more precise evaluation of the adhesive interface . the aim of this study was to compare the apical sealing and gap formation of ah plus / gutta - percha with epiphany system . in addition , the opportunity was taken to assess the effect of the association of epiphany primer and ah plus / gutta - percha on apical sealing and gap formation . the null hypotheses tested were as follows : ( 1 ) there is no difference in apical microleakage or ( 2 ) apical gap formation among the experimental groups and ( 3 ) there is no correspondence between apical microleakage and the presence of gaps . thirty - nine lower single - rooted human premolars with straight root canals and fully developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo , sp , brazil ) , and stored in saline solution at 4c . after endodontic access , the real working length ( rwl ) was established 1 mm short of the apical foramen . a crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5% naocl . the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5 ml ) followed by saline solution ( 15 ml ) . three coats of nail polish were applied to external root surfaces except for the apical 2 mm . the teeth were randomly ( http://www.random.org ) divided into 3 experimental groups ( n=11 ) . the endodontic sealers were prepared according to the manufacturer 's instructions and the cold lateral condensation filling technique ( lc ) was used according with the following description : an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah plus ; dentsply detrey , konstanz , germany ) and placed into the canal to rwl . a size b finger spreader ( dentsply maillefer , ballaigues , switzerland ) was then inserted into the canal to a level approximately 1 mm short of rwl . lc with accessory gutta - percha cones was performed until the entirely filled root canal . the excess gutta - percha was removed with a heated plugger and then compacted vertically . adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a paper point soaked with epiphany primer was used to etch dentin ( 30 s ) and the excess was removed with paper points . the coronal surface of the root filling was light - cured for 40 s ( 600 mw / cm ) . the positive controls ( n=3 ) were left unfilled and coated as described earlier . the negative controls ( n=3 ) were filled and totally coated , including the apical foramina . the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and stored in a chamber held at 100% humidity and 37c for 7 days . next , all teeth were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 ) in the darkness that was buffered using naoh 0.1 n for 24 h at 37c . the silver - impregnated teeth were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the silver ions into metallic silver . lake bluff , il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low speed ( 200 rpm ) with a water - cooled diamond blade . the interfaces were etched with a 35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently air - dried . then , specimen preparation followed the protocol 1 for apical microleakage analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ; or the protocol 2 for gaps analysis using sem according to the following description : specimens were fixed , dehydrated in ascending grades of ethanol and final chemical drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . , louis , mo , usa ) for 10 min , and covered with carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) . the apical 5 mm of the root canal filling were divided into 5 regions of 1 mm to evaluate microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . , cambridge , england ) using back scattered electrons ( bse ) mode ( figure 1a ) . the eds ( inca software , oxford , uk , england ) was performed in lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the identification of silver was made punctually to determine its exact location ( figure 1c ) . each 1 mm region was classified according to the following scores : 0 ( absence of leakage in both interfaces ) and 1 ( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e scanning electron microscopy ( sem ) micrographs using back scattered electrons ( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of agno3 leakage : ( a ) - the apical 5 mm of the root canal filling divided into 1 mm - regions . pointer at the detected metallic silver ; ( b ) area ( 300 m ) scanned for the existence of silver and respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation of its exact location . gp = gutta - percha ; d = dentin impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were made of the interfaces and the surfaces replicated with epoxy resin ( epon - thin , buhler ) . the replicas were covered with carbon to investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as described previously , using secondary electrons ( se ) mode . as the gaps were not continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present . therefore , for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and accordingly classified . scanning electron microscopy ( sem ) micrographs using secondary electrons ( se ) and back scattered electrons ( bse ) mode of the replica and the section of the same specimen , respectively . ( a ) - the apical 5 mm of the root canal filling divided into 1 mmregions . classification of the types of gaps : ( b ) type 1 : gap between dentin / sealer ( pointer ) ; air voids were present within the sealer ( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer interface , into dentinal tubules in a reticular form and granular aspect on the dentin surface ( arrowhead ) ; ( c ) type 2 : gap between sealer / cone ( pointer ) - cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure c at higher magnification : silver deposition into the dentinal tubules in a reticular form and granular aspect within the sealer layer ( arrowhead ) could be seen ; ( the sealer thickness was indicated between black / white arrowheads . gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software program ( minitab inc . the counting of regions analyzed for gaps was also computed and adjusted in a generalized linear model with poisson distribution and logarithmic link function . the sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered as explanatory variables and ah plus and the type 0 as references . thirty - nine lower single - rooted human premolars with straight root canals and fully developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo , sp , brazil ) , and stored in saline solution at 4c . after endodontic access , the real working length ( rwl ) was established 1 mm short of the apical foramen . a crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5% naocl . the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5 ml ) followed by saline solution ( 15 ml ) . three coats of nail polish were applied to external root surfaces except for the apical 2 mm . the teeth were randomly ( http://www.random.org ) divided into 3 experimental groups ( n=11 ) . the endodontic sealers were prepared according to the manufacturer 's instructions and the cold lateral condensation filling technique ( lc ) was used according with the following description : an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah plus ; dentsply detrey , konstanz , germany ) and placed into the canal to rwl . a size b finger spreader ( dentsply maillefer , ballaigues , switzerland ) was then inserted into the canal to a level approximately 1 mm short of rwl . lc with accessory gutta - percha cones was performed until the entirely filled root canal . the excess gutta - percha was removed with a heated plugger and then compacted vertically . adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a paper point soaked with epiphany primer was used to etch dentin ( 30 s ) and the excess was removed with paper points . the coronal surface of the root filling was light - cured for 40 s ( 600 mw / cm ) . the positive controls ( n=3 ) were left unfilled and coated as described earlier . the negative controls ( n=3 ) were filled and totally coated , including the apical foramina . the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and stored in a chamber held at 100% humidity and 37c for 7 days . next , all teeth were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 ) in the darkness that was buffered using naoh 0.1 n for 24 h at 37c . the silver - impregnated teeth were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the silver ions into metallic silver . lake bluff , il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low speed ( 200 rpm ) with a water - cooled diamond blade . the interfaces were etched with a 35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently air - dried . then , specimen preparation followed the protocol 1 for apical microleakage analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ; or the protocol 2 for gaps analysis using sem according to the following description : specimens were fixed , dehydrated in ascending grades of ethanol and final chemical drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . , st . louis , mo , usa ) for 10 min , and covered with carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) . the apical 5 mm of the root canal filling were divided into 5 regions of 1 mm to evaluate microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . , cambridge , england ) using back scattered electrons ( bse ) mode ( figure 1a ) . the eds ( inca software , oxford , uk , england ) was performed in lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the identification of silver was made punctually to determine its exact location ( figure 1c ) . each 1 mm region was classified according to the following scores : 0 ( absence of leakage in both interfaces ) and 1 ( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e scanning electron microscopy ( sem ) micrographs using back scattered electrons ( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of agno3 leakage : ( a ) - the apical 5 mm of the root canal filling divided into 1 mm - regions . pointer at the detected metallic silver ; ( b ) area ( 300 m ) scanned for the existence of silver and respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation of its exact location . impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were made of the interfaces and the surfaces replicated with epoxy resin ( epon - thin , buhler ) . the replicas were covered with carbon to investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as described previously , using secondary electrons ( se ) mode . as the gaps were not continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present . therefore , for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and accordingly classified . scanning electron microscopy ( sem ) micrographs using secondary electrons ( se ) and back scattered electrons ( bse ) mode of the replica and the section of the same specimen , respectively . ( a ) - the apical 5 mm of the root canal filling divided into 1 mmregions . classification of the types of gaps : ( b ) type 1 : gap between dentin / sealer ( pointer ) ; air voids were present within the sealer ( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer interface , into dentinal tubules in a reticular form and granular aspect on the dentin surface ( arrowhead ) ; ( c ) type 2 : gap between sealer / cone ( pointer ) - cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure c at higher magnification : silver deposition into the dentinal tubules in a reticular form and granular aspect within the sealer layer ( arrowhead ) could be seen ; ( the sealer thickness was indicated between black / white arrowheads . gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software program ( minitab inc . the counting of regions analyzed for gaps was also computed and adjusted in a generalized linear model with poisson distribution and logarithmic link function . the sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered as explanatory variables and ah plus and the type 0 as references . the ordinal logistic regression analysis demonstrated that there was no statistically significant difference ( p>0.05 ) among the groups tested and types of gaps over the logit of microleakage . the model was adjusted to identify the effect of the type of gap on microleakage . the type 3 data were not considered isolated in the analysis ( very few regions ) but joined with type 2 data ( figure 2c ; table 1 ) . the adjustment tests of the model presented high p - values for pearson 's test ( p=0.900 ) and deviance ( p=0.852 ) . the adjusted model demonstrated that the effect of the type of gap on the logit of microleakage is significant when compared to the presence of types 1 and 2 ( figure 2b and 2c , respectively ) of gap with the absence of gap ( p=0.047 ) . the correspondence analysis also showed the association between microleakage and both types of apical gaps ( figure 3 ) . correspondence analysis of microleakage vs. type of gap : ( inf.0 ) - absence of leakage ; ( inf.1 ) - leakage up to the 1 millimeter ; ( inf.2 ) leakage up to the 2 mm ; ( inf.3 ) leakage up to the 3 millimeter ; ( inf.5 ) leakage up to the 5 millimeter ; ( f.0 ) absence of gaps ; ( f.1 ) gaps type 1 ; ( f.2 ) - gaps types 1 and 2 . the absence of leakage is associated with the absence of gap ; leakage up to the 1 millimeter is associated with type 1 ; and more extensive leakage is associated with types 1 and 2 frequency and type of gaps in the apical 5 mm of root canal fillings percentage of regions with gaps . type 0 : absence of gaps ; type 1 : gaps between dentin and sealer ; type 2 gaps between sealer and cone the generalized linear model with poisson distribution ( table 1 ) found no difference between ah plus and ah primer ( p=0.497 ) in distribution of each type of gap , but they presented more type 0 regions ( p<0.001 ) . there was a significant interaction between epiphany and the type of gap ( p<0.003 ) and for this group , there were more type 1 regions . based on the findings of this study , the first null hypothesis could not be rejected . recently some studies have shown similar results between ah plus and epiphany on apical sealing ability using the fluid filtration method . consistent with these studies , our results indicate that the epiphany system is as effective as ah plus / gutta - percha in preventing microleakage based on the chemical tracer penetration analysis by sem and the use of epiphany primer does not reduce the microleakage of ah plus . nevertheless , other researches pointed out that the epiphany system provided the greatest resistance to the movement of fluids or dye leakage test , when compared to epoxy resin - based root canal sealers , but others have just indicated the opposite . these apparent discrepancies can perhaps be explained by the methodology used and their variables . these results agree with those of previous studies in which , microleakage markers were used to evaluate apical sealing of the same endodontic sealers . the tracer selected was agno3 , which may penetrate into dentinal tubules due to their physical and chemical characteristics including : concentration , smaller molecular size , its neutral ph , diffusion coefficient and service life up to 168 h post - preparation time of solution . another reason for this choice is that metallic silver deposits may be observed by sem using back scattered electrons ( bse ) mode . on the other hand , the results of the current study disagree with the findings of shipper , et al . an explanation for this would be that their studies have evaluated coronal and not apical bacterial leakage . indeed , coronal seal might be favorably influenced by photoactivation of the material which is unlikely to occur in the apical third . furthermore , it is speculated that in in vivo studies ( dogs ) , the high release of calcium hydroxide ( 41.46 mg / l ) that would occur during the process of epiphany sealer solubilization , would make the medium alkaline , resulting in acceleration of the periapical tissue repair process . this study has shown that the presence of apical gaps at the sealer / dentin or sealer / cone interfaces had an effect on apical microleakage . the epiphany group presented more type 1 gaps than the other groups ( table 1 ) , rejecting the second null hypothesis . these results corroborate with previous observation of gaps in the apical 4 mm of epiphany or ah plus - filled root canals . stress generated during the polymerization shrinkage of epiphany sealer has probably influenced the integrity loss at the sealer / dentin interface . in addition , the cavity configuration factor ( c - factor ) is highly unfavorable for adhesion inside root canals . moreover , it is known that analysis of gap formation of vertically sectioned root filled teeth hides the risk of artifacts during sectioning . therefore , in this study , the sections were made at low speed under water cooling . the resin - epoxy replicas at the dentin / sealer and sealer / cone interfaces were made before the specimens had been prepared for sem examination in order to differentiate genuine gaps from artifactual gaps created after vacuum desiccation in conventional scanning electron microscopes . few studies have focused on apical sealing from two separate perspectives , such as microleakage and apical gap formation . sem was used to evaluate these perspectives , but it was restricted to a descriptive approach . a systematization of the observations , complemented by a statistical analysis , such as the one performed in this study , could provide greater methodological accuracy and impartiality for comparing the experimental groups . the correspondence of analysis between the types of gaps and linear extent of agno3 leakage ( fig . 3 ) confirms the effect of gaps on apical microleakage , expressed by the silver deposition into gaps that extended deeply inside dentinal tubules ( fig . regarding to statistical analysis , the ordinal logistic regression analysis model was used due to microleakage was considered categorized ordinal response variable ( ranging from 0 - no leakage to 5 - leakage up to the fifth millimeter ) . the correspondence analysis is a method that leads to visualize the association between two categorical variables , in this case microleakage and the type of gap . the generalized linear model with poisson which is an extension of usual regression model was performed to quantify the distribution of types of gaps . this result could be explained by two hypotheses : ( 1 ) the low degree of conversion of the epiphany sealer and ( 2 ) the formation of hydrogel at the bond interface resulting from the incomplete evaporation of the epiphany primer solvent . the second hypothesis could also explain the inefficacy of epiphany primer in improving the sealing capacity of ah plus . moreover , there is a chemical incompatibility between these two materials , since the epoxy resin sealers do not copolymerize with methacrylate resin - based adhesives . other studies should be carried out to clarify the sealing ability of the root - filled materials studied . it may be concluded that none of the tested materials completely sealed the apical 5 mm . the presence of gaps had an effect on apical microleakage for all materials . comparing the materials , epiphany system presented more regions containing gaps between the dentin and the sealer ( type 1 ) . in view of these findings , clinically , it can be suggested that ah plus would provide a better apical seal . this study was partially supported by capes ( coordenao de aperfeioamento de pessoal de nvel superior)/institutional qualification program ( pqi n : 0090/03 - 4 ) .

objectivethe aim of this study was to compare the correspondence between gap formation and apical microleakage in root canals filled with epoxy resin - based ( ah plus ) combined or not with resinous primer or with a dimethacrylate - based root canal sealer ( epiphany ) . material and methodsthirty - nine lower single - rooted human premolars were filled by the lateral condensation technique ( lc ) and immersed in a 50-wt% aqueous silver nitrate solution at 37c ( 24 h ) . after longitudinal sectioning , epoxy resin replicas were made from the tooth specimens . both the replicas and the specimens were prepared for scanning electron microscopy ( sem ) . the gaps were observed in the replicas . apical microleakage was detected in the specimens by sem / energy dispersive spectroscopy ( sem / eds ) . the data were analyzed statistically using an ordinal logistic regression model and analysis of correspondence ( =0.05 ) . resultsepiphany presented more regions containing gaps between dentin and sealer ( p<0.05 ) . there was correspondence between the presence of gaps and microleakage ( p<0.05 ) . microleakage was similar among the root - filling materials ( p>0.05 ) . conclusionsthe resinous primer did not improve the sealing ability of ah plus sealer and the presence of gaps had an effect on apical microleakage for all materials .

neonatal diabetes mellitus ( ndm ) presenting within the first 6 months of life includes permanent neonatal diabetes mellitus ( pndm ) , which require lifelong therapy , and transient neonatal diabetes mellitus ( tndm ) where the condition shows remission during infancy but relapses in adolescence . almost all cases of neonatal diabetes have monogenic etiology in contrast to the autoimmune diabetes presenting in children beyond 6 months of age . pndm is associated with defects in pancreatic beta cell development and function . activating mutations in the kcnj11 gene , encoding the subunit kir6.2 , and abcc8 gene , encoding the sulfonylurea receptor 1 ( sur1 ) of atp - sensitive potassium ( katp ) channel , which has a key role in insulin secretion in glucose metabolism , are the most common causes and account for approximately 40% of all cases of pndm12 ) . mutations in the glucokinase ( gck ) and insulin ( ins ) genes have also been reported in patients with pndm . sulfonylureas close the katp channel by an atp - independent route , leading to increase insulin secretion4 ) . therefore , in many patients with pndm with kir6.2 and sur1 mutations , insulin therapy can be replaced by oral sulfonylureas which offer more improvement in glycemic control and better quality of life35 ) . we report a case of pndm caused by a novel p.h186d heterozygous mutation in the kcnj11 gene whose treatment was successfully transitioned from insulin to oral sulfonylurea . her parents were unrelated , and her family was healthy and had no history of diabetes . laboratory results at diagnosis were : blood glucose , 1,041 mg / dl ; ph , 7.025 ; hco3 , 5.1 mmol / l ; pco2 , 19.8 mmhg ; sodium , 147 mmol / l ; potassium , 5.6 mmol / l ; chloride , 113 mmol / l ; blood urea nitrogen , 31 mg / dl ; creatinine , 1.2 mg / dl ; urine ketone , + + + ; glycosylated hemoglobin ( hba1c ) , 7.7% ; and c - peptide , 0.2 ng / ml . after recovery from diabetic ketoacidosis , she had injections of neutral protamine hagedorn ( nph ) insulin two times a day and regular insulin ( ri ) four times a day . her maximal daily insulin dose was at 1.5 u / kg . her insulin requirement was gradually decreased to 0.44 u / kg / day by 4 months of age . although there were several hypoglycemic events , insulin injection for glucose control after the infancy period could not be stopped . genetic study for neonatal diabetes using genomic dna extracted from peripheral lymphocyte was done at 4.5 years of age , and a novel heterozygous mutation of the kcnj11 gene located on chromosome 11p15.1 and encoding kir6.2 , c.556c > g ( p.his186asp ) , was found ( fig . transition of treatment from insulin to sulfonylurea was attempted at the age of 4.8 . before the trial , laboratory findings were as follows : hba1c , 6.9% ; fasting glucose , 205 mg / dl ; c - peptide , 0.84 ng / ml ; 24-hour urine c - peptide , 2.2 g ; negative for islet cell antibody ; anti - insulin antibody ; and anti - gad antibody . she was 103.2 cm in height ( 25th-50th percentile ) , 17 kg in weight ( 50th-75th percentile ) and did not have any specific neurological deficit . oral sulfonylurea ( glibenclamide ) was initiated at a dose of 0.1 mg / kg / day divided into two equal doses . the glibenclamide dose was gradually increased to 0.4 mg / kg / day over 1 week and the insulin was stopped . oral glucose tolerance test ( glucose , 1.75 g / kg ) was done at 6 months and 1 year after completion of the sulfonylurea transition . insulinogenic index [ insulin ( 30 min-0 min)/glucose ( 30 min-0 min ) ] , a marker of beta cell function , became increased from 0.10 to 0.18 , and acute c - peptide response [ c - peptide ( 30 min-0 min)1,000/glucose ( 30 min-0 min ) ] became increased from 6.48 to 8.29 ( table 1 ) . the patient is now 7.5 years of age with a height of 121.4 cm ( 50th percentile ) and weight of 23.4 kg ( 50th percentile ) . blood glucose was well controlled without episodes of hypoglycemia and the hba1c has been lower than during insulin injection ( fig . ndm is a monogenic form of diabetes that presents within the first 6 months of life . heterozygous activating mutations in the kcnj11 and abcc8 gene encoding the subunits kir6.2 and sur1 of katp channel in pancreatic beta cells are the most common and account for nearly half of all cases of pndm12 ) . mutations in the glucokinase ( gck ) , insulin ( ins ) genes have also been reported in patients with pndm . the causes of rare syndromic pndm includes recessive mutations in several genes , such as pdx1 , eif2ak3 , gck , ptf1a , foxp3 , neurog3 , neurod1 , rfx6 , ier3ip1 , hnf1b , glis3 , pax6 , slc19a2 , slc2a2 , and wfs167 ) . in tndm , imprinted locus at chromosome 6q24 seen in more than half of cases8 ) , and kcnj11 and abcc8 gene mutations are also found in some cases . glucose increases intracellular atp level and it induces the closure of katp channels that lead to insulin secretion by pancreatic beta cells . activating mutations in the kcnj11 or abcc8 gene of katp channel lead to katp channels remaining open despite the presence of glucose , thus , insulin secretion can not be increased1 ) . on the contrary , loss of function mutations cause congenital hyperinsulinemia due to the closure of the katp channel and lead to increased insulin secretion . sulfonylureas ( su ) can close the katp channel and increase insulin secretion4 ) . treatment response is not expected in patients with mutations in other genes , such as glucokinase gene , foxp3 , and ipf1 . therefore , molecular diagnosis in neonatal diabetes may help identify patients that are likely to respond to oral sulfonylureas . pearson et al.3 ) reported the responses to sulfonylurea according to mutation types in diabetic patients caused by kcnj11 mutation . there were no differences in blood insulin , c - peptide level , and insulin injection dose between the successful group and unsuccessful group . v59 m , r201c , f35v , h46y , r50q , g53r , r201l , e322k , y330s , f333i mutations also belong to the successful group . switching to su was unsuccessful in patients with q52r , i296l , and l164p mutations . in addition , failure of switching to su was more related with the presence of neurologic features ( 14% in successful group and 80% in unsuccessful group ) and older age at initiation of su . median age was 6 years old ( intraquartile range , 3 - 12 years ) in the successful group and 18 years old ( intraquartile range , 6 - 35 years ) in the unsuccessful group3 ) . the report about a mother and daughter carrying the same kcnj11 mutation showed that the daughter could be switched from insulin to su at age 8.5 years , but her mother had an imcomplete response9 ) . the trial of su switching in a poorly controlled diabetic 19 years old patient with r201h mutation , a mutation type expected to respond successfully , failed10 ) . these observations suggest that factors that can affect the success of switching to su include ; mutation type , severity of mutation , and duration of the diabetes . a long - standing diabetes may lead to islet beta cell exhaustion resulting in nonresponse to su9 ) . ndm caused by kcnj11 gene mutations is inherited in autosomal dominant pattern , but most of these cases result from new mutations without family history . the patient in this case has a novel mutation ( p.h186d ) in the kcnj11 gene and presented with a mild form of pndm . switching to su with an initial dose 0.4 mg / kg / day was successful at 4.8 years of age . oral glucose tolerance test in index patient showed improvement in insulin secretion during the treatment at 6 months and 12 months after the use of su . although she had hypoglycemia every 2 or 3 weeks and experienced a few episodes of hypoglycemia with mental change during insulin treatment , there was no more hypoglycemia after changing to su treatment . oral sulfonylurea therapy is both safe and better than insulin for metabolic control . reported side effects of oral su are transitory diarrhea and tooth discoloration in a few patients311 ) . mean hba1c was 8.1% in patients with insulin injection and 6.4% in patients with su therapy312 ) . chronic su treatment seems to develop no beta cell desensitisation in pndm patients with kcnj11 mutation13 ) . early su therapy at disease onset can permit insulin hypersensitivity and maintained basal insulin secretion , then provide long - term remission in animal subset14 ) . allowing for the potential beneficial effect on neurodevelopmental outcome and glycemic control , empiric tiral of su before genetic testing in neonatal diabetes patients can be considered15 ) . in conclusion , as the mutation type , severity of mutation , and duration of the diabetes are the major factors affecting the success of switching to su , early genetic analysis and trial of sulfonylurea is important in the management of neonatal diabetes . further studies looking at the result of su treatment and long term follow in pndm patients are needed .

permanent neonatal diabetes mellitus refers to diabetes that occurs before the age of 6 months and persists through life . it is a rare disorder affecting one in 0.2 - 0.5 million live births . mutations in the gene kcnj11 , encoding the subunit kir6.2 , and abcc8 , encoding sur1 of the atp - sensitive potassium ( katp ) channel , are the most common causes of permanent neonatal diabetes mellitus . sulfonylureas close the katp channel and increase insulin secretion . kcnj11 and abcc8 mutations have important therapeutic implications because sulfonylurea therapy can be effective in treating patients with mutations in the potassium channel subunits . the mutation type , the presence of neurological features , and the duration of diabetes are known to be the major factors affecting the treatment outcome after switching to sulfonylurea therapy . more than 30 mutations in the kcnj11 gene have been identified . here , we present our experience with a patient carrying a novel p.h186d heterozygous mutation in the kcnj11 gene who was successfully treated with oral sulfonylurea .

radiocontrast - induced nephropathy ( rin ) can lead to acute renal failure ( arf ) , which may require dialysis therapy . arf increases treatment cost due to sepsis , hemorrhage , respiratory failure , and a long hospitalization.[13 ] rin is an important cause of hospital - acquired arf and is responsible for 12% of cases . renal medullary hypoxia and the direct toxic effects of iodinated contrast agents on renal tubules are possible mechanisms responsible for the pathophysiology of rin . identified specific risk factors for rin are current renal insufficiency , diabetes mellitus , and high contrast volume , dehydration , advanced age ( > 70 years ) , congestive heart failure ( chf ) , and nephrotoxic drug use ( angiotensin converting enzyme inhibitor and nonsteroidal antiinflammatory drugs ) . patients at risk for radiocontrast nephropathy are recommended to use nonionic iso - osmolar or nonionic low osmolar contrast agents . increases in serum creatinine levels are useful for detecting rin . in the majority of patients , plasma creatinine levels rise within the first 2448 h after administering the radiocontrast agent , reach a peak within 35 days , and return to normal after 13 weeks . recent studies have reported that urine levels of il-18 ( a pro - inflammatory cytokine ) , kidney injury molecule-1 , and neutrophil gelatinase - associated lipocalin ( ngal ) levels are important for early detection of rin . some reports have shown that il-18 levels start to increase within 46 h and peak at 12 h in patients with acute renal injury . in this study , we aimed to compare the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration . twenty patients ( 11 males and 9 females ) underwent diagnostic and therapeutic contrast - enhanced examinations at the department of internal medicine from january 2009 to march 2009 . the study was approved by the institute ethics committee and written consent was obtained from the selected patients based on a low mehran risk score ( 5 ) . patient demographic characteristics a precontrast - enhanced examination of serum blood urea nitrogen ( bun ) , creatinine , na , k , cl , ca , p , creatinine clearance was analyzed and they were repeated at 24 , 48 , and 72 h following contrast administration . spot urine il-18 levels were measured before and 6 and 24 h after radiocontrast administration with a human il-18 elisa kit ( biosource invitrogen human il-18 , california ) . intravenous iopromid ( 623 mg / ml , 1.5 ml / kg ; ultravist 300 ) , a three - way oral and rectal contrast material for abdominal ct scans , and 650 mg / ml meglumine diatrizoate ( urovist , 100 ml ) were used for every patient . glomerular filtration rate was calculated using the cockcroft 1 h before the procedure , 8.4% nahco3 plus 5% dextrose ( 3 ml / kg / h ) with 1200 mg / day n - acetylcysteine was given to all patients prophylactically . after radiocontrast agent administration , the same prophylactic treatment was continued ( 1 ml / kg / h ) for 6 h. during this time , hydration and urine output were followed by monitoring the intake and release of fluids . patients with no history of kidney disease , plasma creatinine values < 1.2 mg / dl , gfr60 ml / min , nondiabetic , no urinary infection , and no decompensated heart failure were included . urinary il-18 levels were measured with a human il-18 elisa kit ( biosource invitrogen human il-18 , carlsbad , ca , usa ) . the statistical analysis was performed with the ncss pass 2007 and 2008 statistical software ( kaysville , ut , usa ) . serum creatinine levels increased after radiocontrast administration compared with precontrast levels , although the result was not statistically significant [ table 2 ] . a slight increase in creatinine levels occurred at 48 h after radiocontrast administration but they fell to precontrast values at 72 h. a slight increase in plasma creatinine levels at 24 and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant ( p=0.052 and p=0.285 , respectively ) [ table 2 ] . plasma creatinine levels in patients before and after radiocontrast agent administration compared with precontrast urine spot il-18 levels , postcontrast 6 and 24 h urinary levels of il-18 increased significantly ( p=0.048 and p=0.028 , respectively ; table 3 ) . a tendency for postcontrast 24-h urinary il-18 levels to increase was observed compared with 6 h , but the increase was not statistically significant ( p=0.808 ; table 3 ) . spot urine il-18 levels in patients before and after radiocontrast agent administration plasma creatinine levels and spot urine il-18 were weakly but positively correlated with those during the precontrast period , although this finding was not statistically significant ( r=0.246 , p=0.126 ) . similarly , postcontrast 24-h plasma creatinine levels and spot urine il-18 levels were weakly but positively correlated , although this result was also not statistically significant ( r=0.254 , p=0.276 ) . there was no difference between pre- and postcontrast values of serum blood urea nitrogen ( bun ) , creatinine , na , k , cl , ca , p , and creatinine clearance . the most common definition of rin is plasma creatinine levels of 0.5 mg / dl or higher 72 h after contrast administration or 25% higher than the basal plasma creatinine level . plasma creatinine levels began to rise within 24 h in 80% of the patients with rin , peaking at 4872 h , and returning to baseline after 2 weeks . the first 24 h remains unclear in patients with acute renal injury , but il-18 levels start to increase within 46 h , peaking at 12 h. additionally , plasma creatinine is affected by age , body weight , total body volume , gender , race , drug use , muscle mass , and protein intake so researchers are looking for a diagnostic marker for rin that can be measured easily and is not affected by nonrenal factors . most studies related to these parameters include serum and urine cystatin c , serum and urine ngal , and urine il-18 in the analysis . as proinflammatory cytokine il-18 levels increase in urine , tubular inflammation , such as ischemia , reperfusion injury , allograft rejection , cisplatin toxicity , and endotoxemia occur . parikh et al . , found that 72 patients with acute tubular necrosis and delayed graft reaction have significantly higher il-18 levels than other kidney diseases ( urinary tract infection , chronic renal failure , nephrotic syndrome , or prerenal azotemia ) . reported that il-18 generally showed a low sensitivity but high specificity , respectively , for assessing an acute kidney injury diagnosis and risk classification . in our study , we examined spot urinary il-18 levels in comparison with plasma creatinine levels . a weak positive correlation was found between precontrast creatinine and urine il-18 levels , although it was not statistically significant . furthermore , we also found a weak positive correlation between postcontrast 24 h creatinine and urine il-18 levels , although this was not statistically significant either . a slight increase in plasma creatinine levels at 24 h and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant which was regressed to precontrast values at 72 h. a statistically significant increase in the level of spot urinary il-18 levels at 6 and 24 h postcontrast was observed , compared with precontrast spot urine il-18 levels and difference between 6 and 24 hour levels were not statistically significant . one of the limitations of this study is that there are other early biomarkers of acute kidney injury such as ngal and kim-1 . another limitation is that urine il-18 measurement was indexed to serum creatinine instead of urine creatinine since serum creatinine is considered to be a better and commonly used marker in the diagnosis of acute kidney injury . in conclusion , spot urine il-18 levels at sixth hour following radiocontrast administration suggesting that it may be an earlier parameter for identifying kidney injury .

radiocontrast administration is an important cause of acute renal failure . in this study , compared the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration . twenty patients ( 11 males , 9 females ) underwent radiocontrast diagnostic and therapeutic - enhanced examinations . the rin mehran risk score was low ( 5 ) . the radiocontrast agents used were 623 mg / ml iopromid ( 1.5 ml / kg ) , and 100 ml of 650 mg / ml meglumine diatrizoate as three - way oral and rectal contrast material for abdominal computed tomography ( ct ) scans . serum blood urea nitrogen , creatinine , na , k , cl , ca , p , creatinine clearance , and spot urine il-18 levels were analyzed before and repeated at 24 , 48 , and 72 h after radiocontrast administration . six and 24-h urinary il-18 levels were measured with a human il-18 elisa kit following radiocontrast administration . an increase in plasma creatinine 24 and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant ( p=0.052 and p=0.285 , respectively ) . a statistically significant increase in il-18 levels was observed at 6 and 24 h , compared with precontrast values ( p=0.048 and p=0.028 , respectively ) . a tendency for postcontrast 24-h urinary il-18 levels to increase was observed compared with 6 h , but the increase was not statistically significant ( p=0.808 ) . our results show that plasma creatinine starts to increase at 24th hour ; however , spot urine il-18 levels go up at 6th hour following radiocontrast administration implying urine il-18 to be an earlier parameter for kidney injury .

according to the u.s . national institutes of health , up to 80% of human bacterial infections involve biofilm - associated microorganisms 1 . among these , implant - related infections do still have a tremendous impact in orthopaedics and trauma 2 , with high social and economic costs 3 , 4 , posing challenging diagnostic and therapeutic dilemmas 5 . in fact , peri - prosthetic joint infection ( pji ) remains one of the most feared complications in orthopaedic surgery and among the first reasons for implant failure 6 . moreover , given the increasing number of hip and knee arthroplasties performed , the prevalence of this complication is rising , with increasing costs for national health systems and increasing biological costs for the patients , such as loss or reduced joint function and deterioration in their physical and psychological health 7 . according to the widely accepted model of the ' race for the surface ' for pji development , host and bacterial cells compete for surface colonization , with a low probability of bacterial attachment if host cells adhere to implant first , and vice versa . in the event of bacterial adhesion to an implant , in addition , the matrix protects the biofilm cells from various microbicidal agents and stresses , including dehydration , toxins , ultraviolet light , chemical disinfectants , temperature and osmotic shock , and lead them to increased resistance against antimicrobials 9 , 10 . to address the limited efficacy of existing antibiotics in the treatment of established bacterial biofilms , novel approaches are required to prevent bacterial adhesion and biofilm formation 11 . adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new preventive and treatment strategies 12 . bacterial adhesion to surfaces can be divided into a first , reversible phase and a second , irreversible phase . once an implant is inserted into the body , it is covered by a conditional protein layer composed of host proteins , such as albumin and complement , that act as a reservoir of several receptors for bacterial adhesive ligands , mediating adhesion of free - floating bacteria to the surface of the biomaterials 14 , 15 ; these first adhesions are , however mechanically and biologically unstable . few minutes after this first , reversible phase , bacterial clusters attached to the surface starts to express biofilm related genes , produce glycocalyx and form mature biofilm , thus transforming the adhesion from reversible to irreversible 16 . full - formed biofilm can be found few hours after the first bacterial adhesion 17 . antimicrobial surface coatings can be based on an anti - adhesive principle that prevents bacteria to adhere and form biofilms 18 . in fact , some polymer coatings , like the hydrophilic polymethacrylic acid , polyethylene oxide or protein - resistant polyethylene glycol can be applied to the surface of titanium implants and result in significant inhibition of bacterial adhesion 19 - 22 . hydrophobic and superhydrophobic surface treatment technologies have also shown a great repellent antibacterial effect in preclinical studies 23 - 25 . however , clinical application of completely novel coating technologies and compounds , not otherwise previously tested in humans , appears particularly challenging 26 . bacterial colonization can also be blocked by an inhibitor interfering with ligand - receptor interaction for bacterial attachment . one of these inhibitors could be hyaluronic acid ( ha ) , a glycosaminoglycan made up of glucuronic acid and n - acetylglucosamine disaccharide units . it is a uniform , linear and unbranched molecule , with highly variable length and molecular weight ( up to 106 da ) . it is abundant in skin ( up to 56% ) and in connective tissues , with a turnover ranging from several hours to a few days depending on tissues . hyaluronic acid constitutes one of the main components of extracellular matrices . because of its biological properties , ha has several clinical applications ( aesthetic surgery , dermatology , dentistry , orthopedics and opthalmology ) 27 . extensive studies on the chemical and physicochemical properties of ha and its physiological role in humans , together with its versatile properties , such as its biocompatibility , non - immunogenicity , biodegradability , and viscoelasticity , have proved that it is an ideal biomaterial for medical and pharmaceutical applications 28 , 29 . among its various properties , several studies have recently shown the ability of ha to protect against various infectious agents 30 , depending on ha concentration and molecular weight 31 , 32 , while more recently ha interference on bacterial adhesion and biofilm formation has been extensively investigated 33 . given its high biocompatibility and well known safety profile and the anti - adhesive capabilities , ha and its composites represent an attractive , non - antibiotic , option to mitigate the impact of biofilm - related infections in various clinical settings including implant - related infections . aim of this review is to provide an update of the current evidence concerning ha ability to reduce / prevent bacterial adhesion and biofilm formation . nearly two decades ago , pavesio et al . 34 were probably the first to describe the ability of ha to resist bacterial adhesion , with particular reference to staphylococcus epidermidis , and its non - fouling properties 35 , proposing coated polymeric medical devices ( e.g. , intraocular lenses , stents and catheters ) to reduce implant - related infections . in particular , a hydrophilic ha overlayer , linked to the surface of polymethylmethacrylate intraocular lenses ( iols ) , was shown to be able to prevent fibroblasts adhesion and to greatly reduce staphyloccous epidermidis adhesion to the implant surface 36 . the impact of slime dispersants and anti - adhesives on in vitro biofilm formation on iols was further investigated by kadry and co - workers 37 , using a staphyloccous epidermidis wild strain , isolated from a patient with endophtalmitis ; the authors reported the ability of hyaluronan to reduce bacterial adhesion to iols to 30% , compared with untreated control cells . the authors suggested the use of adjuvant therapy such as dispersants or anti - adhesives , in addition to the antibiotics in irrigating solutions for bacterial ocular infections . more recently , the in vitro antiadhesive and antibiofilm activity of hyaluronic acid towards bacterial species commonly isolated from respiratory infections was investigated by drago et al . 33 . in this study , the interference exerted on bacterial adhesion was evaluated by using hep-2 cells , while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet . the experimental findings clearly demonstrated how hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration - dependent manner . moreover , staphylococcus aureus biofilm was found to be more sensitive to the action of ha , compared to that produced by haemophilus influenzae and moraxella catarrhalis . concerning more specifically the antimicrobial activity , ha has also been shown to exert varied bacteriostatic , but not bactericidal , dose - dependent effects on different microorganisms in the planktonic phase 31 , 38 . in this regard , radaeva et al . reported the inhibiting activity of ha with respect to some pseudomonas species 39 , while ardizzoni and co - workers 30 investigated the effects of ha on 15 atcc bacterial strains , representative of clinically relevant bacterial and fungal species . their results showed that different microbial species and , sometimes , different strains belonging to the same species , are differently affected by ha . in particular , staphylococci , enterococci , streptococcus mutans , two escherichia coli strains , pseudomonas aeruginosa , candida glabrata and c. parapsilosis displayed a ha dose - dependent growth inhibition , while no ha effects were detected in e. coli atcc 13768 and c. albicans and s. sanguinis was favoured by the highest ha dose . comparing the potential bacteriostatic effect of some of the most commonly used biomatrix materials ( collagen type i , hyaluronic acid , hydroxyapatite , polylactic acid and polyglycolic acid ) on the growth over the first 12h of exposure of some of the most common orthopaedic bacterial pathogens ( staphylococcus aureus , staphylococcus epidermidis , -hemolytic streptococcus , pseudomonas aeruginosa ) , carlson and co - workers 38 found that ha had the most significant bacteriostatic properties on the studied organisms . 31 investigated the potential bacteriostatic effect of hyaluronic acid in different concentrations and molecular weight on oral and non - oral microorganisms ( staphylococcus aureus , propionibacterium acnes , actinobacillus actinomycetemcomitans , pavotella oris and porphyromonas gingivalis ) with potential application in dentistry surgery ; the results showed that different hyaluronan solutions exerted varied bacteriostatic effects on all the bacterial strains . the authors concluded that the clinical application of hyaluronan in form of membranes , gels , or sponges during surgical therapy may reduce bacterial contamination of the surgical wound , thereby lessening the risk of postsurgical infection and promoting more predictable regeneration . concerning possible orthopaedic applications , in 2004 harris and richards 40 showed the visualization and quantification of s. aureus adhering to a variety of different treated / coated titanium surfaces . in their study , coating titanium with sodium hyaluronate significantly decreased the density of s. aureus adhering to the surfaces and its potential use in osteosynthesis , orthopaedics or dental applications was suggested out . in a very recent review on polysaccharide - based coatings , that have been proposed over the last ten years to impede biofilm formation on material surfaces exposed to bacterial contamination , hyaluronic acid was discussed as one of the most studied , with demonstrated non - fouling properties on glass surfaces 41 ; displaying hydrophilic characteristics ( contact angle of 22 ) , this coating was in fact reported to reduce adhesion of s. epidermidis and e. coli by several orders of magnitude compared to the unmodified glass slide . similarly , adhesion of s. aureus on ti foils functionalized with hyaluronic acid - catechol was lower than on pristine substrates . based on ha antiadhesive properties , a novel ha - based hydrogel has been recently proposed , in order to protect implanted biomaterials in orthopaedics , trauma and dental surgery from bacterial colonization 42 ; this fast - resorbable hydrogel coating , composed of covalently linked hyaluronan and poly - d , l - lactide ( defensive antibacterial coating , dac , novagenit srl , mezzolombardo , italy ) , has been found to have a synergistic antibiofilm activity with various antibacterials and able to be effectively manually spread onto the surface of various biomaterials commonly used in orthopaedics , trauma and dental surgery 43 ( fig . 1 ) . the ability to completely cover even sand - blasted titanium surface and resist scraping has in fact been confirmed by scanning electron microscopy ( sem ) analysis ( cf . this is an important requirement in order to reduce the exposed surface of a biomaterial , thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria . in unpublished experiments ( novagenit srl , data on file ) , in order to evaluate dac ability to prevent bacterial adhesion , 200 mg of hydrogel were homogenously spread on the surface of sterile titanium discs . hydrogel - coated substrates and uncoated substrates ( controls ) were then placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells for 15 , 30 , 60 and 120 minutes . the remaining adhered cells were detached by adding a solution of 0.1% w / v dithiothreitol ( dtt ) ( sigma - aldrich , milan , italy ) to each well and stirring for 15 minutes at room temperature . then , 100 l of each sample were plated onto tryptic soy agar ( tsa ; merck , darmstadt , germany ) and incubated at 37c for 24 hours for cfu counts . the results showed that the adhesion density of s. aureus on titanium discs pre - treated with dac , was significantly lower than adhesion on untreated controls at each time point ( fig . 2 ) . in particular , reductions of adhered bacteria equal to 86.8% , 80.4% , 74.6% and 66.7% vs untreated discs were observed after 15 , 30 , 60 and 120 minutes of incubation , respectively , while an increase of adhesion density during time was observed for both control and pre - treated discs ( fig . further analyses were conducted to show the ability to dislodge previously adhered bacteria ; to this aim , titanium discs were placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells in order to allow the adhesion of bacterial cells . afterwards , 200 mg of hydrogel were spread on the surface of contaminated titanium discs in order to remove previously adhered bacteria . non - adherent bacteria were removed by rinsing with sterile saline , while the remaining adhered cells were detached by adding 0.1% dtt as previously described . then , 100 l of each sample were plated onto tsa and incubated at 37c for 24 hours for cfu counts . the results showed that dac hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15 , 30 , 60 and 120 minutes of 84.0% , 72.8% , 72.3% and 64.3% , respectively ( fig.4 ) . once again , an increase of adhesion density during time was observed for both control and treated discs ( fig . dac hydrogel showed similar or superior in vitro activity , compared to various antibacterials and a synergistic activity when used in combination ( fig . were grown on chrome - cobalt devices in 6-wells polystyrene plates containing tsb for 24 hours at 37c . then , growth medium was removed together with non - adherent bacteria and new broth added . the plates were incubated at 37c in ambient air , until a visible biofilm was obtained . gentamycin and vancomycin were tested at a final concentration of 20 mg / ml . similarly , when mixed with the hydrogel , 60 mg of gel powder were reconstituted with 1 ml of water for injections containing gentamicin or vancomycin at 20 mg / ml concentration . amount of biofilm at each time was determined before hydrogel and antibiotic agents addition and after 0.5 , 1 , 2 , 4 , 6 , 24 and 48 hours of incubation by a spectrophotometric assay . in particular , at each time , broth was removed and biofilm stained with crystal violet . after elution of the stain from implants with absolute ethanol , the amount of biofilm was quantified by reading optical density ( o.d . ) at a wavelength of 595 nm against blank ( consisting of ethanol ) . amount of biofilm at each time was compared with that formed on the same type of implant before treatment . each assay was performed in duplicate and repeated for three times . at each time point , both for gentamycin and vancomycin showed only a partial inhibition of biofilm formation ( ca . 40 - 50% for vancomycin ) , with minor difference between the two studied microorganisms . on the other side , 50% in comparison to the untreated controls , while a combination of the hydrogel with either antibacterial resulted in a larger reduction of biofilm formation ( approximately 75 to 80% in comparison with untreated controls ) . both these experimental studies show the ability of the dac hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens , thus potentially providing an effective protection of the implant ; however , these data also point out how , in the clinical setting , in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics , a passive antiadhesive coating 18 like ha can be overcome by the remaining bacteria in a time - dependent manner . for this reason , any passive antiadhesive coating of implants 44 should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree , also depending on the local environment , the contaminating bacterial species and initial bacterial load ; this may still provide an additional advantage to the host 's cells to first colonize the implanted biomaterial and win the competition with the microorganisms that may eventually be present , thus contributing to reduce the occurrence of implant - related infections . several clinical local applications of ha to reduce the impact of biofilm - related infections have been reported , in different clinical settings , with favourable results and no adverse events . 45 recently described topical administration of hyaluronic acid in children with recurrent or chronic middle ear inflammations and chronic adenoiditis . in this prospective , single - blind , randomised controlled study , otoscopy , tympanometry and pure - tone audiometry in children which received the daily topical administration of normal 0.9% sodium chloride saline solution ( control group ) or 9 mg of sodium hyaluronate in 3 ml of a 0.9% sodium saline solution was performed . the final analysis was based on 116 children ( 49.1% boys ; mean age , 62.9 17.9 months ) : 58 in the control group and 58 in the study group . at the end of follow - up , the prevalence of patients with impaired otoscopy was significantly lower in the study group ( p value = 0.024 ) compared to baseline but not in the control group . in comparison with baseline , the prevalence of patients with impaired tympanometry at the end of the follow - up period was significantly lower in the study group ( p value = 0.047 ) but not in the control group . the reduction in the prevalence of patients with conductive hearing loss ( chl ) ( p value = 0.008 ) and those with moderate chl ( p value = 0.048 ) was significant in the study group , but not in the control group . the mean auditory threshold had also significantly improved by the end of treatment in the study group ( p value = 0.004 ) but not in the control group . several studies have also reported the beneficial effect of topical ha in chronic urinary tract infections ( uti ) . in contrast to traditional antibiotic therapy , which aims at eradicating pathogens , treatment with ha targets bacterial adherence to the bladder mucosa with the presumption that a damaged glycosaminoglycan mucous layer facilitates bacterial adherence and therefore recurrent uti 46 . among others 47 , 48 , lipovac and colleagues evaluated the efficacy of nine ha bladder instillations over 6 months in 20 women with a history of recurrent uti . their status was assessed prospectively but compared with a retrospective review of patients ' charts . the number of infections per year per patient was significantly reduced ( from 4.990.92 to 0.560.82 , p>0.001 ) and the mean time to recurrence ( from 76.724.6 to 178.325.5 days , p>0.001 ) was prolonged significantly . nevertheless 65% of treated patients were free of recurrences until the end of study ( 47.6 weeks ) 49 . were able to provide a higher level of evidence by reporting a prospective , randomized , double - blind , placebo - controlled study , in which a significant reduction of 77% ( p<0.0002 ) in the uti rate per patient per year versus placebo was observed at the end of the study . moreover , mean time to uti recurrence was significantly prolonged ( 185.278.7 versus 52.733.4 days , p<0.001 ) after treatment compared with placebo . overall urinary symptoms and quality of life measured by questionnaires significantly improved compared with placebo 50 . very recently a multicentre european study confirmed the efficacy of intravesical administration of combined hyaluronic acid and chondroitin sulphate ( cs ) for the treatment of female recurrent urinary tract infections 51 . a total of 276 adult women received intravesical administration of ha+cs or standard of care ( antimicrobial/ immunoactive prophylaxis/ probiotics / cranberry ) . at follow - up , 181 patients treated with ha+cs and 95 patients treated with standard of care from 7 centres were available . the crude and adjusted ors ( 95% ci ) for bacteriologically confirmed recurrence within 12 months were 0.77 ( 0.46 to 1.28 ) and 0.51 ( 0.27 to 0.96 ) , respectively . studies were also undertaken to determine the effect on clinical variables , sub - gingival bacteria and local immune response brought about by application of hyaluronan - containing gels in early wound healing after scaling and root planing ( srp ) in dentistry 52 , 53 . in the study reported from eick et al . the exclusion criteria were : antibiotics intake in the 6 months before the study , periodontal treatment received during the previous year , pregnancy , nursing , smoking , chronic diseases such as diabetes mellitus or rheumatoid arthritis , and allergy to ingredients in the drug . in the test group ( n=17 ) , a 0.8% hyaluronan - containing gels ( ha ) was introduced into all periodontal pockets during srp and a 0.2% ha gel was applied by the patients onto the gingival margin twice daily during the following 2 weeks while the control group ( n=17 ) was treated with srp only ; no placebo was used . probing depth ( pd ) and clinical attachment level ( cal ) were recorded at baseline and after 3 and 6 months , and subgingival plaque and sulcus fluid samples were taken for microbiologic and biochemical analysis . in both groups , pd and cal the changes in pd and the reduction of the number of pockets with pd5 mm were significantly higher in the test group after 3 ( p=0.014 and 0.021 ) and 6 ( p=0.046 and 0.045 ) months . six months after srp , the counts of treponema denticola were significantly reduced in both groups ( both p=0.043 ) , as were those of campylobacter rectus in the test group only ( p=0.028 ) . although to date no surface modification has been reported to be able to fully prevent bacterial adhesion and biofilm formation 55 , available data show that hyaluronic acid has a proven in vitro antiadhesive / antibiofilm effect against some of the most common pathogens and it has been used safely , alone or in combination with other polymers , with satisfactory results in different conditions associated with biofilm - related chronic infections . clinical data in various applications , including dentistry , urology , wound management , dermatology and orthopedics , allow to consider the potential use of ha as a protective coating barrier of implants particularly safe and feasible on a large scale basis . while antibacterial coatings to mitigate the occurrence of implant- and biofilm - related infections are regarded as one of the most needed technology , currently only few and insufficient options are available for clinical use in orthopedics and trauma surgery 18 . considering the pathogenesis of implant - related infections , any protection offered by a fully biocompatible antiadhesive barrier , like ha and some of its derivatives , could be extremely useful to reduce the tremendous burden of implant - related infections . on the other hand , it should be noted that hyaluronic acid as a passive protective barrier has some limits . among others , the antiadhesive / antibiofilm effect is limited and may vary , depending on the type of the microorganism , the bacterial load , the local environment , etc . ; moreover , ha protection may be neutralized by the possible ability of some bacteria to produce hyaluronidase , an enzyme that catalyzes the degradation of hyaluronic acid 56 , while collagen and hyaluronan may even become possible ligands for microbial attachment in particular situations 57 , 58 . to overcome at least some of these limits , possible loading of hyaluronic - based hydrogels with antibiotics is technically feasible and has been proposed by different authors 59 - 62 , being a possible option for future developments and large scale clinical applications , provided that regulatory requirements can be met .

living in biofilms is probably the most common condition for bacteria and fungi and biofilm - related infections account for the majority of bacterial infectious diseases worldwide.among others biofilm - related infections , those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma , cardio - surgery and several other surgical disciplines.given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms , new strategies are needed to protect implants and host tissues , overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix.adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach.among various polymers , tested as antibacterial coatings , hyaluronic acid and some of its composites do offer a well - established long - term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation.aim of the present review is to summarize the available evidence concerning the antiadhesion / antibiofilm activity of hyaluronic acid and some of its derivatives to reduce / prevent bacterial adhesion and biofilm formation in various experimental and clinical settings .

in 2011 , who declared combat drug resistance : no action today , no cure tomorrow . . in recent years presently , antimicrobial resistance ( amr ) poses a major threat to patient 's treatment as it leads to increased morbidity and mortality , increased hospital stay , and severe economic loss to the patient and nation [ 3 , 4 ] . the clinical isolates such as pseudomonas aeruginosa , methicillin resistant staphylococcus aureus ( mrsa ) , enterococci especially vancomycin resistant enterococci ( vre ) , and members of family enterobacteriaceae , for example , klebsiella pneumoniae , e. coli , and proteus sp . in the last two decades , there were so much increase of infectious diseases that the standard of public health in many parts of the world is equivalent to preantibiotic era . as per standardized international terminology created by european centre for disease control ( ecdc ) and centre for disease control & prevention ( cdc ) , atlanta , the multidrug - resistant ( mdr ) , extensively drug - resistant ( xdr ) , and pandrug - resistant ( pdr ) bacteria have been well defined . multidrug resistant ( mdr ) was defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories . extensively drug resistant ( xdr ) was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories ( i.e. , bacterial isolates remain susceptible to only one or two antimicrobial categories ) . pandrug resistant ( pdr ) was defined as nonsusceptibility to all agents in all antimicrobial categories . hence , this short term study was undertaken to detect the incidence of mdr , xdr , and pdr bacterial isolates in a tertiary care hospital of central india . this short term cross - sectional study was conducted in the department of microbiology from 15th of april to 15th of july , 2014 . the bacterial strains were isolated from different clinical samples and were identified by conventional methods . the clinical specimens from indoor patient departments ( ipd ) only were included in the study . antibiotic susceptibility test of bacterial strains was done by kirby bauer disc diffusion method as per clinical laboratory standard institute ( clsi ) guidelines . antibiotics used for gram positive cocci ( gpc ) were penicillin , erythromycin , ciprofloxacin , tetracycline , amikacin , vancomycin , and linezolid and for gram negative bacilli ( gnb ) were amikacin , ceftazidime , ceftazidime - clavulanic acid , ciprofloxacin , imipenem , and colistin , respectively . linezolid and colistin were used as supplemental drugs . for urine sample , instead of ciprofloxacin and tetracycline , for routine quality control of antibiotic susceptibility test , s. aureus atcc 25923 , e. coli atcc 25922 , and pseudomonas aeruginosa atcc 27853 were used . mdr , xdr , and pdr strains were detected as per criteria described by ecdc and cdc . methicillin resistant staphylococcus aureus ( mrsa ) strains were detected by meca - mediated oxacillin resistance using cefoxitin disk ( 30 g ) on mueller hinton ( mh ) agar plate inoculated with test strains as per standard disk diffusion recommendations and incubated at 3335c for 1618 hours . inhibition zone 21 mm with cefoxitin disk was interpreted as meca positive according to clsi guidelines . extended spectrum -lactamases ( esbl ) producing strains were detected by combined disk method using ceftazidime ( 30 g ) and ceftazidime plus clavulanic acid ( 30 g plus 10 g ) . an increase in diameter of 5 mm with ceftazidime plus clavulanic acid as compared to ceftazidime disk alone was considered positive for esbl detection . 138 clinical samples were received from intensive care unit ( icu ) and 742 clinical samples were received from wards of different clinical specialities . 698 clinical samples had single bacterial growth and 182 had mixed bacterial growth . out of these 182 clinical samples , 172 samples had 2 bacterial isolates , 4 samples had 3 bacterial isolates , and 6 samples had 1 bacterial isolate along with candida albicans . figure 1 shows that 314 ( 29.6% ) bacterial strains were gram positive cocci ( gpc ) and 746 ( 70.4% ) were gram negative bacilli ( gnb ) . out of 314 gpc , 252 ( 80.3% ) were coagulase positive staphylococci . amongst 746 gnb , 261 ( 35% ) were e. coli , followed by pseudomonas aeruginosa 212 ( 28.4% ) . during the study period , figure 2 shows the incidence of mdr and xdr strains isolated . out of total 1060 bacterial strains studied , 393 ( 37.1% ) bacterial strains were mdr and 146 ( 13.8% ) strains were xdr . amongst 314 gpc strains isolated , 143 ( 45.5% ) and 56 ( 17.8% ) were mdr and xdr , respectively . out of 746 gnb isolates , 250 ( 33.5% ) strains were mdr and 90 ( 12.1% ) were xdr . out of total 9304 patients admitted , 393 ( 4.2% ) and 146 ( 1.6% ) were positive for mdr and xdr strains , respectively . figure 3 shows the incidence of mdr and xdr gram positive cocci isolated . out of total 252 coagulase positive staphylococci isolated , 125 ( 49.6% ) were mdr and 38 ( 15.1% ) were xdr . 10 coagulase negative staphylococci were isolated and 5 ( 50% ) were mdr , whereas 2 ( 20% ) were xdr . 79 ( 31.3% ) coagulase positive staphylococci strains were mrsa and 2 ( 20% ) coagulase negative staphylococci were mrcons . out of total 45 enterococci isolated , 13 ( 28.9% ) were mdr and 16 ( 35.6% ) were xdr . no vancomycin intermediate staphylococcus aureus ( visa ) , vancomycin resistant staphylococcus aureus ( vrsa ) , or vancomycin resistant enterococci ( vre ) were isolated . all streptococcus species including group a , nongroup a , and pneumococcus were sensitive to penicillin . no mdr or xdr strain was isolated from streptococcus sp . all ( 100% ) gram positive cocci were sensitive to vancomycin and linezolid . figure 4 shows incidence of mdr and xdr strains isolated from each species of gram negative bacilli . in the present study , e. coli was the commonest isolate 261 ( 35% ) , followed by pseudomonas aeruginosa 212 ( 28.4% ) . 79 ( 30.3% ) and 22 ( 8.4% ) e. coli strains were mdr and xdr , respectively . out of 200 klebsiella pneumoniae strains isolated , 75 ( 37.5% ) and 25 ( 12.5% ) were detected as mdr and xdr , respectively . out of 42 acinetobacter and other nonfermenter species isolated , 19 ( 45.2% ) and 8 ( 19% ) were mdr and xdr strains , respectively . amongst 250 gnb - mdr strains isolated , the commonest mdr strains were detected from e. coli 79/250 ( 31.6% ) , followed by klebsiella pneumoniae 75/250 ( 30% ) . similarly , out of 90 gnb - xdr strains isolated , the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 ( 32.2% ) , followed by klebsiella pneumoniae 25/90 ( 27.8% ) . in the present study , 137 ( 18.4% ) others include wards like dermatology , pulmonary medicine , orthopedics , and cardiovascular and thoracic surgery ( cvts ) . the different icus include neonatal icu ( nicu ) , medicine icu ( micu ) , operation theatre icu ( ot icu ) , and paediatric icu ( picu ) . out of total 393 mdr strains detected , 127 ( 32.3% ) ( the highest number ) mdr strains were isolated from surgery wards followed by 72 ( 18.3% ) mdr strains from different icus . amongst total 146 xdr strains isolated , 41 ( 28.1% ) ( the highest number ) were isolated from surgery wards also . out of 72 mdr strains detected from different icus , 29 ( 40.3% ) ( the highest number ) mdr strains were isolated from nicu , followed by 20 ( 27.8% ) and 18 ( 25% ) from ot icu , and only 5 ( 6.9% ) from picu . even in the total 26 xdr strains isolated from different icus , 10 ( 38.5% ) ( the highest number ) the percentage of mdr and xdr strains isolated from different icus was 72/138 ( 52.2% ) and 26/138 ( 18.8% ) , respectively , which were again much more than mdr and xdr strains isolated from wards 321/742 ( 43.3% ) and 120/742 ( 16.2% ) , respectively . 275 patients were admitted to nicu , of whom 29 ( 10.5% ) were positive for mdr strains and 10 ( 3.6% ) were positive for xdr strains . out of total 1907 patients admitted to surgery ward , 127 ( 6.7% ) were positive for mdr strains whereas 41 ( 2.1% ) were positive for xdr strains . in micu , 545 patients were admitted and 20 ( 3.7% ) were positive for mdr strains and 8 ( 1.5% ) were positive for xdr strains . the clinical and financial burden to patients and health care providers for mdros is really challenging . barbara soule , joint commission resources practice leader , infection prevention and control services , has told , patients who are infected with mdros often have an increased risk of prolonged illness and mortality . the cost of care for these patients can be more than double as compared to those without mdro infection . since the year 2000 , only 4 new classes of antibiotics have been approved by food and drug administration ( fda ) , us , for example , linezolid , streptogramins , daptomycin , and tigecycline . the problem is that the bacteria are developing resistance at a much faster pace than the new drug development . regarding public health attention , mdros are described as superbugs having very limited treatment options . for some mdros , only 1 or 2 antibiotics can be effective with toxic side effects . in 2009 , boucher et al . have reported eskape organisms as bad bugs , where e stands for enterococcus faecium , s for staphylococcus aureus , k for klebsiella pneumoniae , a for acinetobacter baumannii , p for pseudomonas aeruginosa , and e for enterobacter species . in the year 2009 only , peterson has reported the escape group of organism , which was the same as the above list but k was replaced by c , that is , clostridium difficile , and the last e stands for enterobacteriaceae . in the present study , amongst 250 gnb - mdr strains isolated , the commonest mdr strains were detected from e. coli 79/250 ( 31.6% ) , followed by klebsiella pneumoniae 75/250 ( 30% ) . similarly , out of 90 gnb - xdr strains isolated , the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 ( 32.2% ) , followed by klebsiella pneumoniae 25/90 ( 27.8% ) . aly and balkhy reported that most prevalent mdro in their study was e. coli followed by klebsiella pneumoniae . in another study , carried out in a tertiary care hospital in riyadh , it has been reported that most frequent mdr pathogens were pseudomonas aeruginosa followed by e. coli . the percentage of mdr e. coli strains was more than klebsiella pneumoniae and even pseudomonas aeruginosa in our study probably because a total number of e. coli strains isolated ( 261 ) were also higher . the slightly increased incidence of drug resistant strains observed in our study may be because our hospital is a tertiary care center in a rural setup and patients from adjoining districts and even villages are admitted for treatment . before attending the hospital , most of the patients get different antibiotics from general practitioners or due to over - the - counter sell of antibiotics often in improper dose . the limitation of this study is that this is a single center study for only three - month period in a tertiary care hospital in central india . to reflect the trend of infections caused by mdr and xdr strains of bacteria in the region , a multicenter study involving all types of healthcare setups for a minimum period of one year there is paucity of data regarding mdros in health care setup not only in india but also worldwide . unless and until multidrug resistant organisms are detected and their incidence is known , the strategies for their control can not be adopted properly in healthcare setup . hence , detection , prevention of transmission of mdros by following infection control practices , antimicrobial surveillance , and stewardship are need of the hour . misuse and overuse of antibiotics , over - the - counter selling of antibiotics without prescription to common people , must be stopped by strict implementations of rules and regulations . we hereby conclude that early detection and close monitoring of mdr , xdr , or even pdr bacterial strains must be started by all clinical microbiology laboratories to reduce the menace of antimicrobial resistance which is now a global problem .

background and objective . antimicrobial resistance is now a major challenge to clinicians for treating patients . hence , this short term study was undertaken to detect the incidence of multidrug - resistant ( mdr ) , extensively drug - resistant ( xdr ) , and pandrug - resistant ( pdr ) bacterial isolates in a tertiary care hospital . material and methods . the clinical samples were cultured and bacterial strains were identified in the department of microbiology . the antibiotic susceptibility profile of different bacterial isolates was studied to detect mdr , xdr , and pdr bacteria . results . the antibiotic susceptibility profile of 1060 bacterial strains was studied . 393 ( 37.1% ) bacterial strains were mdr , 146 ( 13.8% ) strains were xdr , and no pdr was isolated . all ( 100% ) gram negative bacterial strains were sensitive to colistin whereas all ( 100% ) gram positive bacterial strains were sensitive to vancomycin . conclusion . close monitoring of mdr , xdr , or even pdr must be done by all clinical microbiology laboratories to implement effective measures to reduce the menace of antimicrobial resistance .

ureterocele is a congenital urinary abnormality characterized by the presence of an intrabladder hernia or cystic ballooning of the lower end of a ureter lying between the mucosa and muscle of the bladder . the abnormality leads to urinary retention and recurrent urinary tract infection ( uti ) , which can cause irreversible damage to the kidney . this abnormality can be suspected in the fetus by antenatal ultrasonography ( usg ) and confirmed by other x - ray investigations after birth . the incidence of ureterocele is variable with the highest rate of 1:500 and it is generally found in females with duplex system association ( 95% ) . ureteroceles can have different clinical presentations , such as antenatal hydronephrosis , uti , vesicoureteral reflux ( vur ) , bladder outlet obstruction , prolapsed urethral mass , etc . usg and voiding cystourethrography ( vcug ) are essential initial procedures for a child suspected of having a ureteral anomaly . a 5-year - old female child was referred to our department for tc - diethylene triamine pentaacetic acid ( dtpa ) scan . she was a follow - up case of right - sided hydroureteronephrosis with pyonephrosis with percutaneous tube ( pcn ) insertion done on the right side . patient 's blood urea and serum creatinine were 29 and 1.3 mg / dl , respectively . usg of the abdomen revealed left - sided mild hydroureteronephrosis and gross enlargement of the right kidney , reaching up to lower abdomen with dilated pelvis and ureter . the child was referred for evaluation of glomerular filtration rate and excretory function of kidneys . tc - dtpa scan was done in our department with right pcn tube in situ . pcn tube was clamped during the acquisition of dynamic and prevoid static image and clamp released thereafter . on tc - dtpa scan , left kidney showed good perfusion and adequate cortical radiotracer concentration followed by good drainage into dilated ureter . right ureter and right pelvicalyceal system ( pcs ) were visualized in the postvoid image ( indirect evidence of vur ) [ figure 2b , thin arrow ] . retention of radiotracer was noted in dilated pcs and dilated ureter in delayed static images acquired till 4 h [ figure 2c and d ] . faint visualization of radiotracer was noted through the pcn tube after removal of clamp [ figure 2b , bold arrow ] . a large photopenia was noted in the suprapubic region in the urinary bladder ( ub ) during dynamic [ figure 1 ] as well as delayed static images acquired till 4 h [ figure 2a d ] . an usg of the abdomen was done in the radiology department of our hospital to find out the cause of this persistent photopenia in the ub . usg of the abdomen showed evidence of left - sided mild hydroureteronephrosis and grossly enlarged hydronephrotic right kidney with dilated right ureter and an intracystic ureterocele arising from the right side [ figure 3a and b ] . perfusion and dynamic images of 99mtc - diethylene triamine pentaacetic acid scan scintigraphy , posterior view with percutaneous clamp in situ , showing good perfusion and adequate cortical function and good drainage of left kidney . photopenia noted in the urinary bladder ( bold arrow ) ( a ) prevoid static image with right pcn clamp in situ . right ureter and right pelvicalyceal system visualization is noted suggesting indirect evidence of vesico - ureteric reflux ( thin arrow ) . ( c and d ) 3 and 4 h delayed static images showing persistent photopenia in the urinary bladder ( arrow marked ) ( a and b ) ultrasonography images depicting right ureterocele within the urinary bladder ( ub ) and dilated distal right ureter according to literature , 90% of the patients with ureterocele are diagnosed before the age of 3 years . most of the patients with ureterocele are classically diagnosed during the investigation for uti , asymptomatic hydronephrosis , and abdominal mass . although the age of diagnosis is decreasing , uti is still the most common clinical presentation of ureterocele in 50% of the patients promoting physician to make the thorough evaluation of the urinary system . the whole nephronourinary system could have already been negatively affected at the time of diagnosis . in our case , usg is an easy method to perform , noninvasive , and probably the best imaging modality for making the diagnosis . reflux can occur in the ipsilateral lower pole in almost half of the patients , but the contralateral system is also affected at a rate of 25% . the tc - dimercapto succinic acid ( dmsa ) scan should be undertaken routinely to assess the distribution of function in the duplex kidney and for detecting and follow - up of scarred tissue and nonfunctioning upper poles in cases of ureterocele . did a study on pediatric ureteroceles in 19 patients , on the diagnosis , management and treatment options . vcug was able to detect vur in 13 out of 17 patients ( 33% ) . dmsa scintigraphy showed ipsilateral renal scarring and nonfunctioning upper pole images in 7 out of 13 patients . computed tomography and mag3 scintigraphy did a study on the management of 36 patients with varied presentation of complicated ureteroceles . usg , micturating cystourethrogram , isotope renogram were done preoperatively in all the babies . in one of the cases , initial usg was unable to detect ureterocele in most of the cases in this study . they said that unless the usg is done in well - hydrated , cooperative patient , ureterocele is likely to be missed on usg . hence , an intravenous pyelogram , radioisotope renogram , and micturating cystourethrogram are invaluable in the complete understanding of ureterocele . in our patient also , the diagnosis of ureterocele was missed on initial usg . photopenia was noted in the ub on our scan , and this prompted us to investigate the patient further . possible causes for photopenic defects in the ub on nuclear scintigraphic studies include bladder papilloma , bladder polyp , carcinoma bladder , bladder calculus , foreign body in ub , intravesical ureterocele , etc . repeat usg revealed the cause of photopenia in ub in our case to be an intravesical ureterocele . on reviewing the literature we found that tc - dtpa scan is useful in the evaluation of renal function in patient with known ureteroceles . however , in our case on finding the photopenia in the ub on tc - dtpa scan , intravesical ureterocele was detected on usg .

ureterocele is a common ureteric anomaly detected in pediatric population . ureterocele diagnosis and evaluation need a variety of radiological methods . we report a case of 5-year - old female child sent for 99mtc - diethylene triamine pentaacetic acid scan for evaluation of glomerular filtration rate and excretory function of kidneys in view of right - sided hydroureteronephrosis and pyonephrosis with percutaneous tube in situ . incidental photopenia was noted in the urinary bladder . on ultrasonography of abdomen cause of this photopenia was found to be an intravesical ureterocele .

health - related quality of life ( hrqol ) is a multidimensional concept that defines a person 's health based on specific aspects , such as physical , emotional , and social functioning and general welfare ( 1 ) . the assessment of hrqol consists of an evaluation of the degree to which these aspects are decreased by symptoms , incapacities , and limitations caused by disease ( 2 ) . the assessment of hrqol has been used as a measure complementary to bone mineral density to evaluate and monitor the burden of osteoporosis on a patient 's daily life ( 3 ) . there are several instruments that can be used to assess the quality of life of individuals with osteoporosis , including the osteoporosis assessment questionnaire , the quality of life questionnaire for osteoporosis ( optqol ) , the osteoporosis quality of life questionnaire , and the questionnaire of the european foundation for osteoporosis ( qualeffo ) ( 4 - 7 ) . the qualeffo , a specific tool used to evaluate subjects with vertebral fractures and that includes questions on pain , physical functioning , social functioning , general health perception and mental functioning , has been shown to be repeatable and consistent ( 7 - 8 ) . vertebral fractures are the most frequent osteoporotic fractures , occurring in at least 30% of the elderly population ( 9 ) , and have important clinical implications ( 10 - 14 ) . these fractures are associated with increased risks of new osteoporotic fractures and mortality , especially in older women ( 10 - 11 ) . only one - third of vertebral fractures are symptomatic ; therefore , patients may be unaware of their presence . indeed , in studies based on the radiographic screening of populations , the incidence of all vertebral fractures has been estimated to be three times higher than the incidence of hip fractures , and only 30% of people with vertebral fractures were found to have received medical attention ( 12 ) . women with vertebral fractures can also experience decreased hrqol due to physical limitations and psychosocial disabilities ( 13 - 14 ) . some studies have assessed the impact of vertebral fractures on hrqol in older women in many countries ( 15,16 ) , but few such epidemiological studies have been conducted in brazil . moreover , most of the studies conducted in this country have been performed in ambulatory or institutionalized individuals ( 17,18 ) . therefore , this study evaluated the impact of vertebral fractures on the quality of life of healthy , community - dwelling women aged 65 years or older using the qualeffo . this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) . the inclusion and exclusion criteria were the same as those of the core study ( 9 ) . all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer . current or previous bisphosphonate use was also an exclusion criterion ( 9 ) . quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) . the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol . radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 " between the tube and the film . the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures . they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached . the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 . each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height . the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer . the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale . the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight = bmi<25 ; overweight = 25bmi<30 ; and obese = bmi30 ( 20 ) . information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews . women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) . physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) . regarding concomitant diseases , those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination . systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) . participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) . the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc . bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur . anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) . the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) . we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density . thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) . the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level . the study had 95% power to detect a difference of 10 points in the total qualeffo score . the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies . demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables . the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test . correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) . generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score . variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model . this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations . this study was approved by the local ethics and research committee ( research protocol 1110/07 ) . this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) . the inclusion and exclusion criteria were the same as those of the core study ( 9 ) . all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer . quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) . the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol . radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 " between the tube and the film . the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures . they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached . the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 . each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height . the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer . the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale . the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight = bmi<25 ; overweight = 25bmi<30 ; and obese = bmi30 ( 20 ) . information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews . women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) . physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) . regarding concomitant diseases , those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination . systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) . participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) . the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc . bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur . anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) . the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) . we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density . thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) . the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level . the study had 95% power to detect a difference of 10 points in the total qualeffo score . the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies . demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables . the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test . correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) . generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score . variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model . this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations . this study was approved by the local ethics and research committee ( research protocol 1110/07 ) . the demographic , anthropometric and clinical data for all participants in the study , grouped based on the presence ( vertebral fracture ) or absence of vertebral fractures ( no vertebral fracture ) , are shown in table 1 . there were no significant differences with respect to the mean bmi or the percentage of caucasian individuals between the groups ( p>0.05 ) ( table 1 ) . regarding the bmi classification , 22.8% and approximately 38% of the subjects were classified as normal and obese , respectively , with no significant difference between the two groups ( p>0.05 ) . a tendency of older age in the vertebral fracture group was observed ( p = 0.057 ) . the frequencies of hypertension ( p = 0.224 ) , diabetes ( p = 0.672 ) , hypothyroidism ( p = 0.723 ) , and two or more concomitant diseases ( p = 0.216 ) and the average number of medications used ( p = 0.497 ) were comparable between the groups . interestingly , the vertebral fracture group contained a higher frequency of women defined as chronic fallers than the no vertebral fracture group ( 64.7 vs. 32.1% , p = 0.017 ) . as expected , the vertebral fracture group had a higher frequency of osteoporosis ( 73.2 vs. 51.1% , p = 0.012 ) and a lower frequency of osteopenia ( 19.5 vs. 38.1% , p = 0.027 ) than the no vertebral fracture group . the results for each domain of the qualeffo and the total qualeffo score in both groups are shown in table 2 . the total qualeffo score was lower in the vertebral fracture group than in the no vertebral fracture group [ 61.4 ( 15.4 ) vs. 67.1 ( 14.2 ) , p = 0.031 ] . likewise , the physical function domain score of the qualeffo was worse in the vertebral fracture group compared with the no vertebral fracture group [ 69.5 ( 20.1 ) vs. 77.3 ( 17.1 ) , p = 0.018 ] . no difference was observed regarding the other qualeffo domains ( pain , social functioning , health perception , and mental functioning ) ( table 2 ) . the total qualeffo score was inversely related to bmi ( rs = -0.21 , p = 0.005 ) and weight ( rs = -0.22 , p = 0.009 ) . the total qualeffo score was worse in women classified as obese than in those classified as overweight or normal [ 61.7 ( 15.4 ) vs. 66.4 ( 13.8 ) vs. 70.8 ( 13.5 ) , respectively , p = 0.008 ] . a lower total qualeffo score was also observed in women with low physical activity than in those with moderate or high activity [ 51.8 ( 19.1 ) vs. 64.8 ( 14.3 ) vs. 70.3 ( 13.2 ) , respectively , p = 0.010 ] ( table 3 ) . similarly , the physical function domain of the qualeffo was inversely related to the bmi ( rs = -0.24 , p = 0.001 ) . in this domain , women classified as obese were found to have lower scores than women classified as overweight or normal [ 70.7 ( 19.3 ) vs. 76.1 ( 16.1 ) vs. 81.4 ( 17.4 ) , respectively , p = 0.002 ] . finally , lower scores for the physical function domain were found among women with low physical activity compared with those with moderate or high activity [ 49.6(24.9 ) vs. 75.1(18.1 ) vs. 80.7(12.6 ) , respectively , p = 0.002 ] ( table 3 ) . a generalized linear model with gamma distributions and logarithmic link functions was developed to identify patient characteristics that were related to the total and physical function domain scores of the qualeffo . variables with a p<0.10 in the univariate analysis ( age , bmi classification , physical activity , diabetes , presence of at least one fracture ) were included as independent variables . the presence of obesity was negatively associated with the total qualeffo score ( p = 0.001 ) . a high physical activity level was positively associated with the total qualeffo score ( p = 0.001 ) . the presence of at least one fracture was associated with a worse total qualeffo score , independent of age , bmi classification and physical activity level ( p = 0.030 ) . likewise , the presence of at least one fracture was negatively associated with the physical function domain , independent of these same variables ( p = 0.041 ) ( table 4 ) . this study was the first study conducted in brazil that specifically assessed the impact of vertebral fractures on the quality of life in older , community - dwelling women using a specific questionnaire . in this study , we demonstrated that the presence of vertebral fractures in this population is related to worse hrqol , particularly with respect to physical functioning . the major advantage of the present study is the homogenous selection of community - dwelling women , unlike previous studies in which individuals were recruited from clinics or from populations included in clinical trials . studies showing worse hrqol in patients with vertebral fractures have been published in several countries ( 15,16 ) . there are only two studies evaluating the hrqol in patients with vertebral fractures in brazil , but neither was specific to older community - dwelling individuals ( 17,18 ) . the first of these two other studies was performed in 55 outpatient women divided into three groups : 1- women without osteoporosis , 2- women without osteoporosis and no vertebral fractures , 3- women with osteoporosis and vertebral fractures . in that study , the quality of life was assessed with the sf-36 , and no difference was found among the three groups . one of the reasons for this finding was the inclusion of only women who were able to perform the spirometric tests , resulting in the exclusion of women in worse conditions who would most likely belong to the fracture group ( 17 ) . later , de oliveira et al . evaluated the quality of life in ambulatory women with osteoporosis and found similar results for those who had vertebral fractures and those who did not . however , that study was not designed to assess the impact of vertebral fractures on quality of life , and the number of women with fractures was too small to enable an accurate assessment ( 18 ) . the assessment of quality of life in relation to the qualeffo pain domain was similar in women with and without vertebral fractures in our study . some studies have found that the pain domain is worse in women with fractures ; however , the patients included in those studies were recruited based on clinical symptoms related to fractures and were compared with those without back pain ( 28 ) . vertebral fractures do not always manifest with symptoms and are often diagnosed based on radiographs taken for other reasons . evaluated the chest radiographs of older women who had been hospitalized for several causes , and they found that only a few of the vertebral fractures present had been previously identified by clinicians ( 29 ) . indeed , in our previous study performed in brazil , 29.4% subjects had vertebral fractures , and none of these patients had prior knowledge of their vertebral fractures ( 9 ) . as observed in other studies , the mental function , social function , and health perception domains were not significantly different between women with and without fractures ( 30 ) . the relatively small differences between the groups with and without fractures may be the result of the acceptance of poor health conditions due to the natural expectation of physical decline in older women ( 13 ) . in our study , we found that obesity and low physical activity were associated with lower quality of life . other authors have reported that higher bmi and a sedentary lifestyle are factors that influence the quality of life in patients with osteoporosis ( 18,31 - 32 ) . it is important to highlight the fact that both obesity and a sedentary lifestyle are preventable factors and can be controlled by a change in lifestyle . some authors observed that physical exercise is associated with a better quality of life ( 33 ) and have demonstrated that a home exercise program for women with vertebral fractures ( 60 min / d , 3x / week for 12 months ) significantly improves the hrqol ( 34 ) . a study performed at our institution in osteoporosis patients showed that an exercise program improved the quality of life in these women ( 35 ) . therefore , our data reinforce the need for all older women to be advised about the benefits of exercise . these women should be encouraged to exercise regularly to reduce their bmi and improve their general welfare . our study is the first in brazil to conduct a thorough assessment of the relationship between quality of life and vertebral fractures . an important characteristic of this study was the use of standardized and reliable instruments for both the vertebral fracture assessment ( genant semi - quantitative method ) and the hrqol assessment ( qualeffo ) . although the qualeffo has not been validated in brazil , which is a limitation of the present study , this tool is the most frequently used for assessing quality of life in osteoporosis and was recommended for the investigation of vertebral fracture subjects in multicentric studies that included centers in brazil ( 36 ) . this questionnaire is more sensitive in detecting differences between groups , and it provides a better discrimination between individuals with and without vertebral fractures compared with generic hrqol instruments , such as the sf-36 , particularly with respect to physical functioning , which is significantly affected in these patients ( 8) . in conclusion , this study demonstrated the negative impact of vertebral fractures on the quality of life in older women , independent of other factors such as bmi and physical activity . although the clinical relevance of vertebral fractures is well established , these results are important for assessing the burden of this disease and reinforce the need to reduce the underdiagnosis and undertreatment of these fractures . the results of this study also highlight the need for awareness of the importance of maintaining proper weight and promoting changes in lifestyle through physical activity and dietary control . this study was supported by fundao de amparo e pesquisa do estado de so paulo ( fapesp ) # 03/09313 - 0 ; conselho nacional de cincia e tecnologia ( cnpq ) # 305691/2006 - 6 ( rmrp ) and # 119601/2009 - 5 ( lf ) ; federico foundation grants ( rmrp ) ; and coordenao de aperfeioamento de pessoal de nvel superior ( capes ) ( jbl , cpf ) .

objectives : the aim of this study was to investigate the impact of asymptomatic vertebral fractures on the quality of life in older women as part of the sao paulo ageing & health study.methods:this study was a cross - sectional study with a random sample of 180 women 65 years of age or older with or without vertebral fractures . the quality of life questionnaire of the european foundation for osteoporosis was administered to all subjects . anthropometric data were obtained by physical examination , and the body mass index was calculated . lateral thoracic and lumbar spine x - ray scans were obtained to identify asymptomatic vertebral fractures using a semi - quantitative method.results:women with asymptomatic vertebral fractures had lower total scores [ 61.4(15.3 ) vs. 67.1(14.2 ) , p = 0.03 ] and worse physical function domain scores [ 69.5(20.1 ) vs. 77.3(17.1 ) , p = 0.02 ] for the quality of life questionnaire of the european foundation for osteoporosis compared with women without fractures . the total score of this questionnaire was also worse in women classified as obese than in women classified as overweight or normal . high physical activity was related to a better total score for this questionnaire ( p = 0.01 ) . likewise , lower physical function scores were observed in women with higher body mass index values ( p<0.05 ) and lower physical activity levels ( p<0.05 ) . generalized linear models with gamma distributions and logarithmic link functions , adjusted for age , showed that lower total scores and physical function domain scores for the quality of life questionnaire of the european foundation for osteoporosis were related to a high body mass index , lower physical activity , and the presence of vertebral fractures ( p<0.05).conclusion : vertebral fractures are associated with decreased quality of life mainly physical functioning in older community - dwelling women regardless of age , body mass index , and physical activity . therefore , the results highlight the importance of preventing and controlling asymptomatic vertebral fractures to reduce their impact on quality of life among older women .

the majority of women with menorrhagia , postcoital bleeding , intermenstrual bleeding , or postmenopausal bleeding ultimately undergo diagnostic hysteroscopy with endometrial sampling as part of their assessment , particularly if symptoms persist or pelvic imaging suggests a uterine abnormality . dilatation and curettage ( d&c ) has been widely considered to be the method of choice for obtaining endometrial samples for histopathological evaluation . however , the needs for admission and general anesthesia and their associated costs have made this option less favorable . in the outpatient setting , endometrial sampling is an effective and acceptable method for obtaining endometrial samples for histopathological assessment [ 3 , 4 ] . inadequate sampling is more problematic in postmenopausal women , for whom up to 68% of endometrial samples are reported to be inadequate . in our institution , the only sampling tool available to perform the outpatient sampling procedure is the uterine explora model i - mx120 ( http://www.coopersurgical.com/ ) ( figure 1 ) . in addition , the device is sterile and disposable ( one - time use ) . the advantages of using explora rather than d&c as a sampling device include a reduction in hospitalization costs , extra convenience for the patient and physician , and the minimal complications of the procedure . the purpose of this study is to compare the effectiveness of the explora model i tool with the conventional d&c technique for obtaining adequate endometrial samples that are capable of providing specific and informative histopathologic diagnoses . after obtaining the approval of our institutional review board , all endometrial samples received at the histopathology department in king khalid university hospital ( kkuh , riyadh , ksa ) between january 2007 and december 2010 were included in this study . a total of 1270 endometrial samples were included ( table 1 ) . two hundred seventy - four samples ( 21.6% ) were obtained by conventional d&c in the surgical theater , while the remaining 996 samples ( 78.4% ) were obtained by senior obstetrics and gynecology residents who used a standardized biopsy technique in the outpatient procedure rooms . during the usage of the explora model i , the syringe provided with the instrument was used to create a negative pressure , and the explora was rotated as it was withdrawn . after withdrawal , the tip was cut off , and the tissue was placed in 10% buffered formalin saline fixative and was sent for pathological examination . the pathologists who interpreted the endometrial samples were blinded to the instrument or method used to obtain the samples . an inadequate sample was defined as consisting of only blood , cervical mucus , endocervical epithelium , or blood with fragments of endometrial glands or stroma insufficient for histopathological assessment and diagnosis . the age , gravidity , parity , menstrual history , uterine size , hysteroscopy findings ( when available ) , and the presence or absence of any cervical abnormality were recorded on the request forms , which were reviewed by the investigators . for each of the two methods used ( explora model i and d&c ) , the numbers and percentages of inadequate samples and age group clustering were calculated and statistically analyzed . of the 1270 endometrial samples obtained , 253 samples ( 19.9% ) were scored as inadequate . of these samples , the explora sampler was used to collect 224 samples ( 88.5% ) , whereas 29 samples ( 11.5% ) were obtained by d&c ( figure 2 ) . thus , the insufficient tissue percentage was higher with the explora ( 17.6% ) than with d&c ( 2.2% ) , which was a statistically significant difference ( p < 0.0001 ) . age group clustering ( i.e. , numbers of premenopausal and postmenopausal women ) of inadequate sample results was also calculated ( figure 3 ) . of the 253 inadequate samples , 82.6% were from women 45 years of age and older ( i.e. , postmenopausal ) compared to 17.4% in premenopausal women ; the age difference was significant ( p < 0.0001 ) . the detection rates of endometrial hyperplasia and carcinoma using both methods were assessed and calculated . of the 73 samples with a diagnosis of endometrial hyperplasia , 50 ( 68.5% ) were diagnosed by d&c , and 23 ( 31.5% ) were diagnosed using the explora sampler . however , of the 18 samples with a diagnosis of endometrial cancer , the rates of detection were similar between the two methods . endometrial sampling for the evaluation of dysfunctional uterine bleeding and the diagnosis of endometrial hyperplasia and carcinoma and other indications remains one of the most commonly performed gynecological procedures [ 14 ] . in recent years , less hazardous and more inexpensive and convenient outpatient sampling methods have replaced the traditional , in - hospital , endometrial curettage . the advantages of outpatient endometrial biopsy include reduced cost and less risk for the patient , as no anesthesia is required . furthermore , the discomfort and pain produced by sampling have been reported to be minimal . however , it is essential to ensure that outpatient endometrial sampling is quantitatively adequate and comparably accurate to conventional dilatation and curettage . a sample is judged as adequate if a specific diagnosis can be given from the histological examination of the endometrial fragments obtained . adequacy can be measured by comparison of either outpatient biopsy with curettage histological evaluation or outpatient biopsy with the results of pathological examination of hysterectomy specimens [ 3 , 4 ] . many techniques for obtaining an endometrial sample without the need for curettage have been described in the literature . chicago , il , usa ) and the novak biopsy curette with a 10 ml syringe functioning as an aspiratory device , which have been shown to be equally effective compared to d&c in detecting an endometrial pathology [ 69 ] . however , the vabra aspirator and novak biopsy curette , although widely available and relatively inexpensive , have several disadvantages , including the need for an electric vacuum pump to perform the aspiration in the former technique and the pain caused by both methods . as a result of these drawbacks , smaller inexpensive and self - contained instruments have been developed and the prototype of this class of endometrial samplers is the pipelle . the pipelle has been shown to have a diagnostic accuracy comparable to that of vabra aspiration and the novak curettage while causing less pain [ 911 ] . all of these instruments ( i.e. , the vabra aspirator , the novak biopsy curette , and the pipelle ) have low rates of false - negative and insufficient tissue results for the detection of endometrial abnormalities , as determined by comparison to hysterectomy specimens [ 1113 ] . it was found that pipelle biopsy had a sensitivity of 99.2% in pinpointing high grade cancer and a sensitivity of 93% in detecting low grade malignancies ; the sensitivities defined for d&c were 100% and 97% , respectively . while excellent agreement was generally noted between preoperative histology and grade and the final pathology , pre - operative endometrial sampling more commonly provided underestimates of final grade ( low grade versus high grade ) than overestimates . the explora is somewhat similar in its design to the pipelle , but clinical studies on its effectiveness are scarce , with the effectiveness ranging between 14.6 and 15% according to various studies [ 6 , 15 ] . our own findings revealed that the rate of obtaining inadequate samples using the explora was much higher ( 17.6% ) than the rates reported in the literature . however , most of these cases ( 82.6% ) were obtained from postmenopausal women with atrophic endometrial status . this finding is in keeping with the rates reported by other investigators [ 58 , 16 ] . this retrospective study suggests that traditional d&c produces better endometrial sample adequacy than the explora technique . this finding indicates that clinicians performing endometrial sampling would benefit from more experience and training using the explora technique . additional studies comparing the adequacy of samples obtained with different endometrial sampling techniques and devices are warranted . furthermore , we recommend using the d&c procedure when the explora - obtained samples are inconclusive or when the use of the explora sampler is accompanied by ultrasound findings that are suspicious of endometrial hyperplasia or carcinoma .

aims . our aim is to compare the adequacy and diagnostic yield of samples obtained by the endometrial explora sampler i - mx120 with endometrial specimens obtained by conventional dilatation and curettage ( d&c ) . methods . a total of 1270 endometrial samples were received in the histopathology laboratories at the king khalid university hospital , riyadh , saudi arabia , between 2007 and 2010 . in the outpatient clinic , the uterine explora model i was used to obtain 996 samples . the remaining 274 samples were obtained by conventional d&c . sample adequacy and the clustering of inadequate specimens according to age groups by the two different techniques were compared and statistically analyzed . results . out of 1270 endometrial samples , 253 ( 19.9% ) were inadequate . the uterine explora was used in 88.5% of these inadequate samples ( 253 samples ) , and the remaining 11.5% were obtained by d&c . the insufficient tissue incidence was higher with the explora ( 17.6% ) than with the d&c ( 2.2% ) and the difference was statistically significant ( p < 0.0001 ) . the ages of the patients , as well as the clinical indications for the procedures , were recorded . conclusion . this retrospective study demonstrated better specimen adequacy when d&c was used compared to the higher rate of sample insufficiency obtained with the explora .

the geological and ( palaeo-)biological evolution of lowland amazonia during neogene and quaternary times remains fascinating since the early days of natural sciences ( for summaries of research history see e.g. , loczy , 1963 and wesselingh , 2008 ) . many , but partly highly controversial models have been introduced to explain its historical development ( for recent compilations see e.g. , lundberg et al . , 1998 ; , 2006 ; wesselingh and salo , 2006 ; rossetti and mann de toledo , 2007 ; haffer , 2008 ; hoorn and wesselingh , 2010 ; hoorn et al . , 2010 ; generally , it is widely accepted that around the onset of the miocene ( 23 ma ) a mega - wetland ( pebas system , also called lake pebas ) developed in western amazonia due to the subsiding subandean foreland ( e.g. , hoorn , 1994 , 2006 ; wesselingh et al . , 2002 ; wesselingh and salo , 2006 ; shephard et al . , 2010 ) . short - lived marine incursions or even a transcontinental seaway from the caribbean sea through the venezuelan / columbian llanos basin southwards to the argentinean paran basin are proposed ( e.g. , rsnen et al . , 1995 ; 2009 ) but heavily disputed ( e.g. , campbell et al . , 2006 ; cozzuol , 2006 ; westaway , 2006 ; latrubesse et al . , 2007 , 2010 ; , 2003 ; hoorn and vonhof , 2006 ; wesselingh , 2006 ) . in the late miocene this mega - wetland disintegrated due to enhanced uplift of the northern / central andes . the drainage pattern of northern south america started to reverse completely to today s easterly course and the modern amazon system became established during the early pliocene ( hoorn , 2006 ; figueiredo et al . , 2009 ; hoorn et al . , 2010 ; latrubesse et al . , 2010 ) . beside the vast size of amazonia and the still fragmentary regional coverage with field surveys , there are considerable inconsistencies in palaeoenvironmental reconstructions and , in particular , in the chronology and correlation of scattered outcrops . wesselingh ( 2008 , p. 5 ) stated : the lack of geological data has led to the emergence of many grand theories about the origin of present - day amazon system and its highly diversity , often based on dubious interpretations of the little data available . the present paper aims to contribute basic sedimentological data from a barely studied region ( eirunep , 2.000 km sw manaus ; fig . 1a ) , which is supposed to be placed at the south - eastern margin of the pebas system ( wesselingh and ramos , 2010 ) . we demonstrate that there is no evidence for a long - lived lake ( sensu gorthner , 1994 ) or any marine influx . conversely , we document a well - structured , aggrading fluvial system of late miocene age , which is in agreement with the sedimentation model and chronology proposed by latrubesse et al . the studied sections are located along the juru and tarauac river , ne respectively se of the city eirunep ( state of amazonia , western brazil ; 245 km s of benjamin constant ; fig . delineations and subdivisions of basins in western amazonia diverge notably and several authors attribute the region of eirunep to the solimes basin ( eirunep subbasin ; e.g. , caputo , 1991 ; roddaz et al . , 2005 ; ramos , 2006 ; wesselingh and salo , 2006 ; wesselingh et al . , however , based on subsurface information , obtained by extensive hydrocarbon and coal exploration campaigns during the 1970 s , this region is situated west of a basement high ( miura , 1972 ; iquitos arch ; corresponds to the carauari arch of caputo , 1991 ) on the eastern margin of the acre basin ( e.g. , del arco et al . , 1977 ; maia et al . , 1977 ; latrubesse et al . , 2010 ; fig . beside quaternary deposits ( terraces , alluvium ) , the scattered outcrops along river banks expose sediments of the upper parts of the solimes formation ( del arco et al . , 1977 ; maia et al . , 1977 ; paz et al . , in press ) . the solimes fm . comprises more than 1000 m thick , largely pelitic sandy alternations with lignitic intercalations and covers a huge part of western amazonia ( fig . . uncertainties in its definition , its stratigraphical and geographical extent as well as its depositional environments basically reflect the ongoing debate about amazonia s history through neogene times . ( 1977 ) , hoorn ( 1993 , 1994 ) , latrubesse et al . the sections were vertically logged by visual inspection of the lithofacies ( including colour , grain size , bedding planes , sedimentary structures , macrofossil content ) . for lithofacies coding the scheme of miall ( 1996 ) was used : capital letters = dominant grain size ( g , gravel ; s , sand ; f , fine sand clay ) followed by a lowercase letter = sedimentary structures and/or biogenic features ( c , clast - supported ; m , massive / faint lamination ; h , horizontal bedding / lamination ; t and p , trough and planar cross bedding ; r , ripple bedding ; s , scour fill ; l , lamination ; r , rooted ; c , coal ; p , pedogenic overprint ) . additionally the outcrops were mapped laterally as far as possible . due to poor outcrop conditions an application of the architectural element concept ( miall , 1996 ) was only loosely possible . for micropalaeontological investigations bulk samples ( 12 kg ) were taken from all outcrops . 500 g of dried sediment ( 40 c , 24 h ) were washed by using diluted hydrogen superoxide for disintegration through standard sieves ( h2o2 : h2o = 1 : 5 ; 63/125/250/500 m ) . wet sieve residuals were washed with ethanol prior to drying ( 40 c , 24 h ) . an in - depth taxonomic examination of the obtained microfossils ( including the sieve fractions < 250 m ) will be subject of further studies . for preliminary stable isotope analyses ( o , c ; 18 samples ) two or three ostracod valves ( 4060 g ; cyprideis spp . ) from earlier sampling campaigns were used ( collected by m.i.r ) . for analyses a thermo - finnigan kiel ii automated reaction system and a thermo - finnigan delta plus isotope - ratio mass spectrometer were used ( university of graz ; standard deviation = 0.1 relative to nbs-19 ; results in per mille relative to vpdb ) . more detailed investigations are in preparation ( m. fonseca / belm , m. caporaletti / graz ) . location : 12.6 km ne eirunep ( s 063355.5/w 0694611.7 , altitude : 108 m ) , left cutbank of the juru river ( fig . 2 ) . description : the basal 1.8 m ( layers 19 ) of the section consists of partly ripple bedded sands and laminated silts ; some cm - thick clay intercalations were observed . above follows a 0.65 m thick alternation of clay with incorporated calcareous nodules and laminated silty sand or ripple bedded sand ( 1017 ; fig . the top is rich in vertebrate remains ( crocodiles , turtles ; 17 ; fig . massive to finely laminated , occasionally bivalves - bearing ( silty ) clay with coaly and sandy layers forms the next 1.14 m ( 1823 ; fig . beds 2425 ( 1.85 m thick ) comprise massive , horizontally or trough cross bedded sand and finely laminated or massive ( sandy silty ) clay / fine sand ( fig . grey mottled , rooted and massive clay ( paleosol ) with a calcareous horizon on its top ( 26 ) . up - section , a 4.2 m ( 2739 ) thick alternation of massive or laminated clay , silt and partly ripple bedded fine sand is developed ( a slight coarsening - upward trend is anticipated ) . bed 32 displays a slightly erosive base with intraclasts in the lower part ( mud pebbles ) . layers 3139 are cut by a channel ( 40 ) with a multiphase fill . large scale cross bedded sands ( -cross stratification ) with sporadic intraclasts ( mud pebbles ) on reactivation surfaces and trough cross bedded and ripple bedded fine microfossil contents : samples from the basal part of the section ( pd2 ) yielded only scarce valves of cyprideis spp . and rare fish elements . interpretation : the lithofacies of the basal part of the outcrop ( 117 ) refers to a deposition in a fluvial overbank environment . the diffuse layers of calcareous nodules ( 17 ) are supposed to be largely a diagenetic feature ( groundwater caliche ) . however , it may also hint to soil - forming processes at seasonally dry floodplain areas ( retallack , 2001 ) . up - section the sediments become finer and contain several ( sandy silty ) coal - rich layers ( 1823 ) . these beds are assigned to the formation of a floodplain pond , which was inhabited by opportunistic ostracods , freshwater bivalves and fishes . however , crevasse splays influenced episodically that shallow lacustrine environment ( sandy and organic matter - rich layers ) . later ( layers 2425 ) , the influx of crevasse splays into the pond , which is still populated by ostracod faunas and fishes increased ( more prominent sandy beds , partly scour - fills ) . pedogenic processes are indicated by incipient soil formation in layer 26 ( mottled , roots ) . floodplain pond conditions ended above ( beds 2739 ) due to enhanced clastic input from an approaching channel . finally ( bed 40 ) , the overbank fines were intersected by a channel of at least 23 m in depth , which was filled up by large scale cross bedded sand . more or less perpendicular to the main accretion surfaces oriented ripple bedding indicates deposition by lateral accretion of a point bar . location : 17.9 km ne eirunep ( s 063251.1/w 0694239.4 , altitude : 107 m ) , left cutbank of the juru river ( fig . 4 ) . description : the lower 5 m of the outcrop are formed by ( section 1 ) : ( 1 ) greenish grey , mottled clay with ferran cutans and cm - sized carbonate nodules ( fig . 5a ) ; ( 2 ) dark grey , massive or indistinctly laminated clay with cm - sized carbonate concretions ( fig . 5b ) , thin , pyrite - rich coaly layers , plant fragments , root traces and vertebrate remains ( crocodiles , turtles ) ; ( 3 ) greenish yellowish , mottled clay with ferran cutans ( passes gradually into bed 4 ) ; ( 4 ) violet - red green - grey , mottled clay / paleosol ( fig . 5c ) ; and ( 5 ) green grey , mottled clay with vertebrate remains ( reptiles ) on top . a trough cross bedded sandy layer with coaly interlayers in the se part of the site mentioned by paz et al . ( in press ) was not exposed during our observation ( section 4 ) . in the nw part ( section 1 ) , above layer 5 , ripple bedded fine sand with intraclasts ( mud pebbles ) at its base is recorded , which contains bivalves and coalified wood remains ( 6 ) . laminated sand alternations ( 7 ) and massive sandy clay , which is rich in molluscs in its lower and upper part ( 8) . up - section ( 9 ) , an alternation of clay and coal layers , rich in molluscs ( i.e. , mycetopoda sp . ) , as well as a thinly bedded succession of laminated sand and thin clay interlayers ( 10 ) follow . towards the se ( section 24 ) an up to 2 m thick , large scale cross bedded sandy unit is developed ( fig . occasionally , intraclasts and vertebrate fragments are found at the base of this element or on accretion surfaces , which dip around ne . the dip of ripple foresets is approximately perpendicular oriented to these surfaces ( sw ) . this unit is overlain by a layer of calcareous nodules ( 11 ; section 3 ) and laminated clay ( silt)coal alternations with bivalve and vertebrate remains ( 13 and 14 = equivalent to 9 ) . bed 12 ( plastic , strongly weathered clay ) is an equivalent of layer 10 . microfossil contents : samples mn1 and mn2 lack microfossils , in mn8a and mn8b only scattered valves of cypria sp . and heterocypris ? mn9 and mn 14 yielded many fish , gastropod and bivalve ( sphaeriids ) remains as well as abundant ostracod valves ( cyprideis spp . ) . earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) documented the following ostracod taxa : cyprideis graciosa , cyprideis lacrimata , cyprideis longispina , cyprideis machadoi , cyprideis pebasae , cyprideis olivencai , cyprideis sp . 1 and 2 . further findings ( plants , fishes , reptiles ) are mentioned in del arco et al . the later authors also reported the occurrence of cyathecidites annulatus , echitricolporites spinosus and grimsdalea magnaclavata pollen . greenish to yellowish and dark grey colouration might indicate more or less water - logged conditions , whereas reddish colours may hint to rather well - drained , oxidizing environments . however , diagenetic alteration can hamper such an interpretation significantly ( retallack , 2001 ) . layer 2 is less affected by pedogenic processes ( faint lamination , coaly layers , plant and reptile fragments ) and perhaps represents floodplain lake / backswamp deposits . the paleosols ( overbank fines ; 15 ) are overlain by a sandy channel fill ( point bar ) with lag deposits at its base and repeatedly occurring intraclasts at its lateral accretion surfaces ( channel depth > 2 m based on the preserved height of this unit ) . in the nw part of morada nova layers 68 represent an abandoned channel fill , which could be the fill of a subordinate channel or the fill of a chute channel . up - section , the influx of the active channel decreased and a floodplain lake developed ( 9 , 13 , 14 ) , which enabled the establishment of plentiful ostracod and aquatic molluscs faunas . the pond itself or its surroundings were richly vegetated ( coaly layers ) and settled by semi - aquatic reptiles . finally , enhanced input of sandy matter points to an increasing influence of the channel , probably due to crevasse splays . ( s 063140.8/w 0693952.0 ; altitude : 106 m ) , left cutbank of the juru river ( fig . description : section 1 ( w part ) starts with a 1.6 m thick succession of laminated , ripple and cross bedded silty sand with coaly intercalations ( layers 16/1 ) . up - section ( 714/1 ) , a 2.5 m thick sequence of ripple bedded silty fine sand layers with commonly erosive basal boundaries ( incl . convolute bedding and m - thick concretions are frequent in its upper part ( 1114/1 ) . silty clay ; 1520/1 ) and comprise coal and mollusc - rich pelitic ( 16/1 , 19/1 ) and coaly layers ( 18/1 ) as well as carbonate - cemented clay ( 20/1 ) . layer 21/1 ( 0.75 m thick ) is composed of greenish violet , mottled or poorly laminated clay with scattered bivalve shells . intercalated are several layers ( 10 cm - thick ) , which consist of 12 mm large , rounded clay clasts ( fig . it is overlain by 1 m of massive clay with abundant mollusc ( e.g. , sheppardiconcha septencincta ; fig . leaves ; 22/1 ) and a > 0.5 m thick ( weathered ) coaly layer ( 23/1 ) with mollusc fragments ( e.g. , anodontites ? section 2 ( e part ; 120 m downstream of section 2 ) starts with > 0.35 m thick , laminated fine sand with coaly interlayers ( layer 1/2 ) . it is cut by a 4 m wide and 0.8 m deep channel fill ( 25/2 ; fining - upward , from base to top : laminated , coal - rich fine sand with rare mollusc and plant remains ; ripple bedded fine sand , rich in plant fragments ; massive clay , rich in bivalves ( sphaeriids ) , several wood remains ; fig . this channel fill is topped by a 0.5 m thick , massive poorly laminated clay with thin fine sand interlayers ( 6/2 ) and 0.2 m of massive , partly indurated ( cemented ) clay ( 7/2 ) . coalified wood remains are rare ; bivalves ( sphaeriids ) are abundant ( especially upper part of 6/2 ) . layer 6/2 and 7/2 taper off towards the w. up - section ( 1 m thickness ) , follow massive , silty sandy clays ( 89/2 ) and ripple bedded silty fine sand with silt interlayers ( 1014/2 ) . especially layer 8/2 is rich in molluscs ( sphaeriids ) , 1314/2 contain coaly fragments ; clay intraclasts are observed at the base of 13/2 . these beds are topped by 2.7 m of mottled silty fine sand with coaly fragments in the lower 0.3 m ( 15/2 ) . . layer 16/2 ( > 1.2 m thick ) comprises coal coaly fine sand finely laminated clay alternations ( fig . 7f ) and is rich in bivalves ( sphaeriids ) in the lowermost 57 cm . whereas samples aq5/1 and aq6/1 contain only rare ostracod remains , aq16/1 yields , beside fish fragments , some valves of cytheridella sp . and darwinulids , accompanied by rare specimens of cypria sp . , cyprideis spp . and , darwinulids , cypria sp . , ilyocypridids ) and fish bones . several ( aq22a/1 ) respectively rare ( aq22b/1 ) cyprideis spp . valves are recorded in the samples of section 1 above , together with fish remains and gastropods ( aq22b/1 ) . sample aq3/2 of section 2 delivered only rarely cyprideis spp . and some fish remains . in the samples aq 5 - 6 - 8 - 13/2 plentiful ostracod faunas ( cytheridella sp . , ilyocyprinids , darwinulids , cyprideis spp . are documented along with fish remains and sphaeriid bivalve shells ( aq8/2 and aq13/2 ) . aq15/2 and aq16/2 are almost barren of ostracods but contain fish and bivalve ( sphaeriids ) fossils . earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) recorded the following ostracod taxa : alicenula ( darwinula ) fragilis , cypria aqualica , c. longispina , c. pebasae , cyprideis sp . 3 , cytheridella purperae , heterocypris ? ( 2006b ) re - examined mollusc material studied by roxo ( 1937 ) from aquidab and described : ampullariidae sp . sheppardiconcha septemcincta . additional findings ( including vertebrates and plants ) are mentioned by del arco et al . the basal , sandy silty layers ( 16/1 = 1/2 ) possibly represent crevasse splay deposits , which are followed by a series of crevasse splay and/or crevasse channel sediments ( 714/1 respectively 25/2 ) . alternatively , these layers could represent channel deposits of an avulsive river arm . in section 2 a subordinate abandoned channel fill ( floodplain pond ; 67/2 ) is developed . afterwards ( 1523/1 and 816/2 ) the influx of crevassing respectively the active channel successively decreased and led to the formation of a floodplain lake , which was only influenced by flash floods in more proximal settings ( section 2 ; e.g. , 12/2 ) . aquatic faunas ( bivalves , ostracods , fishes ) and semi - aquatic crocodilians inhabited that lake , which was surrounded by a densely vegetated backswamp . these beds display a mottled appearance and contain ( 21/1 ) layers of crumb peds ( clast - supported layers of rounded but not interlocked tiny mud balls ) , which indicate bioturbation due to invertebrate activity ( retallack , 2001 ) . finally , the lake became replaced by a swampy environment ( 23/1 and 16/2 ) . location : 27.4 km ne eirunep ( s 063122.0/w 0693542.8 ; altitude : 105 m ) , left cutbank of the juru river ( fig . description : at the base > 2 m of slightly southwards inclined ( < 10 ) rippled bedded silty fine sand ( layer 1 ) are followed by 2.7 m thick violet - brown ( 2 ) and yellowish ( 3 ) mottled paleosols with calcareous nodules ( between 2 and 3 a pedogenically overprinted clay plug is intercalated ) . layers 13 are cut by a channel , whose fill starts ( 4 ) with > 1.5 m thick , massive poorly laminated silty clay with gastropods , rare bivalves and plant ( leaves ) remains . bed 4 grades into a 0.8 m thick , brownish - grey , blocky structured silty clay ( 5 ) with a calcareous crust on its top . it is overlain by 2.4 m of violet - brown , mottled clay ( 6 ) with intercalated layers of calcareous nodules . strata 13 as well as the channel fill ( 46 ) are topped by a 0.4 m thick alternation of laminated silty clay and laminated or rippled bedded fine medium sand with a high amount of plant detritus ( also wood remains ) and abundant vertebrates ( turtles ; 7 ) . the badly exposed , 7.2 m thick beds up - section ( 8) consist of cross and ripple bedded fine sand with intercalations of clayey lenses ( 2 m lateral extension , 20 cm thickness ) , which are formed by clay intraclasts ( mud pebbles , 1 cm diameter ) . repeatedly coaly layers are intercalated and rarely vertebrate ( crocodiles ) remains were found in the intraclast conglomerate . layer 9 represents a 1.2 m thick alternation of horizontally or ripple bedded fine the topmost 1.2 m thick layer 10 consists of ripple bedded silty fine sand and silty clay alternations , which form cm - thick , fining - upward couplets . microfossil contents : sample re4 yielded only a few valves of cyprideis spp . as well as some fish and gastropod remains . interpretation : layer 1 could represent a crevasse splay or levee deposit , which is overlain by pedogenically altered floodplain fines ( 23 ) with intercalated minor channel fills . these overbank deposits are cut by a channel ( ? 20 m wide and 5 m deep ) , which became filled by pelitic , afterwards pedogenically overprinted sediments . the rare ( a matter of preservation ? ) mollusc and ostracod record indicates at least at the beginning ( 4 ) aquatic life in the abandoned channel . above , a crevasse splay ( 7 ) points to rising proximity of the active channel . large wood remains and vertebrate fragments were accumulated by this splay . the sandy succession up - section ( 8) can only be attributed to a sandy bedform sensu miall ( 1996 ) due to insufficient outcrop conditions . intercalated shallow scours , filled up with mud balls indicate deposition of reworked material and variations of the flow regime . the more or less rhythmically stratified , sandy - pelitic alternations at the top of remanso ( 910 ) mark a shift towards overbank environment and might represent a bar - top assemblage . location : 17.5 km se eirunep ( s 064923/w 0694704 , altitude : 117 m ) , left cutbank of the tarauac river ( fig . description : the basal 1.7 m ( layers 13 ) consist of massive or laminated pelite- and fine sand with rare bivalve remains . they are topped by 0.2 m of reddish - brown , massive clay ( 4 ) . the sandy silty layers 59 display a general coarsening - upward trend and contain only rarely bivalve fragments . up - section ( 10 ) , a 0.25 m thick intraclast conglomerate ( mudstone pebbles ) with abundant mollusc ( e.g. , ampullariidae ? this stratum is overlain by thin sand and clay layers ( 1117 ) with abundant mollusc remains ( 12 , 14 , 16 , lower part of 17 ) . layer 14 is rich in coal fragments ( wood , leaves ) at its top . the 2.57 m thick succession above ( 1823 ) starts with thin , ripple bedded fine sand beds ( 1819 ) , followed by massive silty / sandy clay with coaly fragments ( 20 ) and cm - thick alternations of fine sandy silt and silty fine sand ( 21 ; forming couplets of small - scale fining - upward cycles ) . beds 22 and 23 comprise alternations of massive , fine sandy silt and ripple bedded , silty fine sand . 24 consists of 10 cm - thick alternations of indistinctly ripple bedded silty fine sand and massive or poorly laminated pelite . the upper part ( 1.5 m ) is mottled and primary sedimentary structures are obscured due to pedogenic processes . these samples contain several fish remains and in to12 rare characean gyrogonites were found additionally . the samples to20 and to 23 delivered only rare ostracod ( cypria sp . earlier reports of fossil material and facies : ramos ( 2006 ) mentioned the following ostracod taxa : c. aqualica , c. graciosa , c. lacrimata , c. longispina , c. pebasae , c. purperae , cytheridella sp . , darwinula fragilis as well as additional vertebrate and crab findings . interpretation : the lowermost sandy - pelitic layers ( 14 ) might be deposited in a floodplain pond / lake , which was influenced by crevasse splays . the coarsening - upward trend of layers 59 hints to a progressive influx of a crevasse channel ( layer 10 ) . it remains unclear if the ostracods of this layer are allochthonous or if they are incorporated in that layer due to subaquatic inflow of the crevasse splay ( crevasse delta ) into a floodplain pond / lake . up - section ( 1124 ) , plentiful ostracod and mollusc faunas ( especially in the lower part : 1117 ; compare ramos , 2006 ) indicate the return to an aquatic environment and the abandonment of the crevasse channel . small - scale fining - upward alternations of ripple bedded sand and pelite document individual flooding events or surges . location : 22.1 km sse eirunep ( s 065218.3/w 0694705.1 ; altitude : 120 m ) , left cutbank of the tarauac river ( fig . description : layers 16 represent a 3.2 m thick succession of greenish ( 1 ) , reddish ( 2 ) , yellowish ( 3 , 5 , 6 ) or violet grey ( 4 ) coloured , mottled , frequently calcareous nodules - bearing paleosols . in layer 6 vertebrate remains ( crocodile teeth ) and gastropods were found . above follows a 1.1 m thick , indistinctly bedded clay to fine sand ( 7 ) , which contains in its lower half coaly plant fragments and becomes towards the top and laterally progressively mottled ( paleosol ) . the 0.65 m thick sediment column up - section ( 811 ) is composed of massive clay ( 8) , ripple bedded fine sand ( 9 ) , laminated silty fine sand to silt ( 10 ) and fine sand ( 11 ) . each of these layers displays a slightly erosive base and an internal fining - upward trend . the beds 8 , 9 and 11 each start with a basal layer of concretionary mud clasts in which ( layer 8 and 11 ) abundant vertebrate remains were found . layer 12 is formed by 1.2 m thick , ripple bedded fine sand with cm - thick , laminated silty clay interlayers . up - section a 0.7 m thick , orange - grey , mottled paleosol with mud clasts and rare vertebrate remains at its base ( 13 ) as well as a > 1.5 m thick , violet - purple coloured , mottled paleosol are developed ( 14 ) . microfossil contents : the samples ba7a and ba7b contained several ostracod valves ( mainly cyprideis spp . , some specimens of cypria sp . interpretation : the basal layers ( 16 ) represent a succession of paleosols , which could be seriously altered floodplain deposits . due to the occurrence of ostracods , probably bed 7 was deposited in a floodplain lake environment . afterwards , a series of crevasse splay ( maybe partly also crevasse channel ) deposits follows ( 813 ) , which is topped by pedogenically overprinted floodplain fines ( 14 ) . the sedimentary record of the investigated outcrops documents vertically as well as laterally highly variable fine - grained clastic successions ( predominantly clay fine sand , along with fine medium sand ) . based on the lithofacies and , as far as possible , lateral observations , these sediments represent fluvial deposition within active channels as well as in overbank environments . macroforms , generated within the active channel include lateral accretion deposits ( sandy point bars ; figs . 2 and 4 ) and undifferentiated sandy bedforms ( stacked dune fields ? ; fig . 8) , which are probably partly intersected by chute channels or are overlain by overbank fines . these comprise pelite sand - alternations of levee , crevasse splay and crevasse channel deposits . frequently , a lag of mud pebbles and vertebrate fragments is found at the base of crevasse channels / splays . in part , some splays may have entered floodplain ponds / lakes via crevasse deltas ( e.g. , figs . 6 and 9 ) . abandoned , fine - grained channel fills ( mud plugs ) were observed ( fig . decimetre - thick , clay silt beds with rich ostracod and mollusc faunas indicate the presence of short - lived , shallow ponds or lakes within the floodplain ( floodbasin ; e.g. , fig . 6 ) point to partly swampy , poorly - drained conditions and a densely vegetated floodplain , which is , however , influenced by high clastic input . intensively mottled paleosols with root casts and calcareous nodules are ubiquitous ( e.g. , fig . differences in colour and occasional calcic horizons may reflect diverging water - logging due to e.g. , local topography and/or seasonal fluctuations in discharge or climate ( kaandorp et al . nevertheless , diagenesis may have altered significantly primary features and more specific investigations are required ( retallack , 2001 ) . we are aware that restricted outcrop conditions and the limited areal and stratigraphical extent ( 30 m stratigraphical thickness ) of the current investigation hamper conclusions about the nature of fluvial style . reconstructions of fluvial geomorphology , based solely on lithofacies assemblages and fragmentary known fluvial elements , remain to some degree tentative ( miall , 1996 ; bridge , 2003 ) . however , it seems obvious that we are dealing with a suspended load river system due to the high amount of overbank fines , probably within a tropical to subtropical wet dry climate ( kaandorp et al . , 2005 ; latrubesse et al . we observed : ( a ) a large proportion of overbank deposits , ( b ) laterally extensive , heterogeneous , fine - grained avulsion deposits ( including crevasse splays ) , ( c ) predominantly sandy channel deposits , which change laterally and vertically rapidly into overbank deposits as well as ( d ) lacustrine and coaly deposits . some sandy point bars ( of secondary channels ? ) indicate subordinate lateral sediment accretion . these features are not exclusive to anastomosing river deposits , however , they largely coincide with the standard model of an anastomosing river sensu miall ( 1996 ) as well as with characteristics of such a system as reviewed by makaske ( 2001 ; compare also smith , 1983 ) . the general setting ( subsiding foreland basin , extensive sediment input from the andes , possibly incompletely framed by tectonically active basement highs towards the east ) as well as a wet dry seasonal climate ( seasonal floods , channel banks stabilized by vegetation ) would provide the conditions for anastomosing river systems ( smith , 1983 ; nanson and knighton , 1996 ; makaske , 2001 ; latrubesse et al . ( late miocene ) in the state of acre ( sw brazil ) led to similar assumptions ( latrubesse et al . these authors proposed a megafan system , which originates in the andes and encompass a complex mosaic of avulsive rivers and associated wetlands ( including abandoned channels , floodplain lakes , floodbasin deltas , backswamps and well - drained floodplains ; see also wilkinson et al . this system is suggested to reach to the east at least as far as 67 w ( iquitos arch sensu del arco et al . , 1977 ) and probably also beyond ( purus arch ; latrubesse et al . , 1997 , 2010 ; fig according to that , the sections around eirunep were located in a more distal position , which could be the reason for a more pronounced anastomosing - anabranching pattern due to a lower gradient ( compare also interpretations of the late miocene tariquia formation , southern bolivia ; uba et al . ( 2007 , 2010 ) compared the depositional environment of the late miocene solimes fm . in acre with i.e. , the quaternary megafans of the chaco plain and the pantanal wetland ( iriondo , 1993 ; horton and decelles , 2001 the mollusc fauna , recorded from floodplain pond / lake deposits , is dominated by sphaeriids and the pachychilid sheppardiconcha ( fig . 7b ) and indicates freshwater conditions within a fluvio - lacustrine environment ( wesselingh et al . , 2006b ; wesselingh and ramos , 2010 ) . typical freshwater ostracods ( darwinulids , ilyocyprinids , cypria , cytheridella ) are associated with diverse species of cyprideis . this genus is a holoeuryhaline taxon , able to cope with fluctuating salinities , aberrant water chemistries , variable temperatures and oxygenation ( e.g. , meisch , 2000 ; keyser , 2005 ) . as it could adapt to freshwater conditions ( e.g. , lake tanganyika ; wouters and martens , 2001 ) and co - occurs here with exclusively freshwater taxa , there is no constraint to conclude a brackish water environment ( ramos , 2006 ) . preliminary stable isotope data ( o , c ) measured on cyprideis valves from aquidab , morada nova and torre da lua , yielded very negative values ( o : 5.7 to 9.7 , c : 10.3 to 12.5 ) , which exclude any marine influx . there is still intensive debate on definition and timing of palynological biozonation concepts ( e.g. , hoorn , 1993 , 1994 ; latrubesse et al . , 2007 , 2010 ; jaramillo et al . , 2010 ; silva - caminha et al . , however , palynostratigraphy remains a cornerstone for correlation across amazonia up to now and provides at least a rough stratigraphical framework . ( 2006a ) are highly linked to the concept established by hoorn ( 1993 ) . based on the presence of some index taxa ( c. annulatus , e. spinosus , g. magnaclavata ) a late miocene age ( probably asteraceae zone sensu lorente , 1986 ) is proposed by paz et al . ( in press ) for the outcrops morada nova and torre da lua recently . the vertebrate fauna from torre da lua ( ramos , 2006 ) is related to the so - called acre fauna , which is one of the best documented faunas of northern south america ( cozzuol , 2006 ; latrubesse et al . the acre vertebrate assemblage displays significant affinities with the mesopotamian faunas of argentina and uruguay as well as with the urumaco assemblages ( venezuela ) and is dated to the late miocene ( huayquerian south american land mammal age ; cozzuol , 2006 ; latrubesse et al . is still poor and has to be extended by later works ( wesselingh and ramos , 2010 ; see roxo , 1937 ; wesselingh et al . , 2006b ; wesselingh , 2008 , and outcrop descriptions herein ) . from a stratigraphical point of view the mollusc faunas of aquidab and torre da lua ( 2006b ) to be of late miocene origin and post - date the pebas fauna ( compare celestino and ramos , 2007 ; wesselingh , 2008 ; wesselingh and ramos , 2010 , which suggested a late middle miocene age based on the ostracod record ) . a late miocene age seems to be in agreement with the lack of typical endemic pebasian molluscs ( wesselingh and ramos , 2010 ) . the recorded cyprideis - species from aquidab , morada nova and torre da lua ( ramos , 2006 ; celestino and ramos , 2007 ; wesselingh and ramos , 2010 ) have generally long ranges according to muoz - torres et al . ( 1998 , 2006 ; see also whatley et al . , 1998 ) . the occurrence of c. lacrimata and c. pebasae might be indicative for an age not younger than the ( early ) late miocene . nonetheless , due to some contradictions ( e.g. , c. lacrimata is supposed to appear first after the last appearance of c. longispina but both are co - occurring at morada nova and torre da lua ) and the lack of key taxa , the current ostracod zonation needs some adjustment and is not further stressed here . however , to date , a late miocene age seems most plausible for all outcrops described herein by considering the palaeontological record . moreover , this fits well to the palaeogeographic context of this region as proposed by latrubesse et al . sedimentological observations derived from outcrops around eirunep ( south - western part of the state of amazonia ) document various subenvironments of a fluvial system ( upper part of the solimes formation , late miocene ) . beside sandy channel deposits , the main part comprises overbank sediments of levees , crevasse splays / channels / deltas , abandoned channels , backswamps and floodplain paleosols . based on the lithofacies and the general geological setting , this system can be possibly related to an anastomosing river style . there is not any indication for a long - lived lake ( lake pebas ) or any marine influx in this region during the late miocene . ( 1997 , 2007 , 2010 ; compare also cozzuol , 2006 ; westaway , 2006 ) .

in miocene times a vast wetland existed in western amazonia . whereas the general development of this amazing ecosystem is well established , many questions remain open on sedimentary environments , stratigraphical correlations as well as its palaeogeographical configuration . several outcrops located in a barely studied region around eirunep ( sw amazonas state , brazil ) were investigated to obtain basic sedimentological data . the observed deposits belong to the upper part of the solimes formation and are biostratigraphically dated to the late miocene . vertically as well as laterally highly variable fine - grained clastic successions were recorded . based on the lithofacies assemblages , these sediments represent fluvial deposits , possibly of an anastomosing river system . sand bodies formed within active channels and dominant overbank fines are described ( levees , crevasse splays / channels / deltas , abandoned channels , backswamps , floodplain paleosols ) . lacustrine environments are restricted to local floodplain ponds / lakes . the mollusc and ostracod content as well as very light 18o and 13c values , measured on ostracod valves , refer to exclusively freshwater conditions . based on palaeontological and geological results the existence of a long - lived lake ( lake pebas ) or any influx of marine waters can be excluded for that region during the late miocene .

with increasing availability , widened indications , and technical refinements of computed tomography ( ct ) and magnetic resonance imaging ( mri ) , the number of incidentally discovered adrenal lesions , such as adrenal incidentalomas and incidental adrenal enlargement , is increasing . in a recent study by tang et al . , among a total of 564 eligible ct studies ( patients undergoing ct without prior known malignancy , trauma , or endocrine disease ) , adrenal hyperplasia was detected in 64 cases , giving a prevalence of 11.3% . this indicated that incidental adrenal enlargement had a significant prevalence and has become a common clinical problem . it should be emphasized that the term incidental adrenal enlargement represents the way the lesion was detected ( incidentally ) , rather than the etiology or diagnosis . it is a common term for a variety of adrenal disorders , but its cause must be properly assessed so that patients needing treatment , such as those with hormone hypersecretion or malignant disease , can receive appropriate care . however , there is a lack of literature on functional status and its follow - up to provide comprehensive insight to these findings . patients with incidental adrenal enlargement were evaluated in a tertiary referral hospital with endocrinological departments in china . this study aimed to determine the primary clinical presentation that most frequently leads to the discovery of adrenal enlargement , to evaluate clinical characteristics and functional status of these patients , and to analyze risk factors for functional lesions . this retrospective study included 578 patients with adrenal imaging features showing adrenal enlargement who were hospitalized at the department of endocrinology in pla general hospital ( beijing , china ) between january 1993 and july 2013 . data retrieved included patient demographics , final functional diagnosis , adrenal imaging features , and concomitant diseases . patients were classified as having incidental adrenal enlargement when abdominal imaging was performed for indications unrelated to adrenal disease . patients with diseases known to cause adrenal enlargement , such as known endocrine disorders which could affect adrenal size , trauma , and underlying malignancy , were excluded . among all enrolled patients , 78 presented with incidental adrenal enlargement . the ct imaging technique used was not standardized due to the various clinical indications . however , to be included in their entirety on a maximum of 5 mm section thickness , the upper limit of normal was set as 10 mm for the body of the gland and 5 mm for each limb as documented by vincent et al . . the type of enlargement , based on subjective evaluation of the adrenal glands , was recorded as either smooth or nodular . nodular enlargement was diagnosed if the adrenal gland had an irregular contour , contained nodules , and had normal adrenal tissue interspersed between the nodules . smooth enlargement was defined as enlargement of the gland with a smooth contour and no measureable or diffuse nodules . after obtaining patient history and physical examination , all patients underwent biochemical evaluation to assess their functional status . patients with elevated 24-hour urine - free cortisol level ( 2 times ) or those in whom plasma cortisol levels did not decrease after an overnight low - dose dexamethasone test ( 1 mg dst ; cutoff 50 patients with an aldosterone - rennin ratio ( arr ) > 20 underwent any 1 of 3 confirmatory tests ( saline infusion , captopril challenge , or postural stimulation ) to confirm or exclude definitively primary hyperaldosteronism ( pa ) . categorical data such as gender and clinical / radiologic features were compared using -test or fisher 's exact test . variables that resulted in a p < 0.05 in the univariate analyses were entered into logistic regression analysis to assess the risk factors of functional lesions . the hospital ethics committee approved this study , and written informed consent was obtained from all patients or their parents . of 578 patients with adrenal enlargement , 78 cases ( 13.49% ) were detected incidentally . every 2 years , the numbers of total cases were 17 , 11 , 14 , 24 , 33 , 31 , 54 , 55 , 114 , and 225 , respectively . the numbers of incidental adrenal enlargement cases were 0 , 0 , 0 , 1 , 1 , 2 , 5 , 4 , 16 , and 49 , respectively . in addition , the proportion of incidental adrenal enlargement gradually increased ( 0 , 0 , 0 , 4.17% , 3.03% , 7.32% , 9.26% , 7.27% , 14.04% , and 21.33% ) . 39 cases had unilateral enlargement on the left side and 3 on the right side , and the remaining 36 were bilateral enlargement . as shown in table 1 , routine medical checkup was found to have the greatest chance ( 43.59% ) of revealing clinical onsets leading to the discovery of adrenal enlargement . predominant complaints included low back pain ( 10.26% ) and abdominal pain ( 3.85% ) . in addition , there were 30 ( 38.46% ) cases in which the lesions were incidentally detected during hospitalization for underlying diseases , such as diabetes mellitus , hypertension , and coronary heart disease , among others . biochemical and functional evaluation revealed that 54 ( 69.23% ) cases were nonfunctional and 12 ( 15.38% ) were subclinical cushing syndrome ( scs ) ; among these patients , 10 cases were diagnosed as aimah , the other 2 were diagnosed as adenomas , and they were all confirmed by pathology results . primary hyperaldosteronism 6 ( 7.69% ) , metastatic 1 ( 1.28% ) , the primary cancer was gastric cancer . there were 5 patients ( 6.41% ) whose functional status remained unclear because of failure to finish the functional evaluation ( figure 2 ) . nodular adrenal enlargement ( or 7.306 ; 95% ci , 1.72728.667 ; p = 0.006 ) was the risk factor for functional lesions . as outlined above , incidental adrenal enlargement is detected with increasing frequency , most likely due to widespread increase in cross - sectional imaging , and is gradually emerging as a common clinical problem . our study shows that the proportion of incidental adrenal enlargement has gradually increased by year . mean age at diagnosis was 50.32 years , which is in line with other incidentally detected adrenal findings , namely , adrenal incidentaloma . the increasing age of the general population and a research trend towards more advanced investigations in the elderly population may be contributing to the high detection rate in this age group . our results indicate that , for the elderly patients , it is essential to place emphasis on these incidental findings . 's study indicated that , of the total 64 patients , of which 40 ( 63% ) were men and 24 ( 37% ) were women , 43 ( 67% ) cases were bilateral enlargement and 21 ( 33% ) cases were unilateral . in addition , smooth enlargement was more common , in 53 ( 83% ) cases , and together these statistics reflect the likelihood that adrenal enlargement will be bilateral , smooth , and found in men . however , our study did not show this tendency , likely because the research goals and thus , study populations , differed between the 2 studies . 's study aimed to explore prevalence , while the present study aimed to evaluate functional status . in addition , patients enrolled in our study were hospitalized at the department of endocrinology . it should be noted that admitting was more or less selective , especially in tertiary referral hospitals , and that economic considerations in parts of china were still a problem . clinically , upon discovery of incidental adrenal enlargement , 2 issues arise : functionality and malignancy . in the relevant literature [ 48 ] , adrenal enlargement can result from endocrine disorders , such as adrenocorticotropic hormone- ( acth- ) dependent or independent cushing syndrome , pa , multiple endocrine neoplasia type 1 ( men-1 ) , and congenital adrenal hyperplasia . other potential causes include nonfunctional lesions , defined as a radiographic adrenal enlargement without clinical or biochemical manifestations , inflammation , neoplastic processes , obesity , or depression . 12 patients were found to have subclinical cushing syndrome ( originated from the adrenal gland ) and 6 patients were diagnosed with pa . however , the variety of disease spectrum in the study was only moderate , perhaps due , in part , to the limited number of included cases . it is important to note that even though reported prevalence was up to 11.3% , patient referrals to endocrinologists are relatively rare . this is likely related to the poor radiological awareness of this issue and its potential clinical significance . the clinical significance of lesion location and patient gender is smaller . in the present study , acth - independent macronodular hyperplasia ( aimah ) and primary pigmented nodular adrenal hyperplasia often manifest as adrenal hyperplasia . the clinical features of aimah tended to be atypical . nodules usually distorted and completely obscured the normal adrenal glands and were characteristically ginger - like . as for pa , adrenal glands affected by idiopathic hyperaldosteronism ( iha ) may be normal on the ct scan or show nodular changes , and small aldosterone producing adenoma ( apas ) may be interpreted incorrectly by the radiologists as iha on the basis of ct findings of bilateral nodularity or normal - appearing adrenal glands [ 1013 ] . thus , it is a simple matter to explain why nodular enlargement could be a predictive factor of functional lesions . meanwhile , this also suggests that if incidentally detected lesions were nodular enlargements , evaluating its functional status should be a priority . in addition , the present study suggests that lesions on the left side were likely to be nonfunctional . in addition , there was only one patient with malignant lesion in the present study , and thus we were unable to analyze incidence of malignancy . incidental adrenal enlargement is a frequent radiographic finding and it is accompanied by diverse clinical factors that require proper diagnostic evaluation and management . in functional evaluation ,

aim . to investigate incidental adrenal enlargement clinical characteristics and functional status and analyze functional lesion risk factors . materials and methods . this retrospective study included 578 patients with adrenal imaging features showing enlargement . incidental adrenal enlargement cases ( 78 ) were considered eligible . demographics , functional diagnosis , adrenal imaging features , and concomitant diseases were analyzed . results . the number of adrenal enlargements and proportion of incidental adrenal enlargement increased each year . mean patient age was 50.32 years . thirty - nine cases had unilateral enlargement on the left side and 3 on the right side ; 36 had bilateral enlargement . routine medical checkup was found to have the greatest chance ( 43.59% ) of revealing clinical onsets leading to discovery . biochemical and functional evaluation revealed 54 ( 69.23% ) cases of nonfunctional lesions , 12 ( 15.38% ) of subclinical cushing syndrome , 6 ( 7.69% ) of primary hyperaldosteronism , 1 ( 1.28% ) of metastasis , and 5 ( 6.41% ) of unknown functional status . nodular adrenal enlargement ( or , 7.306 ; 95% ci , 1.72728.667 ; p = 0.006 ) was a risk factor for functional lesions . age and lesion location were not significant factors . conclusion . incidental adrenal enlargement is a frequent radiographic finding and is accompanied by diverse clinical factors that require proper evaluation and management . nodular adrenal enlargement was a risk factor .

pursuing the goal of improved health literacy requires more alliances between health and education sectors to improve literacy levels in the population . it is important for health educators to know about knowledge transfer to meet their needs in transferring their knowledge to public . knowledge transfer , which means the synthesis , exchange and ethically application of knowledge within a complex system of relationship among researchers and users , has become one of the recent priorities in research centers . the word the method of knowledge transfer , apart from its characteristics , requires active interactions between researchers and users . caplan proposes the two- communities theory indicating a gap between researchers and policymakers , whereas these days another gap has been formed between researchers and other users . in addition to illiteracy in developing countries that hampers effective health education , there are multitudes of non - medical specialists who do not have enough information about daily health affairs , and this lack is more remarkable in countries that welcome immigrants . immigrants often have significant language and health literacy difficulties , which are further exacerbated by cultural barriers . alongside with sophisticated methods like computer and internet , using simple methods help us bring to achieve our health goals[68 ] . also , the lack of health literacy needs more attention when it is manifested in caregivers . it is believed that some of the undesirable health outcomes in children are because of inadequate health knowledge among caregivers . in the usa , therefore researchers have considered different methods to come up with this deficiency by having children to cooperate in their health programs . a research group in india endeavored to transfer knowledge on leprosy in cooperation with children and informed their parents through them . jacob et al ( 1994 ) started conducting a similar research that yields promising results . rimal and flora ( 1998 ) express that parental dietary behavior is partially affected by children . the findings from studies in this field have encouraged other experts to apply similar methods to conduct their research projects , allahverdipour and bashirian , onyango - ouma and mwangagain from the influence of children in order to teach different parts of their society[1316 ] . despite the researcher 's trends in using novel methods in this field , none of them have used childish poem as a medium for knowledge transferring . in this study , we try to raise families health knowledge about infectious diseases by a method that uses children as health agents . infectious diseases are still the leading cause of mortality in children less than 60 months in developing countries . juvenile age is accompanied by learning childish poems , which can be sung contnuously at home and potentially become as part of both children 's and parents memories . we decided to transfer health knowledge to families through altering this information to childish poems and teaching children at kindergartens . in this method the outcome of transferring health knowledge through childish poems has been assessed in this study . in this study , we use the method of interventional pre and post series to conduct our research . the proposal of this non - invasive project was approved by the research committee of hamadan medical university in january 2009 . it was also accepted by hamadan welfare organization , which is the responsible organization of kindergartens in hamadan . our methodology is briefly explained as follows . at first , a sub specialist in pediatric infectious diseases provides seven short texts about health and common pediatric infectious diseases prevention . then , a poet transfers these conceptions to childish poems for the first time ( appendix 1&2 ) . in the process of preparing an accurate questionnaire , at first 30 questions with three choices of , false and do not know were made , but 24 items of those are confirmed as valid questions by two experts of health education and infectious diseases . to assess the reliability of the questionnaire a pilot study on a 40 participant sample was performed and cronbach 's alpha of the questionnaire was determined as 83 percent . in addition , we defined knowledge mark ( km ) as a parent mark in both the pre - test and/or post - tests . among 35 kindergartens in hamadan , we selected 7 kindergartens by simple random method . the parents ( either father or mother , the one who is responsible to take her / his child ) of all five to six year old children of the selected kindergartens are asked to participate in the survey and a verbal consent is obtained from those who accepted . we excluded the following children from our sample : those whose parents are physicians , nurses , health - care workers , and those children whose parents work at the kindergarten ( exclusion criteria ) . we asked the parents of the sample survey to participate in a pretest by answering to a questionnaire when they are in the kindergartens . also , they were asked to come to the kindergartens personally one more time when it is required ( to answer to the post test ) . then , we asked our kindergarten tutors to teach children seven musical poems about hydatid cyst , antibiotic misuse , botulinum toxin in home - canned foods , dysentery , the importance of sixth tooth , brucellosis and tetanus . in this step , children were not allowed to take home these texts , but they were asked to sing the poems at home and want their parents to re - write the poems on a paper and give them the papers to take to the kindergarten . the teaching stage took between three to four months . during the next stage , parents took a post - test survey with the same 24 questions . parents of 115 kids participated in the pre - test and 103 of them completed the post - test . statistical analysis is done on 103 who completed both tests by paired t - test . the sample data consists of 103 parents who provided solutions to both the pre- and post - test question survey . of the participants 77 were female and 26 male . from the level of education point of view , 19 were below high school diploma , 45 had high school diploma , 8 had associate degree , 27 had ba , 3 had ma , and 1 had phd . the results of the survey show a significant difference between the correct answers in both pre - test and post - test stages ( fig . 1 ) . the frequency of correct answers to the tests table 1 shows the mean of the correct answers in the pre - test stage is 59.22 while the same figure for the post - test step is 81 ( p < 0.001 ) . in addition , we define knowledge mark ( km ) as a parent mark in both the pre - test and/or post - test stages . the mean km shows a significant increase ( 5.01 ) from 13.62 in the pre - test to 18.63 in the post - test ( table 2 ) . mean percent of correct answers to pre - test and post - test categories analyzed by paired t - test sd : standard deviation mean of knowledge mark in pre - test and post test analysed by paired t - test the difference of km promotion among females and males was not significant ( 21.6120.17 in males and 20.331.61 in females , p=0.8 ) . the km was increased to 19.9325.06 among parents who at most had high school diploma and 23.4720.84 among parents with upper educational levels ( p=0.5 ) . health has been the concern of poets such as fiona sampson for years , however , these poems never had educational purposes . the results of this study confirm our hypothesis about the role of children in raising family 's knowledge on health . the results show that neither gender nor the grade of knowledge degree ( below and upper high school diploma or below and upper ba ) affect answers . therefore , people with different educational status have received the information similarly implying that this method can be extended to general cases in a straightforward manner . 1 shows that the percentage of correct answers has increased in 23 out of 24 questions . the percent - age of correct answer to post - test has decreased only in one item ( item 11 ) which compares the risk of brucellosis transmission via unpasteurized milk and yogurt . although the term yogurt has not been mentioned directly in the related poem , but there is a hint about boiling mechanism and we expect that the audience discover the answer by logical thinking about the necessitation of boiling milk for preparing yogurt . the decrease in answering is 2 percent which is not significant ; however , it shows that it would be better to mention the conceptions directly in these types of poems in order to avoid any misconceptions . moreover , the percentage of correct answers to the questions of selected topics in pre - test and post - test has been compared ( table 1 ) and shows that parents have done better in post - test about all topics except one . the difference of correct answers to the questions about brucellosis does not show a significant increase in post - test . this finding is probably due to higher basic knowledge about brucellosis among parents . in other words , parents have had acceptable information about brucellosis even without our educational program , so we observe lower contrast between pre - test and post - test about brucellosis . it should be taken into consideration that brucellosis is an endemic disease in hamadan and people have been educated in different ways about this disease in recent years . although the lowest contrast between pre - test and post - test correct answers belongs to a question about botulism , the overall assessment shows that parent 's knowledge about this topic has been promoted significantly ( p<0.001 , table 1 ) . the significant difference between the pre - test and post - test km , based on the results of table 2 , is the sign of successful knowledge transfer through childish poems . the researcher 's method is impressively successful ; however , the fact that he mentions that some mothers can not distinguish the semantic difference between information and advice , needs serious attention . in addition , since daughters may misunderstand the information , it would be probable to transfer the misconceived information to their mothers and this fact threats the successfulness of study in applying . while , in our study ; firstly , childish poems do not imply sententious , secondly , if the poem is sung wrong , the disturbance in rhythm will be appeared , so the information which is transferred through poems is not at risk of alteration . in evans research , parents get familiar with educated topics with the help of their children in homework assignments designed for asthma management , and are taught about asthma indirectly . the advantage of our study in comparison with evans is that in evans method knowledge transfer brings to bear via homework assignment , while in our study there is no obligation for doing home works , and parents are educated while enjoying their child 's singing . on account of the fact that making communication with younger children is not as difficult as with adolescents for parents , we are sure that knowledge transfers within a family through a continuous and friendly communication will work . christensen expressed the importance of children 's role in promoting the family health status . in his article , he emphasized on the activities that children can perform to enhance their health and promote their family health situation via the health efforts for themselves . we showed that children , additionally to what christensen mentioned , are able to affect their family 's health status directly . they can improve their family 's health as little teachers in health . it should be taken into consideration that the limitations of this study was the impact of other media on participant 's knowledge , which was not preventable . one of the acceptable results of this survey was publishing the poems as a book for children which was republished and welcomed by public . this study 's results suggest that health knowledge transfer to families through childish poems is an applicable method that has many advantages . by applying this joyful , cost - effective and easy - to - use method however , it should be taken into consideration that this method is applicable for families with trainable children , for teaching in larger scales more general ways should be added to this method . moreover , the simplicity of poems and expressing the topics in a less elaborative form are the key factors that affect the successfulness of this way .

objectivethe purpose of this study is to propose an innovative method of knowledge transfer that aims to improve health literacy about pediatric infectious diseases prevention in families . children have an appreciable role in this scheme.methodsthis study is a before and after trial that has been conducted in hamedan in 2009 . after changing seven infectious disease topics into childish poems , we selected five kindergartens randomly and taught these poetries to the children . teaching process held after a pretest containing 24 questions that examined 103 of parents about mentioned topics . the same post - test was given after 4 months of teaching process.findingsthe mean of correct answers to the pretest was 59.22% comparable with 81.00% for post - test ( p<0.00 ) . gender and knowledge degree could not change the results significantly . assuming one 's correct answers to the questions as his / her knowledge mark , the mean of this variable increased to 5.32 by this method.conclusionthis cost - effective and joyful method had successful results in promoting health knowledge . children are able to play an active role in family 's health situation . learning within family atmosphere without any obligations makes our scheme a solution for paving the knowledge transferring way .

septic arthritis of the shoulder is uncommon in adults . it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality . a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder . on examination , his shoulder movements were severely restricted . further evaluation with mri revealed septic arthritis of left gleno - humeral joint for which emergency arthroscopic debridement was done . septic arthritis of shoulder may not present with classical clinical features . hence , a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock , osteomyelitis . patients with septic arthritis usually develop moderate to severe joint pain , warmth , tenderness , effusion , restricted active and passive motion , and sometimes redness . we report an unusual presentation of shoulder septic arthritis in a 43 years old man with no other clinical signs and symptoms of classical septic arthritis . a 43-year - old man presented to the orthopaedic outpatient clinic with 2 months history of pain and limited range of motion in his left shoulder . his pain was insidious in onset , mild to moderate in intensity , aggravated by activities and associated with moderate rest and night pain . patient was diagnosed as frozen shoulder at an outside facility and had been given intraarticular depomedrol 40 mg injection for the same 40 days ago with no improvement in his symptoms . he is diabetic and has liver cirrhosis and is on treatment . on physical examination , the skin colour and temperature of the left shoulder were normal , but the shoulder was tender to touch over the anterior joint line . mri was obtained [ fig 2 ] which showed extraosseous soft tissue enhancements around left shoulder with soft tissue abscess in inter muscular planes of supraspinatous , infraspinatous , subscapularis . blood test revealed normal white cell count with normal differential count , crp-13.4 mg / l ( normal<5.0 mg the erythrocyte sedimentation rate was raised , at 88 mm / hour ( normal range , 0 - 20 mm / hour ) aspiration of shoulder joint was performed and fluid was sent for aerobic and anaerobic culture , afb staining , mycobacterium culture , and mycobacterium genetic testing . arthroscopic lavage of the joint was done and articular fluid sent for repeat aerobic and anaerobic culture . joint visualization was markedly limited because of severe inflammation and fibrinous changes of the joint . the articular cartilages of glenoid and humeral head were completely eroded down to the bone . susceptibility of the isolate was determined with the disk diffusion method and it was susceptible only to colistin . 3 ) fibrocollagenous and fibrovascular inflammed connective tissue containing proliferated blood vessels and perivascular mixed inflammatory cells and lymphoplasmacytoid cells . based on preoperative and intraoperative culture report , a definitive diagnosis of polymicrobial septic arthritis of the shoulder was established and patient was treated with combination antimicrobial treatment . our patient had muted inflammatory response probably due to immunocompromised state ( cirrhosis and diabetes ) which is not uncommon . most often it is hematogenous seeding of shoulder joint , however it can also happen after intraarticular steroid injections . septic arthritis of the shoulder is more common in immunocompromised patients and intravenous drug abusers . in this case report , the patient had atypical clinical symptoms and is immunocompromised and his primary source of infection seems to be hematogenous . our experience with this patient highlights the importance of maintaining high index of suspicion for septic arthritis in immunocompromised patients presenting with atypical shoulder pain associated with stiffness . in the setting of suspected septic arthritis , our initial pre - investigation diagnosis was atypical shoulder pain and stiffness of non specific etiology in an immunocompromised patient and infection was part of the differential diagnosis not the only diagnosis . esenwein et al in his report highlighted the importance of early intervention to prevent chondral damage , osteomyelitis and also to prevent systemic spread . various authors have highlighted the importance of early diagnosis and management of septic arthritis failing which could lead to osteomyelitis and septic shock . in our patient , the diagnosis was delayed due to atypical presentation and over reliance on clinical findings at an outside center . we strongly recommend early imaging studies in immunocompromised patients presenting with shoulder pain and practitioners should avoid loosely diagnosing as shoulder pain with associated stiffness as frozen shoulders . klinger et al . did a retrospective study on 21 patients who underwent surgical treatment for septic arthritis of the shoulder joint between 2000 and 2007 , and he concluded that patients with symptoms for less than 2 weeks did well with arthroscopic approach and early infection can be managed arthroscopically . our reports show that arthroscopic washout can give good result even after 2 weeks of clinical symptoms if there is no evidence of osteomyelitis . septic arthritis of the shoulder is very often due to hematogenous spread and diagnosis is often clinical . laboratory investigations and imaging studies like mri and usg may be useful in establishing the diagnosis but confirmation is usually by joint aspiration . patients who are immunocompromised and have insidious onset of moderate to severe pain and which fails to respond to trial of conservative treatment should be subjected to either ultrasound or mri instead of x - ray because very often in these group of patients it is primarily a soft tissue pathology like impingement , rc tear , calcific tendinitis or rarely infections and malignancies . we recommend that moderate pain of more than 4 weeks duration with severe stiffness in immunocompromised patient ( liver cirrhosis , renal failure , steroid treatment ) should be further evaluated with mri or ultrasound in the setting of normal x - ray and should not be loosely diagnosed as frozen shoulder . goldenberg in his report emphasized the role of local as well as systemic factors that predispose patients with cirrhosis to gram - negative bacterial joint infections . malnick also reported a case of spontaneous septic arthritis in a cirhottic patient that was due to e.coli . our study as well as other studies by goldenberg and malnick highlight the importance of including broad spectrum antibiotics with gram negative cover whilst waiting for final culture sensitivity . septic arthritis of shoulder may not present with classic clinical features . a through clinical and radiological evaluation should be executed and treatment initiated promptly to prevent complications like septic shock and osteomyelitis . the case highlights the importance of establishing anatomical and pathological diagnosis using mri in patients with shoulder pain instead of loosely diagnosing them as impingement or frozen shoulder . the case also challenges the practice of routine shoulder depomedrol steroid injection , in the setting of secondary frozen shoulder , atleast in immunocompromised individuals . primary idiopathic frozen shoulder is a rare condition and secondary frozen shoulder cases are often due to underlying shoulder pathology .

introduction : septic arthritis of the shoulder is uncommon in adults . it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality . accurate diagnosis can be particularly challenging in patients with underlying liver disease . mri is a useful adjunct in early detection of atypical causes of shoulder pain.case report : a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder . on examination , his shoulder movements were severely restricted . further evaluation with mri revealed septic arthritis of left gleno - humeral joint for which emergency arthroscopic debridement was done.conclusion:septic arthritis of shoulder may not present with classical clinical features . hence , a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock , osteomyelitis .

split - liver transplantation ( slt ) is an attractive alternative procedure to expand the donor pool in patients waiting for liver transplantation . the standard split - liver procedure for a child and an adult ( adult / pediatric split liver ttransplantation , a / p slt ) , by splitting segment ii - iii for pediatric recipient and segment i - iv - v - vi - vii - viii for an adult , is an accepted surgical option with good results both for the adult and for pediatric recipient . biliary complications continue to be the major cause of morbidity after slt with a reported high incidence ranging between 10% and 32% [ 1 , 2 ] . as a consequence of bile duct anatomic variations , slt requires a precise knowledge of the liver anatomy [ 3 , 4 ] . the challenge of this procedure is represented by a preoperative radiological assessment of the biliary anatomy often unavailable at the donor 's hospital . thus the risk of biliary duct injury during the splitting procedure is usually considered higher than during living donor procedure . in this report we describe an uncommon late biliary complication that occurred after slt and was successfully treated by a multidisciplinary approach . a 63-year - old male with hepatitis c - related cirrhosis was referred for liver transplantation to our institution . we performed a conventional a / p slt with in situ technique providing the left lateral segment for a child ( segments ii - iii ) and leaving the right lobe graft ( segment i - iv - v - vi - vii - viii ) for an adult recipient . the celiac trunk was left on the left graft while the right hepatic artery remained on the right graft . the patient was transplanted using the piggy - back technique without a veno - venous bypass . the biliary tract was reconstructed performing a duct - to - duct anastomosis using a t - tube by our standard technique previously described . a cholangiography through the t - tube was performed on postoperative day 14 , and the t - tube was clamped before patient discharge . three months after a / pslt the t - tube was removed after a cholangiography with normal findings . one year after transplant the patient showed abnormal liver function tests , hyperbilirubinemia , leukocytosis , and elevated g - glutamyl transpeptidase ( ggt ) , and mild elevation in alanine transaminase ( alt ) . the patient underwent a doppler ultrasound that showed ( a patent hepatic artery ) an intrahepatic bile duct dilatation and an anastomotic biliary stricture . the anastomotic stricture was treated by stenting the main biliary duct during an endoscopic retrograde cholangiopancreatoghaphy ( ercp ) without any evidence of intrahepatic biliary dilatation . after this procedure the patient was submitted to a percutaneous transhepatic cholangiography ( ptc ) showing a complete obstruction of segments vi and vii biliary branches near the duct - to - duct biliary anastomosis ( figure 1 ) . the patient was discharged leaving the external biliary drain open allowing bile drainage and an easy access for repeated radiologic treatment and an internal stent in the common bile duct . three months later the patient underwent a surgical revision because of repeated episodes of cholangitis . during surgery an intraoperative cholangiography was performed through the biliary drain confirming a bile duct dilatation at the level of segments vi and vii . it was impossible to cross the biliary stricture by a torque catheter and by hydrophilic guide wire ( figure 2 ) . we supposed that the segmentary bile duct branch of posterior segments vi and vii draining in the left bile duct was unidentified and tied at the time of the in situ split - liver procedure during the parenchymal transection . a primary biliary reconstruction by biliary repair or biliodigestive anastomosis was considered at high risk because of the presence of postsurgical adhesions sand due to fibrotic tissue replacing the parenchymal transected area . a liver resection of the dilated segments vi and vii was considered at high risk of leaving an inadequate liver mass . we decided to perform a permanent intraoperative obliteration of the dilated intrahepatic ducts by a percutaneous embolization using a nonresorbable agent . with a fluoroscopic guidance through the transhepatic access we positioned a 5-french polyethylene catheter inside the ducts , preliminary flushed by a nonionic dextrose solution . we then injected the tissue adhesive agent n - butyl cyanoacrylate ( nbca , glubran 2 , gem , viareggio , italy ) mixed with ionized oil ( lipiodol , guebert , aulnay - sous - bois , france ) for opacization in a ratio of 1:5 . this solution completely filled the biliary duct , and the occlusion was totally accomplished in a few seconds ( figure 3 ) . during the first 3 days after the chemical bile duct embolization , the patient had a low fever with a slightly abnormal liver function test . a computed tomography ( ct ) scan performed 6 months later showed no sign of hepatic abscesses , and the bile duct dilatation was completely occupied by the nbca - lipiodol mixture . one year after the procedure patient showed normal liver function tests without no episodes of cholangitis . chemical bile duct embolization treatment could represent a valuable solution to treat uncommon biliary complications . these tissue adhesive glues are low - viscosity liquid monomers that undergo rapid polymerization and solidification when they come into contact with organic fluids such as bile . nbca is a permanent liquid embolic material that produces long - term occlusion in vessels of various size through an inflammatory tissue response resulting in vessel thrombosis or tissue atrophy . little is known about the use of cyanoacrylate compounds , and unlike european countries the use of glubran has not been approved by the food and drug administration yet . other authors have described the efficacy of biliary duct ablation by nbca in patients with persistent postsurgical bile leaks after lobectomy or cholecystectomy . vu et al . treated six patients with persistent postsurgical bile leaks as a complication after hepatic lobectomy or cholecistectomy using nbca glue for the obliteration of isolated segmental bile ducts in four cases . endoscopic treatment of biliary leakage by nbca has been described by seewald in nine patients in whom primary stent placement or nasobiliary drain was unsuccessful . described the use of a cyanoacrlylate in the treatment of a pancreatic fistula after distal pancreatectomy . the percutaneous interventional technique represents an effective valuable approach to reduce mortality and morbidity in the treatment of biliary complications after liver transplantation . the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection above all in high - risk patients with posttransplant bile duct injuries , decreasing the morbidity associated with chronic external biliary drainage . futher studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and costs .

biliary complications continue to be a major cause of morbidity after split - liver transplantation ( slt ) . in this report we describe an uncommon late biliary complication . one year after slt the patient showed an intrahepatic bile dicy dilatation with severe cholangitis episodes . the segmentary bile duct of hepatic segment vi - vii draining in the left duct was unidentified and tied at the time of the in situ split - liver procedure . we perform a permanent obliteration of the dilated intrahepatic ducts by a percutaneous embolization using an n - butyl cyanoacrylate ( nabc ) . the management of biliary complications after slt requires a multidisciplinary approach . the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection and decreasing the morbidity associated with chronic external biliary drainage . further studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and cost .

ribonucleotide reductase ( rr ) inhibitors have been studied as radiation sensitizers for over 30 years in both the lab and the clinic . the first of these , hydroxyurea , has been studied in both cervical and head and neck cancers , among others . although initially promising , many of the clinical trials produced negative results , or those that were difficult to interpret . there has recently been a significant advance in our understanding of this pathway from a number of well - done pre - clinical studies . in addition , the discovery of new rr inhibitors with increased potency and improved binding characteristics has produced a significant increase in interest in this area . this review will synthesize the data detailing the response of rr to ionizing radiation ( ir ) and will provide a perspective on the use of rr inhibitors as radiosensitizers in the treatment of human cancers . over the years , many groups have explored the use of rr inhibitors as chemotherapeutics in their own right , or as adjuncts to dna damaging molecules , particularly in hematologic malignancies . while this area looks promising , this subject will not be reviewed here . ribonucleotide reductase is the rate limiting enzyme in the synthesis and repair of dna and is the only enzyme responsible for the conversion of ribonucleoside diphosphates to deoxyribonucleotide diphosphates , the fundamental building blocks of dna synthesis and repair . r1 ( also called rrm1 or m1 ) is the larger , regulatory subunit that is constitutively expressed throughout the cell cycle . it binds allosteric modulators , ribonucleoside diphosphates , and the nucleoside analogs gemcitabine and fludarabine . there are currently two known smaller subunits that bind r1 to form the active enzyme ; r2 ( also called rrm2 or m2 ) and a more recently discovered p53 inducible homolog of the r2 subunit , known as p53r2 . both contain a tyrosine free radical stabilized by a non - heme iron complex that is critical in the reduction of ribonucleotides . r2 is known to be cell cycle regulated , with the highest levels during s phase , however the precise roles of the r2 and p53r2 subunit in the response to ir are an area of active debate , as outlined below ( figure 1 ) . given the pivotal role of rr in dna synthesis and repair , many studies have investigated the effect of dna damaging agents , including ir , on rr and its subunits . even so , there is currently still a great deal of controversy surrounding the exact mechanism of rr response to ir . the first is that the small subunit r2 is up - regulated and provides dntps for dna repair in addition to its usual role in dna synthesis during s phase . this is supported by a number of studies , including one by kuo et al . , who characterized the response of rr in the human cervical cancer cell line , caski . they demonstrated an increase in r2 protein levels after treating cells with ir , which was correlated with an increase in rr activity . however , there was no increase in the transcription of r2 , implying that the protein increase was due to post - transcriptional regulation ( kuo and kinsella , 1998 ) . this finding was reinforced by studies that demonstrated dna damage dependent stabilization of the r2 protein without any change in mrna after ir exposure in mouse balb/3t3 cells ( chabes and thelander , 2000 ) ; however it was in contrast to earlier work showing transcriptional activation of the r1 and r2 promoters after exposure to ir in the same cell line ( filatov et al . , 1996 ) . even though the precise mechanism of r2 response to ir damage is unclear , work has shown that human nasopharyngeal cancer cells overexpressing r2 demonstrate a significant increase in radioresistance and fewer dna double strand breaks ( dsb ) after exposure to ir ( kuo et al . , 2003 ) , confirming the concept that r2 is important in the cellular response to ir the second theory involves the p53 inducible subunit , p53r2 , first reported by tanaka et al . they showed that p53r2 was not cell cycle regulated ( unlike r2 ) , but was significantly induced after exposure of normal fibroblasts to ir ( tanaka et al . , 2000 ) and was found to translocate to the nucleus , the proposed site of most important dntp production ( r2 did not ) . in cells that lacked wild - type ( wt ) p53 , there was no induction , indicating that functional p53 was necessary for p53r2 up - regulation . cells transfected with p53r2 were resistant to dna damage induced cell death . in agreement with other studies , they found no increase in r2 mrna , but did not measure protein levels . additional studies have subsequently shown that p53r2 forms an active complex with r1 ( guitett et al . , 2001 ) and rr activity increased in correlation with the increase in p53r2 , however the response of r2 was variable , with decrease in some lines and a moderate increase in the others ( yamaguchi et al . , 2001 ) . finally , other models are possible , including one described by xue and colleagues . in their study , both subunits were found to bind p53 in human oropharyngeal carcinoma cells , and in response to ir , were released to bind r1 in the nucleus ( xue et al . , 2003 ) , highlighting a third possibility in contrast to those previously presented . clearly , there is still work to be done in elucidating the response of rr to ir . many of the differences seen in the studies discussed can likely be attributed to the use of different techniques , materials , and especially cell lines . what should be clear is that rr is involved in the cellular response to ir and targeting it is both rational and likely desirable in enhancing the treatment of cancers with ir . it is likely that both r2 and p53r2 are involved to a different degree in different cancers , with cellular phenotype likely playing a key role in determining their relative significance . hydroxyurea ( hu ) is a hydroxylated analog of urea and was the first rr inhibitor to be extensively studied . it has directed activity at the tyrosine radical moiety of r2 and was first found to be active against cancer cells in 1963 ( stearns et al . , 1963 ) . subsequent experiments in vitro showed that in addition to its direct inhibition of dna synthesis , it was also a sensitizer of cell killing by x - rays , particularly if given before or after ir ( sinclair , 1968 ) . later experiments showed that this was also the case in in vivo animal tumor models using isotransplants of spontaneous c3h / he mouse mammary carcinomas ( piver et al . , 1972 ) . the total dose of ir to cure 50% of tumors was reduced when hu was combined with fractionated ir , although this effect was nt seen with single fraction ir treatments . given these encouraging pre - clinical results during the 19601970s , hu was subsequently examined in a number of clinical trials in a variety of human cancers . the majority of these trials have occurred in cervical cancer , most commonly in locally advanced disease . in particular , there were a number of prospective randomized controlled trials in the 1970s and 1980s that examined the effect of hu plus radiotherapy vs. radiotherapy alone . and the gynecologic oncology group ( gog ) enrolled 190 women with figo stage iiib or iva cervical carcinoma . hu was administered orally at 80 mg / kg starting on the first day of irradiation and every 3 days thereafter for 12 weeks . patients received at least 50 gy minimum tumor dose to the whole pelvis followed by a single brachytherapy treatment of 20 gy to point a. in spite of the large number of patients enrolled , only 90 were eligible for assessment of response . this was due to ineligibility ( wrong stage , wrong cell type , etc . ) and those that were inevaluable ( refused treatment , periaortic node irradiation , improper field , etc . ) . the data were impressive , with a complete response ( cr ) of 68.1% in the hu group vs. 48.8% in the placebo ( p = < 0.05 ) , and a median progression free survival ( pfs ) of 13.6 vs. 7.6 months ( hreshchyshyn et al . , 1979 ) . however , myelosuppression was more common in the hu group , with seven grade iii or iv myelotoxicities . they recruited 148 women with figo iib or iiib cervical carcinoma and again compared hu to placebo in the setting of conventional radiotherapy , with hu given every 3 days for 12 weeks . of the stage iib patients , 74% receiving hu had no evidence of disease at the completion of therapy compared with 43.5% of the placebo ( p = < 0.01 ) . of the stage iiib patients , the cr rates were 52.5 vs. 33% ( p = 0.22 ; piver et al . , 1977 ) . again , 78% of patients in the hu group developed leucopenia vs. 11% in the placebo group indicating a significant toxicity in addition to the improved clinical effect . at the time these studies were published , it was felt that hu added significant benefit to the treatment of locally advanced cervical carcinoma , and hu plus radiotherapy became the standard of care for the gog . however , over time , much of the data in these studies has been challenged , particularly in a systematic review by symonds et al . they found a number of methodological problems with the studies such as small sample size , large numbers of exclusions post randomization , subgroup analyses of already small groups and questionable censoring . they concluded that hu appears to add to acute toxicity and probably increases late complications and that there is no convincing evidence of sufficient quality to suggest a therapeutic effect of this drug although the gog initially used hu as its standard of care in the treatment of locally advanced cervical cancer , it was nt long before this combination was supplanted by a new adjunct to radiation therapy . one of the most important studies prompting this paradigm shift was the gog 120 trial , first reported in 1999 ( rose et al . , 1999 ) and recently updated ( rose et al . , 2007 ) . gog 120 was a randomized phase iii study comparing cisplatin alone vs. cisplatin , fluorouracil , and hu vs. hu alone , in conjunction with pelvic irradiation for patients with locally advanced cervical cancer and pathologically negative para - aortic nodes . they reported a significant improvement in pfs and overall survival ( os ) in both cisplatin containing arms ( p < 0.001 ) , with relative risks for progression of disease or death of 0.57 and 0.51 for cisplatin alone and cisplatin , fluorouracil and hu , respectively , when compared with hu alone . in addition , toxicities were similar in all groups . further , a similar study by whitney et al . compared the efficacy of standard radiotherapy ( rt ) plus hu with standard rt plus fluorouracil ( 5-fu ) and cisplatin in a randomized controlled trial . in 368 women with figo stage iib , iii , or iva cancer of the uterine cervix , there were significant improvements in pfs and os with the 5-fu and cisplatin combination ( whitney et al . , 1999 ) . adverse effects included leucopenia : 4% of 5-fu / cisplatin patients and 24% of hu patients experienced grade iii or iv toxicity . given the findings of these studies , concurrent cisplatin and radiotherapy became the standard of care in locally advanced cervical cancer , spelling the end of hu use in the treatment of cervical cancer . in addition to its study in cervical carcinoma , hu has also been extensively investigated in other cancers , particularly head and neck . while early studies had significant flaws in methodology , a phase i study showed a 90% response rate in patients treated with hu , 5-fu , and palliative dose fractionated ir ( vokes et al . , 1989 ) , prompting a series of trials by the same group and others . this included work by mantz et al . , who examined the benefit of hu when added to a more extensive chemoradiation protocol including cisplatin , 5-fu , leucovorin , and interferon-2b induction chemotherapy followed by 5-fu and hu with fractionated radiotherapy in 32 laryngeal cancer patients . median follow up was 44.5 months , and , after completion of all therapy , the cr rate was 94% . median os was 44.5 months , and median pfs was 86 , 78 , and 78% at 1 , 3 , and 5 years , respectively , which compared favorably with other published data and for the first time saw patients with stage iv head and neck cancer failing distantly more often than locally ( mantz et al . the increased local control , unfortunately , was associated with increased toxicity , which has also been seen in other studies . one phase i study with prolonged infusion of hu with hyperfractionated , accelerated , external radiation in patients with advanced squamous cell cancer of the head and neck showed an increase in the severity of swallowing toxicity compared with previous trials , with severe edema , and reductions in motility and mobility of pharyngeal and laryngeal structures ( beitler et al . , 1998 ) . the same group later published a study examining the long term impact on swallowing that showed persistent , severe swallowing dysfunction ( smith et al . , 2000 ) . interestingly , these studies examined continuous hu infusions based on pre - clinical data that suggested that hu should always be given concurrently with ir to maximize effect . even though local control was excellent , with just 4 of 26 patients experiencing recurrence , quality of life is of great importance in these patients , and the late follow up reported esophageal strictures for the first time after chemoradiotherapy , suggesting that this particular regimen may be too aggressive . the state of hu in head and neck cancer is well reviewed by argiris et al . ( 2003 ) and while the authors are optimistic about the future of chemoradiation in head and neck cancer , it remains to be seen where hu and other rr inhibitors will fit in the future treatment of this disease site . even as hu has slowly progressed in the clinic , work has continued on examining the mechanism by which it sensitizes cells to the effects of ir . in particular , work by kuo et al . has shown that the sequence with which cells are treated with hu plays an important role . they demonstrated that in the caski cervical cancer cell line , clinically relevant concentrations of hu had a significant interaction with ir , with post - ir exposure > pre - ir ( kuo et al . , 1997 ) . this was associated with increased g2 delay , suggesting a decrease in the repair of damaged dna . in addition , in cells overexpressing the r2 subunit , hu is able to return ir sensitivity to baseline ( kuo et al . , 2003 ) , demonstrating that r2 inhibition is the likely mechanism for hu radiosensitization in these cells . these findings are potentially informative about the failure of hu to become established as a radiosensitizer in cervical cancer . in the majority of the early trials , given that hu works best in vitro when dosed immediately after ir exposure , one could conclude that these trials were not optimized for best effect . in addition , hu has recently been shown to have a significant effect on the mechanism of dna dsb repair employed by cells after exposure to ir . burkhalter et al . showed that cells pre - incubated with hu were unable to use homologous recombination ( hr ) to repair dsb , and instead relied on non - homologous end joining ( nhej ) . in addition , cells that were nhej deficient had significantly more dsb after hu treatment ( burkhalter et al . , 2009 ) . given that nhej is thought to be the dominant dsb repair mechanism in cells treated with hu , rr inhibitors are likely to have enhanced activity in tumors that are nhej deficient . even with new studies on its mechanism of action , hu will likely remain consigned to history due to the many inadequacies it has as a drug molecule . while its oral absorption is almost complete and it is completely distributed in the water compartments of the body , hu has a short half - life ( between 1.6 and 4.45 h ; gwilt and tracewell , 1998 ) and its effectiveness is limited by relatively low affinity for rr and by the development of resistance . one area where it could potentially find use in the future is in cns neoplasms , as it does cross the blood recent studies have examined its use in progressive meningioma in combination with 3d - conformal radiotherapy and adjuvant chemotherapy . in one trial , pfs at 1 and 2 years was 84 and 77% , which is similar to other adjuvant studies ( hahn et al . in spite of the mixed clinical data for hu , there is sufficient proof of concept to suggest that a rr inhibitor can be efficacious as a radiosensitizer in human cancers . thus , there has been a concerted effort to discover more potent molecules with more favorable pharmacokinetics and pharmacodynamics for this purpose . one of the more promising of these is triapine , a thiosemicarbazone that destroys the tyrosyl radical in r2/p53r2 by forming a redox active complex with iron , producing reactive oxygen species . in studies comparing it with hu in vitro , triapine was shown to have significantly higher potency in both enzyme and cell assays . in addition , it was fully active against hu and gemcitabine resistant cells and was equally potent against r2 and p53r2 , whereas hu was approximately threefold less potent at binding p53r2 ( zhu et al . , 2009 ) . in addition , in in vivo models , triapine was active against hu resistant l1210 and kb cell lines and caused significant inhibition of solid tumor growth in mouse xenograft models ( finch et al . further studies have examined the radiosensitizing properties of triapine in a number of human cell lines . used a panel of three human tumor cell lines , including glioma , pancreatic , and prostate cancer cells , with triapine enhancing radiosensitivity when delivered 16 h before or immediately after ir by 1.5- to 2-fold . ir interaction was associated with a reduction in the repair of dna dsb as evidenced by a persistence of h2ax foci at 24 h ( barker et al . , 2006 ) . a similar effect was seen in mouse tumor xenografts , again , with greater effect if triapine was dosed just after ir . the most effective temporal relationship between triapine dosing and ir is similar to that seen with hu in earlier pre - clinical studies . interestingly , normal human fibroblasts were only sensitized when triapine was given before , not after ir , suggesting a potential for an improved therapeutic index for ir triapine sequencing that may be incorporated into future clinical studies . are ic50 ( concentration of compounds producing 50% inhibition of recombinant rr activity ) values for both compounds in an in vitro rr activity assay with r2 or p53r2 bound to r1 ( zhu et al . , 2009 ) . also shown are elimination half - lives ( t1/2 ) for both compounds ( gwilt and tracewell , 1998 ; kunos et al . , 2010b ) . of note , many cancers have virally or mutationally silenced p53 that allows rr activity to continue unchecked . in these cancers , it is potentially of increased importance to inhibit the r2 and p53r2 subunits that have lost p53 regulatory control . this is the case in cervical cancer , where the vast majority have dysfunctional p53 due to hpv infection . in one study , three cervical cancer cell lines with mutated or dysfunctional p53 were irradiated 6 h after triapine exposure . in all cases , the cell lines were sensitized to ir and sustained dna damage as measured by persistence of h2ax foci . in addition , by measuring dctp levels , the investigators were able to show reduced rr activity in cells with and without functional p53 , demonstrating that the inhibition of rr is p53 independent ( kunos et al . , 2009 ) . these findings were reinforced by further work of the same group that also showed a synergistic effect when cisplatin was added in addition to triapine and ir ( kunos et al . , 2010a ) . these promising pre - clinical data have prompted the initiation of a number of clinical trials . indeed , triapine has so far been studied in 27 clinical trials in the usa at various stages of recruitment ( www.clinicaltrials.gov ) , including those in both solid and hematologic malignancies . in particular there are three trials investigating the radiosensitizing potential of triapine , with data from one phase i study being published by kunos et al . the purpose of their study was to assess the safety / tolerability , pharmacokinetics , and clinical activity of triapine three times weekly in concert with once weekly cisplatin and pelvic radiation in 11 patients with gynecologic malignancies . triapine was dosed in 2 h infusions and the half - life was found to be 2 h. all 10 patients with advanced stage ib to ivb cervical cancer achieved complete clinical response , with a median 18 month follow up showing no disease progression in any of the patients . five of the 10 patients had pet / ct evident pelvic or para - aortic lymphadenopathy before treatment , with complete resolution on follow up imaging after treatment ( kunos et al . in addition to the clinical data , the authors also examined objective markers of disease response . late responders had a significantly higher rr activity on day 10 vs. day 1 and there was no temporal change in rr activity in the early responders , indicating that the expected spike in rr activity after ir was suppressed by triapine . the fact that the late responders experienced durable responses in spite of elevated rr activity at day 10 suggests that there may be other mechanisms of triapine activity that require further study . the promising results from this phase i trial prompted a phase ii trial which is now underway . over the last 10 years , there have been a number of major leaps forward in the field of radiation oncology that allow us to deliver higher doses of radiation in ever more specific ways to our patients . still , in the vast majority of cases , we are constrained by dose limiting toxicities that must be accounted for when designing treatment plans and balanced against the therapeutic benefit realized . therefore , the field of radio modulation and specifically radiosensitization will continue to grow in importance in the coming years as we search for ways to maximize the therapeutic index of our treatments . while there are many different radiosensitizers currently being studied , the rr inhibitors are among the oldest of targets , and are getting a new lease of life in recent times with the development of more modern drug molecules . as outlined in the review above , the story began with the study of hu in combination with ir in a number of pre - clinical trials in the 1960s , leading to a large amount of interest in the clinic , culminating with the gog adopting hu and ir as its standard of care in locally advanced cervical cancer . while its success in this arena was short lived , many parallel studies investigating the mechanism of action of hu were carried out , resulting in a far clearer understanding of the biological interactions between ir and rr inhibitors . even as hu was fading from view , investigators were working on successors including the intriguing molecule , triapine . as shown above , this drug works in a similar fashion to hu , but has significantly improved potency , pharmacokinetics , and pharmacodynamics . the phase i study in cervical cancers that was recently published includes some very encouraging data , and the field waits in anticipation for the publication of further trials . it remains to be seen what the best dosing regimen for triapine will be , however the weight of pre - clinical data , including that with hu , suggests that daily dosing shortly after radiation therapy will be most effective and provide the greatest therapeutic window . these authors would encourage studies incorporating this type of dosing schedule in the clinic , although this must obviously be weighed carefully against the potential for increased toxicity . in addition to triapine , there are other new rr inhibitors being tested at various stages , including trimidox and motexafin gadolinium . ( 1994 ) , in a paper that reported 100-fold more potency at rr than hu in a cell based assay . subsequent in vitro studies have demonstrated that while it acts as a radiosensitizer in panc-1 human pancreatic cancer cells ( ahmed and hassan , 2000 ) , it is less potent than hu and further work is ongoing to fully assess its potential . motexafin gadolinium is a texaphyrin molecule that targets thioredoxin reductase in addition to rr . in in vitro experiments , it was shown to inhibit rr with an ic50 of 26 m ( hashemy et al . , 2006 ) , although given that it has a number of other cellular effects , it is unclear how much of its potency as a radiosensitizer is due to rr inhibition alone . it is likely that the coming years will continue to see the emergence of new rr inhibitors with unique properties . the recent advances in the understanding of rr biology and the development of new inhibitors places us at an important crossroads in the story of rr inhibitors . there are sufficient data to provide proof of concept for the target from a biological standpoint , however clinical trials to this point have not been wholly convincing . it will be interesting to follow the development of the field in the next 510 years , particularly with the clinical trials of triapine in cervical cancer , and hopefully at least one of the many rr inhibitors being studied will eventually bring additional therapeutic benefit to patients in the near future . 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 .

ribonucleotide reductase ( rr ) , the rate limiting enzyme in the synthesis and repair of dna , has been studied as a target for inhibition in the treatment of cancer for many years . while some researchers have focused on rr inhibitors as chemotherapeutic agents , particularly in hematologic malignancies , some of the most promising data has been generated in the field of radiosensitization . early pre - clinical studies demonstrated that the addition of the first of these drugs , hydroxyurea , to ionizing radiation ( ir ) produced a synergistic effect in vitro , leading to a large number of clinical studies in the 19701980s . these studies , mainly in cervical cancer , initially produced a great deal of interest , leading to the incorporation of hydroxyurea in the treatment protocols of many institutions . however , over time , the conclusions from these studies have been called into question and hydroxyurea has been replaced in the standard of care of cervical cancer . over the last 10 years , a number of well - done pre - clinical studies have greatly advanced our understanding of rr as a target . those advances include the elucidation of the role of p53r2 and our understanding of the temporal relationship between the delivery of ir and the response of rr . at the same time , new inhibitors with increased potency and improved binding characteristics have been discovered , and pre - clinical and early clinical data look promising . here we present a comprehensive review of the pre - clinical and clinical data in the field to date and provide some discussion of future areas of research .

the study of long bones and the cells of the bone marrow is central to a myriad of research disciplines , including , but not limited to , bone biology , cancer biology , immunology , hematology , and biomechanics . the bone is a highly dynamic organ that together with the cartilage forms the skeleton to provide mechanical support against loading and protection of the internal organs . in addition , the mineral components of bone are a storage sink for the critical signaling molecules calcium and phosphorus , as well as other factors . finally , bones house the bone marrow and , together with metabolically active bone forming osteoblasts and bone resorbing osteoclasts , provide the stem cell niche necessary for the maintenance of hematopoietic and lymphoid cell populations . bone and bone marrow are affected in many disorders , often leading to bone marrow dysfunction , severe bone pain , and pathologic fracture . bone is a common site of metastasis in many solid tumors , most notably breast cancer and prostate cancer , where tumor cells directly engage the bone marrow niche to initiate the vicious cycle of bone metastasis and displace hematopoietic stem cells . hematopoietic malignancies including myeloma and leukemia are characterized by bone marrow dysfunction as well as deregulation of healthy bone remodeling . other non - malignant skeletal disorders are also active areas of research , such as osteoarthritis , osteoporosis , scoliosis , and rickets . even in an otherwise healthy individual , biomechanical failure in a bone all of these disorders represent active areas of research with the goal of identifying new preventative measures and treatment regimens to reduce morbidity and mortality . to research the plethora of roles of the bone and the bone marrow , both under physiologic and pathologic conditions , it is critical for researchers to have a simple and efficient standardized method for dissection of the mouse long bones for rapid processing of large in vivo experiments . the dissection protocol outlined here is suitable for all long bone analyses including ex vivo imaging , histology , histomorphometry , and strength testing , among others . similarly , a standardized bone marrow isolation method with high bone marrow cell recovery and low inter - user variability is important for experimental analysis such as fluorescence - activated cell sorting ( facs ) or quantitative pcr ( qpcr ) as well as downstream applications such as primary cell culture of bone marrow cells . all animal work was approved by the institutional animal care and use committee in accordance with the recommendations outlined in the guide for the care and use of laboratory animals of the national institutes of health . position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint . spray the mouse with 70% ethanol , thoroughly dousing the legs . make a small incision to the right of midline in the lower abdomen , just above the hip . pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the blade of the scissors against the posterior side of the femur , cut the hamstrings away from the knee joint . pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the forceps , hold the distal end of the femur , just above the knee joint . guide the blades of the scissors on either side of the femoral shaft towards the hip joint , being careful not to cut into the femur itself . after reaching the femoral head , indicated by the scissors opening slightly , twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur , being careful not to snap the bone below the femoral head . grasp the top of the femoral shaft with the forceps , cut the soft tissue away from the femoral head to release it from the acetabulum . pull the entire leg bone , including femur , knee , and tibia , up and away from the body , carefully cutting away the connective tissue and muscle connecting the leg to the skin . overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia . grasping the distal end of the tibia , taking care not to sever the tendons , pull the tibia up and away from the body and the pin board . cut any remaining connective tissue attaching the long bone to the mouse at the knee . remove any additional muscle or connective tissue attached to the femur and the tibia . for any applications that require the bone to remain intact ( histology , histomorphometry , biomechanical testing , etc . ) , proceed with standard in - house protocols ( as in ) . to isolate bone marrow , proceed to section 2 . using the forceps , grasp the femur with the patella facing away and the proximal end ( femoral head ) down . overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur . grasp the femur with the anterior side facing away and the proximal end ( femoral head end ) down . rotate the scissors back and forth to remove the condyles , the patella , and the epiphysis to expose the metaphysis . remove any additional muscle or connective tissue attached to the femur using forceps , scissors , and kimwipes . using the forceps , grasp the tibia with the anterior side facing away and the distal end ( ankle end ) down . if the tibial epiphysis is intact , guide the scissors up the tibia shaft to the condyles . gently rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis . remove any additional muscle or connective tissue attached to the tibia using forceps , scissors , and kimwipes . push an 18 g needle through the bottom of a 0.5 ml microcentrifuge tube . place the long bones ( maximum of 2 femurs and 2 tibiae ) into the tube , knee - end verify that the bone marrow has been spun out of the bones by visual inspection . the bones should appear white and there should be a large visual pellet in the larger tube . suspend the bone marrow in appropriate solution ( e.g. , pbs , culture media , facs buffer ) and proceed with experimental protocol ( dna , rna , or protein isolation , facs analysis , or primary cell culture ) . euthanize the mouse in accordance with institutional guidelines . position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint . spray the mouse with 70% ethanol , thoroughly dousing the legs . make a small incision to the right of midline in the lower abdomen , just above the hip . pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the blade of the scissors against the posterior side of the femur , cut the hamstrings away from the knee joint . pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the forceps , hold the distal end of the femur , just above the knee joint . guide the blades of the scissors on either side of the femoral shaft towards the hip joint , being careful not to cut into the femur itself . after reaching the femoral head , indicated by the scissors opening slightly , twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur , being careful not to snap the bone below the femoral head . grasp the top of the femoral shaft with the forceps , cut the soft tissue away from the femoral head to release it from the acetabulum . pull the entire leg bone , including femur , knee , and tibia , up and away from the body , carefully cutting away the connective tissue and muscle connecting the leg to the skin . overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia . grasping the distal end of the tibia , taking care not to sever the tendons , pull the tibia up and away from the body and the pin board . cut any remaining connective tissue attaching the long bone to the mouse at the knee . remove any additional muscle or connective tissue attached to the femur and the tibia . for any applications that require the bone to remain intact ( histology , histomorphometry , biomechanical testing , etc . ) , proceed with standard in - house protocols ( as in ) . to isolate bone marrow , proceed to section 2 . using the forceps , grasp the femur with the patella facing away and the proximal end ( femoral head ) down . overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur . grasp the femur with the anterior side facing away and the proximal end ( femoral head end ) down . rotate the scissors back and forth to remove the condyles , the patella , and the epiphysis to expose the metaphysis . remove any additional muscle or connective tissue attached to the femur using forceps , scissors , and kimwipes . using the forceps , grasp the tibia with the anterior side facing away and the distal end ( ankle end ) down . if the tibial epiphysis is intact , guide the scissors up the tibia shaft to the condyles . gently rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis . remove any additional muscle or connective tissue attached to the tibia using forceps , scissors , and kimwipes . push an 18 g needle through the bottom of a 0.5 ml microcentrifuge tube . place the long bones ( maximum of 2 femurs and 2 tibiae ) into the tube , knee - end verify that the bone marrow has been spun out of the bones by visual inspection . the bones should appear white and there should be a large visual pellet in the larger tube . suspend the bone marrow in appropriate solution ( e.g. , pbs , culture media , facs buffer ) and proceed with experimental protocol ( dna , rna , or protein isolation , facs analysis , or primary cell culture ) . the protocol described here is optimized for rapid dissection of the mouse femur and tibia with a minimum of damage to the bone tissue . this technique is suitable for a number of downstream analyses , including biomechanics studies , histomorphometry ( figure 1a - b ) , and histology ( figure 1c ) . the representative histomophometric micoct 3d reconstruction ( figure 1a - b ) demonstrates that both the cancellous bone and cortical shell are maintained that allows for accurate quantitation of the standardized structural parameters for bone histomorphometry , including trabecular number , thickness , and spacing ; bone volume ; and cortical thickness , among other measures . the representative histologic section shows an h&e stained formalin - fixed and decalcified tibia ( figure 1c ) . the image demonstrates the integrity of both the calcified bone and cellular bone marrow for histologic analysis . the bone marrow isolation procedure preserves the sterility of the bone marrow space , has low handling to reduce contamination , and does not require cutting of the long bone , thus reducing loss of bone marrow yield . this bone marrow is suitable for many downstream applications , including flow cytometry and pcr analyses . in addition , this procedure can be used to isolate bone marrow for primary cell culture of bone marrow cells , including osteoclasts and osteoblasts ( figure 2a - b ) . three - dimensional microct reconstruction of a mouse tibia showing ( a ) the outer cortical shell and ( b ) trabecular bone ( scale bar = 0.5 mm ) . ( c ) histological h&e stain of a decalcified and sectioned tibia ( 4x ) . ( a ) trap staining for multinucleated osteoclasts after 7 days in osteoclastogenic media ( 4x ) . ( b ) alkaline phosphatase ( purple color ) for osteoblasts and alizarin red ( red color ) stain for mineralization after 21 days in osteogenic media . we present a simple and efficient method for removal of mouse hind long bones and subsequent bone marrow isolation . this method maintains the high structural and cellular integrity of the bones and bone marrow and has low handling time , minimizing the likelihood of user - induced fracture or bone scoring that may influence downstream analyses . in addition , the centrifugation method for isolating bone marrow does not require cutting the bone to expose the bone marrow space or fluid to flush the bone marrow , reducing potential points of contamination . moreover , the centrifuge technique is relatively high - throughput with lower hands - on time than other methods , thus reducing processing time . high variation is inherent to in vivo mouse studies due to high mouse - to - mouse phenotypic variation . in order to maximize the research impact of expensive and labor - intensive mouse studies , it is critical to minimize technical experimental error . time from animal sacrifice to downstream analysis or tissue fixation introduces experimental variation that may overcome subtle changes and reduce large differences between groups . the long bone dissection and bone marrow isolation techniques described here are optimized for rapid processing of animals and samples to reduce technical variation . this protocol can be widely applied to many research fields , including investigation of the bone tissue itself or interrogation of the cells of the bone marrow . in addition , this straightforward approach to long bone dissection will enable researchers in related fields to directly interrogate bone contributions in order to expand our knowledge of bone marrow dysfunction in otherwise understudied pathologies .

investigation of the bone and the bone marrow is critical in many research fields including basic bone biology , immunology , hematology , cancer metastasis , biomechanics , and stem cell biology . despite the importance of the bone in healthy and pathologic states , however , it is a largely under - researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation . mice are a common model organism to study effects on bone and bone marrow , necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts . we describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications , including but not limited to histomorphologic analysis and strength testing . in addition , we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time , ideal for cell sorting , primary cell culture , or dna , rna , and protein extraction . the protocol is streamlined for rapid processing of samples to limit experimental error , and is standardized to minimize user - to - user variability .

tuberculosis ( tb ) is a major health issue in developing countries with over two billion people being infected with tuberculosis bacilli world wide ( 1 ) . although tb was recently regarded primarily as a disease of the developing countries it is now also on the increase in developed countries such as the united kingdom , mainly as a result of globalisation as well as an increase in immunosuppressed patients . figures by the health protection agency ( hpa ) show that the cases of tb in the uk increased by 5.5% in 2009 when compared to those in 2008 ( 2 ) . this caucasian patient presented to the emergency department with sudden pain localised to the ulnar aspect of her wrist . x - ray of the forearm was normal and she was treated conservatively but was lost to follow up . she presented again at the orthopaedic clinic 26 months later with continuing swelling and pain . ultrasound showed a synovial tumour on the dorso - medial aspect of the wrist ( figure 1 ) . ultrasound showing a synovial tumour on the dorsal medial aspect of the wrist however , special stains for fungi and acid fast bacilli were negative . subsequently the patient developed a sinus and continued to have a chronic discharge from her left wrist . x ray this time showed destruction of the ulnar styloid consistent with a bony infection ( figure 2 ) . mri showed destruction localised around the ulnar head with oedematous changes extending up the shaft of the ulna ( figure 3 ) . ap xray of the wrist showing destruction of the ulnar styloid consistent with a bony infection . soft tissue swelling can also be noted over the dorsum of the wrist mri showing localised destruction around the ulnar head . the mri also shows thickening around the extensor carpi ulnaris tendon in keeping with marked tenosynovitis at this site . the patient agreed to a repeat biopsy and excision of the necrotic bone ( figure 4 ) . microscopy of the specimen again showed a florid granulomatous inflammatory process with areas of fibrinoid necrosis . post - operative xray of the wrist showing excision of the ulnar styloid further enquiry revealed that our patient had been experiencing lethargy but no other features to suggest tuberculosis . following the diagnosis of tb , the patient recalled that in her former post as a nursing assistant , when she was much younger , she was exposed to patients with tb . although improvements in its management and treatment are continuously occurring an estimated 1.8 million people died of tb in 2008 ( 1 ) . extra - spinal tuberculous osteomyelitis is rare and comprises about 2 - 3% of all cases of osteoarticular tuberculosis , with the hip and knee joints being the most common following spinal involvement ( 3 ) . tuberculous involvement of the ulna is uncommon and tends to be more common in the diaphysis . a high index of suspicion of tb is important to obtain the correct diagnosis . in chronic soft tissue and bony infection the diagnosis of tb may not be evident from the history and examination and indeed no obvious risk factors may be present as in our case . in our patient the infection could have been contracted at a much younger age , when she was caring for patients infected with tuberculosis , and remained latent . however no other risk factors were evident that could explain why the disease was activated after such a long period of latency . it is also interesting to note that the actual distal radio - ulnar joint was not involved , most of the destruction being situated around the ulnar process . although localisation of the tuberculous infection in the bone without joint involvement can occur , this is rare ( 3 ) .

tuberculosis ( tb ) is a major health issue in both developing and developed countries such as the uk . healthy individuals who contract the infection have only 5 - 10% chance of converting to the active disease over their lifetime . we present the clinical history of a 76 year old female who attended the emergency department complaining of wrist pain , and was only diagnosed with tb after three years . this case report emphasises the importance of including tb in the differential diagnosis when treating patients with an infection resistant to conventional antibiotics , even though risk factors for tb may not be evident or special stains for tb are negative .

the value of the ketogenic diet ( kd ) has been recognized in the treatment of epilepsy , although the exact mechanisms by which it exerts its effect remain an enigma . they seem to be different from those of regular antiepileptic medications ( aeds ) , and discovering what they are may lead to its use in clinical situations other than epilepsy as well . the kd is comprised of four elements , the changes of any of them can potentially lead to losing its anticonvulsant effect : ( 1 ) increased amount of fat , usually in a ratio of 3 to 4 grams of fat for each gram of protein and carbohydrates , ( 2 ) as low a consumption of glucose as possible , ( 3 ) caloric restriction , and ( 4 ) fluid restriction . although there is some debate about the last component , clinical practice has shown that stopping fluid restriction can lead to seizure recurrence much in the same way as when stopping glucose restriction . the most important result of observing the diet is the increased blood level of free fatty acids ( ffas ) . the ffas are transferred into the mitochondria , a process which requires the presence of an appropriate amount of carnitine , where they are degraded into ketone bodies through oxidation . these ketone bodies include hydroxybutyrate , acetoacetate , and acetone ( figure 1 ) . the effect of the increased amount of ketone bodies seems to be the most prominent of all other suggested antiepileptic mechanisms of the kd . the degradation of the ketone bodies delivers acetyl - coa directly to the tricarboxylic acid cycle , thus increasing its turnover while bypassing the need to depend on acetyl - coa coming from glycolysis to produce atp . unlike glucose , which requires a transporter to cross the blood brain barrier , the ketone bodies penetrate it easily . when this transporter is deficient , as in glut 1 deficiency , the kd is the preferred way of antiepileptic treatment , since it allows for bypassing the need for glucose . children in whom the conversion of pyruvate to acetyl - coa is blocked , for instance , in pyruvate dehydrogenase ( pdh ) deficiency , will also benefit from bypassing this route . hydroxybutyrate is the predominant ketone body measured in the blood , and it is used to monitor the degree of ketosis during therapy . the breath of a patient who is ketotic while on the diet will often even smell of acetone . acetone is one of the ketone bodies that have an anticonvulsant effect in several types of mouse seizure models . the mechanism of this effect is unknown , although an effect on k2p channels has been proposed . by means of the tca cycle , acetyl - coa increases the level of the neurotransmitters glutamate and -aminobutyric acid ( gaba ) and the major excitatory and inhibitory neurotransmitters in brain , respectively . another product of an elevated level of free fatty acids is polyunsaturated fatty acids ( pufas ) . the potential ability of pufas to block seizure activity in the brain is speculated to be associated with some rather more complicated mechanisms , including ( 1 ) directly inhibiting voltage - gated sodium and calcium channels , ( 2 ) activating a lipid - sensitive potassium channel , ( 3 ) enhancing the activity of the sodium pump to limit neuronal excitability , ( 4 ) activating peroxisome proliferator - activated receptor- ( ppar ) , and ( 5 ) inducing the expression and activity of brain - specific uncoupling proteins in the mitochondria , thereby inducing a neuroprotective effect . neuroprotection can contribute to the anticonvulsant effect , but it may have other effects as well , which can lead to other clinical uses of the kd . appleton and de vivo reported that the kd increased the total quantity of bioenergetic substrates ( adenosine triphosphate ( atp ) ) and elevated the energy charge in rat brain . acetoacetate , a product of hydroxybutyrate dehydrogenation , is transferred into acetyl - coa which enters the tricarboxylic acid ( tca ) cycle . the increased turnover of the tca cycle generates protons and electrons that are channeled to the electron transport chain . this , in turn , drives the formation of atp from adenosine phosphate ( adp ) by atp synthase . enhanced atp can either be converted to phosphocreatine for energy storage or broken into adenosine . enhanced atp levels provide energy reserves for a neuron to continue functioning under stress . increased extracellular adenosine offers a neuroprotective buffer against insults , reduces excitation , and averts excessive atp demands , thus providing local seizure control and neuroprotection . it was also suggested that the kd influences toward an upregulation of transcripts encoding energy metabolism enzymes and increase in the density of mitochondria in neuronal process , leading to heightened energy reserves . an improved energetic status can support seizure prevention , for instance , by supporting gabaergic inhibition . it is suggested that adaptive processes to the metabolic changes induced by the diet lead to changes in gene expression which in turn result in some of the above - noted changes . other path of neuroprotection is modulated through decreased generation of ros which is considered to be related to pufas effect on uncoupling proteins . the fact that the kd is considered a proven therapy with relatively few adverse affects and wide clinical experience , particularly in children , led to recent studies investigating new potential uses for other neurological disorders . one of the most intriguing and active fields of research is the effect of a high - fat caloric - restricted diet on the survival of brain tumors cells . it is hypothesized that mitochondrial abnormalities impair the ability of brain tumors to generate energy from ketone bodies . unlike normal cells , malignant tumor cells have impaired genetic adaptability due to their genetic abnormalities and , therefore , increased susceptibility to environmental stress , such as fasting or caloric restriction . the same genomic defects that are involved in the creation of brain tumors can be exploited for their destruction [ 3 , 10 , 11 ] . in 1995 , nebeling et al . reported two young girls with unresectable advanced stage brain tumors who had poor response to radiation and chemotherapy . they were treated with a kd and their response was remarkable , both clinically and according to positron emission tomography follow - up scans . described a patient with glioblastoma multiforme whose tumor , which is very malignant , improved on the kd . surprisingly , despite the appealing efficacy of this treatment , no further human studies or clinical trials on the kd as a therapy for brain tumors have been conducted . several laboratory studies in mouse and rat models have recently confirmed that inhibition of brain tumor growth is directly related with reduced levels of glucose and elevated levels of ketone bodies . moreover , the kd was shown to reduce reactive oxygen species ( ros ) in the brain . cancer cells need high levels of ros for the induction of angiogenesis and the production of tumor growth factors , thus , through this mechanism the kd can be protective . the clinical manifestations were mainly intractable seizures that necessitated repeated admissions to the intensive care unit , as well as severe cognitive and alertness decline . after the oncologists decided that antitumor treatment would be ineffective , she was started on the kd . after four weeks of trial , it did not have any effect on the tumor progression . however , it did have a notable improving effect on cognition , alertness , and mood of the girl , in spite of her devastating condition . the beneficial effect of the kd on cognition , alertness , and mood is well recognized , and clinical experience shows that in many times it is not less important than the anticonvulsant effect . the potential neuroprotective effect of the kd was what motivated investigations into its potential as a treatment option in other neurologic disorders . there are increasing numbers of reports that ketosis achieved by starvation or administration of a kd has a consistent neuroprotective effect after various brain injuries in animal models . one human pilot study and several animal model studies have shown improvement in autistic behavior parameters with kd treatment . it remains to be further clarified whether this improvement is related to reduced epileptic activity found in up to 30% of these patients or to a primary effect of the kd . a factor that can be crucial for the application of the kd in medical conditions other than intractable epilepsy is the inherent difficulties in its use . dietary restriction can pose a significant problem in a child with a progressive tumor that undergoes massive chemotherapy , who may already be cachectic . the kd may not be an option in a grown - up hyperactive autistic child . thus , the neurologist must evaluate the appropriate clinical , familial , and environmental situations very cautiously prior to the recommendation of the kd . in conclusion , the major metabolic effect of the kd is in supplying the brain with an increased amount of free fatty acids . their degradation into ketone bodies , together with the load of pufas , leads to major changes in the metabolic , bioenergetic , mitochondrial , and even genetic constellation . whether it may also be effective in other pathologies , especially in treating malignancies , awaits future research .

although the ketogenic diet ( kd ) has been widely accepted as a legitimate and successful therapy for epilepsy and other neurological disorders , its mechanism of action remains an enigma . the use of the kd causes major metabolic changes . the most significant of them seems to be the situation of chronic ketosis , but there are others as well , for instance , high level of polyunsaturated fatty acids ( pufas ) . these primary influences lead to secondary , in part adaptive , effects , for instance changes in mitochondrial density and gene expression . clinically , the influences of the diet are considered as anticonvulsive and neuroprotective , although neuroprotection can also lead to prevention of seizures . potential clinical implications of these mechanisms are discussed .

the endotracheal tube ( ett ) should be placed at the optimal level to avoid inadvertent complication . if the ett is too deep , it increases the risk of unintended single lung ventilation . on the other hand , if the ett is too shallow , it may cause vocal cord injury by the ett balloon or accidental extubation . there are many methods for determining the appropriate depth of ett in adults ; fixed insertion depth according to sex ( 23 and 21 cm from the upper incisors in adult males and females , respectively ) , the use of depth marks on the ett , suprasternal palpation of the ett tip or cuff , and bilateral auscultation . although chest radiography and bronchoscopy are considered an accurate method , they are not always feasible and the costs are considerable . considering the individual variation in the length of the trachea , using fixed depths or marks on the ett may result in inadequate placement of the ett . to reduce the risk of single lung ventilation or vocal cord injury , the segment between the proximal edge of the cuff and the ett tip should be placed at the mid - trachea level . if the length of the trachea can be predicted at bedside before intubation , the ett can be placed at a safe depth for each patient . the purpose of this study was to determine whether surface anatomical landmarks can be used to predict the mid - tracheal level in adult patients . neck computed tomography ( ct ) images of adults obtained between 2009 and 2014 were reviewed by a single reviewer after obtaining approval form seoul national university hospital institutional review board ( july 17 , 2015/no . patients with laryngeal , tracheal , or thoracic abnormalities , tracheostomy or significant tracheal deviation caused by mass lesions were excluded . in addition , poor ct image quality , absent sagittal ct images , ct images that did not cover the entire trachea , or ct images taken in neck hyperextension ( boidin angle > 130 on scout view ) or hyperflexion ( boidin angle < 110 on scout view ) position were also excluded . levels of subglottic and tracheal airway segments that include the vocal cords ( vc ) , the cricoid cartilage ( cc ) , and the carina were identified using the method described by sirisopana et al . the vocal cords were identified as the most cranial level of the upper airway , with a teardrop shape below the laryngeal ventricle . anatomic level of suprasternal notch ( ssn ) the level of manubriosternal junction ( msj ) was defined as the middle level between the manubrium and the body of the sternum where the second rib attaches to the sternum . the level of carina was defined as the bifurcation of right and left main bronchus and identified by the figure 8 shaped lumen . the external locations of the cc , ssn , and msj were defined on the skin surface . the distances between the external points of the cc and the ssn ( extcc - ssn ) , and those of the ssn and the msj ( extssn - msj ) were measured . the distances between the subglottic structures at the levels of the vc , the cc , ssn , and carina were measured . tracheal length was calculated as the summation of the distances measured from the vc to the carina . the mid - trachea level was calculated from tracheal length and the ideal position of ett is shown in fig . 2 . identification of subglottic segments on axial view ( right ) , and defining the distances between subglottic segments on sagittal view ( left ) in a 45-year - old male patient . cc = cricoid cartilage , extcc - ssn = the surface distances from the cricoid cartilage to the suprasternal notch , extssn - msj = the surface distances from the suprasternal notch to the manubriosternal junction , msj = manubriosternal juction , ssn = suprasternal notch , vc = vocal cord , tracheal length = a + b + c + d. the ideal position of endotracheal tube ( ett ) to minimize the risk of endobronchial intubation and vocal cord injury . the mid - point between the proximal edge of the cuff and the ett tip was matched to the level of the middle of the trachea ( mtl ) , which was calculated from the tracheal length . cc = cricoid cartilage , ett = endotracheal tube , msj = manubriosternal juction , mtl = the level of the middle of the trachea , ssn = suprasternal notch , vc = vocal cord . the correlations between age , height , weight , and tracheal length were analyzed using linear regression . the relationships between surface measurements and the mid - tracheal level were analyzed with bland the distance between the cc and the ssn was longer in females ( p < 0.001 ) , whereas distances between the vc and the cc , the ssn and the carina , and the tracheal length were longer in males ( p < 0.001 ) . the extcc - ssn was longer in females ( p < 0.001 ) , whereas extssn - msj was longer in males ( p < 0.001 ) . demographic data and measured distances between subglottic structures of trachea , external landmarks , and calculated tracheal length . the calculated tracheal length was 131.9 ( 10.3 ) mm and ranged from 106.0 to 169.8 mm . tracheal length showed weak correlation with height ( r = 0.2188 ) and weaker correlation with age ( r = 0.0004 ) and weight ( r = 0.0466 ) ( fig . the calculated mid - tracheal level was 66.0 5.1 mm from the vc . altman plots showing the difference between the mid - tracheal level and the surface measurements are shown in fig . the bias was calculated by subtracting the mid - tracheal level from the surface measurements . the difference between the extcc - ssn and the mid - tracheal level was 6.6 ( 12.5 ) mm , and the difference between the extssn - msj and the mid - tracheal level was 19.2 ( 6.1 ) mm . altman plots showing the agreement between the mid - tracheal level ( mtl ) and the surface measurements of surface anatomical landmarks . ( left ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 6.6 [ 31.1 to 17.9 ] ( mm ) . ( right ) the bias ( subtraction of the mid - tracheal level from the surface distance from the suprasternal notch to the manubriosternal junction ) and 95% limit of agreement as the mean difference were 19.2 [ 31.1 to 7.2 ] ( mm ) . extcc - ssn = the surface distances from the cricoid cartilage to the suprasternal notch , extssn - msj = the surface distances from the suprasternal notch to the manubriosternal junction , mtl = the level of the middle of the trachea . altman plots of the difference between the extcc - ssn and the mid - tracheal level in female and male patients . the calculated mid - tracheal level was 64.5 ( 4.6 ) mm in females and 67.9 ( 5.0 ) mm in males . the difference between the extcc - ssn and the mid - tracheal level was 1.7 ( 11.7 ) mm in females and 12.8 ( 10.7 ) mm in males ( p < 0.001 ) . altman plots of female ( left ) and male ( right ) patients showing the agreement between the mid - tracheal level ( mtl ) and the surface the surface distance from the cricoid cartilage from the suprasternal notch . ( left ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 1.7 [ 24.6 to 21.2 ] ( mm ) in females . ( right ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 12.8 [ 33.8 to 8.2 ] ( mm ) in males . extcc - ssn = the surface distances from the cricoid cartilage to the suprasternal notch , mtl = the level of the middle of the trachea . this study showed that mid - tracheal level could be estimated by the surface distance between the cc and the ssn ( extcc - ssn ) . compared with males , extcc - ssn more accurately predicts the mid - tracheal level in females . in clinical practice , physicians can use extcc - ssn to determine the depth of ett so that the segment between the proximal edge of the cuff and the ett tip lies at the mid - tracheal level . there are several studies that used the distance between the surface anatomical landmarks to estimate the airway length and the optimal insertion depth of endotracheal tube . lee et al showed that the distance between the upper incisor and the manubriosternal angle in the neck extension position correlates with the distance between the upper incisor and the carina in the neutral neck position . evron et al used the sum of the 2 distances from the mouth angle to the jaw angle and from the jaw angle to the sternal manubrium to determine the depth of ett . however , these methods are complicated than single measurement of extcc - ssn due to the difficulty in locating the sternal manubrium , more than 1 measurement , and the need for full neck extension . the physicians can easily apply our method with a simple measurement of the extcc - ssn to estimate the mid - tracheal level , and apply this depth during endotracheal intubation . however , according to previous studies in adults , flexion and extension of the neck from neutral position are associated with the inward and outward movements of the ett . conrardy et al showed that the orally intubated ett can move 1.5 ( range 0.52.0 ) cm toward the carina during flexion and 2.4 ( range 1.34.3 ) cm away from the carina during extension . kim et al showed that the ett can migrate 1.3 ( sd 0.6 ; range 0.52.5 ) cm toward the carina during flexion and 1.7 ( sd 0.8 ; range 0.43.1 ) cm during extension . therefore , the position of the neck should be considered when determining the optimal depth of the ett . first , this study was a retrospective analysis of ct images and the distances were measured in the neutral position . second , since the vc is used as a baseline of the depth of intubation in our method , it can be difficult to apply our method to patients with high cormack grades . lastly , the efficacy of this method is yet to be demonstrated in clinical practice . in conclusion , the mid - tracheal level , which is considered the optimal depth of ett , can be predicted by the surface distance between the cc and ssn in adults , especially in females .

abstractendotracheal tube ( ett ) should be placed at the optimal level to avoid single lung ventilation or accidental extubation . this study was performed to estimate the mid - tracheal level by using surface anatomical landmarks in adult patients.neck computed tomography images of 329 adult patients between the ages of 16 and 79 years were reviewed . in the midline sagittal plane , the levels corresponding to the vocal cords , cricoid cartilage , suprasternal notch , manubriosternal junction , and carina were identified . the surface distances from the cricoid cartilage to the suprasternal notch ( extcc - ssn ) and that from the suprasternal notch to the manubriosternal junction ( extssn - msj ) were measured . the relationship between mid - tracheal level and the surface distances was analyzed using bland altman plot.the difference between the extcc - ssn and the mid - tracheal level was 6.6 ( 12.5 ) mm , and the difference between the extssn - msj and the mid - tracheal level was 19.2 ( 6.1 ) mm . the difference between the extcc - ssn and the mid - tracheal level was smaller in females compared with males [ 1.7 ( 11.7 ) mm vs 12.8 ( 10.7 ) mm ; p < 0.001].the mid - tracheal level , which is helpful in planning the insertion depth of an ett , can be predicted by the surface distance between the cricoid cartilage and suprasternal notch in adults , especially in females .

embryos generated through assisted reproductive technologies ( art ) are deemed surplus when , for some reason , they are not used for reproductive purposes ( svendsen and koch 2008 ; haimes and taylor 2011 ) ; instead , they are then disposed of or , where this is allowed by a country s art regulation , may be donated for a variety of research purposes . thus , the generation and subsequent fate of embryos that are designated surplus are determined as much by legislation as by embryo biology or technological constraints . for example , legislatures differ in whether cryopreservation of embryos is permitted , meaning that in some countries any ivf embryos not immediately transferred for pregnancy are surplus , while in others they may be frozen for later transfer . in some countries , the law considers surplus embryos as the primary source of material for human embryonic stem cell ( hesc ) research , while in others this use of embryos is expressly forbidden . although hesc research has been subjected to exhaustive ethical scrutiny , the lack of consensus on the ontological or ethical status of surplus embryos means that the uses to which they may then be put remain contested ( deckers 2007 ; guenin 2008 ; moller 2009 ) . even if using human embryos for stem cell research is deemed permissible , ethical issues continue to emerge as new social practices and roles develop around the act of embryo donation . the relative novelty of these practices and roles , complicated by the pace of change in technical possibility and regulation , mean that there is still uncertainty about how to conceptualise embryo donation , not just in terms of systematic ethics but as a sociomoral practice in everyday social life and morality ( banks , scully , and shakespeare 2006 ) . from a sociological perspective , and also to understand more generally how everyday morality deals with unprecedented ethical dilemmas , it is important to examine which conceptualisations emerge as salient and how they are stabilised and used . for example , one way of thinking about the donation of embryos to research is as a bodily gift relationship , one of the many established by modern biomedicine such as the donation of eggs and sperm for reproductive purposes , organs or tissues for transplantation , blood for transfusion , and other biological tissues for research . drawing analogies to existing practices in this way has proved helpful in other instances of bioethical novelty ( hofmann , solbakk , and holm 2006 ) . however , analogies can equally well be misleading , if there are morally relevant but unacknowledged differences between situations . the contexts in which different organs and tissues are donated vary significantly , and these differences influence the sociomoral understanding of donation in each case and make them noncomparable in ethical terms . reproductive tissue , for example , is generally distinguished from other types of donated tissue because eggs , sperm , and embryos have the potential to give rise to new individuals , not just to prolong the lives of existing individuals , or to be used for research . because embryos are generally considered to have a different moral status from other tissues , the use of surplus embryos in research raises moral unease about the instrumentalization of human life that is not raised in quite the same way by the donation of either ova or sperm . it is therefore unclear whether an embryo can meaningfully be treated as a gift ( donation ) without blurring the morally relevant differences between embryos and other tissues . similarly , hesc research , to which the embryo may be given , is a domain of biomedical practices and not an entity . as such , hesc research is not a subject with which a gift relationship can be established ( unlike , for instance , an organ recipient ) . understanding the social and ethical meanings that are emerging for the practices associated with embryo donation calls for a detailed empirical examination of people s reasoning behind donation decisions . however , most empirical studies of embryo donation have not focused on people s donation rationales in depth ( with some exceptions ; for example , haimes et al . 2008 ; de lacey 2003 ) . the scarcity of data on donor rationales means there is a corresponding lack of information on contextual and other factors that influence donor decisions and how these can be related to the evaluations of theoretical ethics . the authors of this paper have been involved in a series of linked qualitative studies of practices of embryo donation , first in the united kingdom ( researcher haimes and colleagues ) , then switzerland ( scully , rehmann - sutter , and porz ) , and in a smaller pilot project in china ( mitzkat , rehmann - sutter , and haimes ) . all three studies shared an interest in understanding reasons for the donation or non - donation of embryos . while the studies in switzerland and china drew upon the original u.k . design , each study was conducted independently , and the details of each project , including interview design , differed in light of the varying regulatory , clinical , and cultural contexts . however , by looking across the three data sets , we hope to gain cross - cultural insights into donation and non - donation rationales and the moral understandings on which they are based . the three studies all used open - ended , semi - structured , one - off interviews between the researcher and people who had been in the position of deciding whether to donate their surplus embryos to research . interviews were designed to explore in depth not just the interviewees decision about donation but also the background to that decision , such as their ivf story , their family and other relationships , their relationship with the clinic and its staff , and so on . the interviews were transcribed , coded , and initially analysed to identify the reasons for donation decisions given by participants in each study , using an interpretative approach that all of us have previously found useful for identifying key themes around decision - making and implicit or explicit ethical judgements ( charmaz 2006 ; smith , flowers , and larkin 2009 ) . study ran for more than three years ; after some familiarisation and observation in the collaborating clinic , 44 in - depth interviews were conducted with couples who had been asked to donate their surplus fresh embryos , generated through ongoing ivf treatment , to hesc research ( haimes and taylor 2009 ) . in the united kingdom , it has been possible for some years to donate unused embryos to stem cell and other kinds of research and also to other couples . the swiss study was carried out shortly after a change in the law that permitted fresh or cryopreserved surplus ivf embryos , under strictly defined conditions , to be donated to stem cell research only . in this study , 17 individuals who had variously chosen to donate or not to donate were interviewed ; thus , the decision concerned the fate of cryopreserved embryos some time after the ivf treatment that had produced them ( scully , rehmann - sutter , and porz 2010 ) . the chinese work involved a much smaller three - month pilot study carried out in a large art hospital . it was designed as a pilot to provide a comparison with the u.k . and swiss material and is , therefore , included here despite the low number of participants , but with no attempt to draw general conclusions from it . legislation in china does not allow the donation of embryos to other couples , but consent can be given for a surplus embryo to be used in stem cell research or for it to be discarded . the study included participant observation of the information and consent procedures and qualitative interviews with five ivf patients , three choosing to donate to stem cell research and two refusing ( mitzkat , haimes , and rehmann - sutter 2010 ) . for the purposes of this paper , the authors involved in the studies jointly compared the rationales for donation decisions given by their participants . we also independently reexamined interview transcripts for material relevant to the discussion of gratitude . in the rest of this paper , we first identify and compare the main reasons given by participants in these three studies for their decisions to donate or to refuse to donate their surplus embryos to research . we then look in more detail at the implications of our findings for one area of potential ethical concern : the possible role of gratitude in making embryo disposition decisions . in this way , we not only collect empirical data to help understand the emerging moral meaning of a new practice , but also give an example to show how empirical data can be used to question and then refine the conceptual basis of bioethical theory . participants who chose to donate their surplus embryos to research had a background premise that donation is fundamentally permissible because embryos do not have the sort of ontological or moral status that would forbid it . the swiss study discovered people stating this explicitly : [ embryos ] are not , they are not yet people ; they are nt beings with souls as far as i m concerned , as one swiss participant said.1 but this did not mean that the surplus embryos were considered as morally equivalent to any other tissue , and participants in both the swiss and u.k . interviewees stopped themselves midway through sentences referring to the left over embryos , which they clearly felt , on reflection , was an inappropriate phrase . so the embryos did not have the sort of moral status that would have made it wrong to donate them , but neither were they completely disposable . to some of our participants , embryos had a value ; they were a precious resource ( haimes et al . 2008 ) that made simply throwing them out an unjustifiable waste . as another u.k . [ i]t just really seemed a waste , really , not to have them used . it was not always clear , however , quite how interviewees were using this notion of precious : whether with reference to economic bio - value ( waldby 2000 ; waldby and mitchell 2006 ) or because they had been obtained at intense personal and emotional cost , or because they had high moral value , or possibly a combination of all these aspects . particularly in the swiss study , which involved cryopreserved embryos stored for some time , participants referred to embryos moral status in a nuanced way that was highly sensitive to the developmental stage of the embryo , its physical location ( whether inside or outside the body ) , and its state ( whether cryopreserved or not ) ( see scully , rehmann - sutter , and porz 2010 ) . studies seemed less interested in explicitly defining the moral status of an abstract embryo than working out what their own particular embryos meant for them at defined points in their own story , and what this meaning then indicated it was morally permissible for them to do with that embryo . across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research . such research was seen as a valuable endeavour by those like the swiss participant who said , i feel that , fundamentally , research has to go on , and i support that . donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k . interviewee said , it was quite simply that we ve been helped by the system , we should help the system back. not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress . in the u.k . study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science : i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it . this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility . a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children . [ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research . note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland , the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies were participants asked outright if they thought their donated embryo was being used in infertility research . however , the fact that they used phrases such as helping others as we have been helped ( italics added ) indicates a potential misunderstanding . it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation . the u.k . participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues . these participants , as in the chinese study , were current patients donating fresh embryos . the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k . study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing . it is possible that this level of engagement with the detail of the research underlies the difference . it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial . as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it . for example , one interviewee said : if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it . i feel a little bit guilty sometimes , not very much , but a little bit like if we can at least give these embryos for a good reason , then it was nt completely in vain . using similar wording , another said , if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it . our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in creating life but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong . ( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . ) they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal . for these swiss participants , then , their donation decision appears to have been driven in part by what we could call a reparative urge . no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below . across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research . such research was seen as a valuable endeavour by those like the swiss participant who said , i feel that , fundamentally , research has to go on , and i support that . donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k . we ve been helped by the system , we should help the system back. not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress . in the u.k . study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science : i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it . this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility . for instance , one participant said , a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children . [ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research . note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as scientific research for the ivf treatment . one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland , the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies were participants asked outright if they thought their donated embryo was being used in infertility research . however , the fact that they used phrases such as helping others as we have been helped ( italics added ) indicates a potential misunderstanding . it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation . participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues . these participants , as in the chinese study , were current patients donating fresh embryos . the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k . study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing . it is possible that this level of engagement with the detail of the research underlies the difference . it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial . as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it . for example , one interviewee said : if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it . i feel a little bit guilty sometimes , not very much , but a little bit like if we can at least give these embryos for a good reason , then it was nt completely in vain . using similar wording , another said , if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it . our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in creating life but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong . ( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . ) they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal . for these swiss participants , then , their donation decision appears to have been driven in part by what we could call a reparative urge . no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below . approximately half of the swiss participants who were interviewed turned out to have decided against donation and instead have their surplus cryopreserved embryo(s ) destroyed . none of these explicitly used the moral status of the embryo as the fundamental basis for rejecting donation to research . the scientists would do with their embryo , which was articulated either as disapproval of specific types of research ( e.g. , not if it s for cloning ) or as a more general unwillingness to relinquish responsibility of the embryo to unknown others . in both sets of reasoning , therefore , participants were expressing unease about the loss of control over what would happen to their embryo . the two chinese interviewees who had opted against donation also did so because of reservations about lack of knowledge of exactly what the embryos would be used for , explicitly mentioning the fear that they would be donated to ( i.e. , used to generate pregnancy in ) other women . in addition , two of the swiss participants gave financial reasons , that is , they expressed anger that they would not be compensated for giving up their embryo and that through donation researchers were getting something for nothing . study , speculation about why some people might refuse to donate raised concerns about what might be done with the embryos and whether a baby would be developed through experimentation ; others wondered if refusers were worried that another infertile couple would receive their embryos and have a baby when they , the embryo providers , failed to do so . sample contained only two actual refusers , who objected on the grounds of possible use in animal research ( haimes and taylor 2009 ; hug 2008 ) . these observations provide material for an empirically grounded bioethics of the ivf stem cell interface ( franklin 2006 ) . in the rest of this paper , we consider a single aspect in detail : the potential problem of gratitude influencing informed consent . empirically , the main reasons given by our participants for donation were : ( 1 ) to avoid the waste of a precious resource , ( 2 ) to give something back to research , and ( 3 ) to compensate for a perceived moral wrong . in none of the three countries did our participants indicate that their decision arose out of any sense of gratitude to the physician who had given them ivf treatment . this is an important observation , because the possible impact of gratitude on informed consent in embryo donation has previously been raised by bioethical commentators . it has been suggested that if there is any institutional or procedural link between ivf treatment and hesc research , the potential donors gratitude toward the clinicians who have helped them to conceive may steer them toward opting for donation ( haimes and luce 2006 ; parry 2006 ; mcleod and baylis 2007 ; also discussed in roberts and throsby 2008 ) . the bioethical concern is that parents who receive the gift of a baby will feel a sense of gratitude ; feeling grateful might then cause parents to feel that they should respond with a gift in return ; and if a request for donation of surplus embryos is made in that context , potential donors might inadvertently be encouraged to see donation as an appropriate form of return gift . in this way the moral emotion of gratitude could compromise the potential donor s freedom to weigh the pros and cons of the request , which in turn means compromising the capacity to give fully voluntary consent . even where the clinicians and researchers themselves are scrupulous about not using persuasion , gratitude might be persuading potential donors to do something they would not otherwise choose to do . to avoid this possibility , countries that allow embryo donation for research may attempt a strict separation of ivf treatment and the stem cell research domain . ( not all legislatures do this , however : china is one country that does not . ) in the united kingdom , for example , it is accepted good practice that requests for donation of spare embryos should not be made by the physician(s ) who delivered the original ivf treatment . in switzerland , a predominant interpretation of the current law on embryo donation is that a couple undergoing ivf treatment should not be told even of the theoretical possibility that a surplus embryo might be generated unless it actually is ( porz et al . regulators hope to rule out any conflict of interest on the part of the treating physician as well as the possibility of gratitude on the part of the patient . there are several theoretical questions that can be asked about a claim that the risk of gratitude affects potential donors decisions . for example , moral psychologists might want to examine the emotional exceptionalism in which gratitude is seen as potentially compromising , while other emotions , or even the absence of emotion , is not . there is also an obvious empirical question which does not seem to have been closely examined : whether potential embryo donors actually do experience a sense of personal gratitude to the physician or one strong enough to sway their donation decision . however , they do indicate that participants rationales for donation indicate a desire to give back in a more complex way . first , our empirical results show the importance of distinguishing between the three moral emotions of gratitude , indebtedness , and solidarity . what we normally mean by gratitude is an emotion primarily associated with gifts or with help that is undeserved . gratitude has been defined as a feeling of thankful appreciation for favours received ( guralnik 1971 , 327 ) and is experienced as a positive emotion . ( 2006 ) provide a basis from experimental psychology for distinguishing between indebtedness and gratitude . one important distinction is that indebtedness is an emotion of exchange , whereas gratitude is not ( watkins et al . even if a debt of gratitude is felt , it does not appear to be analogous to an economic debt indebtedness , however , is , literally , a debt : a state of obligation to repay another ( mauss 2001 , 2 ) . a further feature associated with indebtedness but not with gratitude is the inherent imbalance of power , so that a hierarchy is created in which those who are indebted are rendered more vulnerable . the empirical social psychological work of watkins et al . indicates that , in experimental settings at least , a person s feelings of gratitude diminish as the expectation of return ( indebtedness ) increases . the more reciprocity is expected or demanded , importantly for considering the effects of these social emotions on donation decisions , watkins et al . found that the greater the expectation that something will be given back in return , the less motivated the participants were actually to comply with the norm of reciprocity ( watkins et al . these results suggest that if patients were to sense any expectation of return by an individual physician or researcher , it would if anything lessen any gratitude they might have felt . in the ivf context , what people have received ( the thing for which they might feel grateful ) is their baby or pregnancy , or at the very least , treatment . donors who achieve this have not received a gift , but a medical service , which will have been paid for in one way or another . in switzerland , where ivf is not covered by the mandatory health insurance , this will often be direct payment from the patients to the clinic , as it will also often be in china . in the united kingdom , patients who do not already have children commonly receive two free cycles of nhs treatment ( paid for through taxes on the population ) , and then they pay for future cycles directly through fees to the clinics . whatever the system of payment , however , the point is that the ivf physician or clinic has already been paid , directly or indirectly , for the treatment which produced the outcome . so in the case of embryo donation , it could be argued that neither gratitude nor indebtedness should be anticipated . gratitude would not be expected , because what patients have received was not a favour or unexpected gift ; and indebtedness would not be expected , because there is no debt if payment has already been made . in our three studies , although we found appreciation and high esteem were expressed for the work of the treating hospital or team , we found no evidence of gratitude being expressed toward an individual physician . this is despite the fact that in both the united kingdom and china mention was made of the well - known head physicians who led each clinic ; the mentions were in appreciation of their reputations , not as a debt of gratitude . participants wanted to do something with the surplus embryo that would be of benefit to research and medical treatment for infertility , but this was not directly associated with the physician who provided it originally . indeed , some of our interviewees were critical of aspects of their own ivf treatment , while still wanting to support the infertility research enterprise overall . in this context , it should be remembered that about half the swiss participants interviewed in fact chose not to donate , primarily out of anxiety about losing control over the fate of their embryos , but also in some cases because it meant researchers were getting something for nothing . study , a few patients were suspicious that research was being prioritised over treatment and that embryos might have been kept back for the research rather than frozen . although only a small number of participants expressed this view , it has particular relevance here as it indicates clearly the absence of either gratitude or indebtedness : indeed , it suggests that these participants felt that , if anything , donation would mean the researchers were receiving something beyond their entitlement or were indebted to the patients rather than the reverse . social exchanges may entail giving back not to an individual , because the individual may be unknowable or because the benefit may not have come from a single identifiable person , but to the pattern of social life ( becker 1986)for example , giving back to the institutional biomedical research that had helped the participants . in serial reciprocity , person a receives something from person b , but pays back not to person b but to some other third party ( moody 2008 ) . serial reciprocity accounts for the way that altruistic blood donors often do describe their action in terms of reciprocity , even though they have not received anything from the person to whom their blood eventually goes . what drives serial reciprocity is not gratitude or indebtedness to an individual or an organisation but a sense of solidarity with unknown others in the community . in the case of embryo donation , what appears to be a directly reciprocal act of gratitude a surplus embryo in return for treatment may thus be understood as something quite different . participants in our studies used a rationale for donation based not on reciprocal exchange between individuals , but on giving back to a research enterprise from which they had benefited and which they hoped would benefit unknown others in the future . for the swiss and chinese donors who choose to donate because they think they are supporting infertility research , these unknown others are not completely anonymous : they are known because of imaginatively shared experience . giving something back to the research that had helped them ( as they think they are doing ) was not considered an obligation but , rather , a form of return that was meaningful and seemed especially fitting to them because of the experiences they had gone through . in the u.k . study , although there was greater clarity about the purpose of the research being contributed to , there was still a sense among embryo providers that they were benefiting from the fact that others had participated in research in the past which had improved ivf ; they were clear in expressing solidarity with this imagined community of previous ivf research contributors by making their own contributions to research , albeit research in another domain . at least some of our participants , then , also see their donation to research as an indirect way of passing on the benefit they have received from research to someone else , whose situation in some way resembles their own . the social meaning of this indirect reciprocity is neither gratitude nor indebtedness , but solidarity with other present and future patients . to summarise : our interpretation of the material suggests that donors reasons for donating were not connected in any straightforward way to either gratitude or indebtedness . for some people the desire not to waste a valuable ( in more than one sense ) resource is foremost . a generally positive stance toward biomedical science means that donation is a good use of a valuable resource . with some of the swiss participants , we also identified a reparative urge coming from a sense of moral unease , and here donation seems to offer a route through which the urge to make reparation can be satisfied . all of this suggests that neither gratitude nor indebtedness per se are present in decision - making at the ivf stem cell interface , at least not in terms of undue inducement or of compromising the capacity for informed consent . the reparative urge foregrounds a different set of ethical questions about the sociomoral meaning of the generation of spare embryos and the act of donation . for example , the perceived need for reparation that appears to drive some of the swiss participants donation choices arises because people felt some sense of wrongdoing at creating embryos that are not used for pregnancy . the spirit of the swiss legal framework strongly reinforces the sense that the occurrence of embryos not used for pregnancy is to be considered as wrong . from this point of view , one possible conclusion could be that the felt desire for reparation is in fact an ethically appropriate response : if the act of deliberately creating an embryo is a morally significant one , which needs to be justified by the good of its intended outcome ( pregnancy ) , then not using it for the purpose that justifies it does indeed present a genuine ethical difficulty . on the other hand , the novelty of surplus embryos in the swiss legal and social context ( scully and rehmann - sutter 2006 ) meant that many patients would have been unaware at the time of their ivf of the possibility of generating a surplus embryo . moreover , in most cases there were perfectly valid clinical or legal reasons why the embryos could not be transferred , which fully justified the failure to use them as originally intended . that these factors were not in the participants control the swiss participants were quite aware of the valid reasons why their embryos could not be transferred , and yet some of them still said that they had fallen short of some moral standard . we suggest that this persistent unease ( which does not appear so prevalent in the u.k . these are captured in swiss legislation s highly defensive attitude toward embryos in general and the generation of surplus embryos in particular . swiss law operates from the presumption that a surplus embryo is an exceptional event that will happen only through the failure of regulation and practice specifically set up to prevent it ( porz et al . in addition , as we discussed earlier , the generation of surplus embryos is still an unfamiliar social practice and the role of the embryo donor is one that lacks widespread cultural recognition . in the swiss context of legal exceptionalism , then , producing one of these culturally unfamiliar entities may more readily be understood as something wrong for which reparation is in order . in the more permissive cultural and legislative context of the united kingdom , although some interviewees expressed guilt about other aspects of the process ( for example , inappropriate styles of speech about embryos ) , there was no sense that not using the embryos as intended in itself constituted a moral failing . if our interpretation is correct , as the creation of surplus embryos becomes normalised in swiss society and the role of embryo donor becomes normalised in both the u.k . and swiss ( and other ) societies , there will be a shift in ideas about the moral meaning of surplus embryos , and this should be empirically testable . for the majority of the participants in switzerland and the united kingdom who chose to donate their surplus embryos , donation decisions were not driven by a reparative urge but by the feeling that donation expressed solidarity with research and/or other patients . what our empirical work shows is that gratitude to an individual must be distinguished from a solidarity - based desire to give something back to medical research . however , this does not necessarily mean that solidarity - based reciprocity has no relevant impact on the capacity for voluntary consent . one possible consequence , for example , is that if potential donors feel solidarity with the research enterprise , or with present and future patients , then they might also experience an obligation to support it . however , in all three studies there were people who chose not to donate ( about half of the swiss interviewees did not donate , while in the united kingdom approximately 46 percent of those asked have been shown to opt against donation [ choudhary et al . 2004 ] ) , which suggests either that there is no sense of obligation , or that it is neither universal nor irresistible . there is a second problem if solidarity - based reciprocity is taken as something to be prevented . if there is a risk that gratitude felt by an individual could influence donation decisions , then the risk can be minimised by separating ivf and stem cell research physically and procedurally , as bioethicists have suggested and some clinics and guidelines have implemented . but if instead there is a desire to benefit research or to show solidarity with collective others rather than an individual , then what sort of physical or procedural barriers could be set up to prevent it having an influence ? there are further theoretical questions to be explored here about the assumption that contextual emotions such as gratitude or solidarity compromise the decision to donate . the situation in which donation of a surplus embryo is a possibility comes about as a result of a variety of social , cultural , political , legal , and emotional features that combine to make the situation what it is . it will be important to identify carefully the features that are constitutive of the situation of having a surplus embryo ( including a sense of solidarity to similar others , emotional bonds with the embryo that change over time , regret over the failure to use all embryos for pregnancy , and so on ) and to consider whether these constitutive features can or should also be treated as factors that compromise the responsible , autonomous decision - making capacity of the people involved . this introduces the question of the extent to which any decision to donate can be detached from its context and the contextual factors that shape how people respond to donation requests . finally , we should not forget that the possibility that some donors misunderstand the kind of research they are donating to raises some ethical difficulty . it means that donors may be deciding for donation out of a misplaced sense of solidarity a different ethical problem . the deliberative processes of the donor participants in our three studies incorporated their appreciation of the particular moral value of their own embryos and of the value of biomedical research . these deliberations are complex , and the participants own dependency on pre - existing medical research and their affiliation with others in similar situations must be recognised , we suggest , in order to understand how they might configure donation as a means to cope with the moral ambivalence of the situation in which they find themselves . their decisions are not driven by some unacknowledged sense of debt , but rather are responsive to the emerging social and moral reality of embryo donation in the first decade of the 21st century .

this paper is based on linked qualitative studies of the donation of human embryos to stem cell research carried out in the united kingdom , switzerland , and china . all three studies used semi - structured interview protocols to allow an in - depth examination of donors and non - donors rationales for their donation decisions , with the aim of gaining information on contextual and other factors that play a role in donor decisions and identifying how these relate to factors that are more usually included in evaluations made by theoretical ethics . our findings have implications for one factor that has previously been suggested as being of ethical concern : the role of gratitude . our empirical work shows no evidence that interpersonal gratitude is an important factor , but it does support the existence of a solidarity - based desire to give something back to medical research . thus , we use empirical data to expand and refine the conceptual basis of bioethically theorizing the ivf stem cell interface .

thyroid cancer is the most common endocrine neoplasia which accounts for about 1% of human cancers . mtc originates from calcitonin - producing cells ( c - cells ) of the thyroid gland and accounts for 35% of thyroid cancers . mtc is relatively slow - growing tumor but , if metastasized or relapsed , it becomes very aggressive causing more than 13% of all thyroid cancer - related mortality . in the united states , mtc has an incidence in children of 0.03 per 100 000 population per year with a fairly equal female to male ratio [ 4 , 5 ] . mtc occurs either sporadically or in an inherited autosomal dominant manner . in adults , sporadic mtc accounts for 6575% of mtc , but in children , sporadic mtc is very rare ; the vast majority of mtc diagnosed in the childhood is hereditary . hereditary mtc occurs as a familial mtc ( fmtc ) or as a part of multiple endocrine neoplasia ( men ) type 2a and b syndromes , wherein other endocrine glands are also affected . mtc diagnosed during childhood almost always results from a dominantly inherited or de novo activating mutations in the ret proto - oncogene , which encodes the ret receptor tyrosine kinase [ 69 ] . advances in predictive genetic testing for ret mutations have enabled early diagnosis of hereditary men syndromes and prophylactic thyroidectomy in presymptomatic patients to prevent mtc . the early onset of mtc in hereditary syndromes makes it an important endocrine disease that is increasingly managed by pediatric providers [ 1012 ] . in this review , we discuss the etiology of pediatric mtc and currently available therapeutic modality for the cancer . ret encodes a receptor tyrosine - kinase which is expressed in the neural crest - derived cell types , including thyroid parafollicular cells , neuronal cells , and adrenal medullary chromaffin cells . in these cell types , ret plays a central role in regulating cell proliferation , growth , differentiation , migration and survival . in humans after alternative splicing at the 3 end , ret transcripts encode three protein isoforms with distinct c - terminal ends that contain either 9 ( ret9 ) , 51 ( ret51 ) , or 43 ( ret43 ) amino acids . ret exon 19 is present in all transcripts and its differential splicing at the 3 end produces distinct transcripts wherein exon 19 is either unspliced , spliced to exon 20 , or spliced to exon 21 . all three resulting ret isoforms commonly contain a tyrosine ( tyr1062 ) whose phosphorylation is critical for their activation . the major ret isoforms in vivo are ret9 and ret51 , which consist of 1072 and 1114 amino acids , respectively , and are usually co - expressed . ret consists of an extracellular ligand binding domain , a trans - membrane domain , and an intracellular kinase domain ( figure 1 ) . the extracellular domain includes four cadherin - like repeats and a highly conserved cysteine - rich region , which is located near the cell membrane . the intracellular domain consists of two tyrosine - kinase subdomains , tk1 and tk2 , which contain multiple tyrosine residues that are phosphorylated during receptor activation and are required for the activation of different downstream signaling pathways of ret [ 19 , 20 ] . the ligands for ret are the glial cell line - derived neurotrophic factor ( gdnf ) family proteins , including gdnf , neurturin , artemin , and perseptin . activation of ret also requires the formation of a heterodimeric complex recruiting a gdnf - family receptor alpha ( gfr ) . when unbound by a ligand , ret is monomeric , unphosphorylated , and inactive . when a ligand and the gfr co - receptor bind to the extracellular domain of ret , ret undergoes dimerization and autophosphorylation of the tyrosine residues in their kinase domains . this generates the docking sites for their downstream effectors that contain the src homology 2 domain . for example , gdnf - mediated stimulation of ret results in activation of the pathways regulated by phosphatidylinositol 3-kinase ( pi3k ) and different mitogen - activated protein kinases ( mapks ) , including the extracellular regulated kinases ( erks ) , c - jun amino - terminal protein kinases ( jnks ) , the p38 mapk and the big map kinase ( bmk1 ) erk5 [ 22 , 23 ] . ret is one of the first receptor tyrosine - kinases ( rtks ) that have been found to play a role in neoplasia , being most well - known as a key etiological factor for thyroid cancer [ 6 , 24 ] . activating mutations of ret abnormally enhance ret activity and can trigger tumorigenesis in certain organs although the exact underlying mechanisms are as of yet unclear . first , mutations of the six cysteine residues ( cys609 , 611 , 618 , 620 , 630 , and 634 ) in the extracellular domains can promote ret dimerization via disulfide bonds and result in constitutive ligand - independent activation of ret . second , mutations affecting the tyrosine kinase domains can also confer ligand - independent catalytic activity to monomeric ret . these ret mutants exhibit different patterns of autophosphorylation and altered substrate specificity [ 2628 ] . indeed , activation of different downstream signaling pathways is associated with different clinical features of ret mutant thyroid cancers , as observed in men2 syndromes discussed below . for example , the hirschprung disease , a congenital disorder of neural crest development is caused by a loss - of - function ret mutation . of note , the hirschprung disease is closely associated with men2a , demanding a genetic screening for men2a for children with familial hirschsprung 's disease . a strict correlation exists between specific ret mutations and the onset of hereditary mtc ( table 1 ) [ 31 , 32 ] . the detailed and up - to - date information of ret sequence variations can be obtained from the men2 ret database ( www.arup.utah.edu/database/men2/men2_welcome.php ) , which also contains links to selected men2 literature reviews , gene and protein information , and ret reference sequences . the men2a subtype , accounting for 9095% of the men type 2 cases , is a highly penetrant , autosomal dominant endocrine tumor syndrome characterized by the development of mtc in > 90% of ret mutation carriers . in association with mtc in addition , rare variants of men2a are also associated with cutaneous lichen amyloidosis and hirschsprung disease [ 35 , 36 ] . patients with men2a usually have mutations in the extracellular cysteine - rich region of the ret tyrosine kinase receptor , usually in exon 10 ( codons 609 , 611 , 618 or 620 ) or exon 11 ( codon 634 ) ( table 1 , figure 1 ) [ 31 , 37 ] . more than 80% of men2a patients exhibit a specific substitution , i.e. , cys634arg , on exon 11 . mtc is generally the first manifestation of men2a syndrome and develops during early childhood , usually before age six and sometimes before age two . the men2b subtype accounts for approximately 510% of the men type 2 cases . men2b patients typically feature early coincident onsets of mtc , pheochromocytoma , and gastrointestinal mucosal ganglioneuromas . visible physical symptoms include mucosal neuromas of lips ( bumpy lips ) and tongue , and asthenic marfanoid body habitus [ 36 , 40 ] . men2b patients usually have mutations in the tyrosine kinase domain 2 ( tk2 ) in the intracellular region of ret , which almost always ( > 95% ) lead to a single substitution , i.e. , met918thr , in exon 16 ( figure 1 ) [ 31 , 37 ] . de novo mutations , which usually occur on the paternal allele , are also common in men2b . men2b is characterized by the early development of an aggressive form of mtc in all affected individuals , typically during the first year of life . thus , apart from the genetic testing of ret mutations in children born to a parent with men2b , early diagnosis of men2b remains challenging . individuals with men2b are likely to develop metastatic mtc at an early age if they do not undergo prophylactic thyroidectomy before age one . without this intervention , fmtc is considered as the least aggressive clinical variant of men2a with decreased penetrance and/or delayed onset of the other endocrine pathologic manifestations [ 39 , 43 ] . similarly to sporadic cases , familial mtcs are isolated and are not associated with other endocrine tumors . patients with fmtc harbor mutations similar to men2a in either the extracellular or intracellular region of the ret tyrosine kinase receptor [ 6 , 44 ] . the onset of fmtc is relatively late , not appearing until the second or the third decade of life , and its penetrance is lower than the mtc caused by men2a and men2b [ 31 , 39 , 45 , 46 ] . therefore , it is often difficult to determine fmtc based upon a family history and only careful genetic screening can distinguish between inherited and sporadic forms of mtc . mtc cells secrete the polypeptide hormone , calcitonin , and the glycoprotein carcinoembryonic antigen ( cea ) , and these are used as diagnostic biomarkers for mtc . mtc is most commonly diagnosed by immunohistochemical staining of fine - needle aspiration of a new thyroid nodule for calcitonin , chromogranin a , or cea . serum calcitonin is the primary biochemical marker used for detection , staging , postoperative management , and prognosis for mtc patients . however , in very rare cases , certain mtc cells do not secrete calcitonin , which makes diagnosis and patient follow - up difficult . symptoms of mtc include neck pain , a palpable neck mass , and/or diarrhea resulting from hypercalcitoninemia . the clinical course of mtc in men2 patients is variable and is determined by the codon specific mutations . in hereditary form , an age - related progression of malignant disease is observed , with lymph - node and distant metastases being typically detected years after the onset of tumorigenesis . metastatic spread to cervical and regional lymph nodes ( i.e. , parathyroid , paratracheal , jugular chain , and upper mediastinum ) or to distant sites including the liver , lungs , or bone is common and is frequently present in individuals with a palpable thyroid mass or diarrhea . positive lymph - node status and higher stage at diagnosis predict lower disease - free survival and higher mortality [ 5 , 5456 ] . men2 is one of few hereditary cancer syndromes for which predictive genetic testing is recommended at childhood . genetic testing for hereditary mtc syndromes has had an enormous impact on reducing the incidence of mtc in the families affected by these hereditary syndromes [ 57 , 58 ] . genetic counseling is indicated for all children diagnosed with mtc and others who either carry or are at risk of inheriting a ret mutation . children of patients with men2b should undergo ret analysis at birth , and children of patients with men2a or fmtc should undergo ret analysis before age six [ 59 , 60 ] . even 610% of apparently sporadic cases of mtc demonstrate de novo germ - line ret mutations , thus making genetic testing worthwhile in all patients with mtc . thus , all children with an affected parent in this setting retain a 50% risk of mtc , and surgical decisions must rely solely on clinical testing . the standard treatment for mtc is surgical removal of all thyroid tissue including the posterior capsule [ 39 , 62 ] . early thyroidectomy in all men2 patients can change the course of disease , either in a preventive or a curative fashion . the american thyroid association guidelines task force has classified mutations based upon a model that uses the genotypephenotype correlations to rank the mutations into risk levels for the development of aggressive mtc from the lowest a to the highest d ( table 1 ) . this classification may be used to predict phenotype , to recommend the timing of prophylactic thyroidectomy and the extent of surgical intervention , and to begin biochemical screening for pheochromocytoma and hyperparathyroidism . the ages at which the prophylactic thyroidectomy is recommended for the children tested positive for the ret gene mutation are as follows : ages 01 for ret mutations that carry the highest risk for aggressive metastatic mtc at young ages , i.e. , classified as ata - d ; before age 5 for ret mutations that carry a lower , yet still high risk of aggressive mtc at any age , i.e. , classified as ata - c ; after age 5 for ret mutations that carry a lower risk of aggressive mtc , i.e. , classified as ata - b or ata - a , so long as the affected children have no other clinical signs of mtc development . there is ongoing debate on what age the thyroidectomy should be recommended for fmtc patients . some clinical institutes suggest the prophylactic surgery at age 1015 , depending upon the exact mutation and family history , while recommending yearly test of calcitonin levels prior to deciding the surgery . indeed , the ata management guideline for mtc has been very recently revised . in the setting of a prophylactic thyroidectomy , the lymph - nodes are not routinely removed since metastases are not expected to occur at this stage . in the case of clinically apparent mtc , whether sporadic or hereditary , thyroidectomy and concomitant central and compartment - oriented lateral neck dissection should be performed to increase clinical outcomes . primary hyperthyroidism is rare during childhood ; therefore , parathyroidectomy is usually avoided , particularly during a prophylactic procedure . dissection and autotransplantation of parathyroid tissue is not typically performed at the time of thyroidectomy unless there is enough biochemical evidence for hyperparathyroidism . thyroidectomy in children is usually associated with a higher rate of complications , such as recurrent laryngeal nerve injury and hypoparathyroidism , as compared to the surgery in adults . therefore , pediatric thyroidectomy must be performed by highly experienced thyroid surgeons . for individuals with a ret mutation who have not had a thyroidectomy , annual biochemical screening of calcitonin levels is recommended and , if the results are abnormal , immediate thyroidectomy is required . annual serum calcitonin screening should begin at age six months for children with men2b and at age 35 for children with men2a or fmtc . biochemical evidence of disease recurrence includes elevation of calcitonin and cea levels . all individuals who have undergone thyroidectomy need thyroid hormone replacement therapy along with annual screening for pheochromocytoma and hyperparathyroidism depending upon the ret mutation present in the patients . mtc does not respond well to radiation therapy or the standard cytotoxic chemotherapeutic agents , including doxorubicin , dacarbazine , capacitabine , and 5-fluorouracil [ 43 , 71 ] . of note , the mechanism - based targeted therapies that inhibit ret and other receptor tyrosine kinases have become available for the treatment of surgically inoperable progressive mtc . these include the multi - kinase inhibitors , vandetanib ( zd6474 , caprelsa ) and cabozantinib ( xl-184 , cometriq ) , which have been recently approved by the us food and drug administration [ 72 , 73 ] . a recent phase i / ii trial of vandetanib in children with mtc reported partial responses in 47% patients . in general , the drug efficiency and the primary side effects , i.e. , diarrhea , rash , headache , hypertension , and nausea , were similar between children and adults . phase iii trial of cabozantinib demonstrated a 28% response rate in adults with significant adverse effects . therefore , there is a critical need for more effective therapies for patients with advanced mtc . characterization of additional molecular pathways responsible for mtc development may allow the discovery of therapeutic targets that can be exploited to induce reduction of tumor size , disease stabilization , and symptomatic improvement [ 7782 ] . mtc and the men type 2 syndromes are rare but significant endocrine diseases that are increasingly encountered by pediatricians . our understanding of mtc has been greatly increased by the discovery of ret and the genotype genetic tests according to the established guidelines should be performed whenever diagnosis of mtc is made . due to limited adjuvant treatment options , adequate surgical treatment is critical for initial control of the disease and prophylactic thyroidectomy is recommended for children with men2a and men2b at an early age , sometimes during infancy . emerging newer treatments are expected to better treat this rare but life - threatening malignancy .

medullary thyroid carcinoma ( mtc ) , which originates from thyroid parafollicular c cells , accounts for 3 to 5% of thyroid malignancies . mtc occurs either sporadically or in an inherited autosomal dominant manner . hereditary mtc occurs as a familial mtc or as a part of multiple endocrine neoplasia ( men ) type 2a and b syndromes . a strong genotype - phenotype correlation has been observed between hereditary mtc and germ - line gain of function mutations of the ret proto - oncogene . most cases of pediatric mtc are hereditary whereas sporadic mtc is rare in children and is usually diagnosed in adults . therefore , mtc in children is most often diagnosed in the course of a familial genetic investigation . the standard treatment of mtc mainly requires surgery involving total thyroidectomy and central neck node dissection before extrathyroidal extension occurs . to prevent mtc development in hereditary syndromes , prophylactic thyroidectomy is performed in presymptomatic patients . an appropriate age at which the surgery should take place is determined based upon the data from genotyping , serum calcitonin measurements , and ultrasonography . for the treatment of advanced mtc cases , the broad spectrum receptor tyrosine kinase inhibitors vandetanib and cabozantinib , which also inhibit ret , are used although they are not always effective .

neurosurgery is characterized by the delicate balance between surgical success and potential for devastating side effects . thanks to multiple technological improvements , the morbidity of neurosurgical interventions has substantially decreased over the last decades , allowing for the resection of previously inoperable lesions . in particular , image - guided neurosurgery ( igns ) devices allow the use of coregistered and fused multimodality 3d images to guide the surgeon 's hand and help define preoperatively the boundaries of pathological and predefined functional structures . meanwhile , new modes of medical imaging have also improved the localization of pathological lesions and their characterization . medical imaging nowadays includes a wealth of different techniques , such as computed tomography ( ct ) , structural and functional magnetic resonance imaging ( smri and fmri ) , diffusion tensor imaging ( dti ) , and positron emission tomography ( pet ) . although the overall accuracy of igns is estimated to be 12 mm , current neuronavigation systems can not , however , adapt to changing conditions over time . skull - opening brain shift , brain retraction , cerebrospinal fluid suction , lesion resection , perfusion , and pharmacological manipulation during surgery indeed all alter the 3d morphology of the structures [ 25 ] . these changes can lead to localization errors that are one order of magnitude larger that igns accuracy [ 1 , 2 , 6 ] and may result in incomplete resections or unexpected damage to normal brain . such inaccuracies could be reduced if one could acquire , throughout surgery , fresh images of the same modalities and quality as the preoperative ones intraoperative images such as intraoperative mr ( imr ) images are with the exception of a handful surgical facilities usually acquired using low - field mri scanners that provide lower resolution and contrast than their preoperative counterparts , and , to this date , several useful imaging modalities , such as pet and possibly meg , can not be acquired intraoperatively . one solution is to bring over the high - quality preoperative multimodality images into the intraoperative configuration of the brain using a nonrigid registration technique [ 710 ] . one category of nonrigid registration techniques uses physics - based models , where landmarks are tracked in successive reduced - quality intraoperative images , and their displacement fields drive the deformation of a biomechanical model . so far , most of the mechanical conditions of the brain can not be estimated in the operating room , such as the volume of cerebrospinal fluid flowing out of the skull cavity , intercellular fluid volume changes that result from mannitol injection , or changes in blood volume and vessel permeability . the fact that an intraoperative image can provide the knowledge of the current state of the brain after some deformation partly eliminates the need for a complete evaluation of these mechanical conditions . the nonrigid registration technique replaces them with the landmark displacements evaluated from successive intraoperative images . using a nonrigid registration technique based on a biomechanical model , three types of brain deformations have been identified that require specific modeling , although they depend on common parameters , such as csf suction , perfusion , or pharmacological manipulation . the first deformation is the brain shift , which appears at the beginning of surgery with the opening of the skull and dura . the suction or leakage of csf , as well as the release of intracranial pressure caused by tumor growth , generally cause such shift of the brain ( note that in this work , we name brain shift the specific shift of the brain that occurs after the opening of the skull and dura , before any other surgical act has happened ) . however , for these deformations , we will consider that the shift is a part of these two deformations . the second deformation is the retraction ; when target tissues are located deep inside the brain , the surgeon incises brain tissues and inserts a retractor to spread out the tissues , and to create a path towards the target . the third deformation is the resection , that is , the removal , of lesion tissues . three deformations can thus be defined in terms of the two elemental actions that change the topology of the brain : the introduction of a discontinuity and the removal of some tissues . most studies of brain deformation based on biomechanical models have focused on shifts ( the topology of the brain is not modified ) , that occurs just after the opening of the skull and dura [ 1125 ] . a good review of these different studies can be found in [ 24 , 2628 ] . resection and retraction are more complex to model than ( brain ) shift . until recently , their modeling for the specific application of preoperative image update has received much less attention . one of the difficulty for modeling resection and retraction is that both induce a topological change of the brain because some tissue are cut . a method of mesh adaptation [ 2931 ] or remeshing [ 3235 ] must be used in conjunction with fem if an accurate representation of the location of the cut , for example , the resection cavity or retraction path , is needed to deform the model . indeed , fem can not directly handle discontinuities that go through the fes , and requires to realign the discontinuity with fe boundaries . in the field of fracture mechanics , which studies the growth and propagation of cracks in mechanical parts , some methods were developed to avoid using fem in conjunction with mesh adaptation or remeshing . the extended finite element method ( xfem or x - fem ) appeared in 1999 and has been the object of considerable research since then . xfem works by allowing the displacement field to be discontinuous within some fes of the mesh . the mesh does not have to conform to the discontinuities , so that these can be arbitrarily located with respect to the underlying fe mesh . because xfem allows an accurate representation of the discontinuities while avoiding mesh adaption or remeshing , and because of the similarity between cracks in mechanical parts and cuts in tissue , we proposed the use of xfem for handling cut , resection , and retraction in the updating of preoperative images . this paper presents a complete 3d framework for updating multimodal preoperative images with respect to surgical brain deformations , due to brain shift and successive resections , followed and quantified using imr images . our approach is modular , and is applied iteratively each time a new intraoperative image is acquired . we take into account successive deformations based on a linear elastic biomechanical model which is deformed using fem or xfem , depending on the type of deformation occurring between the pair of imr images under consideration , namely , brain shift or resection . while some 3d results have already been presented for brain shift , and initial 3d results for resection modelings , this paper is the first complete and detailed account of the generalization to 3d of our 2d previous work . we present the state - of - the - art of resection modeling for preoperative image update . in section 3 , we describe our basic strategy for updating preoperative images based on successive intraoperative images . in section 4 , we give detail about our methods and algorithms . in section 5 , we consider two patient cases that illustrate our approach for handling brain shift followed by successive resections . among studies that take into account resection for preoperative image update , one should distinguish two categories . the first category of studies models brain deformation using two time - point images , the first image being acquired before surgery has started , the second image being acquired after resection . in this category , the methods that existed for a second image showing some brain shift are adapted for a second image showing some resection . the second category of studies models brain deformation using more than two time - point images , and models at least two successive resections . among the first category of studies , hagemann et al . developed a 2d method for modeling brain deformation between a preoperative mr image and a postoperative mr image , the postoperative image showing a complete resection . the 2d mesh of the biomechanical model corresponded to the underlying pixel grid of the 2d image . the biomechanical model included four different linear elastic laws for the skull / skin region , the whole - brain region , the csf region , and the image background . they computed the correspondence of the skull boundary , the whole - brain region boundary in the neighborhood of the tumor , and the posterior midline between the two images . they also computed the correspondence between the internal tumor region boundary visible in the preoperative image , and the resection cavity boundary visible in the postoperative image , both boundaries corresponding under the assumption that the resection is complete . the displacements fields of these landmarks drove the deformation of the biomechanical model . as a result , the biomechanical model presented high deformation in the tumor region , which is not physically plausible . however , the resection was complete , and , thus , they were not interested by the displacement field of the biomechanical model in the tumor region itself . clatz et al . developed a 3d method for modeling the brain deformation between a preoperative mr image and an imr image , the latter showing partial or complete resection . the biomechanical model was deformed based on a sparse volume displacement field evaluated from the two images , using a block matching algorithm . in their algorithm , blocks of voxels that presented discriminant structures were selected in the preoperative image . the blocks were then matched to blocks in the imr image using a similarity criterion , for example , a coefficient of correlation . the value of the similarity criterion was used as a value of confidence in the displacement measured by the block matching algorithm . the biomechanical model was then deformed iteratively , driven by the sparse displacement field of the matched blocks , where a block rejection step was included for measured block displacements initially selected but considered as outliers . in the imr image , a part , or the totality , of the tumor tissues the blocks were thus selected and matched in only the healthy - brain region of the two images . they tested their algorithm on six patient cases , and used for validation nine landmarks picked up manually in each image . they found a mean and maximum error on displacements of 0.75 mm and 2.5 mm , respectively . they explained this phenomenon by the fact that a substantial number of block matchings were rejected in the tumor neighborhood . the deformation of the biomechanical model in the tumor neighborhood was thus essentially governed by the linear elastic law , and the result might show the limitation of this model . archip et al . also tested the nonrigid registration method presented in on eleven patient cases , and used the 95% hausdorff distance for evaluating the alignment of the nonrigidly registered images . as a result , they obtained a mean error of 1.82 mm . among the second category of studies , miga et al . they built a linear poroelastic biomechanical model and preoperatively tagged the tetrahedron fes that were going to be removed to simulate the brain deformation due to successive resections . second , a boundary condition was applied to the new boundary of the resection cavity , in order to model the relaxation of strain energy , induced by preoperative tumor growth or surgery acts , stored in the resected tissues , and released after their removal . in this approach , the tissue discontinuity was represented as best as possible with a jagged topology defined by the fe facets defining the boundary of the resection cavity . [ 45 , 46 ] also modeled the removal of tetrahedra in order to model the action of an ultrasonic aspirator in the context of real - time surgery simulation . [ 13 , 47 , 48 ] modeled successive resections based on several time - point imr images . between two successive images , they deformed the biomechanical model , in its current state of update , to take into account the ( partial ) resections(s ) that took place between these two images . first , the biomechanical model , in its current state of update , was deformed in accordance with the displacement field of the healthy - brain boundary between the pair of images under consideration . second , the fes that fell into the resection cavity in the second image of the pair were removed , while the fes that laid across the resection - cavity boundary were cut . to ensure the link between the successive deformed configuration of the biomechanical model , their algorithm kept track of the topology modification between fes and nodes of the mesh before and after the removal of fes . they tested their algorithm on one patient case including five imr images ( the first two imr images being used for brain shift modeling ) , and used for validation thirty - two landmarks picked up manually in each image . they found a mean and maximum error on the displacements of 0.9 0.7 mm ( mean standard deviation ) and 3.7 mm , respectively . they explained this phenomenon by the limited accuracy in the process of picking landmarks in that region , and because the retraction occurring between the second and third images was modeled as a resection , that is , a removal of tissues , even though the tissues were not removed but simply spread out . the methods described above have been all developed using an fem - based biomechanical model for intraoperative image registration . the objective of a surgical simulator is to provide an interactive manipulation with force feedback of the anatomical part to be operated using various surgical instruments . in order to model a large range surgical procedures , jebkov and kuhlen have applied nonlinear xfem for simulating cut , and have shown that xfem is successfully efficient for such purpose . the block diagram of figure 1 shows our global approach for updating preoperative images using successive imr images acquired at different critical points during surgery . although the principles of the approach are quite general , they are tailored for use based on images acquired with a 0.5 tesla intraoperative ge signa scanner , which guarantees that the full volume of brain tissues is included in the image field of view . in our present strategy , the preoperative images are updated incrementally . at the end of each update , the preoperative images should be in the best possible alignment with the last imr image acquired . prior to surgery , a patient - specific biomechanical model is built from the set of preoperative images . because the patient does not necessarily lie in the same position during the acquisition of each of the preoperative images , one may need to perform a rigid registration ( involving translations , rotations , and scales ) to bring these images into correspondence , assuming , in first approximation , there is no local , that is , nonuniform , brain deformation between preoperative images . once the 1st imr image has been acquired prior to the opening of the skull , the set of registered preoperative images and the biomechanical model are registered to the 1st imr image via a rigid transformation . in the present situation , it is assumed that the patient 's brain imaged in the 1st imr image has the same physical shape as the brain imaged in the preoperative images ( note that in the following , when an imr image is defined by a number , this number is the index of the imr image in the series for a specific patient case . the 1st imr image thus corresponds to the very first imr image of the series ) . as each imr image is acquired , this new image and the preceding imr image are used to estimate the deformation of the brain . the update of the preoperative images is done incrementally with each new pair of successive imr images . for each pair , we proceed as follows . a set of common anatomical landmarks the use of surface structures rather than volume structures seems more appropriate given the reduced - quality of typical intraoperative images , and would be more easily adapted to intraoperative modalities other than imr , such as ius . the landmark surface displacement fields resulting from the matching are then applied to the biomechanical model , which is deformed using fem or xfem , depending on the type of deformation occurring between the acquisition times of the imr images in the pair under consideration , namely , brain shift , or resection . the resulting displacement field of the biomechanical model is finally used to warp the set of preoperative images in their current state of updating . note that , for each deformation modeling , the biomechanical model is deformed in accordance with the landmark displacements tracked between the pair of successive imr images under consideration . because intraoperative deformation can follow a reverse direction , it is important to track the landmarks between the next - to - last and the last acquired imr images , rather than track the landmarks between the first and the last acquired imr images . for the patient cases treated in section 5 , we assume that the brain undergoes relatively small deformations ( small strains and small displacements ) , and we use a linear finite - element formulation in the biomechanical model . a consequence of using this linear formulation ( linear elasticity ) is that the equations of solid mechanics can be solved based on the initial configuration of the solid . actually , knowing the displacement field increment un = u u at the anatomical landmarks between configuration n and increment n + 1 , one can apply this constrained displacement field increment un to the initial configuration , and the finite element analysis will lead to the deformation tensor increment n between the configuration n and n + 1 . the final deformation tensor or the body is thus simply obtained from = k=0k . remark that rigorously , the increment of constrained displacement field at the landmark should be applied to the balanced solution of the solid reached after increment n , but as we are using a linear elasticity model , this step can be skipped owing to the superposition principle : if = c , then = k=0k = ck=0k = c , where c is the hooke tensor . as a summary , with this approach , the process of deformations is modeled as a succession of deformations k , for example , brain deformation composed of shift followed by successive resections and the current configuration of the brain biomechanical model , after a specific deformation can then be recovered by adding the computed volume displacements for all successive incremental deformations . remark that this is not a limitation of the method as we could easily extend it to nonlinear model by simply keeping in memory the previous deformed configuration n and adding the constrained displacement field increment un to compute the new deformed configuration at increment n + 1 , simply this would be less computationally efficient . because we use a linear formulation ( and , thus , the incremental volume displacement fields can be added to recover the current configuration of the biomechanical model ) , we could theoretically obtain an identical deformed configuration of the biomechanical model using the two following approaches . the first one would consist of computing and adding the successive incremental deformations of the biomechanical model based on the landmarks tracked between the next - to - last and the last acquired imr images . the second approach would consist in computing directly the deformed configuration of the biomechanical model based on the landmarks tracked between the first and the last acquired imr images . however , the landmarks selected to drive the deformation of the biomechanical model vary depending on the type of deformation , namely , brain shift or resection . in addition , part of the biomechanical model is cut , using xfem , to model resection . consequently , we would not get an identical deformed configuration of the biomechanical model by these two approaches . in order to use a maximum of information from the imr images , we track , as explained for the first approach , the landmarks between the next - to - last and the last acquired imr images . the problem of updating preoperative images between more than two critical points during surgery , that is , based on more than two imr images , is addressed in only a small number of studies . in our previous work [ 39 , 41 ] , and in , the biomechanical model was successively deformed , and this was done using a linear formulation . the framework proposed here , where the initial biomechanical model is always used , instead of using it in its successive states of deformation , has the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively deteriorates with each successive deformation modeling , and which would require remeshing or mesh adaptation for getting back good fe quality . to summarize , for each deformation , the landmarks are tracked between the two successive imr images under consideration . because we use a linear formulation , the displacement fields of these landmarks are applied to the initial , rather than current , configuration of the biomechanical model . the resulting volume displacement field corresponds to the deformation that the brain undergoes between the two imr images . this volume displacement field is used to deform the preoperative images in their current state of update , that is , registered ( at the previous step , if any ) to the first imr image of the pair . after the deformation , the preoperative images are thus in as good as possible registration to the second imr image of the pair . in all the rest of this work , we make a simplification of the approach just presented , by using the 1st imr image as a substitute for the preoperative images . the biomechanical model is thus built based on structures visible in the 1st imr image , instead of in the preoperative images , and the structures used in the model are limited to the ones visible in the intraoperative image . except for the rigid registration between the preoperative images , the biomechanical model , and the 1st imr image , this simplified approach allows us to discuss , illustrate , and test all key aspects of the system . the above strategy allows us to focus on the main issue of this paper , that is , the estimation and handling of 3d deformations . even though the issues involved in the update of preoperative images will need to be addressed in a operational image update system , the present strategy of deforming the imr images remains useful for calibration purpose , even in the operating room . this section details the different methods that are commonly used for updating preoperative images in presence of brain shift and resection . more specifically , the block diagram of figure 2(a ) shows the building of the biomechanical model from the preoperative images . specific regions from the preoperative images are segmented , meshed , and assigned appropriate constitutive laws . the block diagram of figure 2(b ) shows , for any pair of successive imr images , a detailed view of the calculation of the volume displacement field of the initial biomechanical model that corresponds to the deformation that has occurred between the acquisition time of these images . all along surgery , the patient is lying inside the 0.5 tesla intraoperative ge signa scanner . although the patient 's head is fixed , one can not totally rule out the possibility of slight head motion . imr images thus have to be rigidly coregistered to take into account this potential rigid motion . the rigid registration that we use is the point - based landmark transform available in vtk ( http://www.vtk.org/ ) . the segmentation of imr images into specific regions , for example , healthy - brain and tumor regions , is first performed manually using 3d slicer ( http://www.slicer.org/ ) and then smoothed to minimize the dependance of the results on segmentation roughness . it is clear that performing a manual segmentation in the operating room is not acceptable , and that this process needs to be automated as completely as possible to test the feasibility of our framework online . however , while there exist sophisticated segmentation algorithms that could be used [ 5052 ] , in particular for extracting the whole - brain region ( skull and external cerebrospinal fluid masked out ) , the segmentation of the tumor region is still challenging . as mentioned above , the biomechanical model is built , in the present context , from the 1st imr image rather than from the preoperative images . thanks to the use of xfem instead of fem for modeling discontinuities , this biomechanical model can be built offline before the operation starts and does not need to be repeated ( through remeshing ) during the surgery . with respect to fem - based approaches , the execution time thus ceases to be a limiting parameter , which is a remarkable advantage of our approach . it thus requires specific techniques , and we use the meshing software tool isosurf ( http://svr-www.eng.cam.ac.uk/~gmt11/software/isosurf/isosurf.html ) . our goal is to model the boundaries of healthy - brain and tumor regions as two connected surfaces meshes . however , isosurf can only mesh the boundaries of one or several separate regions , and , thus , does not allow one to mesh connected region boundaries with common nodes at their intersections . we thus start by building two separate surfaces meshes that we connect using our own routines based on vtk . the two connected triangle surfaces are then jointly meshed into a single volume mesh of tetrahedra that conform to the two surface meshes using gmsh ( http://www.geuz.org/gmsh/ ) . further details on the building of the biomechanical model , in particular the building of the connected surface meshes , can be found in . a linear elastic law is assigned to the biomechanical model , with young modulus e = 3000 pa and poisson ratio = 0.45 . because displacements , rather than forces , are applied to the model using a linear formulation , the fem or xfem solution is independent of young modulus e . we choose as surface landmarks the whole - brain and internal tumor region boundaries . to evaluate the surface deformations of these region boundaries between two imr images , we use an active surface algorithm [ 55 , 56 ] . because these region boundaries to match must be closed surfaces , we thus use as surface landmarks the whole - brain and healthy - brain region boundaries . the surface deformation of the internal tumor region boundary will be derived from the active surface algorithm of the healthy - brain region boundary . in our active surface algorithm coming from [ 13 , 47 , 48 ] , the external forces f(x ) are computed using a gradient descent on a distance map of the region boundary . with such external forces , the active surface algorithm is not able to take correctly into account local rigid motion due , as an example , to lateral or tangential movement depending on the head orientation . for the whole - brain region , any rigid transformation that could have occurred has already been taken into account by the rigid registration of the imr images ( section 4.1 ) . however , for the healthy - brain region , it can happen that the internal tumor region boundary moves partly in a rigid way . therefore , the active surface , initialized from the healthy - brain region boundary in the first imr image , is first locally transformed in a rigid way along the internal tumor region boundary using the iterative closest point transform available in vtk . then , this resulting surface is deformed using the active surface algorithm as explained above . further details on the local rigid registration of the healthy - brain region boundary can be found in . before applying the displacements whole - brain and internal tumor region boundaries to the biomechanical model nodes , the two surface displacement fields are smoothed based on a weighted - distance average , that is , the displacement of each node is averaged with the displacements of its n closest neighbor nodes . this smoothing will make them consistent with each other , and compatible with the volume mesh in order to avoid element flipping , in particular at the intersections between whole - brain and internal tumor region boundaries . the displacement fields of the surface landmarks are applied to the biomechanical model , which deforms according to the laws of solid mechanics . the equations of solid mechanics are solved using fem or xfem , depending upon the type of circumstances , namely , brain shift or resection . we use the fem - software tool metafor ( http://metafor.ltas.ulg.ac.be/ ) developed in our mechanical - engineering department , to which we have added an xfem module . the initial stress state of the brain is unknown and is thus set to zero for each fem or xfem computation , as in [ 10 , 13 ] . fem discretizes the solid of interest into a mesh , that is , into a set of fes interconnected by nodes , and approximates the displacement field u(x ) by the fem displacement field u(x ) defined as ( 1)ufem(x)=iii(x)ui , where i is the set of nodes , the i(x ) 's are the nodal shape functions ( nsfs ) , and the ui 's are the nodal degrees of freedom ( dofs ) . each i(x ) is defined as being continuous on its compact support i , which corresponds to the union of the domains of the fes connected to node i . in our approach , we use linear nsfs . in contrast , xfem handles a discontinuity by allowing the displacement field to be discontinuous within fes [ 37 , 5860 ] . the xfem displacement field generalises the fem displacement field ( 1 ) with ( 2)uxfem(x)=iii(x)ui+iji(x)j=1neigj(x)aji . the first term corresponds to the fem displacement field ( 1 ) , where i is the set of nodes , the i(x ) 's are the fem nsfs , and the ui 's are the nodal fem dofs . the heart of xfem is the enrichment that adds a number , n , of dofs aji to each node i of the set j , which is the subset of nodes of i whose support is intersected by the discontinuity of interest . these dofs are multiplied by the nsfs i(x ) and the discontinuous functions gj(x ) . the use of specific xfem enrichment functions gj(x ) for a node i j depends on the type of discontinuity , for example , crack , hole , material interface , and so forth , to be modeled . suppose that our goal is to model a crack , characterized by a discontinuity in the displacement field ( as opposed to a material interface for instance , characterized by a discontinuity in the derivative of the displacement field ) . when the crack fully intersects the support of the node , a simple choice is a piecewise - constant unit function that changes sign at the boundary across the crack , that is , the heaviside function ( 3)h(x)={1for ( xx)en>0,1for ( xx)en<0 , where x is again the position of a point of the solid , x * is the position of the point on the crack that is the closest to x , and en is the outward normal to the crack at x * . in case of resection deformation , the goal is to model a discontinuity such that the part of tissues corresponding to tissue removed by the resection has no influence on the deformation of the remaining part of the tissues . one is actually interested in the deformation of the remaining part of the tissues only . in that sense , the hole function as the following equation : ( 4)v(x)={1for ( xx)en>0,0for ( xx)en<0 , could be used as xfem enrichment function , instead of the heaviside function , and would be totally sufficient . the results that we would obtain on the remaining part of the tissues would be identical . however , because the heaviside function is necessary for retraction modeling , we have used the same function for the resection modeling even if it was not strictly necessary . when minimizing the total deformation energy , the resulting xfem equations remain sparse and symmetric as for fem . whereas fem requires a remeshing and the duplication of the nodes along the crack to take into account any discontinuity , xfem requires the identification of the nodes whose support is intersected by the crack and the addition of dofs : ( 1 ) any node whose support is not intersected by the discontinuity remains unaffected and thus possesses three dofs ; ( 2 ) any node whose support is fully intersected by the discontinuity is enriched with three heaviside dofs and thus possesses six dofs . to qualitatively estimate the similarity between two images , we compare the edges extracted from these images using the canny edge detector available in itk ( http://www.itk.org/ ) . indeed , although potentially useful for the sake of comparing methods on a mathematical basis and defining unique correspondences , landmark - based target analysis presents several relevant limitations in the present setting . having experts picking landmarks introduces significant intra- and interobserver variability . picking landmark points , as ferrant et al . did , is rather difficult when it comes to define enough visible landmarks especially in the tumor region on the 5 different images ( and not 2 images only , as majority of studies focusing on brain shift are using ) . rather than point targets , linear tumor contours , and limits between structures and potential eloquent structures matter most in the practical case of tumor ablation neurosurgery . these are the reason why we chose to use the canny edges in order to evaluate the registration . besides , while it is true that these edges do not necessarily physically correspond between the successive imr images , these images have been acquired with the same image protocol ( mr sequence , voxel size , grayscale value range ) , which should limit this problem . to quantitatively estimate the similarity of the two edge maps , we compute the modified hausdorff distance between the sets of edge points , that is , voxels representing the edges , in these two images . the modified hausdorff distance (a , b ) between two sets of points a and b is defined as ( 5)(a , b)=max(h(a , b),h(b , a ) ) with h(a , b)=1naaad(a , b ) , where the directed hausdorff distance h(a , b ) is a measure of the distance of the point set a to the point set b , na is the number of points in set a , and d(a , b ) is the distance of point a a to the closest point in b , that is , d(a , b ) = minbb||a b|| , where ||a b|| is the euclidean distance . the directed hausdorff distance h(a , b ) thus computes the average distance of points of a to points of b. the averaging minimizes the effects of outlier points , for example , due to image noise . the value of the modified hausdorff distance (a , b ) increases with the amount of difference between the two sets of edges points . in the following , we denote by (ia , ib ) the modified hausdorff distance of the edges extracted from the whole - brain region of the images ia and ib , that is , with the skull and external cerebrospinal fluid masked out from them . in this section , we apply our methods , respectively , of brain shift and resection ( imr images are acquired with the 0.5 tesla intraoperative ge signa scanner of the brigham and women 's hospital , boston , usa . imr image size is 256 256 60 voxels , and voxel size is 0.9375 0.9375 2.5 mm ) . two patient cases , each including five imr images , are treated to illustrate our modeling and brain shift followed by successive resections . in both cases , the 1st imr image was acquired prior to the opening of the skull ; the 2nd imr image was acquired after the opening of the skull and dura , and shows some brain shift ; the 3rd , 4th , and 5th imr images were acquired after successive resections . the modelings of brain shift , 1st , 2nd , and 3rd resection are performed using different techniques , as detailed below . except where otherwise noted , the following discussion applies to both patient cases ( the result of each deformation modeling is shown for the two patient cases at the end of section 5.2.3 ) . to model brain shift based on the 1st and 2nd imr images , we estimate the surface displacement fields of the whole - brain region boundary and the internal tumor region boundary from the two imr images . no tissue discontinuity is involved in the brain shift deformation , so the biomechanical model is deformed using fem . this results in the volume displacement field of the biomechanical model , which is illustrated in figure 3 for the first patient case . this volume displacement field is used to warp the part of the 1st imr image corresponding to the whole - brain region . in the following sections , the three successive resections are modeled separately , because they require different types of processing . matching two region boundaries to get a displacement field makes sense only if they correspond to the same physical entity . once the resection has started , we can no longer rely on the entirety of the whole - brain region boundary , since a part of it is now missing . for modeling the successive resections , we thus evaluate the displacement field for the boundary of the healthy - brain region only . the 1st resection occurs between the times the 2nd and 3rd imr images are acquired . however , since the corresponding removal of tissues is most likely accompanied by deformation , one can not exactly determine what tissue is removed based just on the two imr images . we thus decided to model the 1st resection by still relying on the displacement fields of key surfaces , here the healthy - brain region boundary , to deform the biomechanical model . this indeed appears to be the only reliable information concerning the deformation due to resection that we can extract from the 2nd and 3rd imr images . consequently , we do not model explicitly the removal of tissue , but we model directly the deformation resulting from it , without introducing any tissue discontinuity . using the surface displacement field of the healthy - brain region boundary , we compute the deformation of the biomechanical model via fem . then , using the resulting volume displacement field , we warp the part of the 2nd imr image corresponding to the whole - brain region , in the same way as we did in the case of for brain shift . the image resulting from the 1st resection modeling is now registered to the 3rd imr image , except outside of the healthy - brain region boundary , that is , for the tumor region . finally , we alter the resulting image to reflect the effect of resection . for this , we assign the background color to the voxels corresponding to the resected tissue volume the significant feature of the 2nd resection is that some tissue has already been removed by the 1st resection , which means that this tissue can not have any physical influence on subsequent brain deformations because it does not recall that the biomechanical model has been deformed to model the brain shift and the 1st resection and is thus registered to the 3rd imr image . so , using the 3rd imr image , we can define the boundary of the 1st resection , that is , the tissue discontinuity to include in the deformed biomechanical model ( figures 4(a ) and 4(b ) ) . we then enrich the nodes whose supports are intersected by the discontinuity with heaviside dofs . consequently , when the xfem - based biomechanical model deforms , the part corresponding to tissue removed by the 1st resection has no influence on the deformation of the remaining part of the brain . for the first patient case illustrated in figure 4 , the tetrahedron mesh consists of 3,317 nodes , which corresponds to 9,951 fem dofs . the biomechanical model is deformed in accordance with the displacement field of the healthy - brain region boundary evaluated from the 3rd and 4th imr images . the bottom part of the mesh , representing the tissue remaining after the 1st resection , has been deformed according to the displacement field of the healthy - brain region boundary , while the top part , representing the tissue removed by the 1st resection , has been subjected to a translation , but only for visualization purposes . even though the mesh is displayed as two separate parts , it is , in fact , a single entity . indeed , a main feature of xfem is its ability to handle the effect of a discontinuity without modifying the underlying mesh , that is , without remeshing . for modeling the 2nd resection , the edges of fes straddling the discontinuity have been made discontinuous and their nodes moved apart . using the xfem volume displacement field , we warp the part of the 3rd imr image corresponding to the whole - brain region . the resulting image is then masked out with the whole - brain region segmented out from the 4th imr image . one significant feature of the procedure described for modeling the 2nd resection is that it can be applied repetitively for each subsequent resection visible on successive imr images , no matter how many there are . the modeling of the 3rd resection is thus identical to the modeling of the 2nd resection . the tissue discontinuity due to the 2nd resection is defined from the 4th imr image , and used to appropriately enrich the nodes of the biomechanical model . then , this biomechanical model is deformed using xfem , in accordance with the displacement field of the healthy - brain region boundary evaluated from the 4th and 5th imr images . for the first patient case , a simplification for the modeling of the 3rd resection can be made because , by the time the 5th imr image is acquired , the resection is complete . this means that we only need to compute the volume displacement field of the healthy - brain region . since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem will be identical . using the fem ( for the first patient case ) or xfem ( for the second patient case ) volume displacement field , we warp the part of the 4th imr image corresponding to the whole - brain region . the resulting image is then masked out with the whole - brain region segmented out from the 5th imr image . figures 5 and 6 show the results of warping the imr images , as well as the edges extracted from them , after brain shift and each successive resection modeling for the two patient cases . as explained in section 5.2.3 , since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem are identical in the healthy - brain region . one can deduce that using xfem for modeling resection is interesting when the neurosurgeon needs to have an accurate displacement field of the remaining tumor tissues . in this case , it is interesting to evaluate the impact of using fem , instead of xfem , to model the resection as if no resection was performed before . using fem for modeling resection is equivalent to ignoring the presence of resection on intraoperative images . to illustrate the comparison between fem and xfem results , we choose the 3rd resection modeling of the second patient case indeed , it is the deformation with remaining tumor tissues that shows the largest magnitude , and , thus , that is likely to give a maximum difference between the two computations . the healthy - brain and tumor regions segmented out from the 4th and 5th imr images are respectively shown in figures 7(a ) and 7(b ) . the volume displacement fields of the biomechanical model using xfem and fem are respectively shown in figures 7(c ) and 7(d ) . the part of the 4th imr image corresponding to the whole - brain region is warped , first with the volume displacement field obtained via fem , and then with that obtained via xfem . the difference between the two warped images is shown in figure 7(e ) . as expected however , the difference between the two volume displacement fields is smaller than the image resolution ( although the difference between the two volume displacement fields is smaller than the image resolution , the difference between the images resulting of the warping using these two volume displacement fields is nonzero . this is explained by the fact that the ( gray ) value of each voxel of the warped image is defined as a weighted - value of voxels of the original image . the weights are defined based on the overlapping ratio of the voxel of the warped image , with voxels ( determined using the volume displacement field ) of the original image ) . in addition , the deformed 4th imr images , using the xfem- and the fem - based deformations of the biomechanical model , show the same similarity , computed based on the modified hausdorff distance , with the 5th imr . two reasons explain that the differences between the fem and xfem results are so small . first , the brain deformation itself due to the 3rd resection is small , and , thus , it is expected to obtain small differences between the two resulting brain deformations . second , in the case the remaining tumor tissues are close to the healthy - brain region boundary , it implies that they are close to the boundary where surface displacement fields are applied to drive the deformation of the biomechanical model . although this comparison between fem and xfem should be done on more patient cases , we suggest that , in first approximation , fem could be used for modeling resection cases with small brain deformations . nevertheless , the presentation of the successive resections using xfem shows the generality of our framework , and details how xfem is implemented . note that in section 6 devoted to validation , the warped images are the ones deformed with xfem . for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images that are already rigidly registered , we compare the similarity between these ik and ik+1 images , as well as the similarity between the ik and ik+1 images , where ik is the result of warping ik . this gives us an estimate of how well we are able to capture , and compensate for , the local deformations between ik and ik+1 . the goal of the nonrigid registration is , however , to deform the preoperative images . by warping ik for each deformation modeling , we do not take into account the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . to evaluate the effect of this error amplification on the results , we also perform the required succession of warpings on i1 , and we denote the resulting image by i1,k . we then compare , for each deformation modeling , the similarity between i1 and ik+1 , together with the similarity between i1,k and ik+1 . this allows one to evaluate the propagation , that is , the amplification , of alignment error on the results . the modified hausdorff distance computed for each pair of imr images are given in tables 1 and 2 . table 1 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (ik , ik+1 ) and (ik , ik+1 ) . these values are computed using the canny edges extracted from the pair of images ( ik , ik+1 ) ( figures 5 ( d ) and 6 ( d ) ) and ( ik , ik+1 ) ( figures 5 ( e ) and 6 ( e ) ) . we observe that the values for the images nonrigidly registered are relatively constant , that is , 1 mm , for each deformation modeling . six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are ( rigidly and subsequently ) nonrigidly registered . however , the modified hausdorff distance increases for the 3rd resection modeling of the first patient case , as well as for the brain shift modeling of the second patient case . to understand if the nonrigid registration is responsible for the increase of the misalignment of the two imr images everywhere in the whole - brain region , or if this effect is localized , we compute the modified hausdorff distance in the region and neighborhood of the tumor only ( volume region that extents by 25 mm the tumor region segmented in i1 for both patient cases ) . the modified hausdorff distance decreases from (i4 , i5 ) = 1.70 mm to (i4 , i5 ) = 1.37 mm for the first patient case , while it decreases from (i1 , i2 ) = 1.36 mm to (i1 , i2 ) = 1.28 mm for the second patient case . this indicates that the nonrigid registration enhances the alignment of the two imr images within the tumor region and its neighborhood , which is in fact the location requiring the best modeling accuracy . this behavior could be explained by the fact that a maximum of information from the imr images is used in this region , that is , one or two ( in case of brain shift modeling ) surface displacement fields are applied around it . the increase of misalignment elsewhere in the brain volume could be explained by two reasons . first , the landmarks tracked from the imr images are surfaces . as a consequence , the nonrigid registration is expected to give better results near the tracked surfaces than far from them in the volume . the volume misalignment could point out the need for better parameters values and/or other constitutive laws . table 2 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (i1 , ik+1 ) and (i1,k , ik+1 ) . so far , igns systems allow one to rigidly register preoperative and successive imr images . (i1 , ik+1 ) thus represents the navigation accuracy that we can obtain with an igns system at the present time . the comparison of (i1 , ik+1 ) with (i1,k , ik+1 ) gives the improvement that could be practically achieved in the alignment with our approach . as expected , table 2 shows that the igns accuracy decreases through the successive deformations . indeed , the modified hausdorff distance increases from (i1 , i2 ) = 1.24 mm to (i1 , i5 ) = 1.78 mm for the first patient case , and from (i1 , i2 ) = 1.01 mm to (i1 , i5 ) = 1.68 mm for the second patient case . six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are nonrigidly registered . to understand if the modified hausdorff distance increases everywhere in the whole - brain region for the brain shift and 1st resection modeling of the second patient case , we compute the modified hausdorff distance in the neighborhood of the tumor region ( in the same way as explained for table 1 ) , and observe the improvement of the alignment within the tumor region and its neighborhood . as opposed to the values of the modified hausdorff distances in table 1 , the values for the images nonrigidly registered in table 2 increase through the successive resection modeling . this amplification error is due to the fact that , after having modeled brain deformation between a pair of imr images , the deformed biomechanical model is not in perfect alignment with the second image of the pair . since , for the subsequent deformation modeling , the surface landmarks are initialized based on the deformed biomechanical model , this can thus ampliy a misregistration error . we developed a complete 3d framework for serial preoperative image update in the presence of brain shift followed by successive resections . the nonrigid registration technique used an homogeneous linear elastic biomechanical model , driven by the deformations of whole - brain and internal tumor region boundaries for brain shift modeling , and healthy - brain region boundary for resection modelings , tracked between successive imr images . the biomechanical model was deformed using fem for brain shift modeling , and fem or xfem for resection modeling , depending upon whether some brain tissues were previously resected or not . we showed that our approach was modular , and could be applied each time a new imr image is acquired . we used a linear formulation to characterize the deformation of the brains of both patients because the brains underwent relatively small deformations and displacements . while nonlinear biomechanical models have proven effective to decrease yet do not abolish the inaccuracies of fem - based modeling methods of large brain deformations , the deformations observed in our patients during surgery remained moderate ( 47 mm ) , thus reducing the theoretical benefit of using nonlinear models . this allowed us to use simpler linear models and focus on the added value of xfem to simultaneously account for surgical deformations , namely , shift and resection . using a linear formulation implied that , for each new deformation modeling , one could use the initial configuration rather than the last - deformed configuration of the biomechanical model . this had the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively degraded with each successive deformation modeling . this also had the advantage that we did no longer need to reconnect the deformed mesh for each new xfem calculation , which was one drawback of our previous method , presented in [ 39 , 41 ] , where the biomechanical model was successively deformed . we also showed how xfem could handle a discontinuity for modeling resection without any remeshing or mesh adaptation while the representation of the discontinuity remained accurate , that is , the representation of the discontinuity was not based on a jagged topology using fe facets . xfem thus also avoided making the mesh resolution richer in the neighborhood of the resection - cavity boundary for improving the accuracy of the representation of the discontinuity for that purpose only . we showed that our nonrigid registration technique improved the alignment of the successive imr images for most of the deformation modeling of both patient cases . when our nonrigid registration failed , it still improved the alignment locally , that is , within the tumor region and its neighborhood . we tested the explicit modeling of the lateral ventricles ' region with a soft , compressible law in addition to the whole - brain region law used in the homogeneous biomechanical model . in addition to the validation that is usually performed for successive deformation modelings , that is , validation between pairs of successive intraoperative images , shown in table 1 of section 6 or in the work of ferrant et al . [ 13 , 47 , 48 ] , we also evaluated the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . as a result , shown in table 2 of section 6 , we showed that our approach suffered from the propagation of misregistration through the successive deformation modelings . we expected that this was due , at least partly , to the algorithms used to evaluate intraoperative surface displacements fields from the whole - brain and healthy - brain region boundaries . the surface displacement fields were computed using active surface algorithms , and smoothed to make them compatible with the biomechanical model . because of these two smoothings , the deformed biomechanical model was likely to not be in a perfect alignment with the imr image to which it was registered . because the surface displacement fields evaluated for the next deformation modeling were initialized based on the deformed biomechanical model , we expected to observe an amplification of the misregistration , which was confirmed by our quantitative evaluation . at the present time though , commercial igns systems allow one to register preoperative images and successive imr images , but in a rigid way only . consequently , although the effect of error amplification exists , our technique still enhances the current capabilities of commercial igns systems . future work on modeling of brain shift followed by successive resection is required in five main areas . first , the effect of error amplification through the successive brain deformation modelings calls for further research . consequently , the segmentation , and the subsequent smoothing , as well as the evaluation of surface displacement fields , should be improved to minimize the effect of error amplification . second , further research is required to include additional structures in the biomechanical model in general , and to study the best way to include the lateral ventricles in particular . the use of a poroelastic model in order to model the cerebrospinal fluid filling the ventricles could be considered [ 17 , 18 ] . indeed , these images provide volume information ( rather than surface information only ) , are of good quality in comparison to other intraoperative modalities , and possess a field of view that includes the full volume of brain tissues ( for the 0.5 tesla ge signa scanner ) . these images thus allow one to evaluate what , and how , new structures of the brain could be used , to enhance the modeling of brain shift . some regions , for example , the lateral ventricles ' region , could be extracted from the two imr images , and used as surface landmarks to drive the deformation of the biomechanical model [ 13 , 62 ] . indeed , the workflow presented in this paper has the advantage of being easily adaptable . in case the tumor region would not be visible ( enough ) on the imr images , these new structures , easier to segment , could also adequately replace the tumor for driving the deformation . fourth , our global approach should no longer be based on the 1st imr image used as a substitute for preoperative images , but on the preoperative images themselves . fifth , we should implement , for the surgery cases involving large deformations of the brain , a nonlinear formulation of fem [ 63 , 64 ] , and , particularly , a nonlinear formulation of xfem , which is the subject of recent research [ 65 , 66 ] .

current neuronavigation systems can not adapt to changing intraoperative conditions over time . to overcome this limitation , we present an experimental end - to - end system capable of updating 3d preoperative images in the presence of brain shift and successive resections . the heart of our system is a nonrigid registration technique using a biomechanical model , driven by the deformations of key surfaces tracked in successive intraoperative images . the biomechanical model is deformed using fem or xfem , depending on the type of deformation under consideration , namely , brain shift or resection . we describe the operation of our system on two patient cases , each comprising five intraoperative mr images , and we demonstrate that our approach significantly improves the alignment of nonrigidly registered images .

the contribution to human health of the specific fatty acid ( fa ) composition of the diet has received considerable attention in the literature . fatty acids are key nutrients that affect early growth and development as well as the prevention of chronic disease in later life.1 among the fas , omega-3 polyunsaturated fatty acids ( n-3 pufa ) and omega-6 polyunsaturated fatty acids ( n-6 pufa ) have been suggested to decrease and increase several human diseases , respectively . pufa that contains more than one carbon double bond consists of two major classes such as n-6 and n-3 ( fig . linoleic acid ( la ) is a representative n-6 pufa , serving as a substrate to be converted into an arachidonic acid ( aa ) affecting the prevalence and severity of inflammation . n-6 pufa can induce cardiovascular disease , diabetes , cancer , and age - related disease.1 -linolenic acid ( ala ) , eicosapentaenoic acid ( epa 20:5 ) and docosahexaenoic acid ( dha 22:6 ) are important n-3 pufa involved in human . they are essential fatty acids that can not be synthesized by mammals , by which must be obtained from dietary sources such as cold - water fish , certain seeds ( flax ) and nuts ( walnuts ) . a lot of studies suggest that n-3 pufa , as diet - dependent factors , may be critical to preventing disease backed up with authentic antioxidative and anti - inflammatory actions . especially , n-3 pufa has been shown to exert beneficial effects on some chronic degenerative diseases such as cardiovascular disease,2,3 rheumatoid arthritis,4 diabetes,5 other autoimmune diseases,6,7 and cancer.8,9 increased fat consumption by western diet has been associated with the development of cancer such as breast , colon , pancreatic , and prostate cancers with the notable exception of n-3 pufa , which show to have multiple beneficial anti - tumor actions that affect the essential alterations that dictate malignant growth in a number of studies.10 a diet rich in n-3 pufa may protect from cancer , at least at certain sites . studies on the fatty acid status of patients with several cancer types including bladder , pancreatic , lung and esophageal cancer show low concentrations of plasma phospholipid n-3 pufa , ranging from 55 to 88% of amounts in healthy individuals.1113 recent studies have found a positive association between n-6 pufa and cancer risk , whereas in the same model , n-3 pufa were shown to reduce the development of cancer . epidemiological studies suggest that a high n-3 pufa to n-6 pufa ratio may be the optimal strategy to decrease breast cancer risk.14 solid epidemiological study shows that consumption of n-3 pufa appears to protect against the development of hepatocellular carcinoma , even among patients with hepatitis b virus ( hbv ) and/or hepatitis c virus ( hcv ) infection.15 recently , zhennan et al . has reviewed prospective studies investigating the possible protective effects of the dietary intake of n-3 pufa on prostate cancer development.16 fasano et al also has reviewed a lot of in vivo and in vitro experimental studies providing strong indications of the anti - tumor action of n-3 pufa against lung cancer.17 the purpose of this review is to discuss the potential role of n-3 pufa in gastrointestinal ( gi ) cancer development . we believe that increased consumption of n-3 pufa may lower the risk of gi cancer development via various chemopreventive activities . future studies should include combination treatment of n-3 pufa and nutrients with different and complementary mechanisms of chemopreventive action . among various cancers , most of the gi cancers including esophageal cancer , stomach cancer , and colon cancer , have a natural history of multi - step transition from precursor lesions to malignant lesions , inflammation , adenoma formation , dysplastic changes.18 therefore , gi cancers usually have premalignant lesions before developing invasive cancers , for instances , barrett s esophagus for esophageal cancer , chronic atrophic gastritis accompanied with intestinal metaplasia for gastric cancer , and adenoma or dysplasia originating from chronic ulcerative colitis for colon cancer . because the western diet contains disproportionally high amounts of n-6 pufa and low amounts of n-3 pufa , denoted as a high n-6 to n-3 pufa ratio , n-3 pufa may feasibly play a role in several stages of gi cancers management . esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) , esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades . suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus . in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) . moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease . some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer . recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23 h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer . it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach . colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer , crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer . recently there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer . although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk . dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer . kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) . there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer . the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours . studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies . similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc . the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9 esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) , esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades . suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus . in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) . moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease . some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer . recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23 h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer . it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach . colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer , crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer . there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer . although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk . dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer . kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) . there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer . the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours . studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies . similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc . the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9 n-3 pufa , especially , epa and dha , have been shown to have multiple anti - tumor actions . current knowledge of the anti - tumor activity of n-3 pufa has been comprehensively reviewed elsewhere.9,16,39 recently , stephenson et al.10 has reviewed more recent underlying mechanisms providing strong indications of the anti - tumor actions of n-3 pufa on the hallmarks of cancer . n-3 pufa appear to down - regulate epidermal growth factor receptor ( egfr ) , protein kinase c ( pkc ) , ras , and nf-b , insulin like growth factor ( igf ) , which are important cell signaling mediators often found to be elevated in carcinogenesis . secondly , n-3 pufa induces cancer cell apoptosis via modulation of peroxisome proliferator - activated receptors ( ppars ) , the bcl-2 family , and nf-b cell signaling . thirdly , n-3 pufa decreases sprouting angiogenesis by suppressing vascular endothelial growth factor ( vegf)- and platelet derived growth factor ( pdgf)-stimulated endothelial cell proliferation , migration , and tube formation and by inhibition of mmps via no production and nf-b and -catenin cell signaling . fourthly , n-3 pufa also decreases cell - cell adhesion via down regulation of rho - gtpase , which inhibits cytoskeleton reorganisation , and reduction in intercellular adhesion molecule ( icam)-1 and vascular cell adhesion molecule ( vcam)-1 expression . cockbain et al.9 has also proposed the four main anti - tumor actions of n-3 pufa ( i ) modulation of cox activity ; ( ii ) alteration of membrane dynamics and cell surface receptor function ; ( iii ) increased cellular oxidative stress and ( iiii ) derived anti - inflammatory lipid mediators . firstly , n-3 pufa can act as an alternative substrate for cox-2 , instead of aa , leading to a reduction in formation of pro - tumorigenic secondly , incorporation of n-3 pufa into cell membranes alters the fluidity , structure and/or function of lipid rafts or calveolae . especially , the localization of cell surface receptors , such as g protein - coupled receptors ( gpcrs ) , toll - like receptors ( tlrs ) , and epidermal growth factor receptor ( egfr ) , in lipid rafts is believed to be crucial for downstream receptor signaling , controlling proliferation and apoptosis . thirdly , n-3 pufa may have an anti - tumor effect through alteration in the cellular redox state . n-3 pufa can increase reactive oxygen species ( ros ) because it is highly peroxidisable . therefore , n-3 pufa can induce cancer cell apoptosis via elevation of intracellular ros levels . fourthly , n-3 pufa can be metabolized novel anti - inflammatory lipid mediators including resolvins , protectins and maresins . it is known that resolvins exhibit antineoplastic activity via anti - inflammatory and inflammation resolution activity in animal models of acute inflammation . besides these multiple anti - tumor actions , it is also reported recently that n-3 pufa can activate nrf2 and induced nrf2-directed gene expression40,41 and can suppress lipopolysaccharide - induced inflammation through induction of nrf2 expression.42 nuclear factor erythroid 2-related factor 2 ( nrf2 ) is a redox - sensitive master regulatory transcriptional factor that plays an important protective role in cells by regulating cellular redox balance.43 moreover , n-3 pufa significantly reduces oxidative stress - induced endothelial cell ca influx . this effect might be associated , at least in part , with altered lipid composition in membrane lipid rafts.44 recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate . this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations . hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis . since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice . nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties . song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice . recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice . they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers , the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) . fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice . a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion , several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development . the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55 recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate . this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations . hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis . since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice . nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties . song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice . recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice . they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers , the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) . fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice . a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion , several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development . the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55 gi cancer incidence and mortality are increasing in the eastern world and a high n-6 to n-3 pufa ratio in the western style diet may be a contributing factor . there is much evidence to suggest that higher consumption of dietary n-3 pufa is associated with a lower risk of gi cancer in animal models and humans . especially , recent studies suggest that endogenous n-3 pufa delay the progression of colon and stomach cancer and elevating n-3 pufa may be an important strategy to delay / prevent gastrointestinal cancer in high - risk patients via various mechanisms mediating cancer prevention by n-3 pufa ( fig . 2 ) . in addition , using n-3 pufa in combination with other agents with complementary antitumor action may improve their efficacy in gi cancer prevention . recently , manson et al . has started the vitamin d and omega-3 trial ( vital ) , a large randomized , double - blind , placebo - controlled , 22 factorial trial of vitamin d and n-3 pufa supplements in the primary prevention of cancer among a multi - ethnic population of 20,000 u.s . men aged 50 and women aged 55.56 we expect that new findings in combination consumption of n-3 pufa with other nutrients will provide new approaches to public health implications with regard to prevention of gi cancer through dietary and lifestyle interventions .

omega-3 polyunsaturated fatty acids ( n-3 pufas ) , particularly eicosapentanoic acid ( epa ) and docosahexanoic acid ( dha ) , has been acknowledged as essential very long - chain fatty acids contributing to either achieving optimal health or protection against diseases , and even longevity . recent high impact studies dealing with epa and dha have sparked a renewed interest in using n-3 pufas for cancer prevention and cancer treatment , for which n-3 pufas may exert their anticancer actions by influencing multiple targets implicated in various stages of cancer development , including cell proliferation , cell survival , angiogenesis , inflammation , and metastasis against various cancers . however , gastrointestinal cancers develop implicated with the close connection between inflammation and cancer and n-3 pufas especially imposed excellent actions of antiinflammation and antioxidation as well as their restorative actions . in detail , these beneficial lipids can restore or modify inflammation - associated lipid distorsion and alteration of lipid rafts . although the chemopreventive effect of n-3 pufas has been studied in various experimental models , our understanding regarding the underlying mechanisms of n-3 pufas against gi cancer is still limited . in this review article , we described the in - detailed perspective and underlying mechanism of n-3 pufas application for gi cancers and added in vivo efficacy of n-3 pufas with fat-1 transgenic mice experience . we suggest that future work should consider the n-6/n-3 fa ratio , combination treatment of other nutritions and alteration of lipid rafts to be a key element in experimental design and analysis .

sexual development in males is a multistep process , implying a delicate network of molecular events that directs the bipotential gonad to differentiate into testis ( sex determination ) and consequent differentiation of internal and external genitalia in the presence of testicular hormones ( sex differentiation ) . alteration of any genetic or endocrine factors involved in testicular development may lead to 46 xy disorders of sexual development ( dsd ) . genital ambiguity in patients with 46 xy dsd may either occur due to the disorders in gonadal development or due to the disruption in androgen synthesis or action . patients with disorders of gonadal development may present with either complete or partial forms of gonadal dysgenesis . swyer 's syndrome or complete gonadal dysgenesis ( cgd ) is characterized by unambiguous female genitalia , bilateral streak gonads , and elevated gonadotropins . partial gonadal dysgenesis ( pgd ) may have a wide spectrum of phenotypes associated with ambiguous genitalia , varying degree of hypospadias and testis of variable size and echotexture present along the path of decent . ovotesticular dsd ( ot - dsd ) is a rare condition marked by the presence of both ovarian and testicular tissue in an individual while congenital bilateral anorchia ( cba ) is characterized by the complete absence of testicular tissue in genotypic males . disorders of androgen synthesis may occur due to deficiency of various enzymes involved in the steroidogenesis . congenital adrenal hyperplasia ( cah ) due to 17-hydroxysteroid dehydrogenase 3 ( 17-hsd3 ) is caused by the mutations in hsd17b3 gene which is required for the conversion of androstenedione to testosterone . deficiency of this enzyme results in feminized genitalia in newborns due to the low testosterone levels . mutations in srd5a2 gene impairs the activity of enzyme 5-reductase and hence the conversion of testosterone to its more potent form dihydrotestosterone ( dht ) . individuals with 5-reductase deficiency ( 5-rd ) are often raised as females due to undermasculinized genitalia but the gender role changes soon after attaining puberty . mutations in androgen receptor gene may lead to the androgen insensitivity syndrome ( ais ) which can be partial androgen insensitivity syndrome ( pais ) or complete androgen insensitivity syndrome ( cais ) and is the most common cause of 46xy dsd . the spectrum of phenotypes associated with ais may range from completely female through mixed male / female to completely male type . in 1995 , charmian quigley and frank french proposed new grading system for the phenotypic features in ais based on prader classification for cah . individuals diagnosed with 46xy dsd show an overlap in clinical and biochemical parameters which emphasizes the need for comprehensive evaluation with a multidisciplinary approach . this is an ambispective study where all new and old follow - up patients diagnosed with 46xy dsd , being managed at endocrine opd in a period of 16 months ( june 2014october 2015 ) were included . external masculinization score ( ems ) were calculated based on the external genitalia of patients . abdominopelvic ultrasound was done for the localization of gonads ( magnetic resonance imaging ( mri ) also , when required ) . for patients on follow - up , previous records were reviewed to establish a diagnosis as they were on medication while in new patients , samples were collected and hormonal measurements included serum luteinizing hormone , follicle - stimulating hormone , testosterone , androstenedione ( a ) , cortisol , dehydroepiandrostenedione , and plasma adrenocorticotropic hormone levels by electrochemiluminescence immunoassay using commercial kits ( roche , germany ) . radioimmunoassay kit - based method was used for evaluating 17-hydroxyprogesterone ( 17-ohp ) levels ( diagnostic systems laboratories , inc . , webster , tx , usa ) . 5dht was measured using radioimmunoassay ( immunotech ; prague , czech republic ) after extraction from other hormones with diethyl ether and celite chromatography . the short - term human chorionic gonadotropin ( hcg ) stimulation test was carried out to check the functioning of testicular tissue . testosterone levels were measured before and 2448 h after the last injection of a series of three intramuscular injections of hcg on alternate days ( < 1-year - old , 500 units ; 110 years , 1000 units ; > 10 years , 2000 units ) . testosterone : androstenedione ( t : a ) and testosterone : 5 dht ( t:5dht ) ratios were calculated as and when required to rule out 17 beta hsd deficiency and 5 alpha reductase deficiency respectively . on first evaluation , 11 patients had presented with the primary complaint of ambiguous genitalia , three ( who were being reared as female ) with primary amenorrhea , three with undescended gonads and one with fever and vomiting . the presenting complaints , clinical , hormonal , and radiological findings are summarized in table 1 . five ( 26% ) of them were reared females and 14 ( 74% ) as males . ten patients ( s.5 , s.7 , s.8 , s.9 , s.10 , s.14 , s.15 , s.16 , s.17 , and s.18 ) had undergone corrective surgeries for ambiguous genitalia . clinical profile of patients patient s.1 reared as female presented with primary amenorrhea , delayed development of secondary sexual characteristics and unambiguous female genitalia . patients s.2 , s.3 , and s.5 had ambiguous genitalia , undescended gonads , raised fsh and low testosterone levels while patient s.4 had unilateral undescended testis with raised fsh and low testosterone levels , all three were suspected to be cases of pgd . abdominopelvic ultrasonography ( usg ) and mri could not locate gonads in s.7 while in patient s.6 , a nubbin was seen in the scrotal area . inguinal exploration was done to locate testis but it revealed the absence of any testicular tissue . fsh was raised in both [ table 2 ] and there was no testosterone response to hcg stimulation . hormonal profile of patients patient s.8 being reared as female had presented with ambiguous genitalia and raised gonadotropins . she had undergone bilateral gonadectomy and on histological evaluation ot tissue was found , she was diagnosed with ot - dsd . patient s.9 being reared as female presented with primary amenorrhea , genital ambiguity and delayed development of secondary sexual characteristics . she underwent bilateral gonadectomy , histopathological examination revealed testicular tissue and was diagnosed with cais . hormonal profile showed normal testosterone levels with t : dht ratio of 9.7 and t : a ratio of 5.8 . patient s.11 being reared as female came with a complaint of hirsutism and delayed development of secondary sexual characteristics . bilateral gonadectomy was done and histological studies showed the presence of testicular tissue which confirmed the diagnosis of pais as t : dht and t : a ratios were also normal . testosterone levels were undetectable , but showed a good response to hcg stimulation with t : dht ratio of 7.25 , t : a ratio of 5.8 and was diagnosed with pais . patients s.13 , s.14 , s.15 , and s.16 came with ambiguous genitalia and low baseline testosterone levels . testosterone to dht ratio in s.13 , s.14 , and s.15 were 31.4 , 18 , and 13.9 , respectively . due to high t : dht ratio post - hcg stimulation , all three were diagnosed with 5rd while in s.16 , t : dht ratio was not calculated as b / l gonadectomy was done at 2 years of age . molecular analysis of srd5a2 gene in this patient revealed r246q mutation commonly seen in 5-rd . records show that his post hcg serum t : a ratio was 0.06 , he was thought to be a case of 17hsd deficiency . at 2 years of age he had fever , vomiting and loose stools with hyponatremia , hyperkalemia and hypoglycemia . patient s.18 was admitted in pediatrics with a complaint of poor feeding and poor activity since day one of life . on evaluation , he was found to have hypernatremia , hypokalemia , and genital ambiguity with bifid scrotum and hypospadias . in view of electrolyte imbalance , incomplete masculinization , low cortisol , and elevated 17-ohp , he was suspected to be a case of cah and treatment was started . an accurate diagnosis could not be established when he came to us as he was already on steroids and had a history of salt wasting , so steroids could not be stopped for evaluation of the cause of cah . he had undergone three genital surgeries for hypospadias correction but still had difficulty in urination . the patient is kept on antihypertensives , but no definitive diagnosis could be made as the baseline hormonal reports are missing . with the help of clinical , biochemical and radiological evaluation , accurate diagnosis was made in 16 patients while rest 2 are on follow - up and managed in our clinic . discordance between genetic ( xx or xy ) , gonadal ( testis or ovaries ) , external genital ( vulva or penis ) , and internal sex ( wolffian or mullerian ducts ) results in dsd . in humans , the bipotential gonad differentiates into testis in the presence of sry gene present on y - chromosome . testicular differentiation is followed by the development of internal sex ducts and external genitalia in the presence of fetal hormones produced by the testis . sertoli cells secrete anti - mullerian hormone , which causes irreversible regression of paramesonephric or mullerian ducts around the sixth week of gestational age . testosterone produced by leydig cells of fetal testis promotes the development of mesonephric or wolffian ducts and hence male internal genitalia ; epididymis , vas deferens , and vesicular seminalis . the development of external male genitalia depends on the conversion of testosterone to a more potent steroid dht in the presence of 5-reductase enzyme . in 2006 , the term intersex or hermaphrodite was replaced by dsd and 46xy dsd was subcategorized as disorders of testicular development and disorders of androgen synthesis or action . first - line testing in newborns includes karyotyping , imaging ( usg / mri pelvis ) , serum electrolytes , and serum gonadotropins . molecular diagnosis has been made in approximately 20% of cases with 46xy dsd , although confirmatory , but is limited by cost and accessibility . the inability of bipotential gonad to differentiate into testis results in testicular dysgenesis or agenesis which occurs due to the disruption of various genetic factors involved in the process of sex determination . in our study , four patients were diagnosed with pgd , one with cgd , one with ot - dsd , and two with testicular agenesis or cba . in the absence of gonadal development , patients with cgd usually present with unambiguous female genitalia due to the absence of gonadal steroid production while in pgd , the external phenotype depends upon the degree of testicular tissue present . in our study , out of four patients with pgd , three ( s.2 , s.3 , and s.5 ) presented with ambiguous genitalia and one with undescended gonad ( s.4 ) . in such cases , corrective surgeries are done if required , and testosterone supplementations are given at pubertal age . patient s.5 being reared as female had presented with ambiguous genitalia at 4 years of age . with parents consent clitorovaginoplasty and bilateral orchidectomy orchidopexy and biopsy are usually recommended for undescended gonads as the risk of malignancy ranges from 8.3% to 54% in patients with xy pgd . in 46xy cba is a rare condition and is marked by the absence of testis since birth . previous studies have shown the occurrence of pure or partial forms of 46xy gonadal dysgenesis in families of patients with cba , but the genetic cause is still not known . in our study , two patients ( s.6 and s.7 ) with cba were born out of nonconsanguineous marriage and had no family history of genital ambiguity but the father of patient s.7 gave history of oligospermia and assisted reproduction . 46xy ot - dsd is a very rare condition marked by the presence of ot tissue and is confirmed by histopathological examination . parents of patient s.8 had sought medical attention for ambiguous genitalia at the age of 13 years . bilateral gonadectomy was done , and the corrective surgeries for hypospadias and phallus are awaited . the complete or partial resistance to the action of androgens may result in cais or pais in xy individuals . patients with cais may present with primary amenorrhea in adolescence or inguinal swellings in infants . patient s.9 was being reared as female , parents sought medical attention at the age of 18 years for primary amenorrhea and delayed development of secondary sexual characteristics . bilateral gonadectomy was recommended after confirming the gender identity of the patient to be female . a recent study done on 102 phenotypic women with y - chromosome showed an incidence of 17.6% malignancy . of these 9 out of 30 patients ( 30% ) of cais with a mean age of 20.7 years were found to have gonadoblastoma on histological analysis . therefore , adult cais patients who receive late intervention are recommended to undergo immediate gonadectomy in order to avoid the risk of malignancy . the phenotype of patients presenting with pais depends on the responsiveness of genital tissues to androgens . infants with pais present with the genital ambiguity of varying degrees while adults may present with gynecomastia . however , the risk of malignancy in individuals with pais is 15% if the testis is not scrotal in position . hormonal profile showed normal testosterone levels with t : dht ratio of 9.7 and t : a ratio of 5.8 . patient s.11 being reared as female sought medical attention for hirsutism at the age of 19 years . biochemical examination showed normal t : dht and t : a ratios with testosterone levels in male range . gonadectomy was done and histopathological examination showed the presence of testicular tissue , she was then diagnosed with pais . she had female gender orientation and is now on estrogen replacement therapy for breast development . his t : dht ratio was 7.25 and t : a ratio was 5.8 post - hcg stimulation , which excluded the diagnosis of 5rd and 17hsd . though the presumptive diagnosis of patients s.10 , s.11 , and s.12 was pais , as t : dht ratio and t : a ratio were normal , [ figure 1 ] diagnosis with needs further confirmation by molecular studies . flow chart for diagnosis of 46xy disorders of sexual development the interrupted conversion of testosterone to dht due to the mutations in gene encoding 5-reductase enzyme may lead to 5rd . the differentiation of internal and external male genitalia occurs in the presence of testosterone ( t ) and dht . the t : dht ratio is used to diagnose this condition but interpreting these results is not always straightforward . the diagnosis of 5rd is suspected in newborns with undermasculinized genitalia ( depending upon the activity of enzyme 5-reductase ) , low serum testosterone levels , good response to hcg stimulation and high testosterone to dht ratio ( > 1012 ) . a good response to hcg stimulation ( rise by more than twice the baseline value ) is observed in these patients . the patients may present with microphallus , inguinal gonads or gonads in labioscrotal folds and varying degree of hypospadias . treatment with percutaneous dht increases the size of the phallus in infants and children with 5-reductase 2 deficiency . most of them are reared as males and gender reassignment is required in those who are reared as females due to the spontaneous virilization at puberty . the decisions regarding management of masculinizing puberty need to be taken before puberty with a multidisciplinary approach . female gender assignment in these patients by surgery should be undertaken postpuberty with full caution and counseling with parents . in our study , patient s.16 had severely undervirilized genitalia , bilateral gonadectomy had been done at 2 years of age and female gender had been assigned . at the age of 17 years , he sought medical attention for gender dysphoria . at this time , the presence of common r246q mutation on molecular analysis of srd5a2 gene confirmed the diagnosis of 5rd . 46xy patients with deficiency of 17-hsd enzyme usually present with female - like genitalia at birth with clitoromegaly and blind - ending vagina or male type genitalia with micropenis and hypospadias . at puberty , these patients experience significant virilization due to the abnormally elevated levels of androstenedione or extragonadal synthesis of testosterone , the exact cause of which is unknown . the diagnosis of 17-hsd3 can be suspected if testosterone to androstenedione ratio is < 0.8 . the overlapping phenotype of individuals with 17-hsd and pais can be differentiated by molecular analysis or on the basis of hcg stimulated t : a ratio , which is < 0.8 in cases of 17-hsd . patient s.17 initially predicted to have 17 hsd deficiency , but later presented with features of mineralocorticoid and glucocorticoid deficiency at 2 years of age . patient s.19 is presently 49 years of age and gave a history of ambiguous genitalia . he was on dexamethasone , sustanon and had undergone three corrective surgeries for hypospadias but still has a problem in urination . routine examination revealed raised blood pressure for which he is on hypertensives . limitations of this study are that , being an ambispective study , all tests were not done in all patients and baseline records / tests to make a diagnosis could not be obtained in two patients . nonetheless , this gives an insight into how to diagnose and manage 46xy dsd , who may present at different ages , with different etiologies and different gender of rearing in our set up . there is overlap in the clinical , hormonal and anatomic profile of different subsets of 46xy dsd . the diagnosis of 46xy dsd needs to be confirmed by cytogenetic , hormonal , radiological , and genetic tests . phenotype varies depending on the degree of genetic , hormonal and anatomic defect . at times , in spite of all the tests a definitive diagnosis may not be possible . the pediatrician , endocrinologist , surgeon , psychologist , and clinical geneticist need to work as a team for multifaceted management of these patients . this study was supported by department of biotechnology , ministry of science and technology ( bt / pr7681/med/12/597/2013 ) and indian council of medical research ( dhr / gia/1/2014 ) , new delhi , india . this study was supported by department of biotechnology , ministry of science and technology ( bt / pr7681/med/12/597/2013 ) and indian council of medical research ( dhr / gia/1/2014 ) , new delhi , india .

background and objectives:46 xy disorders of sexual development ( dsd ) cover a wide spectrum of phenotypes ranging from unambiguous female genitalia to ambiguous male genitalia with hypospadias or dysgenetic gonads . management of these patients depends on the cause of dsd , degree of feminization , age at presentation , and gender orientation . the aim of this study was to evaluate the presentation and management of patients with 46xy dsd at our center.patients and methods : all new and old patients of 46xy dsd attending the endocrine opd in a period of 16 months were included in this study . clinical , cytogenetic , hormonal , and radiological evaluation were done to identify the cause of dsd.results:among 19 patients , eight were diagnosed with disorders of gonadal development ( one with complete gonadal dysgenesis , four with partial gonadal dysgenesis , two with congenital bilateral anorchia , and one with ovotesticular dsd ) and eight with disorders of androgen synthesis and action ( one with complete androgen insensitivity syndrome [ ais ] , three with partial ais and four with 5 reductase deficiency ) . in three patients , a definitive diagnosis could not be made.conclusions:management of patients with dsd depends on etiology , gender assignment , gender orientation , hormonal treatment , genital surgery , and consequent psychosocial implications . due to the overlapping clinical and biochemical parameters in different subsets of dsd , only a preliminary etiological diagnosis can be made in some cases . genetic studies with long - term follow - up are required for an accurate diagnosis .

urinary tract infection ( uti ) is a frequent cause of morbidity both in the community and in the hospital setting . the causative pathogen can vary greatly geographically , and so it is prudent to identify those with resistant strains and have current data on the appropriate empirical therapy within a region . the most frequently encountered organisms associated with utis include enteric gram - negative bacteria ( with escherichia coli being the most predominant ) , coagulase negative staphylococcus saprophyticus along with proteus mirabilis , klebsiella , and enterococcus , which account for less than 5% . however , recent studies have reported the increasing prevalence of staphylococcus aureus ( sa ) in utis . sa is an opportunistic pathogen affecting both immune competent and immunocompromised individuals , frequently resulting in significant morbidity . many strains of sa carry a wide variety of multidrug - resistant genes on plasmids , which aid the spread of resistance among species . methicillin - resistant sa ( mrsa ) is widespread in many irish hospitals and is increasingly seen in community health care units such as nursing homes . globally , it is considered that there has been an epidemic of mrsa within health care institutions . bacteriuria with sa is hypothesized to occur through a number of mechanisms that includes catheterization , urologic procedures , or seeding of the genitourinary tract including nephrologically excreted bacteria in overt bacteremia . bacteremia itself is associated with bacteriuria in patients infected with sa , which suggests that bacteremia is an important precursor for bacteriuria in some patient groups . herein , we chronologically assessed the source , patient demographics , and antimicrobial susceptibilities of mrsa - positive isolates from the hospital and community setting over a 5-year period in university hospital waterford a tertiary referral hospital with the primary microbiology laboratory for 4 acute hospitals , serving a total catchment area of almost 500,000 people . the microbiology laboratory in university hospital waterford processes all inpatient urines in the catchment area of almost 500,000 , excluding 2 small private elective inpatient institutions . it also processes all urine samples sent from the community ( i.e. , all samples from general practice and nursing homes ) and all specimens from the catchments emergency departments . laboratory diagnosis of mrsa bacteriuria was performed using accredited microbiological microscopy analysis with culture identification criteria and standardized susceptibility testing protocols . our laboratory utilized clinical and laboratory standards institute ( clsi ; 20102014 ) and european committee on antimicrobial susceptibility testing ( eucast ; 2014 ) methodologies . a retrospective data extraction was performed , from the hospital 's electronic microbiological system cognos and de - duplication was executed . we considered a separate episode as a urine specimen culturing mrsa at least 6 months following a previous positive culture . further urine specimens sent within a 6-month period following an initial laboratory diagnosis mrsa bacteriuria were excluded . resistance rates were then calculated for the pathogen 's susceptibility to 5 commonly used antimicrobial agents ciprofloxacin , coamoxiclav , flucloxacillin , nitrofurantoin , and trimethoprim . for unwell patients , that is , those in whom there was clinical suspicion of sepsis or invasive infection , teicoplanin and vancomycin were also tested for sensitivity profiling . in order to obtain further clinical information from our patient group , we also assessed those patients who had an mrsa - positive swab ( usually from groin , nose , or perineum ) , those who had documented mrsa bacteremia , and we analyzed their demographic details . ethical approval was waived for this retrospective observational study in light of its non - interventional nature . over a consecutive 5-year period , the laboratory cultured 425,013 urine samples from all sources , with a mean number of 85,003 specimens per year received ( table 1 ) . a total of 542 unique urine isolates cultured sa and 151 of species ( 27.9% ) were methicillin resistant , meaning that 0.04% of all urine samples tested cultured mrsa . number of urine cultures by year . of these 151 samples that tested positive for mrsa , 92 ( 61% ) were from mid - stream urine ( msu ) samples , 50 ( 33% ) from catheter specimen urine ( csu ) samples , and 9 ( 6% ) were from an unspecified source ( fig . csu = catheter specimen urine , mrsa = methicillin resistant staphylococcus aureus , msu = mid - stream urine . mrsa was slightly more common in msu samples ; however , the findings were not statistically significant ( 18.2% vs 13.9% , p = 0.388 ) . forty - nine ( 32.5% ) specimens were from an inpatient cohort , 9 ( 6% ) were from the emergency department , 79 ( 52.3% ) from general practitioners , 12 ( 7.9% ) were from nursing homes , with 2 ( 1.3% ) from an unrecorded source ( fig . mrsa was seen in a similar proportion of inpatient and outpatient samples indicating that this is not solely a hospital - acquired phenomenon ( 29.1% vs 26.9% , p = 0.587 ) . mrsa = methicillin resistant staphylococcus aureus . the mean age of our patients who cultured mrsa within their urine was 72.7 years ( range 10.9790.2 ) and most patients were aged between 70 and 90 years ( table 2 ) . patients who cultured an mrsa bacterium were older than patients with mssa , and this was statistically significant ( 53 vs 73 years , p 0.001 ) . the number of sa isolates grown over the 5-year study period doubled from 77 in 2010 to 161 in 2014 . the number of mrsa isolates had also increased ; however , the percentage of sa isolates that were methicillin resistant has decreased ( p = 0.145 ) . this is true for both inpatient and outpatient samples ( p = 0.313 , p = 0.254 , figs . mrsa = methicillin resistant staphylococcus aureus , mssa = methicillin sensitive staphylococcus aureus , sa = staphylococcus aureus . inpatient trends in sa mrsa = methicillin resistant staphylococcus aureus , mssa = methicillin sensitive staphylococcus aureus , sa = staphylococcus aureus . mrsa = methicillin resistant staphylococcus aureus , mssa = methicillin sensitive staphylococcus aureus , sa = staphylococcus aureus . regarding the clinical stance of patients within the cohort , 9 patients ( 7% ) with mrsa - positive urine cultures had a documented history of mrsa bacteremia at that time . each of these patients cultured mrsa isolates that were sensitive to nitrofurantoin , and 8 of the 9 patients ( 88.9% ) were sensitive to trimethoprim . it was noted that all 9 patients in this cohort were aged 70 years or above ( p = 0.079 ) , although this did not reach statistical significance . further antimicrobials agents were introduced to the testing panel in 59 ( 39% ) samples , where there was a clinical concern regarding invasive infection or sepsis including the 9 patients with mrsa bacteremia . all 59 samples ( 100% ) were sensitive to both vancomycin and teicoplanin . eighty - eight ( 66.8% ) of our 128 patients cultured mrsa - positive isolates on routine swabs from either mucosal or skin sites . all 3 patients with resistance to nitrofurantoin in their urine had a positive swab from either groin or nose . eight of the 10 patients ( 80% ) who had trimethoprim - resistant mrsa bacteriuria and 87 of the 125 ( 69.6% ) of the ciprofloxacin - resistant mrsa bacteriuria also had mrsa - positive mucosal or skin swabs . there was no association between age and having an mrsa - positive swab in our cohort . throughout the study period , there was 100% resistance of all mrsa isolates to flucloxacillin and coamoxiclav , as expected . ninety - eight percent of isolates were resistant to ciprofloxacin , but of these samples , there was 97.2% sensitivity for nitrofurantoin and 92.4% sensitivity for trimethoprim ( fig . overall , there was only 2.7% resistance for nitrofurantoin , but all of these samples were sensitive to trimethoprim . trimethoprim itself had a 7.4% resistance level , but all of these samples were sensitive to nitrofurantoin , meaning that no sample was resistant to both oral agents , that is , nitrofurantoin and trimethoprim . there was a statistically significant association between nitrofurantoin resistance and younger patients , in that 75% of patients with nitrofurantoin resistance were younger than 70 years ( p = 0.025 ) . there was no statistically significant association between age and resistance profile in any other of the antimicrobial panel . the impact of mrsa is considerable ; in ireland , approximately 40% to 50% of isolates sa recovered from bloodstream infections are methicillin resistant . despite available publications and guidelines on the management of mrsa skin - colonized patients , little has been published on the colonization of urine from patients with indwelling urinary catheters . the identification of an mrsa isolate in a urine culture has important ramifications for patients , both in the community and in the hospital setting . a recent study demonstrated that 22% of patients with mrsa bacteriuria went on to develop invasive mrsa infection within 12 months . mrsa in urine clearly warrants treatment in symptomatic patients , but even in asymptomatic patients , it may require eradication before certain elective procedures such as endourological surgery . it also has consequences for patients and health care providers when it comes to providing isolation and other barrier precautions that should be administered in a nosocomial setting . the european association of urology guidelines outlines that colonization with microorganisms is a special risk factor for urological procedures ( http://uroweb.org/guideline/urological-infections/ ) , and furthermore , that an indwelling catheter is one of the most important risk factors for complications . patients with asymptomatic bacteriuria who undergo traumatic genitourinary procedures associated with mucosal bleeding have a high rate of post - procedure bacteremia and sepsis . bacteremia occurs in up to 60% of bacteriuric patients who undergo transurethral prostatic resection , and sepsis is clinically confirmed in 6% to 10% of these patients . retrospective analysis and prospective , randomized clinical trials support the effectiveness of antimicrobial treatment in preventing these complications in bacteriuric men undergoing transurethral resection of the prostate . there is little information relevant to other genitourinary procedures , but any intervention with a high probability of mucosal bleeding should be considered as a risk for post - procedure sepsis and treatment of mrsa bacteriuria should be considered . some studies have documented that eradication of asymptomatic bacteriuria is not required before nonurologic procedures , such as arthroplasty but s. aureus is the most pathogenic of all staphylococci and this study did not include patients with mrsa bacteriuria . our study would suggest that when it comes to urine - colonizing mrsa eradication , or for treating a patient who is not critically unwell , oral therapy with nitrofurantoin or trimethoprim would be a suitable first - line agent , as none of the patients in our cohort were resistant to both trimethoprim and nitrofurantoin . in the unwell or septic patient , vancomycin or teicoplanin interestingly , less than one - third ( 32.5% ) of our mrsa urine samples came from hospital inpatient sources , implying that mrsa bacteriuria diagnosis is more frequently a community - based phenomenon . national recommendations dictate that hospital patients colonized with mrsa should be isolated in single rooms and expert opinion would surmise that there is an increased risk of spread from mrsa - colonized catheterized patients due to increased interventions and manipulations required from staff ( e.g. changing catheter drainage devices ) . however , within the community , these patients are not recommended to be isolated as would happen within the nosocomial environment and this could contribute to further bacterial spread . the limitations of our study include its retrospective nature which meant that causation was not addressed , and not all key information was available to our research group . also , acting as a potential confounder is the fact that mrsa bacteriuria is not a common pathology meaning we had only small number of urine isolates ( 167 from 106 patients ) despite the large sample size ( 425,013 ) . to this author 's knowledge , there are no prospective mrsa bacteriuria studies underway and a supra - regional or national study of this type could provide further relevant data in this field .

abstracturinary tract infections are one of the most common infectious diseases diagnosed in the community and in the hospital setting . their treatment is complicated by drug - resistant pathogens and the colonization by microbes of indwelling urinary catheters . this study assessed the occurrence and antimicrobial susceptibility of methicillin - resistant staphylococcus aureus ( mrsa ) uropathogens isolated for 5 consecutive years at university hospital waterford between 2010 and 2014 . we created 4 clinically relevant subdivisions , based on urine source : hospital inpatients , patients from the emergency department , patients referred from their general practitioner , and nursing home patients . we performed a retrospective review from the hospital 's electronic microbiological system and calculated resistance rates for each of the standard antimicrobial agents . during the 5-year study period , we studied 151 urine isolates obtained from 128 patients who had an mrsa cultured in their urine sample . there was 100% resistance of all mrsa isolates to flucloxacillin and coamoxiclav . ninety - eight percent of isolates were resistant to ciprofloxacin . the resistance rate for trimethoprim was 7.4% and there was only 2.7% resistance for nitrofurantoin . for a clinical subset of patients , we also demonstrated 100% sensitivity for samples tested against teicoplanin and vancomycin . urinary mrsa is an infrequently studied phenomenon , but with the rising trend of hospital superbugs nationally , its management is of critical importance . suitable agents to address this within our population include nitrofurantoin in the well patient requiring urinary mrsa eradication or vancomycin / teicoplanin in the unwell patient requiring intravenous therapy . in all groups , fluoroquinolones should be avoided due to significant resistance rates .

acquired angioedema ( aae ) , an acquired deficiency of c1esterase inhibitor , is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen . this disorder is strongly associated with chronic lymphocytic leukemia ( cll ) and other indolent lymphoplasmacytic disorders . we describe a patient with known cll who developed incapacitating , recurrent severe abdominal pains , culminating in partial bowel resection . wider appreciation of the possibility of aae , particularly in patients with lymphoproliferative disorders , could lead to preventive therapy and spare unnecessary surgery . acquired angioedema ( aae ) is due to acquired deficiency of c1 inhibitor ( c1inh ) , resulting in excessive complement and bradykinin activities . blood vessel permeability is increased ; thus , angioedema occurs . just as with hereditary angioedema ( hereditary c1inh deficiency ; hae ) , common clinical manifestations are skin swelling , laryngeal edema , and/or abdominal pain.1,2 aae often occurs in the context of lymphoplasmacytic disorders , such as monoclonal gammopathy of unknown significance ( mgus ) , non - hodgkin s lymphoma , or chronic lymphocytic leukemia ( cll).1,3 among 32 patients with aae , castelli found that 13 ( 40% ) had mgus and 9 ( 28% ) had lymphoproliferative disease.3 therefore , all cases of aae should be evaluated for the possibility of underlying lymphoplasmacytic disorder . conversely , when patients with known lymphoproliferative disease manifest compatible symptoms , aae should be expeditiously considered . this is important because aae can be effectively treated medically , but delayed diagnosis can lead to unnecessary diagnostic procedures , therapeutic interventions , or life - threatening complications , well - illustrated by our case . a 78-year - old woman with atherosclerotic vascular disease was transferred to our hospital with abdominal pain and underwent emergent laparotomy . one year earlier , she had been diagnosed with rai stage i cll which had been observed without treatment . two months earlier , she presented with severe abdominal pain , nausea , and vomiting . over the next 8 weeks , she had six emergency room and/or hospital admissions for identical symptoms . pains would begin at rest in the lower abdomen , spread to the upper abdomen , described as gas - like , non - radiating , constant . extensive evaluation included colonoscopy , endoscopic retrograde cholangiopancreatography , magnetic resonance angiography , and abdominal aortography . benign colon polyps and small gallstones were removed , mesenteric stenoses ruled out , yet pains recurred unabated . there was no dysphagia , change in bowel habits , gi bleeding , fever , or sweats . physical examination on transfer revealed blood pressure 130/88 mmhg , pulse 88 beats per minute , respiratory rate 20 cycles per minute , pulse oximetry of 95% on ambient air , and temperature 97.0f . hemoglobin was 18.1 g / dl , hematocrit 55% , platelets 146 10/l , and leukocytes elevated to 34,500 cells/l with 47% neutrophils and 48% lymphocytes . ct scan of abdomen and pelvis with iv contrast showed multiple abnormal loops of small bowel with contrast - enhanced bowel wall edema ( fig . . 1a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema . b a section of small bowel shows massive submucosal edema . there is no infiltration of the wall by lymphocytes a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema . b a section of small bowel shows massive submucosal edema . there is no infiltration of the wall by lymphocytes at laparotomy , massively swollen small bowel was encountered and resected . c4 was 3 mg / dl ( normal 1746 ) , c3 66 mg / dl ( 85200 ) , and c1inh activity reportedly 83% ( 68200% ) . serum protein electrophoresis revealed two faint bands immunofixing as monoclonal igm kappa and igg kappa . lymphocytosis and lymphadenopathy improved and c1inh activity increased to 110% . over 3 years of follow - up , abdominal symptoms never recurred . approximately 145 cases have been reported of aae,3 and this is one of four cases that we have diagnosed in the last decade with abdominal pains from aae with associated cll . table 1 shows characteristics of patients with cll and aae we have seen ( some briefly mentioned in a prior report).4 this is our only case to undergo surgery , allowing unique and dramatic demonstration of massive bowel edema visible radiographically , on surgical inspection and on histopathology . an initial c1inh activity was reported low normal , but there can be no doubt about the diagnosis based on radiologic / surgical / histologic findings , further laboratory results , and the clinical course . characteristic are very low c4 level and low c3 ; diagnostic recommendations currently add c1q and anti - c1inh antibody levels . autoantibody is demonstrable in up to 70% with aae.1 our patient had monoclonal gammopathy , which frequently corresponds to the c1inh autoantibody . patients with the hereditary form ( hae ) usually manifest before age 20 and give a family history of symptoms . in all our patients with cll and aae , chemotherapy and androgens increased c1inh and produced durable remission.table 1characteristics of patients with aaepatientunderlying diagnosismanifestations of acquired angioedemamonths before diagnosislaboratory valuesinterventions prior to diagnosistreatmentangioeedema episodes post - treatment ( years of follow - up)pretreatmentpost - treatment78 years old / fcllrecurrent abdominal pain2c1 inh activity : 83% ( 68200)c1 inh activity : 110% ( 68200)exploratory laparotomy with small bowel resectionchemotherapydanazolnone ( 3 years)c4 : 3 mg / dl ( 1746)ct scanc3 : 66 mg / dl ( 85200)colonoscopysmall monoclonal gammopathyercpmraabdominal aortography74 years old / fsll / cllrecurrent abdominal pain24c1 inh activity : 1% ( 68200%)c1 inh activity : 117% ( 68200%)ct scanchemotherapynone ( 6 years)episodic oropharyngeal swellingc1 inh quantitative : 6.7 mg / dl ( > 11 mg / dl)c1 inh quantitative : 21 mg / dl ( 1025)colonoscopydanazolc4 : 13 mg / dl ( 1647)c3 : 70 mg / dl ( 75161)small monoclonal gammopathy61 years old / mcllrecurrent abdominal pain5c1 inh activity : 4% ( 68200%)not availablect scanchemotherapynone ( 10 years)small monoclonal gammopathycolonoscopydanazol67 years old / mcllrecurrent abdominal pain3c1 inh activity : 6%(68200)c1 inh quantitative : 38 mg / dl ( 2139)colonoscopychemotherapynone ( 3 months)oropharyngeal swellingc1 inh quantitative : 3 mg / dl ( 2139)laryngoscopydanazolc1q : 3.6 mg / dl ( 58.6)c4 : < 2 mg / dl ( 1746)small monoclonal gammopathy characteristics of patients with aae angioedema should be borne in mind among medical illnesses that can mimic acute surgical abdomen , along with such other disorders as porphyria , familial mediterranean fever and sickle cell disease ( fig . 2 ) . one may need to discern whether a true surgical emergency might supervene even when one of these disorders is present . our patient had typical symptoms of acute bowel edema , including diffuse abdominal pain , occasionally rebound tenderness and vomiting , with spontaneous resolution within 15 days.1,2 some patients with aae have cutaneous or upper respiratory edema in addition , or instead of , bowel symptoms . because of cardiovascular comorbidities , there was a high suspicion for ischemic bowel in our patient , but radiography and endoscopy did not support this . in the differential diagnosis , angiotensin - converting enzyme inhibitors rarely precipitate angioedema , but our patient had taken benazepril many years and continued it without incident after aae therapy.fig . 2algorithm for diagnosis and treatment for suspected aae algorithm for diagnosis and treatment for suspected aae in 2009 , the us fda approved the c1inh concentrate berinert p and the kallikrein inhibitor ecallantide ( kalbitor ) for treatment of acute attacks , and the c1inh concentrate cinryze for prophylaxis in severely affected patients.5 fresh frozen plasma can be given when these are unavailable . c1inh concentrates are highly safe and effective ; the standard of care for decades in other countries , but us approval was delayed by concerns of virus transmissibility.1,59 doses used in the clinical hae trials may need to be higher for aae because of increased enzyme clearance . a bradykinin b2 receptor antagonist icatibant ( firazyr ) is under investigation . for long - term control , commonly used for prophylaxis are anti - fibrinolytic drugs or the attenuated androgen danazol , which increases c1inh synthesis at low cost.10 in conclusion , earlier suspicion for aae in our known cll patient could have spared her the morbidities of recurrent abdominal pains , hospitalizations , morbid interventions , and bowel resection . wider appreciation of this disorder takes on added importance as our ability to effectively treat the problem has grown .

introductionacquired angioedema ( aae ) , an acquired deficiency of c1esterase inhibitor , is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen . this disorder is strongly associated with chronic lymphocytic leukemia ( cll ) and other indolent lymphoplasmacytic disorders.discussionwe describe a patient with known cll who developed incapacitating , recurrent severe abdominal pains , culminating in partial bowel resection . signs , symptoms , laboratory and pathologic findings demonstrated aae.conclusionwider appreciation of the possibility of aae , particularly in patients with lymphoproliferative disorders , could lead to preventive therapy and spare unnecessary surgery . this is more important now that more effective medical therapies are available .

distal clavicle fractures associated with partial and complete coracoclavicular ligament disruption are potentially unstable , and non - operative treatment of these fractures is associated with a high non - union rate.[13 ] partial ligament disruptions ( conoid or trapezoid ) retain some inherent stability , and should be distinguished from complete dual ligament disruptions that result in an uncommon and highly unstable subgroup . internal fixation of these fractures necessitates the use of acromioclavicular joint spanning implants ( clavicle hook plate , synthes , usa ) that overcome distraction forces by subacromial leverage ; however , subacromial / acromioclavicular joint encroachment with these implants is associated with subacromial pathology , and this may negatively influence the final outcomes . to overcome the shortcomings of the joint - spanning implants , acromioclavicular joint - sparing implants ( 2.4 mm lcp distal radius plates , synthes , usa ) have been suggested . additionally , a combination of this implant with some form of coracoclavicular fixation using sutures / anchors / endobutton devices ( tightrope , arthrex , fl , usa ) has been recently described for use in the highly unstable fracture subgroup . however , comparative clinico - radiological outcomes of the two techniques are lacking in literature . the purpose of this study was ( 1 ) to analyze the overall and comparative clinico - radiological outcomes ( radiographic and ultrasonographic ) of surgical treatment of distal clavicle fractures associated with complete coracoclavicular ligament disruption , and ( 2 ) to identify and analyze radiographic patterns of fracture and comminution that are associated with this injury . the hospital records of all patients who were surgically treated for lateral clavicle fractures between 2005 and 2008 at three hospitals were retrospectively evaluated to identify the study group . inclusion criteria for this study was defined as : ( i ) acute , isolated , non - comminuted and comminuted fractures of the lateral clavicle , ( ii ) complete disruption of both coracoclavicular ligaments , confirmed on radiographs and at surgery , ( iii ) absence of concomitant or pre - existing subacromial pathology ( rotator cuff tears , acromial undersurface degeneration ) , ( iv ) surgical treatment with either a joint - spanning implant alone ( group 1 ) , or combination of a joint - sparing implant and coracoclavicular fixation with either endobutton device , suture anchor , or coracoid cerclage ( group 2 ) [ figures 1a and b ] , and ( v ) follow - up period of at least 6 months for radiographic evidence of union . exclusion criteria was defined as : ( i ) partial disruption of the coracoclavicular ligaments at surgery , ( ii ) subacromial pathology ( iii ) concomitant injuries to the ipsilateral shoulder girdle , ( iv ) surgical treatment with any technique other than those described in the inclusion criteria , and ( v ) follow - up period inadequate for complete union . surgical treatment of an unstable distal clavicle fracture with a joint - spanning implant ( group 1 ) . ( hk : hook plate , cl : clavicle , m : medial fragment , l : lateral fragment , arrow : fracture line , ac : acromion , h : humeral head , co : coracoid process ) surgical treatment of an unstable distal clavicle fracture with a joint - sparing implant and coracoclavicular ligament reconstruction using endobutton device ( group 2 ) . ( rp : locking distal radius plate , arrow : acromioclavicular joint , ac : acromion , cl : clavicle , en : endobutton , h : humeral head ) the clinical records and operative notes were analyzed to identify patients that satisfied the criteria described above , and all other lateral clavicle fracture patients were excluded . pre - operative radiographs of the study group were obtained and analyzed with a graphic analysis software ; fracture lines were traced to identify involvement of the acromioclavicular joint , lateral fragment integrity , and presence / absence of comminution . the follow - up clinical protocol included a subjective evaluation of pain and functional status by interview , evaluation of active and passive range of motion by clinical examination , and evaluation of muscle strength in elevation and external rotation using a portable dynamometer . clinical acromioclavicular joint tests ( tenderness and paxinos sign ) and rotator cuff tests ( lag signs , bear - hug test , impingement signs ) were used to evaluate these structures.[811 ] clinical outcome measures included ( i ) simple shoulder test , ( ii ) constant and murley shoulder score , and ( iii ) walch acromioclavicular joint score . the follow - up radiographic protocol consisted of standardized radiographs that included a true glenohumeral anteroposterior view ( neutral rotation , elbow by the side ) , and a weight bearing comparative cephalic - tilt view ; these were analyzed for implant migration , acromioclavicular joint pathology ( degeneration , instability ) , subacromial changes ( degeneration , osteolysis ) , acromiohumeral interval measurement ( normal interval = 7 mm or more ) , peri - coracoid changes ( abnormal bone formation ) , and glenohumeral changes . in addition , in those cases where implant had been previously removed , past radiographs were obtained from the records and individually analyzed as described above . the follow - up ultrasonographic protocol included evaluation of the acromioclavicular joint , subacromial space , glenohumeral joint , bicipital groove and biceps long tendon , and suprascapular and spinoglenoid notches ; ultrasound assessment was performed by a senior radiologist experienced in shoulder ultrasound . statistical analysis was performed using a statistical software ( spss inc , chicago , usa ) to determine mean values and range of measured parameters of the overall and implant specific groups . significant differences between clinical and radiological parameters were determined using the non - parametric mann - whitney / wilcoxon rank - sum test for numerical data , and the fisher 's exact test for categorical data . the average age of the patients in the study group was 34.5 years ( range , 20 to 57 years ) . the mechanism of injury was a direct impact to the ipsilateral shoulder sustained during a fall ; in 8 patients , the fracture was sustained during a cycling - related sporting activity ( competitive or recreational ) . ten patients were operated using a joint - spanning implant ( group 1 ) and 5 were operated using a joint - sparing implant technique ( group 2 ) . the mean follow - up period was 26.1 months ( range , 12 to 40 months ) . the overall and group - specific clinico - radiological outcomes and the types and distribution of fracture patterns are summarized in table 1 . overall and group.specific clinico.radiological outcomes and fracture patterns clinical outcomes : the mean cs was marginally higher in group 2 , while the mean sst score was marginally higher in group 1 ; none of these differences were statistically significant . acromioclavicular joint signs were positive in half of the group 1 patients , and in none in group 2 ; however , the walch scores for the acromioclavicular joint were not significantly different for the two groups . return to pre - operative level of recreational and competitive sports was seen in approximately two - thirds of the patients of group 1 , and all patients of group 2 ; this difference was not found to be statistically significant . reoperation rates related to implant removal were significantly higher ( p < 0.05 ) in group 1 patients ( 90 % ) as compared to group 2 ( 0% ) . radiographic outcomes [ figure 2a - d ] : radiographic union was observed in 93.3% of fractures ( 9 in group 1 , 5 in group 2 ) with only one non - union with subsequent fragment resorption ( group 1 ) . radiographic acromioclavicular joint degeneration was present in 3 patients ( group 1 ) ; all 3 patients had a clinically symptomatic ac joint . radiographic acromioclavicular joint superior subluxation was present in 4 patients ( group 1 = 1 patient , 10% ; group 2 = 3 patients , 60% ) ; none of these were clinically symptomatic . subacromial osteolysis and hook migration were seen in 5 patients ( group 1 ) ; implant removal was necessary in 4 of these 5 patients , and at final follow - up , resolution of the radiographic subacromial osteolytic lesions was observed . signs of progressive implant loosening ( screws and/or plate disengagement ) were seen in 3 patients ( group 1 ) . abnormal bone formation was present in 7 patients ( group 1 = 6 , group 2 = 1 ) . ( b ) acromioclavicular joint subluxation ( arrows ) , ( c ) hook migration and osteolysis of acromial undersurface ( arrow ) , ( d ) peri - coracoid ossification ( arrows ) . ( ac : acromion , cl : clavicle , co : coracoid , p : plate , an : suture anchor , g : glenoid , h : humeral head , hk : hook plate , ahi : acromiohumeral interval ) ultrasonographic outcomes [ figure 3a and b ] : supraspinatus lesions were seen in 3 patients ; these included partial articular - side tears ( group 1 = 1 patient , 10% ; group 2 = 1 patient , 20% ) , and supraspinatus bursal - side degenerative changes ( group 1 = 1 patient , 10% ) . acromioclavicular joint screw penetration was observed in 1 patient ( group 2 ) ; this penetration was not apparent on plain radiographs and the patient was asymptomatic at the final follow - up . no abnormalities were detected in the subacromial bursa on static ultrasound evaluation ; dynamic testing revealed abnormal subacromial bursal " bunching " with arm abduction in 2 patients ( group 1 = 1 , group 2 = 1 ) . ( a ) partial articular - surface supraspinatus tendon avulsion ( black arrows ) ( b ) screw penetration into the acromioclavicular joint ( arrows ) . ( gt : greater tuberosity , h : humeral head , ss : supraspinatus , ac : acromion , cl : clavicle , asterix : acromioclavicular joint ) radiographic evaluation of fracture comminution revealed 5 non - comminuted fractures ( group 1 = 2 , group 2 = 3 ) , and 10 comminuted fractures ( group 1 = 8 , group 2 = 2 ) . analysis of the fracture lines and fragment cortices revealed repetitive patterns in the fracture geometry , and four types could be identified : ( i ) type 1 pattern ( n = 5 , 33% ) involved a clean vertical or a short oblique type bicortical fracture line without any comminution [ figure 4a ] , ( ii ) type 2 pattern ( n = 4 , 27% ) involved a zone of segmental comminution , between intact bicortical lateral and medial fragments [ figure 4b ] , ( iii ) type 3 pattern ( n = 5 , 33% ) involved propogation of at least one fracture line into the juxta - articular cortex of the ac joint [ figure 4c ] and ( iv ) type 4 pattern ( n = 1 , 7% ) consisted of fracture line propogation into the acromioclavicular joint [ figure 4d ] . radiographic fracture patterns : ( a ) type 1 , ( b ) type 2 , ( c ) type 3 , and ( d ) type 4 . ( ac : acromion , cl : clavicle , co : coracoid , g : glenoid , h : humeral head , arrows : fracture lines ) the present study is the first study that describes overall and comparative outcomes ( clinical , radiographic , and ultrasonographic ) of surgical treatment of an uncommon lateral clavicular bony - ligamentous injury with joint - spanning and joint - sparing implants . in addition , four radiographic patterns of bone injury were identified to develop guidelines for choice of implants in these injuries . clinical outcomes and union rates after operative treatment of this injury are satisfactory , and this has been shown in other studies in literature . in the present study , both types of implants resulted in satisfactory and comparable clinical outcome scores , irrespective of the radiographic outcomes . however , a significant difference in the reoperation rate for implant removal ( p < 0.05 ) in patients treated with a joint - spanning implant probably implies that a joint - sparing implant could be preferentially used whenever fracture geometry permits . radiological outcomes , especially those associated with joint sparing techniques , have not been well documented in other studies . subluxation of the acromioclavicular joint , ( present in 60% of group 2 patients ) may be related to either concomitant injury to the acromioclavicular ligaments , or to a partial failure of the coracoclavicular ligament reconstruction ; transfer of distraction forces to ac ligaments after fracture union may then induce gradual subluxation of this joint . this complication was rare in the other group , probably due to the joint spanning implant design ; however , other radiological abnormalities like hook migration , subacromial osteolysis , implant loosening , and new bone formation were frequent . the acromioclavicular joint spanning hook plate is a commonly used implant , and most studies have utilized this as the sole implant of choice . although clinical outcomes in these studies have been satisfactory , the effect of the subacromial hook on bursal tissues has been debated and use of the distal clavicle plate suggested . by using ultrasonographic evaluation , this study demonstrated the safety of the joint - spanning hook plate in relation to rotator cuff and subacromial bursa . persistent pain in the post - operative period probably resulted from acromial undersurface irritation ; early plate removal after fracture union is recommended , and long - term clinical outcomes seem to be satisfactory after implant removal . an important finding of this study is the grouping of the radiographic fracture patterns into four surgically relevant types . types 1 and 2 are ideal indications for use of a joint - sparing implant ; adequate bone stock in the distal fragment in these types will theoretically permit secure fixation across the fracture site . in types 3 and 4 , inadequate distal fragment size may not permit secure bicortical fixation , thereby necessitating use of joint - spanning implants . . the retrospective nature and small sample size does not permit identification of all possible fracture configurations , and statistical analysis may differ with larger numbers . however , the clinical results are similar to other literature studies that have analyzed joint - spanning implants . also , the current radiographic patterns described provide guidelines for implant choice , and are not intended to be used as a prognostic classification . we attribute the small size of the study group to the stringent inclusion and exclusion criteria that eliminated several other patients with lateral clavicle fractures , and this was necessary to obtain meaningful conclusions . lateral clavicle fractures with complete disruption of the coracoclavicular ligaments should be regarded as a distinct entity that is perhaps biomechanically and prognostically different from similar fractures with partial ligament disruption . a combination of a locking distal radius plate with coracoclavicular ligament reconstruction resulted in stable fixation and significantly lower reoperation rates , and should be used preferentially in lesser comminuted fractures ( types 1 and 2 ) .

purpose : distal clavicle fracture associated with complete coracoclavicular ligament disruption represents an unstable injury , and osteosynthesis is recommended . this study was performed ( 1 ) to retrospectively analyse the clinico - radiological outcomes of two internal fixation techniques , and ( 2 ) to identify and analyse radiographic fracture patterns of fracture that are associated with this injury.materials and methods : a total of 15 patients underwent osteosynthesis with either ( 1 ) acromioclavicular joint - spanning implants ( group 1 , hook plate device , n = 10 ) or ( 2 ) joint - sparing implants ( group 2 , distal radius plate , n = 5 ) ; these were reviewed at a mean period of 26.1 months ( 12 to 40 months ) . clinical outcomes were measured using constant score ( cs ) , simple shoulder test ( sst ) , and walch acj score ( ws ) . radiographs and ultrasonography were used to assess the glenohumeral and acromioclavicular joints , and the subacromial space . preoperative radiographs were analyzed for assessment of fracture lines to identify radiographic patterns . statistical analysis of the data was performed to determine any significant differences between the two groups.results:the overall clinical outcome was satisfactory ( cs 80.8 , sst 11.3 , ws 17.6 ) and a high union rate ( 93.3% ) was observed . radiographic complications ( acromioclavicular degeneration and subluxation , hook migration , abnormal ossification ) did not negatively influence the final clinical outcomes . four distinct radiographic fracture patterns were observed . a statistically significant difference ( p < 0.05 ) was observed in the reoperation rates between the two groups.conclusions:internal fixation of this fracture pattern is associated with a high union rate and favorable clinical outcomes with both techniques . a combination of distal radius plate and ligament reconstruction device resulted in stable fixation and significantly lower reoperation rates , and should be used when fracture geometry permits ( types 1 and 2).design : retrospective review of a consecutive clinical case series.setting:level 1 academic trauma service , public hospital .

a 68-year - old woman visited the clinic with the chief complaints of pain , swelling , and a warm sensation in her left thigh for over 2 days prior to her visit . the patient also complained about a warm sensation during the nighttime but body temperature was normal when measured in the clinic . she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to the current visit and was wearing compression stockings to prevent the occurrence of a deep vein thrombosis . she had been taking aspirin 100 mg daily , fexonadine 180 mg daily , and hydrea 500 mg twice a day for a diagnosis of polycythemia vera for 1 year and was phlebotomized in a hemato - oncology setting on an irregular basis . in advance of the operation , her erythrocyte sedimentation rate ( esr ) , and c - reactive protein ( crp ) levels were 30 mm / hr and 4.99 mg / dl , respectively . three days after her hip surgery , her esr and crp levels were 23 mm / hr and 6.35 mg / dl , respectively . medical care was administered in close cooperation with the hemato - oncology department and the level of hemoglobin was maintained at < 12 g / dl to prevent a thrombosis prior to her operation . the patient did not have a fever and her skin color seemed normal despite swelling and a slight warm sensation in the left hip and proximal femur . results of a blood test showed that her complete blood count , electrolyte levels , and liver function were all within normal ranges ( white blood cell count [ wbc ] : 4,700/l , hemoglobin : 10.6 g / dl , hematocrit : 32.3% , platelet : 213,000/l , neutrophil count 67.2% , lymphocyte 24.1% , monocyte 6.8% ) whereas the esr and crp level were 38 mm / hr and 8.25 mg / dl , respectively . when compared to tests performed previously , no further observations of additional fractures , signs of infection , or loosening of inserts after the surgery acute or sub - acute inflammation was diagnosed given the perfusion image of soft tissue in the left hip area as well as the blood pool image ( fig . the patient was injected intravenously with antibiotics for 1 week while staying in the hospital , however , symptoms were not alleviated . in order to prevent a deep vein thrombosis , the patient started to move with the aid of a wheelchair and exercised her knee joints 3 days after the operation . g / dl , a color doppler ultrasonography was performed due to her history of polycythemia vera . results revealed a deep vein thrombosis in the common femoral vein as well as in the superficial femoral vein ( fig . symptoms were alleviated following the administration of low - molecular - weight heparin ( enoxaparin , 40 mg ) and warfarin ( 5 mg ) for 5 days . additional warfarin was prescribed ( 2.5 mg daily ) for maintenance at discharge . during her first follow - up visit 2 weeks later , no symptoms were noted and all blood test parameters were within normal ranges . it has been reported that patients with polycythemia vera present with various complications related to thrombosis due to excessive blood viscosity1 ) . several putative reasons have been postulated to explain such complications including hypervolemia , an increase in the volume of red blood cells , telangiectasia due to decreased blood flow velocity as well as vascular elasticity , thrombocytosis , and complex hemostatic disorders attributable to inefficient blood clotting processes2 ) . thrombosis is the leading cause of death in those with polycythemia vera and has been associated with high morbidity as well . approximately 12 - 49% of patients with polycythemia vera experience thrombosis and 20 - 40% will die as a result2 ) . thromboembolism is one of the major complications of hip surgery and contributes to its poor prognosis . warwick et al.3 ) reported that the incidence rate for a deep vein thrombosis was about 1.89% if proper medication was not provided after total hip arthroplasty . therefore , in the case of the patient described in the current case report , it was reasonable to expect that she would be susceptible to the high incidence rate of thrombosis as she underwent the hip surgery based on her history of polycythemia vera . for prophylactic purposes , aspirin was administrated ( 100 mg daily ) and early exercise and active movements were recommended for the patient in order to lower the risk of venous thromboembolism . in the current case report , a patient with polycythemia vera exhibited clinical symptoms and signs of a postoperative infection following bipolar hemiarthroplasty due to femoral neck fractures . there was , however , a deep vein thrombosis which rendered the intravenous administration of antibiotics ineffective in the alleviation of symptoms . early symptoms of a deep vein thrombosis , including swelling , pain , oppressive pain , and a warm sensation , can mimic those of a postoperative infection . furthermore , features of systematic inflammatory responses such as increased levels of interleukin-6 , interleukin-8 , and crp may be observed in patients with a deep vein thrombosis . collectively , these findings suggest that appropriate clinical examinations are warranted at an early stage in order to avoid making an inaccurate diagnosis4,5 ) . on the first visit after the operation , the patient was suspected to have had a postoperative infection due to the simultaneous increase in esr and crp levels . however , the specificity of esr and cpr levels pertaining to the diagnosis of infections is questionable , as most patients display elevated levels of esr and crp following a hip surgery6 ) . these inflammatory responses stimulate the production of cytokines due to the fractures and operation per se , thus biochemical parameters for inflammation are known to be elevated in a non - specific manner . in cases without postoperative infections , elevated crp levels recover to the normal range within 3 weeks of the operation6,7 ) . in the current case report , the elevated crp level that was maintained for up to 24 days after the operation led us to suspect a postoperative infection . however , the abnormally increased level of acute phase reactants ( e.g. , crp ) may be observed in patients with chronic myeloproliferative diseases ( e.g. , polycythemia vera ) and therefore further specific clinical examinations are required for an infectious diagnosis8 ) . regarding the diagnosis of a postoperative infection , elevated level of serum procalcitonin may be more valuable than wbcs and crp levels7 ) . regrettably , the level of serum procalcitonin was not measured in the current case but could have been informative in distinguishing between postoperative infection and a deep vein thrombosis . despite the presentation of similar clinical symptoms between a postoperative infection and a deep vein thrombosis , their etiological mechanisms are completely distinct . therefore , different treatments are warranted , as patients receiving inappropriate medical treatments may experience exacerbated symptoms and conditions . the potential for a deep vein thrombosis requires continuous thought and attention regarding relevant examinations for patients at high risk for thrombosis as well as for embolism ( e.g. , polycythemia vera ) .

a 68-year - old woman who suffered from polycythemia vera presented at our clinic with the chief complaints of pain , swelling , and a warm sensation in her left thigh . she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to this presentation . her erythrocyte sedimentation rate and c - reactive protein ( crp ) levels were elevated . in addition , a postoperative infection was suspected in the 3-phase bone scan ; therefore , she received intravenous antibiotic therapy . this approach proved to be ineffective and she was subsequently diagnosed with a deep vein thrombosis via color doppler ultrasonography . it is interesting to note that a deep vein thrombosis can present with symptoms similar to those of a postoperative infection . furthermore , an elevated crp level is frequently observed in patients suffering from polycythemia vera . therefore , the two conditions , which require completely different treatments , can be confused . we report on this case with a review of the relevant literature .

brachial plexus surgery using the da vinci surgical robot is a new procedure . to evaluate the advantages and the restrictions of the technique , a cadaveric study of supraclavicular and axillary approaches was conducted . we found that the axillary approach was useful and advantageous for lower roots , particularly for thoracic outlet syndrome ( tos ) . this report will focus on the evaluation of axillary robotic approach as the advantages and disadvantages of supraclavicular robotic intervention have been widely discussed in the literature . a human cadaver was subjected to this experiment in paris university ecole europenne de chirurgie anatomy laboratory and da vinci robot system was used . the left arm was tucked along the side and the right arm was placed in a semiflexed position extending toward the anesthesia location near the head , supported by foam and blankets ( figure 1 ) . a 6 cm long incision was made at the right axillar line , lateral to the edge of the pectoralis major muscle ( figure 2 ) . a self - retaining chung retractor was placed into the incision to elevate the pectoralis major muscle flap . the robot was docked as a camera ; right and left robotic arm were adapted in the incision area ( figure 3 ) . a 10 mm 0 downlooking scope , maryland forceps , and a curved scissors the working space was maintained with the self - retaining retractor , without co2 insufflation ( figure 4 ) . the subclavian artery was seen in front of the truncus and was positioned to the posterior of the working space . subclavian artery was dissected from the plexus and truncus of the lower plexus was exposed with blunt dissection . the plexus was exposed thoroughly from t1 to c7 levels . in this surgical setting , the operating surgeon , who has a wide experience in open brachial surgery of the brachial plexus , reported that lower brachial plexus exposure was easier from the axillary working area and a more wide range of motion was achieved to manipulate the robotic tools compared to the supraclavicular exposure for lower part of the brachial plexus . the development of robotic - assisted minimally invasive techniques began in urology , general surgery , and gynecology because of the generally large working spaces available in the abdomen for these types of surgeries [ 14 ] . since then , other surgeons have sought to use robotic devices in other areas , such as the brachial plexus [ 5 , 6 ] . brachial plexus dysfunction can be the result of shoulder trauma [ 7 , 8 ] . it can also occur with tos , which encompasses three separate disorders involving compression of the subclavian artery , subclavian vein , or brachial plexus in the triangular space bordered by the first rib , clavicle , and scalene muscles [ 9 , 10 ] . compression of the vessel - nerve package at the thoracic inlet has been treated with soft - tissue ( scalene muscle ) release and/or bone ( first rib ) resection . surgical approaches to first rib resection may be transthoracic , transaxillary , supraclavicular , infraclavicular , or thoracoscopic [ 9 , 10 ] . however , these approaches are typically associated with incomplete resection of the most medial portion of the first rib and neurovascular complications . theoretically , a minimally invasive transthoracic approach can obviate these problems , enabling complete resection of the offending portion of the first rib without neurovascular complication . , respectively , reported successful results of robotic en bloc first rib resection for tos treatment via transthoracic and transaxillary approaches [ 9 , 11 ] . 's techniques were only bony interventions and as being intrathoracic these need to be lung collapsed and lung complication can be waiting risk . although liverneaux et al . reported techniques and results of upper brachial plexus injury intervention via robotic surgery with a supraclavicular approach , they described the disadvantages as a narrow working space and difficulty to expose the c7 vertebra [ 1 , 5 , 12 ] . to our knowledge , this report is the first to objectively describe robotic axillar brachial plexus exposure . thus , we discuss the theoretical and clinical advantages and disadvantages of the axillary approach in the present report . the development of robot - assisted surgery has revealed new perspectives in peripheral nerve microsurgery . minimally invasive robot - assisted surgery could lead to modification of the classic algorithm for the treatment of traumatic brachial plexus lesions [ 6 , 8 ] . to date , exploration of these lesions has not been attempted less than 3 months after the traumatic event because clinical examination can not provide an accurate diagnosis or reliable prognosis in these first weeks . early intervention may enable initial assessment of the lesion and repair of potentially graftable nerve roots . several robotics properties are particularly adapted to microsurgery , such as high - resolution three - dimensional ( 3d ) visualization with up to 40 magnification , up to 10-fold magnification of surgical movements , elimination of physiological tremors , and the provision of ergonomic work conditions for otherwise uncomfortable surgery . robotic surgical systems allow high - definition magnified 3d visualization of the operative field , provide significant instrument maneuverability , even within a confined space , and may overcome the shortcomings of conventional approaches [ 2 , 5 ] . axillary ( infraclavicular ) brachial plexus intervention via robotic surgery has not been described previously . axillary intervention was previously performed as an open procedure to expose the plexus or resect the first rib for the treatment of tos . martinez et al . described first rib resection via robotic surgery but not to address plexus injury without transthoracic exposure , a novel minimally invasive approach to the first rib from inside of the chest . in addition , gharagozloo et al . reported first rib resection via transthoracic robotic surgery for paget - schroetter disease . 's techniques were considered more useful for lower brachial plexus viewing and assessing according to gharagozloo et al . the experience of the whole surgical team with robotic technology is important for the procedure . during learning curve period , two staff surgeons are required to participate in all procedures to ensure the safety of the program [ 5 , 9 ] . martinez et al . reported importance of the learning curve , not only for the surgeon but also for the entire surgical team and 180 minutes for the initial 10 cases . reported exclusion criteria include a history of previous incision in the same area and obesity , which present difficulties in robotic surgery initiation . other drawbacks of this new surgical approach are the increased cost of surgical equipment and longer operating time , especially during the learning curve period . however , we believe that the avoidance of a classic incision leads to significant patient satisfaction for cosmetic reasons and we believe that demand for this procedure from a select group of patients justifies the exploration of alternative ways to avoid classic brachial plexus exposure . this report presents our initial experience with robot - assisted axillary exposure of the brachial plexus region . in our opinion robotic surgery will be used routinely in the future for brachial plexus surgery and particularly for tos that is caused by bone and/or soft tissue . however , newer dedicated surgical instruments need to be developed and further studies should be conducted to evaluate in vivo application and results of this novel approach .

brachial plexus surgery using the da vinci surgical robot is a new procedure . although the supraclavicular approach is a well known described and used procedure for robotic surgery , axillary approach was unknown for brachial plexus surgery . a cadaveric study was planned to evaluate the robotic axillary approach for brachial plexus surgery . our results showed that robotic surgery is a very useful method and should be used routinely for brachial plexus surgery and particularly for thoracic outlet syndrome . however , we emphasize that new instruments should be designed and further studies are needed to evaluate in vivo results .

older people have particular spiritual needs that are distinct from those of others , particularly in times of ill health and death and dying . it has been reported that spirituality has a positive effect on health outcomes and welfare of the elderly . nowadays , aging phenomenon and relevant problems with its complexities are important issues that have attracted the attention of social and medical scientists to investigate the spiritual needs of the elderly . spiritual health is a dimension of health that is portrayed as the validation of life in relation to god , fellow human beings , and the environment . spirituality is the representative of the basic values that guide a person in searching to find answers to the crucial questions of life , such as the purpose and meaning of life , reality , love , good and bad , disease , and death . over the past three decades , spirituality , religion , and prayer have turned into a subject of large interest for researchers in medicine , nursing , gerontology , physical health and mental health disciplines , often in conjunction with complementary and alternative medicine . this tendency has been stimulated by research demonstrating physical and psychological benefits of spiritual and religious involvements . in fact , approximately 96% of adults in the united states expressed their belief in god , and 72% of identified religion and spirituality as having the most important influence in their lives . saying prayers is a regular daily task for muslims which begins at the age of puberty and continues throughout the life . it is a time getting rid of the material world and approaching the world beyond , as well as , to the inner self . in iran , elderly people are more religious than younger ones , and this is a norm of this culture . a recent national survey of the iranians values and attitudes found that over 80% of the iranians practice prayer regularly as a part of their religion . one might argue that this is a true reflection of individual culture where prayer and religious beliefs are a part of people 's everyday life . however , prayer can go beyond just performing a religious duty , and based on one 's wishes , take different forms of formal and informal practices . it has been shown that disease prevalence , incidence and mortality rates differ from one population to another . this is partly ascribed to their disparities in lifestyle , dietary habits and other characteristics that are somehow affected by culture , especially religious traditions and experiences . during the past two decades some studies have been carried out and the associations between religious beliefs or experiences and various types of physical illnesses , mental health , and mortality have been examined . spirituality is viewed as personally important matter to the older people and it has been proved that it is highly related to older people 's health outcomes . however , there has not been adequate experiential research on the relationship between religious or spiritual factors and healthy lifestyle behaviors , and their impact on the adult 's well - being . scientists believe that probing the effects of religious and spiritual factors as well as healthy lifestyle behaviors may be decisive . additionally , social science and medical research have concurrently reported that older adults are more inclined to take spiritual chase than younger people are . kotrotsiou - barboutal et al.(2006 ) indicated that older adults are remaining members of religious associations without necessarily participating regularly in services , as there are numerous issues that can diminish the active participation in religious events , such as transportation intricacies , bad sound , or the illegibility of small font text in books of prayers available at mosques or religious centers . in spite of those complexities , many elderly manage to find out a way to come up to god through prayer . it is stated that prayer increases the feelings of personal value of an elderly by reducing the feelings of loneliness and desertation , while television and radio provide support for spiritual life . in this regard , researchers have shown that some groups of elderly people with inadequate and difficult livelihood situations communicate their problems by putting their trust in god . moreover , more than half of the elder patients practiced spiritual needs throughout their hospitalization period , with the most prominent need of being in the search of the meaning of life . extensive epidemiological studies have shown that higher levels of religiousness are related to lower mortality rates . studies identified the multidimensional nature of religiousness and spirituality and investigated various religious dimensions , for instance private religious behaviors , devotion , spiritual transcendence , and religious adaptation . for example , it was indicated that some aspects of religiousness predicted lower rates of disability and illness , alcoholism , cardiovascular disease , hypertension , and myocardial infarction . furthermore , clinical research has suggested that spirituality can play a vital role in the recovery of patients suffering from physical and psychological diseases . it seems that religious obligation has a crucial role in averting physical and mental diseases , recovery promotion , and disease adaptation . numerous studies have revealed the positive involvement of spirituality and religious indexes on different aspects of health such as cardiac surgery , mortality , immunity system function and self esteem . above all , religious involvement can be useful for psychological well - being and physical health . additionally , supporting the effect of spirituality may become more and more prominent in later life as a result of declining health , social and financial sources . researchers have proposed numerous psychological , social , and physiological interveners that may explain the spirituality - health association . quantitative measurements and the evaluation of spiritual beliefs are difficult because quantitative studies neither reveal the content , components of the beliefs , and its factors , nor the contexts that might be included . therefore , implementing qualitative research that explores the personal and experiential aspects of beliefs seems crucial , and may shed light on a range of important issues which can be investigated in detail on larger samples by further quantitative studies in future . spiritual or religious beliefs may affect the decisions of individuals that make about their health and illness . thus , recognizing and understanding the spiritual and religious behaviors within the context of cultural influence by a qualitative research is critical for nurse and other health - care providers . the main strengths of the present study are that it focuses on the spiritual life of healthy elderly people . therefore , the present study aimed to explore the concept of spirituality from the perspectives of iranian healthy elderly people . the central question of the paper was what characterizes the spirituality in the iranian healthy older people ? in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 . it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects . initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data . the study consisted of 17 healthy elders within the age range of 65 - 86 . criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study . all participants were shia muslims . in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions . based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection . interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences . the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about spirituality in elderly adults . what is your experience concerning the spirituality in later age? and what is the meaning of spirituality in your experience ? each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next . coding was carried out line by line , and comparative analysis of the quotations was carried out . all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions . each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes . data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories . the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories . credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking . the findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data . this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability . permission to conduct this study was obtained from the ethics committee of tarbiat modares university . other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses . all participants were aware of the purpose of the study , and their participation in the study was optional . informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki . in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 . it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects . initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data . the study consisted of 17 healthy elders within the age range of 65 - 86 . criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study . in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions . the interview was scheduled according to the participant 's agreement . based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection . interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences . the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about spirituality in elderly adults . what is your experience concerning the spirituality in later age? and what is the meaning of spirituality in your experience ? each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next . coding was carried out line by line , and comparative analysis of the quotations was carried out . all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions . each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes . data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories . the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories . credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking . the findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data . this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability . permission to conduct this study was obtained from the ethics committee of tarbiat modares university . other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses . all participants were aware of the purpose of the study , and their participation in the study was optional . informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki . the participants of the study were 11 males and 10 females , within the age range of 65 - 86 . also , 15 out of 21 were married , four were widowed , and one was single . five participants were illiterate , four were primary degree holders , eight had a diploma , two had bachelor degrees , and two had msc degree in human sciences . in terms of their occupation status , two were employees , 13 were pensioners , two was manual worker , and three were housewives . three main categories emerged from the data and 3 - 4 distinctive subcategories within each category were identified . these categories and their subcategories represent the main factors influencing the spirituality in iranian elderly people . main categories and subcategories one of the main categories that emerged from data analysis was spiritual health . four subcategories ; saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , emerged from the participants experiences . also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them . i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day . ( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers . they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives . they said that man would receive god 's blessings as a consequence of his / her deeds in the world . additionally , they believed that the ultimate consequence of good deeds was good in return . if she asks me to fix her air conditioner you can be sure that i ll do it . this is because i think that by doing this i make her happy and she prays for me . , the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support . when i think about my life , i feel that i do n't have any support from my children . also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . ( female ) the elderly participants lived experiences disclosed the subcategory of faith , a way to happiness in life . one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs . also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness . the one who has strong faith and beliefs is always happy and confident . in my opinion worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free . ( male ) spiritual beliefs was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including seeking help from god in difficulties doing good deeds is the god 's will . regarding the subcategory of seeking help from god in difficulties , the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses . this desire results in god 's attention to elderly and also creates sense of trust in older adults . some statements said by participants are : i really believe in having trust in god . when i m trapped in a complicated situation , i believe that this is god who helps me and i only trust in god . ( male ) one participant believed that spirituality was a vital component of older people lives . also , he believed that god was the ultimate source to affect my life or death and he controls everything . we re all waiting to see when the angel of death arrives and takes us away . ( male ) concerning the subcategory of doing good deeds is the god 's will , the participants believed that trust in god guided them to many positive activities in their lives . they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity . one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems . when i want to get out of my house for work , e first thing that comes to mind is that my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody . ( male ) another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage . the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities . for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness . we go to quran classes on friday mornings also.(male ) i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced . i go to the religious rites with the my friends and peers , and i want to get out of loneliness . ( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque . one of the main categories that emerged from data analysis was spiritual health . four subcategories ; saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , emerged from the participants experiences . also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them . i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day . ( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers . they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives . they said that man would receive god 's blessings as a consequence of his / her deeds in the world . additionally , they believed that the ultimate consequence of good deeds was good in return . if she asks me to fix her air conditioner you can be sure that i ll do it . this is because i think that by doing this i make her happy and she prays for me . ( female ) moreover regarding loss of psychological and spiritual support , the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support . when i think about my life , i feel that i do n't have any support from my children . also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . ( female ) the elderly participants lived experiences disclosed the subcategory of faith , a way to happiness in life . one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs . also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness . the one who has strong faith and beliefs is always happy and confident . in my opinion worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free . spiritual beliefs was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including seeking help from god in difficulties , only god 's power over life and death , and doing good deeds is the god 's will . regarding the subcategory of seeking help from god in difficulties , the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses . this desire results in god 's attention to elderly and also creates sense of trust in older adults . some statements said by participants are : i really believe in having trust in god . when i m trapped in a complicated situation , i believe that this is god who helps me and i only trust in god . ( male ) one participant believed that spirituality was a vital component of older people lives . also , he believed that god was the ultimate source to affect my life or death and he controls everything . we re all waiting to see when the angel of death arrives and takes us away . ( male ) concerning the subcategory of doing good deeds is the god 's will , the participants believed that trust in god guided them to many positive activities in their lives . they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity . one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems . when i want to get out of my house for work , e first thing that comes to mind is that my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody . ( male ) another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage . the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities . for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness . we go to quran classes on friday mornings also.(male ) i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced . i go to the religious rites with the my friends and peers , and i want to get out of loneliness . ( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque . this qualitative study describes the concept of spirituality and its impression on iranian elderly people daily living behaviors . the findings are significant as they reveal that the living experiences of saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , are the most important domain and subcategory of spiritual health of elderly people , which can positively affect their health . on the other hand spiritual health is an important dimension of human health and it can determine the individual integrity . also spiritual health is a power that coordinates physical , mental and social dimensions and is necessary in coping with diseases . when spiritual health will is at serious risk , individuals may be stricken by mental disorders such as loneliness , depression , and loss of meaning . there are a number of studies that support the beneficial effects of spirituality on health . behaviors such as reliance on god , pilgrimage and praying create hope and positive attitudes that lead to internal serenity , peacefulness and meaningful life . most faithful people describe their relationship with god as a relationship with an intimate friend and believe that they can control the effect of irrepressible situations by having recourse to god . all in all , religious adaptation relies on beliefs and religious activities and help people to control their stress and physical disorders . having meaning and goal in life , sense of belonging to a sublime source , hope for gods support in time of trouble and social and spiritual support are among the sources that help religious people to suffer less from hardships of life . in the iranian society context , where the majority of the population are muslim , and a religious culture is dominant in the society , it is expected that tendency to spirituality could be affect their health . praying is the expression of soul and a deep human instinct that arises from human soul and is uttered with profound words . it is neither dependent upon any particular spirituality nor entailed in any time and place . the content of praying includes confession of sins and weakness , pleading for forgiveness , bliss and closeness to god . praying reduces mental and psychological pains , releases personal emotions , leading individual to attain eternal source of power . weeping that often comes with praying greatly helps a person to release his or her distress and emotions . also prayer helped participants to cope with disease and gave them calm , hope and inner strength . in this regard , quran has put it magnificently : those who have faith in god and inundate their hearts with name of god , surely god remembrance ascertains their hearts ( ( quran , chapter 13 : sureh 13 , al - rad ) verse 28 ) . many americans , regardless of their health status , rely on their religious and spiritual beliefs to cope with stressful life events . the present study has demonstrated that one of the most important factors that help elderly in confronting with difficulties was seeking help from god . several studies noted that spirituals beliefs had positive influence on welfare and personal and social life of elderly people . effective spiritual coping strategies help individuals find meaning and purpose in their life , problems and difficulties . for instance , one study showed that healthy older adults believed that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being . thus , spirituality plays an important role in the lives of healthy individuals , too . believing in support from spiritual / religious resource and connectedness with a higher power is beneficial and it can affect issues of control , quality of life , spiritual well - being , coping , depression , decision - making , and possibly health outcomes . for instance , it has been reported that healthy older adults believe that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being . thus , spirituality plays an important role in the lives of healthy individuals . in the result of our study a sense of god 's power over life and death and doing good deeds is the god 's will were derived from participants experience around spiritual beliefs . all the participants talked about the importance of god and belief to god 's power in their life . moreover , the findings reveal that god 's power was very important in personal and private experiences . in this regard , roff et al.(2009 ) in a qualitative study through a thematic content analysis identified god 's power in the lived experiences of women who were suffering from breast cancer . also , in another qualitative study by taleghani et al . ( 2006 ) belief to god 's will was a important key in acceptance of disease . 2002 ) suggested that personal variables such as the concept of god and perception of others beliefs played important mediating roles in religious coping with stress situations . religious practices were a subcategory that emerged from the lived experiences of elderly participants in this research . furthermore , the results of study by keefe et al . ( 2001 ) showed that individuals who reported frequent daily spiritual experiences had higher levels of positive mood , lower levels of daily negative mood , and higher levels of all of the social support domains . another subcategory that emerged from participants experience was going to mosque and religious ceremonies . in this regard , palinkas ( 1982 ) found that religious ceremonies are supportive for individuals because liberating and curative friendships relevant to spiritual states brings about psychological health for them . attending religious ceremonies and shrines also reduces individuals anxieties and sense of loneliness . these results are in accordance with our study findings which show that elderly women who participate in religious ceremonies , are relieved from loneliness . also koenig ( 1998 ) suggested that religious practices , attitudes and coping behaviors are prevalent among hospitalized medically ill older adults and are related to social , psychological and physical health outcomes . morris ( 1983 ) examined the relationship between anxiety and pilgrimage and found that people with religious devotion experienced significantly lower rates of anxiety disorder compared to the non - religious group . every muslim learns their prayers from a young age and prays five times a day . in addition , those who seek guidance from the holy quran and believe that it has power over every aspect of their lives , expect and welcome any eventualities in their lives ( quran , chapter balad , verse 4 ) . it seems that elderly people have more tendencies to religious and spiritual subjects than young people . koenig et al . ( 2004 ) in his study showed spirituality and religion as an important factor in their lives more often than younger ones . several experimental studies have been conducted that showed positive effects of prayer on the management of physical symptoms of illness . kwilecki ( 1986 ) assessed the effect of prayer on stress and anxiety and found that 42% of participants report that saying prayer can diminish the stress and anxiety . also salehi ( 2001 ) found that individuals who pray regularly , experienced significantly lower rates of anxiety disorder and depression , psychological balance and hopefulness , compared to the non - prayer group . azizi ( 1996 ) found that more than 90% of prayers attained psychological tranquility and relaxation after saying prayer . several studies noted moderately positive correlations between indicators of physical health and prayer activities . also several studies have shown that prayer can play an important role in the recovery of patients who suffer from cardiac disease , hiv , arthritis rheumatoid , and cva attack , cancer cases , treatment of addiction , and physical , mental and social well - being . furthermore , evidence suggests that patients with strong religious beliefs and high levels of religious activity experience lower levels of pain , have better immune function , lower death rates from cancer , fewer incidences of heart disease , lower blood pressure and levels of cholesterol , better health behaviors , and greater compliance with medical treatment . the practical relevance of this study may conduct future work to further explicate and clarify the linkages between spirituality and physical and mental health and life style . future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area , future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects . it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) . future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area , future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects . it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) . in summary , we found that elderly people describe several elements in their illustration of spirituality in healthcare settings . spiritual health , spiritual beliefs , and religious practice intentionality were the main components of concept of spirituality in this group experiences . also , the findings of this study made it clear that spirituality has a considerable effect on the health and life of iranian elderly people and is a major supportive resource for their physical and psychosocial health . it can reduce mental distress and induce inner peace and hopefulness . because of the significant influence of spirituality on all domains of health of elderly people , it is critically important that health care providers understand how spirituality can considerably influence elderly people throughout their life . these findings will assist health professionals such as nurses , physicians , and social workers to recognize the spiritual needs and value the role of spirituality in promoting health and well - being among elderly people from different religions and cultures worldwide .

background : spirituality is recognized as a personally important matter to the elderly , and there are evidences of its impact on their health . the aim of this study was to explore the concept of spirituality from the perspectives of iranian healthy elderly individuals.materials and methods : a conventional qualitative content analysis of carried out with 21 healthy elderly people from both male and female genders were chosen using a purposive sampling method in tehran in 2010 - 2011 . data collection was done through semi structured interviews . a qualitative content analysis was used to analyze the participants experiences and perceptions on spirituality , using a central question what characterizes the spirituality in the iranian healthy elderly people?results : the following categories emerged from the data analysis : ( 1 ) spiritual health , with four sub categories including saying prayer as a calming factor ; beneficence as a way to god ; loss of psychological and spiritual support ; faith as a way to happiness ; ( 2 ) spiritual beliefs , with three sub categories including seeking help from god in difficulties ; god 's power over life and death ; doing good deeds is the god 's will ; and ( 3 ) religious practice with three sub categories including saying prayer ; reading quran ; and going to mosque , religious ceremonies and pilgrimage.conclusions:in this study was found that spirituality was a fundamental element in elderly individuals lives that help them to adapt with daily living conditions .

according to the national regulations regarding diagnostic criteria of brain death ( bd ) , instrumental confirmatory tests should be used in certain clinical situations , such as intoxications , infratentorial processes , extensive facial damage , children younger than 2 years of age , or any case in which clinical examinations are inadequate . electrophysiological tests are often unavailable due to limited access to proper equipment and competent specialists . therefore , two of them , catheter angiography and computed tomographic angiography ( cta ) , are based on the detection of contrast enhancement of cerebral vessels . in these examinations , first is a non - filling phenomenon , which means a complete absence of contrast above the carotid siphons and foramen magnum . second is a delayed , weak , and persistent opacification of proximal segments of the cerebral arteries not reaching cortical branches . stasis filling causes a significant problem in interpretation of cta results in the diagnosis of bd . since 1998 , when cta was proposed as the new imaging technique in the diagnostics of bd , a consensus on its interpretation criteria has not been reached . an analysis of stasis filling in a dynamic series of computed tomographic perfusion ( ctp ) can provide valuable information on the interpretation of cta results and relation of this phenomenon to brain perfusion . the aim of this prospective study was to characterize stasis filling phenomenon in the diagnosis of bd . to achieve this , we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and control subjects with the use of the 40-s series of ctp scans . we also confronted stasis filling pattern with ctp findings to assess the relevance of this phenomenon to brain perfusion . we examined 67 patients who fulfilled clinical bd criteria according to the national regulations ( coma , apnea , and no brainstem reflexes ) . the examination protocol consisted of cta , followed by ctp and catheter angiography . thirty - seven subjects were excluded due to lack of contrast enhancement in the intracranial arteries in ctp series . finally , 30 ( 44.8 % ) patients , in whom analysis of ctp series enabled us to identify contrast - enhanced intracranial arteries , were enrolled in the study . the population consisted of 18 men and 12 women with a median age of 54.5 years ( range , 2284 years ) . initial causes of coma were intracerebral hemorrhage ( n = 16 ) , subarachnoid hemorrhage ( n = 11 ) , cerebral edema ( n = 2 ) , and ischemic stroke ( n = 1 ) . decompressive craniectomy was performed in 19 ( 63 % ) included patients . among 37 excluded patients , all patients were managed in the intensive care units of two university hospitals and one multi - profile provincial hospital . during all the examinations , the patients were normoventilated and mean arterial blood pressure ( mabp ) maintained at greater than 80 mmhg . the elapsed time between the onset of bd symptoms and the radiological examination ranged between 6 and 48 h. delay was sometimes caused by a prolonged initial observation due to prior use of sedatives and sometimes the limited availability of an angiographic team . the majority of patients became actual organ donors . in some cases , organ donation was not possible for medical reasons and occasionally because of a failure to obtain permission from relatives . in such situations , families were informed about the termination of futile therapy , and ventilators were legally switched off . the demographic and epidemiological characteristics of bd patients are presented in table 1.table 1demographic and clinical characteristics of bd patientsnumbersexage ( years)diagnosiscraniectomyexamined arterystasis filling in angiography1m56ichmca2m71sahmca+3m73ich+mca+4f49sah+aca5f29sahaca6m40isaca7f66ichmca8m84sah+mca9m53ichmca10f67ich+mca+11m63cemca12f58sah+mca13m34sah+aca14m34sah+mca+15m78ichmca+16m39ich+mca+17f38sah+mca18f74sahaca+19f75ichmca+20f40sah+mca+21f22ich+mca+22f34ich+mca23m50ich+aca+24m44sah+mca+25m51ich+pca+26m71ich+mca27f41ce+mca28m59ichmca29m62ich+mca30m56ich+mca+ is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery demographic and clinical characteristics of bd patients is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery the control group consisted of 30 patients who underwent surgical clipping of an intracranial aneurysm . the population consisted of 14 men and 16 women with a median age of 53 years ( range , 1865 years ) . ctp was performed 810 days after neurosurgical clipping of the middle cerebral or anterior communicating artery aneurysms . during all the examinations , mabp stayed above 80 mmhg . none of the control subjects presented any signs of bd , with normal pco2 and po2 . all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] . we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta . injection of 80 ml of iodinated contrast medium at a flow rate of 4 ml / s . then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60 s after contrast injection . siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15 min . administration of 50 ml of the contrast medium at a flow rate of 5 ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns . detailed protocol is presented in table 2.table 2technical parameters of ctp examinationacquisition tube voltage ( kvp)80 tube current ( mas)270 rotation time ( s)1.0 collimation ( mm)24 1.2 scan range ( mm)28.8 cycle time ( s)1.0 scan time ( s)40 automatic dose modulationoffreconstruction slice width ( mm)3 9.6 fov ( mm)220 matrix512 512 recon algorithmh30scontrast injection volume ( ml)50 flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing , two small circular 0.250.3-cm regions of interest ( rois ) the second roi covered the cerebral artery in the most distal visible segment ( see fig . 1 ) . this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table 1 ) . rois were automatically propagated over the entire series of scans . for each roi , time calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig . 1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) . rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) . rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) . this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation . therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases . we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs . cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels . angiography was performed with a delay of 15 min to 3 h after ctp . we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) . after positioning a pigtail 45-f catheter in the ascending aorta , 30 ml of contrast was injected at a flow rate of 15 ml / s . we registered 3050 s series with a frequency of 2 f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines , cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow demographic data recorded for bd patients and control subjects were age and sex . in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series , the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling . whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p < 0.05 , shapiro all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] . we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta . injection of 80 ml of iodinated contrast medium at a flow rate of 4 ml / s . then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60 s after contrast injection . we used the same scanner siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15 min . administration of 50 ml of the contrast medium at a flow rate of 5 ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns . detailed protocol is presented in table 2.table 2technical parameters of ctp examinationacquisition tube voltage ( kvp)80 tube current ( mas)270 rotation time ( s)1.0 collimation ( mm)24 1.2 scan range ( mm)28.8 cycle time ( s)1.0 scan time ( s)40 automatic dose modulationoffreconstruction slice width ( mm)3 9.6 fov ( mm)220 matrix512 512 recon algorithmh30scontrast injection volume ( ml)50 flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing , two small circular 0.250.3-cm regions of interest ( rois ) were positioned in each ctp series of scans . the second roi covered the cerebral artery in the most distal visible segment ( see fig . 1 ) . this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table 1 ) . rois were automatically propagated over the entire series of scans . for each roi , time density curve ( tdc ) was plotted . calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig . 1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) . rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) . rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) . this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation . therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases . we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs . cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels . angiography was performed with a delay of 15 min to 3 h after ctp . we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) . a typical femoral approach was used in all cases . after positioning a pigtail 45-f catheter in the ascending aorta , 30 ml of contrast was injected at a flow rate of 15 ml / s . we registered 3050 s series with a frequency of 2 f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines , cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series , the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling . whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p < 0.05 , shapiro for cerebral arteries , tdcs in bd patients represented flat curves in contrast to tdcs in the control group , which formed steep and narrow gaussian curves ( see fig . 2 ) . baseline density for cerebral and extracranial arteries was significantly higher in bd patients in comparison to the control group ( 60.5 vs. 41.5 hu ( hounsfield unit ) ; we found significantly longer time to peak enhancement in cerebral arteries in bd patients than in controls ( median , 32 vs. 21 s ; p < in bd patients , peak enhancement in cerebral arteries occurred with a median delay of 14.5 s to peak in extracranial arteries while practically no delay was noted in controls ( it occurred 1 s earlier , which is represented as median 1 s value ; p < cerebral arteries in bd patients showed significantly lower peak enhancement in comparison to the control group ( 34.5 vs. 81.5 hu ; p < 0.0001 ) ( see fig . density curves in a bd patient ( a ) and in a control subject ( b ) . 3distribution of baseline density values in bd patients and controls in cerebral and extracranial arteriesfig . 4distribution of time to peak in cerebral and extracranial arteries in bd patients and controlsfig . 6distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls time density curves in a bd patient ( a ) and in a control subject ( b ) . time to peak ( ttp ) enhancement distribution of baseline density values in bd patients and controls in cerebral and extracranial arteries distribution of time to peak in cerebral and extracranial arteries in bd patients and controls distribution of delay of cerebral peak values in bd patients and controls distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls for extracranial arteries , tdcs represented a similar shape in both groups . however , we observed higher enhancement in bd patients at 20 , 30 , and 40 s ( see table 3 ) . peak enhancement was significantly higher and earlier in bd patients compared to controls ( p = 0.0087 and p < 0.0001 , respectively ) ( see figs . 4 and 6 ) . the intensity of peak enhancement in cerebral arteries in relation to extracranial arteries expressed as c / e peak ratio appeared to be significantly lower in bd patients in comparison to the control group ( 0.31 vs. 0.87 ; p < 0.0001 ) ( see fig . 7).table 3variables calculated from dynamic ctp series in bd patients vs. controlsparameterbd ( n = 30)controls ( n = 30 ) p valueenhancement at 20 s ( hu ) cerebral artery7.5 ( 24)41 ( 25)<0.0001 extracranial artery53.5 ( 40)41.5 ( 30)nsenhancement at 30 s ( hu ) cerebral artery18.5 ( 14)25 ( 19)ns extracranial artery36 ( 23)26 ( 20)0.0225enhancement at 40 s ( hu ) cerebral artery20 ( 15)16.5 ( 8)ns extracranial artery36.5 ( 18)17 ( 10)<0.0001values expressed as median ( interquartile range ) ns not significantfig . 7distribution of cerebral / extracranial peak ratio values in bd patients and controls variables calculated from dynamic ctp series in bd patients vs. controls values expressed as median ( interquartile range ) distribution of cerebral / extracranial peak ratio values in bd patients and controls analyzing the influence of demographic and clinical features on dynamic ct parameters , we revealed significant differences in time to peak and delay of peak enhancement in cerebral arteries between subgroups of bd patients with and without craniectomy . in the subgroup with craniectomy , time to peak and delay of cerebral peak were shorter in comparison to the subgroup without craniectomy ( see table 4 ) . a statistically insignificant trend was found in the analysis of tdcs in bd patients with subarachnoid hemorrhage ( sah ) and intracerebral hemorrhage ( ich ) . we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich ( see table 5 ) . we did not observe any significant differences between ct parameters in relation to sex or examined artery.table 4variables calculated from dynamic ctp series in bd patients without vs. with craniectomyparameterbd patients without craniectomy ( n = 11)bd patients with craniectomy ( n = 19 ) p valuetime to peak in cerebral artery ( s)34 ( 6)26 ( 9)0.007delay of cerebral peak ( s)18 ( 8)13 ( 9)0.04values expressed as median ( interquartile range)table 5variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhageparameterbd patients with ich ( n = 16)bd patients with sah ( n = 11 ) p valuetime to peak in cerebral artery ( s)27.5 ( 10)33 ( 9)0.34 ( ns)delay of cerebral peak ( s)13.5 ( 8)18 ( 13)0.16 ( ns)values expressed as median ( interquartile range ) ns not significant variables calculated from dynamic ctp series in bd patients without vs. with craniectomy values expressed as median ( interquartile range ) variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhage values expressed as median ( interquartile range ) in all 30 bd patients , ctp revealed zero values of cbf and cbv in all rois . stasis filling pattern was observed in 14 ( 46.7 % ) and non - filling in 16 ( 53.3 % ) cases . in 37 excluded bd patients , angiography revealed a non - filling pattern in all cases ; no stasis filling was noted . the term stasis filling for describing a delayed , weak , and persistent intracranial opacification in patients with bd was used for the first time by kricheff et al . in 1978 . munari et al . reported stasis filling in 5 % ( one out of 20 ) , braun et al . in 11 % ( 15/140 ) , bradac et al . in 12.5 % ( two out of 16 ) , kricheff et al . in 15 % ( three out of 20 ) , and savard et al . in 28 % ( nine out of 32 ) of cases [ 2 , 69 ] . noted stasis filling in 43 % ( six out of 14 ) and welschehold et al . in 59 % ( 37/63 ) of cases [ 3 , 10 ] . stasis filling , as specific angiographic pattern of cerebral circulatory arrest , is a consequence of two major factors : raised intracranial pressure ( icp ) and high cerebrovascular resistance ( cvr ) . they both cause reduction of cbf , according to poiseuille s law : cbf = cpp / cvr = ( mabp icp)/cvr , where cpp stands for cerebral perfusion pressure . increased cvr in extreme cerebral hemodynamic disturbances was revealed in studies using a transcranial doppler ( tcd ) [ 11 , 12 ] . this is caused mainly by altered cerebral autoregulation mechanisms , which are sufficient to preserve almost constant cbf when cpp is above 50 mmhg . cessation of capillary circulation is consistent with cerebral circulatory arrest while proximal arterial segments are still patent . at this stage , tcd frequently shows to and fro or narrow systolic spike patterns , which reflect blood movement [ 11 , 12 ] . in such circumstances , heartbeat driven slow propagation of contrast column in proximal cerebral arteries is possible . in the present study , recently revealed a potential efficacy of ctp for assessing brain death . in this material , ctp detected complete absence of brain perfusion reflected by zero values of cbf and cbv in all cases . these findings clearly show that stasis filling is not an indicator of any residual brain perfusion but only ineffective propagation of contrast in cerebral vessels ; thus , it does not preclude diagnosis of brain death . ctp results are complementary to the recent report of selcuk et al . , which revealed global reduction of adc consistent with necrosis of neurons using diffusion - weighted mri technique . the findings showed two features of stasis filling phenomenon : delay and weakness of intracranial opacification . tdcs for cerebral arteries in bd patients were characterized by significantly longer times to peak ( median , 32 s ) compared to controls ( 21 s ) . moreover , intracranial peak enhancement occurred with a median delay of 14.5 s to extracranial peak . this is consistent with observations in cta studies , in which intracranial filling in bd patients is usually detected in the late phase ( performed 60 s after contrast injection ) and very rarely in the early phase of scanning [ 3 , 10 , 17 ] . correlation with ctp results shows that such delayed vascular opacification does not provide any brain perfusion , thus can not preclude diagnosis of brain death . typical cerebral tdc in bd patients was a flat curve with a low peak ( median value of 34.5 hu ) ; about three times lower compared to the peak in the extracranial artery . in contrast , extra- and intracranial tdcs in the control group represented steep and narrow gaussian curves , and both peaks were of similar height . such low peak values could be the reason of lower detectability of stasis filling by catheter angiography compared to ct ( 14/67 = 21 % vs. 30/67 = 45 % cases ) observed in the present study . these findings are consistent with results of others as was presented in the first paragraph of discussion . also observed underestimation of intracranial filling in catheter angiography compared to cta in four out of seven cases . although , a larger amount of contrast was used in dynamic ct than in catheter angiography ( 50 vs. 30 ml ) , but in angiography , it was injected intra - arterially , which minimizes the effect of dilution . this discrepancy could be a result of advanced reconstruction algorithms , systems of noise reduction , and signal amplification used in ct scanners , which reduce noise , improve spatial resolution , and low contrast detectability . the other explanation could be the time sequence in performing ctp and catheter angiography . angiography was carried out with a delay from 15 min to 3 h after ctp . during this period , slow rising icp could stop the propagation of contrast at the level of skull base . analyzing the influence of demographic and clinical features on tdc s parameters , we found a statistically significant trend towards shorter time to peak and shorter delay of peak in the cerebral arteries of bd patients with craniectomy compared to those without it . moreover , the incidence of craniectomy was much lower in bd patients excluded from the study due to lack of intracranial opacification ( four out of 37 = 11 % ) compared to included patients who presented intracranial filling ( 19/30 = 63 % ) . several authors previously reported a relationship between skull defect and preserved intracranial filling in bd patients [ 7 , 18 , 19 ] . this can be explained by a decreased icp enabling propagation of contrast in the cerebral vessels . a statistically insignificant trend was found in the analysis of tdcs in bd patients with sah and intracerebral hemorrhage . we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich . this difference was probably caused by vasospasm after sah , which independently increases cvr and additionally contributes to slowing down the propagation of contrast . the major drawback of this study is a consequence of performing ctp shortly after cta in bd patients . the influence of residual contrast injected for cta was reflected by higher baseline density in the cerebral and extracranial arteries , higher and earlier peak enhancement , and shorter time to peak in the extracranial vessels in bd patients compared to controls . however , this contrast contamination should not significantly change values of delay of cerebral peak or c / e peak ratio as they were calculated on the basis of both cerebral and extracranial tcds . in this study , we assessed the characteristic features of intracranial filling in bd patients delay and weakness of cerebrovascular opacification . it led to the conclusion that delayed and weak opacification of cerebral arteries do not necessarily mean the presence of cerebral perfusion , thus does not preclude diagnosis of bd .

introductionstasis filling , defined as delayed , weak , and persistent opacification of proximal segments of the cerebral arteries , is frequently found in brain dead patients . this phenomenon causes a major problem in the development of reliable computed tomographic angiography ( cta ) protocol in the diagnosis of brain death ( bd ) . the aim of our study was to characterize stasis filling in the diagnosis of bd . to achieve this , we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and controls.methodsstudy population included 30 bd patients , who showed stasis filling in computed tomographic perfusion ( ctp ) series . thirty patients , after clipping of an intracranial aneurysm , constituted the control group . the study protocol consisted of cta , ctp , and angiography . time density curves ( tdcs ) of cerebral and extracranial arteries were generated using 40-s series of ctp.resultscerebral tdcs in bd patients represented flat curves in contrast to tdcs in controls , which formed steep and narrow gaussian curves . we found longer time to peak enhancement in bd patients than in controls ( 32 vs. 21 s ; p < 0.0001 ) . in bd patients , peak enhancement in the cerebral arteries occurred with a median delay of 14.5 s to peak in extracranial arteries , while no delay was noted in controls ( p < 0.0001 ) . cerebral arteries in bd patients showed lower peak enhancement than controls ( 34.5 vs. 81.5 hu ; p < 0.0001 ) . in all bd patients , ctp revealed zero values of cerebral blood flow and volume . angiography showed stasis filling in 14 ( 46.7 % ) and non - filling in 16 ( 53.3 % ) cases.conclusiona confrontation of stasis filling with ctp results showed that stasis filling is not consistent with preserved cerebral perfusion , thus does not preclude diagnosis of bd .

gastric cancer is the second most frequent neoplasm of the alimentary tract after the large intestine . 5,103 people in poland were affected by it in 2008 . the case - to - death ratio of around 1 indicates unfavourable prognosis as to recovery from this disease [ 1 , 2 ] . this poor result is determined by the fact that it is rarely ( only in around 8% ) detected in the form of early gastric cancer , in the mildly symptomatic or asymptomatic phase . in most patients it is diagnosed at a higher degree than the 1st degree of disease progression and its classic symptoms are weight loss , continuous and dull pain in the epigastrium , loss of appetite , nausea , vomiting and chronic bleeding [ 3 , 4 ] . the existence of gastric cancer metastasis to the ureter has been described twice in the literature to date . a female patient , age 67 , was diagnosed at the district hospital ( 22.04 - 30.04.2010 ) because of intensified symptoms of left - sided renal colic . based on the conducted usg and single - phase computed tomography tests of the abdominal cavity and the pelvis , dilation of the ureter was found because of its infiltration by a pathological focus with the dimensions of 28 mm 15 mm . another lesion was located nearby at the level of the left iliac muscle 30 mm 27 mm 20 mm , adhering to the sigmoid colon . tissue infiltration of the pelvis minor wall was found descending in the direction of the left appendages . colonoscopy was conducted and in this test the large intestine was described without pathology , while the gynaecological usg test confirmed the presence of fluid in the pelvis . during her stay the patient was treated with analgesic and diastolic medication and then referred for further treatment at the regional oncology centre . because of reported pain complaints , she was immediately admitted to the department of oncological surgery on the day of her visit to the outpatient clinic ( 04.05.2010 ) . the urologist consulting the patient indicated the possibility of kidney damage due to ureteral obstruction with recommendation for an accelerated operation . therefore , pre - operative diagnostics were not extended beyond the tests received from the district hospital . the patient underwent surgery on 10.05.2010 and intraoperatively , besides the expected neoplastic tumour of the left ovary with infiltration of the ureter , numerous neoplastic foci were also found : a sigmoid colon tumour , a caecum tumour , a tumour of the body of the stomach and two single tumours in the omentum . because of the resectability of the neoplastic foci described above , the operation plan adopted earlier was changed and the following were performed in succession with palliative intention : partial gastrectomy by the rydygier method , right - sided hemicolectomy , left ovariectomy and sigmoid resection . after restoring the continuity of the alimentary tract , the tumour occluded the lumen , which is why segmental ureterectomy was performed with end - to - end anastomosis over a pigtail catheter . the patient passed the post - operative period without complications , except a two - day fever . because of the expected alimentary tract failure , parenteral nutrition was included on the 1st day after the procedure and blood deficits were supplemented with 2 units of erythrocyte mass and 7 units of plasma . on the 9th day after the operation , the patient was discharged home in a good general condition with recommendations for further treatment . the received postoperative histopathological protocol indicated that the stomach was the origin of the neoplastic process . to lauren ) , type i ( acc . to goseki ) , of a stomach adenocarcinoma with a g2 malignancy degree , with occupation of the whole thickness of the stomach wall , with the following immunohistochemical characteristics : ck7(+++ ) , ck20(++ ) , ca125(+ ) , mucicarmine(+ ) . neoplastic infiltrations along nerves and neoplastic embolisms of blood vessels , as well as metastatic foci in the greater omentum , were observed in the specimen . because of the palliative partial gastrectomy type , only 3 lymph nodes were described in this specimen and all of them contained neoplastic cells . besides the above , histopathological confirmations were obtained of the metastatic character of the foci in the ovary , caecum and sigmoid colon . the cancer occupied large and small intestine walls without infiltration of the mucous membrane and the immunohistochemical characteristics were identical as for the primary lesion . according to the above protocol , excision within tissues with a healthy margin of these organs was achieved . the widening of the left ureter is visible ( marked by arrow ) focus of metastatic carcinoma in the ureter wall . microscopic section , he staining , magnification 40 neoplastic invasion in the ureter wall . microscopic section , he staining , magnification 100 it is interesting biologically and constitutes the basis for this paper that the ureteral tumour was described by the pathologists not as a neoplastic infiltration encroaching on the ureter , but as a metastatic focus to the ureteral wall . after the operative treatment , the patient was qualified for palliative chemotherapy and received it from 28.07.2010 . the first treatment course was according to the eox regimen ( oxaliplatin and capecitabine ) , but because of the occurrence of neutropenic fever the regimen was changed to pf ( cisplatin and 5-fluorouracil ) . she again received only one course and the treatment was changed once more because newly formed metastatic foci in the liver were located in imaging tests . next , three chemotherapy courses were administered according to the folfiri regimen ( irinotecan , leucovorin and 5-fluorouracil ) , but only until 11.11.2010 because due to progression of changes in the liver described in examinations the chemotherapy was discontinued and the patient was qualified for symptomatic treatment . according to our knowledge treatment of gastric cancer in the disseminated phase of the disease aims at extension of life and achieving a good palliative effect . the recommended methods , allowing the above to be achieved , are chemotherapy or combination radiochemotherapy . in an advanced stage , palliative operations on this organ are performed for life reasons and serve to eliminate complications such as bleeding , perforation or obstruction of the organ . it is indicated that the above complications forced surgical intervention in 1/4 of the patients previously disqualified from operative treatment because of the presence of metastatic foci . according to literature data , patients operated on by palliative resection in the disseminated phase of the disease achieve a survival time of between 9 and 15 months . the survival time is limited by the number of metastatic foci ; when their number is higher than two foci , no statistically significant differences are observed in this scope . the value of these procedures is increasing because of the reported low perioperative mortality and the observed significant improvement in the further quality of life for these patients [ 513 ] . it seems that the operative procedure conducted in the presented patient allowed a good palliative effect to be achieved along with loss of severe colic and maintenance of kidney function . the literature often presents , as characteristic of gastric cancer , blood - borne metastasis to the ovary , termed a krukenberg tumour . this is a term generally defining metastasis to the ovary , mainly gastric cancer and next colon cancer . metastases from other organs , such as the lungs , the mammary gland and the uterus , are also possible , though much rarer . krukenberg tumours are encountered in the course of 2 - 4% of disseminated neoplastic processes . this is a negative prognostic factor , with varying median survival time after its diagnosis : 12 - 13 months in the course of gastric cancer compared to 17 - 29 months in the course of large intestine cancer . this undoubtedly results from biological differences in the course of these neoplasms [ 1423 ] . in summary , the available literature sources have described this only twice to date ( in 1976 and 2000 ) . however , nephroureterectomy was conducted in these quoted cases [ 24 , 25 ] . in the presented patient the kidney was spared and healing of the ureter after end - to - end suture was achieved . the dissemination of a neoplasm with extremely rarely encountered symptomatology described above is an interesting experience which we wanted to share . the beginning of the symptomatic disease as a left - sided renal colic is , in itself , a previously unreported case .

the typical symptoms of advanced cancer of the stomach are well known in clinical practice . the presented case concerns a patient with symptoms of left - sided renal colic , caused by a malignant tumour involving the ureter , which was diagnosed with a ct scan . the multifocal process , involving the stomach , two parts of the colon , the left ovary and the side of the pelvis , was confirmed only during surgery.the resection or partial resection of the above - mentioned organs involved by the malignant process and reconstruction of the alimentary tract as well as the ureter were performed at time of this operation . the patient 's recovery was without any complications . the histopathological findings support the diagnosis of this malignant process as disseminated stomach cancer.in the available literature only two cases of stomach cancer metastasis to the ureter have been described . in both cited examples resection of the ureter with nephrectomy was performed . the review of the literature supports the value of stomach palliative resection in prolonging life and improving quality of life .

eosinophilic gastritis is an extremely rare disease that is characterized by eosinophilic infiltration of the various layers of the gastrointestinal tract in the absence of any definite causes of eosinophilia . patients with eosinophilic gastritis have diverse symptoms , including abdominal pain , emesis , abdominal distension , and weight loss . these symptoms are associated with eosinophilic infiltration of the various layers of the gastrointestinal tract . the disease has to be distinguished from generalized eosinophilic disorder presenting with involvement of other organs . a 52-year - old female patient visited another hospital because of epigastric pain and tenderness . she had no history of allergic diseases such as eczema or atopy , or food or drug allergies . , she had undergone esophagogastroduodenoscopy ( egd ) , which revealed multiple focal ulcerative lesions with diffuse discoloration and edematous change of the rugae in the gastric fundus , cardia , and upper body ( fig . the symptoms had been recalcitrant to treatment with proton pump inhibitors , fasting , and fluid . she had reported no nausea , vomiting , hematemesis , or melena . on admission , her blood pressure was 110/60 mm hg , pulse rate 76 beats per minute , respiratory rate 22 breaths per minute , and body temperature 36.2. physical examination revealed tenderness of the epigastric area . her white blood cell count was 22,770/mm with markedly increased eosinophils ( 5,009/mm , 22% ) . the c - reactive protein concentration was 13.95 mg / dl . her other blood chemistry test results were normal . the tests for viral markers included hepatitis a , b , and c virus and human immunodeficiency virus ; those for autoimmune antibodies included anti - nuclear , anti - double stranded dna , and anti - neutrophilic cytoplasmic antibodies ; and those for tumor markers included -fetoprotein , carbohydrate antigen 19 - 9 , and carcinoembryonic antigen . the serological test for toxocara antibodies ( igg ) was positive , whereas those for echinococcus , paragonimus westermani , sparganumi , and trichinella antibodies were negative . the chest and abdomen radiographic examinations were normal . to investigate the cause of the epigastric pain and tenderness , abdominal computed tomography was performed , which revealed severe edematous wall thickening with focal localized low attenuation of the fundus and cardia of the stomach ( fig . she underwent a repeated egd , which showed diffuse necrotic change in the fundus , cardia , and upper body ( fig . a biopsy specimen was obtained during egd ; a rapid urease test ( clotest ) revealed no evidence of helicobacter pylori . on histopathologic evaluation , the gastric surface was found to be eroded and the underlying lamina propria showed dense eosinophilic infiltration . she was treated with empirical intravenous antibiotics ( cefoperazone and metronidazole ) immediately after her transfer because infectious gastritis was not ruled out . although she was treated with a broad - spectrum anthelmintic ( albendazole ) , antibiotics , proton pump inhibitor , and prokinetic agent during the next 5 days , her symptoms became worse . eosinophilic gastritis was diagnosed according to the clinical pictures , laboratory findings , and endoscopic findings . she was immediately started on methylprednisolone ( 62.5 mg / day ; this dose was maintained for 7 days ) . therefore , she underwent a repeated short - term follow - up egd that showed regenerative epithelial tissue with peeling off , of the necrotic tissue ( fig . she underwent a repeated egd that showed the replacement of white color scar tissue ( fig . currently , she is being treated with 30 mg / day prednisone and is showing considerable clinical improvement . eosinophilic gastroenteritis is an uncommon and rarely reported disorder characterized by eosinophilic infiltration of the various tissue layers of the digestive tract in the absence of any definite causes of eosinophilia , without eosinophilic infiltration in other organs . the diagnosis of eosinophilic gastroenteritis is based on the following criteria : the presence of gastrointestinal symptoms , histological presentation of eosinophilic infiltration in tissue layers of the digestive tract , presence of high eosinophil count in ascites , and no evidence of parasitic infection or eosinophilic involvement of extraintestinal organs . eosinophilic gastroenteritis can involve any portion of the gastrointestinal tract from the esophagus to the rectum , and the stomach is the most commonly involved organ , especially the antrum . in eosinophilic gastritis , the endoscopic features are rather extensive : rugal fold thickening , erythema , friability , nodularity , gastric outlet obstruction , gastric ulcer , and even a normal mucosa . although not clearly defined , the common pathophysiological mechanism of this disease is associated with eosinophilic infiltration and degranulation in specific tissue layers of the digestive tract . this eosinophilic recruitment and activation regulated by diverse cytokines is a part of the host immune mechanism in the gastrointestinal mucosa ; however , it can be a type of serious allergic or inflammatory reaction in the deeper tissue layers of the gastrointestinal tract . although there is no treatment consensus on eosinophilic gastroenteritis , several studies report good results with steroids in dosages from 20 to 40 mg / day , for 6 to 8 weeks [ 7 - 10 ] . in some case studies , leukotriene modifiers such as montelukast or mast cell stabilizers such as sodium cromoglycate have been proposed to be helpful for symptomatic improvement . antihistamines such as ketotifen or immunosuppressants such as mycophenolate mofetil are also used in the treatment of eosinophilic gastroenteritis ; however , their therapeutic effects are not clear and would require more studies . although rare , eosinophilic gastritis should be considered in the differential diagnosis of patients with gastrointestinal symptoms and peripheral blood eosinophilia . lymphoma of the stomach , gastric cancer , and crohn disease involving the stomach may demonstrate endoscopic features similar to those of eosinophilic gastritis . gastrointestinal parasitic infection should be considered in patients with abdominal discomfort , weight loss , and peripheral eosinophilia . in particular , infestations by hookworms , ascaris , strongyloides , toxocara , trichuris , and intestinal capillaria should be considered in patients from endemic areas . in this case , the concentration of the antigen - specific ige to the a. simplex was 0.57 moreover , the serology for toxocara antibodies ( igg ) was positive . however , this is not clinically significant in korea because koreans often consume raw fish , which causes repeated exposures to anisakis . in addition , she had not had contact with any animals , including dogs and cats , at least for several years . moreover , she was treated with albendazole for 5 days , and her symptoms had become worse . after steroid treatment , the symptoms disappeared and the eosinophil count decreased to the reference range . moreover , the follow - up egd showed regenerative epithelial tissue with peeling off , of the necrotic tissue . she had undergone several egds that showed diffuse necrotic change in the fundus , cardia , and upper body . the etiology includes thromboembolism and occlusion of major arterial supply , ingestion of corrosive agents , volvulus of the stomach , endoscopic hemostatic injections , and infectious gastritis . in this case thus , the possible cause of gangrene could be infection , and she was treated with empirical intravenous antibiotics ( cefoperazone and metronidazole ) . however , she was treated with broad - spectrum antibiotics during the next 5 days , and her symptoms became worse . the necrotic portion of the gastric high body is very vulnerable site of retching injury . this retching injury is called prolapse gastropathy syndrome , a clinical syndrome involving the invagination of part of the gastric mucosa into the lower esophagus . direct trauma to the mucosa occurs when the gastric mucosa becomes incarcerated through the lower esophageal sphincter . in addition , the endoscopic findings and histopathologic results were not compatible to prolapse gastropathy syndrome . on the basis of the clinical picture , laboratory findings , and therapeutic results , we concluded the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis in our patient . this case highlights the reality of eosinophilic gastritis presenting as necrotizing gastritis , and that endoscopy and histopathological examination of the biopsies are the most useful tools for the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis . eosinophilic gastritis should be considered in the differential diagnosis in patients with necrotic gastritis who do not respond to empirical treatment .

eosinophilic gastroenteritis is very rare disorder that is characterized by eosinophilic infiltration of the gastrointestinal tract in the absence of any definite causes of eosinophilia . it is associated with various clinical gastrointestinal manifestations , and depends on the involved layer and site . we report a case of eosinophilic gastritis presenting with severe necrosis . the symptoms disappeared immediately after beginning steroid treatment , and the eosinophil count decreased to the reference range . the patient showed eosinophilic gastritis characterized by necrotic change such as necrotizing gastritis . it is a unique presentation of eosinophilic gastritis . to the best of our knowledge , no case of eosinophilic gastritis characterized by necrotic change such as necrotizing gastritis has been previously reported in korea .

the skin is the largest organ of the body and protects the organism against external physical , chemical , and biological insults such as wounding , uvb radiation , and microorganisms . this major barrier resides in the upper layers of the epidermis ( for review see segre , 2006 ) . the epidermis is the upper part of the skin that is continuously renewed . the basal layer , or stratum basale , of the epidermis contains proliferating keratinocytes ( fig . 1 ) . upon withdrawal from the cell cycle , these basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis . the cells reinforce their cytoskeletal keratin filament network , and adjacent cells interact via many desmosomes , a specialized type of cell junction , to resist physical trauma . in the stratum granulosum , the keratinocytes become more flattened and express certain proteins such as profilaggrin and loricrin , which aggregate to form the typical keratohyalin granules of the stratum granulosum . in addition , lipids are produced and stored in lamellar bodies . at the final stage of differentiation , the keratinocytes lose their organelles , including the nucleus , and become the dead , flattened corneocytes of the stratum corneum . during cornification , proteins are cross - linked at the inner side of the cytoplasmic membrane to form a cornified envelope ( for review see candi et al . , 2005 ) . in the transitional layer between the stratum granulosum and the stratum corneum , lipids are extruded to form a water - repelling envelope around the cornified envelope , thereby assuring an adequate permeability barrier function of the mammalian epidermis . improper formation of these envelopes results in an impaired epidermal barrier that can not protect against dehydration , uvb , and infection . the signaling cascades involved in epidermal barrier formation are largely unknown , but the many proteases that seem to be involved are currently being intensively studied . see introduction for details . since the cloning of caspase-14 in the late nineties , it has become clear that this protease is a unique member of the caspase family . unlike apoptotic caspases , which evolved in common ancestors such as hydra , echinodermata , insects , nematodes , and chordates , caspase-14 has so far been found only in terrestrial mammals ( lamkanfi et al . , 2002 ) . in contrast to the ubiquitously expressed other members of the caspase family , caspase-14 is expressed and activated mainly in the epidermis and is absent from most other adult tissues ( eckhart et al . recently , caspase-14 was found to be involved in epidermal barrier formation ( denecker et al . , 2007 ) . in this review , we discuss current knowledge of the expression , regulation , and function of caspase-14 . the expression pattern of caspase-14 is unique among the caspases , as it is present mainly in cornifying epithelia , such as the epidermis , the hassall 's bodies of the thymus , and the forestomach of rodents ( lippens et al . , 2000 , 2005 ; in skin , caspase-14 is expressed only in the differentiating and cornifying layers of the epidermis and the hair follicle ( lippens et al . , 2000 ; this is consistent with the observation that , in vitro , caspase-14 is only expressed in differentiating but not in proliferating keratinocytes ( lippens et al . remarkably , nail matrix keratinocytes that differentiate into specialized nail corneocytes , the building blocks of the nail plate , do not express caspase-14 ( jager et al . in addition , caspase-14 is not expressed in the noncornifying keratinocytes of the sweat gland or the mouth epithelium ( lippens et al . , 2000 ; ultrastructural analysis demonstrated that spatial distribution of caspase-14 in the epidermis and hair follicles is strongly conserved among several mammalian species ( alibardi et al . caspase-14 was found to be associated with the nucleus , the keratohyalin granules , and the desmosomes , whereas in corneocytes , caspase-14 was found in the cytoplasm and was associated with corneodesmosomes ( a modified version of desmosomes ) and nuclear remnants . these observations suggested a role for caspase-14 in nuclear degradation during cornification , but nuclear degradation was not affected in caspase-14deficient mice ( denecker et al . , 2007 ) . the expression of caspase-14 in the hassall 's bodies of the thymus and in the forestomach of rodents is somewhat expected , as they are cornifying structures and express the typical late differentiation markers , such as profilaggrin and loricrin , which are also found in the epidermis ( laster and haynes , 1986 ; favre , 1989 ; jarnik et al . , 1996 ) . protein expression of caspase-14 has also been reported in several noncornifying tissues ( lippens et al . , 2003 ; krajewska et al . , 2004 , 2005 ; kam et al . , 2005 however , these observations should be interpreted carefully , as we have recently shown that the reported expression of caspase-14 in such tissues can be the result of aspecific staining ( denecker et al . , 2007 ) . remarkably , so far caspase-14 has been found only in terrestrial mammals but not in birds or reptiles . whereas birds and reptiles have a stiff , dry , scaly epidermis , mammals have a soft stratum corneum because of the larger amounts of histidine - rich late differentiation markers ( e.g. , profilaggrin ; alibardi , 2003 ) . interestingly , profilaggrin is a direct substrate of caspase-14 ( denecker et al . , 2007 ) . this could indicate that the occurrence of a soft stratum corneum and the caspase-14 gene are associated during evolution . although the expression of caspase-14 is very restricted , little is known about the transcriptional regulation of its gene . in vitro , caspase-14 is only expressed when keratinocytes are forced to differentiate by growing them postconfluently or in suspension or by adding vitamin d3 ( eckhart et al . in contrast , adding ca at high concentrations to the medium , a method frequently used to induce differentiation , did not induce caspase-14 expression ( eckhart et al . retinoids , which suppress keratinocyte differentiation , down - regulate caspase-14 expression ( rendl et al . , these results indicate that transcription factors that are specifically active during terminal differentiation are required to regulate caspase-14 expression . whether caspase-14 expression levels can be regulated at the posttranscriptional level is not known . down - regulation of several differentiation - associated genes by retinoids has been shown to be mediated by the transrepression of activator protein 1 ( ap-1)mediated gene activation ( fisher and voorhees , 1996 ) . indeed , the caspase-14 promoter contains at least two potential ap-1binding sites ( unpublished data ) . the green tea phenol ( )-epigallocatechin-3-gallate ( egcg ) is a potent activator of ap-1 and has been shown to up - regulate caspase-14 in a p38- and jnk - dependent way ( hsu et al . , 2005 , 2007 ) . ap-1 alone is probably not sufficient to drive caspase-14 expression because tnf and 12-o - tetradecanoyl - phorbol 13-acetate , two potent activators of ap-1 in keratinocytes ( arnott et al . , 2002 ) , did not induce caspase-14 expression in keratinocytes ( lippens et al . , 2004 ) . differentiation - dependent expression of caspase-14 in keratinocytes could also result from a strong transcriptional repression in proliferating keratinocytes . this possibility is supported by the observation that mice deficient in nuclear receptor corepressor hairless ( hr ) had 510-fold higher levels of caspase-14 and profilaggrin mrna , starting from postnatal day 6 and progressing during development ( zarach et al . , 2004 ) . these alterations in gene expression were detected mainly in the keratinocytes of the utricle , an abnormal pouch - like structure at the upper part of the hair follicle . increased gene expression occurred before the morphologically distinct utricle could be identified , indicating that the up - regulation was probably a cause rather than a consequence of utricle formation . it would be interesting to analyze both caspase-14 activation and filaggrin processing in these mice . multiple mutant hr alleles in mice and in humans show phenotypic variations that include congenital hair loss , skin wrinkling , and papular rash ( cichon et al . , 1998 ; 1998 ) . whether caspase-14 overexpression is important for the observed phenotypes could be addressed by generating epidermis - specific caspase-14 transgenic mice or by crossing the hr mice with caspase-14deficient mice . procaspases consist of a prodomain , a large subunit ( p20 ) , and a small subunit ( p10 ) . activation of caspases is induced by dimerization , ( auto-)proteolytic cleavage at asp residues , and/or conformational changes ( lamkanfi et al . , 2003 ) . so far , maturation of caspase-14 by proteolytic cleavage into p20 and p10 subunits has been consistently observed only in cornifying epithelia such as the epidermis and the rodent forestomach ( lippens et al . , 2000 ; although some investigators suggested that caspase-8 and -10 can activate caspase-14 in vitro ( ahmad et al . , 1998 ; van de craen et al . , 1998 ) , this could not be confirmed by others ( lippens et al . furthermore , caspase-14 is probably not proteolytically activated by a caspase in vivo , as other caspases are not activated during epidermal differentiation ( eckhart et al . 2000 ; raymond et al . , 2007 ) , and caspase-14 is not processed at an aspartate residue like other caspases but is processed at ile in man and presumably at leu in the mouse ( chien et al . , 2002 ) . alignment of the protease - sensitive loop between the p20 and p10 subunits of the known mammalian procaspase-14 amino acid sequences ( fig . 2 ) reveals a conserved hydrophobic patch that is n terminal of the caspase-14 cleavage site . this patch contains p1-preferred amino acids of elastase - like serine proteases , such as val , ala , leu , and ile ( mallory and travis , 1975 ; vered et al . , 1985 ; takahashi et al . , 1989 ) , suggesting that a serine protease with elastase - like properties could be involved in caspase-14 activation ( unpublished data ) . all together , these data indicate that during skin homeostasis , the caspase-14activating protease is not a caspase , separating caspase-14 activation from the apoptotic and inflammatory caspase cascades that could be detrimental to epidermal integrity . the precise epidermal layer in which caspase-14 is processed and activated is unknown because antiserum specifically recognizing activated caspase-14 is not yet available . however , the following findings indicate that activation of caspase-14 occurs at the interface between the granular and cornified layers of the epidermis or early during cornification . first , both the proform and activated form of caspase-14 can be found in total epidermal extracts , whereas in the cornified layer only activated caspase-14 is found ( fischer et al . , 2004 ) . second , caspase-14 activation coincides with stratum corneum formation both during embryonic development and in organotypic skin cultures ( eckhart et al . this implies that the main biological function of caspase-14 is exercised in the stratum corneum , as proven by the phenotype of the caspase-14deficient mice ( see the next section ) . sequence analysis indicates that a hydrophobic patch in the protease - sensitive loop is conserved . only part of the alignment is shown here , including the c - terminal part of the p20 subunit , the protease - sensitive loop , and the n - terminal part of the p10 subunit . the darker the yellow , the more the amino acids are conserved between species . the catalytic qacrg box is delineated with a green box . the conserved hydrophobic patch is delineated with a red box , and the cleavage site in human caspase-14 is indicated with a red arrow . the alignment was performed using clustalw ( mega version 3.1 ; kumar et al . , 2004 ) and identification of caspase-14 substrates has been hampered for a long time by the unavailability of enzymatically active caspase-14 . however , it was recently shown that proteolytically processed caspase-14 requires high concentrations of kosmotropic salt to be active in vitro , such as sodium citrate , in addition to proteolytic cleavage between the p20 and p10 subunit ( mikolajczyk et al . , 2004 ) . these salts induce both dimerization and ordering of active site loops by partial desolvation of the protein to a more compact , catalytically active protease . the cellular environment of the stratum corneum of the epidermis probably favors caspase-14 activity in a similar way . indeed , the water content decreases from 45% at the transitional layer to 1525% at the skin surface ( warner et al . , 1988 ; caspers et al . , 2001 ) . human caspase-14 preferentially accommodates tryptophan or tyrosine in the s4 subsite , whereas mouse caspase-14 is more tolerant , with almost equal preferences for -branched and aromatic amino acids ( mikolajczyk et al . , 2004 ) . for example , both human and mouse caspase-14 efficiently cleave the fluorescent peptide substrate wehd - amc , but only mouse caspase-14 cleaves ietd - amc as efficiently ( fischer et al . , 2004 ; mikolajczyk et al . , these substrate preferences would classify human caspase-14 as an inflammatory caspase and mouse caspase-14 as an inflammatory and apoptotic initiator caspase ( thornberry et al . , 1997 ; thornberry , 1998 ) . however , human caspase-14 can not proteolytically activate the inflammatory cytokines pro 2004 ) , and there are no data supporting a direct role for caspase-14 in apoptosis ( lippens et al . , 2000 ; denecker et al . , 2007 ) . whether the substrate preferences of human and murine caspase-14 observed in vitro on peptide substrates also occur in vivo is not clear . importantly , profilaggrin , a major structural protein in the differentiating epidermis , has been shown to be a physiological substrate of caspase-14 ( denecker et al . , 2007 ) . identification of additional substrates and determination of the cleavage sites will provide more insight into the preferred recognition sequence of caspase-14 in the context of a protein . keratinocytes can die by two different processes : apoptotic cell death induced by damaging agents such as uvb , chemicals , and cytotoxic cytokines or by a continuous process of differentiation leading to the formation of corneocytes . these processes are clearly distinct pathways executed by different players ( for review see lippens et al . , 2005 ) , but the role of caspases in these two cell death programs has long been controversial . although almost all procaspases are constitutively expressed in the epidermis , only caspase-14 has consistently been shown to be activated during epidermal cornification ( eckhart et al . , 2000b ; lippens et al . , 2000 ; raymond et al . , 2007 ) . in addition , knockouts for apoptotic caspases were not reported to have a phenotypic skin anomaly except for caspase-3deficient mice , in which keratinocyte differentiation is delayed in the embryo but normalized at birth ( okuyama et al . , 2004 ) . however , others could not confirm the activation of caspase-3 during embryonic epidermal development ( fischer et al . although they are not activated during cornification , apoptotic caspases , in contrast to caspase-14 , become activated during uvb- , staurosporine- , tnf- , and tnf - related apoptosis - inducing ligand induced apoptosis of keratinocytes and , thereby , play a role in the apoptotic cell death of keratinocytes ( for review see lippens et al . , 2005 ) . thus , we conclude that apoptotic caspases are not involved in the physiological keratinocyte cell death program leading to cornification . furthermore , caspase-14deficient epidermal cells can undergo classical apoptosis , which genetically demonstrates that caspase-14 is dispensable for the apoptosis of keratinocytes . during development , caspase-14 protein expression is detectable from embryonic day ( e ) 15.5 on , and its processing is observed from e17.5 ( hu et al . , 1998 ; van de craen et al . , 1998 ; fischer et al . , 2005 ) , which coincides with stratum corneum formation and establishment of the epidermal barrier . however , no differences in outside - in barrier formation of the skin of caspase-14deficient mice during embryogenesis were observed ( denecker et al . , 2007 ) . furthermore , caspase-14deficient mice were born at the expected mendelian ratios , were fertile , and had long survival rates . detailed analysis of caspase-14deficient mice indicated that caspase-14 has an important role in cornification , hydration , and uvb protection . the skin of caspase-14deficient mice was shinier , characterized by deeper skin lines , and had larger scales ( denecker et al . , 2007 ) even though the shape and size of the cornified envelopes themselves were not altered . biochemical analysis indicated that caspase-14 was responsible for the correct processing and degradation of ( pro)filaggrin , as epidermis lacking caspase-14 was characterized by an altered profilaggrin processing and staining pattern ( fig . 3 ) and by the presence of aberrant keratohyalin granules , the profilaggrin storage granules . profilaggrin is a large , insoluble protein consisting of a calcium - binding a domain , a b domain , and several tandem repeats of filaggrin units . in the transitional layer 4 ) , which aid in the bundling of keratin intermediate filaments and formation of the cornified envelope ( for review see candi et al . , 2005 ) . subsequently , filaggrin is deiminated ( conversion of arginine to citrulline by elimination of the imino group of arginine by peptidylarginine deiminases ) , causing its release from keratin and allowing its degradation into free hygroscopic amino acids that act as natural moisturizing factors of the stratum corneum ( scott and harding , 1986 ; rawlings and matts , 2005 ) . therefore , filaggrin plays an important role in skin hydration . although the filaggrin unit was detected in caspase-14deficient epidermis by western blot analysis , lower molecular weight filaggrin fragments were also present ( denecker et al . , 2007 ) . immunofluorescence analysis showed that in these mice , filaggrin immunoreactive fragments accumulated in the upper layers of the stratum corneum ( fig . this indicates that the correct degradation of filaggrin into free amino acids was affected in caspase-14deficient skin . interestingly , caspase-14 was found to be associated with keratohyalin granules in the stratum granulosum and to remain cytoplasmic in the stratum corneum ( alibardi et al . , 2004 ) , which could correlate with its possible involvement in the generation of free amino acids . expression of caspase-14 and ( pro)filaggrin in wild - type and caspase-14deficient skin . immunofluorescence staining for caspase-14 ( red ) and ( pro)filaggrin ( green ) on paraffin sections of 5.5-d - old skin of both wild - type ( + /+ ) and caspase-14deficient ( / ) mice ( denecker et al . , 2007 ) . fluorescence microscopy was performed on a cellm system ( olympus ) with an upright microscope ( bx61 ; olympus ) . a specific dapi emission band - pass filter ( 450470 nm ) and a gfp emission band - pass filter ( 510550 nm ) were used . image acquisition and processing were performed with the cellm software using a cooled ccd camera with a 1,344 1,024 pixel resolution . image intensity scaling and color conversion were completed in imagej ( national institutes of health ) . caspase-14 is expressed mainly in the spinous , granular , and cornified layers of wild - type mice and is absent in caspase-14deficient mice . ( pro)filaggrin is expressed in the granular layer and in the lower cornified layer in wild - type skin . in caspase-14deficient skin , additional filaggrin caspase-14 protects the skin against uvb photo damage and water loss and is involved in the processing of ( pro)filaggrin . caspase-14 expression starts in the spinous layer ( indicated in shades of red ) , and cleavage into its p20 and p10 subunits occurs at the transition of the granular to the cornified layer . caspase-14 is active in the dehydrating environment of the cornified layer , where it has an important function in formation of the epidermal barrier leading to protection against uvb and water loss ( denecker et al . , 2007 ) . profilaggrin is a large structural molecule consisting of an n - terminal a domain and a b domain followed by multiple filaggrin repeats and a unique c - terminal sequence ( for review see candi et al . , 2005 ) . profilaggrin undergoes many posttranslational modifications , eventually leading to release from the keratin intermediate filaments ( see the section on the function of caspase-14 for details ) . in the lower stratum corneum these amino acids compose 40% of the natural moisturizing factors present in the stratum corneum and are important for maintaining epidermal hydration ( rawlings and matts , 2005 ) . in caspase-14deficient skin , accumulating filaggrin fragments are present ( denecker et al . , 2007 ) , indicating that an unidentified protease ( asterisk ) cleaves the filaggrin monomer into these fragments and that caspase-14 is responsible for the further processing and degradation of these fragments into free amino acids . as it is very unlikely that caspase-14 is directly responsible for degradation of the filaggrin fragments into free amino acids , we propose two possible mechanisms : ( 1 ) caspase-14 could first cleave these filaggrin fragments , leading to further degradation into free amino acids by another endo- and/or exopeptidase ; or ( 2 ) caspase-14 could directly or indirectly ( by inactivating an inhibitor ) activate an endo- and/or exopeptidase that further processes the smaller filaggrin fragments . kg , keratohyalin granule ; kif , keratin intermediate filament ; nmf , natural moisturizing factors ; sb , stratum basale ; sc , stratum corneum ; sg , stratum granulosum ; ss , stratum spinosum ; tg , transglutaminase . these results , together with the finding that caspase-14 can directly cleave ( pro)filaggrin in vitro , demonstrate that caspase-14 has a critical role in the correct processing of ( pro)filaggrin during cornification . whether the degradation of other differentiation - associated proteins is also affected in caspase-14deficient mice first , caspase-14 may cleave the filaggrin fragments and , thereby , expose cleavage sites that can be recognized by other endo- and/or exopeptidases for further degradation . second , caspase-14 may be the activator of an endo- and/or exopeptidase that cleaves and degrades filaggrin . direct degradation of filaggrin fragments into free amino acids by caspase-14 can be ruled out , as caspases only cleave after aspartate residues . the lack of filaggrin processing into free hygroscopic amino acids in caspase-14deficient mice may lead to the reduced epidermal hydration and increased trans - epidermal water loss observed in these mice ( denecker et al . , 2007 ) . these results point to an important function of caspase-14 in the maintenance of epidermal hydration . although a profilaggrin - deficient mouse has not been generated , it has been demonstrated that flaky tail ( ft / ft ) mice , which have an autosomal recessive mutation in the flaky tail gene ( probably the profilaggrin gene ) , lack a functional filaggrin monomer ( presland et al . , 2000 ) . these mice have been proposed as a model for the filaggrin - deficient skin disease ichthyosis vulgaris because they have dry , flaky skin and irregular scales of variable size . the importance of filaggrin has been underscored recently by human genetic studies demonstrating that loss - of - function mutations in the profilaggrin gene are the primary cause of the skin disease ichthyosis vulgaris ( smith et al . , 2006 ) , which is characterized by silvery scales on the abdomen and palmar hyperlinearity . 2006 ) , possibly as a result of a defect in epidermal barrier function that allows the increased entry of allergens and infectious agents . two of the important functions of the skin are prevention of water loss and protection against environmental stress , such as protection against uvb radiation , which are essential for terrestrial life . the development of caspase-14deficient mice revealed that the absence of caspase-14 enhances sensitivity toward uvb - induced photo damage and apoptosis of the skin ( denecker et al . , 2007 ) . importantly , this is not caused by cell - autonomous differences in dna damage sensitivity and apoptosis between wild - type and caspase-14deficient keratinocytes . instead , the uvb - filtering capacity of the stratum corneum is severely reduced in caspase-14deficient skin , as higher levels of cyclobutane pyrimidine dimers are detected immediately after uvb irradiation . this indicates that caspase-14 has an indispensable role in the photoprotective function of the stratum corneum . interestingly , topical application of egcg , an inducer of caspase-14 expression , has been shown to be photoprotective ( elmets et al . , 2001 ) . how caspase-14 alters the structural and biochemical properties of the stratum corneum is currently under investigation . caspase-14 was shown to be expressed at the protein level in several cancer cell lines ( pistritto et al . , 2002 ; koenig et al . , 2005 ; krajewska et al . , 2005 ) . in addition , caspase-14 mrna , together with keratin 1 and profilaggrin mrna , was decreased in murine uvb - induced squamous cell carcinoma , possibly reflecting reduced differentiation in the tumor ( rundhaug et al . , 2005 ) . furthermore , in some cases , caspase-14 protein expression was associated with highly differentiated cornified areas of lung squamous cell carcinoma and cervix carcinoma ( koenig et al . , 2005 ) . however , caspase-14 activation in tumors has not been shown , and so it might not be responsible for the tumor phenotype . presumably , the ectopic caspase-14 expression is caused by the changed transcriptional activity in these epithelial tumors . mutations in the caspase-14 gene have not been found in human carcinomas except very rarely in colorectal tumors , which most probably were not the cause of altered caspase-14 expression ( koenig et al . , 2005 ; we as well as other investigators demonstrated that caspase-14 expression is substantially down - regulated in psoriatic lesions but is unaffected in the nonlesional epidermis ( lippens et al . , 2000 , 2004 ; psoriasis is an autoimmune disease characterized by the uncontrolled proliferation of keratinocytes and impaired cornification , which results in the aberrant presence of nuclei in the cornified layer , also called parakeratosis . although caspase-14 is absent in these parakeratotic regions , this is probably not the cause of the development of parakeratotic plaques , as caspase-14deficient mice did not show spontaneous parakeratosis ( denecker et al . , 2007 ) . more likely , caspase-14 down - regulation results from the impairment of terminal differentiation or up - regulation of transcriptional repressors . the absence of caspase-14 in psoriatic plaques may lead to the formation of a defective barrier and , therefore , to the aggravation of psoriatic lesions . treating the parakeratotic plaques of patients with a vitamin d3 analogue results in the up - regulation of caspase-14 and likewise , in the flaky skin ( fsn / fsn ) mouse model of psoriasis , topical egcg treatment causes the up - regulation of caspase-14 and the amelioration of psoriasis ( hsu et al . , 2007 ) . interestingly , the expression of junb is strongly down - regulated in psoriatic lesions , and inducible epidermal deletion of both junb and c - jun in mice results in a psoriatic phenotype ( zenz et al . , 2005 ) . because caspase-14 might be regulated by these transcription factors , it would be interesting to elucidate whether caspase-14 is down - regulated in junb / c - jun deficient mice . recent evidence sheds light on the crucial role of caspase-14 in the skin , but several major questions remain . activation of caspase-14 occurs most probably at the interface between the granular and cornified layer , implicating a role for caspase-14 in the stratum corneum . it is now clear that the caspase-14activating protease is not a caspase but probably an epidermis - specific serine protease with elastase - like properties . this is not surprising , as it has been known for a long time that serine proteases are of major importance in epidermal homeostasis . determination of the in vitro conditions for caspase-14 activity that mimic stratum corneum conditions , together with the generation of caspase-14deficient mice , led to the identification of ( pro)filaggrin as the first known physiological caspase-14 substrate . importantly , caspase-14 seems to be involved in the correct processing of filaggrin preceding its degradation into free hygroscopic amino acids , which might explain its role in the prevention of water loss from the epidermis . proteomic approaches could lead to the identification of additional caspase-14 substrates , which would contribute to understanding the role of caspase-14 in the skin . caspase-14 also protects against uvb - induced damage , which means that it is involved in the establishment of the biochemical or structural properties of the stratum corneum as a uvb filter . how caspase-14 establishes the uvb - filtering capacity of the corneum is not completely understood . an extensive biochemical analysis of caspase-14deficient epidermis could reveal these mechanisms . whether caspase-14 , its activating protease , or its substrates could be used as therapeutic agents or as targets to improve formation of the epidermal barrier is a challenging research goal .

caspase-14 is a unique member of the evolutionarily conserved family of cysteinyl aspartate specific proteinases , which are mainly involved in inflammation and apoptosis . however , recent evidence also implicates these proteases in proliferation and differentiation . although most caspases are ubiquitously expressed , caspase-14 expression is confined mainly to cornifying epithelia , such as the skin . moreover , caspase-14 activation correlates with cornification , indicating that it plays a role in terminal keratinocyte differentiation . the determination of in vitro conditions for caspase-14 activity paved the way to identifying its substrates . the recent development of caspase-14deficient mice underscored its importance in the correct degradation of ( pro)filaggrin and in the formation of the epidermal barrier that protects against dehydration and uvb radiation . here , we review the current knowledge on caspase-14 in skin homeostasis and disease .

gossypiboma ( retained surgical sponge ) describes a mass of cotton or sponge that is left behind in the body cavity during a surgical operation . these foreign bodies can often mimic tumors or abscesses when detected clinically or with radiation . in fact , surgical sponges without opaque markers are the leading cause for the difficulty of a correct diagnosis . here , we present a case in which a foreign body , gauze without radiopaque markers , was left behind during a procedure that involved the removal of an intrapelvic tumor . after a computed tomography ( ct ) scan had revealed information leading to an inaccurate diagnosis , an accurate diagnosis of gossypiboma was successfully made using endoscopic ultrasound - fine - needle aspiration ( eus - fna ) . a 60-year - old female patient was referred with an unexpected detection of an intrapelvic tumor during a routine ct scan . the patient 's medical history included a hysterectomy and a left side ovariectomy at the age of 27 . the ct scan revealed a 45 mm tumor in the patient 's pelvis [ figure 1a and b ] , which was diagnosed as a gastrointestinal stromal tumor ( gist ) , a chronic expanding hematoma or leiomyoma . a forward - view eus ( tgf - uc260j , olympus optical co. , tokyo , japan ) identified a low echoic lesion when scanned from the sigmoid colon . fna was performed with a 22-gauge needle ( ezshot2 , olympus optical co. , tokyo , japan ) by using the forward - view eus [ figure 2 ] . examination of the fna specimen revealed a foreign body ( string of gauze ) displaying macrophage cells and necrotic tissue [ figure 3 ; hematoxylin and eosin , original magnification 40 ] . during the surgery , histological examination of the tissue adjacent to the foreign body revealed granuloma formations with fragmented silken threads [ figure 4 ; hematoxylin and eosin , original magnification , 100 and 200 ] . a : sagittal section ( black arrow ) ; b : horizontal section ( black arrow ) . endoscopic ultrasound ( eus)-fine - needle aspiration using forward - view eus a foreign body ( string of gauze ) obtained using the 22-gauge needle , with macrophage cells and necrotic tissue granuloma formations with fragmented silken threads gossypiboma describes a mass of cotton or a sponge that is retained in the body after a surgery . gossypiboma causes foreign body reactions of the surrounding tissue since they are inert and show no specific decomposition . in this case , radiopaque sponges were not used , and there were no adverse symptoms in the 30 years postsurgery . the typical appearance of gossypiboma shows a spongiform pattern with gas bubbles . in typical cases , however , it can be difficult to diagnose the disorder with ct scans in cases without gas bubbles . in the case described in this paper , radiologists using findings from a ct scan , initially and incorrectly diagnosed the lesion as gist with fibrosis . however , the disorder was correctly diagnosed by a team of gastroenterologists using eus - fna . the eus - fna procedure revealed a segment of string in a fixed foreign body in the pelvis . the application of eus using curved linear array ( cla ) endoscopes in the lower gi tract has been limited to the rectum and distal sigmoid colon because of the oblique - viewing optics . there were case reports using oblique - viewing cla echoendoscopes to evaluate lesions proximal to the sigmoid colon , but advancement of the cla echoendoscope beyond the sigmoid required the use of an overtube or a guide wire previously placed with a colonoscope . binmoeller et al . have reported the feasibility and safety of the front - view forward - array echoendoscope for evaluating right colon lesions . thus , we used a front - view forward - array echoendoscope to safely evaluate the intrapelvic tumor and sample the tissue . a correct diagnosis was obtained using this method . in summary , this is the first known case report diagnosing gossypiboma by eus - fna .

gossypiboma is a term used to describe a foreign mass within the body that is composed of a cotton matrix . gossypiboma can cause infection or abscess formation from an early stage , or it can remain clinically silent for many years . gossypiboma can be diagnosed using several methods including abdominal x - ray films , us of the abdomen , contrast - enhanced computed tomography of the abdomen , and during surgery . however , the variable appearance of gossypiboma can lead to diagnostic misinterpretations . here , we report a successful diagnosis of gossypiboma using a novel method , that is , endoscopic ultrasound - fine - needle aspiration .

to demonstrate the usefulness of enhanced depth imaging optical coherence tomography ( edi - oct ) in investigating choroidal lesions inaccessible to ultrasound sonography . in a 60-year - old woman with an asymptomatic choroidal nevus , normal oct was used to observe the macula and edi - oct to image the choroidal nevus that was inaccessible to ultrasound . the exact location of the lesion in the choroid and the dimensions of the nevus were measured . the lesion was located in the superior macula , and the nevus was homogeneous in its reflectivity . we observed a thickened choroid delineated by the shadow cone behind it , measuring 1,376 325 m in the larger vertical cut and 1,220 325 m in the larger horizontal cut in an image with a 1:1 pixel mapping and automatic zoom . edi - oct appears to be an excellent technique for measuring choroidal nevi and all choroidal lesions accessible to oct imaging by depicting their exact location in the choroid , their dimensions , and their demarcation from the surrounding healthy tissue , thus allowing for a more efficient and accurate follow - up . choroidal nevus is the most common tumor of the ocular fundus , present in 7% of the white population [ 1 , 2 ] . despite its benign nature , it presents a risk of visual loss and visual field loss , and can rarely transform into malignant melanoma . optical coherence tomography ( oct ) and ultrasonography have been used for imaging choroidal nevi . recently , enhanced depth imaging oct ( edi - oct ) has been used in order to measure choroidal thickness in normal and pathologic eyes [ 3 , 4 ] . here , we report a case of a flat choroidal nevus inaccessible to ultrasound sonography that was evaluated by edi - oct . a 60-year - old woman was referred to our department with an asymptomatic choroidal nevus in her left eye . her best - corrected visual acuity was 20/20 in both eyes . slit lamp and fundus examinations of the right eye were normal . in the left eye , the anterior segment was normal ; however , on fundoscopy a choroidal nevus located in the superior macula was observed ( fig . spectral - domain ( sd ) oct ( heidelberg engineering , heidelberg , germany ) revealed normal foveal thickness ( fig . 2 ) . the flat nevus was inaccessible to ultrasound sonography , and edi - oct was used to image its posterior surface ( fig . edi - oct is a new technique that consists of positioning a heidelberg sd - oct close enough to the eye in order to obtain an inverted image , averaged for 100 scans . the advantage of this technique is that the sensitivity of the imaging in deeper tissue layers is increased , and thus the obtained measurements are more accurate . we employed this technique to image the location of the nevus in the choroid and obtain its dimensions as a reference measure for the follow - up examination ( fig . 3 ) . heidelberg sd - oct provides an exact correlation between the oct image and the infrared photograph . as infrared light is absorbed by melanin , the nevus appears brighter . in the edi - oct image , the lesion appears homogeneous and hyperreflective at the level of the large choroidal vessels , masking the underlying choroidal vasculature . the choroidal thickness at the level of the nevus appears greater than the neighbor choroid ( fig . the diameter of the nevus was calculated based on the presence of a shadow cone created by the lesion 's lateral borders . the choroidal thickness was measured based on the hyperreflectivity between the bruch 's membrane and the beginning of the shadow cone ( fig . the choroid measured 1,220 325 m in the larger horizontal cut and 1,376 325 m in the larger vertical cut in an image with a 1:1 pixel mapping and automatic zoom ( fig . edi - oct appears to be an excellent technique for evaluating flat choroidal nevi that are inaccessible to ultrasound sonography and accessible to oct imaging , displaying simultaneously the lesion and the retinal changes . it is a reproducible examination that allows depiction of the exact location of the nevi in the choroid , their dimensions and their demarcation from the surrounding healthy tissue . to date , the software available for sd - oct does not allow for objective measures . the subjective measures obtained are useful upon comparison with the follow - up images . in order to limit the possibility of bias , these follow - up images must be obtained and compared with the same sd - oct machine . we believe that this technique is clinically useful and , together with color fundus photography , can provide a more efficient and accurate follow - up of choroidal nevi .

purposeto demonstrate the usefulness of enhanced depth imaging optical coherence tomography ( edi - oct ) in investigating choroidal lesions inaccessible to ultrasound sonography.methodsin a 60-year - old woman with an asymptomatic choroidal nevus , normal oct was used to observe the macula and edi - oct to image the choroidal nevus that was inaccessible to ultrasound . the exact location of the lesion in the choroid and the dimensions of the nevus were measured.resultsthe lesion was located in the superior macula , and the nevus was homogeneous in its reflectivity . we observed a thickened choroid delineated by the shadow cone behind it , measuring 1,376 325 m in the larger vertical cut and 1,220 325 m in the larger horizontal cut in an image with a 1:1 pixel mapping and automatic zoom . the macular profile and thickness were both normal.conclusionsedi-oct appears to be an excellent technique for measuring choroidal nevi and all choroidal lesions accessible to oct imaging by depicting their exact location in the choroid , their dimensions , and their demarcation from the surrounding healthy tissue , thus allowing for a more efficient and accurate follow - up .

kelch - like ech - associated protein 1 ( keap1)-nuclear factor e2-related factor 2 ( nrf2 ) is a major cellular pathway that protect normal cells against oxidative and xenobiotic damage.1 nrf2 is an essential transcription factor for antioxidant and detoxification genes and is crucial for the chemopreventive effect of various phytochemicals against carcinogenesis . representative chemopreventive agents that induce nrf2 include carotenoids , curcumins , cyclic lactones , diterpenes , dithiolethiones , epithionitriles , flavonoids , indoles , isothiocyanates , organosulfides , and phenols.2,3 accumulating evidences have demonstrated that phytochemicals can protect cells from oxidative stress related diseases including inflammatory , cardiovascular , and neurodegenerative diseases and cancer , through nrf2-dependent responses . paradoxically , recent researches demonstrated the dark side of nrf2.4 cancer cells acquire a growth advantage by eliminating keap1-mediated negative control of nrf2 , which leads to activation of the nrf2-dependent defense response.4 constitutive upregulation of nrf2 has been found in many types of cancers , including skin , breast , prostate , lung , head and neck , and endometrial cancer.5 overexpressed nrf2 provides a growth advantage for cancer cells by protecting those cells from oxidative stress and anticancer agents , thus contributing to chemoresistance.5 in this review , we summarize the therapeutic development of nrf2 inhibitors that enhance the efficacy of the current cancer treatments . nrf2 is a basic leucine zipper transcription factor with 7 domains from neh1 to neh7 . neh1 has dna binding motifs.6 neh3 , neh4 , and neh5 have domains involved in the transactivation of nrf2 target genes by binding coactivators . neh2 has a major regulatory domain with 2 binding sites for keap1 named etge and dlg ( fig . keap1 has 5 functional domains including kelch , intervening region ( ivr ) , broad complex / tramtrack , bric - a - brac domain ( btb ) , an n - terminal region ( ntr ) , and a c - terminal region ( ctr).3 cysteine residues of ivr play as a sensor for reactive oxygen species ( ros ) . the btb domain has a cys151 residue and is responsible for nrf2 ubiquitination through binding to cullin e3 ubiquitin ligase ( cul3)-based ubiquitin e3 ligase ( fig . 1a ) . under unstressed conditions , nrf2 forms a complex with keap1 , leading to polyubiquitination and subsequent degradation by the 26s proteasome ( fig . 2a).7 upon exposure to a variety of stressors , including ros , toxicants , and carcinogens , nrf2 is released due to modification of the cysteine residues of keap1 ( fig . 2b).8 two mechanistic models have been proposed for the regulation of nrf2 : the hinge and latch model and the keap - cul3 dissociation model.810 in the hinge and latch model , 2 binding motifs of the neh2 domain in nrf2 have different affinities.9 for the ubiquitination , both the dlg and etgf motifs should be occupied by keap1 proteins . ros modify the cysteine residues of keap1 , leading to the release of the dlg motif ( latch ) without changing the etgf motif ( hinge ) on neh2 . another model is the keap - cul3 dissociation model in which oxidative stress disrupts the keap1-cul3 e3 ligase interaction without changing the conformation of keap1.8 nrf2 stabilization with de novo synthesis increases the cytoplasmic level of nrf2 . nrf2 translocates into the nucleus and binds to an antioxidant response element ( are ) as a heterodimer with musculo - aponeurotic fibrosarcoma ( maf ) to recruit a transcriptional coactivator and promote thetranscription of various cytoprotective genes that are associated with antioxidant and detoxification enzymes . upon recovery of cellular redox homeostasis , keap1 translocates to the nucleus to dissociate nrf2 from the are resulting in degradation of nrf2.11 nrf2 binds to ares in promoter regions of various genes that regulate the cellular response to oxidative and xenobiotic stress . nrf2 target genes include antioxidant genes and phase ii enzymes such as heme oxygenase-1 ( ho-1 ) , nad(p)h : quinone oxidoreductase 1 ( nqo1 ) , glutathione s - transferase ( gst ) , and glutathione peroxidases . the are - bearing genes that are regulated by nrf2 in humans have been studied using microarray analysis of genes induced by chemopreventive agents or knockout of keap1 in human cell lines.2 these genes can be categorized into 5 groups : antioxidant genes ( gclc , gclm , gpx2 , gsr , slc7a11 , srxn1 , and txnd1 ) , nadph - generating enzymes ( g6pd , me1 , and pgd ) , metal - binding proteins ( fth1 , ftl , mt1 , and mt2 ) , drug - metabolizing enzymes and drug transporters ( akr1b1 , akr1b10 , akr1c1 , akr1c2 , akr1c3 , akr1c4 , cbr1 , gstm3 , mrp2 , nq1 , and ptgr1 ) , and stress response proteins ( gadd45 , hmox1 , hsp40 , and hsp70).2 knockdown of keap1 in human keratinocytes resulted in upregulation of 23 mrnas while akr1b1 , akr1b10 , and akr1c1 were induced to the greatest extent , showing increases of between 12- and 16-fold indicating that aldo - keto reductases could be useful biomarkers for nrf2 activation.12 as keap1 is responsible for inhibitor kappa b ( ib ) kinase beta ( ikk- ) ubiquitination , upregulation of some of these genes could be attributable to the increase of nuclear factor - kappa b ( nf-b ) activity.13 expression profiling in mice has revealed that nrf2 regulates approximately 100 genes and that two - thirds of nrf2-target genes are not involved in detoxication or antioxidation . instead , many of the target genes are associated with inflammation and immunity proteins.2 the aryl hydrocarbon receptor ( ahr ) is a transcription factors that mediates the biological effects of its xenobiotic ligands including dioxin.14 the ahr activates the transcription of nqo1 and other genes through the xenobiotic - responsive element ( xre).15 ahr induces the direct binding of nrf2 to promoters or indirectly produces reactive intermediates that trigger the nrf2 signaling pathway.16 the cross - talk between nf-b and nrf2 is more complex . several chemopreventive agents , such as sulforaphane , epigallocatechin-3-gallate , and curcumin , induce nrf2 signaling with concomitant repression of nf-b.14 the nf-b p65 subunit re - presses the nrf2-are pathway by preventing creb binding protein ( cbp ) from binding nrf2 through competitive interaction or by promoting the recruitment of histone deacetylase 3 ( hdac3 ) , a corepressor , to the are.17 in chondrocyte apoptosis , shear - induced cyclooxygenase-2 , an nk-b target gene , can suppress phosphatidyl inositol 3-kinase ( pi3k ) activity , causing a reduction in thenrf2-mediated transcriptional response.18 in contrast , nrf2 inhibits the activity of nf-b by attenuating phosphorylated ib . nrf-2 target genes , including ho-1 have been reported to negatively affect nf-b activation.19,20 p53 has been reported to suppress the nrf2-dependent transcription of antioxidant response genes by directly interacting with are - containing promoters . previous studies have suggested that the function of this negative regulation is to prevent the formation of antioxidant environment , which could hinder the induction of apoptosis by p53.21 in contrast , direct targets of p53 could upregulate nrf2 activity . studies have shown that p21 inhibits the degradation of nrf2 by interacting with the dlg motif , leading to upregulation of antioxidant genes.22 these dual functions of p53 toward nrf2 indicate that p53 may decide the fate of damaged cells whether to repair the damaged cells or apoptosis . nrf2 is also involved in autophagy by inducing p62 which is a receptor for autophagic degradation . as p62 could block binding between nrf2 and keap1 , p62 contributes to the activation of nrf2 target genes in response to oxidative stress creating a positive feedback loop.23 the association of nrf2 overexpression and chemoresistance has been reported in many cancers including non - small cell lung cancer , stomach cancer , endometrial cancer , and osteosarcoma.2427 overexpression of nrf2 target genes was also found in a variety of solid tumors.28 ho-1 overexpression was found in brain cancer , prostate cancer , and renal cancer.29,30 nqo1 is known to be overexpressed in hepatoblastoma , colon cancer , breast cancer , and non - small cell lung cancer.31 several mechanisms have been proposed for the activation of keap1-nrf2 signaling in cancer . mutation of keap1 can result in accumulation of nrf2 in nucleus through decreased degradation or inhibition of nuclear export.28 in non - small cell lung cancer , heterozygous mutation of keap1 ( 8% of patients ) was reported to be sufficient for nrf2 overexpression.32 in addition to lung cancer , mutations in the keap1 gene were found in various cancers including breast ( 2% ) , colon ( 8% ) , gastric ( 11% ) , liver ( 9% ) , ovary ( 19% ) , and prostate ( 8%).3336 mutations in the neh2 domain of nrf2 were found in lung ( 11% ) , cancer ( 6% ) , and head and neck cancers ( 25%).37,38 all of these mutations were somatic mutations . mutation of egfr , kras , braf , myc , and the bcr - abl fusion can activate nrf2 , resulting in enhancement of ros detoxification and other oncogenic roles of nrf2 , including chemoresistance.3941 furthermore , posttranslational modifications can activate the keap1-nrf2 signaling pathway . hypermethylation of the keap1 promoter was found in 47% of lung cancer patients and this feature was associated with poor outcome.42 epigenetic regulation of keap1 was also found in malignant glioma , colon cancer , and breast cancer.4345 in addition to the upregulation of cytoprotective genes , constitutive expression of nrf2 may confer a survival advantage to cancer cells by promotion of cell proliferation , chemoresistance and inhibition of apoptosis . an active pi3k - akt pathway augments the nuclear accumulation of nrf2 which then re - directs glucose into the anabolic pathway to increase metabolism , indicating the reinforcement of metabolic reprogramming by nrf2.46 nrf2 could mediate cell proliferation with dual regulation through epidermal growth factor receptor ( egfr ) signaling and keap1 interactions.39 in cells with the keap1 gene mutation ( a549 cells ) , activated nrf2 promotes cell proliferation independent of egfr signaling . therefore , egfr tyrosine - kinase inhibitors are intrinsically ineffective in these types of non - small cell lung cancer.39 cancer metastasis and tumor progression requires the epithelial - mesenchymal transition ( emt ) and the loss of e - cadherin is considered to be a main event in emt . in hek293 cells , complex of e - cadherin and beta - catenin could bind to c - terminus of nrf2 preventing nuclear translocation of nrf2.47 as e - cadherin inhibits nrf2-mediated transcription , loss of e - cadherin could promote nrf2 translocation and confer an additional survival advantage to cancer cells.47 the keap1-nrf2 pathway is involved in the inhibition of apoptosis by interacting with p53 and b cell lymphoma-2 ( bcl-2 ) . p53 inhibits the activation of nrf2 target genes by direct interacting with are - containing promoters or activating p21.21 considering that p53-induced apoptosis requires the accumulation of ros , increased activity of antioxidant genes by nrf2 in cancer cells can inhibit p53 dependent apoptosis . bcl-2 can repress cell death by dimerization with bcl-2-associated x protein ( bax ) and the bcl-2 homology 2 ( bh2 ) domain of bcl-2 is required for this heterodimerization.48 it was found that keap1 binds to the bh2 domain and facilitates the ubiquitination of bcl-2 leading , to bax accumulation and enhanced apoptosis . thus , mutations in the keap1 binding site for bcl2 are responsible for the anti - apoptotic effect as well as overexpression of nrf2.49 moreover , it has been reported that nrf2 can directly activate the transcription of bcl-2 and bcl - xl.50,51 many studies have reported the association between nrf2 upregulation and chemoresistance in various cancers , including gastric cancer , osteosarcoma , non - small cell lung cancer , endometrial cancer , bladder cancer , and neuroblastoma.2427,52 platinum drugs generate electrophilic molecules that damage cancer cells . chemoresistance to these drugs can be explained by high expression of antioxidant nrf2 target genes.4 another mechanism of chemoresistance is the induction of the drug efflux pump family , which includes the mdr , by nrf2.53 some drugs , including hdac inhibitors , oxaliplatin , and proteasome inhibitors can induce nrf2 and thus decrease the effectiveness of chemotherapy.5456 in light of the data presented in this review , nrf2 is an attractive molecular target for the inhibition of cancer . in contrast to nrf2 activators , including numerous phytochemicals , only a small number of nrf2 inhibitors have been identified ( table ) . trigonelline , a coffee alkaloid , efficiently decreased basal and tertiary butylhydroquinone ( tbhq)-induced nrf2 activity in chemoresistant pancreatic carcinoma cell lines ( panc1 , colo357 , and miapaca2 ) which have high nrf2 activity . along with inhibiting nrf2 the sensitivity of all cell lines to anticancer drugs and tumor necrosis factor - related apoptosis inducing ligand ( trail)-induced apoptosis was enhanced by trigonelline.57 chrysin ( 5,7-dihydroxyflavone ) is a natural flavonoid that is found in many plant extracts . in doxorubicin resistant hepatocellular carcinoma cell line ( bel-7402/adm ) , chrysin significantly reduced nrf2 expression by downregulating the pi3k - akt and erk pathways.58 in that study , chrysin restored chemosensitivity by downregulating the nrf2-downstream genes such as ho-1 , akr1b10 , and mrp5 . apigenin ( 4,5,7-trihydroxyflavone ) is a natural flavone that is present in many fruits and vegetables and has various biological activities , such as anti - inflammatory and antioxidant properties . in a study using bel-7402/adm cell lines , apigenin reduced nrf2 expression as well as the expression of nrf2-downstream genes . when cells were treated with doxorubicin , apigenin showed a synergistic anti - tumor effect on bel-7402/adm cell lines , resulting in reduced cell proliferation and more substantially induced apoptosis.59 brusatol is a quassinoid that is extracted from brucea javanica . brusatol reduced nrf-2 protein level without changing the keap1 level and sensitized various cells including hela , mda - mb-231 , and a549 to chemotherapeutic agents such as carboplatin , 5-fluorouracil , etoposide , and paclitaxel.60 the anticancer effect of brustalol mediated by nrf2 was confirmed in a xenograft model indicating that brusatol could be used to combat intrinsic resistance . ascorbic acid is an antioxidant that can suppress the level of nrf2 . in the imatinib - resistant chronic myelogenous leukemia cell line kcl22/sr , binding of nrf2 to the are of the gamma - glutamylcysteine synthetase gene promoter was much stronger than in the parental imatinib - sensitive cell line kcl22.61 furthermore , addition of ascorbic acid to kcl22/sr cells resulted in a decrease in nrf2-dna binding and partial restoration of imatinib sensitivity to kcl22/sr.61 luteolin ( 3,4,5,7-tetrahydroxyflavone ) is a polyphenolic flavonoid found in high concentrations in celery , green pepper , and parsley . in a study using a cell - based are - reporter assay , luteolin was found to be a potent nrf2 inhibitor.62 in that study , luteolin significantly sensitized a549 cells to the anticancer drugs oxaliplatin , bleomycin , and doxorubicin indicating the potential application of luteolin as a natural sensitizer in chemotherapy . constitutively expressed nrf2 can promote cancer cell proliferation and protect cells against oxidative stress and therapeutic agents . it must be kept in mind that chemopreventive agents including various phytochemicals can induce chemoresistance and tumor progression by activating the keap1-nrf2 pathway . however , it will be necessary to understand the molecular regulation of nrf2 and identifying the individualized status of nrf2 expression .

nuclear factor e2-related factor 2 ( nrf2 ) is a transcription factor that upregulates expression of a battery of genes to combat oxidative and electrophilic stress . modification of kelch - like ech - associated protein 1 ( keap1 ) by reactive oxygen species stabilizes nrf2 by escaping from degradation . nrf2 then binds to antioxidant response elements ( ares ) on the promoter region of various genes . activation of the keap1-nrf2-are pathway plays critical roles in the chemopreventive effect of various phytochemicals . however , nrf2 can protect cancer cells from oxidative stress and promote cell proliferation . moreover , recent studies reveal that activation of the nrf2 pathway is critical for resistance to chemotherapeutic agents . the aim of this review is to provide a molecular basis for the use of nrf2 inhibitors in overcoming chemoresistance .

a 3-year - old male , known to have isolated speech delay , presented to the emergency department with severe body aches and inability to walk . few days prior to his presentation , the child started to have flu - like symptoms in the form of rhinorrhea and low - grade fever . this was followed by irritableness , hypoactivity , and inability to move his legs and arms . he was seen in a private hospital where computed tomography of the brain and a lumber puncture were done that showed no abnormalities . in the next day , his condition continued to worsen . he developed difficulty in swallowing and dark - colored urine , so he was transferred to our institution . upon physical examination , the laboratory tests showed elevated creatine kinase level ( 1 778 856 u / l ; normal range 20 - 200 u / l ) , elevated serum transaminases ( serum glutamic - pyruvic transaminase [ sgpt ] = 1857 u / l [ normal range 0 - 50 u / l ] , serum glutamic - oxaloacetic transaminase [ sgot ] = 8626 u / l [ normal range 0 - 50 u / l ] ) , and creatinine 0.72 mg / dl ( 0.3 - 0.7 mg / dl ) . the child started on intravenous hydration and received 1 dose of intravenous immunoglobulin . on the second day , the creatine kinase and serum transaminase levels improved , with creatinine reaching 0.94 mg / dl , the highest level reached . the creatine kinase and serum transaminase levels continued to improve gradually over the following days , with slow improvement in the clinical condition . he received another 2 doses of intravenous immunoglobulin on days 13 and 14 of admission . muscle biopsy showed endomysial inflammatory infiltrates , mostly composed of cd8 and cd4 lymphocytes ; myofibers necrosis mostly ventral portion of myofibers ; residual granular microcalcifications ; and terminal complement complex deposits that outline necrotic myofibers , all features compatible with necrotic myopathy and early rhabdomyolysis ( figure 2 ) . the arrows showed the degenerated myofibers . upon discharge , the child had mild muscle aches , was able to sit , and move his arms and feet . he had persistent weakness in the lower limbs , but was unable to walk , with a follow - up examination 1 month after discharge , revealing that the child was able to walk normally with positive deep tendon reflex . two months after discharge , the creatine kinase level dropped to 193 iu / l . rhabdomyolysis results from skeletal muscle breakdown due to various causes . viral myositis , trauma , and inherited disorders are the most common causes in the pediatric age group . the breakdown releases the normal cell contents into the bloodstream , including creatine kinase , myoglobin , phosphorus , and potassium . rhabdomyolysis is suspected clinically when muscle aches , muscle weakness , and tea - colored urine are present . the diagnosis is based on the high creatine kinase level , which is the most sensitive marker . although there is no established cutoff levels , a concentration 5 to 10 times higher than the upper limit of normal reference range ( ie , 500 - 1000 other laboratory indicators include serum and urine myoglobin concentrations that may be useful but not essential for the diagnosis . the most common complication of rhabdomyolysis is kidney damage . in recent large pediatric studies reported by mannix et al and wu et al , it was shown that the rate of acute renal failure in the pediatric patients with rhabdomyolysis ranged from 5% to 8.7% . the risk factors that predispose a patient with rhabdomyolysis for developing acute renal injury are creatine kinase level concentration more than 5000 u / l , creatine kinase value upon admission , and slower decline in the serum creatine kinase level . other factors include high myoglobin level , persistent or abrupt increase in the potassium or calcium level , as well as persistent metabolic acidosis . on the other hand , a recent study by fernandez et al showed that the most reliable predictor of acute renal failure and the need for dialysis is the creatinine level above 1.7 , despite the peak creatine kinase . the mainstay for the prevention and treatment of acute kidney injury is early and aggressive volume resuscitation . other management options include alkalization of the urine , forced diuresis with mannitol , and loop diuretics . in severe cases or in our case , the child developed extremely high creatine kinase levels that were not reported in the literature . a thorough literature review was done searching for high creatine kinase levels in patients with acute rhabdomyolysis . in 2014 , a case report described a pediatric patient with mcardle disease who developed rhabdomyolysis with a high 6-digit creatine kinase level reaching 500 000 2 pediatric cases were reported . in both , the creatine kinase levels reached were 60 000 a case report of an adolescent with rhabdomyolysis due to undiagnosed hypothyroidism was reported with a creatine kinase level that reached around 34 000 a research article in 2013 examined the clinical spectrum of patients with rhabdomyolysis presenting to the pediatric emergency department , in which the peak serum level of creatine kinase was 9825.1 23 079.1 the only risk factor for developing acute renal injury that our patient had was a high peak creatine kinase level upon admission . on the other hand , he did not develop metabolic acidosis and had a normal albumin and electrolytes levels throughout his hospitalization . he responded very well to volume resuscitation with rapid decline in the creatine kinase level . although he received intravenous immunoglobulin for the fear of guillain - barre syndrome or other underlying immune - mediated diseases , neither the creatine kinase level decline nor the clinical status improvement was affected by the intravenous immunoglobulin administration . the sharp decline in the creatine kinase level there are no clues in the family history or the patient s personal history that may suggest an underlying inherited metabolic , muscular , or genetic disease . , this could be the first presentation of lipin-1 mutation that causes recurrent rhabdomyolysis in children . lipin-1 gene encodes the muscle - specific phosphatidic acid phosphatase , a key enzyme in triglyceride and membrane phospholipid biosynthesis . the episodes of rhabdomyolysis are mostly triggered by intercurrent infections and fever and to a lesser extent by fasting or exercise . the prognosis of lipin-1 deficiency is poor , with up to one - third of patients dying during an episode of rhabdomyolysis . in about 60% of patients with recurrent rhabdomyolysis ,

rhabdomyolysis is an acute life - threatening condition that can occur in childhood secondary to many causes . the authors report the case of a 3-year - old male child who presented with acute rhabdomyolysis . the peak plasma creatine kinase level was extremely high . the 2 main causes of rhabdomyolysis in childhood are viral myositis and trauma , which can sometimes lead to acute renal failure . the highest creatine kinase levels reported in the literature so far was a 6-digit level in 2014 case report . in this study , the authors report the case of a 7-digit creatine kinase level in a child secondary to viral myositis who did not require renal dialysis .

the study was approved by the institutional ethics committee , and informed consent was obtained from all patients in accordance with the tenets of the declaration of helsinki . the donors were selected based on these inclusion criteria : myopic spherical refractive error ranging from 0.5 d to 10.00 d with no astigmatism or astigmatism less than 0.5 d ; healthy eyes free from significant systemic and ocular diseases ; and ability to understand the content of the consent form and willingness to undergo serological testing . eligible candidates underwent the relex smile procedure with the standard technique11 by the same surgeon ( s.g . ) using the visumax fs laser . the cap thickness was 100 m , and the optical zone varied from 6 to 6.5 mm . after dissection of both anterior and posterior planes , the lenticule was extracted through a superior 2-mm incision . bites were taken from one end to the diagonally opposite end , with the knot tied on the superior aspect of the lenticule to determine the side at the time of subsequent implantation . the lenticule was immediately transferred to a sterile plastic vial containing phosphate - buffered solution , labeled with the patient 's identification number , eye , and date , and sent to the cryobank for processing and cryopreservation . two types of solutions ( washing solution and cryopreservation solution ) were prepared . the washing solution comprised 10 ml of phosphate - buffered saline ( 1 ) , 200 l of penicillin streptomycin ( 100 ) , and 200 l of amphotericin b ( 100 ) . the cryopreservation solution comprised 9 ml of tissue culture media ( cryobank wash media ) and 1 ml of 20% dimethyl sulfoxide ( dmso ) . the tissue culture medium is a commercially available medium used for semen processing in reproductive biology ( cryocell india pvt ltd , new delhi , india ) . the medium contains sodium chloride , potassium chloride , magnesium sulfate , anhydrous potassium phosphate monobasic , calcium chloride , anhydrous sodium bicarbonate , hepes , glucose , sodium pyruvate , sodium lactate , phenol red , and human albumin in company - specified compositions . extracted lenticules were washed twice for 10 minutes each in the washing solution ( fig . the lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . using a liquid dropper , a stock freezing solution containing 10% human tissue culture medium and 20% dmso ( sigma - aldrich , st louis , mo ) was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso . the cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers ( ibp ; indian oil corporation limited , nasik , india ) . , the tissue was transported from the cryobank to the hospital in a liquid nitrogen container . the vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied . the lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents . a visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision . the incision was opened with a seibel spatula and the plane of the pocket was dissected . the cryopreserved lenticule ( which was matched for the refractive error after correcting for back vertex distance ) was placed on the patient 's cornea with its center marked with gentian violet dye . after insertion , the lenticule was spread out , and the center was aligned with the pupillary center . a depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes , the novelty of the nomograms , and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required . postoperatively , a topical steroid ( 1% prednisolone acetate ; allergan , irvine , ca ) was prescribed for 3 months in a tapering dosage . in addition , 5% hypertonic saline drops ( hypersol-5 ; jawa pharmaceuticals , india ) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing . patients were examined postoperatively on days 1 and 15 and at 1 , 3 , and 6 months . a slit - lamp examination was performed to check for lenticule clarity , position , and wound healing . postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward , the following assessments were performed : uncorrected visual acuity , best - corrected visual acuity ( bcva ) , retinoscopy for residual refractive error , topography using orbscan(bausch & lomb - technolas , munchen , germany ) and sirius ( schwind eye - tech solutions , kleinostheim , germany ) , anterior segment optical coherence tomography ( optovue , fremont , ca ) , clinical photography , specular microscopy ( tomey , japan ) , dry eye assessment ( schirmer 's i ) , and aberrometry ( itrace ; tracey technologies , houston , tx ) . two types of solutions ( washing solution and cryopreservation solution ) were prepared . the washing solution comprised 10 ml of phosphate - buffered saline ( 1 ) , 200 l of penicillin streptomycin ( 100 ) , and 200 l of amphotericin b ( 100 ) . the cryopreservation solution comprised 9 ml of tissue culture media ( cryobank wash media ) and 1 ml of 20% dimethyl sulfoxide ( dmso ) . the tissue culture medium is a commercially available medium used for semen processing in reproductive biology ( cryocell india pvt ltd , new delhi , india ) . the medium contains sodium chloride , potassium chloride , magnesium sulfate , anhydrous potassium phosphate monobasic , calcium chloride , anhydrous sodium bicarbonate , hepes , glucose , sodium pyruvate , sodium lactate , phenol red , and human albumin in company - specified compositions . extracted lenticules were washed twice for 10 minutes each in the washing solution ( fig . the lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . using a liquid dropper , a stock freezing solution containing 10% human tissue culture medium and 20% dmso ( sigma - aldrich , st louis , mo ) was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso . the cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers ( ibp ; indian oil corporation limited , nasik , india ) . on the day of surgery , the tissue was transported from the cryobank to the hospital in a liquid nitrogen container . the vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied . the lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents . a visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision . the incision was opened with a seibel spatula and the plane of the pocket was dissected . the cryopreserved lenticule ( which was matched for the refractive error after correcting for back vertex distance ) was placed on the patient 's cornea with its center marked with gentian violet dye . the lenticule was held with 2 forceps and inserted into the pocket . after insertion , the lenticule was spread out , and the center was aligned with the pupillary center . a depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes , the novelty of the nomograms , and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required . postoperatively , a topical steroid ( 1% prednisolone acetate ; allergan , irvine , ca ) was prescribed for 3 months in a tapering dosage . in addition , 5% hypertonic saline drops ( hypersol-5 ; jawa pharmaceuticals , india ) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing . patients were examined postoperatively on days 1 and 15 and at 1 , 3 , and 6 months . a slit - lamp examination was performed to check for lenticule clarity , position , and wound healing . postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward , the following assessments were performed : uncorrected visual acuity , best - corrected visual acuity ( bcva ) , retinoscopy for residual refractive error , topography using orbscan(bausch & lomb - technolas , munchen , germany ) and sirius ( schwind eye - tech solutions , kleinostheim , germany ) , anterior segment optical coherence tomography ( optovue , fremont , ca ) , clinical photography , specular microscopy ( tomey , japan ) , dry eye assessment ( schirmer 's i ) , and aberrometry ( itrace ; tracey technologies , houston , tx ) . table 2 provides the details of donors and lenticules used for fili in the 9 eyes treated . demographic details of patients who underwent fili ( 7 patients , 9 eyes ) details of donors and lenticules used for fili ( n = 9 ) on day 1 , in the first 2 consecutive eyes , the implanted lenticules had mild to moderate descemet membrane folds that resolved spontaneously within 1 week . subsequently , the treated eyes were prescribed hypertonic saline drops , which showed clear lenticules within 48 hours and until the last follow - up time point . figure 2 shows serial clinical photographs and anterior segment optical coherence tomography of the eye treated with fili at day 15 and at 6 months post - fili showing clear lenticules with good centration . a , serial digital clinical photographs ( 16 ) of 32-year - old woman operated for + 6.5 d hyperopia in the right eye with fili . distance from edge of the lenticule to limbus was measured at 3 points and verified at every visit to check for centering and any shift in position . b , six - month postoperative anterior segment optical coherence tomography of an eye treated for + 6.5 d hyperopia showing a clear and well - centered lenticule . all eyes had an uncorrected visual acuity equal to or better than the preoperative bcva . the refractive predictability for hyperopia was fairly accurate , and all eyes had a residual spherical equivalent within 1.0 diopter ( d ) , although in the aphakic eye , the residual spherical equivalent was + 4.1 d. there was an average of 0.75 to 1.00 d against - the - rule astigmatism in all eyes due to the superior 4 mm incision , which reduced and stabilized over time . postoperative results of eyes treated with fili ( n = 9 ) table 4 provides changes in anterior and posterior curvature , pachymetry and asphericity ( q value ) following fili . all eyes had central corneal steepening with a mean change in anterior keratometry of 3.5 d in the central 3-mm zone . there was an average 0.33 d flattening of posterior corneal curvature in all eyes , which was seen more in thicker lenticules compared with the thinner ones . figure 3 illustrates changes in anterior and posterior curvature post - fili in an eye , over time . changes in anterior and posterior topography , pachymetry , and asphericity post - fili ( n = 9 ) orbscan anterior corneal surface topography ( top ) and sirius posterior elevation ( bottom ) over the 6-month postoperative course after fili for eye with + 6.5 d hyperopia . the postoperative q values in all eyes became more negative , suggesting a hyperprolate shift . table 5 provides analysis of the epithelial thickness profile , which suggested mild epithelial thinning in the 5-mm zone in all hyperopic eyes , except for the aphakic eye , which showed mild epithelial thickening in the corresponding zone . figure 4 illustrates the epithelial thickness map of an eye treated with fili at 6 months . epithelial thickness profile on anterior segment optical coherence tomography in the central 5-mm zone epithelial thickness profile with anterior segment optical coherence tomography of an eye 6 months post - fili for + 6.5 d hyperopia . higher order aberrations ( hoa ) did not show a significant increase in root mean square values postoperatively in the 8 hyperopic eyes ( p > 0.05 ) . endothelial cell count and schirmer i test scores were normal and the changes were not significant at the last follow - up ( p > 0.05 ) ( table 6 ) . total hoas at 5-mm pupil size ( n = 8) , ecd , and schirmer 's i scores , pre- and post - fili ( n = 9 ) the potential advantages of cryopreservation of refractive lenticules have been largely described for use in the same patient , in the event of future ectasia or presbyopia by restoring corneal volume.3 with the increasing number of eyes undergoing relex smile for myopia , and thus , the extracted lenticules as a by - product , it may be a novel idea to preserve these lenticules on a long - term basis using cryopreservation for potential future use and research . following establishment of one such tissue bank , we wanted to explore the possibility of use of such cryopreserved refractive lenticules for potential treatment of hyperopia and study the feasibility , safety , efficacy , and reproducibility of this new treatment . initially , the amblyopic eyes were studied mainly for changes in refraction and topography following tissue addition . there was also the goal of evaluating any loss of bcva and complications , such as haze and tissue rejection . even these patients benefited from the surgery , as their refractive error was corrected to their best spectacle - corrected visual acuity and reduced their dependence on eyeglasses . after reviewing the results for 6 months , the indications were extended to normal eyes and bilateral surgery . unlike myopia , the correction of hyperopia involves creation of a furrow - like ring zone in the paracentral zone of the cornea . both photorefractive keratectomy and lasik are subtractive procedures that steepen the cornea by ablating the midperipheral tissue.14,15 this creates an abnormal hyperprolate shape of the cornea that is associated with significant regression , especially in higher degrees of refractive errors.16 it potentially leads to the induction of hoas like coma and spherical aberrations and loss of bcva.17 fili is an innovative technique based on barraquer 's law of thickness,18 whereby the cornea is made steeper by addition of a lenticule of known thickness and power into a pocket created in a patient 's cornea using the fs laser . the concept is similar to epikeratophakia , used to treat aphakia in the past , which had its own disadvantages.19 a fs laser overcomes these problems by creating precise lenticules and incisions at an accurate depth . the lenticular tissue processing technique described by mohamed - noriega et al10 was modified in this study by replacing bovine serum albumin with human tissue culture media to eliminate the risk of transmission of zoonotic diseases or unexpected adverse reactions due to bovine serum . the protocols of gradual staged thawing , which are essential for the whole cornea to prevent endothelial cell injury due to sudden thawing , were not strictly followed in this study . microbiological and cellular cultures were not deemed necessary , as the tissue processing and preservation were conducted under strict sterile conditions , and the tissue did not contain vital endothelial cells . also , previous studies have suggested that stromal keratocytes within the extracted lenticules remain viable and can be stimulated to proliferate in appropriate cell culture conditions , even after a prolonged period of cryopreservation.9,10 the change in asphericity after fili suggests an increase in the prolateness of the cornea , which is expected after tissue addition , although the observed change was not seen as much as it is after hyperopic lasik.20 the shape of the cornea is more natural after tissue addition compared with tissue subtraction , which does not lead to significant induction of hoas.21 the total hoas after fili remain within normal acceptable values and may be better than hyperopic lasik / photorefractive keratectomy.22,23 also , tissue addition may be more forgiving for mild decentration , which is not the case with hyperopic lasik . the rate of enhancement following hyperopic lasik can be up to 10% or more,24 especially in younger patients . after tissue addition , significant changes in topography or regression are not expected ; thus , retreatment rates may be lower . epithelial thickness profile is more favorable , which , unlike hyperopic lasik , does not cause midperipheral epithelial hyperplasia to compensate for loss of stromal tissue , hence reducing chances of regression.25 problems of haze and flap - related issues like delayed healing , infection , and dislocation are eliminated , thereby leading to greater patient comfort and faster recovery . a fairly good refractive predictability was observed with this technique in the treatment of moderate hyperopia , although results were not highly accurate for correcting very high hyperopia ( eg , aphakia ) . in a case study reported by pradhan et al,4 a 10 d lenticule was implanted to achieve correction of aphakia but had a high residual refraction of + 5 d. there are possible explanations for this result . first , the technique of pradhan et al of pocket creation was different from the technique used in this study . in the said study , a smile lenticule was first created , followed by dissection of the anterior plane to create a pocket , thus making 2 planes that could possibly cause weakening of the cornea . second , the optical zone of the lenticule in the study of pradhan et al was 5.7 mm compared with 6 mm in the current aphakia case , causing comparatively less volume of tissue addition . the correction is expected to be relatively higher in larger lenticules because there is more volume of the tissue added . in this hyperopic series , the lenticule size ranged from 6.3 to 6.8 mm . also , the posterior curvature changes as described in the case study of pradhan et al reduced the effect on anterior curvature . this was also probably the cause of the high + 4.1 d residual refraction in the current study . after compensation , implantation of lenticule of approximately 12.5 d is required , and this is currently not possible with visumax . the biomechanics of corneas in these patients were not studied due to lack of equipment such as the corvis ( oculus optikgerte gmbh , wetzlar , germany ) . however , because most of the bowman layer is preserved and tissue is being added , it is assumed that the biomechanics are more stable than lasik , where a flap is cut and tissue is subtracted from the periphery.26 the procedure certainly amounts to corneal transplantation and thus , theoretically carries the risk of future rejection . however , unlike traditional full thickness grafts , which have a graft host junction proximal to host limbus , in this technique , the lenticule was well protected in the host corneal pocket and did not come in direct contact with the host 's limbal vasculature and immune system , making the risk of allogeneic graft rejection remotely possible.27 also , the lenticule was mainly stromal collagen tissue ; the incidence of rejection is significantly low in such a scenario compared with endothelial rejection . because no sutures are applied , there are no suture - related complications such as loosening , vascularization , or infection that would potentially trigger graft rejection.28 nevertheless , there is a need to monitor for signs of rejection and treat it like allogeneic graft rejection should rejection occur . because it is a reversible procedure , the lenticule can always be explanted in the event of any adverse effects or rejection in future . the preliminary results of fili in hyperopia indicate that this may be a better technique than hyperopic lasik for moderate hyperopia in terms of stability , regression , aberrations , and postoperative dry eye . although it is a bit early to exactly predict the refractive outcomes , initial data indicate that the cryopreservation technique used is safe in heterologous individuals . further studies with a larger cohort and modification of the surgical technique with a goal of reducing postoperative astigmatism and refining nomograms are suggested to determine the long - term safety and refractive effects on the cornea and establish the technique as a valid option to treat hyperopia .

purpose : to describe the technique of cryopreservation of corneal lenticules extracted after small incision refractive lenticule extraction ( relex smile ) and initial results of femtosecond laser intrastromal lenticular implantation for hyperopia.methods:lenticules were collected from patients undergoing relex smile for the correction of myopia and subjected to a tissue processing technique and cryopreservation . these lenticules were subsequently used to treat 8 hyperopic eyes and 1 aphakic eye . a femtosecond laser was used to create a pocket into each patient 's cornea , followed by implantation of a cryopreserved lenticule . the patients were monitored through follow - up examinations for a mean 155.4 days ( 38310 days).results : the mean interval from storage of lenticules to removal from liquid nitrogen was 96 days ( range , 19178 days ) . mean spherical equivalent of hyperopic eyes treated was + 4.50 1.1 diopter ( d ) . mean keratometry and pachymetry changed from preoperative 43.9 d and 531.6 m to 47.4 d and 605.2 m , respectively , postoperatively . mean residual spherical equivalent for hyperopic eyes was + 0.6 d and + 4.1 d for the aphakic eye . none of the eyes showed evidence of rejection or loss of best - corrected visual acuity at the end of the follow - up period.conclusions:the cryopreservation technique seems to be a safe method of long - term storage of refractive lenticules extracted after relex smile for use in allogeneic human subjects . it may potentially be a safe and effective alternative to excimer laser ablation for hyperopia because of the low risks of regression , haze , flap - related complications , postoperative dry eye , and higher - order aberrations.clinical trial registration url : http://www.clinicaltrials.gov . unique identifier : ctri/2014/01/004331 .

sepsis is the major cause of death in the intensive care unit . despite improvement of antibiotics treatment and supportive techniques , the mortality of septic shock increases to approximately 60% . , it not only helps doctors to make an early diagnosis of sepsis , but also predicts outcomes . there have been some biomarkers and cytokines used in both the clinical practice and laboratory including soluble triggering receptor expressed on myeloid cells-1 ( strem-1 ) , procalcitonin ( pct ) , n - terminal probrain natriuretic peptide ( nt - pro - bnp ) , c - reactive protein ( crp ) , interleukin-6 ( il-6 ) , and interleukin-10 ( il-10 ) . trem-1 is a recently discovered member of the immunoglobulin superfamily of receptors that is specifically expressed on the surfaces of neutrophils and monocytes . strem-1 is a soluble form of trem-1 and is upregulated when exposed to infectious diseases . pct is a polypeptide consisting of 116 amino acids and is the precursor of calcitonin ; it was proven useful to identify nonsystemic inflammatory response syndrome and was firstly used in sepsis . nt - pro - bnp is a biologically inactive form that is cleaved from the prohormone probrain natriuretic peptide ( pro - bnp ) by proteolytic enzymes before secretion . crp is a widely used biomarker to discriminate the inflammatory response to sepsis . il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines during sepsis course . . indicated that strem-1 was more accurate than pct and crp in the diagnosis of sepsis , but others showed that the prognostic utility of serum strem-1 in septic shock was inferior to that of pct . the purpose of the study was to compare the prognostic value of biomarkers and cytokines versus clinical severity scores and improved death risk prediction . a total of 102 patients with sepsis from single centre hospital intensive care unit were enrolled from december 2010 to august 2012 according to the 2001 international sepsis definition conference . exclusion criteria included : age younger than 18 years , preexisting thyroid disease and lung cancer that influence procalcitonin levels , patients with acute coronary syndromes and renal dysfunction , and patients staying in icu less than 24 hours . the study was approved by the hospital 's ethics committee and either the patients or their relatives provided informed consent . demographic and disease data of patients included age , gender , chief complaints for admission , vital signs , length of stay in icu , infection sites , microorganisms , routine blood test results , liver and kidney functions , coagulation indicators , blood gas analysis , acute physiologic assessment and chronic health evaluation ( apache ) ii scores , and sequential organ failure assessment ( sofa ) scores . these were recorded on 3 days ( days 1 , 3 , and 5 ) . serum was collected at these same time points and pct , strem-1 , nt - pro - bnp , crp , il-6 , and il-10 levels were determined in the end . pct was measured using an enzyme - linked fluorescence analysis kit ( elfa , vidas brahms pct kit , biomerieux sa , france ) . strem-1 was determined using a double antibody sandwich elisa ( quantikine human trem-1 immunoassay elisa kit , r & d systems , minneapolis , mn , usa ) . nt - pro - bnp was measured with an available immunoassay analyzer ( elecsys 2010 ; roche diagnostics , mannheim , germany ) . crp was determined using scattering using a nephelometric assay ( dade - behring , sa paris , france ) . il-6 and il-10 were determined using elisa ( immulite ; diagnostics products corporation , los angeles , ca ) . quantitative data with normal distributions are given as means standard deviations ( sd ) . quantitative data that were not normally distributed were summarized as medians ( interquartile ranges ) and compared by nonparametric tests ( mann - whitney u test ) . we made a logarithmic conversion for the nondistribution data when we did dynamic comparison in figure 2 . proportions were used to express qualitative data and the differences in proportions between groups were compared using a chi - square test . we compared the characteristics of survivors versus nonsurvivors using univariate analysis and used receiver operating characteristics ( roc ) curves to evaluate prognostic value of the biomarkers and cytokines predicted 28-day mortality . those variables with p values less than 0.05 on univariate analysis were then entered into a multivariate logistic regression analysis to further identify the independent predictors of 28-day mortality . there were no significant difference in age and sex of these two groups ( p > 0.05 ) . the apache ii and sofa scores of patients in the nonsurvival group were higher than those of patients in the survival group ( p = 0.000 , p = 0.000 , resp . ) , ( table 1 ) . serum pct , strem-1 , il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ( p < 0.001 ) . there were no differences in nt - pro - bnp and crp , il-10 levels between the two groups ( p > 0.05 ) ( table 2 ) . the roc analysis showed that the accuracy of the pct , strem-1 , il-6 , apacheii , and sofa scores on day 1 for the prediction of 28-day mortality was moderate ( auc > 0.7 , p < 0.01 ) , whereas the accuracy of nt - pro - bnp , crp , and il-10 was low ( auc < 0.7 , p > 0.05 ) ( figure 1 ) . comparing auc of pct , strem-1 , and il-6 , we found that there was no significant difference of auc between strem-1 and pct ( p = 0.2910 ) , and the auc of the two markers were higher than that of il-6 ( p > 0.05 ) . meanwhile , there was no significant difference of auc between apacheii and sofa scores ( p = 0.3753 ) . the auc of apacheii and sofa scores were higher than those of strem-1 and pct ( p < 0.05 ) ( table 3 ) . the baseline day 1 variables that were found to be significantly different between survivors and nonsurvivors on univariate analysis ( pct , strem-1 , il-6 , apacheii , and sofa scores ) were entered into a logistic regression model . among these variables , three variables remained independently associated with 28-day mortality : strem-1 , pct , and sofa score ( table 4 ) . median serum biomarkers and cytokines levels were determined on days 1 , 3 , and 5 and were compared between the survival and nonsurvival groups . serum pct , strem-1 , and il-6 levels in the nonsurvival group were higher than those in the survival group on days 1 , 3 , and 5 ( p < 0.01 ) . there was no difference in nt - pro - bnp levels on day 1 ( p > 0.05 ) , but later the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 ( p < 0.05 ) . there were no differences in crp and il-10 levels on days 1 , 3 , and 5 . serum pct , strem-1 , il-6 , and nt - pro - bnp levels showed a decrease trend in the survival group ( p < 0.05 ) , but there was no decrease tendency in the nonsurviving group for these four biomarkers ; strem-1 even had a increase trend ( p < 0.05 ) . serum crp levels in both surviving and nonsurviving groups had decrease tendency ( p < 0.05 ) ( figure 2 ) . recently , pct , strem-1 , crp , and nt - pro - bnp cytokines were widely used to diagnose sepsis and reflect the severity , but the results were not the same . meanwhile , there were few studies to put so many biomarkers in one study , particularly how to combine the biomarkers , and clinical severity scores remained unclear . the present study showed that the serum levels of strem-1 and pct in nonsurvival group were higher than those in the survival group ; meanwhile , they decreased in survival group , but stayed in high levels even increased in the nonsurvival group during sepsis time course . many previous studies have shown that dynamic changes in strem-1 levels could predict survival and mortality of patients at the early stage of sepsis [ 10 , 11 ] . strem-1 is widely used to diagnose sepsis [ 7 , 12 ] . in the present study , serum strem-1 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ; it decreased in survival group , but it even increased in the nonsurvival group . some studies failed to find the association between strem-1 and poor outcome [ 8 , 13 ] . at a cutoff of 252.05 pg / ml , strem-1 measurements yielded a sensitivity of 85.7% , specificity of 75.7% , positive predictive value of 70.6% , negative predictive value of 88.2% , and an accuracy of 79.4% for differentiating nonsurvivors from survivors . pct is normally produced in the c cells of the thyroid gland ; plasma pct levels in healthy humans are approximately 550 pg / ml in normal state ; its half - time is about 2233 hours in serum . many tissues and cells except thyroid gland produce and release that pct on systemic inflammation . several previous studies reported pct could serve as a useful tool to distinguish sepsis from systemic inflammatory response syndrome [ 15 , 16 ] . on the other hand , pct could reflect the severity of sepsis and outcome . a study by christophe clec'h and coworkers found that serum pct on day 1 was significantly higher in patients with than without septic shock . meanwhile , among patients with sepsis , pct concentrations were significantly higher in those who died than in the survivors , at all four measurement time points . similar results were drawn from other investigations [ 16 , 18 ] . very few studies failed to find the prognostic value . at a cutoff of 10.64 ng / ml , procalcitonin measurements yielded a sensitivity of 76.2% , specificity of 81.7% , positive predictive value of 53.5% , negative predictive value of 67.8% , and an accuracy of 61.8% for differentiating nonsurvivors from survivors . nt - pro - bnp has been found to be a useful markers in the diagnosis , management , and prognosis of patients with congestive heart failure and was secreted into blood in response to atrial or ventricular wall stretch . a recent meta - analysis suggested that an elevated nt - pro - bnp level may prove to be a powerful predictor of mortality in septic patients . in our study , there was no difference in nt - pro - bnp level between groups on day 1 , but the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 . meanwhile , serum nt - pro - bnp level showed a decreased trend in the survival group , but there was no decrease tendency in the nonsurvival group . we concluded that nt - pro - bnp may predict sepsis 28-day mortality in different stages . one research demonstrated that elevated serum nt - pro - bnp value represented an independent predictor for poor icu outcome in the presence of clinical severity scores ; the cut - off in admission nt - pro - bnp that best predicted outcome was 941 pg / ml . crp is an acute phase protein and a sensitive systemic marker of inflammation and tissue damage . the secretion of crp begins within 46 h after stimulus , doubles every 8 h thereafter , and peaks at 3650 h . the role of crp in sepsis prognostic value seemed different . in our study , there was no significant difference between survivors and nonsurvivors during the three measures , similar to previous study , indicating that crp was just an inflammatory biomarker and failed in reflecting sepsis severity . have reported that crp levels in severe sepsis were lower than those in sepsis , suggesting that crp levels did not reflect the severity of sepsis . il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines in sepsis . in our study , serum il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on days 1 , 3 , and 5 . the above results showed that il-6 had the prognostic utility for sepsis , whereas il-10 did not show the power . surez - santamar and coworkers enrolled 253 hospitalized septic patients ; they found that il-10 and il-6 were the best predictors , whereas pct showed only moderate predictive value for mortality . another study investigated the prognostic value of il-6 , pct , and crp in critically ill patients during the first increase of fever ; only il-6 levels were significantly higher in nonsurvivors compared with survivors , in which prognostic value was superior to pct and crp . in contrast , kawczyski and polakowska indicated that the predictive value of il-10 plasma concentration was better than that of il-6 . to sum up , strem-1 , pct , and il-6 serum values attribute to the prognosis of sepsis during the time course . the dynamic changes of biomarkers and cytokines were more meaningful for predicting the sepsis procession . schneider and coworkers retrospectively analyzed the relationships between serum pct , il-6 , and apacheii score and prognosis of 220 patients on the first day after operation . they found that pct was the sole independent predictor of 28-day mortality , in which prognostic ability was superior to those of il-6 and apacheii score . zhang et al . suggested that serum strem-1 levels reflected the severity of sepsis more accurately than those of pct and crp and were more sensitive for dynamic evaluations of sepsis prognosis . facing the results , we wonder which was the best predictor and how to combine them together and which was more valuable compared to clinical severity scores . apache ii and sofa scores have been widely used to validate mortality risk stratification . in our study , we used roc and logistic regression model to search for the best predictor . based on roc analysis , strem-1 and pct showed the equal prognostic ability ( 0.792 for pct , 0.862 for strem-1 , p = 0.291 ) , whereas their prognostic utility was inferior to that of apacheii and sofa scores which had equal power to predict outcome ( 0.923 for apacheii score , 0.953 for sofa score , p = 0.375 ) . logistic regression model showed that serum strem-1 , pct , and sofa score were the independent predictors of 28-day mortality , which was supported by other result . as far as we know , the interrelationship between strem-1 , pct , nt - pro - bnp , cytokines , and clinical severity scores for mortality prediction in general icu patients has not been previously evaluated . our research firstly discovered that strem-1 and pct had the equal prognostic ability for sepsis mortality and were superior to other parameters . previous study has indicated that the iteraction of trem-1 and interact adaptor protein dap12 can stimulate neutrophil and monocyte - mediated inflammatory response via the triggering and release of pro - inflammatory cytokines and chemokines . strem-1 increases quickly when exposued to infection , and its half - time is short . in bacterial infections , serum pct levels start to rise at 4 h after the onset of systemic infection and peak at between 8 and 24 h ; it decreased 50% every 24 hours along with therapy . in contrast , crp rises slowly and peaks 36 h after an endotoxin challenge . the mechanism of nt - pro - bnp release in sepsis is complex , and kinetics characteristic is unknown . il-6 and il-10 rise quickly and peak at 24 hours and maintain a short time . the patients admitted to icu often delayed more than 24 hours , either crp or cytokines serum concentration was unable to reach the peak at the period of sepsis . of course , the exact roles of biomarkers and cytokines in sepsis process are not clear , and need to be further studied . firstly , our study chosen a part of sepsis biomarkers and did not put all biomarkers in the research . of course , it was a costly and unnecessary task to do so . secondly , every biomarker has its own dynamic characteristics ; meanwhile the patients were not in the same sepsis stages in the study ; thus the explanation for the results would be influenced . thirdly , we excluded patients with previous heart diseases history , but we did not perform the ucg to evaluate cardiac function . finally , the sample size of the study was small and larger studies are needed . in summary , elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers . combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic for sepsis mortality . in the future , in order to improve the accuracy of the prognosis of sepsis , the combination of novel biomarkers and traditional markers of sepsis , reflecting different aspects of sepsis , is an attractive advice and is worthy of further investigation .

objective . to investigate the prognostic significance of serum soluble triggering receptor expressed on myeloid cells-1 ( strem-1 ) , procalcitonin ( pct ) , n - terminal probrain natriuretic peptide ( nt - pro - bnp ) , c - reactive protein ( crp ) , cytokines , and clinical severity scores in patients with sepsis . methods . a total of 102 patients with sepsis were divided into survival group ( n = 60 ) and nonsurvival group ( n = 42 ) based on 28-day mortality . serum levels of biomarkers and cytokines were measured on days 1 , 3 , and 5 after admission to an icu , meanwhile the acute physiology and chronic health evaluation ii ( apache ii ) and sequential organ failure assessment ( sofa ) scores were calculated . results . serum strem-1 , pct , and il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ( p < 0.01 ) . the area under a roc curve for the prediction of 28 day mortality was 0.792 for pct , 0.856 for strem-1 , 0.953 for sofa score , and 0.923 for apache ii score . multivariate logistic analysis showed that serum baseline strem-1 pct levels and sofa score were the independent predictors of 28-day mortality . serum pct , strem-1 , and il-6 levels showed a decrease trend over time in the survival group ( p < 0.05 ) . serum nt - pro - bnp levels showed the predictive utility from days 3 and 5 ( p < 0.05 ) . conclusion . in summary , elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers . combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic utility for sepsis mortality .

nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively . they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) . all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study . the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) . they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) . the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) . the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat . the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility . two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) . the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) . cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue . the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt , the riders performed a self - paced warm - up ( approximately two laps on the tt course ) . the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt , the riders had access to hr , speed , distance , and power output data . wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively . environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively . relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air ) , the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively . power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt . hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) . rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) . in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter . body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) . continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova . anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) . effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) . data are presented as mean sd along with the mean differences ( 95% confidence interval ) . nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively . they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) . all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study . the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) . they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) . the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) . the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat . the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility . two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) . the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) . cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue . the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt , the riders performed a self - paced warm - up ( approximately two laps on the tt course ) . the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt , the riders had access to hr , speed , distance , and power output data . wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively . environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively . relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air ) , the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively . power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt . hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) . rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) . in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter . body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) . continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova . anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) . effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) . data are presented as mean sd along with the mean differences ( 95% confidence interval ) . nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively . they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) . all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study . the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) . they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) . the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) . the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat . the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility . two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) . the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) . cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue . the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt , the riders performed a self - paced warm - up ( approximately two laps on the tt course ) . the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt , the riders had access to hr , speed , distance , and power output data . wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively . environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively . relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air ) , the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively . power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt . hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) . rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) . in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter . body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) . continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova . anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) . effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) . data are presented as mean sd along with the mean differences ( 95% confidence interval ) . there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) . speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) . speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) . average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) . this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) . 1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction . the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig . 1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig . power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig . power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig . power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively . 2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) . the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) . consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) . hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig . 2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig . 2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 , the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance . there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) . speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) . speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) . average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) . this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) . 1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction . the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig . 1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig . power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig . power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig . power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively . 2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) . the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) . consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) . hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig . 2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig . 2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 , the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance . there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) . speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) . speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) . average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) . this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) . 1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction . the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig . 1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig . power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig . power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig . power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively . 2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) . the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) . consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) . hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig . 2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig . 2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 , the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance . the current study is the first to determine the effects of acute heat exposure and heat acclimatization on performance and pacing during outdoor cycling tt in experienced cyclists . the cyclists initiated all tt in the heat with a similar power output as maintained during the first 20% of ttc . however , while maintaining similar hr , they subsequently experienced a marked decrease in power output and speed in the heat . these decrements were progressively restored with heat acclimatization , despite core temperature in all tt in the heat increasing significantly more than that in cool conditions . our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) . in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants . these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) . however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined . our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 ) relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement , our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) . one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) , this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig . 1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig . our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity . indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate . although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) . it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig . moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) . rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) . given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) . this similar work rate adopted seems to correspond to a critical power ( 16 ) . given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) . however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . initial power output was similar between trials and decreased thereafter in relation to acclimatization state . indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) . consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow . the current study is the first to investigate cycling performance during outdoor tt in a hot environment . . however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study , the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) . the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% . consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study , air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) . thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different . our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) . in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants . these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) . however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined . our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 ) relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement , our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) . one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) , this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig . 1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig . our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity . indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate . although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) . it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig . moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) . rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) . given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) . this similar work rate adopted seems to correspond to a critical power ( 16 ) . given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) . however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . initial power output was similar between trials and decreased thereafter in relation to acclimatization state . indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) . consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow . the current study is the first to investigate cycling performance during outdoor tt in a hot environment . however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study , the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) . the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% . consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study , air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) . thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different . our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) . in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants . these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) . however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined . our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 ) relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement , our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) . one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) , this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig . 1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig . our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity . indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate . although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) . it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig . moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) . rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) . given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) . this similar work rate adopted seems to correspond to a critical power ( 16 ) . given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) . however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . initial power output was similar between trials and decreased thereafter in relation to acclimatization state . indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) . consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow . the current study is the first to investigate cycling performance during outdoor tt in a hot environment . however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study , the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) . the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% . consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study , air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) . thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different . the novel findings of this investigation are that competitive cyclists performing an outdoor tt undertake their effort at the same power output , irrespective of the environmental conditions or previous experience . consequently , non heat - acclimatized cyclists are incapable of sustaining this absolute effort . however , the decrement in power output is partly recovered after 1 wk of heat acclimatization and almost fully restored after 2 wk . furthermore , our data seem to confirm that sustainable power output is related to a given relative intensity ( 24 ) , which is partly reflected in the maintenance of hr within a certain range . finally , because of the reduction in air density associated with cycling in hot ambient conditions , speed was not different between tth-3 and ttc .

abstractpurposethis study aimed to determine the effects of heat acclimatization on performance and pacing during outdoor cycling time trials ( tt , 43.4 km ) in the heat.methodsnine cyclists performed three tt in hot ambient conditions ( tth , approximately 37c ) on the first ( tth-1 ) , sixth ( tth-2 ) , and 14th ( tth-3 ) days of training in the heat . data were compared with the average of two tt in cool condition ( approximately 8c ) performed before and after heat acclimatization ( ttc).resultstth-1 ( 77 6 min ) was slower ( p = 0.001 ) than tth-2 ( 69 5 min ) , and both were slower ( p < 0.01 ) than ttc and tth-3 ( 66 3 and 66 4 min , respectively ) , without differences between ttc and tth-3 ( p > 0.05 ) . the cyclists initiated the first 20% of all tt at a similar power output , irrespective of climate and acclimatization status ; however , during tth-1 , they subsequently had a marked decrease in power output , which was partly attenuated after 6 d of acclimatization and was further reduced after 14 d. hr was higher during the first 20% of tth-1 than that in the other tt ( p < 0.05 ) , but there were no differences between conditions from 30% onward . final rectal temperature was similar in all tth ( 40.2c 0.4c , p = 1.000 ) and higher than that in ttc ( 38.5c 0.6c , p < 0.001).conclusionsafter 2 wk of acclimatization , trained cyclists are capable of completing a prolonged tt in a similar time in the heat compared with cool conditions , whereas in the unacclimatized state , they experienced a marked decrease in power output during the tth .

worksites in glass and metal works , foundries , rolling mills , in the vicinity of hardening furnaces , rotary kilns in the cement industry , high - temperature laboratory furnaces , as well as firefighting , are associated , in addition to common mechanical hazards ( e.g. , due to splashes of molten metals and slag ) , sparks and direct contact with open fires , with hazards caused by emission of harmful optic radiation within the visible ( vis ) and infrared ( ir ) spectrum range . excessive exposure to ir radiation leads to dryness of the mucous membranes , burns of the eyeball and development of cataracts . protection against thermal hazards due to excessive amounts of ir radiation reaching the eyes is provided by protective optic filters installed in spectacles , goggles or face protections . besides protecting the user against hazardous ir radiation , such filters should ensure that the object / area of work can be seen clearly . if the level of ir radiation is very high , the use of filters reflecting radiation is recommended , as it allows the temperature increase in the filter itself to be reduced . the filters currently used for the protection of the face and eyes against hazardous ir radiation are of metallic reflective type , made of glass or organic material ( mainly polycarbonate ) bases coated with a single metallic layer for reflectance of ir radiation or absorption filters . absorption filters are manufactured from dyed glass obtained in a mass staining process by introduction of a factor modifying the radiation within the vis and near infrared ( nir ) spectrum range passing through the filter . the effect of a protective optical filter obtained as a result of this process is based on absorption of a large proportion of optic radiation , whereas only a small proportion of radiation the filter is exposed to is reflected . this is an undesirable phenomenon , contributing to deterioration of the filter user 's comfort . reflective metallic filters are obtained by deposition of a metallic layer of copper ( cu ) or gold ( au ) on mineral or organic bases . such filters take advantage of the characteristic properties of the metals used in their production , i.e. , significant reflection coefficient for ir radiation and significant transmittance for the vis spectrum . despite the fact that the technique of deposition of interference layers has been known for many years the application of thin film ir filters in eye protections to block the previous papers published by the authors of this study contain descriptions of the structure of interference filters designed for application in eye protections highly effective in reduction of ir radiation with high level of transmission of the vis spectrum . the authors investigated also the optical and mechanical parameters of the newly developed protective optic filters manufactured with the use of interference technologies . the analysis of the results of the conducted tests demonstrated numerous advantages of interference technology application in the construction of protective filters , including : improved resistance to damage of the external protective filter surface ( cracking , scratching etc . ) as well as high reflection coefficients for the nir spectrum . the aim of the present study was to compare the optic properties of protective filters manufactured by taking advantage of the interference technology and those coated with a single metallic layer reflecting ir radiation . the coefficients characterizing their optic parameters , allowing to assess the protective properties within the vis and ir spectrum range were compared . the following coefficients were analysed : luminous transmittance ( v),ir transmittance within the 7801400 nm range ( a),ir transmittance within the 7802000 nm range ( n),ir reflectance within the 7802000 nm range ( r n),ir absorptance within the 7802000 nm range ( a n),shade number ( n ) . luminous transmittance ( v ) , ir transmittance within the 7801400 nm range ( a ) , ir transmittance within the 7802000 nm range ( n ) , ir reflectance within the 7802000 nm range ( r n ) , ir absorptance within the 7802000 nm range ( a n ) , the level of protection ( shade number ) is determined on the basis of luminous transmittance ( the higher protection level correlates with the higher level of ir radiation blocked and appropriately lower vis light transmittance ) . protection level consists of a code number ( e.g. , 4 ) for ir filters and a shade number ( e.g. , 3 , 5 , 7 ) calculated according to the following equation : ( 1 ) where v = luminous transmittance . for the particular level of protection , the mean ir transmittance within the 7801400 and 7802000 nm ranges as well as the ir reflectance and absorptance within the 7802000 nm range are determined . for comparative analysis of the metallic reflective filters available in the market versus the interference filters developed by the authors , the following samples were prepared for the tests : off the shelf metallic reflective filters on a polycarbonate base ( flat parallel plate of 50 mm diameter and 2 mm thickness with a metallic layer of copper ( cu ) protection levels : 4 - 3 , 4 - 5 , 4 - 7);interference filters on a polycarbonate base ( flat parallel plate of 50 mm diameter with interference coating made up of the following materials : aluminium , h4 latio3 substance and silicon dioxide ) . off the shelf metallic reflective filters on a polycarbonate base ( flat parallel plate of 50 mm diameter and 2 mm thickness with a metallic layer of copper ( cu ) protection levels : 4 - 3 , 4 - 5 , 4 - 7 ) ; interference filters on a polycarbonate base ( flat parallel plate of 50 mm diameter with interference coating made up of the following materials : aluminium , h4 latio3 substance and silicon dioxide ) . the protection levels , luminous transmittance and symbols used for marking the tested filters are presented in table 1 . characteristics of the filters.symbolluminous transmittance v ( % ) protection levelfilter typecu 4 - 314.1334 - 3metallic reflectiveitf 4 - 39.0824 - 3interferencecu 4 - 52.0214 - 5metallic reflectiveitf 4 - 51.4704 - 5interferencecu 4 - 70.3534 - 7metallic reflectiveitf 4 - 70.1604 - 7interferencenote : protection level consists of code number ( 4 ) for infrared filters and shade number ( 3 , 5 , 7 ) calculated according to equation ( 1 ) . note : protection level consists of code number ( 4 ) for infrared filters and shade number ( 3 , 5 , 7 ) calculated according to equation ( 1 ) . optical properties of the filters were measured with a cary 5000 spectrophotometer ( varian , australia ) . the spectrophotometer enabled transmittance or reflectance of optical radiation within the vis and nir spectrum to be recorded with 1 nm steps and 0.001% resolution level . spectral transmittance and reflectance measurements were carried out within the wavelength range of 7802000 nm . the quartz halogen lamp was used as a source of radiation . to detect transmitted or reflected radiation the photomultiplier tube and lead sulphide detector were used for vis and nir spectrum , respectively . spectral transmittance was measured with incident radiation falling normally on the filter surface in the visual centre ( defined as in ) . for spectral reflectance the measurements were taken every second owing to the high absorption of investigated filters within the nir region . since the minimum averaging time of the cary 5000 spectrophotometer was 0.0125 s , the values of transmittance or reflectance were measured 125 times for a given wavelength . the aforementioned coefficients were calculated from the following equations [ 1012 ] : luminous transmittance ( 2 ) where ( ) = spectral transmittance , v( ) = spectral visibility function of the average human eye for daylight or night vision , s d65( ) = spectral energy distribution of standard illuminant d65 ; ir transmittance within the 7801400 nm range ( 3 ) where ( ) = spectral transmittance ; ir transmittance within the 7802000 nm range ( 4 ) where ( ) = spectral transmittance ; ir reflectance within the 7802000 nm range ( 5 ) where ( ) = spectral reflectance ; ir absorptance within the 7802000 nm range ( 6 ) where r n = ir reflectance within the 7802000 nm range , n = ir transmittance within the 7802000 nm range . luminous transmittance ( 2 ) ir transmittance within the 7801400 nm range ( 3 ) ir transmittance within the 7802000 nm range ( 4 ) ir reflectance within the 7802000 nm range ( 5 ) ir absorptance within the 7802000 nm range ( 6 ) the factor before integrals corresponding to the value of the measurement step at the measurement of spectral characteristics . transmittance and reflectance characteristics of the studied filters.note : cu 4 - 3 = metallic reflective filter ; itf 4 - 3 = interference filter ; cu 4 - 5 = metallic reflective filter ; itf 4 - 5 = interference filter ; cu 4 - 7 = metallic reflective filter ; itf 4 - 7 = interference filter . table 2 presents the values of coefficients determined on the basis of equations ( 3)(6 ) . infrared transmittance within the 7801400 and 7802000 nm range , infrared reflectance within the 7802000 nm range and infrared absorptance within the 7802000 nm range . infrared transmittance a and n ( % ) infrared reflectance rn ( % ) infrared absorptance an ( % ) symbola 7801400 ( nm)n 7802000 ( nm)rn 7802000 ( nm)an 7802000 ( nm)cu 4 - 31.4860.94283.51615.542itf 4 - 31.0030.56382.08417.353cu 4 - 50.2160.13487.31912.547itf 4 - 50.0380.00691.0378.957cu 4 - 70.0040.02388.30811.669itf 4 - 70.0040.03994.3125.649 optical radiation within the 7801400 nm spectrum range passes through the anterior compartment of the eye , the lens and reaches the retina . exposure to an excessive amount of radiation from this range can result in burns of the retina and , in extreme cases , its permanent damage . , chronic long - term exposure may lead to the development of cataracts , referred to as ir or glass worker 's cataracts . in view of the above the results presented on the graphs in figure 1 and the determined transmittance values ( see table 2 ) indicate unequivocally more effective blocking of ir radiation provided by interference filters . the values of a and n for interference filters are lower even by an order of magnitude than those obtained for the metallic reflective filters . for instance , the mean value of ir transmittance within the 7802000 nm range for interference filters with protection level 4 - 5 ( one of the most common protection levels used in the metallurgic industry ) equals 0.006% and is significantly lower than the maximum acceptable value specified in , which is 10.6% . for all the investigated filters , the values of r n exceed 60% , which classifies them in the group of filters with increased ir reflectance . the levels of the ir absorptance for metallic reflective filters and interference filters of 4 - 3 and 4 - 5 protection class are similar . for the highest of the investigated protection levels 4 - 7 the above findings are important for selection of a filter appropriate for the radiation source . the selection of filter protection level is dependent on the temperature of the radiation source . the 4 - 7 level is used for radiation sources whose temperature exceeds 1600 k ( e.g. , occurs in the metallurgical industry ) . with such high temperatures , reduction of filter heating as a result of exposure to ir radiation is important . as it follows from comparative analysis of the optic properties of filters taking advantage of the interference technology and filters coated with a single metallic layer reflecting ir radiation , the interference filters provide a more effective blocking of ir radiation in comparison with the currently used protective filters .

the paper analyses the selected optical parameters of protective optic filters used for protection of the eyes against hazardous radiation within the visible ( vis ) and near infrared ( nir ) spectrum range . the indexes characterizing transmission and reflection of optic radiation incident on the filter are compared . as it follows from the completed analysis , the newly developed interference filters provide more effective blocking of infrared radiation in comparison with the currently used protective filters .

this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) . the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010 the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s . veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas . it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history . as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) . the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems . adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 . patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date . all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements . the final sample included patients who were enrolled in the database for at least 12 months after the index date . average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) . patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status . demographic information included age on index date , sex , race , bmi , and year of index date . the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes . medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits . characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and tests for categorical variables . clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) . for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle . the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use . all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle . the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle . diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) . dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications . the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis . the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a distribution , and adjusted results are reported as annualized incremental cost differences . all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use . sas software version 9.2 was used to conduct statistical analyses , and a two - tailed level of 0.05 was used to determine statistical significance . this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) . the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010 the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s . veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas . it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history . as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) . the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems . adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 . patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date . all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements . the final sample included patients who were enrolled in the database for at least 12 months after the index date . average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) . patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status . demographic information included age on index date , sex , race , bmi , and year of index date . the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes . medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits . characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and tests for categorical variables . clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) . for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle . the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use . all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle . the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle . diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) . dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications . the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis . the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a distribution , and adjusted results are reported as annualized incremental cost differences . all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use . sas software version 9.2 was used to conduct statistical analyses , and a two - tailed level of 0.05 was used to determine statistical significance . of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis . as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m . almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) . rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) . patient baseline characteristics , demographics , comorbidities , complications , medications , and resource use the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone , was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig . dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) . dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg . in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers . after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig . 2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) . similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal . dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig . diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal . dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis . as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m . almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) . rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) . the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone , was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig . dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) . dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg . in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers . after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig . 2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) . similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal . dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig . diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal . dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . * the study results showed that , compared with the achievement of only the ldl - c goal , achievement of both hba1c and ldl - c goals is associated with a lower risk of microvascular complications , acs , and cardiovascular surgeries ( pci or cabg ) , lower utilization of health care resources , and lower costs of care , but no additional effect was observed for the composite cardiovascular - related end point . in addition , dual - goal achievement relative to hba1c goal achievement is associated with a lower risk of the composite cardiovascular - related end point , cabg , and outpatient visits . to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label , randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) . a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study , lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) . these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement . in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c . the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) . we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements . current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration . our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals . the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) . this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) . our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers . our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study , achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) . this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes . our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone . our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings . for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s . patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) . a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm , compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) . first , the data used for this study include laboratory measurements for patients over time . second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements . this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models . first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model . second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions . third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records . fourth , because we used vha data , the patients in our study were predominantly male . although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed . finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c , ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion , veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone . these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes . to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label , randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) . a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study , lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) . these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement . in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c . the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) . we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements . current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration . our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals . the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) . this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) . our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers . our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study , achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) . this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes . our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone . our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings . for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s . patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) . a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm , compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) . first , the data used for this study include laboratory measurements for patients over time . second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements . this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models . first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model . second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions . third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records . fourth , because we used vha data , the patients in our study were predominantly male . although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed . finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c , ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion , this retrospective study among u.s . veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone . these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes .

objectivethis study compared the clinical and economic benefits associated with dual - goal achievement , glycated hemoglobin ( hba1c ) < 7% ( 53 mmol / mol ) and ldl cholesterol ( ldl - c ) < 100 mg / dl , with achievement of only the ldl - c goal or only the hba1c goal in veterans with type 2 diabetes mellitus ( t2dm).research design and methodsthis retrospective cohort analysis evaluated electronic medical records ( veterans integrated service network 16 ) in adult t2 dm patients with two or more measurements of ldl - c and hba1c between 1 january 2004 and 30 june 2010 ( n = 75,646 ) . cox proportional hazards models were used to compare microvascular and cardiovascular outcomes by goal achievement status ; generalized linear regression models were used to assess diabetes - related resource utilization ( hospitalization days and number of outpatient visits ) and medical service costs.resultsrelative to achievement of only the ldl - c goal , dual - goal achievement was associated with lower risk of microvascular complications ( adjusted hazard ratio [ ahr ] 0.79 ) , acute coronary syndrome ( 0.88 ) , percutaneous coronary intervention ( 0.78 ) , and coronary artery bypass graft ( cabg ) ( 0.74 ) ; it was also associated with fewer hospitalization days ( adjusted incidence rate ratio [ airr ] 0.93 ) and outpatient visits ( 0.88 ) , as well as lower diabetes - related annual medical costs ( $130.89 ) . compared with achievement of only the hba1c goal , dual - goal achievement was associated with lower risk of the composite cardiovascular - related end point ( ahr 0.87 ) and cabg ( ahr 0.62 ) , as well as fewer outpatient visits ( airr 0.98).conclusionsachieving both hba1c and ldl - c goals in diabetes care is associated with additional clinical and economic benefits , as compared with the achievement of either goal alone .

endotracheal intubation is one of the most important skills for anesthesiologists in securing the airway during general anesthesia and resuscitation . failure to secure the airway can cause anesthesia - related life - threatening morbidity and mortality . however , the difficult laryngoscopy and tracheal intubation rate still remains at 1.513% due to poor reliability of traditional protocols , algorithms , and combinations of screening tools in identifying a potentially difficult airway . due to the portable , noninvasive characteristics , point - of - care ultrasound ( us ) technique has been widely used in the operating room for ultrasound - guided nerve block , central venous access , and pneumothorax diagnosis . with improved visualization of airway structures , prasad et al . first found that us can reliably image all of the structures visualized by ct , and the infrahyoid airway structure parameters measured by ultrasound agree well with the parameters measured by ct . adhikari et al . further demonstrated that the anterior neck soft tissue thickness measured by ultrasound at hyoid bone and thyrohyoid membrane levels can be used as an index to predict difficult laryngoscopy , but only the anterior neck soft tissue thickness at thyrohyoid membrane levels can be used as an independent predictor of difficult laryngoscopy . interestingly , they did not find a correlation between us measurements and clinical screening tests . . also found that an abundance of fat tissue at the anterior neck region as detected by ultrasound in israeli obesity patients was a very good independent predictor of difficult laryngoscopy , being a much better predictor than body mass index ( bmi ) per se . however , a similar study in the unites states showed that us quantification of anterior soft tissue and general bedside screening tests failed to predict difficult laryngoscopy in american obese patients , suggesting that racial or body shape differences might exist . to define the role of airway us in predicting difficult laryngoscopy , we evaluated the feasibility of ultrasound in predicting difficult laryngoscopy in a chinese han population . after approval of the research protocol by the local hospital ethics committee for human studies and obtaining personal informed consent , 203 american society of anesthesiologists ( asa ) grade i and ii adult patients undergoing elective surgeries and receiving general anesthesia were included in this study . exclusion criteria included patients who had facial , cervical , pharyngeal and epiglottis surgical or trauma history , patients with most teeth lost , and patients with arthritis . the modified mallampati score ( mms ) was specified according to the visibility of pharyngeal structures with the patient in an upright sitting position , head in neutral position , mouth wide open , and tongue protruding to its maximum without phonation ( figure 1a ) . class i is visualization of the hard palate , soft palate , fauces , uvula , and pillars . class ii is visualization of the hard palate , soft palate , fauces , and base of uvula . thyromental distance ( tmd ) was measured from the mental prominence to the thyroid cartilage with the patient s neck extended fully . interincisor gap ( iig ) was measured from the upper central incisors to the lower central incisors while the patient s mouth was fully opened . ultrasound measurements were performed by the primary investigator with the patient supine and the head and neck in neutral position . the thicknesses of anterior neck soft tissue at hyoid bone , thyrohyoid membrane , and anterior commissure levels were obtained transversely across the anterior surface of the neck with a 136 mhz hfl38 linear array ultrasound probe attached to a sonosite s - nerve machine ( sonosite inc . , the minimal distance from the hyoid bone to skin surface ( dshb ) was measured ( figure 2a , 2b ) . at thyrohyoid membrane level , the distance from skin to epiglottis midway ( dsem ) between the hyoid bone and thyroid cartilage was measured ( figure 2c , 2d ) . at anterior commissure level , the minimal distance from skin to anterior commissure ( dsac ) was obtained ( figure 2e , 2f ) . after anesthesia induction with midazolam 0.04 mg / kg , propofol 22.5 mg / kg , fentanyl 24 g / kg , and succinylcholine 2 mg / kg , endotracheal intubation was carried out by anesthesia providers with a minimum of 2 years of endotracheal intubation experience . the macintosh blades were used to expose the target larynx , and no external laryngeal pressure was used to facilitate this process . classification of laryngoscopic views was based on the method described by cormack and lehane ( figure 1b ) . continuous data are expressed as means ( standard deviation [ sd ] ) ; categorical data are expressed as numbers of occurrences ( percents ) . comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests , as appropriate , for non - continuous variables . the best fit lines of dshb , dsem , dsac , mms , iig , and tmd were determined using a least - squares regression technique . receiver operating characteristic ( roc ) analyses were used to calculate the comparable threshold values of dshb , dsem , dsac , tmd , iig , and mms . optimal cutoff values were calculated using the youden index ( calculated as : sensitivity + specificity 100 ) . statistical analyses were performed using medcalc for windows , version 12.6.1.0 ( medcalc software , ostend , belgium ) . the level of statistical significance was p<0.05 , and p<0.0001 was considered to be very statistically significant . continuous data are expressed as means ( standard deviation [ sd ] ) ; categorical data are expressed as numbers of occurrences ( percents ) . comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests , as appropriate , for non - continuous variables . the best fit lines of dshb , dsem , dsac , mms , iig , and tmd were determined using a least - squares regression technique . receiver operating characteristic ( roc ) analyses were used to calculate the comparable threshold values of dshb , dsem , dsac , tmd , iig , and mms . optimal cutoff values were calculated using the youden index ( calculated as : sensitivity + specificity 100 ) . statistical analyses were performed using medcalc for windows , version 12.6.1.0 ( medcalc software , ostend , belgium ) . the level of statistical significance was p<0.05 , and p<0.0001 was considered to be very statistically significant . a total of 203 eligible patients ( 120 females , 83 males ) were included in this study . there were 28 of 203 patients ( 13.8% ) categorized as difficult laryngoscopy , and 50% of patients with difficult laryngoscopy in our study were males . no differences were noted in sex , age , and height , but bw and bmi values in the difficult laryngoscopy group were higher . the bmi value was 25.632.80 kg / m for the difficult laryngoscopy group , and 23.613.43 kg / m for the easy laryngoscopy group ( p<0.05 ) . airway evaluation parameters , including mms , tmd , iig , dshb , dsem , and dsac , are shown in table 2 . the thicknesses of anterior neck soft tissue measured by us were greater in the difficult laryngoscopy group compared to the easy laryngoscopy group at the level of the hyoid bone , thyrohyoid membrane , and anterior commissure . however , no statistically significant difference was found between easy and difficult laryngoscopy groups for tmd and iig . a strong positive correlation existed between dsem and dshb ; moderate positive correlations between dsem and dsac , dshb and dsac ; small positive correlations between mms and dshb , mms and dsem , mms and dsac ; and very small positive correlations between tmd and iig . the correlation coefficients along with 95% confidence interval ( ci ) between dsem and dshb , dsem and dsac , dshb and dsac , mms and dshb , mms and dsem , mms and dsac , and between tmd and iig were 0.74 ( 0.670.80 , p<0.0001 ) , 0.60 ( 0.500.68 , p<0.0001 ) , 0.69 ( 0.610.75 , p<0.0001 ) , 0.32 ( 0.190.44 , p<0.0001 ) , 0.27 ( 0.140.40 , p=0.0001 ) , 0.32 ( 0.190.44 , p<0.0001 ) , 0.18 ( 0.05 to 0.31 , p=0.0089 ) , respectively ( figure 3a3 g ) . small negative correlation were found between iig and mms ( r=0.27 , p=0.0001 ) ; and very small negative correlations were found between mms and tmd ( r=0.20 , p=0.0042 ) , iig and dsac ( r=0.18 , p=0.0113 ) , and iig and dshb ( r=0.15 , p=0.0344 ) ( figure 3h3k ) . no correlations were found between tmd and dsem , tmd and dshb , and tmd and dsac ( data not shown ) . to further assess the roles of mms , iig , tmd , dshb , dsem , and dsac in predicting difficult laryngoscopy , the roc curves ( figure 4 ) were drawn using medcalc software with laryngoscopy grade over ii as the threshold of difficult laryngoscopy . as determined by the youden index , the optimal cutoff values ( with sensitivity and specificity in parentheses ) for mms , iig , tmd , dshb , dsem , and dsac to predict difficult laryngoscopy were over 2 ( 50.0% , 82.3% ) , 3.8 cm ( 75.0% , 48.0% ) , 5.9 cm ( 21.4% , 93.7% ) , 1.28 cm ( 85.7% , 85.1% ) , 1.78 cm ( 100.0% , 66.3% ) , and 1.1 cm ( 75.0% , 80.6% ) , respectively . the areas under the roc curve ( aucs ) were significantly different from the area under the reference line ( area=0.5 ) for mms , dshb , dsem , and dsac ; and the p values of mms , dshb , dsem , and dsac were all less than 0.0001 . no difference between the aucs of tmd , iig , and the area under the reference line were identified , suggesting that mms , dshb , dsem , and dsac might be better than tmd and iig in predicting difficult laryngoscopy ( table 3 ) . with the rapid expansion of portable us in anesthesia for guiding the placement of central venous catheters and the performance of regional block [ 1113 ] , some preliminary results exploring the feasibility of portable us in airway evaluation and management have been reported , even though the air attenuates us transmission and thereby creates artifacts [ 35,14,15 ] . in the present study , we compared the role of the us measurements and traditional screening tests , including iig , tmd , and mms , in predicting difficult laryngoscopy in a chinese han population . we found that strong positive linear correlations existed among the thicknesses of anterior neck soft tissue measured by us at hyoid bone , thyrohyoid membrane , and anterior commissure levels . the aucs of mms , dshb , dsem , and dsac are all over 0.7 , indicating they are all good parameters in predicting difficult laryngoscopy . the aucs of tmd and iig were less than 0.7 , suggesting that tmd and iig were poor parameters in predicating difficult laryngoscopy . to clearly visualize the glottis and smoothly finish the intubation procedure , anesthesiologists need to place the laryngoscope blade to the base of the epiglottis , and then lift it up therefore , in addition to the performer s skills and experience , many factors , including dental status , mouth opening , oropharyngeal space , mandibular space , and neck motility , are all involved this complicated procedure . mms mainly reflects the size of the tongue relative to the oral cavity , or the oropharyngeal space [ 1820 ] , iig means the mouth opening , and tmd can reflect the length of the neck . the mallapati score was first introduced by mallampati et al . in 1985 , and then modified by samsoon and young in 1987 . due to the simple and clear evaluation standards , mms has become one of the most commonly used tools to predict a difficult airway . however , mms mainly depends on patient cooperation and body position , which can greatly affect the accuracy of airway evaluation . therefore , mms has been reported to be a good predictor by many , but was found to be of limited value by others . a recent meta - analysis involving 177 088 patients demonstrate that mms is inadequate as a stand - alone test to predict difficult laryngoscopy or tracheal intubation , but it may well be a part of a multifactorial model for the prediction of a difficult tracheal intubation . iig has been repeatedly reported to be associated with difficult intubation , but our results showed that there was no significant difference in mean iig between difficult and easy laryngoscopy groups ( 3.740.43 vs. 3.860.47 cm ) . furthermore , the auc of iig was 0.580.06 , which was far less than 0.7 , suggesting that iig might be not a good screening parameter for predicting difficult laryngoscopy in our study . our iig conclusion was similar to the previous report by savva , but different from results of other studies . whether this difference was really caused by racial factors requires further investigation because the measurements of iig need patients to fully cooperate . tmd is also used as a screening test to predict difficult airway , but our results suggest that tmd is not a useful screening test . however , we could not determine whether this was because of limited patient numbers or because tmd was not useful in predicting difficult airway due to the small sample size . ct , mri , and other imaging techniques can accurately measure the thickness of anterior neck soft tissue , but they are expensive and unavailable in many operating rooms . portable us is inexpensive , rapid , and convenient to perform in the operating room , and most importantly , it can quantify the neck fat thickness as accurately as mi . however , the results of 2 prior studies focusing on soft tissue thickness at and below the level of the vocal cords in a specific group of obese patients are inconsistent . further measured the anterior neck soft tissue at the hyoid bone and thyrohyoid membrane levels , which is important to displace the glottis by the laryngoscopic blade , and found that us measurements of anterior neck soft tissue thickness at the level of hyoid bone and thyrohyoid membrane can be used to predict difficult laryngoscopies , and a 2.8-cm us measurement at the thyrohyoid membrane was a good independent predictor of difficult laryngoscopy . our results show that the thicknesses of anterior neck soft tissue at the level of the hyoid bone ( 1.51 [ 95% ci=1.401.61 ] cm vs. 0.98 [ 95% ci=0.941.02 ] cm ) , the thyrohyoid membrane ( 2.39 [ 95% ci=2.172.62 ] cm vs. 1.49 [ 95% ci=1.431.55 ] cm ) , and the anterior commissure ( 1.30 [ 95% ci=1.181.42 ] cm vs. 0.92 [ 95% ci=0.870.94 ] cm ) were greater in the difficult laryngoscopy group and were significantly correlated . furthermore , the ranges of anterior neck soft tissue for those with difficult laryngoscopy were mutually exclusive from those patients with an easy laryngoscopy , indicating that they are independent predictors of difficult laryngoscopy , even though the ranges of these 3 parameters were smaller than in a previous report . glottis exposure by place laryngoscope is a very complicated procedure , and many subjective and objective factors such as the provider s skills and experience , airway secretions , and abnormalities of anatomical structures are involved in this procedure . the investigators were not totally blinded to the study purpose , and some clinical signs might indicate the possibility of difficult laryngoscopy , which can cause some bias during us measurements . due to the low incidence of difficult laryngoscopy , it is impossible for us to randomly assign an equal number of patients to both groups , thus there were 28 patients in the difficult group and 175 patients in the easy group . anterior neck soft tissue thicknesses measured by us at the hyoid bone , thyrohyoid membrane , and anterior commissure levels are independent predictors of difficult laryngoscopy . the commonly used screening tests for difficult intubation have only poor - to - moderate predictive power when used alone . combinations of these screening tests or risk factors with us measurements might increase the ability to predict difficult laryngoscopy .

backgroundthe aim of this study was to determine if ultrasound ( us ) measurements of anterior neck soft tissue thickness at hyoid bone ( dshb ) , thyrohyoid membrane ( dsem ) , and anterior commissure ( dsac ) levels can be used to predict difficult laryngoscopy.material/methodswe included 203 patients age 2065 years scheduled to undergo general anesthesia in this prospective observational study . correlation analysis and receiver operating characteristic curve ( roc ) analysis were used to determine the roles of screening tests [ interincisor gap ( iig ) , thyromental distance ( tmd ) , modified mallampati score ( mms ) ] and us measurements ( dshb , dsem , dsac ) in predicting difficult laryngoscopy.resultsthere were 28 out of 203 patients categorized as difficult laryngoscopy . dshb , dsem , dsac , and mms were greater in the difficult laryngoscopy group ( p<0.0001 ) . there was a strong positive correlation between dsem and dshb ( r=0.74 ) ; moderate positive correlations between dsem and dsac ( r=0.60 ) , dshb and dsac ( r=0.69 ) ; small positive correlations between mms and dshb ( r=0.32 ) , mms and dsem ( r=0.27 ) , mms and dsac ( r=0.32 ) , all p values 0.0001 ; very small positive correlation between tmd and iig ( r=0.18 , p=0.0089 ) ; small negative correlation between iig and mms ( r=0.27 , p=0.0001 ) ; and very small negative correlations between mms and tmd ( r=0.20 , p=0.004 ) , iig and dsac ( r=0.18 , p=0.011 ) , iig and dshb ( r=0.15 , p=0.034 ) . the areas under the roc curve ( aucs ) of mms , dshb , dsem , and dsac were significantly larger compared with the reference line ( p<0.0001).conclusionsanterior neck soft tissue thicknesses measured by us at hyoid bone , thyrohyoid membrane , and anterior commissure levels are independent predictors of difficult laryngoscopy . combinations of those screening tests or risk factors with us measurements might increase the ability to predict difficult laryngoscopy .

w celu oceny wpywu perikardiotomii tylnej na wystpowanie wysiku osierdziowego , tamponady oraz migotania przedsionkw przeprowadzono prospektywne badanie kliniczne z randomizacj . badana grupa skadaa si z 96 pacjentw ( 77 mczyzn i 19 kobiet ) w wieku rednio 58,1 9,8 roku . pacjenci zostali losowo przydzieleni do jednej z trzech grup : grupy pacjentw poddanych perikardiotomii tylnej ( grupa i , n = 30 ) , grupy kontrolnej ( grupa ii , n = 33 ) oraz pacjentw , u ktrych zastosowano tylny drena osierdziowy , ktrzy jednak nie zostali poddani zabiegowi perikardiotomii tylnej ( grupa iii , n = 33 ) . czas trwania hospitalizacji pooperacyjnej ( p = 0,03 ; 11,56 10,64 ) oraz czsto reoperacji z powodu tamponady ( p = 0,019 ; 12,1% ) byy znaczco wysze w grupie ii . znaczny wysik osierdziowy wykryto u jednego pacjenta ( grupa ii , n = 1 ) w pierwszym dniu po operacji , u jednego pacjenta w pitym dniu po operacji ( grupa iii , n = 1 ) oraz u jednego pacjenta w 30 dniu po operacji ( p = 0,028 ) w grupie i odnotowano regresj wysiku osierdziowego . i stwierdzono rwnie wyszy wspczynnik pooperacyjnego migotania przedsionkw , jednake w tym wzgldzie midzy grupami nie byo znaczcych rnic . pacjenci , ktrzy nie zostali poddani perikardiotomii tylnej lub u ktrych nie zastosowano tylnego drenau klatki piersiowej , wykazywali szcztkowy wysik osierdziowy , wymagali duszej hospitalizacji i musieli by ponownie operowani z powodu tamponady . zarwno perikardiotomia tylna , jak i tylny drena klatki piersiowej okazay si skuteczne we wczesnym okresie pooperacyjnym . pericardial effusion , which is a common complication after coronary artery surgery ( cas ) , may be the cause of significant morbidity . it is often benign and small in volume , but can lead to life - threatening events , such as cardiac tamponade . delayed postoperative cardiac tamponade following an open - heart operation is an infrequent , but potentially serious complication . although previous reports have dealt with the clinical features and management of this problem , an optimal approach to prevent postoperative pericardial effusion and related problems has not been clearly defined [ 3 , 5 ] . this study aims to investigate the effects of posterior pericardiotomy ( pp ) on the development of pericardial effusion , tamponade , and atrial fibrillation following cas . this prospective randomized case - controlled study was carried out on 96 patients ( 77 men and 19 women ; age : 35 - 78 years ; mean : 58.1 years ) . the patients , undergoing cas performed by the same surgical team between march 2012 and january 2013 in istanbul mehmet akif ersoy thoracic and cardiovascular surgery research and training hospital , were assigned to 3 groups . after the study had received the approval of the institutional review board , informed consent was obtained from all patients . the treating physician did not know the groups to which individual patients had been assigned . patients with renal failure , hyperthyroidism , emergency cas , history of cardiac operations associated with valvular heart disease , low ejection fraction ( < 35% ) , and preoperative atrial fibrillation or other rhythm disorders , as well as patients who did not provide research authorization , were excluded from the study . the study groups included patients after pp ( group i , n = 30 ) , a control group ( group ii , n = 33 ) , and patients without pp who received posterior pericardial tubes ( the tube was placed behind the heart and sutured to the adjacent pericardial tissue to avoid tube - induced ventricular arrhythmias ) ( group iii , n = 33 ) . i , a 4-cm longitudinal incision was made parallel and posterior to the left phrenic nerve , extending from the left inferior pulmonary vein to the diaphragm , as described by mulay et al . . in group i , the relationship between the location of the distal part of circumflex anastomosis and pp was also carefully considered . moreover , all patients underwent pleurotomy and lateral pericardiotomy ( approximately 3.5 - 4.5 cm in size ) in which the internal thoracic artery ( ita ) was used for anastomosis and positioned medially to the lung , which allowed the ita to run without kinking or twisting . the inferior part of the pericardium was left open ( 2 cm ) in our study . a straight tube was placed in the anterior mediastinum and an angled tube was placed in the left hemithorax in all patients . the same postoperative pain management protocol was provided for all patients . after a routine closure of the chest , drains were frequently milked and stripped to ensure tube patency during the intensive care unit stay . the chest tubes were removed on the 2 postoperative day when the drainage became serous ; otherwise , they remained in use for one or more days . pericardial effusion was defined as the accumulation of fluid in the pericardial space determined by transthoracic echocardiography ( tte ) . the presence of pericardial effusion on 2-dimensional tte was assessed using the criteria previously described by bakhshandeh et al . . two - dimensional echocardiography was performed on postoperative days 1 and 5 , as well as 1 month after surgery . the maximum diastolic separation between the pericardium and epicardium was measured at the tip of the mitral valve leaflet . when the diastolic echo - free space between the left ventricular posterior wall and the pericardium was < 10 mm , the pericardial effusion was classified as small . when the space was 10 - 20 mm , the effusion was classified as moderate , while for spaces > 20 mm , the effusion was classified as severe . if the diagnosis of evident pericardial effusion ( moderate or severe ) was established , tte was performed daily , and the volume of the effusion was observed closely . we compared the groups with regard to the volume of effusion on postoperative days 1 , 5 , and 30 . patients with no or small pericardial effusion and an uneventful postoperative course were discharged from the hospital on postoperative day 5 . patients with moderate and severe pericardial effusion were treated with non - steroidal anti - inflammatory drugs . during hospitalization , all patients were continuously monitored with the use of a portable electrocardiogram ( ecg , telemetry ) to detect atrial fibrillation . atrial fibrillation was considered to be persistent and clinically significant when it lasted for more than 5 min . statistical analysis was performed with the ncss ( number cruncher statistical system ) 2007 statistical software ( utah , usa ) . one - way anova and tukey hsd tests were used for the statistical analysis of the groups parameters . the differences were also analyzed using tests and independent t - tests where applicable . the hospital mortality in the study was 2% ( n = 2 ) ; both patients were in group ii . one of these patients experienced low cardiac output during and following reoperation due to cardiac tamponade and died on postoperative day 12 . the other patient had an uneventful postoperative course , but was readmitted to the hospital on postoperative day 11 due to cardiac tamponade and acute kidney failure . pericardial drainage was performed , but the patient died of sepsis on postoperative day 45 . the groups did not differ in terms of demographic parameters , which are summarized in table i. the average total volume of drainage ( mediastinal and thoracic ) during the first 24 hours was similar in all groups ( 536.67 237.873 cc , 656.97 407.634 cc , and 577.27 374.349 cc , respectively ; p = 0.383 ) ( table ii ) . in group i , the volume of the fluid evacuated by mediastinal drainage was lower than in groups ii and iii , but the difference was not statistically significant ( table ii ) . two patients in group ii required reoperation due to excessive mediastinal hemorrhage and cardiac tamponade . the incidence of cardiac tamponade was higher in group ii ( n = 4 , 12% ) , and the differences between the groups were statistically significant ( p = 0.019 ) . two of the patients with pericardial tamponade were managed by subxiphoid pericardial drainage alone ; 400 to 1000 ml of fluid was removed . two patients in group iii were reoperated due to excessive hemorrhage in the early postoperative period . demographic variables in groups differential drainage volume from each chest tube the volume of pericardial drainage was added to the volume of mediastinal drainage for group iii . the average volume of pericardial effusion significantly decreased with time in groups i and ii ( fig . approximately 50% of the patients had small pericardial effusion on the first postoperative day ; no difference between the groups was found . moderate pericardial effusion was detected in 8 patients ( group i , n = 1 ; group ii , n = 3 ; group iii , n = 4 ) on the first postoperative day , in 11 patients ( group ii , n = 7 ; group iii , n = 4 ) on the fifth postoperative day , and in 7 patients on the 30 postoperative day ( group ii , n = 6 ; group iii , n = 1 ) during the study period . severe pericardial effusion was detected in one patient ( group ii , n = 1 ) on the first postoperative day , in one patient ( group iii , n = 1 ) on the fifth postoperative day , and in one patient ( group iii , n = 1 ) on the 30 postoperative day . the incidence of moderate to severe pericardial effusion in group i was significantly lower than in the other groups on the 30 postoperative day ( n = 0 , p = 0.028 ) . figure 1 shows a comparison of the groups with regard to the volume of pericardial effusion . four of these patients ( group ii , n = 4 ) underwent drainage of severe pericardial effusion on postoperative days 0 , 1 , 11 , and 17 ; one patient ( group ii ) underwent sternal dehiscence repair . comparison of the groups with regard to the volume of pericardial effusion atrial fibrillation was detected in 24 of the 96 patients ( 25% : 6 patients in group i , 11 patients in group ii , 7 patients in group iii ) . no statistically significant difference between the groups ( p = 0.392 ) was present in this respect . the length of hospital stay was significantly shorter in group i ( 6.63 2.71 , p = 0.03 ) . there are a number of articles favoring the use of posterior pericardiotomy , but no comprehensive comparison has been conducted between the use of a pericardial tube and pp in patients undergoing cas . pericardial effusion and the associated complications are common after all kinds of cardiac surgery due to postoperative bleeding or post - cardiotomy syndrome . however , clinically significant pericardial effusion occurs rarely and can be a crucial risk factor for cardiac tamponade ( 1% ) . posterior pericardiotomy has been reported to reduce the prevalence of pericardial effusion from 40% to 8% with a simultaneous reduction in the prevalence of svt ( supraventricular tachycardia ) . the semi - sitting position helps thoracic drainage in the early postoperative period by supporting the patient 's back . despite all the above - mentioned measures , the posterior wall of the pericardial sac forms an unprotected space that may cause pericardial effusion to develop septae . this leads to the appearance of adhesions preventing blood drainage , leading to cardiac compression . moreover , the collection of fluid may persist due to bleeding resulting from the use of anticoagulants . in fact , anti - coagulants ( low - molecular - weight heparin ) were used postoperatively in our patients with atrial fibrillation . posterior pericardial effusion can be evacuated by placing a tube in the posterior pericardial space . the placement of the pericardial tube is simple to perform , but it may have certain drawbacks . the tube may come into contact with coronary grafts during cardiac activity or movement . however , the patients included in the study ( in group iii as well as the other groups ) did not exhibit any arrhythmias ( apart from af ) or ischemic changes . this may be due to the fact that we fixed the pericardial tube to the surface of the diaphragmatic pericardium with polypropylene . pp or the use of posterior tubes may lessen the volume of pericardial effusion after cas . moreover , patients in group i exhibited regression of pericardial effusion during 30 postoperative days . this proved that pp had a lasting effect on pericardial effusion , as presented by figure 1 . what stands out in our results is that pericardial effusion may occur if no intervention is performed on the posterior part of the pericardium . this technique is suggested in both cas and valve surgery , but it is not routinely used [ 13 , 14 ] . what is more , pp is recommended by the american college of chest physicians guidelines and by some authors as a preventive technique for postoperative atrial fibrillation [ 15 , 16 ] . supraventricular tachyarrhythmias , mainly in the form of atrial fibrillation , occur in up to 40% of patients undergoing cas . although af is usually benign , it can cause hemodynamic instability , prolonged hospital stay , and increased cost and , in rare cases , predispose to cerebrovascular accidents . the antiarrhythmic effect of pp may , however , be a theoretical issue , as we observed similar results regarding atrial fibrillation in all the groups . in fact , the results of our study were different than expected with regard to atrial fibrillation compared to other researchers [ 4 , 6 , 16 ] . additionally , asimakopoulos et al . also found that the prevalence of atrial fibrillation was not significantly reduced ( 20% ) in comparison to the conventional technique ( 26% ) . another issue is that humans have increasingly longer life spans , which may result in higher numbers of reoperations . ellman et al . found that patent coronary grafts were the most commonly injured structures during reoperation ( 46% ) . we routinely cover the aorta and proximal anastomoses of grafts with thymic fat tissue and the anterior wall of the pericardial cavity . we also place an anterior mediastinal tube over the thymic fat tissue before closing the sternum in all patients . behind the heart , adhesions between the inferior surface of the heart and the diaphragm can cause difficulties when dissecting the tissue layers during reoperation . mulay et al . demonstrated that pp provides an effective drainage pathway from the left pleural cavity , thereby reducing the prevalence of pericardial effusion . for this reason , pp may be advantageous for reoperated patients in terms of reducing adhesions related to pericardial effusion . echocardiography can be useful in confirming and identifying the presence of pericardial effusion , hematoma , and tamponade . according to the results of our study , it is important to diagnose pericardial effusion at an early stage . hemodynamic deterioration due to pericardial fluid can be divided into four stages : pericardial fluid , hemodynamic tamponade , echocardiographic tamponade , clinical tamponade . echocardiographic follow - up of patients with pericardial effusion might be a factor extending the duration of hospital stay . we recommend the pp technique and routine echocardiographic monitoring during the postoperative period in patients undergoing cas . there is not sufficient research regarding the effects of pp on long - term results . the present study also demonstrated that patients undergoing pp required a significantly shorter hospital stay and experienced lower rates of pericardial effusion and cardiac tamponade than the other groups . this research was carried out on a small set of patients ( n = 96 ) ; therefore , there was an imbalance between men and women enrolled in this trial . group ii had a 2-fold elevation in the number of patients going into af postoperatively . in conclusion , pp may be a useful technique in the prevention of pericardial effusion and cardiac tamponade after cas because it is simple , safe , and effective . . oral presentation at the 22 annual meeting of the asian society for cardiovascular and thoracic surgery that will be held in istanbul on april 3 - 6 , 2014 .

introductionpericardial effusion and atrial fibrillation occur commonly after coronary artery surgery.aim of the studya prospective randomized clinical trial was conducted to evaluate the effects of posterior pericardiotomy on the occurrence of pericardial effusion , tamponade , and atrial fibrillation.material and methodsthe study group consisted of 96 patients ( 77 male and 19 female ) at a mean age of 58.1 9.8 years . the patients were randomly assigned to one of three study groups : patients undergoing posterior pericardiotomy ( group i , n = 30 ) , controls ( group ii , n = 33 ) , and patients with additional posterior pericardial drainage tubes ( 28 mm ) who did not undergo posterior pericardiotomy ( group iii , n = 33).resultspostoperative hospitalization ( p = 0.03 ; 11.56 10.64 ) and reoperation due to tamponade ( p = 0.019 ; 12.1% ) were significantly higher in group ii . extensive pericardial effusions were detected in one patient on the first postoperative day ( group ii , n = 1 ) , in one patient on the fifth postoperative day ( group iii , n = 1 ) , and in one patient on the 30th day after the operation ( group iii , n = 1 ) . pericardial effusion exhibited regression in group i on postoperative day 30 ( p = 0.028 ) . a higher rate of postoperative atrial fibrillation was noted in group i , but no significant differences were found between the groups with regard to postoperative atrial fibrillation.conclusionspatients who did not undergo posterior pericardiotomy or did not receive posterior chest tubes exhibited residual pericardial effusion , required longer hospitalization , and had to be reoperated due to tamponade . both posterior pericardiotomy and the use of posterior tubes are effective in the early postoperative period .

cancer places a huge burden on society and has been identified as the leading cause of death in both more and less economically developed countries . projections based on the globocan 2012 estimates predict a substantive increase to 19.3 million new cancer cases per year by 2025 , due to growth and ageing of the global population . south africa , like other developing countries , is also experiencing an increase in the overall burden of disease attributable to cancer , with the number of new cancer cases predicted to increase by 46% by 2030 . this is likely to result in an increase in the use of cancer chemotherapy agents , which assist in preventing the proliferation , invasion , and metastases of the cancer cells . the basis for chemotherapy is anticancer drugs containing platinum , that is , cisplatin ( cis - diamminedichloroplatinum ii ) and carboplatin ( cis - diammine 1,1-cyclobutane dicarboxylatoplatinum ii ) . other chemotherapy drugs include nitrogen mustard , amino - nicotinamide , dichloromethotrexate , bleomycin , and 5-fluorouracil [ 5 , 6 ] . the first of these drugs , that is , cisplatin , consists of a divalent pt ( ii ) central atom and four ligands of cis - positioned pairs of chlorine atoms or amine groups . since its discovery in the 1970s , cisplatin continues to be hailed as one of the most potent cancer chemotherapeutics in children and adults , as it is unique and unmatched in its effectiveness against many cancers , namely , osteogenic sarcoma , medulloblastoma , testicular , cervical , and ovarian cancers . similarly , its toxicity profile is expansive , involving the gastrointestinal , hematologic , renal , and auditory systems . while the use of saline hydration and mannitol diuresis may prevent nephrotoxicity , neurotoxicity is still not curable or preventable . ototoxicity refers to the hearing disorder that results from the temporary or permanent inner ear dysfunction after treatment with an ototoxic drug . other drug classes known to have ototoxic properties include aminoglycosides , loop diuretics , quinine , nonsteroidal anti - inflammatory drugs , and antiretroviral therapy ( art ) . this is of concern in south africa , as it is estimated that 12.2% of the population ( 6.4 million persons ) were hiv positive in 2012 , which is 1.2 million more people living with hiv than in 2008 ( 10.6% , or 5.2 million ) . resultantly , art exposure had almost doubled from 16.6% in 2008 to 31.2% in 2012 . not only will many infected people be at risk for ototoxicity due to arts , but a large number will also be susceptible to hiv - related cancers , such as kaposi 's sarcoma , non - hodgkin 's lymphoma , and cervical cancer , as well as infectious diseases such as tuberculosis , conditions that often require pharmacological therapy with the adverse side effect of ototoxicity . it is possible that their treatments could consist of simultaneous use of more than one ototoxic drug , increasing the likelihood of ototoxicity . all health care professionals managing patients with cancer should therefore be knowledgeable about the ototoxic properties of cisplatin . however , malhotra indicated that most oncologists in india do not make referrals for audiological evaluations of patients receiving cisplatin , while a study in south africa revealed that the effects of ototoxicity , the role of audiologists , and need for their expertise were not fully realized by the oncologists sampled . this is further supported by evidence from the south african study of khoza - shangase and jina which indicated that most general practitioners sampled also do not appear to carry out ototoxicity monitoring strategies , despite being aware of their own role within an ototoxicity monitoring programme . this review therefore aims to serve as resource for health professionals to enhance their understanding of ototoxicity as well as their roles within an ototoxicity monitoring programme by providing an overview and description of this condition in patients diagnosed with cancer and receiving cisplatin chemotherapy . the review identified peer - reviewed articles available from january 1975 to july 2015 on the topic of cisplatin - associated ototoxicity and ototoxicity monitoring and included english articles only . the same researcher conducted the literature search and reviewed the abstracts and articles for inclusion in the study . studies were identified using keyword and mesh term searches of electronic databases depicted in table 1 . a manual search of relevant authors and journals the references cited by each publication , review , or book chapter were reviewed in order to locate additional potential publications . in order to be selected , the article had to present data on either cisplatin ototoxicity and/or ototoxicity monitoring in human participants , and no research designs were excluded . running these searches yielded a total of 2106 records , of which 1581 were excluded based on the title and/or abstract as well as duplication . eighty - five relevant articles , comprising six national and 79 international articles , were selected . a perusal of narrative reviews of other auditory pathologies was conducted in an attempt to determine areas of significance for an overview of cisplatin ototoxicity . this resulted in the following eight areas being included : the mechanisms of cisplatin ototoxicity , clinical presentation , risk factors , incidence rates in adults and children , the effect on quality of life , ototoxicity monitoring , otoprotective strategies , and management of an ototoxic hearing loss . one such mechanism , the antioxidant model , involves the formation of reactive oxygen species ( ros ) within the cochlea and consequent reduction in antioxidant enzymes following exposure to cisplatin chemotherapy [ 1620 ] . another mechanism of cisplatin ototoxicity involves the significant contribution of nicotinamide adenine dinucleotide phosphate oxidase 3 isoform ( nox3 ) to the generation of reactive oxygen species within the cochlea , when activated by cisplatin [ 17 , 21 ] , while a third mechanism relates to the activation of transient receptor potential vanilloid 1 channel ( trpv1 ) [ 2224 ] . the molecular mechanisms of cisplatin ototoxicity therefore include the following:creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation ands - nitrosylation of cochlear proteins .with the cellular mechanisms of cisplatin - associated ototoxicity including damage to the outer hair cells , supporting cells , marginal cells of the stria vascularis , spiral ligament , and the spiral ganglion cells , it is evident that the structures of the inner ear are most susceptible to damage by cisplatin chemotherapy , with apoptotic degeneration of the hair cell in the organ of corti being most prominent . the outer hair cells in the basal turn of the cochlea are most affected [ 27 , 28 ] . this leads to an initial elevation of high frequency audiometric thresholds , followed by a progressive loss into the lower frequencies with continued therapy [ 27 , 28 ] . knowledge of the different mechanisms of cisplatin ototoxicity is important for health care professionals as it will create an awareness of its complexity and the resulting clinical presentation . creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation and s - nitrosylation of cochlear proteins . cisplatin - associated ototoxicity usually manifests as irreversible , progressive , bilateral , high frequency sensorineural hearing loss with tinnitus . the latter may occur with or without a hearing loss and may be permanent or transient , sometimes disappearing a few hours after treatment or alternatively persisting a week after treatment . while most of the hearing loss is permanent , there is sometimes sporadic and partial recovery . in addition , rare cases of unilateral hearing loss have been reported , which are usually explained by tumour location and surgical or therapeutic intervention on the affected side . moreover , the hearing loss is not always symmetrical [ 33 , 34 ] , with jenkins et al . finding that 75% of women on cisplatin chemotherapy displayed an asymmetry of hearing thresholds of at least 10 db between ears posttreatment . schmidt et al . , in their investigation of 55 children on cisplatin chemotherapy , found that the high frequency hearing thresholds were slightly elevated in the left ear and that males had a greater degree of hearing loss than the females . the degree of hearing loss is often variable and is related to the dose ; that is , the higher the cumulative dose , the greater the ototoxic effect [ 35 , 36 ] . the duration , number of cycles administered , and method of administration also influence cisplatin - associated ototoxicity . additional factors that may increase ototoxicity include exposure to concomitant noise , chemicals , and other ototoxic medications . furthermore , evidence also shows that melanin content is related to an increased risk of cisplatin - associated ototoxicity . individuals with dark eyes and therefore a higher melanin content in the cochlear are at greater risk of ototoxic damage , as the melanin causes retention of the platinum within the cochlear and subsequently increases the risk of damage [ 41 , 42 ] . individuals presenting with renal insufficiency , that is , high levels of serum creatinine , are at a greater risk for cisplatin - associated ototoxicity . genetic risk factors , such as megalin and glutathione s - transferases gene polymorphism , have also been reported to influence cisplatin ototoxicity , as do physiological factors such as age , with younger children and older adults ( older than 46 years ) presenting with a greater severity of hearing damage . awareness of these risk factors may assist health care professionals with informational counselling of the patient receiving cisplatin chemotherapy . the incidence of cisplatin ototoxicity is variable in adults ( table 2 ) and children ( table 3 ) . the variations may be due to a number of factors , such as differences in the dose , both within a cycle and the total amount administered over multiple cycles , time interval between courses , method of administration , and treatment duration , as well as differences in patient population . ototoxicity poses a major problem to the cancer patient , as the quality of life after receiving cisplatin chemotherapy may be negatively affected due to hearing loss , resulting in social , emotional , and vocational difficulties , as effective communication is often hindered . tasks that normal hearing persons take for granted may become challenging and frustrating . in addition , an individual 's safety may be compromised due to the hearing loss , as appropriate response to alarms and warning signals may be delayed . furthermore , a hearing loss may also result in psychosocial and physical health problems , as well as depression and social isolation . hence , hearing loss , often referred to as the invisible condition , has serious visible ramifications on the quality of life of a hearing impaired individual . this is particularly relevant if the individual has already experienced the hearing world , as the hearing function is never restored to normal , even though patients may benefit from the use of assistive listening devices , such as hearing aids and cochlear implants . the impact of an ototoxic hearing loss may be more profound for infants and young children who are at a critical stage of their speech and language development . furthermore , the high frequency nature of an ototoxic hearing loss may result in speech recognition and comprehension being compromised , resulting in possible neurocognitive and psychosocial delays . there is also an elevated risk for academic learning problems and psychosocial difficulties in school - aged children and adolescents . literature indicated that childhood survivors of neuroblastoma had twice the rate of difficulties , as indicated by parent reports , with reading and math skills , and/or attention and a higher risk of a general learning disability than those without a hearing loss . there was also poorer self - reported quality of life scores in these children with regard to school functioning . hence , cisplatin - associated ototoxicity further complicates the morbidity of cancer patients , as it would also isolate them from family members and significant others at a time when they require the greatest support . advancements in medical knowledge and technology , such as screening and early detection of several cancers , have resulted in notable improvements in relative five - year survival rates for cancer [ 64 , 65 ] . therefore , improving the quality of life after cisplatin - based chemotherapy becomes increasingly important , and resulting comorbidities such as ototoxicity can be managed appropriately and immediately if adequate monitoring is in place . the nature of ototoxicity is such that it often goes undetected until speech intelligibility is affected and is usually detected when a communication problem becomes evident . communication problems , such as constantly asking for repetition or not responding when spoken to , signify that the hearing loss has progressed to the frequencies important for understanding speech . in this case , an audiological monitoring programme can avert , to a large extent , the reduced quality of life as a result of hearing loss , as patients on cisplatin chemotherapy can be identified early , counselled , monitored , and managed appropriately through interventions in a logical , systematic , and coherent manner . audiological monitoring should aim to identify the hearing loss early and reduce its impact on the individual 's life by means of proper medical and hearing intervention . prospective audiological evaluations remain the only reliable method for detecting ototoxicity before it becomes symptomatic . an ototoxicity monitoring programme should involve a health care team comprising of an oncology nurse , oncologists , audiologist , and pharmacist to ensure effective sustainability of such a programme , if implemented , with the patient being the central focus . the audiologist is involved in identifying an ototoxic hearing loss , informing the oncologist of such a development , counselling the patient and their family , and prescribing amplification devices , such as hearing aids and cochlear implants . early identification of an ototoxic hearing loss provides oncologists with an opportunity to adjust the chemotherapy regimen in order to reduce or prevent further deterioration of hearing . the oncologist and nurses should also counsel patients on the side effects of cisplatin , including ototoxicity , in an attempt to prepare them for treatment outcomes and help them set realistic expectations . pharmacists who have access to a patient 's medication list may also alert the oncologists and audiologists to those who are on other ototoxic medication and therefore at a greater risk for cisplatin - induced ototoxicity . effective management of such patients using evidence - based practices may improve management of those with cancer , ensuring that they and their families are counselled and appropriate interventions are timeously implemented . the principles of early identification and early intervention are a part of ototoxicity monitoring , and the audiologist can manage such a programme . in countries without ototoxicity management guidelines , the guidelines for the audiological management of individuals receiving cochleotoxic drug therapy developed by the american association of speech - language - hearing association may , consequently , guide the audiologist in the implementation of an ototoxicity monitoring programme within a local , regional , or national setting . for widespread acceptance and use , ototoxicity monitoring programmes need to incorporate efficient and cost - effective ototoxicity identification techniques , while considering the health care system and demographics of the patient population being managed . for any population receiving ototoxic medication , the following should be considered : ( 1 ) the patient 's level of alertness or ability to respond reliably ; ( 2 ) the most appropriate times during the treatment protocol for test administration , and ; ( 3 ) the test should comprise the baseline , monitoring and post - treatment evaluations . appropriate time intervals for audiological assessments may differ depending on the type of cancer as well as the frequency and dose of cisplatin ( figure 2 ) . the audiological assessments should incorporate a detailed case history , otoscopic examination , immittance audiometry , speech audiometry , dpoaes , and conventional and extended high frequency audiometry ( i.e. , up to 20 000 hz ) ( hfa ) [ 69 , 73 ] . these procedures are all conducted for the baseline assessment and the six - month follow - up evaluation [ 69 , 73 ] . while auditory brainstem response may be used , it is not considered a standard procedure for monitoring ototoxicity . monitoring audiological evaluations during treatment and the one- and three - month follow - up evaluations include case interview , otoscopy , and immittance audiometry as well as air conduction pure tone and objective testing . however , full - frequency threshold testing is impractical for many patients on cisplatin chemotherapy , as these individuals are often extremely ill and easily fatigued . the use of abbreviated threshold monitoring procedures that are clinically practical for these patients is therefore recommended . one such method involves the use of the sensitive range for ototoxicity ( sro ) . this is the highest frequency with a threshold at or below 100 db spl followed by the next six lower adjacent frequencies in 1/6-octave steps or the one octave range near the highest audible frequency . sro is usually determined during the baseline evaluation and is dependent on each patient 's hearing threshold configuration . during monitoring evaluations however , full - frequency testing should be conducted within the same session if an asha significant hearing change is noted within the sro . if a patient on cisplatin chemotherapy is still responsive and alert , the protocol presented above would be suitable . however , a patient who has limited responsiveness may be required to undergo the same audiological evaluations , except speech audiometry . patients who are responsive as well as those who have limited responses can undergo both behavioural and objective testing . however , those patients who are too ill or too young to respond should undergo only objective testing , such as otoscopy , tympanometry , acoustic reflexes , and dpoaes or abrs . while pure tone audiometry in the conventional frequency range is suitable for evaluating hearing in the range responsible for speech understanding , as well as for differential diagnosis , it is less sensitive to detecting early ototoxic change [ 11 , 70 ] . the two tests identified as being the most important for the early detection of cisplatin ototoxicity are hfas and oaes , each also having limitations ( see table 4 ) . therefore , using each test in isolation may not be as effective as utilizing a test battery approach , as it increases the chances of obtaining reliable audiologic monitoring data over time . in addition , these two tests could be used to complement one another in every cycle of chemotherapy to ensure the earliest detection of ototoxicity . the ototoxicity monitoring protocol proposed by asha represents an aggressive and ideal approach for monitoring ototoxicity and is dependent on a country 's infrastructure and resource constraints . the asha guidelines may therefore not be generalized to a country without considering the contextual factors that may influence its applicability to that country . however , it does provide guidance towards creating a roadmap that countries , such as south africa , may aspire towards in implementing an ototoxicity monitoring programme . similar to india , no programmes have been formally implemented to identify and monitor ototoxicity in patients on cancer chemotherapy in south africa . as a result , there is no contextually relevant research to steer the implementation of an accountable and effective ototoxicity monitoring program in the country . this is probably one of the main reasons for ototoxicity monitoring programmes not being commonplace in local hospitals and clinics . in addition , the health of south africans is characterized by a quadruple burden of disease , encompassing the occurrence of infectious diseases , the rise of noncommunicable diseases , and perinatal and maternal disorders , as well as injuries and violence , which may result in cancer receiving low priority for health care services . however , the creation of an audiological monitoring programme allows for better control of cancer related comorbidities . over the years , a number of studies have investigated the use of otoprotectants with cisplatin , their purpose being to protect the inner ear from any injury while not interfering with the antitumor effects of cisplatin . otoprotective strategies include reducing the formation of free radicals by maintaining glutathione levels and antioxidant activity . three mechanisms may provide protection against cisplatin , these being endogenous molecules , exogenous agents , or a combination of exogenous agents that trigger endogenous protective mechanisms . however , endogenous agents are not effective against cisplatin when the dose exceeds a certain threshold [ 17 , 81 ] . nearly all of the otoprotective agents are sulfur- or sulfhydryl - containing compounds ( thio compounds ) , known as antioxidants , and potent heavy metal chelators . the numerous otoprotective agents utilized in clinical and animal studies include amifostine , d - or l - methionine , methylthiobenzoic acid , lipoic acid , tiopronin , glutathione ester , sodium thiosulfate , melatonin , vitamin e , n - acetylcysteine , dexamethasone , and resveratrol . however , none of these agents have been found to be unequivocally beneficial in preventing cisplatin ototoxicity and no agent is currently recommended for routine use . further research is therefore needed to find new methods and optimize old ones to prevent and/or treat hearing loss during cisplatin therapy . in addition , intratympanic administration of medication together with gene therapy needs to be further explored . intratympanic administration involves the diffusion of the otoprotective agent across the round window into the inner ear , where its therapeutic effect is exerted . an advantage of this method of administration is that there are higher concentrations of the otoprotective agent in the inner ear , this being in comparison to the use of oral or parenteral administration , without potentially reducing the efficacy of the cisplatin treatment [ 89 , 90 ] . the disadvantage of this procedure , however , is that each ear would have to be treated with a moderately invasive procedure . alternatively , gene therapy may prove to be beneficial in protecting an individual against cisplatin - induced hearing loss as several genes , namely , megalin , glutathione - s - transferases , thiopurine s - methyltransferase , and catechol - o - methyl transferase , may be responsible for susceptibility to hearing loss . if a cisplatin - associated hearing loss results in communication difficulties , it is the audiologist 's ethical responsibility to begin or recommend aural rehabilitation . however , this intervention should not only occur once hearing loss has been detected but before the patient begins the cisplatin chemotherapy . aural rehabilitation techniques such as speech reading and counselling on compensatory communication strategies should be conducted . the counselling should include spouses and significant other , as hearing loss may not only impact the person with cancer but also frequent communication partners . patients with sensorineural hearing loss due to the use of cisplatin may also benefit from the use of assistive listening devices such as hearing aids or cochlear implants . children with ototoxic hearing loss may also require the use of personal frequency modulated systems in the classroom . furthermore , with the recent developments in hearing aid technology , a patient with an ototoxic hearing loss is more likely to receive the desired amplification benefit . these hearing aids are able to amplify sounds at and above 8000 hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping . frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound . while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds . commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) . these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively . these hearing aids are able to amplify sounds at and above 8000 hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology . hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping . frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound . while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds . commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) . these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively . this review has highlighted that cisplatin ototoxicity is a frequent adverse event of cisplatin chemotherapy that may negatively affect the quality of life of patients with cancer . the different molecular and cellular mechanisms involved in cisplatin - associated ototoxicity highlight the complexity of this condition and the consequent difficulty in identifying an effective otoprotective agent . the varying incidence rates reported in both adults and paediatrics may be due to the different audiological tests employed in the monitoring of the cancer patient 's hearing status and therefore highlight the importance of the use of extended high frequency audiometry and dpoaes in ototoxicity monitoring . an audiological monitoring programme comprising a team of health care professionals , knowledgeable about cisplatin ototoxicity , may therefore serve to improve evidence - based service delivery to these patients .

cisplatin is an effective drug used in the treatment of many cancers , yet its ototoxic potential places cancer patients , exposed to this drug , at risk of hearing loss , thus negatively impacting further on a patient 's quality of life . it is paramount for health care practitioners managing such patients to be aware of cisplatin 's ototoxic properties and the clinical signs to identify patients at risk of developing hearing loss . english peer - reviewed articles from january 1975 to july 2015 were assessed from pubmed , science direct , and ebscohost . seventy - nine articles and two books were identified for this review , using mesh terms and keywords such as ototoxicity , cisplatin , hearing loss , and ototoxicity monitoring . this review provides an up - to - date overview of cisplatin - associated ototoxicity , namely , its clinical features , incidence rates , and molecular and cellular mechanisms and risk factors , to health care practitioners managing the patient with cancer , and highlights the need for a team - based approach to complement an audiological monitoring programme to mitigate any further loss in the quality of life of affected patients , as there is currently no otoprotective agent recommended routinely for the prevention of cisplatin - associated ototoxicity . it also sets the platform for effective dialogue towards policy formulation and strengthening of health systems in developing countries .

the synthetic modification of proteins enables the construction of biomolecular hybrids that can be used to study protein function , deliver potent therapeutics to cellular targets , and build new materials . the synthesis of these constructs requires a suite of chemoselective bioconjugation reactions that proceed under mild , aqueous conditions in the presence of the native functional groups that are present on protein surfaces . the most common methods for protein modification target the nucleophilic side - chains of lysine and cysteine . however , these strategies can result in complex product mixtures , as lysine is typically found in high abundance on the protein surface and uniquely reactive cysteine labeling sites can be difficult to install in many instances ( such as thiol proteases and proteins produced via the eukaryotic secretory pathway , for example ) . many newer approaches for the site - selective modification of proteins involve the introduction of artificial amino acids with reactivities that are orthogonal to those of the native amino acids . along these lines , a number of powerful methods have been developed for the selective modification of azide , alkyne , alkene , carbonyl , and aniline moieties . however , the difficulty of introducing a non - canonical amino acid can limit the application of these methods . complementary approaches rely on the site - selective modification of native amino acids by enzymes . in addition , a reliable method for the modification of c - terminal thioesters with n - terminal cysteines , termed native chemical ligation , has been developed by kent and co - workers . this method has been used for the semi- and total synthesis of complex protein substrates , including the chemical synthesis of a single glycoform of human erythropoietin . as an alternative strategy , we and others have developed methods for the selective modification of the n - terminal amino group . methods that target the n - terminus can offer significant advantages for bioconjugate preparation , as they can be used for a wide range of protein targets produced by virtually any expression system . conceptually powerful as they are , however , these methods can be hampered by long reaction times , often require large excesses of reagent , and/or involve at least two - steps for the attachment of synthetic molecules . we have therefore sought to develop new techniques that can achieve n - terminal modification with similarly high positional selectivity , but with significantly improved efficiency . herein , we report an oxidative coupling pathway that can preferentially modify the n - terminus of proteins with fast kinetics . peptide substrates were first used to screen reaction conditions and identify the site of modification . a peptide panel with varying n - terminal residues was then evaluated to determine the sequence specificity of the reaction , leading to the identification of proline as the optimal n - terminal amino acid . the reaction was next applied to protein substrates , showing similarly high levels of conversion when an n - terminal proline residue was present . this mild bioconjugation reaction enables the facile , rapid modification of proteins to create a well - defined and stable linkage in a single position , and thus should be useful for many different applications in chemical biology and the construction of biomolecular materials . we have previously reported the chemoselective coupling of aniline moieties on proteins to electron - rich aromatic rings , such as o - aminophenols , at slightly acidic ph ( 6.06.5 ) . these reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant , with the latter reagent exhibiting improved compatibility with glycoproteins and substrates with free sulfhydryl groups . the use of ferricyanide as the oxidant also yields a single reaction product ( 1 ) , whereas periodate leads to the formation of a ring contracted species as a competing pathway . the ferricyanide - based reactions are presumed to involve an o - iminoquinone as the reactive intermediate , as suggested in scheme 1 , or could involve the corresponding o - quinone after imine hydrolysis . taken together , the oxidative coupling strategies have demonstrated excellent functional group compatibility and the ability to join large unprotected biomolecules at low concentrations , as demonstrated for the coupling of peptides , polymers , and nucleic acids to specific locations on viral capsids and antibody fc domains . while these coupling reactions were found to be highly aniline - selective under the conditions used , several studies have reported the reaction of o - aminophenols and o - catechols with native amino acids , dating back to 1949 . in addition , recent work by messersmith has shown the ability of proteins to be coupled to o - quinone moieties present on polydopamine - coated surfaces . these reports suggested that secondary coupling pathways could be developed to achieve the modification of native amino acids with o - aminophenols ( scheme 1 ) , and thus initial experiments were designed to identify the optimal reaction conditions for achieving this with complex molecules . in our previous work , we noted that low amounts of background reactivity could be observed in aniline - based oxidative coupling reactions when higher ph conditions ( > ph 6.5 ) were used . in an initial effort to characterize this alternative reaction pathway , conditions and reaction times were first screened to increase the reaction yields for peptides that did not contain aniline groups . angiotensin i and melittin were used as substrates , as they contain many reactive amino acids , including lys , arg , his , trp , and tyr . the peptides were exposed to 2-amino - p - cresol using k3fe(cn)6 as the oxidant . the reaction ph was varied from 5.5 to 8.5 , and the reaction mixtures were analyzed using maldi - tof ms ( supporting information figure s1 ) . the level of modification increased with the basicity of the reaction , with near quantitative modification of angiotensin i after 20 min at ph 7.5 and higher . throughout these initial investigations , it was noted that angiotensin and melittin showed significant differences in reactivity , with angiotensin consistently demonstrating better conversion . ms / ms analysis of the angiotensin product was used to identify the participating residue , and revealed that the n - terminal amino group was responsible for the observed reactivity ( supporting information figure s2 ) . as further confirmation of the site - selectivity , several peptide substrates were screened for reactivity ( figure 1a and supporting information figure s3 ) . consistent with the n - terminal reaction selectivity , the only peptide that did not react had a pyroglutamate in this position , and therefore no free amino group . ( a ) modification of commercially available peptides was monitored by maldi - tof ms . ( b ) a positional scan of the n - terminal amino acid was evaluated . peptides with the sequence xadswag were tested for reactivity with 2-amino - p - cresol . the reactions were run with 100 m peptide , 200 m aminophenol , and 5 mm ferricyanide at ph 7.5 and analyzed by lc ms . shown is the average percent modification with error bars representing the standard deviation of three reactions . the same peptides were used for a screen of coupling partner equivalents ( c ) and a time screen ( d ) . most buffers did not alter the reactivity , but imidazole and buffers containing a morpholine or piperazine ring ( pipes , hepes , hepps , and mops ) significantly impeded the reaction ( supporting information figure s4 ) . this is possibly due to small amounts of buffer impurities that react competitively with the oxidized intermediate . the time course of the reaction was also investigated ( supporting information figure s5 ) . in addition , it was found that the peptides could be modified using naio4 as the oxidant , or 4-methylcatechol as the coupling partner ( supporting information figures s67 ) . both of these reactions showed the same dependence on ph ; however , moderate levels of modification were still observed at acidic ph ( 5.56.5 ) when using these alternative coupling conditions . these observations suggested that the peptides were reacting with the o - quinone intermediate formed in situ from either the catechol or the iminoquinone precursor ( after imine hydrolysis ) . given the differential reactivity observed on peptide substrates , we synthesized peptides with varied n - terminal residues ( xadswag ) to determine the specificity of the reaction . the base sequence was selected to increase the mass of the peptide , impart water solubility , and include a tryptophan residue for quantitation using uv monitoring . the peptides were synthesized on the solid phase using standard fmoc synthesis , cleaved from the resin , and purified by hplc . after purification , the peptides were resuspended in phosphate buffer , ph 7.5 , adjusted to a concentration of 1 mm , and stored at 20 c until use . to assess the effect of the n - terminal residue on reactivity , the peptides ( 100 m ) were reacted with 2 equiv of 2-amino - p - cresol ( 200 m ) in the presence of k3fe(cn)6 ( 5 mm ) in phosphate buffer , ph 7.5 ( figure 1b ) . ms ( see supporting information figure s8 for representative ms data for the modified peptides ) . most n - terminal amino acids showed good - to - high levels of conversion ( 6090% ) , but proline stood out as the only residue that showed nearly complete modification ( 90100% ) . a second observation of this screen was the fact that tryptophan , tyrosine , and methionine residues were not oxidized by the ferricyanide reagent , consistent with our previous report of oxidative coupling with this oxidant ( see supporting information figure s8 ) . however , free cysteine residues can be oxidized to various species , potentially including disulfides and sulfenic acids , and thus it is recommended that they be protected as disulfides before oxidative coupling is attempted ( vide infra ) . to optimize the reagent ratios ( specifically the equivalents of o - aminophenol ) , the peptides ( 100 m ) were reacted with 110 equiv of the o - aminophenol ( 1001000 m ) in the presence of ferricyanide ( 10 mm ) . after 30 min , the reactions were quenched with excess tris(2-carboxyethyl)phosphine ( tcep ) . it was demonstrated that conversion was highest using 25 equiv of the coupling partner ( figure 1c ) . using more than 5 equiv of this was most likely due to the ability of the aminophenol to react with itself at higher concentrations ( 1 mm ) . consistent with this , when using 10 equiv of the o - aminophenol , a byproduct was observed with a mass that corresponded to the condensation of 3 aminophenols ( 344 da ) . we also investigated the differences in coupling rates for representative n - termini . the reaction of 2-amino - p - cresol with three different peptides was monitored over the course of 1 h ( figure 1d ) . the peptides ( 100 m ) were reacted with 2 equiv of the o - aminophenol ( 200 m ) in the presence of ferricyanide ( 5 mm ) , and aliquots were quenched with excess tcep at the indicated time points . the proline terminal peptide not only reached the highest level of conversion , but also did so in a significantly shorter time than the other termini . despite efforts to optimize conditions for all n - termini the reaction of n - terminal amines with o - aminophenols was characterized using small molecule mimics . the methyl esters of phenylalanine ( h - phe - ome ) and proline ( h - pro - ome ) were coupled to 2-amino - p - cresol using ferricyanide at ph 7.5 . the crude products were characterized using two - dimensional nmr and high - resolution mass spectrometry . the primary amine of h - phe - ome formed p - iminoquinone product 2 , which was analogous to the one formed with aniline coupling partners ( scheme 1 , supporting information figure s9 ) . however , the secondary amine of proline prevented the formation of the p - iminoquinone tautomer , and thus favored o - quinone product 3 ( scheme 1 , supporting information figure s10 ) . given the different linkage obtained with proline , we verified the stability of the product to a variety of conditions . the proline terminal peptide , padswag , was first modified with 2-amino - p - cresol . after purification , the modified peptide ( 100 m ) was exposed to reductants , nucleophiles and acidic and basic ph ( 10 mm additives or buffer ) . no loss of product was observed under any of the conditions tested , demonstrating the hydrolytic stability of the product ( supporting information figure s11 ) . the ability of the linkage to withstand these conditions renders this method quite useful for the construction of biomolecular materials for a variety of applications . with a view toward in vivo applications , current efforts are examining the stability of the linkage in blood plasma , as well as evaluating the intrinsic immunogenicity of the o - quinone group . in the process of characterizing the reaction products , it was observed that the colored products absorbed light at wavelengths greater than 500 nm ( with max between 505 and 525 nm depending on the amine coupling partner ) . as the starting coupling partners and ferricyanide did not absorb at these wavelengths , this unique absorbance provided a means to monitor the reaction progress . the different amine coupling partners ( p - toluidine , h - pro - ome , and h - phe - ome ) were reacted with 4-methylcatechol in the presence of 10 mm ferricyanide , and the absorbance of the resulting solution was monitored at 520 nm to determine the relative rates of reactivity ( figure 2a ; for unnormalized data see supporting information figure s12 ) . the catechol substrate was used for these studies to simplify the reaction pathway by eliminating the imine hydrolysis step . the reactions were run under pseudo - first order conditions with 0.1 mm catechol and 1 mm amine coupling partner . when the reaction was carried out at ph 6.0 , the reaction with the proline analogue reached completion nearly as rapidly ( 2 min ) , but the reaction with the primary aliphatic amine of phenylalanine required longer reaction times ( 10 min ) . ( a ) amine coupling partners were reacted with 4-methylcatechol as a model substrate . reactions were run under pseudo - first order conditions with 100 m catechol , 1 mm amine , 10 mm ferricyanide in 50 mm phosphate buffer . ( b ) a peptide containing both an n - terminal proline and a p - aminophenylalanine residue ( pad(paf)swag ) was tested for reactivity with 2 equiv of 2-amino - p - cresol at ph 6 . the remainder of the reaction was purified and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed by lc ms . by quantifying product formation by absorbance , we were also able to measure the second - order rate constant for the proline - based coupling ( supporting information figure s13 ) . the reaction of 1 equiv of h - pro - ome ( 100 m ) with 1 equiv of 4-methylcatechol ( 100 m ) and 100 equiv of k3fe(cn)6 ( 10 mm ) was performed in triplicate at 25 c . the second - order rate constant for the coupling was determined to be 44 4 m s. while proline reacted rapidly with the electron - rich coupling partner , the small molecule studies indicated that aniline should react faster . the rate for the aniline reaction was too fast under these conditions to determine the second - order rate constant accurately . given the differences in reactivity observed at ph 6.0 and 7.5 , we hypothesized that it would be possible to modify the aniline side chain of p - aminophenylalanine ( paf ) and the n - terminal proline amine sequentially . to test this hypothesis , we synthesized a peptide containing both reactive moieties ( pad(paf)swag ) . only one modification was observed when the peptide was reacted with 2-amino - p - cresol at ph 6.0 ( figure 2b ) . ms / ms analysis of the modified peptide confirmed that the single modification occurred on the aniline side chain ( supporting information figure s14 ) . after this step , the peptide was purified and subsequently reacted with 2-amino - p - cresol at ph 7.5 . reaction at the higher ph enabled a second modification of the peptide substrate , at the n - terminal proline residue . the differential reactivity was investigated by comparing the reactivity of a protein containing a paf residue to proteins without the artificial amino acid . the paf residue was introduced into the coat protein of bacteriophage ms2 , which self - assembles into a spherical , hollow protein shell . myoglobin and a mutant of the tobacco mosaic virus ( tmv ) coat protein were used as native protein substrates . reactions with 2-amino - p - cresol were either performed on the isolated , individual proteins or with the aniline containing protein mixed with the native protein substrate ( supporting information figure s15 ) . addition of the aniline containing protein to the native protein decreased the n - terminal reactivity , indicating that the aniline residues react more rapidly than n - terminal residues with the o - aminophenols . in addition , ms2 showed significantly higher reactivity at all of the phs tested , confirming preference for aniline residues . the oxidative coupling reaction with n - terminal amino groups was first tested on proteins with native n - termini . several proteins were reacted with o - aminophenol - functionalized 5 kda peg under the optimized reaction conditions ( figure 3 ) . the native proteins showed moderate levels of reactivity , which could be attributed to inaccessible n - termini or simply to the less reactive n - terminal residues . to test if proline terminal proteins were more reactive , a proline residue was introduced to the n - terminus of gfp and the tobacco mosaic virus ( tmv ) coat protein . the n - terminus of tmv was also slightly extended from the native sequence ( addition of pag ) . the proline - gfp was treated with a variety of conditions to determine the specificity of the reaction ( figure 4a ) . only at basic ph in the presence of both the o - aminophenol substrate and the oxidant was modification observed . additionally , the proline - terminal variant showed significantly improved reactivity compared to that of the wild - type n - terminus . these high levels of modification were maintained even when only 12 equiv of the o - aminophenol peg was used . the site of modification was confirmed to be the n - terminal proline by lc ms / ms analysis of a tryptic digest of proline - gfp modified with 2-amino - p - cresol ( supporting information figure s16 ) . ( a ) the n - terminus of several proteins was pegylated using o - aminophenol - functionalized 5 kda peg and ferricyanide . ( b ) modification of wild type proteins with 5 kda o - aminophenol - peg was monitored by sds - page . ( a ) a proline was introduced to the n - terminus of gfp . the proline terminal variant showed much higher levels of modification than the wild - type protein . the band doubling is due to a gel artifact , and appears in all lanes . ( b ) mutants of tmv were reacted with 5 equiv of 2-amino - p - cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc ms . reaction conditions for both native and proline terminal proteins were optimized by evaluating reactivity with myoglobin and proline - gfp . the reaction time , buffer , and ph were screened ( supporting information figures s17 and s18 ) . similar to the results obtained with peptide substrates , the reaction reached its highest level of conversion after about 1530 min . in addition , most buffer salts tested were compatible with the reaction with the exception of buffers containing morpholine ( mops ) or piperazine moieties ( hepes ) , as was observed with peptide substrates . these buffers decreased the level of modification slightly , but did not completely inhibit reactivity . the effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants . little reactivity was observed at acidic ph , and the level of conversion increased between ph 7.0 and 8.0 . at higher reaction ph ( 8.0 ) a second modification was observed , indicating that lysines may also participate in the reaction . however , it is also possible that under more forcing conditions , such as higher reaction ph or increased concentration of aminophenol substrate , the aminophenol reacts with both itself and the n - terminal amino group resulting in double modification of the n - terminus . n - terminal mutants of tmv were also evaluated for their reactivity with o - aminophenols . the tmv monomers assemble into well - known double disk structures , displaying 34 copies of the n - terminal groups on their peripheries . two n - terminal mutants ( pag and ag ) were reacted with 5 equiv of 2-amino - p - cresol ( 100 m ) and 1 mm k3fe(cn)6 for 30 min . ms demonstrated that the proline terminal mutant reached nearly complete conversion , while the alanine terminal mutant showed low levels of modification under these coupling conditions ( figure 4b , see supporting information figure s19 for wider mass range and ion series ) . the compatibility of the reaction with cysteine residues was also tested using tmv . a single cysteine residue ( s123c ) was introduced into the tmv coat protein with a proline n - terminus ( pag s123c tmv ) . this mutant was reacted with 2-amino - p - cresol and analyzed by lc the cysteine residue also reacted with the o - aminophenol , resulting in two modifications . however , it was found that the n - terminal proline could be modified selectively if the cysteine was first capped ( figure 5a , b , see supporting information figures s2122 for wider mass range and ion series ) . to do this , the cysteine residue was protected as a disulfide bond by reaction with 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb , ellman s reagent ) . after the oxidative coupling step the disulfide bond was readily reduced by tcep , leaving the free cysteine and the modified n - terminus . alternatively , the cysteine residue was modified with a maleimide , followed by modification at the n - terminus with an o - aminophenol reagent . a ) pag s123c tmv was reacted with small molecule substrates and analyzed by lc cysteine residues were protected as a disulfide using ellman s reagent ( dtnb ) before oxidative coupling . subsequent reduction of the disulfide resulted in selective modification of the n - terminus . the n - terminal proline was then modified with a rhodamine - functionalized o - aminophenol . this strategy allowed for the direct , dual modification of the protein at both the cysteine residue and the n - terminus . two fluorophores paired for frster resonance energy transfer ( fret , alexa fluor 488 c5-maleimide and o - aminophenol functionalized rhodamine b ) were thus conjugated to tmv using this strategy to create a templated array of chromophores for light harvesting applications . the free cysteine was first quantitatively labeled with an alexa fluor maleimide ( supporting information figure s22 ) . the n - terminal proline was then coupled to a fluorescent o - aminophenol resulting in 50% modification of the tmv monomers with both fluorphores ( figure 5b ) . complete modification of the n - terminus was not observed as tmv precipitated from solution with increasing levels of modification with the rhodamine dye . in current experiments , we are using this dual - labeling strategy to introduce more soluble chromophores . the oxidative coupling reaction was also compared to the reaction of protein amines with activated esters . this acylation methodology is commonly employed , and can be targeted to the n - terminus by controlling the reaction ph in some cases . the reactions were compared on creatine kinase , a protein with a native proline n - terminus . reaction with 15 equiv of o - aminophenol peg resulted in good levels of modification of creatine kinase ( 5060% ) , while reaction with 15 equiv of n - hydroxysuccinimide ( nhs ) peg resulted in low levels of modification ( 525% , figure 6 ) . only when a vast excess of the nhs peg was used were moderate levels of modification achieved . as was the case with proline - gfp , some over modification was observed under the oxidative coupling conditions when using five or more equivalents of aminophenol . this could result from dimerization of the oxidized species before protein coupling , but has yet to be characterized due to the low abundance of this product . in any case , lowering the reaction ph slightly or using fewer equivalents of the o - aminophenol substrate prevented the over modification from occurring . the modification of the n - terminus of creatine kinase with aminophenol peg was compared to the reaction of creatine kinase with nhs peg . in this study , we have identified conditions for the oxidative coupling of o - aminophenols to n - terminal amino acids . proline residues work particularly well with this strategy , and are therefore strongly recommended when using it . these groups can be introduced readily in n - terminal positions using site - directed mutagenesis and escherichia coli expression , especially since the methionine residue resulting from the start codon is cleaved when proline is in the second position . the fast kinetics of the reaction allow it to be successful even at low reagent and substrate concentrations , and suggest that it can be used for sterically demanding bioconjugations . the oxidative coupling strategy reported here offers two distinct advantages over other n - terminal labeling methods . first , the modification occurs in a single step and does not require initial oxidation of the n - terminus . second , the fast second - order kinetics allow for low concentrations of the coupling partners to be used . however , to achieve high levels of modification on protein substrates , proline was required as the n - terminal residue . this new protein modification strategy is currently being explored in our lab for the generation of protein - based materials . in the larger context , new techniques for the introduction of a single functional group in a specific position on a protein surface are always in demand . the ability of the n - terminal oxidative coupling method to achieve this in a single , brief reaction step is highly advantageous , and the fact that it can be combined with cysteine modification chemistry provides new opportunities for complex bioconjugate synthesis .

the synthetic modification of proteins plays an important role in chemical biology and biomaterials science . these fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces . in this work , an oxidative strategy is demonstrated for the efficient modification of n - terminal residues on peptides and n - terminal proline residues on proteins . the strategy uses o - aminophenols or o - catechols that are oxidized to active coupling species in situ using potassium ferricyanide . peptide screening results have revealed that many n - terminal amino acids can participate in this reaction , and that proline residues are particularly reactive . when applied to protein substrates , the reaction shows a stronger requirement for the proline group . key advantages of the reaction include its fast second - order kinetics and ability to achieve site - selective modification in a single step using low concentrations of reagent . although free cysteines are also modified by the coupling reaction , they can be protected through disulfide formation and then liberated after n - terminal coupling is complete . this allows access to doubly functionalized bioconjugates that can be difficult to access using other methods .

alcohol withdrawal syndrome ( aws ) is characterized by varied symptoms that range from mild to severe intensity depending on several factors including the quantity , frequency and duration of alcohol intake , and the number of prior withdrawal episodes , as well as individual differences in the vulnerability [ 14 ] . symptoms usually present themselves within 6 to 24 hours after cessation of alcohol intake [ 5 , 6 ] . subtyping of the aws has been attempted in the past , as gross conceptualized and proposed 3 constellations of alcohol withdrawal symptoms : factor 1 hallucinogenic that consisted of nausea , tinnitus , visual disturbance , pruritus , parasthesia , muscle pain , agitation , sleep disturbance , tactile hallucinations , and hallucinations which are auditory or visual or both ; factor 2 affective and physiological that consisted of anxiety , depression , tremor , and sweats ; and factor 3 delirium that consisted of clouding of the sensorium , impairment of consciousness , and impairment of contact with the observer . a cluster analytic study identified three different symptoms clusters of alcohol withdrawal , namely , cns excitation , adrenergic hyperactivity , and delirium . several rating instruments have been used to measure severity of alcohol withdrawal . among them , the most commonly used observer - rated scale is the 10-item clinical institute withdrawal assessment - alcohol , revised ( ciwa - ar ) . it has been proposed that alcohol withdrawal symptoms in ciwa - ar appear multidimensional . a pubmed search supplemented with the study by pittman et al . was to explore the relationship between awsc and ciwa - ar , for which they carried out study on 127 male inpatients of alcohol dependence with principle components factor extraction with varimax rotation of ciwa - ar , a self - rated alcohol withdrawal symptoms checklist ( awsc ) and on combined items of ciwa - ar and awsc . they found three , five , and seven factor solution , respectively , for ciwa - ar , awsc , and combination of ciwa - ar and awsc . the analysis of ciwa - ar was done on 7 items as 3 items had zero variance . the first factor ( variance 23.9% ) was tension / anxiety which consisted of anxiety , agitation , and tactile disturbances . the second factor ( variance 22.9% ) was autonomic arousal which consisted of paroxysmal sweats , tremor , and headache or fullness in head , whereas the third factor ( variance 17.4% , eigenvalue less than 1 ) was nausea and vomiting which consisted of a single item , nausea , and vomiting . in our setup , we use ciwa - ar as part of the measure for the management of alcohol withdrawal symptoms . it is generally observed that alcohol withdrawal symptoms fluctuate in presentation and severity across time . the present study was carried out to explore the dimensionality of this scale in an attempt to identify a set of underlying factors that exist and can explain the interrelationships among various manifestations of acute alcohol withdrawal symptoms . the knowledge of these underlying factors may enhance our understanding of aws and better prediction of complications thus management plans . this was a cross - sectional hospital - based study , conducted at centre for addiction psychiatry , central institute of psychiatry , ranchi , india , a tertiary care referral centre during may 2005 to june 2006 . study sample included 201 , only male fulfilling icd-10 dcr ( world health organization ) for alcohol dependence with currently withdrawal state , aged between 18 and 60 years , admitted within 24 hours of abstinence and patient himself or his guardian consenting for the study . information on patient 's demographics , treatment history , past history , and family history was obtained from interviews with patients and accompanying person . detailed physical and neurological examinations were done to exclude any comorbid general medical condition , comorbid other psychiatric disorder , and any other comorbid substance use disorders except caffeine and tobacco . the sociodemographic data sheet included age , marital status , religion , community , education , and economic status . whereas clinical variables recorded were age of onset of drinking alcohol , duration of dependence , past history of detoxification , number of previous detoxification , past history of withdrawal seizure , past history of delirium tremens , family history of alcohol or substance dependence , degree of relationship if family history of alcohol or substance dependence is present , and family history of mental illness . it is the most widely used and studied 10-item alcohol withdrawal monitoring scale , which excludes vital sign abnormalities . it was developed from the 18-item clinical institute withdrawal assessment for alcohol and it has been studied across various geographic locations . it has a good reliability , validity , and it is considered as one of the most widely used alcohol withdrawal assessment scale for symptom - triggered therapy . each sign and symptom item of ciwa ar is evaluated on a 07 point likert scale except for one item orientation and clouding of sensorium , which is scored on a 04 point likert scale . a score of 8 points or less indicates mild withdrawal and patients scoring less than 10 do not usually need additional medication for withdrawal . patients admitted with alcohol dependence syndrome with acute withdrawal were evaluated with ciwa - ar immediately after admission then every six hours , as a routine protocol of the ward . detailed physical examination , mental status examination , and planned screening laboratory investigation were done to ensure conformity of study criteria . the patients get admitted with varying lengths of abstinence , ranging from hours to days , so we initially took all the ratings of all the patients and arranged them as rating at first 6 hours of abstinence and at every six hours like at 6 , 12 , 18 , 24 , 30 , 36 , 42 , 48 , 52 , 58 , and 64 hours till ciwa - ar scoring reaches below 10 . the averages of each rating of ciwa - ar scores were computed to see the severity of withdrawal symptoms across time span of abstinence . many patients were admitted after overnight , 12 to 18 hours of abstinence , so we included the patients who were admitted with at least 24 hours of abstinence . meanwhile management and medications were continued as per ward protocol and no adjustment for study purpose was done . the standard detoxification protocol included thiamine supplementation , benzodiazepines either lorazepam or diazepam , and correction of fluids and electrolytes if any and other symptomatic treatment of associated conditions like dyspepsia or concurrent injury , wound , and infections . the collected data on 201 patients was statistically analyzed , using statistical package for social sciences ( spss , inc . , exploratory factor analysis ( maximum likelihood method ) was carried out to identify factor structure on all items of ciwa - ar for day three . kaiser - meyer - olkin measure of sample adequacy and bartlett 's test of sphericity were also done to assess appropriateness of conducting factor analysis . two criteria for retaining the number of components were considered : kaiser 's criterion to retain eigenvalues greater than unity and cattell 's scree plot inspection for the point of inflexion . the mean age of the group was 37.18 ( sd 9.35 , range 1869 ) years . most of them were married ( 83.3% ) , educated ( 90% ) , residing in urban background ( 64.7% ) , belonging to middle socioeconomic status ( 60.3% ) , and earning a livelihood ( 80.4% ) . the mean age of starting alcohol was 21.63 ( sd 4.99 ) years , whereas mean duration of dependence on alcohol was 6.49 ( sd 5.06 ) years . 22.5% had history of withdrawal seizures , in their course of alcoholism , and only 2% reported history of delirium tremens in the past . family history of substance dependence was present in 33.3% of the total sample , out of which alcohol dependence was present in 56.9% of subjects and cannabis dependence in 8.3% . the item frequency and mean of all six hourly ciwa - ar ratings were calculated ; the mean scores of ciwa - ar at 24 hours and at 36 hours are shown in table 2 . the mean ciwa - ar score at 24 hours was 13.32 ( sd 9.27 ) and 20.4 ( sd 9.09 ) at 36 hours . based on the frequency variance and total ciwa - ar score we decided to carry out factor analysis with the 10 items of ciwa - ar as on scoring at 36 hours of abstinence . bartlett 's test of sphericity was significant ( 2 = 1.044 , df = 45 , p < .001 ) , indicating that a factor analysis is appropriate . factor analysis ( extraction method - maximum likelihood ) with the 10 items of ciwa - ar for day three , resulted in initial three factors with eigenvalues greater than unity . the scree plot was also showing clear inflexion , supporting three factors ( figure 1 ) . therefore , three factors were retained , which captured 68.74% of variance . following varimax rotation with kaiser 's standardization , the factors and their item loadings , with absolute values greater than 0.1 , are shown in table 3 . none of the items loaded on more than one factor . the first factor named as delirious factor , which had highest loading from tactile disturbances ( .999 ) , followed by auditory disturbances , orientation and clouding of sensorium , and agitation . it explained 34.34% of variance and showed good internal consistency ( cronbach 's alpha = .91 ) . the second factor named as autonomic factor reflected four - item loading , highest from anxiety , followed by paroxysmal sweats , tremor , and headache or fullness in head . it explained 24.25% of variance and showed moderate internal consistency ( cronbach 's alpha = .66 ) . the third factor named as nonspecific factor reflected two - item loadings , nausea , and visual disturbances that explained 10.04% of variance and a cronbach 's alpha of .26 . we examined the factor structure of the ciwa - ar in a population of adult men hospitalized to a tertiary psychiatric institute for treatment of alcohol dependence . the ideal study of aws could have been the assessment before starting medication , but this was practically not possible for many reasons . firstly , many of the patients come for admission after 12 to 18 hours of abstinence and severe withdrawal , so keeping them drug free was ethically not possible . thus natural aws presentation and its severity were masked by routine benzodiazepam administration and thiamine supplement . secondly , many other patients were referred from primary care centers with initial management , including long acting benzodiazepines like diazepam that masks the aws . thirdly , many other patients came even before onset of withdrawal and in a state of intoxication ; the aws was not fully evolved in terms of range of symptoms and severity . for all these reasons , the mean ciwa - ar score for the initial 24 hours of abstinence ( first day ) of admission was only 13.32 ( sd 9.27 ) . later the sequential rating found more prominent withdrawal symptoms reaching highest mean score of 20.4 at thirty - six hours then gradually decreased . as drug free aws was not possible , we consider that the higher mean score of ciwa - ar represents the aws better than low score . also as both aws medications and alcohol itself are cns depressant and act in a similar way , either medication or alcohol intake should not make much difference in clinical picture . so we decided to proceed for factor analysis with highest mean ciwa - ar scoring at the 36th hour . the severity of withdrawal symptoms and appearance of complete sets of withdrawal symptoms at the 36th hour may have been influenced by plasma half - life of benzodiazepams being used for detoxification . in this study , choice of medication was with treating team of the hospital ; however , only either intermediate acting lorazepam or long acting diazepam was used for this purpose . there was no use of short acting benzodiazepines , which causes varying and rebound withdrawal symptoms across different time frame with its dosing and changing plasma concentration . another more important influencing reason for varying withdrawal symptoms across different time frame could have been the dose of benzodiazepines , but we did not interfered with any medications or dosing and it was continued as per ward protocol to ensure naturalistic conditions . however , the equivalent benzodiazepines mean dose at 36 hour was 30 mg of diazepam per day . but this equivalent dose may not be accurate as many patients were on oral medications and others were on parenteral benzodiazepines . we excluded any other comorbid substance use disorders or polysubstance dependence but a few patients may also have undisclosed benzodiazepine or organic inhalant abuse or addiction . we also excluded any comorbid general medical condition especially epilepsy for that reason , patients on antiepileptic either taking it regularly or skipping will modify the alcohol withdrawal symptoms . for that matter any psychotropic drugs causing cns depression or any effecting stimulants will alter the withdrawal symptoms . most of the patients needed proton pump inhibitor drugs like pantoprazole or omeprazole for the alcohol induced dyspepsia , peptic ulcer disease , or gastroesophageal reflux disease , but these medications do not impose any effect on alcohol withdrawal symptoms . in a previous study by pittman et al . , the mean ciwa - ar score for day one was 13.2 ( sd 3.7 ) which was similar to our study , 13.32 ( sd 9.27 ) at 24 hours ; they continued with analysis on data collected on the first study day to exclude medication effects . but we analyzed the data as of 36 hours of abstinence , showing higher mean ciwa - ar score and all ten items had variance above 10% , thus representing fully developed withdrawal syndrome . however we could not control the medication effect used to control the withdrawal symptoms for ethical reasons , the used medications were benzodiazepine and thiamine supplementation for all the patients . two items of ciwa - ar , namely , auditory disturbances and tactile disturbances were very infrequent in our sample ( 9.5 and 10% ) on day one which increased to 55.1 and 65.7% at 36 hours , other items scoring raised on day two like agitation , orientation and clouding of sensorium , visual disturbances , and paroxysmal sweats ; few other items remained unchanged on day one and two like anxiety and tremor ; however scoring of only this item headache or fullness in head improved on day two from 28.9 to 12.9% ( table 2 ) . this indicates the importance of time duration of abstinence to study the alcohol withdrawal symptoms . we found a three - factor solution based on rotated eigenvalues and scree plot analysis . the first factor explained 34.34% variance and consisted of four items , namely , tactile disturbances , auditory disturbances , orientation and clouding of sensorium , and agitation . this factor appears to represent perceptual abnormality and delirium and may be considered as subclinical spectrum of delirium . however , the only other factor analytic study of ciwa - ar by pittman et al . found these items ( except agitation ) were infrequent in their sample and were not included in analysis . the difference may be due to the time span of withdrawal on which data was collected . study analyzed ciwa - ar for day one and also our data on day one showed very low variance for these items . this suggests that ratings on alcohol withdrawal symptoms on the very first day may miss certain set of symptoms , which appears on and around day two and are characterized by perceptual abnormality and delirium like picture . further progression of alcohol withdrawal , we could not found beyond 36 hours , may be due to effects of continued medications for withdrawal suppression . surprisingly , visual disturbances item did not have high loading on this factor , whereas tactile and auditory disturbances had maximum factor loading ( 0.999 and 0.873 , resp . ) . the etiological basis for these two disturbances includes cns rebound excitation which alters the perceptual quality . furthermore , some contribution of nutritional and specific vitamin deficiencies , such as thiamine and folate , and associated peripheral neuropathy probably add to these perceptual disturbances . the other two items , orientation and clouding of sensorium and agitation with factor loadings of 0.851 and 0.777 , respectively , represent delirium . the perceptual alteration and delirium being loaded in a single factor may have some predictive association and hence need to be studied for management plan . the second autonomic factor explained 24.25% of variance and consisted of four items : anxiety , paroxysmal sweats , tremor , and headache and fullness in head with factor scores of .998 , .660 , .528 , and .245 respectively . cronbach 's alpha for this factor was 0.66 showing adequate internal consistency . within this factor this factor may be a result of the practice of not using adrenergic medication on routine basis at our institute . all items ( paroxysmal sweats , tremor , and headache or fullness in head ) were loaded similarly . we had additional anxiety to this factor ; hence , we share the name of this factor with pittman et al . as autonomic . the third nonspecific factor explained 10.04% of variance and consisted of two items : nausea and vomiting and visual disturbances , with low factor scores of .51 and .27 , respectively . there are three proposed physiologic bases for the symptom manifestation of alcohol withdrawal symptoms : cns excitation , adrenergic hyperactivity , and delirium , which may be attributed to different neurotransmitters and they respond selectively to different pharmacotherapy . the cns excitation may be secondary to deficiency in gaba activity , whereas increase in cns epinephrine level causes adrenergic hyperactivity . the nmda receptor hypersensitivity and overactivity of certain subtypes of nmda receptors are associated with delirium . though we did not find a factor structure of aws in accordance with very strict pathophysiological manifestation of either autonomic , or cns excitation or psychological / affective , or perceptual / hallucinogenic in our sample , the obtained factors suggest existence of subgroups of patients with different set of symptoms . this was expected as alcohol withdrawal manifests by simultaneous involvement of all mechanisms rather than any mutually exclusive mechanism . this simultaneous involvement of several other neurotransmitters besides gaba and nmda , like noradrenaline , acetylcholine , and dopamine as well as hormones and electrolytes [ 17 , 18 ] will affect the symptom presentation . additionally our data analysis had ciwa - ar ratings of day two ( 36 hours ) of admission with suppression of withdrawal symptoms from detoxification medications , that is , benzodiazepines . this would have differential effect on withdrawal symptoms manifestation in terms of gaba suppression only and selective unopposed action on other neurotransmitters or lacking adrenergic activity . though avoiding pharmacological suppression or modification was not possible on ethical grounds to understand completely natural unmodified withdrawal manifestation . there may also be possible influence of different types of alcoholic beverage and percent content of alcohol . strength of our study includes large sample size and not interfering with any medications or management strategies thus providing setting of naturalistic conditions . the use ciwa - ar is the most widely accepted alcohol withdrawal assessment scale and selection of abstinent hours was important to allow time for full appearance of symptoms , even though under cover of detoxification medication . there was for better coverage and inclusion of withdrawal symptoms at 36 hours as indicated by total ciwa - ar score and item frequency . these results have wider implications for the recognition and management of aws , particularly , for better understanding and identification of the symptom profiles for and differential management plans across subtypes of aws . the use of adrenergic antagonists may have a valuable role in addition to benzodiazepines , in a set of patients with autonomic features . one of the limitations in our study is that it includes male only patients ; however , gender can be an important issue in aws presentation and its severity . even studies found that sex hormone affects the aws by modulating the function of the gaba - a receptor . also low levels of testosterone are associated with symptoms like indecision , excessive worrying , fatigability , and lassitude . another limitation of this study is patients sample with severe aws requiring inpatient management , thus limiting generalizability of our findings across gender and to mild to moderate severity cases . another limitation was that diagnosis was made clinically with icd-10 dcr criteria for alcohol dependence with currently withdrawal state , but not by a validated clinical interview . it is known that autonomic arousal is an important mechanism in aws ; thus , other physiological measures and biological markers for objective assessment may be included in future studies . further studies may also be carried out including cases of mild to moderate severity and in both sex to uncover the differences . also , factor analysis depends heavily on the population studied ; therefore , studies on different population may be required to generalize our findings . for the clinical practice , it is advisable not to overdepend on rating scales and it must not replace a thorough clinical evaluation of the patient 's medical status in prediction of those at risk of severe alcohol withdrawal . the acute alcohol withdrawal symptoms was most severe at 36 hours of abstinence in our sample . this study finds multidimensionality of alcohol withdrawal symptoms as measured with ciwa - ar ; we found three factors explaining 68.74 percentage of variance and named as delirious , autonomic and nonspecific .

objective . to identify the underlying factor structure of alcohol withdrawal syndrome , as measured with ciwa - ar . methods . exploratory factor analysis was conducted on the items of ciwa - ar . on 201 alcohol - dependent male patients seeking treatment for alcohol withdrawal at 36 hours of abstinence . results . a three - factor solution was obtained that accounted for 68.74% of total variance . first factor had loading from four items ( 34.34% variance ) , second factor also had four items ( 24.25% variance ) , and the third had two items ( 10.04% variance ) . conclusions . factor analysis reveals the existence of multidimensionality of alcohol withdrawal as measured with ciwa - ar and we found three factors that can be named as delirious , autonomic and nonspecific factors .

twenty - two workers were followed up from 15th april to 30th april 2008 during working hours , from approximately 6 am to 3 pm , for a total of 160 working hours . this included only workers who had worked more than 50% of each day 's working period and had worked for at least 10 days of the follow - up period . information regarding the amount of liquid intake expressed ( in litres , l ) and daily productivity output ( in tons ) were recorded . the rehydration strategies were re - structured by decision makers participating in the project and the author of this paper . for an eight working hour schedule , all workers were instructed to drink 1 l of the rehydration solutions or tap water at least 30 minutes before they began to work . they were also encouraged to drink water and rehydration solutions ( 250 ml ) every 3045 minutes . in total , they were each asked to drink 10 l of liquid ( tap water and rehydration solutions ) per day . in order to facilitate the monitoring of basic heat stress indexes during the 2007/2008 harvesting season , temperature , humidity and dew point were measured by using a portable temperature monitoring device ( easylog , model el - usb-2 ) . variables such as heart rate and weight were evaluated at the beginning and at the end of the working day . neither the amount of liquid intake nor productivity was validated . wet bulb globe temperature ( wbgt ) values during the 2007 harvest season at montelimar farm descriptive statistics were used to analyse climate data ( temperature ( c ) ; relative humidity ( % ) ; dew point ( c ) ) ; liquid intake and output production . chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 . descriptive statistics were used to analyse climate data ( temperature ( c ) ; relative humidity ( % ) ; dew point ( c ) ) ; liquid intake and output production . chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 . 1 shows in situ hourly climate variation direct from the sugarcane fields while workers harvested the crop . as can be seen , temperature and relative humidity values oscillated from 23.5 to 34.5c and from 40% to 64% , respectively , reaching maximum values as early as 810 am . this meant that water distribution had to be started at around that time on an hourly basis . climate measurements during 160 working hours of follow - up for 22 sugarcane workers at the montelimar farm . table 2 shows the amount of liquid intake ( including both regular tap water and specially formulated rehydration drinks ) . what is of great concern is that , although temperature increased to maximum values early in the morning , many workers did not follow the rehydration measures and drank less than 6 l , a potentially dangerously low volume . comparison between temperature measurements and daily water intake for 22 sugarcane workers at the montelimar farm . this showed that 13 workers who had the highest production output ( range 68 tons , average 7.45 tons ) were those who drank more than 6 l of liquid ( regular tap water and/or specially formulated rehydration solutions ) in comparison to those who drank less liquid and had a smaller production output . relationship between production output and daily water intake for 22 sugarcane workers at the montelimar farm . measurements of heart rate and body weight at the start and end of the working day showed that workers experienced increases in heart rate and loss of body weight as they worked in these hot conditions . historically , monitoring of toxins in the work environment has been the primary focus for identifying risks . some potential biomarkers linked to cell injury are immunological factors , lymphokines , growth factors , prostaglandins , endothelins , collagen , adhesion molecules , thromboxanes , leukotrienes , platelet activating factors and heat shock proteins ( 10 ) . as mentioned earlier , heat illness is a major cause of preventable morbidity worldwide ( 1 ) and although human beings possess considerable ability to compensate for naturally occurring heat stress , many occupational environments and/or physical activities expose workers to heat loads which are so excessive as to threaten their health and productivity ( 11 ) . it is important to remember that the normal human body contains approximately 60% of water , the present project evaluated climatic conditions at sugarcane plantations located at sea level , on the southwest coast of nicaragua . monitored climate indices ( temperature and relative humidity ) values varied from 23.5 to 34.5c and from 40% to 64% , respectively , reaching maximum values as early as 8 am . according to the nicaraguan institute of territorial studies ( ineter ) ( 13 ) , relative humidity values for the whole month of april varied between 69 and 79% all along nicaragua 's pacific coastline from san juan del sur ( southern region ) to chinandega ( western region ) . managua lies between the two regions ; the montelimar sugar farm is located 62 km from managua . however , the ineter humidity data are quite different from the relative humidity registered on the farm located in western managua . temperature data were not available at ineter 's website . although only 22 subjects were followed - up for a short period of time in this study , important results were obtained . other authors have shown the relationship between heat stress health effects and the ability to perform different tasks , as well as the increased risk of suffering work - related injuries ( 14 ) . in this study , the workers drank more liquid as temperature values increased to maximum peaks . this was part of a rehydration process which was well planned in advance by the company 's decision makers . unlike during past harvesting seasons , when water and rehydration solutions were distributed quite randomly , during this harvesting season cool water and specially formulated drinks were distributed or intended to be distributed to workers , who received 1 l just before they began their working day and then 500 ml every 30 minutes . the basis of this principle is that drinking to satisfy thirst is not enough to keep a person well hydrated . most of the people become aware of thirst once they have lost 12 l of body water and persons highly motivated to perform hard work may incur losses of 34 l before serious thirst forces them to stop and drink . since dehydration reduces the capacity for absorption from the gut , workers must be educated regarding the importance of drinking enough water during work and continuing generous rehydration during off - duty hours ( 14 ) . there was a significant increase of production , with up to 8 tons per worker during the follow - up period compared to the normal 5.5 tons per worker prior to the change in rehydration measures . this important change in rehydration policies and increase in production output is the result of various efforts of training workers , foremen and managers on heat stress prevention , proper hydration measures and quality of ( working ) life carried out by occupational health and safety professionals ( physicians and engineers ) , human resources departments and top management at sugarcane farms . . often workers rejected the new rehydration measures , most of the time because it was difficult for them to understand thoroughly the dehydration and physiological compensatory mechanisms . some of the reasons for this can be attributed to their low educational level , and feeling that nothing bad has ever happened to me before , etc . certainly more effort in terms of intervention strategies and scientific investigation needs to be carried out among workers in nicaragua who perform jobs in which they are exposed to high ambient temperatures . these include farm workers , construction workers , miners and fishermen , especially those employed in the informal sector , which occupies about half of nicaragua 's economically active population . more funds should also be designated by companies decision makers for improving basic working conditions , in order to increase overall productivity ( and workers satisfaction in terms of better wages ) . this would also translate into safer and healthier workers , less absenteeism from sick leave , fewer accidents and other incidents . the author has not received any funding or benefits from industry to conduct this study .

backgroundheat illness is a major cause of preventable morbidity worldwide . workers exposed to intense heat can become unable to activate compensation mechanisms , putting their health at risk . heat stress also has a direct impact on production by causing poor task performance and it increases the possibility of work - related morbidity and injuries . during the sugarcane harvest period , workers are exposed to excessive sunlight and heat from approximately 6 am to 3 pm . a first assessment of heat stress during the 2006/2007 harvesting season served to redesign the existing rehydration measures . in this project , sugarcane workers were provided with more rehydration solutions and water during their work schedule.objectiveto assess heat stress preventive measures in order to improve existing rehydration strategies as a means of increasing productivity.methodsa small group of 22 workers were followed up for 15 days during working hours , from 6 am to 3 pm . selection criteria were defined : to have worked more than 50% of the day 's working schedule and to have worked for at least 10 days of the follow - up period . a simple data recollection sheet was used . information regarding the amount of liquid intake was registered . production output data was also registered . temperature measurements were recorded by using a portable temperature monitoring device ( easylog , model el - usb-2).resultsthe average temperature measurements were above the nicaraguan ministry of labour thresholds . seven workers drank 78 l of liquid , improving their production . output production increased significantly ( p=0.005 ) among those best hydrated , from 5.5 to 8 tons of cut sugarcane per worker per day.conclusionsproductivity improved with the new rehydration measures . awareness among workers concerning heat stress prevention was increased .

choroid plexus papilloma ( cpp ) is a rare benign tumor accounting for 0.5% of all intracranial tumors that is more common in childhood , consisting of 1.5 to 6.4% of pediatric intracranial tumors.1 2 the majority of cpps occur during the first 2 years of life , with 12.5 to 20% ( 10 to 12% ) reported in infants under 1 year of age.1 3 there have been some reports of calcified cpp , cpp in an adult , cpp in the cerebellopontine angle ( cpa ) , and atypical form of cpp.4 5 6 7 however , a case combining with these four features adult , dense calcification , atypical form , and cpa is considered to be extremely rare ; such a case has never been encountered among the 8,540 skull base tumors on which the senior author ( t.f . ) has operated in the past 20 years . we report an adult case of densely calcified , atypical cpp originated from the cpa without connection to the forth ventricle . a 41-year - old man presented with a chief complaint of headache , dizziness , and difficulty with gait that had been gradually worsening for 8 months . neurological examination revealed blurred vision , nystagmus with lateral gaze , and mild right facial weakness . intermittent difficulty with balance and gait were also present ; however , no motor weakness was observed . computed tomography ( ct ) of the head without contrast revealed a high - density multilobular mass in the right cpa causing mass effect and slight ventricular enlargement ( fig . magnetic resonance ( mr ) images demonstrated a trilobed solid mass that measured 4.1 cm in maximum diameter ( fig . the mass was extra - axial in location and revealed isointensity on both t1-weighted images ( t1-wi ) and t2-weighted images ( t2-wi ) . ct and mr images did not reveal any connection between the tumor and the fourth ventricle . the surface of the tumor was roughly irregular and looked grayish pink . since the tumor was friable , gelatinous , vascular , and adherent to surrounding structures , it was hard to separate from cranial nerves . cranial nerves vi , vii , viii , ix , and x were enveloped in the tumor mass , which included calcification . the patient was discharged after recovery from the dysphagia by appropriate rehabilitation . computed tomography without contrast revealed a high - density multilobular mass in the right cerebellopontine angle . magnetic resonance images demonstrated a trilobed solid mass that was in an extra - axial location . the tumor revealed isointensity on both t1-weighted images ( upper left ) and t2-weighted images ( upper right ) . homogeneous intense enhancement was identified ( axial in lower left , sagittal in lower right ) . cpp reported in this article primarily occurred in the cpa with close proximity to the foramen of luschka lacking continuity of the fourth ventricle . the majority of choroid plexus tumors ( cpts ) originate in the ventricular system , 43 to 67% in the lateral ventricle , 24 to 39% in the fourth ventricle , and 9.5 to 11% in the third ventricle , respectively.8 9 the common site of origin differs in each pediatric and adult group . in the pediatric age group , 60 to 80% of such tumors occur in the lateral ventricle , especially in the trigone , because the vascular stalk of the choroid plexus in the lateral ventricle is usually attached to the inferior portion of the trigone . on the other hand , most cpp in adults occurs within the fourth ventricle.9 10 11 a small number of the tumors have been reported to be located in extraventricular regions , the cpa , or the cerebellar hemisphere or the suprasellar cistern.8 10 12 cpp within the cpa was first described by cushing in 1917.13 to date , cpp within the cpa has been reported in 7 to 9% of all cpts and is most exclusively found in adults.9 11 14 15 it is divided into two categories according to the origin of the tumor ; one is primary extraventricular cpts and the other is a result of a direct extension of fourth ventricular tumors through the foramen of luschka.15 because the tumor of our case revealed no connection from the fourth ventricle , it is considered to originate from small choroid tufts that normally project from each recess at the foramen luschka into the cpa or from ectopic choroidal islets unconnected with the choroid plexus.16 the cpp of our case indicated dense calcification with ventricular enlargement . the bulbous tufts extended from the fourth ventricular choroid plexus through the foramen of luschka have been known as the bochdalek flower basket , which could be identified in 75% on ct and 96% on mr imaging.17 calcification of the region was found in 38% on normal imaging anatomy.17 in pathological states , calcification of cpp was reported in 4.1% on plain skull radiographs and 23.8% on ct examinations in patients of all 9 the feature of the calcification was commonly described as a stippled or patchy configuration that may include some degrees of hemorrhage.8 9 10 dense calcification was exceptionally reported by sarkar et al.2 the rate of hydrocephalus in association with cpp is 90% and the rate increases up to 100% in malignant cpps.8 the cause of hydrocephalus was considered a combination of cerebrospinal fluid ( csf ) overproduction ( four to five times that in healthy persons ) , obstruction of csf pathways by the tumor mass , impaired csf absorption , increased protein content of csf around the tumor , subarachnoid scarring or granulations related to recurrent bleeding from the tumor , elevated intraventricular pulse pressure , and adhesions around the exit foramina of the fourth ventricle caused by highly proteinaceous or hemorrhagic csf.2 10 18 the finding of tumor calcification in the cpa generally required consideration of differential diagnosis with papillary or psammomatous meningioma , melanotic schwannoma , and myxopapillary ependymoma . among these tumors , association of ventricular enlargement with the finding of calcification might suggest the possible presence of cpts . gross total removal was performed in our case of atypical cpp , although the tumor adhered to adjacent structures . in the 2007 world health organization classification of tumors of the central nervous system , cpts are classified into cpp as grade i , atypical cpp as grade ii , and choroid plexus carcinoma ( cpc ) as grade iii . among cpts , total removal of cpts is recommended and seems to be feasible since a significant prognostic factor was relevant to the extent of tumor removal , and no definitive evidence of the beneficial effect of radiotherapy has been reported6 18 19 20 . however , it is not always possible because of tumor bleeding and firm adhesion to adjacent structures , especially cranial nerves . the rate of complete resection of benign cpp was 80% and the 5-year survival rate indicated is nearly 100% . the majority of survivors are neurologically intact.19 because of the good prognosis , the preservation of cranial nerve functions should be primarily considered in surgery for benign cpp . as for the atypical cpp , the rate of total removal was decreased to 50 from 63%.11 21 atypical cpp showed a recurrence rate of 29% , which was much higher than the 6% seen in cpp . in addition , seeding within the central nervous system , spontaneous pulmonary metastasis , or rapid regrowth of atypical cpp were also reported.7 22 23 total or gross total removal should be advocated , especially in malignant cpts even though the tumor encases the cranial nerves . the finding of calcification in the cpa should suggest the possible presence of cpp , especially if ventricular enlargement is present .

choroid plexus papilloma is a rare benign tumor accounting for 0.5% of all intracranial tumors . the majority of choroid plexus papillomas occur during the first 2 years of life in the ventricular system . moreover , dense calcification of the tumor is uncommon . we report an adult case of densely calcified , atypical choroid plexus papilloma that originated from the cerebellopontine angle without connection to the forth ventricle . because the case involves a rare combination of four features adult , dense calcification , atypical form , and cerebellopontine angle each characteristic associated with the choroid plexus papilloma was discussed . the finding of calcification at the cerebellopontine angle in an adult should suggest the possible presence of choroid plexus papilloma .

multipath signals occur in numerous microwave and rf applications when an unwanted portion of the original transmission propagates along any alternate path and ultimately couples to the receiver distorting the amplitude and phase of the desired signal [ 14 ] . if the amplitude of the multipath signal is sufficiently large , its impact can be considerable . in multistatic radar and communication systems , these types of interference are most often caused by reflections of either the main beam or side lobes with objects near or actually in the beam path . classic examples include main beam propagation close to the earth 's surface with associated reflections off of the ground or water ( figure 1(a ) ) . various approaches can be used to filter or compensate for these reflections through time - gating and signal time synchronization . the potential for interference from multipath signals increases substantially in near - field applications ( figure 1(b ) ) , especially in situations where the receiving and transmitting hardware are integrated . commercially available multichannel network analyzers ( e.g. , zvt8 by rohde & schwarz ; munich , germany ) utilize robust strategies to minimize these signal coupling problems . we are developing multichannel transceiving arrays for medical microwave imaging which exploit near - field concepts to produce electrical property maps ( permittivity and conductivity ) of tissues of interest [ 8 , 9 ] and have addressed the issue by incorporating ( a ) dedicated mixers for each channel , ( b ) additional solid state switches for isolation , ( c ) double- and triple - braided coaxial cables , and ( d ) compartmentalized rf circuitry . the implementation has proven effective for our application achieving channel / signal isolation greater than 130 db . an alternative data acquisition strategy integrates a commercial , 2-port network analyzer with an electronic switching network to feed an array of antennas [ 1012 ] which is effective but also has limitations because ( i ) dynamic range is constrained by the provisions of the network analyzer , ( ii ) two - way signal loss is incurred through the network , and ( iii ) the switching matrix has relatively poor cross - channel isolation . equally important in near - field imaging is the multiple paths a signal can take within the imaging zone . figures 2(a ) and 2(b ) show a photograph of our clinical breast imaging tank and a schematic diagram of the antenna configuration , respectively . in this situation , the array of 16 monopole antennas surrounds the breast and can be moved to multiple vertical positions . the antennas and target are submerged in a solution of glycerin and water which is lossy over the operating frequency range ( 700 mhz3 ghz ) . early empirical tests have indicated that reflections off the tank side walls do not impact the desired signals for an array with the antennas mounted on a 15.2 cm diameter circle . likewise , analysis of the monopole beam patterns as a function of frequency has shown that artifacts are minimal when the array approaches the liquid interface at the top of the tank . with respect to reflections off the bottom surface , the base of the tank was at least 1.8 wavelengths ( at the lowest frequency ) below the active sections of the antennas in our initial clinical installation when the array was located at its lowest position during an exam . since minimal multipath signals resulted from reflections off the top liquid surface , symmetry would suggest that the same should be true for the base of the tank ( figure 1(b ) ) . however , surface waves can cause multipath signals that can be especially difficult to eliminate in near - field systems . their excitation can be complex , but their propagation characteristics along two material interfaces , whether planar or along cylindrically shaped structures , have previously been studied in depth [ 1520 ] . surface waves can readily propagate at the interface of two dielectric materials or one conductor juxtaposed directly with a dielectric . their propagation and attenuation characteristics are nominally determined by the electrical properties of the two materials . in addition , their amplitude decays exponentially away from the interface in the perpendicular direction as a function of the lossiness of the complementary materials . it should be noted that these investigations have stemmed partially from our efforts to perform microwave tomographic images on patients in an actual mr scanner for the purpose of exploiting the refined spatial resolution of the mr along with the more specific nature of the tissue dielectric properties . the mr bore is quite small and places a significant constraint of space for the microwave system . this was where we first encountered multipath signal corruption which subsequently led to this study . in the following sections , we discuss the theoretical underpinnings of these modes for the geometries present in our system . we demonstrate cases from our current imaging system , where the measurements indicate corruption of the desired signals from multipath signals associated with the base of the tank . we then show experiments that allow us to partially isolate the effects to surface waves propagating along other pathways . we realize that there are a number of propagation modes around the antennas , their feedlines and the tank surfaces , of which the surface waves are only one possible contributor , but understanding these contributions is important . we present an initial strategy for minimizing the effects of these signals which may be instructive for designing other near - field imaging systems , including simulations confirming the earlier theoretical discussion and validating our feedline design strategy . a challenging situation occurs when a portion of the transmitted signal propagates along an unwanted path and recombines with the original signal at the receiver . time - gating strategies can sometimes be effective when the nature of the multipath signal is well understood [ 1 , 2 ] . the potential influence of a multipath signal when the original transmission is a continuous wave can be written as ( 1)resultant signal = acombcos(t+comb ) , where ( 2)comb = acombtan1(b1a1),acomb = a12+b12,a1=[adecosde+ampcosmp],b1=[adesinde+ampsinmp ] . here , ade , amp , de , and mp are the desired and multipath signal amplitudes and phases , respectively , is the operating frequency , and t is time . for example , if the magnitude of the multipath signal is 25 db below that of the desired signal , the maximum possible amplitude and phase errors in the resultant signal would be 0.48 db and 3.22 , respectively . for a 15 db multipath signal , these values increase to 1.42 db and 10.24. clearly , the resultant phase and amplitude errors can become very significant for multipath signals that are on the same order of magnitude as the desired signals . it should be noted that there can be many multipath contributions with a range of amplitude and phase contributions . this single contributor analysis serves to give a flavor that the unwanted effects can be significant and is generalizable to multiple sources . the first surface mode to be considered involves propagation along the interface between the tank base and coupling liquid . following the analysis by stratton , figure 3 shows a plane wave in region 1 impinging on an interface ( x = 0 ) with region 2 . in this case , the magnetic field ( hy ) is only oriented in the y - direction ( out of the page ) . the complex relative dielectric properties in the two regions are 1 = 1 j1 and 2 = 2 j2 , respectively . the classic surface wave solution occurs when the reflection coefficient is zero ( at the brewster angle ) , which is complex - valued in this instance . if a is the amplitude of the incident plane wave , then the magnetic component of the incident and transmitted waves can be represented as ( 3)hy = ae[jh1xjz ] , x>0,hy = ae[jh1xjz ] , x<0 , where h1 + = k1 and h2 + = k2 are needed to satisfy the wave equation , k1 and k2 are the wave numbers for the two regions , and is the propagation constant . the wave impedances for the two regions are given by ( 4)z1=h1k1z1=h1k1z011j1,z2=h2k2z2=h2k2z012j2 , where z1 and z2 are the free space impedances in the corresponding regions , and z1 and z2 are the associated wave impedances [ 21 , 22 ] . this yields ( 5)h1=h2 , where ( 6)=21=2j21j1. from these relationships , we can solve for ( 7)h1=k01(1)12,h2=k012(1)12,=k01(1)12 . in this situation , we are primarily interested in surface waves propagating along the outside of a coaxial cable , and their associated attenuation as a function of distance after the mode has been sufficiently established . for this analysis , we will consider the case of a coaxial line which is abruptly terminated by an open end ( figure 4 ) . a coaxial cable supports a tem - mode electromagnetic field which is incident on the cable opening . part of the signal is partially reflected into the cable , while a second portion is transmitted as a surface wave propagating along the outside of the surrounding cable . the fields transmitted into the surrounding space can be determined from the distribution at the coaxial opening which can be found by solving the integral equation for the radial component of the electric field over the opening : ( 8)14+jcabe(,0)kc(,)d = j1abe(,0)kc(,)d0cosejkrrd , where c and 1 are the complex - valued permittivity of the coaxial cable insulator and the surrounding dielectric materials , respectively , a and b are inner and outer coaxial radii , respectively , is the operating frequency in radians , and k is the wavenumber in the coupling liquid , where 0 is the free space magnetic permeability . and are the radial cylindrical coordinates within and outside of the coaxial cable , respectively , is the angular coordinate within the coaxial cable , and r is defined as r=2+2 - 2cos. the variable kc( , ) is represented as ( 9)kc(,)=jn=0n()n()an2n , where ( 10)n()=y0(na)j1(n)j0(na)y1(n),n={kc2n2,kc>njn2kc2,kc>n , an2=22n2[j02(na)j02(nb)1 ] , n>0,a02=lnba . the eigenvalue n are solutions of the characteristic equation : ( 11)y0(na)j1(nb)=j0(na)y1(nb ) , where jn and yn are bessel functions of the first and second kind of order n , respectively , and kc is the wavenumber inside the coaxial line . once e( , z = 0 ) is determined , then electric and magnetic fields can be found at any point ( , z ) in the surrounding medium from ( 12)e(,z)=1abe(,0)0(jk+1r ) zcosejkrrdd , or ( 13)e(,z)=1abe(,0) 0[1(jk+1r)]cosrejkrrddh(,z)=j1abe(,0) 0cosejkrrdd , where ( 14)r=z2+2+22cos. from these equations it follows that the electromagnetic fields inside the surrounding medium decay approximately as e. figure 2(a ) shows the illumination tank used in our current clinical breast imaging system . each monopole antenna consists of an exposed length ( 3.8 cm ) of 2.2 mm diameter semirigid coaxial cable with only the center conductor and insulating teflon layer intact . for mechanical robustness , the coaxial feed line is enclosed in a 6.4 mm diameter rigid stainless steel tube , and the active section of the antenna is covered with an accompanying length of a delrin cylinder acting as a protective radome . the space between the copper coaxial outer conductor and the stainless steel sleeve is sealed at either end with silver epoxy to eliminate wave propagation along the gap . the antennas have a nominal return loss of 10 db over the bandwidth of 7003000 mhz . the black delrin fittings at the antenna / tank base contain hydraulic seals through which the antenna feeds pass to allow vertical motion of the array while eliminating any coupling fluid leakage . the 16 antennas are positioned on a 15.2 cm diameter circle , and both sets of 8 interleaved antennas are supported by individual mounting plates under the tank which provide independent motion of the array in groups of 8 . the tank is fabricated out of plexiglas with an inner wall diameter of 27.3 cm and thickness of 1.3 cm , and the base has a thickness of 2.5 cm . all connecting cables are double - braided to minimize stray radiation . in these experiments , the antennas were positioned at heights close to the tank base ( that were not used in any clinical exams ) to study the multipath phenomenon in detail . figures 5(a ) , 5(b ) , and 5(c ) show three illumination tanks with different heights , arrays of monopole antennas , and coaxial feed lines that were fabricated from the same plexiglas and had identical diameters and wall / base thicknesses as figure 2(a ) tank . the feed lines passed through holes in the base of each tank and were fastened to sma flange connectors which were bolted to the tank floor . the tanks were filled with an 80 : 20 glycerin / water mixture with the liquid level 1.5 cm above the antenna tips . in figures 5(a ) and 5(c ) , the feed lines were both 10 cm long ; however , the latter was bent in a serpentine shape such that the top height of the feed line was only 5 cm above the tank floor . the feed line in figure 5(b ) was straight and was only 5 cm long . in these experiments , we used plexiglas for the tank materials with a dielectric constant of r = 2.7 that was effectively lossless in this frequency range . the dielectric properties of the 80 : 20 glycerin / water bath are plotted in figure 6 as a function of frequency . utilizing the clinical system described in section 2.2 , a + 5 dbm signal was transmitted at multiple frequencies over the 7002500 mhz range from a single antenna and received at the remaining 15 antennas . this sequence was repeated for the array positioned at multiple heights above the tank base . receive antenna amplitudes are plotted for representative frequencies in figure 7 . at 900 mhz , the measured levels are high for the receivers closest to the transmitter ( relative receiver numbers 1 , 2 , 14 , and 15 ) compared to the rest of the array and do not change dramatically with changes in antenna height . however , the amplitudes are considerably lower for the more distant receive antennas . for antenna heights 7 cm or greater ( above the tank floor ) , the attenuation follows a smooth curve hitting a maximum at antenna 8 ( which is furthest away from the transmitter being located on the opposite side of the array ) . at antenna heights 5 cm and lower , the behavior is consistent with the three frequencies shown in figure 7 . given that the distance from the antennas to the tank side walls did not change during the experiments , and that the antennas were sufficiently far from the liquid / air interface at all times for any array heights ( 10 cm in the worst case ) , the corruption of these most distantly received signals appears to be caused by multipath propagation associated with antenna tip proximity to the base of the tank most probably due to reflections off of the tank base or surface wave propagation along the dielectric interfaces . in this set of experiments figures 8(a ) and 8(b ) show the received signals for a single transmitter over a range of frequencies for straight feed line lengths of 10 cm and 5 cm ( tanks in figures 5(a ) and 5(b ) ) , respectively . for the longer ( 10 cm ) lines in figure 8(a ) , the field patterns appear well - behaved like those in the previous section , when the array was positioned at the largest heights above the tank floor . however , the patterns for the shorter ( 5 cm ) line lengths in figure 8(b ) exhibit corruption of the signals resulting from the longer propagation distances similarly to when the array heights were closest to the base of the tank for the clinical system ( in figure 7 ) . these corrupted signals are nominally between 50 to 70 dbm and occur in uneven patterns relative to the same signals from the longer feed lines which reach 80 to 90 dbm at the furthest antenna . for the shorter propagation distances ( i.e. , the signals received at antennas 14 and 1215 ) , the attenuation patterns from the shorter and longer feed lines are similar ( figure 8(b ) versus 8(a ) ) . these results suggest that the unwanted multipath signal effect is the same in both the experimental tanks and the clinical system tests in the previous section and depends on the antenna height from the base of the tank . however , in figure 8(c ) , the antenna feed length is exactly the same as in figure 8(a ) , but is curved such that the antenna tip is the same height above the tank floor as the antennas in figure 8(b ) ( which led to signal corruption ) ; yet , the measurement results emulate those in figure 8(a ) ( which are not corrupted ) . here , the active part of the antennas is still positioned on the same 15.2 cm diameter circle as in the other two tanks . these findings show that the principle signal corruption observed in figure 8(b ) is not due to reflections off the tank floor ; otherwise it would have appeared in figure 8(c ) since the antennas are at the same position above the base for both figures 8(b ) and 8(c ) . it should be noted that the signal disruptions for this situation appear more substantial than that for the antennas used in the clinical system as discussed in section 3.1 . this is very likely due to differences in the designs of the hydraulic seals , feedline shielding , and antenna radome for the other system . more likely , the multipath signals in figure 8(b ) ( with short feed lines ) result from surface waves traveling along the outside of the coaxial lines , across the plexiglas / liquid and/or plexiglas / air interface and back up the outside of the receiver coaxial feed . the theoretical considerations in section 2.2 indicate that the attenuation along the coaxial lines is far more substantial than from the planar tank - base surface wave modes . figure 9(a ) shows a plot of attenuation as a function of frequency for 15.2 cm of plexiglas / liquid surface waves and indicates that very little attenuation of a surface wave propagating along this interface occurs at the frequencies we used . thus , in our experiments , the real source of surface wave attenuation comes from propagation along the outside of the coaxial lines . figure 9(b ) shows plots of the attenuation that results from single 5 and 10 cm lengths of feed line in the coupling fluid . the theoretical predictions of attenuation along the two 5 cm antenna feed lengths ( transmit and receive ) and the path along the plexiglas base / liquid interface are approximately 70 db ( { 2 34 db } + 2 db ) for the 1 ghz case ( values interpolated from figures 9(a ) and 9(b ) ) . given a transmit power level of + 5 dbm , the resultant 65 dbm multipath signal for the shorter line would easily corrupt the desired 81 dbm signal ( figure 8(a ) ) . only when the feed lines are nearly doubled in length , and the associated surface wave attenuation increased accordingly is the corruption of the desired signals reduced to an acceptable level . along with the analytical discussion in sections 2.1 and 2.2 , we have also performed simulations of the configurations described in section 2.4 . figures 10(a ) , 10(b ) , and 10(c ) show the 900 mhz electric field magnitude distributions for the long and short , straight antenna feed and the longer , serpentine structure , respectively . these simulations were computed using ansys ( burlington , ma , usa ) hfss version 13.0 . for all cases , there is a reasonably broad antenna pattern emanating outwards from the active part of the antennas , and this feature is reasonably similar for all feed line types . for the straight feeds especially the longer one , it is clear that there are considerable surface currents generated along the coaxial lines . for the shorter straight one , there is a high degree of field strength along the coaxial line within the plexiglas volume below the horizontal interface . the fields along the interface generally agree with our previous notion that the surface waves preferentially propagate within the lower - loss medium ( in this case the plexiglas ) as can be seen by the substantially greater amplitudes directly under the line . the results for the serpentine feedlines are consistent with previous results in that the field strength in the plexiglas is lower than that for the short , straight line case . figure 11 shows a plot of the field strength just below the liquid interface from a point directly underneath the antenna extending 140 cm to the right for all three corresponding plots . the field values are considerably less for the longer straight line but are also less for the serpentine cases compared to the short , straight cases . for the serpentine case , there seems to be some signal coupling between the lower feedline bend and the plexiglas . as discussed in section 1 , surface waves do decay exponentially from the surfaces , and given the proximity of the feedlines and the liquid / plexiglas interface , some coupling is expected . the potentially debilitating effects of unwanted multipath signals is a critical consideration in translating near - field microwave imaging approaches into clinical and commercial systems . for our noncontacting antenna approach , surface waves ( relative to signal reflections from the imaging tank walls ) appear to cause the biggest effects as they propagate along the outside of the transmitting coaxial line across the illumination tank floor and back up the coaxial feed lines of the receivers . when the imaging tank is deep and the transmitting / receiving antenna tips are sufficiently far above the tank base , the surface wave signals are adequately suppressed relative to the transmissions through tissue . the results presented here indicate that 9 - 10 cm of distance along the feed line is adequate . however , reducing the tank depth is of interest for practical reasons and is essential in some settings and appears possible because reflections from the floor of the tank are still too small to degrade the measured signals propagating through tissue . indeed , we found that antenna tip distances as little as 5 cm from the tank floor maintain receiver signal fidelity across the array provided the surface wave contributions are attenuated through an equivalent feedline length approaching 10 cm . these findings are significant because they indicate that the antenna array and imaging tank geometry can be altered substantially by manipulating the shape of the antenna feed line , which can be exploited to ensure sufficient surface wave attenuation . there are certainly other mechanisms for multipath propagation including coupling of fields from the feedlines directly to portions of the breast tissue outside the immediate plane of propagation and are certainly good topics for further investigation .

microwave imaging techniques are prone to signal corruption from unwanted multipath signals . near - field systems are especially vulnerable because signals can scatter and reflect from structural objects within or on the boundary of the imaging zone . these issues are further exacerbated when surface waves are generated with the potential of propagating along the transmitting and receiving antenna feed lines and other low - loss paths . in this paper , we analyze the contributions of multi - path signals arising from surface wave effects . specifically , experiments were conducted with a near - field microwave imaging array positioned at variable heights from the floor of a coupling fluid tank . antenna arrays with different feed line lengths in the fluid were also evaluated . the results show that surface waves corrupt the received signals over the longest transmission distances across the measurement array . however , the surface wave effects can be eliminated provided the feed line lengths are sufficiently long independently of the distance of the transmitting / receiving antenna tips from the imaging tank floor . theoretical predictions confirm the experimental observations .

jaw cysts are usually painless , but they can increase in size as they absorb neighboring structures . enucleation generally induces bone regeneration within a cystic lesion less than 3 cm in diameter and eliminates the lesion ; however , it is difficult to postoperatively predict lesion recurrence and bone regeneration in cases of larger cysts . for this reason , a surgeon can choose between conservative and aggressive treatments for a large , aggressive , cystic tumor12 . it has been reported that marsupialization and decompression , both of which are conservative treatments that create an opening to reduce pressure within a cystic cavity and induce bone formation , generate few postoperative complications and produce great therapeutic effects3456 . these procedures not only provide good surgical access to smaller lesions , but they also preserve intraoral tissues , maintain pulp vitality , and reduce recurrence rates . for this reason , many surgeons prefer these methods for treating large cysts . this study aimed to analyze and assess the therapeutic effects of decompression for patients who underwent decompression followed by enucleation . we reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital ( gwangju , korea ) and who had undergone enucleation after decompression . we collected data on their age and gender , location and size of the lesions , preoperative and postoperative histopathological findings , gap in time between decompression and enucleation , and variation in the size of the lesions , complications , and recurrence according to the time gap . patients were excluded from group review if they had no radiological data , if they had cured soft tissues due to the failure to maintain the window , or if they had been observed for less than six months after enucleation . we measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient , measured width and length with a caliper , and multiplied them together to determine the lesion index ( li ) with the aim of simplifying measurement of irregularly sized lesions . to determine the efficacy of decompression , lis were divided into initial and final lis , which were then graded on the basis of nakamura et al . 's formula7 . to assess the extent of reaction to decompression for each lesion , the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period . before conducting a biopsy , clinical and radiographic data were used to determine if patients should undergo decompression . in decompression , the window was formed in the buccal alveolar bone region of the jaw , and a biopsy was conducted after excising some portion of the cyst wall . 1 ) after the treatment , patients were educated on how to wear and remove the device , and they were instructed to clean the device twice a day ( morning and night ) using saline solution . panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph , and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing . we reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital ( gwangju , korea ) and who had undergone enucleation after decompression . we collected data on their age and gender , location and size of the lesions , preoperative and postoperative histopathological findings , gap in time between decompression and enucleation , and variation in the size of the lesions , complications , and recurrence according to the time gap . patients were excluded from group review if they had no radiological data , if they had cured soft tissues due to the failure to maintain the window , or if they had been observed for less than six months after enucleation . we measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient , measured width and length with a caliper , and multiplied them together to determine the lesion index ( li ) with the aim of simplifying measurement of irregularly sized lesions . to determine the efficacy of decompression , lis were divided into initial and final lis , which were then graded on the basis of nakamura et al . 's formula7 . to assess the extent of reaction to decompression for each lesion , the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period . before conducting a biopsy , clinical and radiographic data were used to determine if patients should undergo decompression . in decompression , the window was formed in the buccal alveolar bone region of the jaw , and a biopsy was conducted after excising some portion of the cyst wall . 1 ) after the treatment , patients were educated on how to wear and remove the device , and they were instructed to clean the device twice a day ( morning and night ) using saline solution . panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph , and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing . in total , 17 patients ( 7 males and 10 females ) were reviewed , and their average age was 33.1 years ( range , 13 - 76 years ) . one patient was diagnosed with nevoid basal cell carcinoma syndrome ( nbccs ) and showed multiple keratocystic odontogenic tumors ( kcot ) in the maxilla and mandible . two of these cysts were treated with marsupialization.(table 1 ) jaw cysts were observed in 10 patients ( 58.8% ) in their teens or 20s , which was a higher rate compared to other age groups , and the sex ratio was 1:1.4 with seven males ( 41.2% ) and 10 females ( 58.8%).(fig . 2 ) radiographic results showed that all lesions were unilocular and were either pushing or intruding into the surrounding structures ( maxillary sinus or mandibular canal ) in 15 patients ( 88.2% ) . based on the results of the histopathologic examination , 10 patients were diagnosed with kcot ( 58.8% ) , five patients with dentigerous cysts ( dc ) ( 29.4% ) , one patient with radicular cyst ( rc ) ( 5.9% ) , and one patient with nasopalatine duct cyst ( 5.9% ) . only one patient ( 5.9% ) was treated with marsupialization only , and 16 patients ( 94.1% ) were treated with marsupialization followed by enucleation . there was no case where the preoperative histopathologic results changed after the operation . the average time of decompression was 8.13.4 months , with 5.54.8 months for a cyst in the mandible and 8.33.1 months for the maxilla . the average time of decompression based on lesion type was 8.83.2 months for kcot and 6.02.7 months for dc , suggesting that kcots require approximately three more months of treatment . the li was measured at an average of 1,349 mm before decompression and 463 mm after decompression , showing an average size decrease of 64% after decompression . according to nakamura 's formula , five patients ( 29.4% ) showed a greater than 80% decrease in size , nine patients ( 52.9% ) showed a 50%-80% decrease in size , and three patients ( 17.6% ) showed a less than 50% decrease in size.(fig . 3 ) the mandible decreased by 10% more in size than the maxilla , and it had a 1.7-fold higher reduction rate in the same period . also , when comparing size reduction based on patient age , the decrease was greater in patients in their teens or 20s compared to the other age groups , but the reduction rates were not significantly different . males were observed to have a smaller final lesion size , while the reduction ratio was higher in females ; however , no significant differences were observed . additionally , larger lesions reduced faster that smaller ones , and kcots showed more positive results for decompression than other lesion types.(table 2 ) one patient 's recovery was complicated by hypoesthesia after marsupialization , but this naturally recovered over time . the total follow - up period was 1 to 8 years , and 3 out of 17 patients were suspected recurrence . however , one patient was diagnosed with scar tissue after re - examination , and one patient was diagnosed with periapical cyst due to re - infection of an incomplete root canal treatment . a true recurrence appeared on one nbccs patient 's left maxilla and is currently being followed - up after re - operation . since decompression was introduced as a conservative treatment for odontogenic cyst , many cases have been treated with decompression , and various studies have reported high success rates89 . however , the reaction varies depending on the cyst . anavi et al.10 conducted decompression on 57 patients who were diagnosed with kcot , dc , or rc , and no statistically significant differences in reduction rate were reported based on the histologic diagnosis . in present study , the reduction rate in kcot was higher than the other cysts , with an average of 131.84 mm / month , with the final reduction being the highest . this is because kcots are aggressive , so the size of the lesion tends to be greater than that of other cystic lesions . according to this study , cases of li larger than 1,000 mm showed twice the sensitivity of li smaller than 1,000 mm . therefore , in this study , kcot patients seemed to have a higher sensitivity to decompression since the li was larger than 1,000 mm . there are no formal criteria for the decompression period or change in size ; however , according to previous studies , a 65% reduction was reported when maintained for an average of 8.4 months . additionally , an 81% reduction was observed when decompression was maintained for an average of 17.5 months911 . another study reported that it is preferable to perform enucleation if the size of the lesion after decompression decreases more than 50%-60%8 . in this study , the average decompression period was 8.1 months , and the lesions showed an average reduction of 64% . the effects of decompression based on age are controversial , as it was reported that younger patients had higher reduction rates10 , but it also was reported that decompression is not correlated with age12 . therefore , even at older ages , if a large cystic lesion exists within the jaw , postoperative complications can be reduced by decreasing size via decompression . brndum and jensen13 performed enucleation after decompression in 12 patients with kcot ; during 7 to 17 years of follow - up , no recurrence was reported . such low recurrence rates were reported in many studies , and some of them reported biopsy results indicating that the histological characteristics of the residual cyst epithelium changed from parakeratinization to orthokeratinization or were no longer observed14 . based on these results , august et al.11 reported that histological changes are likely to appear when decompression is maintained for longer than nine months , and he proposed a decompression period of at least nine months . this study used a panoramic radiograph to two - dimensionally evaluate the lesion by measuring the maximum vertical and horizontal widths of the lesion . this method is widely used because of ease , but it has a disadvantage in accuracy , as it two - dimensionally evaluates a three - dimensional lesion . therefore , a recent method of measuring volume via three - dimensional analysis using a computer program was introduced15 . however , this method requires a special program and skilled technique , so it can be difficult to apply clinically . although plane analysis was performed in the present study due to lack of data for three - dimensional analysis , a more accurate analysis is expected once sufficient data is obtained . this study was limited in that statistical analysis was not performed due to lack of samples . thus , when a lesion is large , reducing its size via decompression not only allows for conservative treatment , but it can also minimize complications . according to this study , all patients treated with decompression were reported to have shown a reduction in lesion size and higher sensitivity with a larger lesion size . there was no difference in the effect of decompression based on age , and only one patient experienced recurrence of the cyst .

objectivesthe purpose of this study is to evaluate the treatment efficacy of enucleation after decompression.materials and methodsa total of 17 patients with cystic lesion of the jaw were treated with decompression followed by enucleation . pre- and postdecompression panoramic radiographs were analyzed.resultsthe mean percentage of reduction after decompression was 64% . the reaction was graded as good ( > 80% ) in five patients ( 29.4% ) , moderate ( 50%-80% ) in nine patients ( 52.9% ) , and poor ( < 50% ) in three patients ( 17.6% ) . the reduction rate of larger cystic lesions was faster than that of smaller lesions . however , the reduction rate was not affected by age . the duration of follow - up ranged from one to eight years . there were no complications , and one case recurred.conclusiondecompression is an effective method for the initial treatment of jaw cysts .

for the first time , the presented data suggest that a variant in the retn gene plays a significant role in acne pathogenesis . we also demonstrate an excess transmission of mutant allele g at retn-420 from 130 trio families using tdt analysis . therefore , we suggest that inherited variation in retn at -420 appears to be associated with heritable acne vulgaris .

resistin ( retn ) , recently found to be relevant to inflammation and inflammatory disorders . we , therefore , aimed to investigate the potential role of retn gene polymorphism in pathogenesis of acne vulgaris with familial history . we investigated the retn-420c / g polymorphism in 180 patients with acne vulgaris and 180 healthy individuals in a case - control association analysis . in this study , we also investigated the heritability of the retn susceptible allele from 140 trio families with acne affected offspring . the genotyping was performed by polymerase chain reaction and direct dna sequencing . the retn-420c / g polymorphism was significantly associated with acne in patients compared with healthy controls ( p=0.014 ) . the minor allele g at -420 was more prevalent in cases vs. controls ( p=0.002 ) . the retn-420c / g polymorphism was significantly associated with severity of acne vulgaris in patients ( p=0.0097 ) . the results of a transmission disequilibrium test revealed a significant association between the retn-420c / g polymorphism and acne vulgaris ( p<0.001 ) . for the first time in the literature , to our knowledge , we demonstrate a significant association of the retn-420c / g functional polymorphism with familial acne vulgaris .

many studies over the past 15 years have focused on discovering solvents that help promote efficient cc bond formation under benign reaction conditions . new solvents in dielsalder(2 ) and barbier reactions have been sought to help streamline the one - pot transformations further , reducing synthetic steps and organic waste . polar media often accelerate reactions of this type which has led to the use of water as an acceptable solvent for such organic conversions . recent contributions from our laboratory to this field have centered on coupling reactions between propargyl aldehydes and allyl or propargyl halides under barbier conditions to form homoallylic and homopropargylic propargyl alcohols . these compounds find wide use as synthetic templates and show a propensity for oxy - cope rearrangements ( scheme 1 ) . the use of -chloropropargylphenyl sulfide ( 4 ) , coupled with a propargyl aldehyde should offer an easy route into formation of enediyne and epoxydiyne skeletal structures ( scheme 2 ) . previous studies revealed that formation of 2 or 3 under indium - promoted barbier conditions is solvent dependent . use of an organic solvent for the coupling reaction results in a mixture of 2 and 3 while the use of water as a solvent yields 2 as the sole product . it is possible that water protonates the coupled product to form 2 quickly , quenching the alkoxide intermediate , thereby suppressing the possible oxy - cope pathway shown in scheme 1 . it may also be possible that the increased polarity of the solvent itself stabilizes the anion formed upon coupling , thus raising the relative rate of cope rearrangement in this system . with a less polar , aprotic solvent , the anion will not be quenched , nor will it be stabilized to the same extent as witnessed in water . a combination of these phenomena may be why an oxy - cope rearrangement occurs under less polar conditions , forming product 3 ( scheme 1 ) . a coupling reaction between 1 and 4 ( scheme 2 ) was conducted under aqueous conditions to form 2 ( y = sph ) , with good regioselectivity ; however , the rate of this reaction is surprisingly slow . although reactions of the type shown in scheme 1 are reported in the literature to be accelerated by polar solvents such as water , coupling reactions between 1 and 4 , as shown in scheme 2 , are quite slow . indeed , when water is used as the solvent , reactions often require 1036 h for completion . this oil has a density greater than that of water so it is presumed to be an organometallic species formed by interaction of the indium metal with the halide species . isolation of the oil followed by workup in an acidic thf / h2o mixture leads to isolation of propargylphenyl sulfide and , to a smaller extent , the aldehyde . we surmise that low solubility of this organometallic complex leads to poor reagent mixing , slowing the barbier coupling under aqueous conditions . second , indium - promoted coupling reactions conducted in water become more acidic as the reaction proceeds . we believe it is possible that the aldehyde is in equilibrium with its corresponding hydrate species , reducing the electrophilicity of the carbon center . a combination of these two reasons would lead to a decrease in the rate of reaction between 1 and 4 when conducted in an aqueous solvent . the reaction proceeds more quickly ( 812 h ) in dmf , a less polar solvent , due to better solubility of all reagents , however , a mixture of 2 and 3 was isolated . it was originally expected that a mixture of water and dmf would lead to faster reaction times while still maintaining regiocontrol of product formation . the use of a water / dmf solvent system , however , resulted in a mixture of products with water constituting up to 50% v / v of the solvent system . when water exceeds 50% v / v of the solvent system , only product 2 is isolated , but the rate of reaction slows perceptively once again . to help expedite this reaction sequence , we began a search for solvent with polarity similar to , or greater than that of water . it was expected that an extremely polar organic solvent would promote the same type of acceleration normally witnessed in barbier couplings carried out under aqueous conditions . an organic solvent could also help solubilize the intermediate and reduce possible formation of the hydrate species , thus allowing for a faster coupling reaction to occur . if increased polarity stabilizes the anion formed by coupling of 1 and 4 , oxy - cope rearrangement should also be suppressed . with a dielectric constant of 186.9 at 25 c , significantly greater than that of water ( 78.37(7 ) ) , and dmf ( 38.3(7b ) ) , n - methylformamide ( nmf ) has found occasional use in previous reactions requiring polar organic solvents ; however , it has historically found more widespread use as an organic reagent . with a pka of approximately 24,(7d ) nmf is reactive under a range of organometallic reagent conditions , limiting its use as a solvent . indium - promoted coupling reactions , however , have been shown to be accelerated under aqueous conditions,(1 ) and the relative acidity of nmf , being higher than that of water , should not hinder the proposed barbier couplings proposed within this paper . we were especially intrigued by the fact that the dielectric constant of nmf increases to 220 as the temperature is lowered from 25 to 0 c . we were interested in examining what effect the increase in dielectric constant at lower temperature could have on a barbier - style reaction of the type shown in scheme 2 among others . this paper reports on the use of nmf in barbier - style reactions with a focus on rates of reaction , stereochemistry , and its use to form enediyne and epoxydiyne skeletal structures.(9 ) although nmf has characteristics similar to those of dmf , it also has characteristics similar to water , such as a polar xh bond.(7 ) thus , having found a very polar solvent with properties similar to both water and organic systems , we hypothesized that the 1,2-coupling product could be formed in high yield under short reaction times . a series of reactions was set up to test this hypothesis . as shown in table 1 , use of either water or nmf resulted in formation of the 1,2-product ( 2 ) solely with no trace of 3 detected in the product mixture . although the yield of product was moderate to high in either solvent , product formation in reactions conducted in nmf proceeded at a much greater rate . we believe that there are two possible reasons behind the rate increase of coupling reactions carried out in nmf . the ratio of reagent was as follows : 1.0:1.5:1.1 ( aldehyde / halide / indium ) . the reaction was run at 0.1 m with respect to indium . in the lewis acid catalyzed reaction , 10 molar % of the lewis acid was used . all species completely dissolved in nmf , unlike what was observed for reactions in water . as such , we believe mixing is much more efficient , allowing the reaction to proceed at a faster rate . has also been noted in many literature examples to lead to rate enhancement in indium - promoted barbier reactions . we believe a mixture of better solubility coupled with greater solvent polarity results in an additive effect toward increasing the rate of reactions conducted in nmf when compared to water . h - bonding capabilities of nmf may explain the greater regioselectivity for reactions conducted in nmf when compared to those observed in dmf and thf ; however , the h - bonding capacity of nmf is much lower than that of water.(7d ) increased solvent polarity , thus increasing the stability of the intermediate anion and raising the relative energy of the oxy - cope rearrangement , may seem more likely at this time . in an effort to exclude the solvent as a direct participant in intermediate formation when nmf is used , nmr spectroscopy studies were performed to determine if a possible hemiaminal or imminium species was forming in situ ( figure 1 ) . the imminium functionality carries a positive charge and would increase the electrophilicity of the reactive carbon . mixing of 1 and nmf under sonication for periods of up to 24 h , however , revealed the presence of only 1 and nmf by nmr spectroscopy . possible formation of hemiaminal or imminium species . the presence or absence of a lewis acid has shown to help control stereoselectivity in indium - promoted cc bond - forming reactions . previous studies in our laboratory have shown that the addition of indium(iii ) chloride as a lewis acid favors formation of the syn - product in indium - promoted cc bond - forming reactions between 4 and aldehyde functional groups . in the absence of the lewis acid , the anti - product is favored ( figure 2).(5b ) it was our hope that similar effects would be witnessed in the present system , especially while using nmf as the reaction solvent , and we were pleased to see this trend continue . absence of a lewis acid in the reagent mixture favored a nonchelated pathway , leading to formation of anti-2 . use of indium chloride to control stereochemistry.(5b ) the presence of a lewis acid in the reaction mixture favored a chelated pathway , leading to formation of syn-2 ( figure 3 ) . diastereoselectivities were determined either by direct measurement of vicinal coupling constants in the hydroxyl sulfide or by corresponding conversion to the epoxide compounds as shown in figure 4.(11 ) general j values for hydroxyl sulfides and epoxide structures . entries 1 , 5 , and 13 ( table 1 ) describe reactions run in nmf in the absence of a lewis acid . good anti - selectivity is witnessed with ratios as high as 15:85 ( syn / anti ) . in all cases , with the exception of r = phenyl , systems , with and without incl3 , run in nmf gave better selectivity than systems run in water . both systems showed poor selectivity when r = tms for reasons of which we are not quite sure . when incl3 was added as a lewis acid , the chelated route was favored in ratios as high as 90:10 ( syn : anti , entry 15 ) . n - methylformamide was shown to increase the rate of reaction compared with water and offered better stereocontrol in the systems studied . enediyne and epoxydiyne functionalities contain either a ( z)-3-ene-1,5-diyne or ( z)-3-epoxy-1,5-diyne unit which leads to their noted activity.(9 ) these unique functional groups cyclize to form a p - benzyne- or p - benzyne - like intermediate that interacts with dna by abstracting hydrogen.(9 ) the dna diradical then either couples with itself , or cleaves , stopping replication . natural product enediyne and epoxydiynes have found use as anticarcinogenic compounds.(9 ) this area of study is slowed , however , by complicated access into the skeletal systems required.(9 ) literature procedures that find use in epoxy alkyne formation such as halohydrin cylization,(12)m - cpba,(13 ) or oxone(14 ) have been shown to be successful , but limited examples are known with these systems at present . under the barbier coupling conditions described in this paper , formation of compound 2 offers an efficient and controlled method for the formation of either an enediyne or epoxydiyne skeletal structure under benign conditions by allowing easy conversion to either the cis - epoxydiyne ( from syn-2 , scheme 3 ) or the cis - enediyne ( from anti-2 , scheme 4 ) . facile synthesis of epoxydiyne and enediyne functional groups under the reported conditions would allow for their use as template structures in organic synthesis , opening access to more sophisticated units for further exploration . in the following pages , we describe the transformation of 1 into either an epoxydiyne ( 5 ) or enediyne ( 6 ) skeletal structure in two steps with high regio- and stereocontrol using nmf as a new solvent to form 2 under barbier conditions , followed by a one - step transformation to yield either 5 or 6 . as shown in scheme 3 , syn-2 can adopt the correct conformation to form a cis - oxirane upon reaction with either me3obf4 or tloet . conversion of the hydroxy sulfide starting material ( 2 ) to the epoxyalkyne product ( 5 ) was quite successful as shown in table 2 . in all cases , this fact is worth mentioning as barbier reactions with propargyl halides can lead to rearrangement of the organometallic intermediate into an allenyl product . reagent 4 has been previously shown to offer one of the few examples of a barbier reaction with a propargyl halide where rearrangement does not occur.(5b ) use of tloet(17 ) gave yields similar to that of me3obf4(18 ) in all cases of oxirane formation , with stereoselectivity preserved under both sets of conditions . use of the anti-2 allows formation of the ( e)-alkene ( the alkyne functional groups are cis to each other ) functional group by the pathway shown in scheme 4 . formation of the enediyne product was possible under slightly acidic conditions . a catalytic amount of p - tsoh is added to anti-2 in a mixture of water and thf ( 10% water ) with stirring for 37 h. the reaction was also conducted under basic conditions after transformation of the oh group to a tosylate . yields were quite low under basic conditions , presumably due to the competing acidity of the alkynyl proton , leading to decomposition and side product formation . due to low yields , results of the acid - catalyzed conversion of anti-2 to ( e)-6 are shown in table 3 . a slight amount of isomerization was witnessed under acidic conditions ( about 3% of the product mixture ) , but overall , good stereocontrol was realized in these conversions , allowing for easy entry into the cis - enediyne ( ( e)-alkene ) skeletal structure in a two - step sequence . the assignment of e- and z - isomers was made by comparison of the -vinyl proton shift . alignment of the -vinyl proton and the heteroatom cis to each other gives rise to a lower field proton resonance than trans alignment of these two groups ( figure 5).(19 ) the assignments for the enediyne products are compared correctly with the stereochemistry of compounds 2 and 3 within this study . coupled with the original indium - promoted barbier reaction , the transformation of starting materials 1 and 4 into enediyne or epoxydiyne compounds proceeded in two steps in an overall 6080% overall yield , offering an efficient , benign , and good - yielding formation of these important skeletal structures . the use of n - methylformamide is shown to be a nice alternative to aqueous conditions in indium promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide ( 4 ) . this paper introduces the use of nmf as s viable solvent in a barbier coupling reaction , revealing increased rates and better selectivity when compared to reactions conducted in water and other organic solvents . the increased efficiency of the barbier coupling reaction as described in this paper allows for facile formation of epoxy - and enediyne skeletal structures in an overall two - step procedure . the introduction of nmf as an alternative solvent in barbier reactions will open new pathways to organic synthetic chemists . further studies to test other halides and aldehyde functional groups under barbier coupling reaction conditions in nmf are currently underway and will be reported in due time . tetrahydrofuran was purified by distillation under an argon atmosphere over sodium and benzophenone prior to use . radial chromatography was performed using a chromatatron . to a solution of 30 mmol of alkyne in 80 ml of thf that had been cooled to 60 c under nitrogen was added 15 ml of n - buli ( 2.0 m in thf ) with stirring . to this solution was added n , n - dimethylformamide ( 4.66 ml , 60 mmol ) dropwise over 10 min . after 20 min of further stirring , the solution was added to a mixture of 75 ml of tert - butyl methyl ether and 160 ml of 10% kh2po4 solution which had been previously cooled to 0 c . the entire solution was stirred at 0 c for 30 min and then allowed to warm to room temperature . the organic layer was dried over mgso4 , and the solvent removed under vacuum to give a clear oil . all propargyl aldehydes were used without further purification and could be stored at 0 c for 23 weeks . yields for this reaction ranged from 85 to 95% . a magnetically stirred solution of aldehyde ( 1 ) ( 2.0 mmol ) in deionized water ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) . the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) . the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2 10 ml ) . the combined organic layers were dried over mgso4 and evaporated to leave a dark yellow to brown oil . purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil . purification was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield = 471 mg ( 1.76 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 ) 0.91.5 ( m , 7h ) , 1.82 ( d , j = 1.7 hz , 0.2h ) , 1.87 ( d , j = 1.6 hz , 0.8h ) , 2.13 ( t , j = 6.5 hz , 2h ) , 4.09 ( m , 1h ) , 4.86 ( m , 1h ) , 7.17.8 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 15.5 , 18.2 , 21.3 , 34.2 , ( 41.5 , 42.9 ) , ( 65.0 , 66.1 ) , 71.3 , 78.5 , 80.2 , 84.7 , 125.4 , 126.1 , 126.3 , 127.6 , 128.0 , 136.4 ; ms m / z ( m ) calcd 258.1078 , obsd 258.0998 . calcd for c16h18os : c , 74.38 ; h , 7.02 . found : c , 74.61 ; h , 6.99 . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : total yield = 489 mg ( 1.78 mmol ) = 80% . anti isomer : yield = 391 mg ( 1.41 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 1.94 ( d , j = 1.8 hz , 1h ) , 4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 43.8 , 66.0 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 . h nmr ( 300 mhz , cdcl3 ) 1.83 ( d , j = 1.8 hz , 1h ) , 3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 44.1 , 65.8 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 50:50 syn / anti ratio : total yield = 418 mg ( 1.54 mmol ) = 70% ; anti isomer : yield = 209 mg ( 7.7 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 0.24 ( s , 9h ) , 1.84 ( d , j = 1.6 hz , 1h ) , 4.23 ( dd , j = 1.6 , 5.3 hz , 1h ) , 4.80 ( d , j = 5.3 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 0.28(3 ) , 44.4 , 66.5 , 67.5 , 74.1 , 84.3 , 86.4 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 ; ms m / z ( m ) calcd 274.0848 , obsd 274.0819 . calcd for c15h18ossi : c , 65.64 ; h , 6.61 . found : c , 65.17 ; h , 6.55 . syn isomer : yield = 209 mg ( 7.7 mmol ) = 70% ; h nmr ( 300 mhz , cdcl3 ) 0.24 ( s , 9h ) , 1.79 ( d , j = 1.7 hz , 1h ) , 4.15 ( dd , j = 1.7 , 7.8 hz , 1h ) , 4.69 ( d , j = 7.8 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 0.28(3 ) , 44.1 , 67.9 , 68.1 , 75.0 , 83.2 , 86.9 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides : yield = 633 mg ( 1.69 mmol ) = 77% ; h nmr ( 300 mhz , cdcl3 ) 0.25 ( s , 6h ) , 0.97 ( s , 9h ) , 1.25 ( m , 2h ) , 1.802.00 ( m , 3h ) , 3.83 ( t , j = 6.5 , 2h ) , 4.11 ( m , 1h ) , 4.99 ( m , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 0.28 ( 2 ) , 14.5 , 15.1 , 21.8 ( 2 ) , 21.9 33.4 , 47.0 , 64.6 , 65.1 , 67.9 , 75.2 , 81.9 , 87.3 , 125.4 , 126.6 ( 2 ) , 127.1 , 128.4 , 135.9 ; ms m / z ( m+ ) calcd 374.1736 , obsd 365.1633 ( loss of h2o ) . the general procedure was followed as described in section i.a with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used . separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield = 437 mg ( 1.63 mmol ) = 77% . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 75:25 syn / anti ratio : yield = total = 458 mg ( 1.65 mmol ) = 75% . anti isomer : yield = 115 mg ( 0.41 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 1.94 ( d , j = 1.8 hz , 1h ) , 4.12 ( dd , j = 1,8 , 4.5 hz , syn isomer : yield = 343 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 1.83 ( d , j = 1.8 hz , 1h ) , 3.92 ( dd , j = 1.8 , 8.0 hz , 1h ) , 4.94 ( d , j = 8.1 hz , 1h ) processing and analysis of this reaction proceeded as described in section i.b . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 60:40 syn / anti ratio : yield = total = 434 mg ( 1.58 mmol ) = 72% . anti isomer : yield = 174 mg ( 0.63 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) . syn isomer : yield = 260 mg ( 0.95 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield 583 mg ( 1.55 mmol ) = 71% . analysis as shown in section i.a.4 . a magnetically stirred solution of aldehyde 1 ( 2.0 mmol ) in nmf ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) . the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) . the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2 10 ml ) . the combined organic layers were washed with 1 n hcl ( 3 20 ml ) , dried over mgso4 , and evaporated to leave a dark yellow to brown oil . purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil . separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield = 522 mg ( 2.02 mmol ) = 92% . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : yield = total = 556 mg ( 2.00 mmol ) = 91% . anti isomer : yield = 445 mg ( 1.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) . syn isomer : yield = 111 mg ( 0.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 40:60 syn / anti ratio : yield = total = 566 mg ( 2.06 mmol ) = 94% . anti isomer : yield = 340 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) . syn isomer : yield = 226 mg ( 0.82 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield = 717 mg ( 1.91 mmol ) = 87% . the general procedure was followed as described in section i.c with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used . separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield = 527 mg ( 2.05 mmol ) = 93% . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 85:15 syn / anti ratio : yield = total = 568 mg ( 2.05 mmol ) = 93% . anti isomer : yield = 85 mg ( 0.31 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.13 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.08 ( d , j = 4.7 hz , 1h ) . syn isomer : yield = 483 mg ( 1.74 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 3.92 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.97 ( d , j = 8.1 hz , 1h ) . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 70:30 syn / anti ratio : yield = total = 548 mg ( 2.00 mmol ) = 91% . anti isomer : : yield = 164 mg ( 0.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.23 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.80 ( d , j = 5.5 hz , 1h ) . syn isomer : yield = 384 mg ( 1.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.68 ( d , j = 7.8 hz , 1h ) . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides . stereoselectivity was determined upon transformation to the epoxide product : yield = 732 mg ( 1.96 mmol ) = 89% . a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 100 mg , 0.39 mmol ) in dichloromethane ( 10 ml ) was treated with trimethyloxonium tetrafluoroborate ( 90 mg ( 0.60 mmol ) . the solution was stirred at room temperature for 8 h and diluted with 7% sodium hydroxide solution ( aqueous , 8 ml ) . purification was accomplished by radial chromatography on silica gel ( elution with 80:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 85:15 syn / anti ratio : yield = 52 mg ( 0.35 mmol ) = 90% ; h nmr ( 300 mhz , cdcl3 ) 0.9 1.5 ( m , 7h ) , 1.82 ( d , j = 1.8 hz , 0.15h ) , 1.86 ( d , j = 1.9 hz , 0.85h ) , 2.17 ( m , 2h ) , 3.25 ( d , j = 4.6 hz , 0.85h ) , 3.40 ( d , j = 2.3 hz , 0.15h ) , 3.50 ( dd , j = 1.8 , 2.3 hz , 0.15h ) , 3.61 ( dd , j = 1.9 , 4.6 hz , 0.85h ) ; c nmr ( 75 mhz , cdcl3 ) ( 14.0 , 14.1 ) , ( 17.2 , 17.4 ) , 21.6 , ( 31.7 , 31.8 ) , 44.5 , ( 48.2 , 48.3 ) , 66.7 , ( 77.5 , 77.9 ) , ( 79.5 , 79.7 ) , ( 85.2 , 85.5 ) calcd for c10h12o : c , 81.04 ; h , 8.16 . found : c , 80.99 ; h , 8.19 . a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) . the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) . purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 85:15 syn / anti ratio : yield = total = 110 mg ( 0.64 mmol ) = 89% . anti isomer : yield = 12 mg ( 0.07 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 2.44 ( d , j = 1.8 hz , 1h ) , 3.51 ( dd , j = 1.8 , 2.4 hz , 1h ) , 3.97 ( d , j = 2.4 hz , 1h ) , 7.157.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 48.2 , 53.1 , 66.2 , 80.3 , 87.3 , 91.7 , 123.5 , 128.4 ( 2 ) , 129.0 , 133.1 ( 2 ) . calcd for c12h8o = c , 85.69 h , 4.79 . found : c , 86.00 h , 4.71 . syn isomer : yield = 98 mg ( 0.57 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 2.43 ( d , j = 1.8 hz , 1h ) , 3.46 ( dd , j = 1.8 , 4.7 hz , 1h ) , 4.15 ( d , j = 4.7 hz , 1h ) , 7.157.23 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 48.7 , 53.1 , 66.5 , 80.1 , 87.3 , 92.0 , 123.0 , 128.3 ( 2 ) , 128.7 , 132.3(2 ) . a solution of the hydroxy sulfide mixture ( 70:30 syn / anti ) ( 200 mg 0.73 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) . the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) . purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 70:30 syn / anti ratio : yield = total = 108 mg ( 0.66 mmol ) = 90% ; anti isomer : yield = 32 mg ( 0.30 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 0.13 ( s , 9h ) ; 2.21 ( d , j = 2.0 hz , 1h ) , 3.25 ( dd , j = 2.0 , 2.3 hz , 1h ) , 3.55 ( d , j = 2.3 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 ) 0.10 ( 3 ) , 44.9 , 49.9 , 69.9 , 81.7 , 86.0 , 102.0 ; ms m / z ( m ) calcd = 164.0657 , obsd = 164.0656 . syn isomer : yield = 76 mg ( 0.46 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 0.13 ( s , 9h ) , 2.19 ( d , j = 2.0 hz , 1h ) , 3.32 ( dd , j = 2.0 , 4.9 hz , 1h ) , 3.63 ( d , j = 4.9 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 ) 0.10 ( 3 ) , 45.1 , 49.3 , 70.1 , 81.5 , 85.6 , 101.2 . a solution of the hydroxy sulfide mixture ( 90:10 syn / anti ) ( 250 mg 0.67 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 150 mg ( 1.00 mmol ) . the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 12 ml ) . purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) to give a mixture of two diastereomers as a colorless oil in a 90:10 syn / anti ratio : yield = 148 mg ( 0.56 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 ) 0.22 ( s , 6h ) ; 0.97 ( s , 9h ) , 1.27 ( m , 2h ) , 1.82.0 ( m , 3h ) , 3.23 ( d , j = 4.4 hz , 0.9h ) , 3.31 ( d , j = 2.5 hz , 0.1h ) , 3.53 ( dd , j = 1.9 , 2.5 hz , 0.1 hz ) , 3.69 ( dd , j = 1.9 , 4.4 hz , 0.9h ) , 3.83 ( m , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 0.28 ( 2 ) , 14.5 , 14.7 , 21.8 ( 2 ) , ( 22.0 , 22.3 ) , ( 33.9 , 34.2 ) , ( 43.8 , 44.0 ) , ( 47.1 , 47.9 ) , ( 64.7 , 65.0 ) , 67.3 , ( 78.7 , 79.1 ) , 81.8 , ( 85.4 , 85.8 ) ; ms m / z ( m ) calcd = 264.1546 , obsd = 264.1545 calcd for c15h24osi : c , 68.13 ; h , 9.15 . found : c , 67.79 ; h , 9.4 . a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in chloroform ( 20 ml ) was treated with tloet ( 234 mg , 0.93 mmol ) . after 20 min of stirring , the insolubles were removed by filtration through a short celite pad . the resulting organic solution was washed with a saturated nahco3 solution ( aq ) and a saturated nacl solution ( aq ) . the organic layer was dried and concentrated . purification and analysis of products proceeded in the manner previously described ( section ii.a ) . subsequent hydroxy sulfides were treated in a fashion similar to that in ii.b.1 and analyzed in the manner previously described in section ii.a . a solution of the hydroxy sulfide mixture ( 25:75 syn / anti ) ( 100 mg , 0.39 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid . the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) . after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min . the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated . purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 73:27 cis / trans ratio : yield = 82 mg ( 0.34 mmol ) = 87% ; h nmr ( 300 mhz , cdcl3 ) 0.901.51 ( m , 7h ) , 2.07 ( m , 2h ) , 2.63 ( s , 0.27h ) , 2.71 ( s , 0.73h ) , 5.90 ( s , 0.73h ) , 6.21 ( s , 0.27h ) 7.107.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) ( 13.9 , 14.1 ) , 17.1 , 22.9 , 31.6 , 77.2 , ( 79.5 , 80.3 ) , ( 83.1 , 83.7 ) , ( 90.5 , 91.7 ) , ( 113.3 , 113.9 ) , 125.1 , 128.4(2 ) , 129.3(2 ) , 134.0 , ( 145.9 , 146.2 ) ; ms m / z ( m ) calcd 240.0973 , obsd 240.0973 . calcd for c16h16s : c , 79.95 ; h , 6.71 . found : c , 80.09 ; h , 6.60 . a solution of the hydroxy sulfide ( 200 mg 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid . the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) . after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min . the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated . purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : ield = 156 mg ( 0.60 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 ) 0.2.95 ( s , 1h ) , 6.01 ( s , 1h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 ) 81.7 , 82.9 , 89.3 , 92.6 , 114.0 , 122.0 , 125.7 , 128.1 ( 2 ) , 128.3 , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , 134.0 , 144.6 . a solution of the hydroxy sulfide mixture ( 20:80 syn / anti ) ( 200 mg , 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid . the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) . after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min . the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated . purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 77:23 e / z ratio : yield = 160 mg ( 0.61 mmol ) = 85% ; h nmr ( 300 mhz , cdcl3 ) 0.2.95 ( s , 0.77h ) , 3.01 ( s , 0.23h ) , 6.04 ( s , 0.77h ) , 6.35 ( s , 0.27h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 ) ( 81.5 , 81.7 ) , 83.0 , 89.3 , ( 92.6 , 93.0 ) , ( 114.0 , 119.2 ) , 122.0 , 125.7 , 128.1 ( 2 ) , ( 128.3 , 128.5 ) , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , ( 134.0 , 134.5 ) , ( 144.6 , 145.1 ) . a solution of the hydroxy sulfide ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid . the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) . after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min . the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated . purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : yield = 123 mg ( 0.0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 ) 0.0.11 ( s , 9h ) , 2.87 ( s , 1h ) , 6.20 ( s , 1h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 0.09 ( 3 ) , 78.7 , 81.2 , 95.3 , 100.9 , 112.5 , 124.8 , 127.2(2 ) , 128.7(2 ) , 131.8 , 142.1 . calcd for c15h16ssi : c , 70.25 ; h , 6.29 . found : c , 69.99 ; h , 6.43 . a solution of the hydroxy sulfide mixture ( 40:60 syn / anti ) ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid . the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) . after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min . the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated . purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 50:50 e / z ratio : yield = 120 mg ( 0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 ) 0.09 ( s , 4.5h ) 0.10 ( s , 4.5h ) 2.81 ( s , 0.5h ) , 2.87 ( s , 0.5h ) , 5.77 ( s , 0.5h ) , 6.20 ( s , 0.5h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 0.09 ( 3 ) , ( 78.7 , 79.1 ) , ( 81.2 , 81.8 ) , ( 95.3 , 95.9 ) , 100.9 , ( 112.5 , 116.1 ) , 124.8 , 127.2(2 ) , 128.7(2 ) , ( 131.8 , 132.9 ) , ( 142.1 , 143.5 ) . a solution of the hydroxy sulfide mixture ( 15:85 syn / anti ) ( 250 mg 0.67 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid . the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) . after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min . the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated . purification was accomplished by radial chromatography on silica gel ( elution with 10:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 82:18 cis / trans ratio : yield = 192 mg ( 0.54 mmol ) = 80% ; h nmr ( 300 mhz , cdcl3 ) 0.15 ( s , 6h ) , 0.93 ( s , 9h ) , 1.27 ( m , 2h ) , 2.11(m , 2h ) , 2.71 ( s , 0.82h ) , 2.80 ( s , 0.18h ) , 3.75 ( m , 2h ) , 5.93 ( s , 0.82h ) , 6.16 ( s , 0.18h ) , 7.077.26 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 0.13(2 ) , ( 13.9 , 14.1 ) , ( 15.0 , 15.1 ) , 21.3(3 ) , 33.9 , ( 64.9 , 65.3 ) , ( 77.3 , 78.1 ) , ( 79.7 , 80.1 ) , ( 83.2 , 84.1 ) , ( 93.1 , 93.7 ) , ( 114.1 , 115.0 ) , 125.4 , 127.7(2 ) , 129.1 , 129.3 , ( 133.7 , 134.0 ) , ( 144.9 , 145.4 ) ; ms m / z ( m ) calcd 356.1630 , obsd 355.9891 calcd for c21h28ossi : c , 70.73 ; h , 7.91 . found : c , 70.81 ; h , 8.01 .

indium - promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide are reported . although water has been shown to accelerate indium metal promoted reactions , the reverse pattern was observed in this series . use of n - methylformamide ( nmf ) , which has not previously been a solvent known for use in indium - promoted reactions , afforded an acceleration of these barbier - style reactions compared to water . indium - promoted reactions in this study also showed excellent regiocontrol and good stereocontrol , allowing for easy entry into the formation of epoxydiyne and enediyne skeletal structures . this paper also describes use of the barbier coupled product ( 2 ) as a new , and easy , entry into the formation of enediyne and epoxydiyne skeletal structures .

sepsis is the systemic inflammation caused by infection with bacteria , viruses , fungi , parasites , or toxic products , and has become one of the leading causes of mortality in children . in developed countries , the mortality rate of pediatric sepsis is about 210% and is as high as 50% in developing countries . pediatric sepsis usually leads to tachycardia , tachypnea , peripheral vasodilation , fever or hypothermia , and even multiple organ dysfunction syndrome ( mods ) . frequently - used applications in pediatric sepsis treatment concentrate on maintaining metabolic , circulatory , and respiratory stabilities , and antimicrobial agents , as well as surgical and adjunctive therapies such as nitric oxide and corticosteroids , which show great benefits in the outcome of pediatric sepsis treatment . acute kidney injury ( aki ) is a severe consequence of sepsis and mods , and about 1527% of aki in children is generated from pediatric sepsis . the pathophysiology of septic aki is related to the altered global renal blood flow , as well as the comprehensive effects of inflammation , microvasculature blood flow , and bioenergetics factors . for example , glomerular endothelial injury during aki progression in a lipopolysaccharide - induced mouse sepsis model is closely related to tumor necrosis factor and receptor 1 . due to the increased risk of mortality caused by septic aki , it is imperative to explore effective therapies for controlling aki in pediatric sepsis . baicalin is one of the flavonoid constituents in scutellaria baicalensis georgi , a kind of perennial herb with protective effects against diseases like hydrogen peroxide - induced oxidative stress and cerebral ischemia injury [ 911 ] . specifically , baicalin has been revealed to protect against oxidants , anxiety , acute hepatic injury , experimental periodontitis , and sepsis . it also alleviates reperfusion - induced damage to the myocardium , the mechanism of which may be associated with its anti - apoptotic roles . moreover , a recent study found that baicalin can inhibit inflammation and cell apoptosis , which allows it to protect against ischemia - reperfusion injuries in the kidney . however , few studies have investigated the role of baicalin in septic aki or pediatric sepsis . this study aimed to uncover the effects of baicalin in treating aki in pediatric sepsis patients . the baicalin adjunctive therapy was tested in pediatric sepsis patients , after which the effect was compared based on the renal function assessment from bun and serum creatinine ( cr ) . we also performed experiments in the cecal ligation and puncture ( clp)-induced mouse sepsis model to investigate the effects and potential mechanisms of baicalin in septic aki . this study lays the foundation for future application of baicalin in adjunctive therapies for aki in pediatric sepsis . a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 . all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) . the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure . the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients . no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) . patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions . the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital . these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital . clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report . thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin . the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity . then the cecum was relocated to the abdominal cavity , and the abdomen was closed . the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) . mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling . cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions . briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices . fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark . then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min . the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min . slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) . total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions . dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse , 1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) . then the synthesized complementary dna ( 20 ng ) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) . data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control . protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions . the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes . the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c . after washing in pbs 5 times , the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature . ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals . comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) . a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 . all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) . the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure . the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients . no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) . patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions . the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital . these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital . clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report . thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin . the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity . then the cecum was relocated to the abdominal cavity , and the abdomen was closed . the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) . mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling . cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions . briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices . fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark . then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min . the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min . slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) . total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions . dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse , 1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) . then the synthesized complementary dna ( 20 ng ) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) . data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control . protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions . the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes . the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c . gapdh ( ab181602 ) was used as an internal control . after washing in pbs 5 times , the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature . ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals . comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) . in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies . results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) . it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy . we next performed experiments in the mouse model to analyze the function of baicalin in septic aki . mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp . both bun and cr levels were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model . the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis . tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) . thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model . bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis . qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) . bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) . protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2 are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model . in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies . results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) . it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy . we next performed experiments in the mouse model to analyze the function of baicalin in septic aki . mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp . both bun and cr levels were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model . the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis . tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) . thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model . bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis . qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) . bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) . protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2 are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model . this study performed investigations in pediatric sepsis patients and in a clp - induced mouse sepsis model to reveal the role of baicalin in aki of pediatric sepsis . baicalin adjunctive therapy in the patients and treatment in the mouse model decreased bun and cr levels . further histological and expression analyses suggested the suppressive function of baicalin in renal cell apoptosis . the 50 pediatric patients participating in this study were chosen from pediatric sepsis patients diagnosed with aki , and the mouse model induced by clp was detected with elevated bun and cr levels , which suggested potential kidney injury . previous studies using mouse sepsis models used various baicalin treatment methods , including topical application on the mouse skin to analyze the effect of baicalin on epidermis , intraperitoneal injection to investigate the role of baicalin in mammary glands , and baicalin - containing diets to study liver injury , inflammation in the kidney , and lung carcinoma [ 22,2729 ] . in these studies , the dose of baicalin was usually 100200 mg/(kgd ) , and the duration ranged from 3 to 50 days . thus , in the present study we chose to perform baicalin treatment in the model mouse via intragastric administration of 200 mg/(kgd ) for 6 days , and the significantly elevated bun and cr levels in the clp group compared to the sham group indicated that this treatment method effectively induced kidney injury in the mouse sepsis model . clinical trials with baicalin adjunctive therapy have focused on its protective roles in ulcerative colitis , hepatic fibrosis , and diabetes mellitus [ 3032 ] , and several studies have investigated the potential of baicalin in treating patients with early diabetic nephropathy , in which baicalin inhibits aldose reductase activity and reduces urinary albumin excretion rate and the blood 2-microglobulin . the present study further discovered the protective role of baicalin against aki in pediatric sepsis patients , which was reflected in the reduced bun and cr levels . moreover , the experiments in the mouse model confirmed that baicalin treatment in a sepsis animal model could improve renal functions . these findings show the potential of using baicalin to attenuate aki in pediatric sepsis patients , but the proper dosage and possible complications need to be assessed in future research . tunel results showed that baicalin treatment in the mouse model significantly suppressed renal cell apoptosis , and western blot and qrt - pcr analyses indicated consistent changes in the expression of bax and bcl2 . a similar mechanism has been reported in mouse mammary glands , where baicalin inhibits cell apoptosis via regulating bax and bcl2 . in addition , baicalin inhibits hepatocyte apoptosis in mouse kidney injury induced by concanavalin a ; therefore , suppressing renal cell apoptosis is likely to be one of the mechanisms by which baicalin attenuates septic kidney injury . however , the anti - proliferative role of baicalin has also been reported in studies on lung cancer and mouse embryonic stem cells , and it is converted to baicalin by intestinal gut flora to act as a pro - apoptotic and anti - proliferative substance . baicalin is capable of promoting neuronal differentiation , and thus may benefit therapies for parkinson and alzheimer diseases . we speculate that there are multiple potential mechanisms behind the effects of baicalin in various diseases ; therefore , it is necessary to uncover the detailed mechanisms in order to optimize use of baicalin to control aki in pediatric sepsis patients . the investigations of baicalin in pediatric sepsis patients and the clp - induced mouse sepsis model reveal the protective role of baicalin against aki in pediatric sepsis . baicalin can inhibit renal cell apoptosis , which may be one of its functional mechanisms . this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and future research will concentrate on more detailed mechanisms of baicalin .

backgroundpediatric sepsis has high morbidity in children , may lead to acute kidney injury ( aki ) , and further aggravate the disease . baicalin is a kind of flavonoid in scutellaria baicalensis georgi and has been reported to protect against several diseases , but its roles in septic aki remain unclear . this study aimed to uncover the effects of baicalin in aki during pediatric sepsis.material/methodsblood urea nitrogen ( bun ) and serum creatinine ( cr ) levels were detected in 50 pediatric patients , who underwent basic therapy with or without baicalin adjunctive therapy . mouse sepsis models were constructed by cecal ligation and puncture ( clp ) and treated with baicalin intragastrically , after which bun and cr examination , tunel apoptosis assay , and expression analyses of bax and bcl2 were performed.resultsbaicalin adjunctive therapy significantly decreased bun and cr levels in pediatric sepsis patients ( p<0.05 ) . clp led to elevated bun and cr levels in the mouse model ( p<0.01 ) , indicating kidney injury accompanied by sepsis . baicalin decreased bun and cr levels ( p<0.05 ) , and reduced the apoptotic cell percent in the renal tissue ( p<0.05 ) of the clp model . it inhibited bax and promoted bcl2 in the renal tissue , which was consistent with cell apoptosis changes.conclusionsbaicalin is capable of suppressing renal cell apoptosis and protecting against aki in pediatric sepsis . this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and further research is necessary to reveal its deeper mechanisms .

the main cause of treatment failure and death in cancer patients is metastasis the formation of secondary tumors in organs distant from the original neoplastic cell tissue . adjuvant therapy of proven efficacy is not currently available for cancer patients ; therefore , the search for new targets of therapeutic reagents is required to prevent both proliferation and metastases . during metastasis , it is known that the mode of invasion is one of the markers of the malignancy and prognosis of cancer . the receptor for advanced glycation end - products ( rage ) , a multiligand member of the immunoglobulin superfamily of cell surface molecules , interacts with distinct molecules implicated in homeostasis , development , and inflammation . rage binding by ligands such as advanced glycation end - products ( ages ) triggers the activation of key cell signaling pathways , thereby reprogramming cellular properties . in addition , several reports have suggested that rage is associated with cancer malignancy [ 4 , 5 ] . the advanced stage of the glycation process ( one of the posttranslational modifications of proteins ) leads to the formation of ages and plays an important role in the pathogenesis of angiopathy in diabetic patients , aging , and neurodegenerative diseases [ 69 ] . a growing body of evidence suggests that the interaction of glyceraldehyde - derived ages ( glycer - ages ) , but not glucose - derived ages ( glc - ages ) , with rage elicits oxidative stress generation in numerous types of cells ( vascular wall cells , mesangial cells , schwann cells , and cortical neurons ) , all of which could contribute to the pathological changes seen in diabetic vascular complications of alzheimer 's disease [ 1013 ] . we have recently found that glycer - ages stimulated the growth and migration of cultured human melanoma cells and that anti - rage antibodies inhibited the tumor formation and lung metastasis of melanoma cell xenografts and subsequently improved survival in athymic mice . however , the effects of glycer - ages on other cancer cells remains poorly understood , and the molecular mechanisms behind their effects have not been clarified . in the present study , we examined the effects of glycer - ages on cultured human lung adenocarcinoma a549 cells and showed that glycer - ages enhanced their malignancy rather than their proliferation . n - acetyl - l - cysteine ( nac ) was purchased from sigma - aldrich ( st . louis , mo , usa ) . briefly , 25 mg / ml of bsa ( a0281 , sigma - aldrich ) were incubated at 37c for 7 days under sterile conditions with 0.1 m glyceraldehyde ( ga ; nakalai tesque , kyoto , japan ) and 5 mm diethylenetriamine - pentaacetic acid ( dojindo , kumamoto , japan ) in 0.2 m phosphate buffer ( ph 7.4 ) . the modified albumin was then purified by pd-10 column ( ge healthcare , buckinghamshire , england ) chromatography and dialysis against phosphate - buffered saline ( pbs ) . control unglycated bsa was incubated under the same conditions except for the absence of glyceraldehyde as a negative control . protein concentrations were determined with the dc protein assay reagent ( bio - rad , richmond , ca , usa ) using bsa as a standard . the preparations were tested for endotoxin using the pyrotell - t test ( seikagaku bio - business , tokyo , japan ) , but no endotoxin was detected . human lung adenocarcinoma a549 and hepatocellular carcinoma hep3b cells were grown in dulbecco 's modified eagle 's medium ( dmem ; sigma - aldrich ) supplemented with 10% fetal bovine serum ( fbs ; equitech - bio , kerrville , tx , usa ) under standard cell culture conditions ( humidified atmosphere , 5% co2 , 37c ) . yamagishi , and its mock vector - transfected hep3b cells were maintained in 10% fbs / dmem in the presence of 700 g / ml g418 ( roche , mannheim , germany ) . the cells were washed with ice - cold ca and mg free pbs ( pbs ( - ) ) and subjected to lysis buffer ( 1% tritonx-100/nonidet p-40 , 10 mm sodium fluoride , 1 mm sodium orthovanadate , 5 mm sodium pyrophosphate , 2 mm egta , 5 mm edta , and 1 protease inhibitor cocktail ( complete , mini ; roche ) ) . subsequently , the cell lysates were incubated on ice for 5 min and centrifuged at 10,000 g for 10 min at 4c . their protein concentrations were then measured using the bradford assay ( bio - rad ) . cell lysates ( 30 g of proteins / lane ) dissolved in sds sample buffer ( 62.5 mm tris - hcl ( ph 6.8 ) , 2% sds , 10% glycerol , and 0.01% bromophenol blue ) containing 5% 2-mercaptoethanol ( 2-me ) were boiled for 3 min at 95c , separated by sds - page , and then electrotransferred onto pvdf membranes ( millipore , billerica , ma , usa ) . biotinylated markers ( cell signaling , beverly , ma , usa ) were used as molecular weight markers . the membranes were blocked for 1 h using 5% skimmed milk in pbs containing 0.05% polyoxyethylene sorbitan monolaurate ( pbs - t ) . after being washed twice with pbs - t , the membranes were incubated with goat anti - rage antibody ( n-16 ) or mouse anti--actin antibody ( santa cruz , santa cruz , ca , usa ) for 1.5 h. subsequently , the membranes were washed twice with pbs - t and incubated with anti - goat igg antibody ( santa cruz ) or anti - mouse ig 's antibody ( biosource , camarillo , ca , usa ) and anti - biotin antibody ( cell signaling ) for 1 h. after being washed a further two times with pbs - t , the immunoreactive proteins were detected with ecl plus western blotting detection reagents ( ge healthcare ) using a luminescent image analyzer ( las-1000uvmini ; fujifilm , tokyo , japan ) . the density of the bands was analyzed using a multi gauge version 3.0 ( fujifilm ) . the cells ( 710 cells / cm ) were seeded in various plates or culture dishes ( bd biosciences , franklin lakes , nj , usa ) and incubated for 24 h. the control unglycated bsa and glycer - ages treatments were carried out in 0.1% fbs / dmem for 48 and 72 h , and cell viability was determined by the wst-1 assay . after removing the medium , 100 l / well of 10% fbs / dmem and 10 l / well of wst-1 solution ( 5 mm wst-1 , 0.2 mm 1-methoxy-5-methylphenazinium , and 20 mm hepes buffer ( ph 7.4 ) ) ( dojindo ) were added , and the cells were incubated for 2 h. absorbance was measured at 450 nm and 650 nm using a microplate reader ( labsystems multiskan ascent , model no . 354 ; thermo fisher scientific , kanagawa , japan ) , and the net difference ( a450 a650 ) was used to express cell viability . the migration and invasion capacity were evaluated in 24-well transwell chambers and biocoat matrigel invasion chambers ( bd biosciences ) , respectively . in both assays , the upper and lower culture compartments were separated by polyethylene terephthalate filters with a pore size of 8 m . ten percent fbs was used as a chemoattractant in the lower chamber compartments . before starting the invasion assay , the matrigel matrix of the chambers was reconstituted by adding serum - free dmem for 2 h. in the migration assay , 1 10 cells in serum - free dmem with control unglycated bsa or glycer - ages ( 100 g / ml ) were added into each of the upper chambers for 20 h. in the invasion assay , 2 10 cells in serum - free dmem with control unglycated bsa or glycer - ages were added to each of the upper chambers for 48 h. then , the nonmigrating or noninvading cells on the upper side of the chamber membranes were removed using cotton swabs . the cells that migrated to or invaded the opposite side of the chamber were counted . the active form of rac1 was detected with the ez - detect rac1 activation kit ( pierce , rockford , il , usa ) . this assay uses the ability of a glutathione s - transferase ( gst ) fusion protein corresponding to the p21-binding domain ( pbd ) of human p21 activated protein kinase 1 ( pak1 ) to specifically bind to the active gtp - bound and not the inactive gdp - bound forms of rac1 and cdc42 . cell lysates were incubated with gst - pak1-pbd and swellgel immobilized glutathione discs for 1 h at 4c , leaving an aliquot for measuring the levels of total rac1 . the bound proteins were eluted in 2 sds sample buffer containing 5% 2-me for 5 min at 95c and characterized by western blotting using monoclonal anti - rac1 antibody . rac1 activity was determined by densitometric quantification of the pulled - down rac1-gtp level and normalizing it to the level of total rac1 detected in the sample lysates . total rna was isolated from the cells with isogen ( nippon gene , tokyo , japan ) , and 50 ng of rna were reverse transcribed into cdna with primescript rt reagent kit ( takara , shiga , japan ) using a geneamp pcr system 9700 ( perkin - elmer applied biosystems , foster city , ca , usa ) . real - time pcr was performed with sybr premix ex taq using a smart cycler ii system ( takara ) . the reaction mixture ( 25 l ) contained 1 sybr premix ex taq , 0.2 m pcr forward primers , 0.2 m pcr reverse primers , and 10 ng of cdna as a template . the primers used were as follows : tgf- : 5- gcg tgc taa tgg tgg aaa cc -3 and 5- cgg agc tct gat gtg ttg aag a -3 , mmp-2 : 5-agt ctg aag agc gtg aag-3 and 5- cca ggt agg agt gag aat g -3 , and -actin : 5- tcc acc tcc agc aga tgt gg -3and 5- gca ttt gcg gtg gac gat -3. all processes were performed according to the manufacturer 's instructions . -actin was used as an endogenous control gene in order to normalize target gene expression values . the cells were seeded and incubated for 24 h. the control unglycated bsa and glycer - ages treatments were carried out in serum - free dmem for 48 h. the conditioned medium was collected and used with a mmp-2 biotrak activity assay system ( ge healthcare ) . all experiments were performed in duplicate and repeated at least two to three times , with each experiment yielding essentially identical results . the significance of differences between group means was determined by one - way analysis of variance . to investigate whether rage proteins are present in human lung adenocarcinoma a549 cells , we carried out western blot analysis using anti - rage antibody ( n-16 ) . rage proteins of different molecular weights were detected in a549 cells ( figure 1 ) . in full length rage cdna - transfected human hepatocellular carcinoma hep3b cells , the major band ( 57 kda ) ( indicated by an arrow in figure 1 ) represents the full - length rage protein . likewise , the full - length rage protein was also detected in a549 and mock transfected hep3b cells . no bands were detected in a neutralization experiment using blocking peptide ( data not shown ) so the other bands may represent deglycosylated rage proteins . we examined the effect of glycer - ages on the cell viability of a549 cells . cell viability was determined after 48 h incubation with control unglycated bsa or glycer - ages . twenty , 50 , and 100 g / ml of glycer - ages significantly decreased cell viability to 83 , 64 , and 54% , respectively , ( figure 2(a ) ) . in contrast , control unglycated bsa had no effect . furthermore , glycer - ages significantly attenuated cell proliferation ( figure 2(b ) ) ; however , no cell death was induced by glycer - ages according to cell death detection elisa ( data not shown ) . we examined the effect of glycer - ages on cell migration and invasion . in the migration assay , the glycer - ages - treated cells showed significantly ( 2.7 times higher ) greater migration than the control unglycated bsa - treated cells ( figure 3(a ) ) , and in the invasion assay , the glycer - ages - treated cells showed significantly ( 3.3 times higher ) greater invasion than the control unglycated bsa - treated cells ( figure 3(b ) ) . when glycer - ages were added to the lower chamber , there was no effect on the results of the invasion assay ( data not shown ) . these results suggest that glycer - ages - rage interaction is necessary for cell migration and invasion . rho family small gtpases are widely accepted to be key regulators of actin reorganization and motility . interestingly , rage signaling has been shown to result in the activation of rac1 [ 1720 ] , and the activation of rage by ages has been shown to induce the generation of reactive oxygen species ( ros ) . the glycer - ages - induced rac1 activity was about 2 times higher than that of the control unglycated bsa , and it was suppressed by pretreatment with n - acetyl - l - cysteine ( nac ) , an ros scavenger ( figure 4(a ) ) . likewise , the glycer - ages - enhanced migration capacity was also significantly suppressed by pre - treatment with nac ( figure 4(b ) ) . thus , glycer - ages - rage interaction enhances migration capacity by activating rac1 via ros generation . members of the matrix metalloproteinase ( mmp ) family are widely accepted to be key regulators of tumor invasion [ 2224 ] . in particular , mmp-2 and -9 , which are called gelatinases , are key enzymes for degrading type iv collagen and are thought to play critical roles in tumor invasion and metastasis . we examined the expression of transforming growth factor- ( tgf- ) and mmp-2 mrna and assessed mmp-2 activity with the mmp-2 biotrak activity assay system . neither the mrna expression of tgf- or mmp-2 was significantly increased by the addition of glycer - ages at 24 h ( figures 5(a ) and 5(b ) ) . pro - mmp-2 activity was also not significantly altered by the addition of glycer - ages at 48 h ( figure 5(c ) ) . however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% ( figure 5(d ) ) . rage , which is a multiligand receptor , affects diseases such as cancer and diabetes through its ligands . several reports have suggested that rage and amphoterin are closely associated with invasion and metastasis in cancer cells [ 26 , 27 ] . ages have been implicated in the development and progression of diabetic angiopathies , including skin dermopathy . however , it is still unclear which ages subtypes play a pathogenetic role and which ages receptors mediate the effects of ages on cells . we have provided direct immunochemical evidence for the existence of six distinct ages structures within the ages - modified proteins and peptides that circulate in the sera of diabetic patients . recently , we found that glycer - ages perform a diverse range of biological activities on vascular wall cells , mesangial cells , schwann cells , and cortical neurons [ 1013 ] . we also found that glycer - ages , but not glucose - derived ages ( glc - ages ) , significantly stimulated the growth and migration of human melanoma cells . furthermore , the tumor formation of melanoma cells xenografts in athymic mice was prevented by treatment with anti - rage antibody . in tumor bearing - mice , survival rates were prolonged , and spontaneous pulmonary metastases were inhibited by treatment using anti - rage antibody . in addition , glycer - ages were present in the beds of human melanoma tumors , whereas they were hardly detected in normal skin . these results suggest that glycer - ages are involved in the growth and invasion of malignant melanoma through their interactions with rage . recently , we found that glycer - ages have the strongest binding affinity for rage [ 28 , 29 ] and subsequently elicit oxidative stress generation and evoke vascular inflammation , thereby implicating them in accelerated atherosclerosis in diabetes [ 10 , 11 ] . taken together , glycer - ages - rage interactions play an important role in the progression of melanoma cells to malignancy . however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind these effects have not been clarified . in the present study rage includes full - length , c - truncated , and n - truncated rage . the rage antibody ( n-16 ) used in this study , which recognizes the n - terminus of rage , detects full - length and c - truncated rage . indeed , full - length rage ( 57 kda ) was detected in a549 cells . the other bands may have represented the de - glycosylated form or c - truncated rage . glycer - ages are associated with cell cytotoxicity , but glc - ages are not [ 21 , 31 , 32 ] . in mesangial cells , intracellular ros produced by glycer - ages were found to induce apoptotic cell death . however , no a549 cell death induced by glycer - ages was observed in this study . it is thought that the cytotoxicity of glycer - ages depends on the sensitivity of their target cells to ros . furthermore , glycer - ages enhanced the migration and invasion activity of the a549 cells , both of which are prominent features of cancer malignancy . glycer - ages induced the invasion of a549 cells across matrigel , indicating that matrix degradation and migration mechanisms had been stimulated in these cells . at the molecular level , glycer - ages - induced phenomena resemble the effects of long - term oxidative stress or tgf-. both oxidative stress and tgf- are key regulators of the malignancy rather than the proliferation of cancer cells [ 3335 ] . tgf- strongly induces mmp-2 expression in a549 cells ; however , mrna expression of tgf- was not induced by glycer - ages , and the mrna levels of mmp-2 , which is produced by tgf- , did not change . in long - term oxidative stress , mori et al . showed that intracellularly produced ros activated rac1 and enhanced the invasion capacity of tumor cells by activating mmp [ 33 , 3739 ] . a recent report showed that sustained exposure of cells to h2o2 , but not one time exposure to h2o2 , increased pro - mmp-2 activation through the induction of membrane type 1-mmp ( mt1-mmp ) expression . indeed , our results also showed that glycer - ages enhanced the migration capacity of a549 cells by activating rac1 via ros and increased their invasion capacity by activating mmp-2 . however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% . it is suggested that the activation of other mmp such as mmp-13 may also participate in the above - mentioned changes , and future studies are necessary to clarify this matter . in conclusion , we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation . these results suggest that glycer - ages play a critical role in cancer malignancy and are potential targets for therapeutic intervention .

the receptor for advanced glycation end - products ( rages ) is associated with the malignancy of cancer . a recent study has suggested that glyceraldehyde - derived ages ( glycer - ages ) enhanced the malignancy of melanoma cells , but glucose - derived ages did not . however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind the above - mentioned effect have not been clarified . the present paper aimed to examine the effect of glycer - ages on cultured lung cancer a549 cells . rage was expressed in a549 cells . glycer - ages significantly attenuated cell proliferation . furthermore , glycer - ages enhanced the migration capacity of the cells by activating rac1 via ros and also increased their invasion capacity . we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation . these results suggest that glycer - ages play a critical role in the malignancy of cancer rather than its proliferation and are potential targets for therapeutic intervention .

osseointegrated implants are a practical alternative to traditional prosthodontics ; however , designing an implant - supported prosthesis with function and esthetics is a challenge . malaligned implants often complicate the clinical laboratory procedures employed for fabrication of superstructures . due to improper load distribution , a stent is an appliance used for radiographic evaluation of height and width of the available bone during treatment planning for implant placement or during surgical procedures to provide site for optimum implant placement . the variable type of stents includes vaccuform or acrylic resin templates adapted over duplicated casts of diagnostic wax - up . these include plastic or metal tubes and channels in acrylic resin that dictate the position of implants . stents should be made of transparent material , stable and rigid when in position , should cover enough teeth to stabilize its position , and when no teeth are present , stents should extend onto unreflected soft tissue regions . with the use of computed tomography ( ct ) and computer added designing ( cad)/ computer added machining ( cam ) technology , it is now possible to construct a surgical implant stent that would allow the clinician to predetermine implant locations virtually and surgically place them without reflecting a tissue flap.[58 ] but the surgical stents prepared with ct and cad / cam technology are very much more cost effective than a conventional surgical stent and require enough time and special center and/or laboratories for their fabrication and later on their correction , if required . in this study , we used the conventional surgical stents to evaluate their efficacy in determining the position and diameter of implants . the aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter . the aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter . the study was conducted in the out - patient department of prosthodontics and dental material sciences in collaboration with department of oral and maxillofacial surgery , faculty of dental sciences , csmmu , lucknow . patients were treated in this study with the conventional surgical stents ; all the implants were placed to the desired depth as planned in virtual implant planning . all the patients ( 45 patients for whom 89 implants were placed at different sites ) [ figures 1 , 2 ] visiting the department of prosthodontics and oral and maxillofacial surgery for implant supported prosthesis placement and who followed the standard inclusion criteria for placement of implants were included in the study . first of all , diagnostic cast of the patients was made and acrylic teeth / tooth were waxed - up [ figure 3 ] . then , the impression of that waxed - up cast was taken and poured to reproduce another cast with the missing teeth / tooth replaced by dental stone . then , mesiodistal and buccolingual markings were drawn over these teeth / tooth to find a center point on the occlusal surface [ figure 4 ] . later on , after applying the separating media over these regions and filling the undercuts with wax , transparent self - curing material was applied at the teeth to be replaced along with some adjacent area antero - posteriorly , which confers the stability to the stent during implant placement [ figure 5 ] . the holes were drilled in the stent so formed at the center point for the assessment of the diameter and position of implant and filled with gutta percha [ figure 6 ] . this stent was placed in the mouth and radiograph was taken to evaluate the position of gutta percha . the efficacy of stents was then evaluated at the time of surgical exposure for implant placement and after the surgery , with the help of radiographs [ figures 7 , 8 ] . intraoral view of the patient radiograph of the patients waxed - up missing teeth missing teeth replaced by dental stone in the cast with markings stent made of transparent material stent in place with gutta percha filled in holes post - operative radiograph philip j. described a technique to construct a surgical guide on mounted diagnostic casts . he also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis . murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning . the outer lamina is designed for use in the computed tomography ( ct ) evaluation using radiopaque markers . yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker . the matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy . roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient , when implant - supported restorations are planned . a template that provides radiographic evaluation of the implant site and precise or modified surgical placement kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants . he also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis . murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning . the outer lamina is designed for use in the computed tomography ( ct ) evaluation using radiopaque markers . yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker . the matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy . roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient , when implant - supported restorations are planned . a template that provides radiographic evaluation of the implant site and precise or modified surgical placement kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants . the foremost advantage of the stent used in this study is its surgical ease , simplicity , precise accuracy and low cost . it can be fabricated with minimum laboratory procedures which are used in routine dental practice . on the other hand , the fixed type screw retained implant stents which are fabricated with the help of ct and cad / cam technology are very costly and require more time and laboratory procedures . the stents fabricated with the radiopaque markers provide radiographic as well as clinical ease for optimum implant installation . along with all these advantages the drill holes can directly be made through the stent , so the implant surgery may become less traumatic ( with the preservation of soft tissue including the gingival margins of adjacent teeth and the interdental papilla ) with decreased operative time which results in accelerated post - surgical healing , fewer post - operative complications and increased comfort and satisfaction for the patient . the possibility of displacement of stent during implant surgery was minimized by extension of the stent antero - posteriorly to cover enough area over the teeth / tooth present adjacent to edentulous area . in cases where no teeth are present , stabilization of stent was achieved by extending it over the unreflected areas like retromolar regions . this study shows the extreme accuracy of this conventional surgical stent . if each step of this protocol is followed precisely , it is possible to deliver an optimum implant installation in terms of position and diameter and later on function at a very much reduced rate and time , as taken in ct and cad / cam technology which are the most accepted methods

aim : demand from implant patients for quality and efficient treatment is increasing . fortunately dental implant treatment is evolving with patients expectations.materials and methods : the study comprised of 45 patients for whom 89 implants were placed at different sites . efficacy of the stents is evaluated in determining the position and diameter of the implants.conclusion:this study shows the extreme accuracy of this surgical stents in implant installation in terms of position and diameter .

type 1 diabetes ( t1d ) is characterized by autoimmune destruction of pancreatic -cells , primarily mediated by t lymphocytes , resulting in insulin deficiency and lifelong exogenous insulin dependence ( 1 ) . latent autoimmune diabetes in adults ( lada ) is a subtype of autoimmune diabetes , but the progression of autoimmune -cell destruction is slow ; therefore , patients with lada are not insulin dependent at the initial phase ( at least 6 months ) of disease onset . clinical manifestation of lada shares the features of classical t1d and type 2 diabetes ( t2d ) ( 2 ) . t1d , lada , and t2d present a spectrum of diabetes . they range from patients with classic childhood t1d characterized by autoimmune - mediated destruction of -cells with the lowest c - peptide levels to age - related exacerbation of glucose tolerance with the highest bmi seen in t2d ( 3 ) . furthermore , patients with lada have susceptibility genes for both t1d and t2d ( 4 ) , suggesting that lada lies between t1d and t2d . b cells infiltrate the islets of young nod mice , the commonly used mouse model for human t1d , and play a role in the initiation of -cell destruction by the autoimmune response ( 5 ) . studies have shown that marginal zone b ( mzb ) cells and follicular b ( fob ) cells can effectively activate cd4 t cells and facilitate their proliferation by serving as important antigen - presenting cells , especially as antigen - specific antigen - presenting cells ( 6,7 ) . b - cell targeted therapy has been shown to delay the fall of c - peptide levels in patients with recent - onset t1d ( 8) . clinical trials have identified b cells as a possible therapeutic target for the prevention and reversal of t1d . b - cell targeted therapy also has been successfully used in treating other autoimmune diseases , including rheumatoid arthritis ( 9 ) , systemic lupus erythematosus ( 10 ) , and multiple sclerosis ( 11 ) . moreover , studies have shown that b cells are important in regulating both innate and adaptive immunity ( 12 ) . a subset of b cells expressing cd19cd5cd1d , which have a regulatory function in infection , inflammation , and autoimmune disease , recently has been identified . these regulatory b cells can regulate t - cell responses and inhibit inflammation in an interleukin-10 ( il-10)dependent manner ( 1316 ) . the maturation and expansion of b10 cells require il-21 and cd40 signals both in vivo and ex vivo ( 17,18 ) . considerable evidence shows that patients with autoimmune diabetes have altered frequencies of circulating immune cells , including t cells ( 19 ) , dendritic cells ( 20 ) , natural killer cells ( 21,22 ) , and neutrophils ( 23 ) . although studies have reported no significant change in total b cells in peripheral blood of patients with t1d ( 21,24 ) , little is known about b - cell subpopulations with distinct immune functions that may play a role in the diabetes spectrum of t1d , lada , and t2d . we hypothesized that an altered phenotype of b - cell subsets is associated with autoimmune diabetes . this study investigated a change of peripheral b - cell subsets in patients with t1d , lada , and t2d compared with healthy control subjects . the results show altered frequencies of various b - cell subsets associated with autoimmune diabetes . two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1 3.5 years ) , lada ( n = 82 , 6.1 5.9 years ) , or t2d ( n = 95 , 3.0 3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) . diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) . diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion . lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) . diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment . healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8 exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease . this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol . anthropometric and metabolic data of participants data are % ( n ) , mean sd , and median ( 25th75th percentile ) . compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d . study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure . fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) . hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) . fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) . gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) . the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects . the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) . fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol . the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples . fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . , dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining . 1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) . the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively . the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination . b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells . c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells . anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi . all the significant differences between groups are the results of adjusted analyses unless stated otherwise . associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations . for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software . two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1 3.5 years ) , lada ( n = 82 , 6.1 5.9 years ) , or t2d ( n = 95 , 3.0 3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) . diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) . diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion . lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) . diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment . healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8 exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease . this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol . anthropometric and metabolic data of participants data are % ( n ) , mean sd , and median ( 25th75th percentile ) . compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d . study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure . fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) . hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) . fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) . gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) . the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects . the positive samples were tested twice for confirmation . the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) . fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol . the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples . fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . , dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining . 1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) . the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively . the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination . b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells . c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells . anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi . all the significant differences between groups are the results of adjusted analyses unless stated otherwise . associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations . for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software . the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d . the percentage of males in the lada group was higher than in the ngt group . the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group . patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups . the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt . there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups . we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig . the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig . we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi . fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) . we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig . 2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig . the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells . p values refer to comparison of data after adjustment for age , sex , and bmi . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig . b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig . after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig . however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d . we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells . however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level . correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test . in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d , the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) . finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) . the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d . the percentage of males in the lada group was higher than in the ngt group . the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group . patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups . the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt . there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups . we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig . the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig . we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi . fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) . we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig . 2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig . the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells . p values refer to comparison of data after adjustment for age , sex , and bmi . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig . b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig . after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig . however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d . we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells . however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level . correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test . in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d , the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) . finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) . increasing evidence suggests that b cells are as important as t cells in the immunopathogenesis of autoimmune diabetes . most , if not all , diabetogenic t cells are known to be t - helper 1 subsets , but little is known about b - cell subsets in autoimmune diabetes . we investigated the phenotype and frequency of various b - cell subsets in the peripheral blood across the clinical spectrum of diabetes . the major finding of this study was a distinct alteration of b - cell subsets across the diabetes spectrum and their association with clinical features . we found no significant difference in the frequency of cd19 b cells , which is in accordance with the published studies ( 21,30 ) ; however , we did not find a negative correlation between total b cells and age , as previously reported ( 24,31 ) . the current study went further than the examination of the frequency of total b cells , focusing on b - cell subsets with distinct immunological functions . of note , we found that patients with t1d or lada had an increased frequency of mzb cells but decreased frequency of fob cells compared with control subjects with ngt and patients with t2d . in all groups , we found that fcp was negatively associated with mzb cells but positively associated with fob cells . attanavanich and kearney ( 6 ) reported that mzb cells were more effective at activating naive cd4 t cells than fob cells . mzb cells in nod mice were also shown to be important antigen - presenting cells for the activation of t cells in pancreatic - draining lymph nodes ( 32 ) . given the negative correlation between mzb cells and fcp , it is possible that mzb cells may promote -cell destruction in patients with t1d or lada . decreased frequency of fob cells in the circulation of patients with t1d and lada suggests that some fob cells migrated from the blood to the pancreas or the draining lymph nodes as a result of the local inflammatory response . along with the disease progression , more immune cells are known to infiltrate to the islets of both nod mice and patients with t1d ( 5,33 ) , but the patients with t1d in the current study have a relatively long history of diabetes ( 3.1 years ) . thus , fob cells appear to be more important in initial rather than established pancreatic -cell autoimmunity ( 34 ) . we showed that patients with t2d and lada more frequently had t2-mzp b cells than control subjects with ngt , which associates with parameters of blood pressure , lipidemia , and glycemia but not with hba1c or fcp . these observations are consistent with a previous study showing that glycemia and lipidemia are associated with immune cells ( 35 ) . another interesting finding was the decrease of b10 cell frequency in all types of diabetes , being the lowest in t1d . whether this phenotype is mediated through a common mechanism or different ones is not clear . what is clear is that immune mechanisms are involved and that more studies , especially functional studies , are required . although b cells are generally acknowledged for their function in humoral immune response by producing antibodies , they also have been demonstrated to play an immunoregulatory role in the prevention of autoimmune disease by producing il-10 and downregulating t - cell responses ( 13,14,36 ) . the observed b10 cell reduction could contribute to the breakdown of self - tolerance that leads to -cell destruction in patients with t1d ( 24 ) or lada . antigen - activated il-10producing b cells may selectively inhibit autoreactive t - cell responses to islet - specific antigen to maintain self - tolerance , and hyperglycemic nod mice and patients with t1d lack this population of regulatory b cells ( 37 ) . in the clinical trial where b cells in patients with new - onset t1d were depleted , the patients showed a transient remission ( 8) . however , blanket targeting of all b cells , as used in the clinical trial and in treating other autoimmune disorders , needs to be improved by selectively deleting pathogenic b cells while preserving regulatory b cells to promote tolerance . our preclinical study showed that more regulatory b cells were regenerated after b - cell deletion in an animal model ( 38 ) . furthermore , studies have shown that patients with long - standing t2d could have a secondary autoimmune response toward islet -cells ( 39 ) . the reduction of b10 regulatory cells might contribute to the dysregulation of immune response in t2d . a functional study on il-10producing b cells and further phenotyping will be the goal of future work . in the current study , although we currently can not identify a specific subset that could be used for diagnostic purposes , the results strongly imply that the change of b - cell subtypes is related to the pathogenesis of autoimmune diabetes . therefore , it is important to reconsider the approach of b - cell targeted therapy in patients with t1d and aim to selectively remove pathogenic b cells but promote regulatory b cells . in summary , the current data demonstrate distinct differences in the frequencies of peripheral b - cell subsets in t1d and lada , which are associated with islet function and glycemia . the findings support the notion that these immunological alterations are involved in loss of self - tolerance and -cell destruction . the findings also suggest that , similar to regulatory t - cell therapy , transfusion of ex vivo expanded autologous b10 cells might open a new and effective therapeutic strategy in treating autoimmune diabetes .

objectiveb lymphocytes play an important role in the immunopathogenesis of autoimmune diabetes . we hypothesized that the altered b - cell subset phenotype is associated with autoimmune diabetes.research design and methodspatients with type 1 diabetes ( t1d ) ( n = 81 ) , latent autoimmune diabetes in adults ( lada ) ( n = 82 ) , or type 2 diabetes ( t2d ) ( n = 95 ) and healthy control subjects ( n = 218 ) with normal glucose tolerance ( ngt ) were recruited . we determined the percentage of circulating b - lymphocyte subsets , including cd19+cd23cd21 + ( marginal zone b [ mzb ] ) , cd19+cd23+cd21 ( follicular b [ fob ] ) , and cd19+cd5+cd1dhi ( interleukin-10producing regulatory b [ b10 ] ) cells by flow cytometry.resultspatients with t1d or lada had increased percentages of mzb cells and decreased percentages of fob cells compared with healthy control subjects with ngt and patients with t2d . moreover , patients with t1d showed the lowest frequency of b10 cells compared with patients with lada or t2d , whereas healthy control subjects expressed the highest frequency of b10 cells . of note , the frequency of mzb cells was negatively associated and the frequency of fob cells was positively associated with fasting c - peptide ( fcp ) . the frequency of b10 cells was positively correlated with fcp and negatively correlated with hemoglobin a1c.conclusionsthe data show that patients with t1d or lada express an altered frequency of b - cell subsets , which is associated with islet function and glycemia . these findings suggest that b lymphocytes may be involved in loss of self - tolerance and -cell destruction and could be used as a biomarker and potential target for immunological intervention .

human papillomavirus ( hpv ) , a virus from the papillomavirus family , is capable of infecting humans . about 200 different strains of hpv identified , based on dna homology , have been found to be etiologically linked to cervical , vaginal , vulvar , penile , anal , oral , and plantar infectious lesions and cancers , as well [ 1 , 2 ] . the hpv genome , a double - stranded dna molecule , consists of 8 kilobase pairs ( kbp ) of nucleotides , which comprises 3 regions : 6 early open reading frames ( orfs)-e1 , e2 , e4 , e5 , e6 , and e7 ; 2 late orfs - l1 and l2 ; and an upstream regulatory region . a considerable volume of hpv specific information pertaining to its genome , proteome , structure , and disease association is available scattered on the web with trivial annotations ; however , it is not customized to explore for data analysis , host - pathogen interaction , strain - disease association , drug designing , and sequence analysis , etc . therefore , we proposed to develop a comprehensive reserve on hpv with maximum possible inputs and outputs for the end users . amongst the existing 200 strains of hpv , 150 have been sequenced as of now , and their data available at the national center for biotechnology information ( ncbi ) . genome and proteome information of those viral strains was retrieved from ncbi . besides pubmed , various other online resources and published literature were also explored for validating genomic , proteomic , as well as strain and disease - associated information on hpv strains . hpv strain - specific information , such as strain name , sequencing status , sequencing centre , ncbi accession i d , associated disease information with references , genome statistics ( gc% , at% , a , t , g , c count , genes , and proteins ) , etc . , were curated from various online resources , and protein parameters ( length , molecular weight , isoelectric point ) were calculated using expasy protparam . modeller9v10 and the swiss - model server were used for protein structure prediction . the stereochemistry of each protein was evaluated through procheck analysis , available at the rcsb validation server ( http://deposit.rcsb.org/validate/ ) , and validated using prosa - web ( http://prosa.services.came.sbg.ac.at/prosa.php ) . human papillomavirus proteome database ( hpvpdb ) , the relational reserve , was developed using microsoft sql server 2005 as the back end . html , javascript , and cgi - perl - based web interfaces were employed to execute sql queries . the application layer , the web interface , and the backend relational tables were integrated using cgi - perl . home , about , tools , search , and advanced search interfaces can be explored to obtain strain- and protein - specific information . reserve comprises the strain - specific detailed informationon its name , sequencing status , submission details , date of submission , ncbi ids , disease types and subtypes , type of dna , genome length , molecular weight , nucleotide composition ( a , t , g , c , at , gc content ) , number of genes and proteins , and protein list . a genome map of each strain obtained by geneious 5.4.4 software ( available from http://www.geneious.com/ ) is also integrated in this page . users , through an advanced search option , can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome . each protein entry comprises protein overview ( name , locus , function , etc . ) , protein sequence information ( amino acid sequences with ncbi accession number with provision for direct protein blast against ncbi nr database ) , protein parameters ( length , molecular weight , theoretical isoelectric point [ pi ] , amino acid composition , etc . ) , protein structure ( predicted 3d structure by homology modeling viewed by jmol ( available from http://www.jmol.org/ ) with the java platform , ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web . hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny . the original human papillomaviruses database was developed and hosted by the los alamos national laboratory ( lanl ) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases ( niaid ) . ' human papillomaviruses : a compilation and analysis of nucleic acid and amino acid sequences ' contains four annual data books of papillomavirus information published in both paper and electronic form ( 1994 , 1995 , 1996 , and 1997 ) but has not been updated since 1997 . this contains nucleotide sequences of few hpv strains and other papillomaviruses , amino acid and nucleotide sequence alignments , analysis , related host sequences , and database communication . we did not find any structural information in that database . in hpvpdb , along with updated protein sequence information , genome and protein structure information is also provided . amongst the existing 200 strains of hpv , 150 have been sequenced as of now , and their data available at the national center for biotechnology information ( ncbi ) . genome and proteome information of those viral strains was retrieved from ncbi . besides pubmed , various other online resources and published literature were also explored for validating genomic , proteomic , as well as strain and disease - associated information on hpv strains . hpv strain - specific information , such as strain name , sequencing status , sequencing centre , ncbi accession i d , associated disease information with references , genome statistics ( gc% , at% , a , t , g , c count , genes , and proteins ) , etc . , were curated from various online resources , and protein parameters ( length , molecular weight , isoelectric point ) were calculated using expasy protparam . modeller9v10 and the swiss - model server were used for protein structure prediction . the stereochemistry of each protein was evaluated through procheck analysis , available at the rcsb validation server ( http://deposit.rcsb.org/validate/ ) , and validated using prosa - web ( http://prosa.services.came.sbg.ac.at/prosa.php ) . human papillomavirus proteome database ( hpvpdb ) , the relational reserve , was developed using microsoft sql server 2005 as the back end . html , javascript , and cgi - perl - based web interfaces were employed to execute sql queries . the application layer , the web interface , and the backend relational tables were integrated using cgi - perl . home , about , tools , search , and advanced search interfaces can be explored to obtain strain- and protein - specific information . reserve comprises the strain - specific detailed informationon its name , sequencing status , submission details , date of submission , ncbi ids , disease types and subtypes , type of dna , genome length , molecular weight , nucleotide composition ( a , t , g , c , at , gc content ) , number of genes and proteins , and protein list . a genome map of each strain obtained by geneious 5.4.4 software ( available from http://www.geneious.com/ ) is also integrated in this page . users , through an advanced search option , can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome . each protein entry comprises protein overview ( name , locus , function , etc . ) , protein sequence information ( amino acid sequences with ncbi accession number with provision for direct protein blast against ncbi nr database ) , protein parameters ( length , molecular weight , theoretical isoelectric point [ pi ] , amino acid composition , etc . ) , protein structure ( predicted 3d structure by homology modeling viewed by jmol ( available from http://www.jmol.org/ ) with the java platform , ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web . hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny . the original human papillomaviruses database was developed and hosted by the los alamos national laboratory ( lanl ) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases ( niaid ) . ' human papillomaviruses : a compilation and analysis of nucleic acid and amino acid sequences ' contains four annual data books of papillomavirus information published in both paper and electronic form ( 1994 , 1995 , 1996 , and 1997 ) but has not been updated since 1997 . this contains nucleotide sequences of few hpv strains and other papillomaviruses , amino acid and nucleotide sequence alignments , analysis , related host sequences , and database communication . we did not find any structural information in that database . in hpvpdb , along with updated protein sequence information , genome and protein structure information is also provided . hpvpdb brings together comprehensive information on a total of 1,036 protein sequences and 743 predicted structures . the outcome of this study might provide a platform for simultaneous structural comparative analysis of these proteins and help in finding out variations in their structures to explore why different strains of hpv have causative associations with different types of cancers . further , this might also help in designing specific drugs or vaccines against specific strains of hpv . currently the hpvpdb is updated manually through online resources and available scientific publication review ; however , to sustain the quality , these data are analyzed and checked before incorporation into this reserve . meanwhile , to provide regular updates , our team is committed to searching for newly sequenced hpv strains , updating this reserve , and serving the users .

human papillomavirus ( hpv ) infection is the leading cause of cancer mortality among women worldwide . the molecular understanding of hpv proteins has significant connotation for understanding their intrusion in the host and designing novel protein vaccines and anti - viral agents , etc . genomic , proteomic , structural , and disease - related information on hpv is available on the web ; yet , with trivial annotations and more so , it is not well customized for data analysis , host - pathogen interaction , strain - disease association , drug designing , and sequence analysis , etc . we attempted to design an online reserve with comprehensive information on hpv for the end users desiring the same . the human papillomavirus proteome database ( hpvpdb ) domiciles proteomic and genomic information on 150 hpv strains sequenced to date . simultaneous easy expandability and retrieval of the strain - specific data , with a provision for sequence analysis and exploration potential of predicted structures , and easy access for curation and annotation through a range of search options at one platform are a few of its important features . affluent information in this reserve could be of help for researchers involved in structural virology , cancer research , drug discovery , and vaccine design .

according to numerous estimations , 15% to 29% of patients with cataract have 1.5 diopters ( d ) of refractive astigmatism.1,2 cataract surgeons usually prefer to treat cataract and correct refractive ( spherical and astigmatic ) disorders at the time of the surgery . corneal incision ( astigmatic keratotomy ) and peripheral corneal relaxing incisions ( pcris ) were performed to treat these disorders.3,4 the main drawbacks of these approaches are that the outcome depends on multiple factors as the patient s age , the depth and length of the incision , complications related to wound healing , epithelial defects , or induction of dry - eye symptoms . these parameters affect the visual outcome in a unpredictable way so the corneal incisions are not considered a reliable method for astigmatism correction . toric intraocular lens ( iols ) implantation was introduced in the 1990 s as an option for astigmatism correction in cataract patients . initially they presented the disadvantage of postoperative rotation that decreased the visual outcome.58 new toric iol designs ( acrysof toric iol ; alcon , fort worth , tx ) , approved by the us food and drug administration ( fda ) at the end of 2005 , have been found to be more stable and appear to be the preferred iol for correcting preexisting astigmatism in conjunction with cataract surgery.9,10 the acrysof iols are available in three options : t3 , t4 , and t5 of astigmatic correction 1.5 , 2.25 , 3.00 d , respectively ( at the iol plane ) . the aim of this study is to report the clinical results of acrysof toric iols implantation for preexisting astigmatism correction and compare the postoperative rotation of the iols one and six months postoperatively . this prospective study included eyes that had cataract surgery at the papageorgiou general hospital , thessaloniki , greece , between may 2008 and december 2008 . the study was conducted according to the tenets of the declaration of helsinki and patients gave informed consent after the nature and intent of the study had been fully explained to them . inclusion criteria were : cataract , age 70 years or younger , and preoperative regular corneal astigmatism greater than 1.00 d. exclusion criteria were : glaucoma , corneal disease , previous corneal or intraocular surgery , macular degeneration or retinopathy , and history of ocular inflammation . each patient had a complete ophthalmologic examination , including visual acuity ( va ) , slit - lamp examination , intraocular pressure ( iop ) measurement , and dilated fundus examination . topography ( magellan mapper ; nidek technologies , padova , italy ) , automated refractometry ( rk600 ; reichert , inc . , depew , ny ) , and ultrasonic immersion biometry ( ocuscan rxp ; alcon ) were performed to determine the appropriate iol spherical power . cylindrical power and axis placement to achieve emmetropia were calculated using an online toric iol calculator program ( available from http://www.acrysoforiccalculator.com/ ) . the 0180 axis was marked with the patient in a sitting position to avoid cyclotorsion using the nuijts / lane preoperative toric reference corneal marker ( ae-2791tbl ; asico , westmont , il ) ( figure 1 ) . intraoperatively , the desired implantation axis was marked using an intra - op toric axis marker ii ( ae-2794 ; asico ) ( figure 2 ) . a foldable iol was implanted in the capsular bag through a 2.75 mm limbal incision on 110. the toric iol was injected with a monarch - ii injector ( alcon ) and placed around 1015 off axis before the ophthalmic viscosurgical device ( sodium hyaluronate 1% ) was removed . after ophthalmic viscosurgical device removal , the iol was rotated to its final position by exactly aligning the toric reference marks with the limbal implantation axis marks . all surgeries were performed by the same experienced surgeon ( it ) using topical anesthesia and a standard divide - and - conquer phacoemulsification technique . because of the intraoperative marker design and the pen mark fading during the operation in several cases , it was difficult to assess the proper alignment of the iol after its placement . because of this difficulty , an image was captured from the real - time streaming recording of the surgery and was assessed blindly by a second operator using a commercially available software ( screen protractor ; iconico , ny , usa ) ( unpublished data ) . to overcome this difficulty we consider that the use of the marker set consisting of beveled degree gauge ( ae-1590 ; asico ) and maloney astigmatism axis marker ( ae-2741 ; asico ) is more appropriate for the whole procedure because it allows maximum visibility . the on - screen assessment of the axis remains a useful tool for postoperative evaluation of the iol location ( figure 3 ) . measurements of visual acuity ( using the snellen opto - type at a six - meter distance ) , iop , and comprehensive slit lamp examination were performed at one - day and one- and six - month postoperative visits . at the one- and six - month follow - up , a digital photograph and corneal topography ( only at the one - month visit ) were obtained to estimate the postoperative rotation of the iol using the software tools mentioned above ( figure 1 ) . outcomes of interest included uncorrected va , cylindrical astigmatism power before and after iol implantation , and the possible rotation of the iol one and six months after the operation ( when the initial desired place was at 0 ) . absolute values of the rotation used for the analysis after detecting rotation was not under investigation . completed data forms were analyzed with microsoft excel 2007 ( microsoft corporation , redmond , wa ) and spss software ( version 16.0 ; spss inc . , chicago , il ) . twenty - nine eyes of 19 patients ( mean age 63.03 5.42 years ) were enrolled in this study . uncorrected va was found to be 5/10 or more in 26 of 29 eyes ( 89.7% ) and 8/10 or more in 19 of 29 patients ( 65.5% ) . preoperative and postoperative corneal topography showed significant reduction of refractive astigmatism in all eyes after the surgery . mean power of the astigmatism was 2.38 0.91 d ( range 1.55 d ) preoperatively and 0.64 0.61 d ( range 02.5 d ) postoperatively ( figure 4 ) . one month postoperation , the mean toric iol axis rotation was 2.2 1.5 ( range 0.67.8 ) . six months postoperation , the mean toric iol axis rotation was 2.7 1.5 ( range 0.98.4 ) . the later rotation occurred between one and six months , and was found to be more than one degree ( 1.1 ) only in one eye ( 3% ) and in all other cases was less than this value ( figure 5 ) . it is known that one degree of deviation causes 3.3% reduction of the iol cylindrical power . calculated reduction of the desired correction ( in diopters ) because of the observed rotation ( mean value , one and two standard deviations ) for the three iol models is shown in table 1 . no eye had secondary surgery to reposition the iol axis within the six - month postoperative period . the results of our study corroborate previous studies,11 which demonstrates that proper selection and preoperative examination of patients followed by uncomplicated iol implantation of one - piece hydrophobic acrylic toric iols results in acceptable stability and visual outcome . in particular , the postoperative rotation of toric iols appears to occur in the early postoperative period ( 1 month ) and remains constant later than six months . the lens rotates until a fibronectin and collagen adhesion develops between the iol and the posterior capsule , which prevents any further rotary motion.12 the amount of cylindrical correction reduction due to postoperative rotation was not large enough13 to affect the expected end result in final uncorrected distance va . acrysof one - piece hydrophobic acrylic toric iols implantation shows satisfactory stability , acceptable clinical results , and is an exceptional option for correction of refractive astigmatism .

purpose : to present clinical results of toric intraocular lens ( iol ) implantation for preexisting astigmatism correction and determine the time of any postoperative rotation.patients and methods : twenty - nine eyes of 19 patients underwent uncomplicated phacoemulsification and were implanted with an acrysof toric iol . uncorrected visual acuity , residual astigmatism , and postoperative rotation of the iol were estimated one and six months after the operation.results:uncorrected visual acuity was 0.5 in 26 of 29 eyes ( 89.7% ) and 0.8 in 19 of 29 patients ( 65.5% ) . the mean toric iol axis rotation was 2.2 1.5 ( range 0.67.8 ) one month postoperation and 2.7 1.5 ( range 0.98.4 ) six months postoperation.conclusion:implantation of one - piece hydrophobic acrylic toric iols appears to have acceptable stability , which encourages visual outcome and emerges as an attractive alternative for correction of refractive astigmatism .

song is a complex , learned motor skill that involves the precise coordination of vocal and respiratory musculature in order to produce highly stereotyped renditions of a memorized song model . two pathways contributed to this behavior are identified in the songbird forebrain : the motor pathway that is required throughout life for normal song production , and the anterior forebrain pathway ( afp ) which is necessary for song learning and plasticity [ 13 ] . hvc ( used as the proper name ) is the beginning nucleus of both pathways and projects to the robust nucleus of the arcopallium ( ra ) and afp . the motor pathway is from hvc to ra , and then ra projects to brainstem nuclei controlling the vocal and respiratory muscles . during song , ra neurons in adult birds generate a highly stereotyped sequence of bursts [ 5 , 6 ] and also receive input from lateral magnocellular nucleus of the anterior nidopallium ( lman ) , the output nucleus of afp . notably , recordings from lman neurons projecting to ra revealed highly variable spiking activity across song renditions , suggesting that lman may act as a source of variability . these results indicate that synaptic activity in lman - ra synaptic transmission is crucial to song plasticity [ 8 , 9 ] , but the detailed mechanism is unclear . it has been observed that ra received excitatory glutamatergic inputs from hvc and lman . the former is mediated by a mixture of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid- ( ampa- ) type receptors [ 4 , 10 , 11 ] , and the latter is mediated almost exclusively by nmda - type receptors . lman - ra synaptic transmission is necessary for the variability of song in adult zebra finch , suggesting that the activity of nmda receptor on the lman - ra synapses might induce the activity pattern change of ra neurons and change the bursts that control the vocal and respiratory muscles . the control of gain , which refers to modulation of a neuron 's responsiveness to input , is critically important for normal sensory , cognitive , and motor functions [ 1215 ] . gain control is achieved through a divisive process and is observed as a change in the slope of the input - output relationship . such gain changes are frequently observed in the responses of cortical neurons and are thought to play an important role in neural computations . modulations of gain have been observed in the enhancement of neural responses by attention [ 12 , 16 ] and can also be induced pharmacologically . in a neuron model , nmda receptors caused such membrane potential fluctuations between a hyperpolarized down - state and a depolarized up - state , which may gate the information and gain modulation . here , we test the gain change of ra projection neurons after exogenous nmda was applied to selectively activate nmda receptors , using whole - cell current clamp recording in adult male zebra finch slices . brain slices were prepared from adult male zebra finches ( taeniopygia guttata ) ( > 90 days old ) obtained from commercial suppliers as previously described [ 19 , 20 ] . brains were dissected into ice - cold , oxygenated ( 95% o2 and 5% co2 ) slice solution . slice solution consists of ( in mm ) sucrose 248 , kcl 5 , nahco3 28 , glucose 10 , mgso47h2o 1.3 , and nah2po4h2o 1.26 [ 21 , 22 ] . coronal brain slices ( 250 m thick ) containing ra were cut with a vibrating microtome ( ma752 , usa ) and collected in artificial cerebrospinal fluid ( acsf ) that had been warmed to 37c and subsequently allowed to cool to room temperature . slices were allowed to recover in the holding chamber for at least 1.5 h and were equilibrated to room temperature before recordings were made . standard acsf consists of ( in mm ) nacl 125 , nahco3 25 , nah2po4h2o 1.27 , kcl 2.5 , mgso47h2o 1.2 , cacl2 2.0 , and glucose 25 and was adjusted with sucrose to a final osmolarity of 350 mos . for recording , a slice was transferred to a submerge - type chamber where it was continuously exposed to acsf , saturated with 95% o2 and 5% co2 at room temperature ( 2226c ) at the rate of 2.0 ml / min . ra and the surrounding tissues were distinguishable under bx51wi microscope connected with a dic - ir video camera ( olympus , japan ) . at high magnification ( 40x ) , ra neurons were visualized and the recordings were made from healthy cells . recording pipettes were fabricated from borosilicate glass ( sutter instruments , usa ) using a flaming - brown puller ( model p-97 , sutter instruments , usa ) and were filled with the pipette solution containing ( in mm ) : k meso4 120 , nacl 5 , hepes 10 , egta 2 , mg - atp 2 , and na3-gtp 0.3 ( ph 7.3 - 7.4 , and 340 mos ) . the recording pipettes , which had resistances ranging from 3 to 5 m , were positioned using an integrated motorized control system ( sutter instruments , usa ) . the potential changes after application of nmda were corrected during the input - output stimulation protocols . the series resistance ( rs ) , between 15 m and 30 m , was compensated using the bridge balance . all agents were applied by changing the bath perfusate from standard acsf to modified acsf in which various drugs were simply added . unless indicated otherwise , the clampfit 9.2 ( axon instruments , usa ) , mini 6.0 ( synaptosoft inc . , fort lee , nj , usa ) , and originpro 8.0 ( originlab , nothampton , ma , usa ) were used for analysis . events with peak amplitude of 50 mv or higher and a rise time of about 0.5 ms were detected automatically , and the results were analyzed with origin pro 8.0 . the steady - state spike rate was estimated by counting the number of spikes in 1 second , and the result was plotted versus the intensity of the injected current ( f - i relationship ) . the slope of the f - i relationship ( referred to as gain ) was estimated by linear fitting . slope parameters were estimated separately for individual neurons and that figures 4 and 5 contain mean slope values averaged for the whole groups of neurons . the amplitude of ahp was quantified as the difference between spike threshold and the lowest point of ahp . throughout means were compared using paired two - tailed student 's t - test or one - way anova followed by scheffe 's multiple comparison . stable whole - cell recordings were obtained in 160 ra projection neurons from 51 adult male zebra finches . a neuron with the resting membrane potential < 50 mv and an overshooting action potential was considered viable . ra projection neuron was identified by a larger soma , time - dependent inward rectifier induced by hyperpolarizing current and spontaneous regular spike firing or by depolarizing current pulses . the input - output relationship was examined by injecting current pulses from 0 to 90 pa with an increment of 10 pa and an interval of 15 s , and they were lasted for 1 s. traces of action potentials in response to 1 s current steps of 10 , 50 , and 90 pa were shown in figure 1(a ) . the slopes of the f - i relationships were 246.92 28.00 hz / na before nmda application and increased to 325.49 73.34 furthermore , the input resistances were decreased by 20 m nmda in ra projection neurons ( p < 0.05 , n = 6 ) ( figure 2 ) . the afterhyperpolarization ( ahp ) of action potential underlies the modulation of firing frequency . to test the effect of nmda on spike ahp comparison of spike shape is shown in figure 3(a ) with 90 pa current injection before and after nmda application . in average , there was no significant change in the amplitude of ahp after nmda application ( p > 0.05 , n = 5 ) ( figure 3(b ) ) . then we used 50 m dl - apv , a nmda receptor antagonist , to check the selectivity of nmda receptor , and we found that the slope of f - i was not changed by dl - apv alone ( p > 0.05 ) . the slope was 255.78 66.69 hz / na and 269.34 56.96 hz / na ( p > 0.05 ) before and after application of 50 m dl - apv ( figure 4 ) , respectively . nmda increased the f - i slope 1.32 times ( p < 0.05 ) ; however , the gain modulation induced by nmda was completely abolished by dl - apv ( figure 4 ) . previous studies reported that ra projection neurons received glutamatergic and gabaergic inputs [ 23 , 24 ] , which might increase background synaptic activities and modulate the gain of neurons [ 25 , 26 ] . to determine the effect of ampa receptor and gaba receptor on gain modulation by nmda , we examined the effects of nmda in the presence of cnqx , an antagonist for ionotropic glutamate ampa receptors , or / and bicuculline methiodide ( bic ) , a competitive antagonist of gabaa receptors , respectively . bic ( 10 m ) accelerated the depolarization by nmda application , but bic alone had little effect on the f - i slope . in the presence of bic , the mean initial slopes were 276.46 59.4 and 332.6 40.02 hz / na before and during application of nmda . the f - i slope was increased from 1.32 to 1.39 in the presence of nmda and bic ( p > 0.05 ) ; that is , there is an increase of 105.30% over the nmda treatment ( figure 5 ; p > 0.05 , unpaired t - test ) . cnqx ( 10 m ) decreased the membrane potential and abolished the spontaneous spikes of ra projection neurons sometimes but did not affect the slope of f - i relationship . in the presence of cnqx , nmda application hardly changed the f - i slope of ra projection neurons no matter whether bic was present or not ( figure 5 ; p > 0.05 ) . these results indicated that the ampa receptor - dependent excitability played a crucial role in the gain modulation by nmda . the slope was 0.97 or 1.11 in the presence of cnqx or cnqx and bic , respectively . the gain induced by nmda was significantly decreased by cnqx ( p < 0.05 ) . in this study , we showed that nmda increased the gain of ra projection neurons in adult zebra finch . nmda - induced gain increase was limited to firing frequencies less than 40 hz . the effect of nmda on gain was mediated through nmda receptors and might involve synaptic activities . in songbirds , we found that nmda increased the slope of f - i relationship in ra projection neurons , which was completely blocked by dl - apv , a nmda receptor selective antagonist , suggesting that nmda increased the sensitivity of ra projection neurons . the pharmacology of nmda receptors involved in gain modulation has been examined in mammals . in cat visual cortex , nmda increased the gain of the contrast - response ( c - r ) curve of the neuron in layers ii / iii and v / vi . in mouse , the thalamocortical neurons in the dorsal lateral geniculate nucleus play a key role in the generation of firing patterns through nmda receptors . the neural activity in the afp of songbirds can direct moment - by - moment changes in the primary motor areas responsible for generating song . singing - related activity in lman is more variable in pattern across repeated renditions during undirected song than directed song [ 28 , 29 ] . reported that the fundamental frequency of the syllable was shifted by stimulating the lman - ra pathway , which is mediated by nmda receptors . our results indicated that the activity of nmda receptor changed the sensitivity of ra projection neuron . ra , as a premotor nucleus , occupies an important position in the song system , integrating information from both pathways . ra projection neurons are responsible for transmitting song information to midbrain and brainstem vocal and respiratory structures . temporal pattern of spike bursts in ra projection neurons is associated with the timing of acoustic features of birdsong . precise timing of individual spikes has a close relationship with stereotypic behavior , which suggests that the song is represented in ra by a temporal code [ 5 , 31 ] . thus , the modulation of nmda receptors in the activities of ra projection neurons could be very important for the plasticity of birdsong . previous studies in slices demonstrated that nmda receptor achieves the nonlinear , multiplicative behavior seen in gain modulation by several mechanisms . mel presented results from compartmental modeling studies showing that dendrites containing nmda - type synaptic conductances and voltage - dependent na , k , and ca channels can impose nonlinear interactions between nearby synapses that give rise to approximate multiplications [ 32 , 33 ] . in thalamocortical cell of the rat and cat dlgn , nmda receptors contribute to the burst firing generated by the low - threshold ca potential and depend on the membrane potential range over which this type of firing occurs . in substantia nigra dopamine neurons , nmda receptor - mediated synaptic conductance generates transient high - frequency activity by rapidly but transiently overwhelming the conductance 's underlying action potential ahp and/or engaging postsynaptic voltage - dependent ion channels in a manner that overcomes the limiting effects of ahp . but our results showed that there was no significant change in spike ahp after nmda application . so the gain induced by nmda in ra projection neurons may not be mainly modulated by the change of spike shape , but by synaptic activities . the change in membrane potential has been shown to play an important role in the firing pattern regulation by nmda receptor , supporting the idea that depolarization acutely alters firing frequency . it has been shown that simple excitability or inhibition alone can induce a multiplicative gain change in the cortical neurons . in this model , one set of synaptic inputs provided purely excitatory drive to a target neuron , and another set of combined excitatory / inhibitory inputs ( or balanced inputs ) provided the modulation . in this setup , changing the firing rates of the balanced inputs produced an approximately multiplicative gain modulation of the response to the purely excitatory drive . here , blockade of the excitatory or inhibitory transmission alone had no effect on the f - i slope of ra projection neuron , implying that simple excitability or inhibition alone can not induce a multiplicative gain change . however , our results did not exclude a role of spontaneous synaptic activity on gain modulation in vitro , which , due to the loss of synapses in slice , was not sufficient to produce a significant change in gain . ampa receptors are required for the gain modulation in our study , because blocking excitatory transmission mediated by ampa receptors almost abolished the effect of nmda on gain modulation . two reasons might be involved : one is the two states of nmda - base mechanism due to the special anatomical structure of ra . ra projection neurons receive inputs from two areas , hvc and lman . both are excitatory glutamatergic but have distinct postsynaptic properties . hvc - ra is largely mediated by ampa receptor , whereas lman - ra is almost completely mediated by nmda receptor . ampa / nmda receptor composition of different neuronal pathways is the foundation of gating and gain modulation . nmda receptor - rich pathway can gate ampa - rich input and increase the gain of a neuron responding to the input of ampa - rich pathway . the second is related to the intrinsic properties of nmda receptors , which are uniquely voltage dependent . except binding with neurotransmitter blockade of main excitatory inputs mediated by ampa receptor with cnqx hyperpolarized the membrane potential , which caused nmda receptors not activated . therefore , the ampa receptor - mediated excitatory transmission is essential for the gain modulation induced by nmda . it is well known that the nmda receptor plays a critical role in many physiological and pathological processes , such as learning , memory , neurodegeneration , epilepsy , and ischemia . the nmda receptor - mediated synaptic transmission satisfies the associative property of hebbian learning and in fact plays a critical role in its cellular model , long - term synaptic plasticity . nevertheless , pharmacological agents which block nmda receptors impair a variety of brain processes , suggesting that nmda receptor transmission also plays an important role beyond long - term memory and participates in shaping the dynamic activity of neural networks . in song system , the expression pattern of nmda receptor is related to the development of song learning [ 21 , 3740 ] . recent results show that the nmda receptor on the lman - ra synapses generates variability across different song renditions , thereby facilitating reinforced learning of songs . how nmda accomplishes this role is not clear . our results showed that nmda application increased the gain of ra projection neurons , in which ampa receptor was necessary . the ampa receptor - mediated excitability is derived only from hvc [ 10 , 11 ] . therefore , nmda receptor in lman - ra synapse might regulate hvc - ra synaptic transmission , which implicated that nmda receptor is more effective to modulate the syllable online . these findings suggest a mechanism by which nmda receptor can selectively modulate behaviorally relevant excitatory inputs .

the song of zebra finch is stable in life after it was learned successfully . vocal plasticity is thought to be a motor exploration that can support continuous learning and optimization of performance . the activity of ra , an important pre - motor nucleus in songbird 's brain , influences the song directly . this variability in adult birdsong is associated with the activity of nmda receptors in lman - ra synapses , but the detailed mechanism is unclear . the control of gain refers to modulation of a neuron 's responsiveness to input and is critically important for normal sensory , cognitive , and motor functions . here , we observed the change of gain in ra projection neurons after exogenous nmda was applied to activate nmda receptors using the whole - cell current clamp recording . we found that nmda substantially increased the slope ( gain ) of the firing rate - current relationship in ra projection neurons . the ampa receptor - dependent excitability played a crucial role in the modulation of gain by nmda . these results suggested that nmda receptors may regulate the dynamics of ra projection neurons by input - output gain .

first described by veterinarians , pythiosis is a serious life- and limb - threatening infection endemic to thailand , but rarely seen in the western hemisphere . known to infect immunocompetent horses , dogs , and other large animals,2 , 3 pythium infection was first reported in humans in 1989 in association with thalassemic hemoglobinopathy.4 , 5 human pythiosis is known to present in one of four clinical entities : subcutaneous , ocular , vascular , and disseminated . here , we present a unique case of vascular pythiosis managed with limb - sparing vascular bypass grafts complicated by pseudoaneurysm formation . the patient is a 17 year - old jamaican male with a history of paroxysmal nocturnal hemoglobinuria and severe aplastic anemia refractory to horse anti - thymoglobulin treatment , which was transfusion - dependent . one month prior to admission at our institution , he went fishing in a fresh water pond in jamaica and his left lower leg was caught in a bush . on the following day , he noticed a small bullae with a black center and later developed severely painful left inguinal swelling . he was admitted to a local hospital for the next several weeks , where all diagnostic testing performed was unremarkable . his pain persisted despite antibiotic and antifungal therapy , and he was later transferred to our institution for consideration of bone marrow transplant . on admission , he was a febrile with a white blood cell count of 6.2 ( k/l ) and platelet count of 18 ( k/l ) and was complaining of severe left leg pain . he had a 9 8 cm soft tissue defect with overlying escar on the posterior aspect of his left leg ( fig . 1a ) , and his exam was also notable for an exquisitely tender and erythematous left inguinal mass . motor and sensory exam were intact in both lower extremities without pain upon passive hip flexion . several small , dark macules were noted along the lateral aspect of his left foot ( fig . mri showed a t2 bright left groin mass with edema along the fascial planes , and lower extremity angiogram demonstrated occlusion of the left common femoral artery with distal profunda femoris and mid - superficial femoral artery reconstitution ( fig . the patient was not a candidate for percutaneous intervention , and was taken to the operating room for exploration . visual inspection of the common femoral artery after exposure was significant for several patches of necrotic vessel wall . initial attempt with thrombectomy was unsuccessful , as the fogarty catheter actually perforated the anterior wall at the areas of necrosis . the patient ultimately received an extra - anatomic left external iliac to superficial femoral artery bypass using a nonreversed greater saphenous vein graft , as the external iliac was the closest available inflow that did not visually appear to be involved with the infection . intraoperative wound cultures were positive for pythium insidiosum and the patient was started on a combination of voriconazole , terbinafine , and amphotericin b. given his critical condition , an emergency investigational new drug request was drafted for permission to administer a p. insidiosum antigen vaccine available for compassionate use from the pan american veterinary laboratories . the vaccine was given once daily in one - week intervals in conjunction with subcutaneously injected interferon gamma ( ifn- ) , for a total of four weeks . three weeks after his initial surgery , the patient developed new sites of pain along the graft site and repeat imaging showed a 2.5 cm pseudoaneurysm at the proximal anastomosis to the external iliac artery with contrast extravasation ( fig . he was taken to the operating room for an extra - anatomic left common iliac artery to the previous femoral popliteal bypass graft using a reverse saphenous vein graft . the patient later developed a polymicrobial superinfection of his wounds , and his vascular repair continued to break down leading to increased ischemic pain . after several multidisciplinary discussions with the family that all medical interventions were exhausted , and that even highly aggressive surgical measures including hip disarticulation or hemipelvectomy could not guarantee source control , conservative measures were initiated and ultimately , the patient expired . this patient presented a major diagnostic challenge , and his disease process and subsequent management were previously unknown to our hospital . in retrospect , he presented with a classic case of vascular pythiosis , with an antecedent history of exposure to surface water and exam findings of vascular insufficiency , including pain out of proportion to exam , distal emboli , and an ischemic ulcer . the correct infectious diagnosis was not made until 1 month after exposure , after a vascular bypass was already performed . indeed , in a large multi - institutional study from thailand with a total of 102 cases of pythiosis , 60 patients received a diagnosis of vascular pythiosis , and only the patients who had undergone amputation survived . further review of the literature for reports of vascular pythiosis demonstrated that the majority of patients were treated with amputation without any signs of recurrence , and were followed for 5 to 35 months post - operatively.4 , 6 , 8 , 9 in our case , complete source control was not achieved during the first operation since the common femoral artery and the proximal portion of the superficial femoral artery were oversewn and left in place , standard practice for the treatment of peripheral arterial disease . incomplete source control eventually led to development of a pseudoaneurysm at the proximal anastomosis and additional surgery . while the second repair was cited in previously uninvolved tissue planes , it did not protect against further infection , likely because p. insidiosum is known to invade through proteinase secretion . the authors could find no reported cases of confirmed vascular pythiosis treated with limb - sparing procedures.2 , 7 , 11 , 12 medical therapy in the treatment of vascular pythiosis involves antifungal agents , immunotherapy , and iron chelation therapy . much of the difficulty in eradicating this organism stems from its complicated phylogenesis;2 , 6 , 14 it shares features with true fungi at the same time it is phylogenetically more closely related to algae and diatomeae . p. insidiosum develops mycelium like fungi , but its cell walls do not contain chitin and its cytoplasmic membrane lacks ergosterol , both of which are important targets of traditional antifungal drugs . despite this , drugs that affect cellular permeability , including amphotericin b , terbinafine , and the azoles , have been used with some clinical success.2 , 15 more recently , immunotherapy has been used as an adjunct to surgery and antimycotics . immunotherapy was first successfully used in humans in 1998 in a patient with a vascular p. insidiosum infection refractory to surgery and antifungals.2 , 16 a p. insidiosum vaccine delivered twice with a two - week interval resulted in a cure 1 year later . data collected from clinical trials and other reports estimate vaccine efficacy to be 5560%.2 , 17 the proposed mechanism of action of the p. insidiosum vaccine is to shift host adaptive immune response from a predominantly t helper 2 ( th2 ) response to a t helper 1 ( th1 ) response.2 , 17 pythium exoantigens are typically processed by antigen presenting cells ( apcs ) and presented to nave cd4 helper t cells ( th0 ) . this induces th0 cells to adopt a th2 phenotype secreting il-4 and il-5 , which trigger many downstream effects , including mast cell and eosinophil migration to the site of infection with subsequent degranulation , releasing inflammatory mediators and causing tissue damage . loss of tissue architecture then creates a more favorable environment for these pathogenic hyphae to take hold . conversion of a th2 to th1 response would favor secretion of ifn- and il-2 , leading to increased cytotoxic t lymphocyte recruitment and therefore , direct killing of p. insidiosum . in this case , the purpose of injecting ifn- subcutaneously was to augment a th1 response , and co - administration of gm - csf was to increase the efficiency of exoantigen presentation to apcs . another treatment modality used to control this infection is iron chelation therapy . given the association between pythium infection in patients with hemoglobinopathies with a long - standing history of blood transfusions4 , 7 and the discovery of a ferrochelatase gene in p. insidiosum , other research efforts have focused on elucidating the role of iron overload in p. insidiosum pathogenesis . there is evidence that iron overload from transfusions contributes to derangements in host immunity by impairing lymphocyte function and altering phagocytosis , leading to a more susceptible state for infection6 , 19 and is in fact , a key virulence factor for the survival of p. insidiosum in the host . correcting iron overload has been cited as a treatment in infections common in thalassemia , and has also been used in pythiosis . in our patient , in conclusion , this patient 's vascular p. insidiosum infection was refractory to optimized medical treatment , immunotherapy with cytokine injections , and surgery . attempted source control with limb salvage necessitating vascular bypass was unsuccessful in treating this case of vascular pythiosis . written informed consent was obtained from the patient 's guardian for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request.key learning pointsclinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency . the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency . the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .

introductionpythiosis is a serious life- and limb - threatening infection endemic to thailand , but rarely seen in the western hemisphere . here , we present a unique case of vascular pythiosis initially managed with limb - sparing vascular bypass grafts complicated by a pseudoaneurysm in our repair.presentation of casethe patient is a 17 year - old jamaican male with severe aplastic anemia . he sustained a minor injury to his left leg while fishing in jamaica , which evolved to become an exquisitely tender inguinal swelling . his physical exam and imaging were significant for arteriovenous fistula with limb ischemia . pathology obtained during surgery for an extra - anatomic vascular bypass showed extensive invasion by pythium insidiosum . he later developed a pseudoaneurysm at the site of proximal anastomosis and required urgent intervention.discussionthis patient presented with a rare , but classic case of vascular pythiosis , which was unrecognized at the time of presentation . a variety of therapeutic modalities have been used to treat this disease , including antibiotics , antifungals , and immunotherapy , but the ultimate management of vascular pythiosis is surgical source control.conclusiona high index of suspicion in susceptible patients is needed for timely diagnosis of vascular pythiosis to achieve optimal source control .

tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model ( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) . the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures , then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12 gy ) at the city of hope ( duarte , ca ) facility . subsequently the tissues were incubated for 6 h at 37 c with 5% co2 after irradiation . then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna . the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues . the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280 > 2.0 , and rin > the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) . the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis . for this purpose , a quantity of 0.025 mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42 c using the hybbag mixing system with 1 onearray hybridization buffer ( phalanx biotech ) and 0.01 mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format . the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) . testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver . the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values were calculated to test the null hypothesis that expression is absent ( called p - value detected ) . a p - value detected < 0.05 indicated that the transcript specific to a given probe was truly present or expressed . lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes , rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level . three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes . this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample the scattered plots of raw and normalized log2 data were compared using the r function pairs . pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of < 0.01 . scatter plots showed that technical replicates had high repeatability and all the correlation values were > 0.953 . our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12 gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments . we only used genes with | fold change | > 1.5 and p - value < 0.05 . we input gene symbols into david bioinformatics , while throughout we used the default settings . the threshold for significance was a benjamini - adjusted p - value < 0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation . table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) . that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation . also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division . overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in . the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics . up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results . goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature . other enriched categories are from the interpro , sp pir , smart , and kegg databases . count = the number differentially expressed genes annotated with a given enriched term . adj p - val = benjamini - adjusted p - values . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html . up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results . goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature . other enriched categories are from the interpro , sp pir , smart , and kegg databases . count = the number differentially expressed genes annotated with a given enriched term . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html . tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model ( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) . the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures , then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12 gy ) at the city of hope ( duarte , ca ) facility . subsequently the tissues were incubated for 6 h at 37 c with 5% co2 after irradiation . then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna . the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues . the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280 > 2.0 , and the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) . the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis . for this purpose , a quantity of 0.025 mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42 c using the hybbag mixing system with 1 onearray hybridization buffer ( phalanx biotech ) and 0.01 mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure , the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format . the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) . testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver . the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values were calculated to test the null hypothesis that expression is absent ( called p - value detected ) . a p - value detected < 0.05 indicated that the transcript specific to a given probe was truly present or expressed . lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes , rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level . three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes . this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample the scattered plots of raw and normalized log2 data were compared using the r function pairs . pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of < 0.01 . scatter plots showed that technical replicates had high repeatability and all the correlation values were > 0.953 . our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12 gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments . we only used genes with | fold change | > 1.5 and p - value < 0.05 . we input gene symbols into david bioinformatics , while throughout we used the default settings . the threshold for significance was a benjamini - adjusted p - value < 0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation . table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) . that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation . also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division . overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in . the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics . up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results . goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature . other enriched categories are from the interpro , sp pir , smart , and kegg databases . count = the number differentially expressed genes annotated with a given enriched term . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html . up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results . goterm cc = gene ontology cellular component , mf = molecular function , bp = biological process , and up_seq_feature = uniprot sequence feature . other enriched categories are from the interpro , sp pir , smart , and kegg databases . count = the number differentially expressed genes annotated with a given enriched term . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file = function_annotation.html .

we evaluated a three - dimensional ( 3d ) human oral cell culture that consisted of two types of cells , oral keratinocytes and fibroblasts as a model of oral mucositis which is a debilitating adverse effect of chemotherapy and radiation treatment . the 3d cell culture model was irradiated with 12 or 2 gy , and total rna was collected 6 h after irradiation to compare global gene expression profiles via microarray analysis . here we provide detailed methods and analysis on these microarray data , which have been deposited in gene expression omnibus ( geo ) : gse62395 .

adolescents in sub - saharan africa face high exposure to human immunodeficiency virus ( hiv ) infection . despite all efforts to prevent the disease , prevalence estimates in sub - saharan unaids ( i.e. , the joint united nations programme on hiv and aids ) priority countries range from 1.3 to 15.6 % among young women and 0.56.5 % among young men aged 1524 years ( unaids , 2011 ) . higher prevalence estimates are associated with certain regions ( e.g. , eastern and southern africa ) and subpopulations ( e.g. , adolescent orphaned and/or married girls ; birdthistle et al . , 2008 ; operario , underhill , chuong , & cluver , 2011 ; pascoe et al . , 2010 ) . in addition , united nations children s fund ( unicef ) estimated there were 2 million adolescents aged 1019 years living with hiv globally in 2009 ( unicef , 2011 ) . kenya and zimbabwe were among countries with the highest numbers of infected adolescents with an estimated 136,000 ( prevalence estimate 1.5 % ) 1 and 104,000 ( prevalence estimate 3.1 % ) adolescents living with hiv , respectively ( unicef , 2011 ) . the true measure of effectiveness for such interventions is reduction in hiv and other sexually transmitted infections ( stis ) . however , very few adolescent prevention studies have used biomarker data as outcomes ( michielsen et al . , 2010 ) because of concerns about the difficulties in collecting these data in resource poor settings ( jukes , simmons , & bundy , 2008 ) , even when power analyses suggest that moderate intervention effects could be detected . behavioral prevention scientists have instead relied on self - reported sexual behavior , which is subject to social desirability bias and likely to differ between study arms ( cowan & pettifor , 2009 ; kirby , obasi , & laris , 2006 ) . widespread access to testing and treatment facilitated by the president s emergency plan for aids relief ( pepfar ; http://www.usaid.gov/what-we-do/global-health/hiv-and-aids ) have greatly enhanced opportunities to successfully incorporate biomarker procedures into adolescent hiv prevention research designs , since infected individuals identified through testing offered by studies may access free or subsidized hiv treatment and care ( stringer et al . , 2006 ; wools - kaloustian et al . , 2006 , 2009 ) moreover , including biomarker outcomes can greatly improve the credibility and legitimacy of hiv prevention research . to address the issue of feasibility , we present two case studies of prevention trials with biomarker data collection among adolescents in kenya and zimbabwe . together these studies provide a valuable opportunity to examine the ethical and practical challenges of biomarker procedures within hiv prevention intervention clinical trials in developing countries . specifically , we describe the challenges faced by investigators in the two cases in developing and implementing ethical procedures for informed consent , biomarker testing , and disclosure of test results . we also present the practical challenges of collecting biological samples in resource - limited countries and compare sample collection by blood spots relative to venipuncture . in addition , we examine difficulties with analyzing biological samples when the performance of assays in certain populations is unclear . we also provide specific suggestions for further research and development in collecting and analyzing biomarker data for adolescent prevention research . the primary goal of hiv prevention among adolescents ( ages 1019 years ) is to reduce new infections and related sexual risk factors [ world health organization ( who ) , 2006 ) . although the hiv prevalence of adolescents ages 15 and older is monitored through demographic and health surveys ( dhs)2 in most sub - saharan africa countries , little is known about the prevalence of hiv in adolescents under 15 years . moreover , these adolescents are rarely tested for hiv in clinical settings unless they present in poor health or with recurrent infections ( ferrand et al . , 2009 ) . although a small number of recent randomized controlled trials have used biomarkers ( baird , mcintosh , & ozler , 2010 ; cowan et al . ; pronyk et al . , 2006 ; ross et al . , 2007 ) , most adolescent hiv prevention studies have relied on changes in self - reported sexual behavior to assess intervention effectiveness ( see reviews : cowan & pettifor , 2009 ; michielsen et al . , 2010 ) , with audio computer - assisted self - interviews intended to improve veracity ( mensch , erulkar , & hewett , 2003 ) . however , biomarker comparisons have suggested that self - reported measures have low validity ( cowan et al . , 2002 ; gavin et al . , 2006 ; mensch , hewett , gregory , & helleringer , 2008 ; palen et al . , 2008 ; plummer et al . , 2004 ) . for example , in one study , only one of 16 youth with hiv admitted to ever having sex , and four of the 16 also had either chlamydia or gonorrhea ( cowan et al . , 2002 ) . inclusion of a highly prevalent and reliable biomarker of sexual activity in conjunction with self - reported sexual behavior can help to improve reliability of this outcome measure . hiv infection is not the most useful biomarker of adolescent sexual activity by itself because of the relatively low prevalence among younger adolescents and the potential for transmission through perinatal and non - sexual blood - borne routes ( amornkul et al . , 2009 ; ferrand et al . , 2009 ; stover , walker , grassly , & marston , 2006 ) . herpes simplex virus type 2 ( hsv-2 ) , the primary cause of genital herpes , is a commonly used biomarker of sexual activity because the presence of hsv-2 antibodies is highly associated with past sexual behavior ( tobian et al . , 2009 ; van de laar et al . , 1998 ) . in contrast to other stis such as chlamydia , gonorrhea , syphilis and trichomoniasis , infection with hsv-2 is life - long , and , once established , there is currently no treatment to eliminate it ( geretti , 2006 ; gupta , warren , & wald , 2008 ) . hsv-2 prevalence in the adult general population in sub - saharan africa is high , ranging from 30 to 80 % in women and from 10 to 50 % in men , as assessed by detection of hsv-2 antibodies [ amornkul et al . , 2009 ; , 2005 ; munjoma et al . , 2010 ; national aids & sti control programme ( nascop ) , republic of kenya , 2009 ; tobian et al . , 2009 ; weiss , 2004 ] . prevalence estimates for adolescents are only available through community - based studies and are lower . one study in zimbabwe among adolescent females aged 1519 years reported that 12 % of participants tested positive for hsv-2 ( birdthistle et al . a study in western kenya found hsv-2 prevalence to be 9 % for females and 4 % for males aged 1314 , and 28 % for females and 17 % for males aged 1519 years ( amornkul et al . , 2009 ) . another study conducted in zimbabwe among adolescents whose mean age was 15 years reported a prevalence estimate of 0.2 % , suggesting a later stage of sexual debut ( cowan et al . 2012 ) evaluated the use of hsv-2 as a biomarker of sexual debut among young people aged 1024 years . following a comprehensive review of the literature , the authors concluded that the use of hsv-2 as a biomarker for sexual debut is limited because of its low transmissibility and the fact that not all potential sexual partners are infected with the virus . building on these findings , our study describes the challenges of collecting hiv and hsv-2 biological data as biomarkers for sexual activity among sub - saharan african youth in prevention trials . we present two clinical trial case studies to examine important issues related to adolescent biomarker data collection in two clinical trials in kenya and zimbabwe ( hallfors et al . , both studies were school - based , cluster randomized trials testing school support ( including payment of school tuition and school uniforms ) as a structural hiv prevention intervention for adolescent orphans . in both studies , we collected blood samples from participants for hiv and hsv-2 testing , but they differed as follows : we collected samples by venipuncture with results disclosed to participants and their guardians in kenya , whereas in zimbabwe , we collected samples by finger pricks for dried blood spots ( dbs ) and results were not disclosed . the kenya sample ( n = 837 ) comprised young adolescent male ( 52 % ) and female ( 48 % ) orphans in grades 7 ( 61 % ) and 8 ( 39 % ) enrolled in school in siaya district , nyanza province . their mean age was 14.9 ( sd 1.5 ; range 1121 years ) ; 22 % reported ever having had sex . among girls , hiv and hsv-2 prevalence was 1 % ( n = 10 ) and 3 % ( n = 28 ) , respectively . among those with positive hiv or hsv-2 test results , 70 % ( n = 7 ) and 64 % ( n = 18 ) , respectively , reported never having had sex . the original sample for the zimbabwe study was orphan 6th grade girls ( n = 328 ) in manicaland province primary schools ; surveys were administered annually during 20072010 ( hallfors et al . , 2011 ) . after securing a project renewal grant , we collected biomarker specimens and a final survey in march through june 2012 , when most participants were 1617 years old . from the original sample , we located 287 ( 88 % ) for the 2012 survey . twenty - three percent reported ever having sex ; 18 % had ever been married and/or pregnant . hiv and hsv-2 prevalence was 4 % ( n = 12 ) and 6 % ( n = 16 ) , respectively . among those with positive hiv or hsv-2 test results , 50 % ( n = 6 ) and 44 % ( n = 7 ) , respectively , reported never having had sex . although the protection of adult participants in hiv prevention research is well developed ( national institute of mental health collaborative hiv / std prevention trial group , 2007 ) , we found that clear guidance regarding hiv and other sti testing and disclosure is lacking for children and adolescents . as an example , our two clinical trials were peer - reviewed by two different nih scientific review panels ; one set of reviewers thought that it was an ethical imperative to disclose hsv-2 as well as hiv results to adolescents , whereas the other was concerned that disclosure of biomarker results might lead to stigma and abuse . these divergent opinions led to discrepant protocols for disclosure , with one trial disclosing and the other not disclosing test results to participants . we used the national hiv testing and counseling ( htc ) guidelines [ central statistical office ( cso ) & macro international inc . , 2007 ; nascop , republic of kenya , 2008 ] for hiv in both countries . given the lack of national guidelines for adolescent sti testing in kenya [ ministry of health ( moh ) , republic of kenya , 2006 ) , we developed a protocol in conjunction with the siaya district aids and std control office that closely followed hiv guidelines . study protocols were reviewed and approved by irbs in the us , kenya and zimbabwe . the kenya study was approved by the ministry of education and siaya district education office ; school head teachers identified orphans and organized meetings in which moi university research collaborators and a district education officer explained the study to students and their caregivers . caregivers and students we invited a total of 923 orphans in grades 7 and 8 in 2011 to participate ; 849 ( 92 % ) consented and 837 ( 91 % ) completed both the student survey and biomarker testing . following national guidelines , we required kenya study participants under age 18 to have an adult guardian accompany them for hiv rapid testing ( nascop , republic of kenya , 2008 ) . in some instances , however , the guardian was not available because of work , other commitments , or illness . in such cases we permitted a relative , teacher or neighbor to stand - in for the guardian if they had a note from the guardian or if research staff were able to speak with the guardian by phone . given the sensitive nature of hiv testing , the senior counselor interviewed all unclear or undocumented cases to determine whether or not to proceed with specimen collection and htc . we visited clinics several times to give participants the opportunity to return for testing with their guardian or authorized proxy . although not required , all participants ages 18 and older were accompanied by an adult for support in case of a positive hiv test result . in accordance with national guidelines , we gave referrals to publicly funded hiv care and treatment centers to all adolescents with positive hiv test results , while hiv negative participants were counseled on risk reduction ( nascop , republic of kenya , 2008 ) . we disclosed results to both guardians and participants if the latter were under age 18 and to the participants alone if they were 18 years old according to the study protocol . in zimbabwe , with permission from the provincial education officer , the consortium principal investigator and a research associate held meetings at study schools to explain the continuing study to parents / guardians , participants , and key school staff . we obtained written parent / guardian consent and participant assent for continued voluntary participation in the study , including separate checkbox consent for survey and blood sample collection . guardians raised few concerns , although they did ask why we were not disclosing test results . participants who were married or 18 years or older provided their own consent . in deference to local cultural norms and to prevent potential spousal abuse , we prepared a second consent form for husbands to provide consent for their wives participation . most of the wives gave their own consent for the survey and did not ask their husbands for consent . in four cases , the husband signed a consent form and in five additional cases , the husband refused and the wife did not take the survey . although asking husbands for permission for their wives study participation is not consistent with us culture , it appears to be a useful protocol accommodation for some young wives in rural zimbabwe . we consistently relied on local researchers and key informants to make such culturally sensitive decisions . after 4 months , we found 88 % of the original sample who consented to the survey ; 85 % consented to blood specimen collection . of those not surveyed , two participants had died in childbirth , 2 % refused , and approximately 10 % of the sample could not be found . of those not consenting to blood specimen collection ( n = 8) , six were married and refused for religious reasons or husband s objection . two were consented for the survey by school officials who refused to provide additional consent for the blood collection , and no guardian was available to give permission . hiv testing is technically far more advanced for widespread use in sub - saharan africa than hsv-2 testing . rapid hiv antibody tests are now widely used and accepted ( moh , republic of kenya , 2006 ) , and sequential algorithms for retesting have been adopted in kenya and zimbabwe for rapid testing ( cso & macro international inc . , 2007 ; nascop , republic of kenya , 2008 ; who , 2004 ) . rapid hiv tests meet high performance standards , sample collection procedures are simple , tests are easy to perform , and interpretation of test results is reasonably clear . in contrast , hsv-2 serologic testing presents some important challenges . although a number of hsv-2 type - specific serologic tests are available , only a limited number have been tested among sub - saharan african populations . these include the herpeselect hsv-2 enzyme - linked immunosorbent assay ( elisa ; focus diagnostics , cypress , ca ) and kalon hsv-2 elisa ( kalon biological ltd . , guildford , uk ; biraro , mayaud , morrow , grosskurth , & weiss , 2011 ) . these tests require skilled laboratory staff , specialized equipment , and a constant source of electricity . they are appropriate for use with large batches of samples as in sentinel surveys and are inexpensive , costing about $ 3 per test . although the literature reports variable performance of these tests with respect to sensitivity and specificity in sub - saharan populations , kalon is often found to be superior to focus herpeselect ( see biraro et al . , 2011 for a review of the performance of hsv-2 tests in sub - saharan africa ; 2008 ; ngayo , friedrich , holmes , bukusi , & morrow , 2010 ; smith et al . , 2009 ; van dyck et al . , 2004 ) . when using the manufacturer s instructions ( index cutoff value = 1.1 for both tests ) , kalon tends to have lower sensitivity but higher specificity than focus herpeselect ( delany - moretlwe et al . , 2010 ; gamiel et al . , 2008 ; ngayo et al . , 2010 ; smith et al . , 2009 ; van dyck et al . , the low specificity of focus herpeselect is particularly problematic when disclosing results to research participants because of an unacceptably high rate of false positives when prevalence is low ( gamiel et al . , 2008 ; mark et al . , 2008 ; ngayo et al , 2010 ; van dyck et al . , 2004 ) . increasing the assay cut - off values is one strategy for improving the performance of both the kalon and focus herpeselect tests . a number of studies have shown that increasing the cut - off value for focus herpeselect to 3.5 increases specificity , although sensitivity is necessarily reduced ( delany - moretlwe et al . , 2010 ; smith et al . , other studies similarly improved performance at cut - off values of 3.2 and 3.3 ( delany - moretlwe et al . , 2010 ; gamiel et al . , 2008 ; ngayo et al . , although findings are mixed , increasing the cut - off value for the kalon test to 1.5 has also been shown to increase specificity , with an attendant reduction in sensitivity ( gamiel et al . , 2008 ; ngayo et al . in our studies , the kenya laboratory used the kalon assay , and the zimbabwe laboratory used the focus test . at the time of our studies , neither assay had been validated for dbs by the manufacturer in african populations . the efficiency of dbs for hsv-2 testing with each assay must be evaluated in specific populations prior to use ( hogrefe , ernst , & su , 2002 ) . because the kalon test had not been validated for hsv-2 testing with dbs samples , specimens in the kenya study were obtained by venipuncture . the zimbabwe laboratory had validated the focus test with dbs ( mudzori & mutsogoro , 2006 ) . in both studies , we used a modified algorithm to interpret results : samples with initial index values < 0.9 were reported as negative and no further testing was conducted . samples with initial index values > 2.5 were defined as high positive and were reported as positive without additional testing . samples with initial index values from 1.5 to 2.5 were considered low positive , and those with initial index values between 0.9 and 1.5 were considered equivocal ; all low positives and equivocals were retested in duplicate . if both retest results had index values > 1.5 , the final result was reported as positive . if both retest results were < 0.9 , the final result was negative . if retest results were equivocal ( 0.91.5 ) or discordant , the final result was indeterminate . although test performance was improved by increasing the cut - off and expanding the range of results for repeat testing , the performance characteristics of tests to detect antibodies to hsv-2 are imperfect . based on a recent systematic review of hsv-2 antibody test performance in sub - saharan africa ( biraro et al . , 2011 ) , sensitivity for the kalon test used in kenya was estimated at 94 % and specificity at 92 % with the higher cut - off value of 1.5 . the predictive values of all diagnostic tests are strongly influenced by the prevalence of infection in the population , and in low prevalence situations , even a very good test will have a poor positive predictive value ( ppv ) . using the sensitivity and specificity values for the kalon test under the testing conditions employed in our kenya study , the ppv was estimated at 26 % with a 3 % hsv-2 prevalence . that is , we can be confident that about one in four participants with positive test results truly had hsv-2 antibodies present in their blood sample ; however , as many as three in four could have had a false positive test result . in kenya , we conducted sample collection and htc primarily in local public health facilities near the schools . eighteen facilities participated . in two sites , with permission from the school headmasters and district education officer , we collected blood samples and conducted htc at the school . venipuncture was conducted by six skilled phlebotomists trained in htc . to avoid obtaining two separate blood specimens ( finger prick for rapid hiv testing and venipuncture for hsv-2 testing ) the process , including counseling , blood draw , hiv testing , disclosure of results took on average 20 min . the process was longer for participants with discordant results who needed a tie - breaker test and for participants with hiv positive results . we offered all participants and their guardians a snack either before or after specimen collection . we stored blood samples for hsv-2 testing in labeled vacutainer tubes in a cooler box without ice and transported them to a local laboratory within 24 h for serum processing . sera were stored at the local laboratory in a freezer at 20 c until transport on ice weekly to the hsv-2 testing laboratory . the logistics of specimen collection , processing and transport for this study were challenging but feasible . in comparison , we did not require guardians to be present because hiv results were not provided to participants . sampling and dbs preparation were much less obvious to others nearby , helping to safeguard participant privacy and reduce concerns about witchcraft or other conspiracy theories regarding hiv / aids and blood , which circulate in sub - saharan african communities ( tenkorang , gyimah , maticka - tyndale , & adjei , 2011 ) . blood from the finger prick was dropped on five circles on a dbs filter card labeled with the participant s study i d . some participants may have been dehydrated or not very well nourished , making it difficult to fill all circles , but adequate samples were collected from all participants . specimens were dried , stored with a desiccant , and transported to the testing lab at ambient temperatures . dbs samples are easy to store and transport and require no processing or refrigeration ( see table 1 for a summary of the study procedures).table 1comparison of biomarker testing procedures in two research studieskenyazimbabweethical considerationswritten guardian consent if < 18/adolescent assentwritten guardian consent / adolescent assentresults disclosed to guardian and adolescentno results disclosedreferrals for carenot applicablecollecting and analyzing biological samples methodvenipuncturedried blood spot hiv testrapid testrapid test hsv testkalon elisafocus elisafeasibility of collection methods time20 min3 min temperature for specimen transportcooler with no iceambienttesting costsreference labreference lab bench fee , supplies , storage , labor us$ 5,784bench fee , supplies , labor us$ 8,000field site lab sample processing , labor , supplies , storage us$ 3,611additional fee us$ 480 other supplies us$ 100 transportation us$ 375 total us$ 9,919total us$ 8,480 elisa enzyme - linked immunosorbent assay comparison of biomarker testing procedures in two research studies elisa enzyme - linked immunosorbent assay given the experience we have described , we conclude that it is feasible to obtain hiv and hsv-2 biomarker data for adolescent hiv prevention intervention studies . in particular the us pepfar program has accomplished considerable staff training in sub - saharan africa ; it has also stimulated the development of well - equipped laboratories , as well as useful tools for testing and protocols for improving test sensitivity , specificity , and cost ( stringer et al . , 2006 ) . in addition , it has stimulated the development of national guidelines ( particularly for adults ) for the disclosure of results . for hiv incorporation of hsv-2 serology results poses considerable challenges , particularly if results are disclosed to participants . based on our experience , there are two reasons why we do not recommend disclosing hsv-2 serology results to adolescent human subjects in sub - saharan africa . first , in populations with low or moderate prevalence of infection , as might be expected for adolescents , the potential for false positive test results is substantial in combination with relatively minor imperfections in test specificity . hsv-2 infection is often a silent disease , insofar as only 1025 % of people with antibodies for the condition are aware that they have genital herpes ( fleming et al . , 1997 ; leone , fleming , gilsenan , li , & justus , 2004 ; sizemore , lakeman , whitley , hughes , & hook , 2006 ) . adolescents without symptoms may deny ever having had sexual intercourse3 , further complicating the clinical picture and raising concerns about false positives with the potential for psychological and social harm related to participation in the study . second , local departments of health and sti authorities in sub - saharan african countries were much less familiar with hsv-2 pathology and treatment than with hiv and some other stis . even in cases where subjects acknowledge sexual exposure or genital herpes symptoms , local health clinics may not be prepared to provide appropriate treatment or care . given these vexing problems , the wisdom of disclosing hsv-2 results to adolescents after testing within a clinical prevention trial is questionable . in particular , decisions about whether to inform adolescent participants in prevention trials about hsv-2 test results should be made only after prevalence data are available and the predictive values of the test can be accurately assessed . in light of the feedback from the nih study section that raised concerns about possible stigma and abuse of hiv - positive participants in zimbabwe , as well as our own concerns , we recommend that more research be conducted to study the consequences of disclosure to adolescents in sub - saharan africa . a review of the literature indicates that sub - saharan adolescent prevention studies with biomarker outcomes have taken a variety of approaches to disclosure . in some studies , counselors disclosed participants hiv results immediately after rapid testing ( baird , garfein , mcintosh , & ozler , 2012 ; dalal et al . , 2012 ; medley et al . , 2012 ) . in other studies , participants could choose to receive their results after testing , either immediately or a few weeks later ( amornkul et al . , 2009 ; jewkes et al . , 2006 ; ross et al . , 2007 ) . in still other studies , the research did not provide test results but voluntary counseling and testing ( vct ) was made freely available for study participants ( birdthistle et al . none of these studies , however , examined whether adolescents experienced any untoward outcomes with testing and disclosure , whether hiv positive youth enrolled in care or took steps to prevent transmission , whether hiv negative youth benefited from prevention counseling and engaged in subsequent testing , or if adolescents and their guardians felt it was appropriate to learn their status in the context of a research study . from our kenya study , the problem of stigma did not appear to be a major obstacle to adolescent testing and disclosure in our study area . perhaps this is because hiv prevalence is among the highest in the country , and few families have been unaffected by hiv / aids . moreover , high hiv prevalence has drawn research , education , and hiv counseling and testing campaigns to the area . for example , in our kenya clinical trial , more than 50 % of participants ( all in grade 7 and 8) reported that they had previously been tested for hiv , mostly through a home - based testing initiative of the kenya medical research institute ( kemri ) and us centers for disease control and prevention ( cdc ) . although our study provided hiv test results to youth , we relied on phlebotomist / nurse counselors to refer youth and their guardians for care , following national guidelines . the onus of making contact with the clinic was left up to the individual and was not tracked by the system of care ; and follow up to make sure that hiv positive youth accessed care was well beyond the scope of our study . since it is unknown whether youth experience significant mental trauma and social prejudice , and whether they actually engage in the system of care , we recommend that further research be conducted to examine the perceptions and behaviors of youth and particularly newly diagnosed youth after disclosure of their test results to ascertain the consequences of this aspect of research participation and whether it can be improved . this is particularly important for adolescents , since results are disclosed in the presence of their guardian . regarding procedures for collecting biomarkers , we conclude that finger - sticks and dbs are overwhelmingly superior to venipuncture in sample collection efficiency and reduced burden on participants and community institutions . manufacturer validations of dbs for hsv-2 are urgently needed , however , for this procedure to be more widely accessible to researchers . because blood collection for dbs is minimally invasive , this procedure has excellent potential for widespread acceptability and consequent high participation in school and community research sites . in addition , rapid hiv testing in sub - saharan africa is typically conducted by finger prick , making this the preferred way for trained african health workers and counselors to participate in research data collection ( who , 2004 ) . given the complex decisions required for biomarkers , we recommend that behavioral prevention scientists interested in using biomarker data collaborate from the early planning stages with biomedical scientists who have expert knowledge in hsv-2 , as well as hiv , laboratory tests and testing procedures . we found that team members with such expertise provide valuable assistance in selecting qualified in - country laboratories , developing appropriate budgets , protocols , quality assurance and algorithms for interpreting results , and helping research staff to bridge communication with in - country laboratory staff . further , we recommend that hsv-2 test kit manufacturers determine optimal cut - off standards for sub - saharan populations , and that researchers who conduct hsv-2 testing explicitly define the testing cutoffs they use in publications . there is now a substantial literature documenting validity problems when using manufacturers cutoffs with african populations potentially inflating prevalence findings ( gamiel et al . , 2008 ; ngayo et al . , 2010 ; van dyck et al . , 2004 this suggests the need for action to improve assay validation practices on the part of manufacturers as well as methodological details from researchers documenting their findings . when conducting collaborative international research , it is important to recognize that alternative ethical systems exist . thus , for international research we recommend inclusion of team members who are knowledgeable about local contexts . indeed , successful collaborative research partnerships respect local cultures , values and practices ; negotiate effectively within these systems ; and incorporate into study designs ethical practices that are appropriate and sensitive to local cultural contexts ( e.g. , in our case , providing for husband consent for married participants ; christakis , 1992 ; emanuel , wendler , killen , & grady , 2004 ) . behavioral prevention scientists traditionally have relied on self - reported sexual behavior survey item measures to evaluate adolescent hiv prevention interventions . our experiences conducting research with orphan adolescents in kenya and zimbabwe suggest that collection of hiv and sti test results even in rural , resource - poor settings in sub - saharan africa is a feasible addition to the behavioral research toolkit . the use of sti biomarkers can greatly improve the validity of findings from adolescent behavioral intervention trials . research is urgently needed to examine the risks and benefits of hiv testing and disclosure of test results in the context of a research study for adolescents . further development and implementation of sti biomarker assessment techniques particularly pertaining to using dbs is needed to advance hiv prevention science .

self - report of sexual behavior among adolescents is notoriously inconsistent , yet such measures are commonly used as outcomes for human immunodeficiency virus ( hiv ) prevention intervention trials . there has been a growing interest in the use of hiv and other sexually transmitted disease biomarkers as more valid measures of intervention impact in high hiv prevalence areas , particularly in sub - saharan africa . we examine the challenges , benefits , and feasibility of including hiv and herpes simplex virus type 2 ( hsv-2 ) biomarker data , with details about different data collection and disclosure methods from two adolescent prevention trials in kenya and zimbabwe . in kenya , whole blood samples were collected using venipuncture ; adult guardians were present during biomarker procedures and test results were disclosed to participants and their guardians . in contrast , in zimbabwe , samples were collected using finger pricks for dried blood spots ( dbs ) ; guardians were not present during biomarker procedures , and results were not disclosed to participants and/or their guardians . in both countries , prevalence in the study samples was low . although the standard of care for testing for hiv and other sexually transmitted infections includes disclosure in the presence of a guardian for adolescents under age 18 , we conclude that more research about the risks and benefits of disclosure to adolescents in the context of a clinical trial is needed . notably , current serological diagnosis for hsv-2 has a low positive predictive value when prevalence is low , resulting in an unacceptable proportion of false positives and serious concerns about disclosing test results to adolescents within a trial . we also conclude that the dbs approach is more convenient and efficient than venipuncture for field research , although both approaches are feasible . manufacturer validation studies using dbs for hsv-2 , however , are needed for widespread use .

stroke is rare in children , however , a common cause of neurological disease , and it is a major cause of death ranks in the top ten in pediatric period11,15 ) . documented incidence has been reported as 2.5 - 8/100000 children / year12,13 ) . medical treatment includes maintaining cerebral perfusion pressure with hyperventilation and osmotherapy , barbiturate coma and in case of thrombotic ischemia , thrombolytic procedure , and anticoagulanttherapy . however , if all medical treatment is fail to drop the intracranial pressure ( icp ) and deterioration of patient progressed , alternative treatments , such as surgical decompression could be mandatory . the common rationale of decompressive craniectomy with or without duroplasty is to let the volume expansion of the swelling brain to extracranial space via removed skull flap and prevent cerebral herniation and secondary damage of brain parenchyme . in case of traumatic brain injury , however , there has not been a definite evidence or standard guideline for decompressive craniectomy for pediatric patient with non - traumatic acute stroke with uncontrolled refractory high intracranial pressure . we will introduce our series about the functional and clinical outcome after decompressive craniectomy to control refractory high icp due to non - traumatic acute stroke in pediatric patient . between march 2004 and december 2006 , decompressive - hemicraniectomy and duroplasty was performed in 5 toddlers and preschool children with non - traumatic , malignant refractory high icp . any patient have no trauma history , and refractory high icp was diagnosed as clinical evidence of acute stroke , massive infarction or hemorrhage with midline shift , compression of basal cisterns in conventional radiologic examination such as computed tomography ( ct ) and magnetic resonance image ( mri ) and , neurological deterioration consisting of decreased level of consciousness or increasing levels of sedation , or somnolence or stupor compared with baseline status at admission17 ) . and all operations were performed by one pediatric neurosurgeon and all patients received treatment in pediatric intensive care unit of one medical center . in all patients , ct or mri blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit . clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone . a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression . multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin . the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later . glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge . in all patients , ct or mri was obtained immediately , after stabilization of ventilation and hemodynamicsat first . blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit . clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone . a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression . multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin . the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later . glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge . five pediatric patients were treated with decompressive hemicraniectomy with duroplasty for non - traumatic and refractory intracranial hypertension after unilateral hemispheric stroke . four patients were toddler and one patient was pre - school girl . three were boy and two were girl . four cases were caused by ischemic stroke , and another one case was hemorrhagic stroke . one case of ischemic stroke , patient had atrial septal defect ( asd ) . and including this patient three cases of ischemic stroke patients were taken angiography ; however there was no abnormal findings in angiography . in all patients , conventional laboratory result and special serum anti - body test , including anti - phospholipid antibody were within normal rage . at the time of operation , all patients had a gcs score < 8 ( median 7 , range 6 - 8 ) and all patients hadunilateral mydriasis . surgical decompressive hemicraniectomies were performed at a mean of 12 hours ( range 4 - 19 hours ) . in all cases we had performed supratentorial hemicraniectomies . the mean peak icp was 28.6 mm hg ( range 25 - 30 mm hg ) . and the icp of all patients were dropped to normal range within one and half days after operation . there were no surgical complications , such as cerebrospinal fluid leak or wound infection , even after cranioplasty . one of ischemic stroke patient had got a ventriculoperitoneal shunt operation because of post - stroke hydrocephalus . the mean follow - up period was 47.6 months ( 36 - 68 months).gos and pcpcs for 5 patients had been measured to evaluate postoperative neurological outcome . based on the regular follow up gos scores , 5 patients had shown satisfied recoveries : 4 had good recoveries ( gos score of 5 ) , and 1 had moderate disabilities ( gos score of 4 ) . the pcpcs scores also had revealed contend results : 4 patients received scores of 2 , and only one patient scored of 3 . a 17-month - old girl was transferred to our emergency department with decreased mentality . hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side . in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd . initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and emergency mri sturdy was performed with mr angiography and diffusion - weighted image . but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift . after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days . a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore . the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty . the bone flap was secured without any complications . at the end of follow - up , 41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus . a 17-month - old girl was transferred to our emergency department with decreased mentality . neurological examination revealed a stuporous child without occasional spontaneous eye opening . hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side . in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd . initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift . after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days . a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore . the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty . the bone flap was secured without any complications . at the end of follow - up , 41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus . in stroke patient , most common cause of death is due to uncontrolled icp associated with large hemispheric infarctions10,12 ) . when it is present , it is associated with a dramatic increase in mortality . in addition , early icp elevation in patients with large hemispheric infarctions is highly concluded with high mortality16 ) . it means that the one of most important goal of acute phase stroke treatment is control the icp and prevent a secondary damage due to brain swelling . in spite of many clinical and experimental trials on medical therapeutic method , there has been a widely accepted definite guideline for oxygen saturation , temperature , serum glucose level , blood pressure for adult stroke patient1 ) . however , for pediatric stroke patient , even in many of mostly basic and elementary medical guideline has not been established yet14 ) . to prevent and control icp , thrombolytic therapy such as clot lysis has an accepted role in acute stroke treatment . in adult stroke patient group , there are antithrombotic management of adult ischemic stroke protocol and guideline published by the american heart association1 ) , such as intravenous tissue plasminogen activator , intra - arterial thrombolysis and oral administration of aspirin . but , for pediatric stroke patient , there is no guideline of this useful treatment method either14 ) . to the best of our knowledge , the first report about the benefits of decompressive hemicraniectomy in pediatric patient was described by carter et al.3 ) at 1997 . however , from this time , only a few report deals with decompressive craniectomy for pediatric patient were published . in adult acute stroke patient group , the effect of decompressive craniectomy also has been established . vahedi et al.21 ) reported that in a meta - analysis combining the data of 93 subjects from three small , randomized , controlled trials , mortality for conservative management in adult ischemic stroke patients with early brain edema was 71% as compared with 22% for decompressive surgical intervention . nowadays , some case reports and review has been published favorable outcome after decompressive surgery in childhood acute stroke2,17 ) . it is not often however in some article , decompressive craniotomy have been regarded as emerging procedure can save life , for pediatric stroke patient6,11 ) , even guideline for craniectomy was introduced11 ) . in point of fact widely accepted proper operation timing is immediately after signs of herniation and a midline shift which was defined as a more than equals 5-mm contralateral shift of the midline structures on the preoperative cranial ct scan on septum pellucidum level , compressed basal cisterns , or uncal herniation -- had to be present on cranial ct had been noted . recent studies in adult stroke group have shown that this procedure not only reduces mortality but also improves neurological functional outcome . this procedure may be life - saving if done early in cases of impending herniation , is associated with good outcomes in pediatric stroke patients9,11 ) . maybe if operation was performed earlier , it may minimize the vicious circle of brain swelling , increased icp , ischemia , and infarction12 ) . in our series , four toddlers and one pre - school girl with refractory high icp due to non - traumatic , ischemic and hemorrhagic stroke four patient received pcpcs score of 2 ( mild disability ) and only one received that of 3 ( moderate disability ) after 3 years later post operation . these results showed that decompressive craniectomy can not only play a rescuer therapeutic role but also guaranty acceptable clinical outcome in the treatment regimen in non - traumatic , refractory high icp in pediatric field . in some articles , decompressive aghakhani et al.2 ) report that decompressive craniectomy was performed in pediatric patient with malignant intracranial hypertension due to infectious encephalitis . in adult patient group , there had been not infrequently reported that malignant intracranial hypertension due to encephalitis treated with decompressive craniectomy . now , even in case of pediatric patient with infectious disease in central nerve system , decompressive craniectomy could be considered as life - saving - treatment options . gordon et al.7 ) report that pediatric stroke can affect not only patient itself , but also his or her whole family . over half of stroke survivors have suffer from decrease in quality of life and affects the entire family , and relates to both neurological deficits and psychosocial factors11 ) . hence , if all medical treatment is fail to drop the icp in non - traumatic pediatric acute stroke patient , without hesitation , a decompressive craniectomy could be regarded as next step treatment option . in this study , we describes the young pediatric patients have undergone decompressivehemicraniectomy for a non - traumatic acute hemispheric stroke . it also shows that decompressive hemicraniectomy can be lifesaving and can besafely performed in toddler and pre - school children . and young children can get a survival and relatively independence and family satisfaction.decompressive hemicraniectomy should be considered as an alternative therapy for patients with life - threatening brains welling refractory to medical management .

objectivelife - threatening hemispheric stroke is associated with a high mortality and morbidity . decompressive hemicraniectomy has been regarded as an effective treatment option for refractory intracranial hypertension . here , we reported the clinical course of 5 children with decompressive craniectomy and duroplasty after non - traumatic refractory intracranial hypertension.methodsfour toddlers and one preschool - girl were included in this study ; there were 3 boys and 2 girls with a mean age of 34.6 months ( range 17 - 80 ) . decompressive craniectomy including duroplasty was performed in cases of dilatation of pupil size after intensified standard medical therapy had proven insufficient . all children had a pediatric glasgow coma scale score < 8 at pre - operation state . the mean time - point of craniectomy after stroke attack was 12 hours ( range 4 - 19).resultsduring the long - term follow - up period ( mean 47.6 months ) , no children died . one year later , when we checked their glasgow outcome scale scores , only one toddler received a score of 4 ( moderate disability ) . but the others had good recoveries although they had minor physical or mental deficits . according to the pediatric cerebral performance category scale , 4 children received a score of 2 ( mild disability).conclusiondespite our small cases , we suggest that decompressive hemicraniectomy and duroplasty is an acceptable and life - saving treatment for refractory intracranial hypertension after unilateral hemispheric stroke in toddlers and preschool children .

this was a double - blind , randomized , crossover trial in accordance with the declaration of helsinki and good clinical practice . the primary objective was to assess albuminuria during different treatments compared with that with placebo ; secondary objectives were to assess effect on 24-h blood pressure , glomerular filtration rate ( gfr ) , biomarkers , and raas components . after informed consent , patients attended a screening visit comprising laboratory tests and evaluations of inclusion / exclusion criteria . slow - release furosemide in a fixed dose ( mean dose 109 mg/24 h , range 60360 mg/24 h ) was prescribed to prevent blood pressure elevation and fluid retention . patients used an electronic blood pressure device ( ua-779 ; a&d instruments , abingdon , u.k . ) to measure home blood pressure throughout the study . change in uaer ( percentage ) versus placebo during treatment with 300 mg aliskiren daily , 300 mg irbesartan daily , or the combination ( p < 0.001 vs. placebo for all treatments ) . from a list of prescreened candidates , 41 patients were screened for study participation ( supplementary fig . 1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-0168/dc1 ) . nine of these were screen failures mainly due to albuminuria levels below the randomization requirement . of the 10 randomly assigned patients who left the study before completion , 2 died , 1 was lost to follow - up , 4 had an adverse event that led to exclusion ( diarrhea , severe hypertension , recurrent urinary tract infection , and dizziness ) , and 3 withdrew consent . twenty - six patients had the primary end point , albuminuria assessed after randomization , and were included in the final analysis ; the remaining six patients dropped out shortly after random assignment and were not included in the final analysis . after washout , patients attended a randomization visit before 2 months of treatment with placebo , 300 mg aliskiren once daily , 300 mg irbesartan daily , or the combination of the two , in random order . patients with type 2 diabetes ( world health organization criteria ) aged 3080 years were eligible for randomization with baseline urinary albumin excretion rate ( uaer ) > 100 mg/24 h , hypertension ( baseline office blood pressure > 135/85 mmhg ) , and baseline gfr > 40 ml / min per 1.73 m. exclusion criteria included major cardiovascular disease ( within 6 months ) , heart failure ( new york heart association class ii rather , we used active washout : during the first 14 days of all treatments , every patient received 150 mg aliskiren daily to avoid risk of hypotension or a drastic increase in blood pressure in the switch from placebo to combination treatment or vice versa . during the last 3 days of each treatment , patients collected three consecutive 24-h urine samples for assessment of geometric mean uaer . at the last day of each treatment period , patients attended our clinic for assessment of gfr and mounting of standard takeda 24-h blood pressure devices ( tm2421 , version 7 ; a&d medical , tokyo , japan ) . measurements were performed every 15 min from 7 to 23 h ( daytime ) and every 30 min from 23 to 7 h ( nighttime ) . urinary albumin and creatinine concentrations were determined on a turbidimetric hitachi 912 system ( roche diagnostics , mannheim , germany ) . samples for prorenin , plasma renin activity ( pra ) , high - sensitivity pra ( hs - pra ) , immunoreactive plasma renin concentration ( ir - prc ) , angiotensinogen , ang i , ace activity , ang ii , and aldosterone levels were determined after 30 min of supine rest , and the plasma was frozen after centrifugation ( 80c ) . raas components and biomarkers were measured at baseline and at the end of treatment periods . biomarkers of inflammation , endothelial dysfunction , and cardiovascular risk were measured : high - sensitivity c - reactive protein ( hs - crp ) ( enzyme immunosorbent assay [ eia ] ; dako , glostrup , denmark ) ; serum soluble vascular adhesion molecule-1 and serum soluble intercellular adhesion molecule-1 ( eia ; diaclone , besanon , france ) ; plasma plasminogen activator inhibitor-1 ( hyphen biomed kit ; andresy , france ) ; serum nh2-terminal - probrain natriuretic peptide ( eia kit ; biomedica , wien , austria ) ; fibrinogen ( immunoturbidimetry ) ; and plasma asymmetrical dimethyl arginine ( high - performance liquid chromatography ) . total renin concentration , prorenin concentration , plasma angiotensinogen , pra , and biomarkers of inflammation and endothelial dysfunction were measured using methodology described previously ( 4 ) . ir - prc was measured with an immunoradiometric kit ( renin iii ; cisbio , gif - sur - yvette , france ) . hs - pra was measured by in - house radioimmunoassay of ang i formed during incubation of 25 l plasma and 50 pmol sheep angiotensinogen for 3 h at 37c in a total reaction volume of 100 l . the assay was double calibrated against ang i and the international reference preparation of renin , 68/356 , from nibsc ( hertfordshire , u.k . ) . plasma ang i and ang ii were measured using in - house radioimmunoassays and ethanol extraction of plasma samples . ace activity was determined using a commercial radioenzymatic assay ( ace direct ; bhlmann - laboratories , schnenbuch , switzerland ) . randomization was blinded to all investigators , and the study drugs were packed and labeled before delivery to the site . it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% . this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% . the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . a two - sided p value < 0.05 was considered significant the correlation between changes in albuminuria and changes in hs - pra or ang ii were assessed by a nonparametric spearman correlation coefficient . correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined . fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration gfr ) , and log - transformed levels were compared using a paired t test . statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) . it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% . this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% . the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . a two - sided p value < 0.05 was considered significant . the correlation between changes in albuminuria and changes in hs - pra or ang ii correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined . fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration gfr ) , and log - transformed levels were compared using a paired t test . statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) . baseline demographic data are shown in table 1 . primary and secondary objectives were met . during placebo treatment geometric mean uaer was 258 ( range 842,361 ) mg / day , mean sd 24-h blood pressure was 140/73 15/8 mmhg , daytime blood pressure was 144/76 17/9 , and nighttime blood pressure was 130/68 17/9 . gfr was 89 27 ml / min per 1.73 m and mean serum creatinine was 88 demographics of the 32 randomly assigned patients with type 2 diabetes , hypertension , and albuminuria and the 26 included in the final analysis data are means sd , n , or mean ( range ) . aliskiren treatment led to a significant reduction in albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) but not significantly different from irbesartan , lowering uaer by 58% ( 4270 ) ( p < 0.001 vs. placebo ) . combination treatment reduced albuminuria by 71% ( 5979 ) ( p < 0.001 ) compared with placebo , significantly more than with either monotherapy ( p < 0.001 and p = 0.028 ) . the relative difference between aliskiren and combination treatment was 31% . to adjust for treatment - induced changes in gfr and the potential influence on albuminuria reduction , we calculated fractional clearance , which was reduced by 46% versus placebo during aliskiren treatment ( p = 0.021 ) , by 56% versus placebo during irbesartan treatment ( p = 0.002 ) , and by 67% versus placebo during combination treatment ( p = 0.001 ) . systolic / diastolic 24-h blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) versus placebo . a correlation was found between change in albuminuria and change in 24-h diastolic blood pressure during all treatments ( p = 0.039 ) . seated office systolic / diastolic blood pressure was reduced 7/4 mmhg by aliskiren , 6/4 mmhg by irbesartan , and 12/8 mmhg by the combination , all statistically significant compared with placebo , except for diastolic blood pressure during irbesartan treatment . gfr was significantly reduced 4.6 ( 95% ci 8.80.3 ) ml / min per 1.73 m by aliskiren ( p = 0.037 ) , 8.0 ( 12.33.6 ) ml / min per 1.73 m by irbesartan ( p < 0.001 ) , and 11.7 ( 15.97.4 ) ml / min per 1.73 m by the combination ( p < 0.001 ) compared with placebo . aliskiren significantly reduced hs - pra , ang i , and ang ii by 87 , 75 , and 52% , respectively , compared with placebo ; irbesartan had the opposite effect ( table 2 ) . when combined , the activating effect of irbesartan was counteracted by aliskiren , reducing hs - pra , ang i , and ang ii by 88 , 78 , and 56% , respectively , compared with irbesartan monotherapy . whereas combination treatment caused a 1,068% increase in ir - prc versus a 279% increase during aliskiren monotherapy and a 178% increase during irbesartan monotherapy , hs - pra was reduced 47% compared with placebo after combination therapy . pra measured by a conventional method was affected similarly to hs - pra , although the changes were smaller ( table 2 ) . the renin - specific activity ( renin bioactivity / total renin mass ) was 3 and 4% after aliskiren and combination therapy compared with placebo , respectively , thereby confirming that 9697% of renin was aliskiren bound . during irbesartan monotherapy , changes in raas components and cardiovascular biomarkers versus placebo all treatments were administered once daily . adma , asymmetrical dimethyl arginine ; icam-1 , intracellular adhesion molecule-1 ; nt - probnp , nh2-terminal - probrain natriuretic peptide ; pai-1 , plasminogen activator inhibitor-1 ; vcam-1 , vascular adhesion molecule-1 ; vwf , von willebrand factor . a significant correlation between reduction in albuminuria and increase in ir - prc was observed for all active treatments combined ( r = 0.597 , p = 0.0001 ) . there was a significant correlation between changes in albuminuria and increase in ang ii during irbesartan treatment ( correlation coefficient 0.486 , p = 0.022 ) ; no significant correlations were observed in the other treatment groups . hs - crp was reduced 35% from the placebo level with aliskiren ( p = 0.047 ) and 35% with irbesartan ( p = 0.043 ) . other statistically significant changes from placebo levels were a 6% reduction in soluble intracellular adhesion molecule-1 ( p = 0.017 ) observed with the combination treatment and a 7% reduction in fibrinogen during aliskiren treatment ( p = 0.037 ) . no treatment led to significant changes from placebo levels in any of the other cardiovascular biomarkers ( table 2 ) . the most frequent adverse events were urinary tract infection ( four patients , one male ) , pneumonia ( three patients ) , and cough ( three patients ) , occurring during different treatments . anemia and hypomagnesemia were detected in two patients during the combination treatment . compared with each monotherapy , combination treatment showed an increase in plasma potassium by 0.2 mmol / l ( p = 0.036 ) . one patient dropped out during the placebo period after several systolic blood pressure readings > 180 mmhg . two patients died before the first measurement of albuminuria after the randomization and were not included in the final end point analysis . the first death was that of a 42-year - old obese man ( bmi 42 kg / m ) with a history of ischemic heart disease , myocardial infarction , and hypertension 4 years before study entry . the patient experienced sudden cardiac arrest , seemingly after a myocardial infarction during aliskiren treatment . the second death was that of a hypertensive , obese , 73-year - old man with diabetes duration of 16 years . the deaths were instantly reported to relevant authorities and were not suspected as being related to any of the drugs studied . subsequently , home blood pressure measurement frequency was increased from twice weekly to twice daily , and patients were instructed to contact the investigator by direct phone ( available around the clock ) , if any measurement was > 160/100 mmhg . extra measurements of sodium , potassium , and creatinine were introduced 3 weeks into each treatment period . in this exploratory study , we demonstrated that treatment with 300 mg aliskiren once daily was as efficient in reducing albuminuria as standard therapy with 300 mg irbesartan once daily . when we combined the two treatments at the same doses , the reduction in albuminuria was enhanced . the added antiproteinuric effect with combination treatment compared with aliskiren alone was 31% . given that the reductions in 24-h systolic blood pressure with aliskiren were unexpectedly small compared with those with placebo relative to 24-h diastolic blood pressure changes and were substantially lower than the office systolic blood pressure measurements in this study , we conducted a thorough review of potential flaws in data collection , storage , device calibration , reporting , and calculation . there was no evidence to indicate that the ambulatory data collection , storage , or reporting was flawed , and the unexpected results could be a chance occurrence . our study suggested that the combination of aliskiren and irbesartan had an additional raas blocking effect compared with monotherapy because a synergistic increase in ir - prc was observed with the combination , which was related to the antiproteinuric effect , whereas hs - pra was reduced 50% compared with the reduction with placebo . as opposed to the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study ( 5 ) , which showed an additional 20% albuminuria reduction after 24 weeks of treatment with aliskiren compared with placebo added to the maximal recommended dose of losartan and optimal antihypertensive therapy , this is the first study with a head - to - head comparison between aliskiren and irbesartan treatment . no other antihypertensive drugs except for furosemide were allowed in our study , thereby offering a clearer picture of the effect of the two compounds used , compared with the avoid study , in which aliskiren was combined with losartan and a mixture of other antihypertensive drugs . this study is different with regard to patient population , with a lower mean baseline uaer compared with that in the avoid study . in addition , we assessed the effect of renin inhibition on gfr , a measurement that is more precise than the estimated gfr used in the avoid study . raas blockade is believed to reduce proteinuria through several different mechanisms : the mean transcapillary hydraulic pressure difference , the glomerular surface area , and the size and charge selectivity of the glomerular filter . in diabetic nephropathy several of these variables are abnormal , and raas blockade has been demonstrated to normalize directly measured or estimated glomerular hydraulic pressure ( 79 ) , to reduce the shunt - like defects in the membrane , at least in part ( 10 ) , and to restore the charge - selectivity properties of the glomerular membrane ( 11 ) . aliskiren is thought to reduce albuminuria by the same mechanism as during treatment with ace inhibitors or arbs . recently , fisher et al . ( 12 ) have shown that aliskiren treatment increases renal plasma flow to a larger extent than the ace inhibitor captopril . the increase in renal plasma flow may be a response to angiotensin at1 receptor dependent reduction of the vascular tone in the efferent arteriole . reduced vascular tone in the efferent glomerular arteriole could be responsible for the decrease in intraglomerular pressure , leading to the reduction in albuminuria and gfr as demonstrated in our study . although the combination reduced gfr up to 12 ml / min ( 95% ci 15.97.4 ) , we interpreted this as an reversible hemodynamic change and not as an indication of nephrotoxicity ( 13 ) . in fact , it has been shown that an early hemodynamic reduction in gfr can translate into long - term renoprotection ( 13 ) . when the albuminuria reduction was adjusted during combination treatment for changes in gfr ( fractional clearance ) , it was 11% higher than during irbesartan treatment , a nonsignificant change that was possibly due to a small sample number . signs of more effective raas blockade were evident from the synergistic effect of combination treatment on ir - prc . this conclusion is based on the fact that renin release into plasma is proportional to the interruption of the permanent negative feedback loop of ang ii on renin secretion ( 14 ) . combining aliskiren with irbesartan provided a 12-fold increase in ir - prc , but still with a 50% reduction in hs - pra compared with changes during the placebo period . this renin rise could reflect a high degree of intrarenal raas blockade during combination treatment as compared with that for the monotherapies , as has been suggested in nondiabetic patients ( 15 ) . the reductions in albuminuria in our study were correlated with the rise in ir - prc , supporting the concept of increased intrarenal raas blockade underlying the additional effects observed during combination treatment . compared with other studies of dual raas blockade , the rise in ir - prc is higher in dual raas blockade using aliskiren than in dual raas blockade with an ace inhibitor and an arb ( 5,16,17 ) . such marked increases in renin during aliskiren treatment have been noted before ( 18 ) . apart from reflecting more complete ( intrarenal ) raas blockade , they may also be due to the detection of prorenin as renin ( 19 ) or a change in the renin half - life after its binding to aliskiren ( 20 ) . pra was measured both by a conventional method and by a new high - sensitivity assay ( hs - pra ) , which is independent of endogenous substrate variation ( 21 ) . because high pra levels confer the risk of cardiovascular disease ( 22 ) , it will be interesting to evaluate long - term effects of direct renin inhibition in the ongoing aliskiren trial in type 2 diabetes using cardio - renal endpoints ( altitude ) , providing data on hard cardiovascular and renal end points ( 23 ) . the antihypertensive effect of aliskiren was smaller than that found in previous larger studies ( 16 ) , although the office blood pressure reduction did not differ from that caused by irbesartan . the ongoing telmisartan alone and in combination with ramipril global endpoint trial ( ontarget ) investigators ( 24 ) concluded that in a cardiovascular risk population , dual raas blockade with the arb telmisartan and the ace inhibitor ramipril is equivalent in reducing cardiovascular events compared with either as monotherapy , although with more frequent adverse events , including renal adverse events . almost 3,000 of the participating 25,260 patients had microalbuminuria at baseline , and substudies of albuminuria effects are expected . several short - term studies using dual raas blockade in diabetic nephropathy have shown promising antiproteinuric effects , as reviewed by rossing ( 17 ) , but the largest study so far ( 25 ) did not show additional benefits of a combination of ramipril and irbesartan , compared with ramipril monotherapy , in terms of albuminuria reduction after 20 weeks . the size was , however , sufficient to demonstrate the likely beneficial effect of combination therapy with aliskiren and irbesartan , although we evaluated a surrogate end point . in addition , the discrepancy between 24 h and office blood pressure readings complicates interpretation of the results . studies evaluating mortality and morbidity are ongoing and will provide further information on dual raas blockade with aliskiren . in summary , we demonstrate an antiproteinuric effect of dual raas blockade with aliskiren and irbesartan in combination compared with either treatment alone in patients with type 2 diabetes , hypertension , and albuminuria . the synergistic effect on ir - prc illustrates a higher degree of intrarenal raas blockade during combination treatment .

objectivewe investigated whether the antiproteinuric effect of the direct renin inhibitor aliskiren is comparable to that of irbesartan and the effect of the combination.research design and methodsthis was a double - blind , randomized , crossover trial . after a 1-month washout period , 26 patients with type 2 diabetes , hypertension , and albuminuria ( > 100 mg / day ) were randomly assigned to four 2-month treatment periods in random order with placebo , 300 mg aliskiren once daily , 300 mg irbesartan once daily , or the combination using identical doses . patients received furosemide in a stable dose throughout the study . the primary end point was a change in albuminuria . secondary measures included change in 24-h blood pressure and glomerular filtration rate ( gfr).resultsplacebo geometric mean albuminuria was 258 mg / day ( range 842,361 ) , mean sd 24-h blood pressure was 140/73 15/8 mmhg , and gfr was 89 27 ml / min per 1.73 m2 . aliskiren treatment reduced albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) , not significantly different from the 58% ( 4279 ) reduction with irbesartan treatment ( p < 0.001 vs. placebo ) . combination treatment reduced albuminuria by 71% ( 5979 ) , more than either monotherapy ( p < 0.001 and p = 0.028 ) . fractional clearances of albumin were significantly reduced ( 46 , 56 , and 67% reduction vs. placebo ) . twenty - four - hour blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) . gfr was significantly reduced 4.6 ( 95% ci 0.38.8 ) ml / min per 1.73 m2 by aliskiren , 8.0 ( 3.612.3 ) ml / min per 1.73 m2 by irbesartan , and 11.7 ( 7.415.9 ) ml / min per 1.73 m2 by the combination.conclusionsthe combination of aliskiren and irbesartan is more antiproteinuric in type 2 diabetic patients with albuminuria than monotherapy .

nanobiomaterials are characterized by constituent particles and/or surface features less than 100 nm in at least one dimension . starting with photolithography and dry etching in the 1980 's to high - resolution electron beam lithography and other technologies in the 1990 's , nanotechnology allows for making surface structures for cell engineering and has led to an increasing application in healthcare over the last decades . nanolayers are used to enhance the surface biocompatibility of polymeric drug delivery systems , control the release of substances such as antibiotics or growth factors , act as gene - delivery vehicles , or serve as robust light emitters for cellular labeling and tracking [ semiconductor nanocrystals , quantum dots ( qds ) ] . nanotechnology is also applied to modify and improve the surface structure in orthopaedic implants to promote their osseous integration . however , there are also side effects of nano- and microparticles in vivo . micro- and nanoparticles released by friction of articulating partners from artificial joints are a major reason for aseptic implant loosening in orthopaedic surgery and may lead to severe peri - implant osteolysis ( particle disease ) . in addition , nanoparticles can induce or promote allergic or inflammatory reactions or influence hemolysis and blood coagulation [ 57 ] . although the cytocompatibility of a biomaterial is strongly influenced by its chemical composition , surface topography plays a crucial role for cell - surface interactions . material surface properties have been studied intensively , but still lack from reliable data about cytocompatibility . especially , the superordinate principles of cellular responses to surfaces with a defined topography are not well known and poorly understood . because many variables influence cellular interactions to surface structures , it is difficult to draw conclusions and formulate general principles for nano- and microstructured surfaces . this review summarizes recent data of effects by nano- and microstructured biomaterials and particles in vitro designed for orthopaedic application to get a solid framework outlining the critical interactions that govern the cytocompatibility . because biomaterials in orthopaedics are predominantly applied on bone , this review is focussed on the interactions of osteoblasts and bone - marrow - derived cells with structured biomaterials . osteoblasts and osteoclasts are mainly responsible for the osteointegration of nanostructured biomaterials in orthopaedics . osteoblasts derive from mesenchymal progenitor cells which are localized mainly in the bone marrow and periosteum . they are characterized by cuboidal and flat morphology ( diameter about 20 m ) , present a large amount of rough endoplasmatic reticlum and a large golgi apparatus , and are potent to produce osteoid , a collagen i rich matrix . in addition , these mononuclear cells are also responsible for osteoid calcification ( hydroxyapatite ) . typical marker proteins for osteoblasts are cbfa1/runx2 , osteocalcin , osteopontin , osteonectin , bone sialoprotein ( bsp ) , osteoprotegerin ( opg ) , collagen i , and alkaline phosphates ( alp ) . figure 1 gives a brief summary of the expression of several markers during osteoblast differentiation . osteocytes act in a paracrine and mechanosensory manner , and can activate osetoblasts and osteoclasts . the latter cell type derived from the hematopoietic line , has multiple nuclei and is responsible for bone resorption . its ruffled border is flanked by a sealing zone which facilitates local acidification and removal of bony matrix such as ca , h3po4 , and h2co3 by endocytosis . osteoclasts express high levels of tartrate - resistant acid phosphatase ( trap ) and cathepsin k. the interaction between osteoblasts and osteoclasts is complex . during differentiation , the ostoblast progenitors express receptor activator of nuclear factor ligand ( rankl ) and macrophage colony - stimulating factor ( m - csf ) which are strong stimuli for osteoclastogenesis . in contrast , osteoprotegerin ( opg ) is a potent inhibitor of osteoclasts . moreover , the interactions between osteoblasts and osteoclasts in vivo are regulated by several hormones and cytokines , including parathyroid hormone ( pth ) , calcitonin , and il-6 . it is generally accepted that the three - dimensional surface topography ( size , shape , surface texture ) is one of the most important parameters that influence cellular reactions [ 2 , 1119 ] . although many studies have investigated cellular reaction to different surface pattern , the significance of macro structure studies on bone cell behavior is questionable since in vivo adhesion structures ( e.g. , cell membranes , basement membranes ) are comprised of much smaller nanometer scale features [ 20 , 21 ] . the immature bone is characterized by an average inorganic grain size of 1050 nm whereas mature bone has an average inorganic grain size of 2050 nm ( 25 nm in diameter ) . considering these parameters , modern implants for bone application have been designed with a smooth surface at the nanometer level . it was surprising that some of these have induced the formation of peri - implant fibrous tissue and implant loosening in vivo , while other implants with a higher degree of roughness showed significant better osteoconductive properties [ 2325 ] . there are various methods to modify the degree of roughness as well as surface energy and topography in orthopaedic implants . typically applied techniques to enhance the degree of roughness and promote the osteointegrative properties of biometals ( e.g. , ti , cocrmo , ss ) are chemical etching or anodization and also sand - blasting , sputter - coating , and machine - tooling . the lack of knowledge in cellular reaction to nanostructered biomaterials is based to a great extent on the difficulty in varying surface chemistry and topography independently . moreover , the use of different cell lineages and culture conditions makes it difficult to compare results from different investigators [ 2631 ] ( table 1 ) . there is also a lack of consensus concerning the proper representation of implant surface topography . one major misunderstanding is the practice of defining a surface by its manufacturing process instead of concisely defining the topographic measurements [ 17 , 33 ] . considering these limitations for interpretation , the following review gives an overview of cellular reactions to surface structures of different orthopaedic biomaterials . the first step after exposure of any biomaterial to a biological environment results in the rapid adsorption of proteins to its surface . the composition , type , amount , and conformation of adsorbed proteins regulate the secondary phenomena such as cellular adherence and protein exchange [ 3537 ] and also following cellular reactions such as migration , proliferation , and differentiation . the potency for biomaterials to adsorb proteins is influenced by its physiochemical characteristics such as surface energy or hydrophobicity , and is also dependent on the local environment ( ph , concentration of ions , composition and functional groups of proteins , strength of solution , temperature ) ( vroman effect ) ( figures 2 , and 3 ) . for inorganic nanocrystals and microstructured surfaces there are at least two approaches to change their hydrophobic surfaces : a ligand exchange reaction can replace the original hydrophobic surface with bifunctional coupling molecules or an inorganic coating such as silica ( 1 ) or an encapsulation of nanocrystals in an amphiphile organic coating ( 2 ) . the first phase of protein adsorption onto a biomaterial 's surface is characterized by the attachment of small rapidly diffusing proteins , followed by a progressive replacement by larger proteins with a high affinity to the substrate . here , especially proteins with arg - gly - asp ( rgd ) containing sequences such as fibronectin or vitronectin act as cell receptors and have chemotactic or adhesive properties to bone cells . in addition , these rgd - peptides also have a strong effect on matrix maturation and biomineralization [ 4648 ] . after conditioning of a naked biomaterial by protein adsorption , cells attach rapidly on the protein - coated surface . besides the influence of proteins , the cellular attachement to a nanostructed surface is also influenced by its physiochemical properties , especially by the outer functional groups [ 30 , 50 , 51 ] . schweikl et al . showed on self - assembly monolayers that the osteoblast proliferation on hydrocarbon chains , terminated by ch3 , was as high as on amino groups ( nh2 ) and hydrophilic oxidized surfaces , but significantly lower on fluorocarbon ( cf3 ) groups . mller et al . showed that 3-aminopropyl triethoxysilane ( apts ) presents amine functional groups which allow for grafting rgd tripeptides and that the rgd - apts hybrid promotes cell adhesion , spreading , and cytoskeletal organization . here , the zetal potential ( differences in potentials between the surface of a tightly bounded layer and a diffuse layer ) and the interfacial tension ( wettability ) of a surface is crucial [ 54 , 55 ] . it was demonstrated for cpti surfaces that the contact angle ( ca ) , parameter for wettability , increases linearly with the average roughness when the angles were higher than 45 , but decreases linearly with roughness when the angle was less than 45 . recent data examining osteoblast response to controlled surface chemistries indicate that hydrophilic surfaces ( high number of polar components ) improve cell attachment and matrix synthesis and also the osteogenic potency compared to hydrophobic surfaces [ 5759 ] . compared ti alloys and cocr alloys towards protein absorptive properties and cell attachment with an osteoblast precursor cell line . they found no significant differences between ti alloys and cocr , but significantly greater cell adhesion rates for the ti implants and concluded that cell adhesion is a result of higher hydrophilicity of ti alloys . in contrast , other data showed that a low degree of wettability promotes protein adhesion and also cellular attachment to a biomaterial , and mller et al . found no direct correlation between the wettability of the material surface and the osteoblast attachment and proliferation rate . also qu et al . found no significant differences of cell attachement on various titanium surfaces with different degrees of wettabilities ( hydrophobic acid - etched , coarse - blasted large grit acid - etched , hydrophilic modified acid - etched , and modified coarse - blasted large grit , acid - etched ) on mg68 cells . heating ( oxygen / atm ) or peroxide treatment of biometals result in a thicker oxide layer and a more hydrophilic surface . . showed that heat - treated titanium surfaces changed the wettability ( more hydrophilic ) but does not significantly affect the fibronectin and albumin adsorption as well as the initial osteoblast precursor cell attachment in vitro . emphasized that the rate of protein correlates more with changes in chemical composition than with changes in wettability in metal surfaces . they showed that a preheating of ti6al4v specimen does not only lead to a thicker oxide layer but also results in an enrichment of v and al within the surface oxide . in contrast , post - treatment with butanol after preheating reduces the content of v , but not in al , and significantly increases the rate of fibronectin adsorption up to 2040% . compared to the cellular attachment phase , the following adhesion phase lasts longer and involves various proteins and molecules ( figure 2 ) . as a link between cell and biomaterial , the interactions of a surface topography and serum proteins are crucial for the cytocompatibility of a biomaterial . especially , the adsorption of adhesion proteins , such as fibronectin and vitronectin , from serum containing solutions and integrin - mediated signaling has been demonstrated to mediate cell adhesion and spreading . it has been shown that nanotube or nanoparticle surfaces created by anodization have promoted osteoblast adhesion up to three times compared to unanodized ti . these results were confirmed by the group of webster and other investigators [ 6871 ] who demonstrated that the initial attachment of osteoblasts onto the surface of biometals such as cpti , ti6al4v , and cocrmo is enhanced by submicron to nanometer consistent particles compared to metals composed of respective micron particles . one possible explanation of this phenomenon is the higher amount of particle binding sites for osteoblast adhesion at the surfaces of nanophase metals compared to micron particle size metals . the theory of enhanced protein and cell binding capacities by larger surface areas / roughness degrees was also confirmed for porous ha materials . another example of the significance of surface structures for protein binding and osteoblast attachment is the helical rosette nanotubes ( hrn ) which can build self - assembly surface structures . it was demonstrated that a significant change of hrn coverage by heating correlated with the protein - binding and osteoblast adhesion potency in titanium surfaces [ 73 , 74 ] . it is evident that not only the surface topography influences protein deposition and cell adherence but also proteins and cells modify the surface properties of a defined surface . based on a surface analysis of the different biometal specimen before and after cell cultivation , we showed previously that a cell attachment and/or protein precipitation increase the roughness in polished biomaterials ( steel , ti6al4v , and cocr ) . for porous coated cocr surfaces , we found only slight and no relevant changes in roughness whereas cell cultivation onto sandblasted ti6al4v lead to a strong decrease in specimen roughness . both , the increase in roughness after cell culturing in the different biometals and the decrease in roughness of sandblasted ti6al4v could be explained by the dense cellular growth and accumulation of debris in depth of the structured surfaces and/or protein deposition as shown by other investigators [ 75 , 76 ] . in addition , not only the amount but also the type of protein adsorption by a surface is crucial for cellular adherence and following reactions such as migration and differentiation . as an example , ti surfaces ( ra : 0.370.01 m ) adsorp fibronectin in higher concentrations compared to albumin , and fibronectin - coated ti surface promoted more osteoblast attachments in comparison to albumin - coated ti surfaces . these results correspond to the data of other authors who showed excellent osteoconductive properties after fibronectin adsorption onto a biomaterials ' surface [ 7880 ] . based on irm and tem analysis , the closest distance of cells to a surface ( glass ) was found to be approximately 10 nm [ 81 , 82 ] . historically , results from chicken fibroblasts have lead to a classification of three different types of separation . ( 1 ) focal contacts ( fc ) : approximately 1015 nm separation from the substrate under the peripheral regions of the leading lamellae ( appearing black in tem ) . fc act as an interface between intra and extracellular components and occur linearly beneath the associated cytoplasmic stress fibres [ 83 , 84 ] . they are tenacious adhesion sites that remain attached to the substratum even when cells are forcibly detached , indicating their function as anchorage structures . ( 2 ) close contacts : corresponding to approximately 30 nm separation ( broader grey areas in tem ) . ( 3 ) greater separation : corresponding to approximately 100140 nm ( white regions in tem ) . it is evident that not only fc appear soon after cellular attachment but also that ( -catenin - positive ) adherence junctions occur within 14 hours for grooved ti - based substrates . these observations underline the high significance of an early intercellular communication soon after adherence to a surface . the mechanisms of initial cellular adherence to a surface are different from long - term adherence as shown by a lack of statistical correlation between short - term adhesion ( strength of cell attachment and early adhesion ) and long - term adhesion ( strength of cell - matrix interface ) forces [ 14 , 15 , 86 ] . . showed that the cultivation time has an influence on the long - term adhesion in biometal surfaces according to td ( t ) = atb , a being independent of b ( td : time - dependend adhesion index , a : surface - dependent parameter , b : substrat - independent exponent , 0.5+/-0.03 ) . for polylactides ( plla ) , it was shown on oct-1 osteoblast - like cells that cell adhesion but not the proliferation could be enhanced by nanoscale and microscale roughness compared to smooth surfaces . in addition , there is evidence that fc show a dynamic behavior which allows for cellular migration and motility . linear plla fibres with length scales of 0.52 m , constructed by electrospinning , have shown cellular contact guidance and enhanced osteoblastic differentiation . here , cell morphology revealed that cells grown on fibres had smaller projected areas than those on planar surfaces . also other polymers such as plga have been shown to be effective in enhancing osteoblast differentiation in vitro . diener et al . demonstrated on mg-63 osteoblastic cells that fc adhesion was smaller on ti and ss than on collagen - coated glass coverslips and that all fc showed a mobility of focal adhesions . however , anselme et al . found higher adhesions on ti6al4v substrates than on noncollagen - covered glass samples , and emphasized that substrates with various surface compositions but with the same surface topography did not induce significant differences of adhesion . based on the knowledge of protein adsorption and its effects on cellular attachment and adherence , a selective surface coating of nanostructured surfaces with rgd or collagen proteins offer a promising solution to improve the number of osteoblasts adhered on artificial surfaces [ 53 , 95102 ] . imprinting surfaces technology with deposition of specific protein - recognition sites can help to promote osteoblastic growth and differentiation [ 103106 ] . protein - recognition can be based on a protein - ligand binding and/or electron donor - acceptor interactions or other types of binding forces . integrin 51 and 5 v3 subunits competitively bind to rgd - sites of fibronectin [ 107 , 108 ] . dependent on the surface topography and chemistry of the biomaterial , fibronectin undergoes changes in structure including modulation in functional activity and shift in integrin binding capacity . based on the data of self - assembled monolayers , it was shown that integrin subunits show selective binding capacities to different terminal groups . integrin 51 shows a strong affinity to oh and nh2 surfaces , whereas 51 and 5v3 bind also to cooh but show poor binding capacities on ch3 surfaces [ 109113 ] . furthermore , some data show that oh and nh2 surfaces can up - regulate osteoblast - specific gene expression but also matrix mineralization compared with cooh and ch3 functional groups [ 47 , 112 ] . cell migration and proliferation is the attachment following phase between the cell and the material surface . it is evident for designing nanostructured implants that cells use the nanotopography of a substrate for orientation and migration [ 117119 ] . although it is known that bone cells align along defined substrate morphologies ( contact guidance ) , the detailed relation between ordered nanotopography and cell behavior remains unknown in detail . for the first time , in 1964 it was shown that convex surfaces enhance cellular overlap , while grooves minimize cellular overlap . as pre - requisite to reach a defined cell colonization during directed tissue formation , structured nanophase surfaces lead to a predictable osteoblast orientation and migration on these surfaces [ 17 , 121 , 122 ] . interaction between the ecm and associated changes in the orientation of the cytoskeleton are crucial for cell metabolism of cells and morphology due to actin - myosin tension structures . anisotropic topographies ( e.g. , topographical grooves , chemically patterned stripes , or curved surfaces of a fibre ) are potent to exert morphological as well as physiochemical features on cells at the same time , indicative for the complex environmental influence on cells . focal contacts are important structures for cellular adherence onto a surface but may also delay migration and mobility of the cells . it was shown that bone - derived cells ( mg63 cells ) respond to a nanoscale roughness by a higher cell thickness and a delayed appearance of focal contacts . especially , nanoporous ti - oxide surfaces promote cellular spreading and induce numerous filopods and osteoblastic differentiation [ 124 , 125 ] . on electrochemically microstructured hexagonal pattern , mg63-cells go inside 30100 m but not in 10 m cavities . most authors report a parallel orientation of cells cultured on polished ( smooth ) surfaces [ 57 , 114 , 126 ] ( figure 4 ) . another method to not only enhance cellular adherence but also to promote osteoblastic differentiation and biomineralization of biometals is a surface anodization , for example , by -glycerophosphate sodium and calcium acetate [ 6671 ] . cellular adhesion via fc may strengthen the linkage between cell and ecm and also impair the ability to dynamically remodelling the ecm and influence the migration rate . for collagen - coated coverslips , focal adhesion of mg-63 osteoblastic cells moved with a speed of 60 nm / min , whereas the speed was reduced in ti and more in ss surfaces . another study on nb2o5-coated polished cpti samples showed that mc3t3-e1-osteoblast migration was fastest on smooth surfaces ( ra = 7 nm ) , whereas adhesion strength , spreading area , and collagen - i synthesis were promoted by intermediate roughness ( ra = 15 nm ) . however , it was surprising that higher degrees of roughness ( ra = 40 nm ) were rather peaked and reduced the speed of adhesion process in the same study . besides the surface properties of a biomaterial , the cellular migration rate is dependent on the cell type and its differentiation stage . a higher migration rate is associated with a lower level of osteoblast differentiation . cells with a low motility are characterized by a strong formation of fc while motile cells form less adhesive structures . it was found that mature osteoblasts spread out and form a greater number of fc when settled on smoother surfaces . although cellular spreading is higher on smoother surfaces , some data indicate that the alp - expression is higher for rough isotropic surfaces ( electro - erosion , acid - etching , sandblasted ) compared to smoother substrates ( machine tooling , polishing ) . considering recent publications , there is no or only week statistical significance that there is a difference between the initial number of adherent cells and following proliferation of cells cultured onto a biometal or ceramic nano-/microscale surface in vitro . however , some authors emphasize that the influence of functional chemical groups for cellular migration and proliferation are stronger than general surface properties such as wettability . especially a tio2-layer seems to promote cellular growth and proliferation on nanostructured biometals [ 128 , 129 ] . other examples for a promotion of cell - to - bone contact in vitro and also in vivo are machine - etched ti - surfaces ( e.g. , osteotite ) , defined sand - blasted implants [ 124 , 125 , 131 ] , and hydroxyapatite ( ha ) coatings , for example , by plama - spray techniques [ 132134 ] . recent studies investigating the response of adherent cells to nanography surfaces indicate that different cell phenotypes have different levels of sensitivities [ 117 , 135137 ] . here , osteoblasts react to features as low as to the 10 nm dimensions , which is comparable in size to a single collagen fibre . moreover , the qualitative and quantitative kinetics in gene and protein expression is strongly influenced by topography and physiochemistry of a defined surface . microporous ha surfaces seem to promote a high number of fc and increased levels of alp but short actin stress fibres compared to nonmicroporous ha surfaces [ 72 , 139 ] . there is also evidence that ti and ha surfaces can activate early intracellular signalling pathways as shown by expression of relevant molecules such as - and 1-integrin , fak , erk followed by c - jun and c - fos genes for proliferation and alp for differentiation [ 139 , 140 ] . however , hallgren et al . found no significant histomorphometric and biomechanical differences between nanopatterned and control implants . . showed that microfabricated discontinuous - edge surfaces ( des ) , repeated open square boxes with a depth of 10 m , alter osteoblast adherence and migration but enhance cell multilayering , matrix deposition and mineralization when compared to smooth controls . in contrast to our data , anselme et al . found higher proliferation rates on ss compared to ti6al4v . however , bigerelle et al . demonstrated that neither material composition nor surface roughness amplitude influence cell proliferation , whereas they found a very significant influence on manufacturing process and surface topography for long - term adherence and proliferation in vitro . our in vitro results confirm the well known osteogenic in vivo properties of ti implants , which may be based on surface factors observed on its outer tio2-layer [ 143146 ] . mller et al . demonstrated the ability of osteoblasts to grow into an open - porous ti implant ( metal foam ) and li et al . also demonstrated that mc3t3-e1 cells attach to and are able to divide well in the inner surface of a highly porous trabecular ti6al4v implant . some in vitro studies demonstrated an enhanced total protein and collagen production , as well as increased alp activity of osteoblasts cultured on nanoparticulate metals ( cpti , ti6al4v , and cocrmo ) indicating advantages for nanostructured surfaces for osteointegration [ 1 , 149 , 150 ] . based on the data of redey et al . , it can be concluded that the low attachment and collagen production rates are related to a low wettability of a nanosurface . nanotextured surfaces of ti surfaces prepared by chemical etching have upregulated the expression of bsp and op . as demonstrated by qu et al . , the expression of the bone - associated genes such as alp , oc , type - i - collagen , osteoprotegerin , and glyceraldehyde-3-phosphate - dehydrogenase is promoted by modsla ti surfaces . some data also suggest that fluoride - modified ti surfaces can stimulate osteoblastic differentiation compared to unmodified titanium surfaces [ 151 , 152 ] . . showed in their in vitro experiments that nanophase biometals induce significantly greater calcium and phosphorus deposition by osteoblasts and also allow for calcium and phosphorous precipitation from culture media without osteoblasts in contrast to microphase ti6al4v and cocrmo . furthermore , the authors found advantages in mineral precipitation without osteoblast for tial4v but no differences in dependency to the type of ti ( wrought , microphase , or nanophase ) . it was evident that the increased calcium and phosphorus mineral content correlated to greater amounts of underlying aluminium content on ti6al4v surfaces . although some data indicate that nanostructured ti alloys promote non - cell - mediated ca / po4-mineral deposition from culture media compared to cocrmo substrates , the greatest cell - dependend calcium and phosphorus mineral deposition occurred on nanophase cocrmo . it is evident that micropattern collagen films or scaffolds promote not only cellular adhesion but also allow for an osteoblastic differentiation and biocalcification in vitro [ 153155 ] . for ha- and dcpp - coated , ti surfaces the ca / p ratio influence the biomineralization rate in vitro . besides the osteoblast - promoting effects of defined substrates and surface topographies , some data also allocate an inflammatory response induced by nano- or microstructured biomaterials . it was shown in many studies that cell - biomaterial interactions can activate macrophages which results in the synthesis of proinflammatory agents such as tnf , ifn , il-1 and -6 , rankl and no [ 157159 ] . some data have shown proinflammatory effects of different biomaterials which increase with the degree of surface roughness . here , macrophage inflammatory protein-1 , tnf , monocyte chemoattractant protein-1 , and members of the interleukine and leukotriene family play a crucial role in biometal - induced inflammations [ 160164 ] . most studies report about an enhanced expression of pro - inflammatory cytokines and chemokines by cells attached to rougher surfaces . some data also indicate that anionic and neutral hydrophilic surfaces increase macrophage - monocyte apoptosis and reduce macrophage fusion to modulate inflammatory responses to implanted materials . however , adverse cellular effects seen with metallic implants may also be attributed to corrosion products or to the separation of metal ions ( fe , cr , ni ) which may have a major impact on cellular survival and differentiation [ 166168 ] . those studies which suggest that a cell - mediated metal ion release by biometals that did not affect the cell viability or proliferation are characterized by short cultivation periods or other conditions which limit the reliability of data [ 169171 ] . up to date , only few authors report about no significant influence of the cellular adherence and expression of osteoblast proteins by different biometals and surfaces such as alp expression [ 172 , 173 ] . in contrast to the great opportunity enhancing biocompatibility and osteogenic potency of surfaces applied on bone by nanotechnology , micro- and nanoscaled particles released by friction of artificial joints can induce severe inflammation and may lead to osteolysis and implant failure [ 174 , 175 ] ( figure 5 , table 2 ) . there is a wide range in particles size and morphology produced by simulators for artificial joints . particles released from metal - metal ( crcomo alloys ) are predominantly chromium oxide particles or cocrmo with varying ratios of co and cr . they show a round to oval morphology and also a substantial number of needle - shaped particles were found during the first circles . emphasize the importance of particle size as a critical factor in osteoblasts proliferation and viability in vitro . some data indicate that in contrast to ti - surfaces nano- and mircoparticles induce an inflammatory response although titanium is one of the biometals with the highest degree of cytocompatibility . as shown by miyanishi et al . , the release of vegf may play a crucial role in the pathogenesis of ti - induced osteolysis . some data indicate that phagocytosis of ti particles is not a precondition for an inflammatory response such as a release of tnf or il-6 in cultured macrophages . it is evident that a binding of the macrophage cd11b / cd18 ( macrophage mac-1 receptors / receptor of complement cr3bi , can also bind to icam-1 and icam-2 ) by integrin - specific antibodies also increased the release of tnf and il-6 in macrophages . this finding also suggests that the complement system plays a role in the pathogenesis of particle - induced inflammation , too . especially , uhmwpe particles with a size range of 0.11.0 m have been shown to be most reactive for macrophage activation and cytokine secretion in bone marrow cells [ 179 , 180 ] . however , not only the particle size but also the particle volume ( number ) is a critical factor for particle - mediated release of cytokines by macrophages . green et al . demonstrated for pe that the cell - particle ratios of 1 : 100 ( size 0.497.2 m ) and 1 : 10 ( size : 0.494.2 m ) induced significant stronger release of tnf and il-1 in macrophages . the authors conclude that especially particles in the phagocytosable size range of 0.310 m appear to be the most biologically active ones . the latter statement was also confirmed for silicon carbide ( sic ) particles and biometals such as cpti , ti6al4v and uhmwpe [ 184 , 185 ] . granchi et al . investigated the in vitro effects of al2o3 and uhmwpe particles in an osteoblast - osteoclast co - culture system . both particles did not affect either cell viability or tnf and gm - csf release , whereas il6 release was dependent on the particle concentration . uhmwpe particles increased the release of rankl from osteoblasts and induced large amounts of multinucleated trap - positive giant cells in an osteoblast - osteoclast co - culture system . also , carbon - based particles with low wear factors such as p25-cvd showed a high degree of cytocompatibility in vitro . howling et al . demonstrated on fibroblasts and monocytes that p25-cvd particles < 100 nm were significantly less cytotoxic to both cell types than cocr metal wear particles . while the classical water - suspendable nanoc60 nanocrystal is apparently cytotoxic to various cell lines , the closely related fully hydroxylated , c60(oh)24 , is nontoxic , thus producing no cellular response . also , functionalized single - walled carbon nanotubes are nontoxic to cells in culture [ 198200 ] . there is evidence that not only particle size and chemical content but also the concentration strongly influence cellular reactions in vitro . wilke et al . showed a positive correlation between the release of proinflammatory cytokines ( il-6 , -1 , and tnf ) and amounts of ti6al4v - particles ( 10 , 10 , 10 , and 10 particles / ml ) by human bone marrow cells over 2 weeks . some in vitro data also indicate that ti particles induce a stronger fibroblastic differentiation signal than uhmwpe in monocytes and other cells [ 182184 ] . . showed that particles of high - density polyethylene ( hdp ) and ti6al4v induced significantly more proinflammatory mediators ( il-1 , il-6 , tnf ) and bone resorption compared to al2o3 and zro2 in vivo . based on these data , it can be assumed that ceramics show a high degree of cytocompatibiltiy . for ha especially , particles with a size < 53 m inhibit cellular proliferation , especially in osteoblasts and lead to a decrease in tgf1 and a significant increase in pge2 and ldh concentration , but did not influence the tnf or alp titer in vitro . it could be concluded that larger ha particles may be compatible with bone cells while smaller - sized ha particles can both activate the osteoclasts and decrease the cell population of the osteoblasts in vitro . numerous variables influence the biocompatibility and osteogenic potency of nanostructured biomaterials in vitro and in vivo . besides the locotypical environment in vivo or in vitro , the surface structure and the composition of a biomaterial affects cellular attachment , adherence , proliferation and migration , and also differentiation and survival of defined cell types . here , information about typical parameters such as chemical composition , surface structure ( topography , geometry , roughness , particle size ) , surface energy , hydrophobicity , and the degree of solubility in aqueous solutions of a biomaterial will help to value and grade a defined implant concerning its osteblast promoting potency . considering recent publications , we could assume some general principles of cytocompatiblity and cell - surface interactions in nano- and microstructured surfaces . ( 1 ) wettability of a nanosurface influences significantly protein adsorption , which is a prerequisite of cellular adherence in serum containing solutions . ( 2 ) nanostructured surfaces enhance the surface area of biomaterials and promote cellular adherence . ( 3 ) the chemical outer functional groups of a nanosurface significantly influence cellular migration , proliferation , and differentiation but direct correlations between distinct parameters and cell functions are not entirely cleared . ( 4 ) the formation of fc underly a dynamic process and influence the motility and migration of cells . ( 5 ) a higher degree of differentiation is corresponding to a decreased cellular motility . ( 6 ) phagocytable particles with a size < 10 m induce the strongest cellular response with regard to releasing inflammatory cytokines . ( 7 ) although ti has a high degree of cytocompatibility in vitro , phagocytable ti particles can induce a fibroblastic differentiation .

cell - surface interactions play a crucial role for biomaterial application in orthopaedics . it is evident that not only the chemical composition of solid substances influence cellular adherence , migration , proliferation and differentiation but also the surface topography of a biomaterial . the progressive application of nanostructured surfaces in medicine has gained increasing interest to improve the cytocompatibility and osteointegration of orthopaedic implants . therefore , the understanding of cell - surface interactions is of major interest for these substances . in this review , we elucidate the principle mechanisms of nano- and microscale cell - surface interactions in vitro for different cell types onto typical orthopaedic biomaterials such as titanium ( ti ) , cobalt - chrome - molybdenum ( cocrmo ) alloys , stainless steel ( ss ) , as well as synthetic polymers ( uhmwpe , xlpe , peek , plla ) . in addition , effects of nano- and microscaled particles and their significance in orthopaedics were reviewed . the significance for the cytocompatibility of nanobiomaterials is discussed critically .

six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) . most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries . sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping . separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) . five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) . three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) . six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) . most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries . sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping . separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) . five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) . three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) .

legumes ( fabaceae or leguminosae ) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria , a process that takes place in a specialized structure known as the nodule . legumes belong to one of the two main groups of eurosids , the fabidae , which includes most species capable of endosymbiotic nitrogen fixation 1 . legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago ( mya ) . papilionoids are the largest clade , dating nearly to the origin of legumes and containing most cultivated species 2 . medicago truncatula ( mt ) is a long - established model for the study of legume biology . here we describe the draft sequence of the mt euchromatin based on a recently completed bac - assembly supplemented with illumina - shotgun sequence , together capturing ~94% of all mt genes . a whole - genome duplication ( wgd ) approximately 58 mya played a major role in shaping the mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation . subsequent to the wgd , the mt genome experienced higher levels of rearrangement than two other sequenced legumes , glycine max ( gm ) and lotus japonicus ( lj ) . mt is a close relative of alfalfa ( m. sativa ) , a widely cultivated crop with limited genomics tools and complex autotetraploid genetics . as such , the mt genome sequence provides significant opportunities to expand alfalfa s genomic toolbox .

surgical treatment of avascular necrosis ( avn ) of head of femur include core decompression , osteotomies , nonvascularized bone grafting , free vascularized fibular grafts , hip resurfacing , bipolar hip arthroplasty ( bha ) and total hip arthroplasty ( tha).123 tha is indicated in the young individual in avn with acetabular involvement ; however , its role is unclear in cases without acetabular involvement.4 bha was initially limited to be used in hip osteoarthritis , nonunions and acute neck femur fractures.56 bateman7 and giliberty8 were first to use bha in ficat stage 3 avn based on the hypothesis that acetabular floor retains a regenerative property , which regenerates bone in the subchondral region , if stimulation in the form of weight bearing is given through an accurately fitted cup and theorized that preferential motion at inner bearing will decrease the cartilage erosion . papers have been published with both satisfactory91011121314 and unsatisfactory results using bha.5151617 the main limitations sighted are migration of the outer cup ( secondary to the acetabular erosion ) and unpredictable pain relief.3514 incidence of groin pain varied from 11.5% to 42%181920 and incidence of acetabular erosion and protrusio is reported to range from 0% to 45%.1819 the activity of the patient and the duration of followup determine the erosion of acetabulum and appearance of symptoms.21 however , the main issue seems to be motion between the outer cup and the acetabulum , a nonconcentric acetabulum and particulate wear.16222324 to decrease this motion between the outer cup and the acetabulum a gentle reaming of the acetabulum with insertion of tight fitting acetabular cup can be done as suggested above by bateman and giliberty.78 this is thought to decrease the incidence of groin pain and the acetabular erosion and also revision secondary to these issues.2526 in this retrospective series , we have assessed the midterm outcome of bha in young adults ( age < 60 ) with ficat stages 3 and 4 avn . a retrospective study with a prospective followup was planned . 80 consecutive patients below 60 years of age ( 96 hips ) were operated by the senior author between 1995 and 2010 using bha with tight fitting cup technique . all patients were males with a mean age of 42 6.44 years ( range 30 - 59 years ) . out of a total of 96 hips , 76 were diagnosed as idiopathic avn , 8 were posttraumatic avn while 12 were post alcohol consumption avn . ficat 's classification system was used based on anteroposterior ( ap ) and lateral hip radiograph.26 clinically , the indication for surgery was pain with progressive inability to carry out daily activities . patients with radiographs showing no or minimal acetabular involvement were included to undergo the procedure , while cases with advanced osteoarthritis with ostephytosis or protrusio were excluded . surgeries were done in the lateral decubitus position using the posterolateral approach under epidural anesthesia . the hip joint capsule was excised if diseased . in our study , intraoperatively on gross examination , all the 96 hips ( ficat stage 3 and 4 ) were found to have mild to moderate acetabular involvement as per steinberg et al.2 the cartilage was graded grossly as grade 0 ( normal ) if no abnormalities were observed ; grade i ( mild degeneration ) if there was superficial fibrillation and slight irregularity of the surface ; grade ii ( moderate degeneration ) if there was moderate fibrillation , alteration in color and consistency and thinning of cartilage without complete erosion to bone . gentle superficial reaming of the acetabulum was done making the acetabulum concentric for a tight fitting bipolar cup [ figure 1a ] . the trial cups should fit snuggly enough so that when the hip is taken through a range of motion , no gross movement should be appreciated between the outer cup and the acetabulum . this assures that the prosthesis is tight fitting and movement between the outer cup and the acetabulum is avoided . during the process of making acetabulum concentric , sometimes subchondral bone was exposed . in these cases , pulse lavage of the prepared acetabulum was done to remove all debris before putting the bipolar cup . the size of the cup was equal to the size of the tight trial cup chosen . we did keep instrumentation for total hip replacement ( thr ) ready intraoperatively ; however , we did not have to do thr in any of our selected cases . ( a ) peroperative photograph showing concentric acetabulum after superficial gentle reaming with the large hand reamer . ( b and c ) x - ray taken in neutral and abduction showing movement taking place mostly at inner bearing self - centering cup with 28 mm inner head and uncemented stems were used in 60 hips ( zimmer , warsaw , usa , biomet , warsaw , usa ) . in the remaining 36 hips , normocentric hemispherical cups with 26 mm inner head and cemented stems were used ( inor , mumbai , india and rch , mumbai , india ) . the patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks . all patients were called for a final followup for clinical and radiological assessment . the clinical evaluation consisted of recording the details of thigh or groin pain , range of movements at the hip ( patient was asked to sit cross legged on the bed ) , limb length discrepancy and harris hip score ( hhs ) . the radiological evaluation comprised of looking for subsidence of the stem , recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup . ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [ figure 1b and c ] . the femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler 's line and center of the outer head , whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs . a subgroup analysis was also done , by dividing the cohort according to ficat 's class , to evaluate the effect of acetabular involvement on the outcome of the procedure . the patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks . all patients were called for a final followup for clinical and radiological assessment . the clinical evaluation consisted of recording the details of thigh or groin pain , range of movements at the hip ( patient was asked to sit cross legged on the bed ) , limb length discrepancy and harris hip score ( hhs ) . the radiological evaluation comprised of looking for subsidence of the stem , recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup . ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [ figure 1b and c ] . the femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler 's line and center of the outer head , whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs . a subgroup analysis was also done , by dividing the cohort according to ficat 's class , to evaluate the effect of acetabular involvement on the outcome of the procedure . the mean followup was 7.52 1.80 years ( range , 4 - 16 ) . the hhs improved from a preoperative value of 39.33 6.11 ( 30 - 54 ) to 89.12 6.68 ( 74 - 96 ) ( p = 0.0001 , paired t - test ) in the postoperative period . according to hhs grades , the final outcome was excellent in 52 , good in 28 and fair in 16 hips . hip and groin pain was reported in four hips ( 5% ) but this did not limit activity in any of the patient [ table 1 ] . 22 hips had flexion deformity of approximately 3 - 5 with a final range of flexion of 100.2 8.82 ( 85 - 116 ) . subsidence of the femoral component was seen in 8 hips ( < 5 mm ) ; however , these hips were asymptomatic . shortening of 1 cm was noted in 10 limbs , and 1.5 cm shortening was noted in 1 limb . there was no incidence of hip dislocation or dissociation of the bipolar cup and no case required revision . on ap neutral and abduction x - rays there was no appreciable movements noted between the outer cup and the acetabulum in any of the case . there was medial migration in 5 hips and proximal migration in 12 hips , but none more than 2 mm and all were asymptomatic . comparison of results with various studies on subgroup analysis between the ficat stages 3 and 4 [ table 2 ] it was noted that hips with ficat stage 3 were younger at the time of surgery and also had better final outcome in terms of range of motion . the final hhs score was however not statistically different in both groups indicating good results even in cases with acetabular involvement . none of the ficat stage 3 patients had any groin pain or femoral subsidence at the final followup while 4 cases of groin pain and 8 cases of femoral subsidence were seen in ficat stage 4 group . figures 2 to 5 show case series with final result of four patients in our series . comparison between the ficats 3 and 4 subgroups x - ray right hip joint anteroposterior view in a 35 year old male showing ( a ) preoperative radiograph with avascular necrosis right hip secondary to fracture neck femur . also showing implant in situ ( b ) 4 years postoperative radiograph of the same patient treated with bipolar hip arthroplasty . ( c ) 16 years followup radiograph of the same patient without any loosening or migration and with subchondral sclerosis ( a ) x - ray pelvis with both hip joints anteroposterior view in a 40 year old male showing avascular necrosis ficat stage iii in right hip ( post alcoholic ) ( b ) x - ray of right hip joint anteroposterior view of same patient showing implant in position at 9 years followup without any sign of erosion or migration . ( c ) clinical photograph of same patient showing cross legged sitting ( a ) x - ray pelvis with both hip joints anteroposterior view in a 65 year old male showing posttraumatic avascular necrosis ficat stage iv ( see non concentric acetabulum ) and implant in situ . ( b ) x - ray pelvis with both hip joints anteroposterior view of same patient at 9.5 years followup showing well fitting large cup . ( c and d ) clinical photographs showing range of motion x - ray pelvis with both hip joints anteroposterior view in a 45 year old man with bilateral avascular necrosis of femoral head ( a ) 6 months followup showing right side bipolar arthroplasty left side ficat stage iv avascular necrosis . ( b ) at 5 years followup showing well fixed implant with large cup well placed in acetabulum surface replacement is an option in younger people with avn , but has limited indications and is a demanding procedure with high cost.27 the results published by for surface replacement are not uniform and a longterm followup is lacking.272829 tha is the method of choice for the treatment of advanced avn of the femoral head;53031 however , its overuse may lead to increased wear and need for early revision in young adults.14 various studies have shown that functional utility of tha reduces to 80% at 10 years , 33% at 16 years , subsequently requiring a revision surgery . alternate bearing surfaces like metal on metal or ceramic on ceramic decrease the wear rate but have their own set of complications ( metallosis , squeaking)3233343536373839 and long term results are still awaited.40 when compared with bha , conflicting results have been reported . chan and shih30 have reported that there was no difference in the incidence of osteolysis , thigh or groin pain , dislocation rates and revision rates between bha and tha . they concluded that in young patients with ficat stage 3 avn , bha may be a useful alternative to tha . furthermore , bha is less demanding , blood loss is comparatively less5 and revision is easier as compared with the revision of tha as the acetabulum is still intact.41 lee et al.3 have reported 23% outer cup migration rate , 15% gluteal pain and 20% groin pain in bha . ito et al.15 have reported 42% radiological failures , 42% incidence of groin pain requiring a revision surgery in 25% hip undergoing bha . similar results were shown by cabanela14 and lachiewicz and desman17 with groin pain and acetabular erosion as the two most important reasons for poor results in bha . groin pain has been variably attributed to the preservation of diseased joint capsule , to irritation of the subchondral nerve endings of the acetabulum and to the acetabular erosion.1442 in our study , the diseased capsule was always excised to avoid capsular impingement . furthermore , as nerve endings in the posterior capsule supply the acetabulum , excising it blocks the nerve supply to the acetabulum and thus helps in relieving pain . poor fitting bipolar prostheses can lead to cartilage necrosis giving rise to groin pain and degeneration.3041 acetabular erosion has also been attributed to a fair amount of movement at the outer bearing due to a wide surface of the cup , which was greater than two third of a sphere.4243 reaming in bha technique creates a better fit that reduces movements between the outer cup and the acetabulum41 and ensures that on weight bearing , movements are transmitted to the inner bearing . in effect , this works like a low friction arthroplasty and results in reduced pain and damage to the acetabular bone stock.41 this also explains the low incidence of acetabular migration and low revision rate in our series . dudani et al.25 too used the same technique and reported 80% good result at average follow of 7.2 years . pellegrini et al.19 have reported a higher risk of revision with acetabular reaming while muraki et al.5 concluded that acetabular reaming increases the tendency of superomedial migration . the uniformly good results at the mid term seen in our series may be attributed to the technique and amount ( depth ) of reaming . pellegrini et al.19 reamed the acetabulum till a depth when the osseous floor showed punctuate bleeding . we have used a more conservative reaming without breaching the integrity of the subchondral bone , and this is probably why we have low incidence and magnitude of medial migration . on the contrary [ figure 2c ] the subchondral bone shows sclerosis , asserting the bateman hypothesis regarding the regenerate potential of acetabulum.44 in our series , ten patients showed this sclerosis . nagai et al.43 in 12 - 18 years old followup study of nonself centering bateman bipolar endoprosthesis for nontraumatic osteonecrosis of the femoral head concluded that the original bateman endoprosthesis was effective in delaying the need for tha for more than 10 years in ficat stage 3 avn of the femoral head . however , the sample size was very small , with 4 out of 12 patients having some groin pain and study limited to ficat stage iii avn only . in another study tsumura et al.45 found no difference in clinical results however incidence of migration in ficat 2 and 3 stage was less when compared with stage 4 . based on this they recommended bha only for ficat stages 2 and 3 . in our study , we have used bha for both stages 3 and 4 stages . although the final range of motion was better in ficat stage 3 group , the final hhs was similar in both the groups showing effectiveness of bha with tight fitting cup in both ficat 3 and 4 stages . thus , even in cases with acetabular involvement this technique can be used with good clinical results and can defer the need for tha . bipolar hip arthroplasty using tight fitting cup for avn hip has a low incidence of groin pain , acetabular erosion , and revision in midterm followup . this procedure can be used for treatment in young adults with ficat stages 3 and 4 avn of the femoral head to defer a definitive tha . further large series with long term followups , multicentric randomized studies and reproducibility of results will be needed to establish this method .

background : bipolar hip arthroplasty ( bha ) is one of the options for treatment of avascular necrosis ( avn ) of the femoral head . acetabular erosion and groin pain are the most allowing for gross motion between the common complications . we propose that these complications are secondary to improper acetabular preparation allowing for motion between the bha head and the acetabulum.materials and methods : the current study retrospectively evaluated patientsrecords from case files and also called them for clinical and radiological followup . 96 hips with avn of the femoral head treated with bha were included in the study . all patients were males with a mean age of 42 years ( range 30 - 59 years ) . in all cases , the acetabulum was gently reamed till it became uniformly concentric to achieve tight fitting trial cup . clinical followup using harris hip score ( hhs ) and radiological study for cup migration were done at followup.results:the mean followup was 7.52 years ( range 4 - 16 years ) . the hhs significantly improved from a preoperative value of 39.3 ( range , 54 - 30 ) to a postoperative value of 89.12 ( range 74 - 96 ) . according to hhs grades , the final outcome was excellent in 52 hips , good in 28 and fair in 16 hips . hip and groin pain was reported in four hips ( 5% ) , but did not limit activity . subsidence ( less than 5 mm ) of the femoral component was seen in 8 cases . subgroup analysis showed patients with ficat stage 3 having better range of motion , but similar hhs as compared to ficat stage 4 patients.conclusion:bipolar hip arthroplasty ( bha ) using tight fitting cup and acetabular reaming in avn hip has a low incidence of groin pain , acetabular erosion and revision in midterm followup . good outcome and mid term survival can be achieved irrespective of the ficat stage .

the success of valproic acid ( vpa , 2-propylpentanoic acid ) , a short branched chain fatty acid , for the therapy of absence seizures , partial seizures , tonic - clonic seizures , bipolar disorder , schizoaffective disorder , social phobias , neuropathic pain and for the prophylaxis and treatment of migraine headaches has spurred investigation for its use in the treatment of other conditions . treatments for encephalopathy secondary to hyperammonemia as a side - effect of vpa treatment remain empirical and restoration of possible hepatic carnitine deficiency remains controversial . while some authors have found a positive correlation between plasma ammonia level and vpa dose or concentration others have found none . her elevated vpa levels were associated with elevated ammonia levels as expected of a positive correlation . in contrast , later on in the patient 's clinical course her raised venous ammonia levels were found to be slowly down - trending in the presence of sub - therapeutic serum vpa . the present case report is about a 25-year - old hispanic female with a history of depression since age of 12 years , bipolar disorder , borderline personality disorder , purging type bulimia and history of suicide attempts at age 12 was admitted to the intensive care unit after attempted suicide by consuming an unknown number of vpa pills . this patient had recently started taking vpa ( depakote ) as a mood stabilizer at a dose of 250 mg at bed time about 3 days earlier , after a failed therapy . patient was taking paroxetine 20 mg for depression ; this was incremented to 40 mg at the time vpa was started . she reported that the change to vpa made her listless , lowered her mood , decreased her motivation , worsened her depression and caused suicidal thoughts . she overdoses herself on vpa which she took with alcohol . after ingesting the pills she notified her family who then brought her to the emergency room . on admission , her vitals were stable ( blood pressure : 118/72 mm hg ; temperature : 98.7f ; pulse : 82 beats / min ; oxygen saturation : 99% on room air ) . on initial examination she looked disheveled , although drowsy , was oriented to person , place and time . the complete blood count and comprehensive metabolic panel that was recorded initially and subsequently were unremarkable [ table 1 ] . subsequently , she was administered oral lactulose once and then 1400 mg of levocarnitine orally every 6 h for the next 2 days . her vpa levels which peaked after admission continued to trend down over the next 4 days , the ammonia levels also peaked subsequent to admission then showed an initial dip followed by a very slow down - trend [ figure 1a ] . the normalized log - linear plot of vpa serum concentration over time showed a down - trending linear first - order kinetics with a calculated t = 15.08 h [ figure 1b ] ( expected t for depakote : 9 - 16 h following oral dosing regimens of 250 - 1000 mg . the serum ammonia normalized log - linear plot in contrast showed two different decay kinetics : initial sharp down - trend for vpa levels greater than 90.9 g / ml but much slower downtrend for vpa levels less than 90.9 g / ml down into the subtherapeutic range of 8.5 g / ml . patient continued to complain of lethargy for the next 4 days which subsequently alleviated . at 14 days laboratory values of the patient at the time of admission ( a ) valproic acid ( vpa ) and ammonia levels as a function of time ( days ) . vertical axes : straight line ( left ) : vpa levels ; dashed line ( right ) : venous ammonia levels . ( b ) logarithm of normalized vpa and ammonia levels as a function of time in days . the normalization for each compound , for levels at each recorded time point ( x ) was to its recorded maximum level ( xmax ) . the normalized log - linear plot of vpa showed linear first - order decay kinetics with a calculated t = 15.08 h ( r = 0.994 ) recognition of symptoms of hyperammonemia remains a challenge especially in psychiatric patients . in a review of 11 case reports of symptomatic hyperammonemia in a psychiatric setting it was noted that acute hyperammonemic encephalopathy in psychiatric patients may present in various ways , sometimes with subtle clinical features . the previously reported spectrum of clinical features included : improvement in mood , worsening of psychiatric symptoms , mild complaints of fatigue and delirium . in our patient , hyperammonemia secondary to vpa overdose caused listlessness , lowering of mood , decreased motivation , worsening depression and suicidal ideation . we observed presence of vpa in serum caused increased venous ammonia levels , which eventually trended down to normal after its cessation . increasing vpa levels in serum was associated with increasing venous ammonia levels initially [ figure 1 ] . the peak concentration of ammonia noted was likely a decrement from the real peak based on the time to peak of depakote tablets being 4 h ( the patients initial vpa levels were measured at 2 h after she allegedly consumed the vpa while the next blood draw was 9 h later ) . we also made the following observations : ( 1 ) raised venous ammonia levels may remain symptomatic for a significant period of time beyond discontinuation of vpa . ( 2 ) existence of more than one concurrent mechanism with different decay kinetics in the metabolism of ammonia in presence of high or low concentrations of serum vpa . unmasking of an inherent urea cycle metabolic disorder could have amplified the differential decay rates of venous ammonia . the possibility of more than one mechanism causing hyperammonia secondary to vpa use is suggested by the different proposed mechanisms of vpa metabolites causing hyperammonemia , these include : ( 1 ) propionic acid inhibiting the urea cycle enzyme carbamoyl phosphate synthetase i ; ( 2 ) valproyl - coa or a closely related compound as a proximate inhibitor of mitochondrial ureagenesis ; ( 3 ) valproyl - carnitine ester causing a relative carnitine deficiency and ( 4 ) 2,4-dien - vpa inhibiting b - oxidation . although , we can not speculate on the mechanisms responsible for the different kinetics of decay of ammonia levels in the presence of down - trending vpa levels we can confidently state that non - elevated hepatic enzymes and hyperammonemia despite carnitine replacement indicated that hepatic damage and carnitine deficiency had very little role in causing the slowly down - trending elevated ammonia levels in the presence of subtherapeutic serum vpa levels . hyperammonemia and psychiatric disturbance secondary to valproate use in our case showed a score of 9 by a naranjo algorithm , indicating strong causality with the drug and placing it in causality category of definite . our assessment of a high adverse reaction probability scale was based on the fact that there are previous conclusive reports of hyperammonemia secondary to vpa use with similar psychiatric features as presented in this case report , hyperammonemia occurred only after vpa use and eventually trended down to normal levels with discontinuation of vpa , patients psychiatric disturbance correlated with hyperammonemia , the patient symptomatically improved with drug discontinuation , there are no alternative explanation of hyperammonemia and altered mood in this case , the serum levels of vpa were elevated with concentrations known to be toxic and that toxic effects observed were more severe with high dose and less severe when secondary hyperammonemia decreased . this case report suggests that hyperammonemia , which is known to occur in about 50% patients treated with vpa , may have more than one concurrent etiologic mechanism with different decay kinetics which are not related to hepatic damage or carnitine deficiency .

valproic acid ( vpa ) has successfully been used in the therapy of a number of conditions including absence seizures , partial seizures , tonic - clonic seizures , bipolar disorder , schizoaffective disorder , social phobias , neuropathic pain and migraine headaches . there is a high rise in number of cases of toxicity due to overdose of vpa . hyperammonemia , a common side - effect of vpa , is caused by several proposed etiologies , reported as having uncertain correlation with vpa dose or concentration . we present here a case of a 25-year - old female patient with a past history of psychiatric complaints , presented with elevated serum vpa levels associated with elevated venous ammonia levels subsequent to vpa overdose . later in the presence of sub - therapeutic serum vpa levels her venous ammonia levels remained raised and slowly down - trending . vpa levels and ammonia levels were found to be normal after 14 days . patient was treated with levocarnitine . her liver enzymes were never elevated . different decay kinetics of venous ammonia in presence of high and low concentrations of vpa indicates that vpa can cause symptomatic hyperammonemia via more than one concurrent etiological mechanism . in this patient , the mechanisms causing hyperammonia secondary to vpa use were not related to hepatic damage or carnitine deficiency .

intramammary infection ( mastitis ) is the most common reason for the use of antimicrobials in dairy cows . antimicrobials have been used to treat mastitis for more than fifty years , but consensus about the most efficient , safe , and economical treatment is still lacking . the concept of evidence - based medicine has been introduced to veterinary medicine and should apply also to treatment of mastitis . the impact on public health the aim of this article is to review current treatments of mastitis during lactation and seek for evidence - based , best practice treatment recommendations for bovine mastitis . penetration of substances into milk when administered parenterally or absorption and distribution throughout the udder when infused intramammarily ( imm ) depends on their pharmacokinetic characteristics . these are lipid solubility , degree of ionization , extent of binding to serum and udder proteins , and the type of vehicle . antimicrobial treatment of dairy cows creates residues into milk , and residue avoidance is an important aspect of mastitis treatment . the activity of macrolides , tetracyclines and trimethoprim - sulphonamides has been shown to be reduced in milk . selecting a substance with a low minimum inhibitory concentraton ( mic ) value for the target pathogen the antimicrobial should have bactericidal rather than bacteriostatic action , because phagocytosis is impaired in the mammary gland . however , activity in vitro does not guarantee efficacy in vivo when treating bovine mastitis . antimicrobial resistance amongst mastitis pathogens has not yet emerged as a clinically relevant issue , but geographical regions may differ in this respect . the biggest problem is the widespread resistance of staphylococci , particularly staphylococcus aureus , to penicillin g . cure rates for mastitis caused by penicillin - resistant strains of s. aureus seem to be inferior to those of mastitis due to penicillin - susceptible strains . it is not known if this is due to pharmacologic problems of the drugs used , or virulence factors possibly linked to -lactamase gene of the resistant isolates . using an in vitro -lactamase test for determining resistance to penicillin g of staphylococci before treatment coagulase - negative staphylococci tend to be more resistant than s. aureus and easily develop multiresistance . mastitis causing streptococci have remained susceptible to penicillin g , but emerging resistance to macrolides and lincosamides has been detected . antimicrobial susceptibility of coliform bacteria varies but normally is not a limiting factor for therapy . an important question regarding the treatment of mastitis is whether the antimicrobial should accumulate in the milk or in the udder tissue . the target site may depend on the causative agent : streptococci are known to remain in the milk compartment , but s. aureus penetrates udder tissue and causes deep infection ( table 1 ) . the advantages of this route are high concentrations of the substance achieved in the milk and low consumption of the antimicrobial as the drug is directly infused into the diseased quarter . for example , concentration of penicillin g in milk after imm administration is 100 - 1000 times as high as the concentration after systemic ( parenteral ) administration . a disadvantage of the imm administration is uneven distribution throughout the udder and the risk of infecting the quarter when infusing the product via the teat canal . efficacy of imm treatment varies according to the causative pathogen , with the best therapeutic response being shown for mastitis caused by streptococci , coagulase - negative staphylococci , and corynebacterium spp .. where to target antimicrobial therapy in clinical mastitis due to different pathogens the systemic route of administration has been suggested to be more efficient than imm for the treatment of clinical mastitis as antimicrobials theoretically have better penetration of the udder tissue by this route . however , it is difficult to attain and maintain therapeutic concentrations in milk or udder tissue following systemic administration . many commonly used broad - spectrum antimicrobials such as oxytetracycline , trimethoprim - sulphonamide and ceftiofur , it is difficult to produce and maintain therapeutic concentrations in the milk . macrolides would have ideal pharmacokinetics , but clinical studies have failed to demonstrate efficacy when used for the systemic treatment of clinical mastitis . in streptococcal mastitis , spiramycin and tylosin have shown reasonable efficacy . one additional problem for the bovine practitioner is that the recommended dosage for many antibiotic preparations for adult cattle may be too low when pharmacological aspects are considered , but residue studies have been carried out using the approved dosages . repeated intramuscular injections of large volumes of antibiotics can be irritating and can not be recommended from the animal welfare point of view . one substance used for systemic treatment is penicillin g , which as a weak acid penetrates poorly into the mammary gland , however , due to the very low mic values of susceptible organisms , therapeutic concentrations can be achieved in milk . penethamate is a more liphophilic penicillin g formulation and diffuses better than penicillin g procaine into milk . the efficacy of systemic treatment with penicillin g or penethamate has been shown in clinical trials . combinations of penicillin and aminoglycosides should not be used , as there is no scientific evidence demonstrating a better efficacy for the combination and aminoglycosides are known to produce long - lasting residues . the only type of mastitis where systemic treatment would be clearly advantageous may be mastitis caused by s. aureus . in severe mastitis due to coliform bacteria , parenteral administration of antimicrobials has been suggested to combat bacteraemia . the general benefit of antimicrobial treatment in coliform mastitis has been questioned , but systemic antimicrobial treatment is recommended in cases of severe escherichia coli mastitis with heavy bacterial growth in the udder . fluroquinolones and cefquinome have shown efficacy in experimental trials and ceftiofur in a clinical field trial . there is no evidence that administering bactericidal antimicrobials to cows with severe coliform mastitis causes the release of massive amounts of endotoxin . finally , the antimicrobial used for systemic treatment of mastitis must be approved for dairy cattle . for example , penicillin g procaine or fluoroquinolones are not approved for dairy cattle in the united states . treatment of mastitis should be targeted towards the causative bacteria whenever possible , but in acute situations , treatment is initiated based on herd data and personal experience . rapid or on - farm bacteriological diagnosis would facilitate the selection of the most appropriate antimicrobial . treatment protocols and drug selection for each farm should be made by veterinarians familiar with the farm . the use of on - farm written protocols for mastitis treatment can promote judicious use of antimicrobials . therapeutic response of the cows can be monitored using individual somatic cell count data if available , or using the california mastitis test , and with bacteriological samples in herds with contagious mastitis . in general , the use of narrow - spectrum antimicrobials is preferable ( table 2 ) . first choice antimicrobials for treating mastitis caused by streptococci and penicillin - susceptible staphylococci are -lactam antimicrobials , particularly penicillin g. broad - spectrum antimicrobials such as third or fourth generation cephalosporins should not be used as first alternatives for mastitis , as they may increase emergence of broad - spectrum -lactam resistance . systemic treatment is recommended in clinical mastitis due to s. aureus and in severe cases of coliform mastitis , preferably in combination with imm treatment . too short a duration of standard treatment is probably an important reason for poor cure rates in mastitis therapy . a longer treatment improves cure rates , and duration of treatment should generally be extended in mastitis caused by s. aureus and streptococcus uberis . clinical mastitis should be treated for at least three days ; this recommended treatment duration is longer than label treatments in many countries . all mastitis treatment should be evidence based i.e. , the efficacy of each product and treatment length should be demonstrated by scientific studies . treating subclinical mastitis with antimicrobials is generally not economical during lactation because of high treatment costs and poor efficacy . in a study with a large number of subclinical mastitis cases , the overall bacteriological cure rate for antimicrobial treatment was 75% and that for no treatment 68% . the marginal benefit applied for streptococcal mastitis only ; in mastitis due to s. aureus , antimicrobials were equal to no treatment . treatment of subclinical mastitis will not affect the incidence of mastitis in the herd unless other preventive measures are taken . studies on treating cows based on high somatic cell counts have generally shown that no effect on milk production has been achieved in herd problems caused by very contagious bacteria such as s. aureus or streptococcus agalactiae treatment of subclinical mastitis is advised .

treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account . in acute mastitis , where bacteriological diagnosis is not available , treatment should be initiated based on herd data and personal experience . rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial . treating subclinical mastitis with antimicrobials during lactation is seldom economical , because of high treatment costs and generally poor efficacy . all mastitis treatment should be evidence - based , i.e. , the efficacy of each product and treatment length should be demonstrated by scientific studies . use of on - farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials .

approximately 40 million people are infected with the human immunodeficiency virus ( hiv ) worldwide ( unaids 2006 ) . infections by this lentivirus from the family retroviridae are characterized by a long asymptomatic period after host immune defenses control the initial infection . during this subsequent chronic phase of infection , hiv slowly diminishes the host the virus replicates by hijacking the intracellular molecular machinery to transcribe viral rna , and eventually the productively infected cells die ( fauci 1988 ) . over time , the density of virions in the blood stream increases and the immune system functioning becomes progressively compromised , leaving hiv - infected individuals increasingly susceptible to opportunistic infections . being blood - borne , hiv is transmitted via contact with the blood of an infected individual : through transfusions , needle - sharing , sexual contact or from mother to child during childbirth or breast - feeding . initially there were concerns that hiv might be vector - borne ( e.g. , transmitted via mosquitoes ) but it has since become widely accepted that such transmission does not occur at any significant level ( bockarie and paru 1996 ) . this current belief stems both from epidemiological data and experimental studies that directly examine the potential for hiv transmission via arthropods ( lawrence 1987 ; lifson 1988 ; bockarie and paru 1996 ) . why is hiv not vector - borne ( throughout this article we use the term vector synonymously with arthropod ) ? the majority of medical scientists , when asked this question , will offer the following explanations ( bockarie and paru 1996 ) : ( i ) hiv concentrations in the blood are too low during human infection to permit vector transmission ; ( ii ) hiv is unable to survive long enough outside of humans ( or primates ) for vector transmission ; ( iii ) hiv is not able to replicate within arthropod vectors . each of these explanations has empirical support ( lifson 1988 ; bockarie and paru 1996 ) and thus all three are good explanations for the lack of vector transmission in hiv . if most arthropods pick up very little hiv when feeding on humans , and if the level of hiv in ( or on ) these vectors quickly decays , then no significant vector transmission is expected to occur . the above findings provide a satisfying proximate explanation for the lack of vector transmission in hiv . given the current characteristics of hiv , these three features of the virus make it unlikely that vector transmission will occur . in this article , we approach this question from an evolutionary perspective . therefore , rather than taking the characteristics of hiv as given , we are interested in understanding why hiv has evolved these particular features and not others instead . for example : ( i ) why has hiv not evolved a replication strategy that results in viral concentrations high enough to allow vector transmission ? ( ii ) why has hiv not evolved to be more durable and thus to survive longer outside of humans ? ( iii ) why has hiv not evolved the ability to replicate in arthropod vectors ? asking such questions might strike many readers as strange since much of evolutionary biology proceeds by asking why certain features of organisms are as we observe them rather than by asking why some features are absent . this is only natural , since it is difficult to decide which , among the infinite number of missing features , should be the focus of study . it is useful to view such questions as falling on a continuum , from questions about the lack of traits that are virtually non - existent in all taxa ( e.g. , why do dogs not have wheeled appendages ) to questions about the lack of traits that are common in some populations or species but not others ( e.g. , why do german shepherds not have curly hair ? ) . our contention is that , by asking such questions about the absence of some traits , we can gain deeper insight into biology . to quote philosopher arthur eddington ( 1927 ) , the contemplation in natural science of a wider domain than the actual leads to a far better understanding of the actual . . as in all science , however , not all questions are interesting , and this serves as our primary guide for focusing on some missing features and not others . in particular , we focus on the lack of vector transmission in hiv , because of the profound epidemiological significance of its absence . an arthropod - transmissible form of hiv would clearly exacerbate the already devastating impact of the disease , opening up routes of transmission to groups previously at low - risk ( e.g. , children ) . therefore , it is worth asking why , from an evolutionary standpoint , this has not occurred ( weiss 2001 ) . more to the point , if there are conditions under which hiv could have evolved vector transmission we would do well to understand these , not only from the standpoint of scientific curiosity , but also to prevent such an outcome in the future . in this article , we address the question of why hiv lacks vector transmission , both through a consideration of available empirical data and through the construction of mathematical models . although our results are necessarily speculative , we believe that they shed some light on the evolutionary biology of hiv , and on the evolutionary biology of blood - borne pathogens more generally . there are two broad reasons why a trait of interest ( in this case vector - transmission ) might not evolve . first , the necessary genetic variation for the trait might arise only very rarely ( if at all ) . for instance , the evolution of rna viruses , such as hiv , could be strongly constrained by the size of their genome ( holmes 2003 ) . second , the necessary form and strength of natural selection might not be present for the trait to evolve , at least over the timescale under consideration . thus , from an evolutionary standpoint , hiv is not vector - borne because either the necessary genetic variation for such transmission has never arisen , or the necessary selective factors that would make such variants increase in frequency over the relevant timescale have not occurred . our evolutionary explanation for why hiv is not vector - borne will be sought within these two possibilities . before beginning to consider these explanations there are two different processes that might result in vector - transmission , and that are often lumped under this single heading . this occurs when the arthropod acts solely as a means of physical transport of viral particles between hosts ( e.g. , having viral particles in and around mouthparts ) . this occurs when the virus replicates within the arthropod vector during the time period between feeding events . how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ? the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population . unfortunately , performing such an assay on all genotypes within the population would be next to impossible . furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low . nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened . below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission : a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods . such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) . for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al . 1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus . most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al . this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al . 1993 ) . it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) . other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods . in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al . at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al . the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al . 1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) . interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) . it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al . even if this does occur , however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible . ( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors . evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al . 1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al . furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al . in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al . 1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) . it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al . alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses . the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al . there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al . 1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous . thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ? on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle . this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) . alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv . mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) . the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate . although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans . the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics . this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 ) , v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) . note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this . also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) . the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people . we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) . the parameter is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age . our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al . 2007 ) . results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption . lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age . in particular , we will suppose that (a ) has the form where is the time during the infection at which aids develops . this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv . in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia . similarly , evidence suggests that sexual transmission rate , , also increases with viremia ( operskalski et al . 1997 lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of in untreated patients ( mellors et al . with these specifications , our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms . suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1 0 ) . also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission . assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively . equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate . more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold . empirical evidence in support of this functional form for hiv exists ( quinn et al . , a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig . 1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore , some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission . conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig . 1 ) . sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) . b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text . parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 , = 15 . these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs . for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1 p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig . the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) . solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years . the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b . first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed , we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years . also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act . more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred . unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al . 1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv . biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however , biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve . thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost . there are at least two biologically plausible mechanisms through which such a cost might arise . first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case , the cost stems from an evolutionary trade - off between these two transmission routes . the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8) could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain . there is , however , no a priori reason why this would be expected rather than the reverse . therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well . the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host . it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation . if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread . modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host . this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission . the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host . the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection . specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation . when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) . the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes . first , if rb ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand , if rb ra < ba , then vector transmission will never evolve . in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve . this will be true whenever the mutation rate of the virus is high ( i.e. , large ) and when the selective advantage of sexual transmission in terms of within - host competition is large . the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission . how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ? the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population . unfortunately , performing such an assay on all genotypes within the population would be next to impossible . furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low . nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened . below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission : a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods . such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) . for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al . 1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus . most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al . this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al . it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) . other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods . in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al . at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al . the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al . 1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) . interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) . it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al . 1983 ) , although this has been controversial ( kuno 2004 ) . even if this does occur , however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible . ( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors . evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al . 1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al . furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al . in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al . 1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) . it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al . alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses . the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al . there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al . 1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous . thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ? on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle . this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) . alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv . mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) . the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate . although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans . the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics . this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 ) , v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) . note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this . also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) . the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people . we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) . the parameter is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age . our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al . results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption . lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age . in particular , we will suppose that (a ) has the form where is the time during the infection at which aids develops . this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv . in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia . similarly , evidence suggests that sexual transmission rate , , also increases with viremia ( operskalski et al . lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of in untreated patients ( mellors et al . 1997 ; levy 1998 ; raffanti et al . , our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms . suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1 0 ) . also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission . assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively . equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate . more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold . empirical evidence in support of this functional form for hiv exists ( quinn et al . , a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig . 1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore , some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission . conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig . sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) . b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text . parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 , = 15 . these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs . for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1 p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig . the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) . solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years . the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b . first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed , we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years . also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act . more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred . unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al . 1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv . biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however , biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve . thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost . there are at least two biologically plausible mechanisms through which such a cost might arise . first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case , the cost stems from an evolutionary trade - off between these two transmission routes . the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8) could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain . there is , however , no a priori reason why this would be expected rather than the reverse . therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well . the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host . it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation . if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread . modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host . this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission . the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host . the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection . specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation . when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) . the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes . first , if rb ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand , in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve . this will be true whenever the mutation rate of the virus is high ( i.e. , large ) and when the selective advantage of sexual transmission in terms of within - host competition is large . the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission . experiments and epidemiological data have unequivocally demonstrated , however , that such vector transmission does not occur at any significant level , and various aspects of hiv biology have been implicated as proximate reasons ( bockarie and paru 1996 ) . these reasons do not offer an explanation for why vector transmission has not evolved , however , and as weiss ( 2001 ) points out , we ought to seriously consider whether such evolution might occur in the future ( for a summary , see table 1 ) . main conclusions of the study existing data suggest that the lack of mechanical vector transmission in hiv is not due to genetic constraints . while ecological constraints , such as number of vectors and biting rates , may limit vector transmission in certain areas , these constraints would likely not explain why hiv has not evolved this form of transmission in areas where vector - borne diseases ( e.g. malaria ) are endemic . rather , there must presumably be a reason why such transmission is selectively disadvantageous in hiv . effective mechanical vector transmission can be brought about only through the evolution of higher levels of viremia , and this also results in a more rapid onset of aids . this reduces the duration over which such strains can be transmitted from an infected human , more than is made up for by the occurrence of vector transmission . it also remains possible that insufficient time has elapsed for the evolution of vector transmission to occur , but our calculations suggest that this is not a very compelling possibility . on the other hand , existing data is largely consistent with the hypothesis that biological vector transmission has not evolved in hiv because of genetic constraints . at the same time , it is not possible to rule out a selective explanation instead . in particular , if there is a genetic trade - off between efficient replication in humans and replication in arthropod vectors , then a conflict between selection favoring effective replication within humans , and selection favoring arthropod transmission between humans can readily prevent biological transmission from evolving . this is particularly likely when the mutation rate of the virus is high , and thus might provide an explanation for the lack of biological vector transmission in all retroviruses . our analysis might also be extended to include other forms of transmission , for instance needle transmission ( see bruneau et al . several evolutionary consequences of this are possible depending both on the level of viremia required for such transmission to occur and the resulting transmission rate . for instance , if needle transmission can be achieved with a lower viremia than sexual transmission , and if this leads to a sufficiently high transmission rate , less virulent strains could be favored . conversely , if needle transmission requires a high viremia and leads to a sufficiently high transmission rate , more virulent strains would be favored . the only situation in which enhanced needle use could lead to the evolution of vector - borne transmission would be if effective needle transmission requires a viremia close to that of vector - borne transmission , while leading to a much higher transmission rate than vector - borne transmission . this way , strains with high viremia could be maintained in the population through needle transmission , and vector - borne transmission would then occur largely as a byproduct . our conclusions in this article are necessarily speculative , but such speculation is a necessary part of the initial stages of any research . one of our aims is to stimulate future research into the evolutionary biology of hiv transmission . from the results presented here , a number of different directions might be taken to ground these evolutionary ideas more firmly in empirical data . one possibility would be to examine more closely mechanical vector transmission in immunodeficiency viruses of other species . for example , more data on the epidemiological patterns of siv and its potential for alternative routes of transmission would be enormously useful . since siv is believed to be at the evolutionary ancestor of hiv , it would be very interesting to know if the longer evolutionary history it has had with its host has resulted in different transmission patterns . to the best of our knowledge , there are no empirical studies testing the potential for vector transmission of siv . another fruitful approach might be to conduct artificial selection experiments with hiv in arthropod tissue culture . experiments have demonstrated that hiv can not currently replicate significantly in arthropod cells , but no study to our knowledge has attempted to select for the evolution of hiv replication in such cells . one could even imagine doing such experiments with both mammalian and arthropod cell cultures to determine of the evolutionary trade - off postulated here actually occurs . ultimately , it will require innovative experiments and empirical studies to push the boundaries of our knowledge of hiv , and the use of evolutionary biology as a powerful tool for designing sensible intervention strategies . these kinds of studies are beginning to appear for other aspects of hiv biology ( e.g. , see mller et al . 2006 for an interesting evolutionary analysis of hiv virulence ) but more work on transmission biology would be useful . for example , if further empirical research validated the hypothesis presented here , that mechanical vector transmission has not evolved because of its associated mortality costs , this would then have important implications for how we attempt to stop the spread of hiv . strategies such as condom use , while beneficial for reducing the extent of sexual transmission , could thereby enhance the relative benefit of vector transmission , potentially resulting in the evolution of this new route of transmission . the use of antibiotics against bacterial pathogens has clearly brought home the fact that pathogens can readily evolve the means to circumvent our control measures , and there is no reason to expect things to be any different for other control measures . the use of antiviral medication , on the other hand , not only reduces sexual transmission but also the level of viremia , and therefore would presumably not move the selective balance more towards vector transmission . it is only by asking these kinds of questions , however , that we will have a chance at preventing adverse future outcomes . finally , the question of biological vector transmission addressed here is really a special case of the more general question of the evolution of a pathogen s host range . why do some pathogens have a relatively broad taxonomic host range while others are much more conservative ? this continues to be an interesting and important question in the evolutionary ecology of parasites ( poulin 2007 ) and there are some theoretical results predicting when we might expect different outcomes ( gandon 2004 ) . from the standpoint of human diseases this is also clearly an important question since emerging diseases , such as pandemic influenza , are precisely instances in which a pathogen evolves a different host range . a better understanding of the evolutionary biology of parasite host ranges is an important goal for future research .

abstractmany pathogens of humans are blood borne , including hiv , malaria , hepatitis b and c , west nile virus , dengue , and other viral hemorrhagic fevers . although several of these pathogens are transmitted by blood - feeding arthropods , hiv is not . a number of properties of hiv and its life cycle have been identified as proximate explanations for the absence of arthropod transmission , but little consideration has been given to why hiv has not evolved this form of transmission . we consider the empirical evidence for arthropod transmission , and suggest that mechanical transmission has not evolved in hiv because such strains would induce a faster onset of aids during infection , which would thereby limit their ability to spread . on the other hand , it is not as clear why biological transmission has not occurred . available data suggests that a lack of appropriate genetic variation in hiv is one explanation , but it is also possible that a conflict between natural selection occurring within and between infected individuals has prevented its evolution instead . we discuss the potential significance of these ideas , and argue that taking such an evolutionary perspective broadens our understanding of infectious diseases and the potential consequences of public health interventions .

in the normal kidney , immunohistochemical expression of contractile protein alpha - smooth muscle actin ( asma ) is limited to the vascular smooth muscle cells . in pathological conditions , the expression of asma the first experimental publication on this subject , by johnson et al , involved a series of clinical studies on the role of asma in diffuse renal diseases . in 1992 alpers and colleagues found higher expression of asma in proliferative types of glomerulonephritis gn . in iga nephropathy , asma expression in interstitium is related to damaged renal function and progression of disease . the expression of asma in interstitial myofibroblasts helps to differentiate between minimal change disease and the early phase of idiopathic membranous gn . in patients with iga nephropathy , higher mesangial expression of asma predicts a progressive decline in renal function . in focal segmental glomerulosclerosis , , interest in the asma is increasing due to the fact that the asma in cytoplasm of the podocytes is associated with nephrin and thus is indirectly involved in the glomerular filtration barrier . asma is analyzed in various types of gn together with markers such as metalloproteinase and nestin in investigation of tissue remodulation and epithelial - mesenchymal transition . to our best knowledge , this is the first morphometrical study of the correlation of asma expression in gn and renal function at the time of biopsy . this retrospective study analyzed 142 patients 82 adults and 60 children who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 . patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample , pathological diagnoses were classified into 2 categories proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy . absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults , absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children , bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey . serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low . ccr was determined from sc , the patient s height and proportionality constant using the schwartz method . the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits . asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) . paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient . tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) . hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam . positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer . computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) . a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using analysis software ( analysis soft imaging system , usa ) . all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium . area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) . their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys , nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc . this retrospective study analyzed 142 patients 82 adults and 60 children who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 . patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample , pathological diagnoses were classified into 2 categories proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy . absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults , absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children , bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey . serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low . ccr was determined from sc , the patient s height and proportionality constant using the schwartz method . the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits . asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) . paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient . tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) . hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam . positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer . computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) . a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using analysis software ( analysis soft imaging system , usa ) . all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium . area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) . their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys , nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc . the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) . the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) . the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults , expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively . in children , positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc , the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults , expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) . the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) . significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) . in children , absolute values of ccr negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults , there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) . children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) . no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age . the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) . the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) . the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults , expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively . in children , positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc , the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults , expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) . the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) . significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) . in children , absolute values of ccr negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults , there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) . children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) . no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age . studies of asma expression in renal parenchymal diseases began more than 2 decades ago , when it was noticed that a ) damaged glomerular mesangial cells change their immunophenotype expressing asma and b ) asma - positive myofibroblasts start interstitial fibrosis . a number of papers were published about asma expression in different types of human gn , its connection to proliferation markers and prognostic impact . most authors agree that asma expression in glomeruli was higher in proliferative gn and increases as the disease worsens , which makes asma expression a potential clinical prognostic factor . some authors determined the relationship between iga nephropathy , lupus nephritis and other types of gn with expression of asma . kim in 2001 found correlation between proliferation marker ki-67 and asma in different types of gn . in this study we focused on the connection of asma expression with impaired renal function in glomerulonephritis , measured at the time of biopsy . we did not find correlation with a special type of gn nor proliferation , possibly because of heterogeneity of the sample and the arbitrary method of categorization into proliferative and non - proliferative gn . increased expression of asma in the interstitium can be found in different diseases such as proliferative gn , diabetic nephropathy and renal transplant rejection . the asma has prognostic value due to the association with interstitial fibrosis , urine protein and sc [ 8,2528 ] . asma - positive myofibroblasts are responsible for the increased amount of extracellular matrix and renal fibrosis . this study also found higher expression of asma in interstitium of all patients with higher sc . greater expression of interstitial asma in children and adults was associated with higher grades of ccr and lower absolute values of ccr . we confirm that elevated sc or lower values of ccr are associated with higher expression of asma in interstitium . several studies have attempted to predict the development of progressive renal failure , measuring histomorphometric changes in the tubulointerstitial compartment . the best correlating parameters of interstitial fibrosis with renal function are the ratio of the accumulation of tgf - beta-1 and its antagonist decorin , interstitial expression of asma , and accumulation of interstitial collagen . according to jiang et al . , who analyzed asma production in peritoneal fibroblasts stimulated by tnf - beta-1 , hepatocyte growth factor could be important in blocking postoperative peritoneal adhesion . analyzed 133 biopsies of various human renal diseases , and found tubular epithelial cells with mesenchymal phenotype ( vimentin and asma positive ) whose numbers have been associated with the level of sc and degree of interstitial damage . analyzed 27 patients with iga nephropathy who had normal ccr at the time of biopsy , and found that expression of asma in mesangium predicts a progressive decline in renal function . in our study , a negative connection of asma expression in glomeruli and sc at the time of biopsy was found , statistically significant in adults and non - significant in children , but with the same trend . it is well known that asma expression in the mesangial cell indicates its change to myofibroblastic immunophenotype . our presumption is that asma - expressing mesangial cells are capable of higher contraction activity ; this leads to glomerular hyperfiltration as an early adaptive mechanism to glomerular damage . in a recent experimental study of membranous glomerulonephritis in rats , osteopontin and asma are expressed together in myofibroblasts in the crescents of damaged glomeruli . in children , the absolute values of ccr a possible explanation that children have a better ccr is due to phenotypic modulation of human mesangial cells to more contractile cells , which makes the glomerular function transitory normal or even increased . we analyzed association of expression of asma and hypertension , because it is known that glomerular mesangial cells during hypertensive damage express myofibroblast phenotype and expression of asma increases . the only significant association that we found was between expression of asma in interstitium and increased blood pressure in children . we did not find significant correlation between expression of asma in glomeruli nor interstitium with 24-hour urine protein in our patients . this result confirms previous findings that urine protein is primary linked to the changes in permeability of glomerular filtration barrier . nevertheless , according to some studies asma expression in interstitium is linked to the degree of proteinuria and sc , and higher interstitial asma expression is an indicator of poor prognosis . asma expression in interstitium is associated with sc and ccr and consecutively with decrease of renal function . asma expression in glomeruli is associated with lower values of sc in adults , as well as with normal or higher ccr in children . this correlation suggests that myofibroblastic phenotypic modulation of glomerular cells has a favorable impact on filtration . when calculated with precise computer - assisted quantitative morphometric technology , renal expression of asma the disadvantage of this study is its small sample size with different types of gn ; the next survey should include a much narrower cohort of patients with a specific type of gn , as well as better phenotypic identification of asma - positive glomerular cells .

summarybackgroundin a healthy kidney , contractile protein alpha - smooth muscle actin ( asma ) is immunohistochemically strongly expressed only in the blood vessels , while in pathological conditions it can be visualized in glomerular mesangial cells and interstitial myofibroblasts . the aim of this study was to explore the possible correlation between expression of asma in glomerulonephritis ( gn ) and indicators of renal function.material/methodswe analyzed expression of asma in percutaneous renal biopsy of 142 adult and pediatric patients with gn and its correlation with blood pressure , serum creatinine , creatinine clearance and 24-hour urine protein at the time of biopsy . immunoexpression of asma was analyzed quantitatively using computer - assisted morphometric analysis . relative surface of asma expression in all glomeruli and interstitium was calculated for each patient.resultsin adults and children , greater expression of asma in interstitium was associated with higher serum creatinine and reduced creatinine clearance . conversely , greater asma expression in glomeruli was associated with normal or decreased serum creatinine in adults and increased creatinine clearance in children . in children , correlation was found between high blood pressure and asma expression in interstitium.conclusionswe confirmed that interstitial expression of asma is associated with reduced renal function at time of biopsy . the connection of asma expression in glomeruli with lower serum creatinine and normal or increased creatinine clearance suggests a favorable role of this phenotypic change in glomerular filtration rate ; further investigation is needed .

chondroblastoma accounts for < 1% of all primary bone tumors . usually occurring in young adolescents , however , unusual sites of presentation such as patella , talocalcaneal , and temporomandibular joint has been reported . since last decade , fine needle aspiration cytology ( fnac ) has evolved as a simple , safe , and noninvasive preoperative tool in the diagnosis of bone tumors . it is necessary to differentiate chondroblastoma from other giant cell - rich lesions , as chondroblastoma exhibits predominantly benign behavior , though recurrences and metastasis are on record . we report cytomorphology in two cases of chondroblastoma confirmed by histopathology with a brief review of literature . a 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months . swelling was noted in the upper end of tibia measuring 3 cm 3 cm , and was tender on palpation . x ray showed typically lytic , centrally placed , sharply demarcated lesion with sclerotic border in the epiphysis . a 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration . local examination revealed swelling at the upper end of humerus measuring 4 cm 4 cm . fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case , ultrasound - guided aspiration was done because the lesion was deep - seated . smears were highly cellular , polyhedral cells arranged in sheets and small clusters , giving a pebble stone pattern appearance . the individual cells were monomorphic with a well - defined cell membrane , glassy cytoplasm at places showing microvacuolation . additionally seen was cartilaginous matrix , plenty of scattered osteoclast - type multinucleated giant cells , and bluish granular calcification surrounding individual cells chicken wire calcification [ figure 1a c ] . ( h and e , 40 ) ( b ) cytology smears showing polygonal cells with vacuolization in clusters , with calcification seen in the letilower corner . ( h and e , 100 ) ( c ) cytology smears showing chicken wire calcification . ( h and e , 100 ) ( d ) histopathology section showing cartilage , chicken wire calcification , and polygonal cells with vacuolization of cytoplasm . ( h and e , 40 ) at surgery , curettage and bone grafting was performed . grossly , the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage . histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm . a 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months . swelling was noted in the upper end of tibia measuring 3 cm 3 cm , and was tender on palpation . x ray showed typically lytic , centrally placed , sharply demarcated lesion with sclerotic border in the epiphysis . a 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration . local examination revealed swelling at the upper end of humerus measuring 4 cm 4 cm . fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case , ultrasound - guided aspiration was done because the lesion was deep - seated . smears were highly cellular , polyhedral cells arranged in sheets and small clusters , giving a pebble stone pattern appearance . the individual cells were monomorphic with a well - defined cell membrane , glassy cytoplasm at places showing microvacuolation . additionally seen was cartilaginous matrix , plenty of scattered osteoclast - type multinucleated giant cells , and bluish granular calcification surrounding individual cells chicken wire calcification [ figure 1a c ] . ( h and e , 40 ) ( b ) cytology smears showing polygonal cells with vacuolization in clusters , with calcification seen in the letilower corner . ( h and e , 100 ) ( c ) cytology smears showing chicken wire calcification . ( h and e , 100 ) ( d ) histopathology section showing cartilage , chicken wire calcification , and polygonal cells with vacuolization of cytoplasm . ( h and e , 40 ) at surgery , curettage and bone grafting was performed . grossly , the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage . histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm . unusual sites of location include pelvis , patella , talus , calcaneum , and temporomandibular joint . most of the chondroblastomas are benign in nature requiring simple curettage and bone grafting . however , recurrence is known to occur in 10 - 38% of the cases . metastases are unusual but occur in the lungs at the time of recurrence . as in most of the cases of bone tumors , this , compounded with nonconforming radiological features , leads to the tragic delay in diagnosis . though the age of the patient and location of the lesion in the given bone are key pieces of information , accurate and early diagnosis is a must for planning appropriate treatment . the past decade has witnessed the impressive use of fnac in the diagnosis of bone tumors . despite this , we have a limited case series describing fnac findings in chondroblastoma . fna aspirates of chondroblastoma show mononuclear cells arranged in a dispersed pattern like pebbles . these cells have well - defined cell margins , glassy cytoplasm , and nucleus with indentation and grooving . apart from associated giant cells and chondroid matrix , the chicken wire calcification that has been described in histopathology sections can be readily appreciated in fnac smears and can be a clue to diagnosis . however , errors in the fnac diagnosis of this rare tumor are known to occur , with the major pitfalls being its association with aneurysmal bone cyst ( abc ) leading to a nonrepresentative aspirate and the occurrence of a plethora of giant cell - rich lesions in the differentials . in one large series of 12 cases of chondroblastoma , the authors described the cytomorphology of this tumor in detail . the diagnosis was missed on fnac , among which , one was associated with abc and in another case an erroneous diagnosis of a giant cell tumor was made . the cause of misdiagnosis in one case was due to its association with abc , leading to nonrepresentative aspirate . in the second case , due to the location of the tumor in preauricular region , cartilaginous stroma aspirated , in a study of 110 cases of bone lesions attributed the missed diagnosis in two cases of chondroblastoma at fnac to inadequate sampling and misinterpretation as giant cell tumor . in giant cell tumor , the mononuclear cells are spindle - shaped and occur in clusters . in chondromyxoid fibroma , the smears are sparsely cellular and lack the mononuclear cell component . instead , stellate cells with pleomorphism are noted . aspirates in abc are usually hemorrhagic , scantily cellular , composed of osteoclast giant cells , histiocytes , osteoblasts , and spindle cells . in addition to chondroblastoma , abc can occur secondarily in giant cell tumor and osteosarcoma . this report reiterates the cytomorphologic findings in chondroblastoma and its differential diagnosis from other giant cell containing lesions of bone . the collective application of the knowledge of the cytomorphological features of this rare bone tumor would aid in accurate preoperative diagnosis even in the absence of typical clinical and radiologic presentations .

chondroblastoma is a rare , giant cell - rich , benign neoplasm of bone . since the past few decades fine needle aspiration cytology ( fnac ) has gained momentum in preoperative diagnosis of bone lesions . at cytology , other giant cell - rich tumors and tumorlike lesions such as aneurysmal bone cyst ( abc ) , giant cell tumor , and chondromyxoid fibroma fall under the differential diagnosis of chondroblastoma . due to the difference in the treatment protocol and prognosis , preoperative diagnosis is mandatory . we describe the cytomorphology in two cases of chondroblastoma diagnosed at fnac and confirmed by histopathology . at cytology , the presence of giant cells , chondroid matrix , mononuclear cells with nuclear indentation , and grooving along with glassy , vacuolated cytoplasm are characteristic of chondroblastoma . in addition to this , the presence of chicken wire calcification is a useful clue to the accurate diagnosis of chondroblastoma at fnac .

suicide is a grave public health issue with a global mortality rate of 16 per 100,000 individuals.1 suicide is one of the top three leading causes of death , particularly among individuals aged 1544 years , which is a productive age group.1 suicide is a complex event that is influenced by biological , cultural , psychological , and socioeconomic factors . more than 90% of suicide victims were diagnosed with psychiatric problems at the time of death , and approximately two thirds had been diagnosed with depression.2,3 depressed patients tend to consult physicians other than psychiatrists , and a variety of medical professionals , including general practitioners , the staff of emergency departments , and non - psychiatric nurses , are key interveners for preventing suicide . among medical staff members , negative attitudes toward suicide such as anger could affect nontherapeutic reactions to suicidal individuals.4 furthermore , a belief that suicide is a personal right was negatively related to suicide intervention skills.5 an accurate assessment of the attitudes toward suicide held by medical staff members is relevant . previous studies have shown that difficulties with parental bonding during childhood could be a predisposing factor for the onset of many psychiatric conditions , such as anxiety , depressive states , and maladjusted behaviors.68 parental bonding and premorbid personality traits play an important role in shaping the developmental trajectory of an individual , including his / her ability to adjust to stressful events . although few studies have assessed the relationship between parental bonding and mental conditions among medical staff members,9,10 no study has evaluated the association between parental bonding and attitudes toward suicide . the objective of this study was to investigate whether parental bonding is associated with attitudes toward suicide among medical college students in japan . students in their fifth year of medical school at hirosaki university , hirosaki , japan , participated in the study . the surveys were distributed to 226 medical students . of the distributed 226 surveys , 160 questionnaires ( 116 males and 44 females ) the demographic data ( age and sex ) were obtained from self - questionnaires and interviews . the data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and the subjects provided written informed consent before participating in the project . the parental bonding instrument ( pbi ) was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 . a total of 12 items measure the dimension of care , and 13 items measure protection , both on a likert scale . the care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference . the overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy . higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective . we employed the japanese version of the attitudes toward suicide questionnaire ( atts ) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes , including common occurrence , suicidal expression as mere threat , unjustified behavior , impulsiveness.12,13 each item , with the exception of items 10 and 28 , was scored on a five point scale from 1 ( strongly agree ) to 5 ( strongly disagree ) . items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 ( strongly disagree ) to 5 ( strongly agree ) . multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide . the data were analyzed using the pasw statistics software program for windows , version 18.0 . students in their fifth year of medical school at hirosaki university , hirosaki , japan , participated in the study . the surveys were distributed to 226 medical students . of the distributed 226 surveys , 160 questionnaires ( 116 males and 44 females ) the demographic data ( age and sex ) were obtained from self - questionnaires and interviews . the data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and the subjects provided written informed consent before participating in the project . the parental bonding instrument ( pbi ) was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 . a total of 12 items measure the dimension of care , and 13 items measure protection , both on a likert scale . the care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference . the overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy . higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective . we employed the japanese version of the attitudes toward suicide questionnaire ( atts ) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes , including common occurrence , suicidal expression as mere threat , unjustified behavior , impulsiveness.12,13 each item , with the exception of items 10 and 28 , was scored on a five point scale from 1 ( strongly agree ) to 5 ( strongly disagree ) . items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 ( strongly disagree ) to 5 ( strongly agree ) . multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide . the data were analyzed using the pasw statistics software program for windows , version 18.0 . table 1 presents the items included in the six factor atts with their descriptive data . paternal care was significantly associated with paternal protection , maternal care , maternal protection , and right to suicide . maternal care was significantly associated with maternal protection , right to suicide , and common occurrence . right to suicide was significantly associated with common occurrence , unjustified behavior , and preventability / readiness to help . table 3 shows the multiple regression analysis of the atts sub - scales with the pbi scores . after adjusting for age and sex , maternal care approached a statistically significant association with the right to suicide . under the same conditions , maternal care the present study assessed attitudes toward suicide and their associations with parental bonding among japanese medical college students . the majority of the participants in our study agreed that anyone could commit suicide ( 88.8% ) and that suicide is preventable ( 86.3% ) . in addition , the multiple regression analysis revealed that participants who reported a higher level of maternal care thought that suicide was a common occurrence and tended to think that people do not have the right to commit suicide . domino and takahashi used the suicide opinion questionnaire to investigate the differences in attitudes toward suicide between students in japan and those in the united states , and the results demonstrated that the scores for right to die and normality were higher in japanese students and that those for aggression were higher in american students.14 etzersdorfer et al compared the attitudes towards suicide held by medical students in madras with those in vienna , finding that medical students in madras rejected the right to commit suicide and assisted suicide to a greater degree than the students in vienna.15 in a swedish study to assess whether attitudes differ between students at the beginning and end of their studies , students in the final year of medical school more frequently considered suicide to be an expression of psychiatric disease and thought that people attempting to commit suicide were not responsible for their actions.16 in addition , a study from japan revealed that sympathetic comments increased with the amount of years in school among students while critical comments decreased.17 in japan , mentality on suicide might be more culturally visible and acceptable than other countries . in past years , suicide was regarded as an honorable solution to personal guilt and failure . regarding occupational differences in attitudes toward suicide , onc et al found that medical students and general practitioners held the most permissive attitudes in turkey.18 in addition , they showed that the attitude scores of general practitioners regarding their preparedness to prevent suicide were lower compared to those of other groups . in japan , kodaka et al reported attitudes toward suicide among pharmacists using the atts.13 more than half of pharmacists reported that people understand the wishes of others to commit suicide , and 16.8% of the participants thought that people who mention suicide do not actually take their own lives . a 10 year follow - up study of norwegian physicians ( n=631 ) revealed that a low level of maternal care predicted severe depressive symptoms and partially mediated low self - esteem.9 lung et al assessed the psychological effects on 127 health care workers who had cared for suspected severe acute respiratory syndrome ( sars ) patients . early maternal attachment and neuroticism were found to heavily affect mental health during life - threatening stress.10 distinct patterns of parental bonding may contribute to the development of different dysfunctional schemas which may influence attitudes toward suicide.19 there are several limitations in this study . first , we used pbi to assess the child rearing behavior experienced by our participants . although pbi has shown basic reliability for long periods,20 we could not completely rule out possible influences of the current mood state or recall bias in individuals because pbi is an instrument for measuring recalled parental behaviors . second , certain parameters were not assessed in this study personality traits , a history of psychiatric disorders , suicidal behavior , and current mood state could affect the attitude towards suicidal behavior . third , because the participants in our study are medical students , we can not generalize these results to general practitioners . our study indicated that the majority of participants seriously believe that anyone could commit suicide . participants who have experienced a higher level of maternal care thought that suicide occurs commonly and tended to think that people do not have the right to commit suicide . although parental bonding predisposes these attitudes toward suicide , educational programs could change caretaking practices.21 to achieve more positive attitudes towards suicidal behavior , promoting the best practices for suicide prevention is needed for medical students .

backgroundsuicide is a grave public health issue that is responsible for a high mortality rate among individuals aged 1544 years . attitudes toward suicide among medical staff members have been associated with appropriate therapeutic responses to suicidal individuals . the aim of this study was to examine the effects of parental rearing on attitudes toward suicide among japanese medical college students.methodswe examined the association between parental bonding and attitudes toward suicide in 160 medical college students in japan . the parental bonding instrument was used to assess the attitudes and behaviors of parents . the attitudes toward suicide were evaluated using the japanese version of the attitudes toward suicide questionnaire.resultsthe mean age of the subjects was 25.24.0 years old . the majority of the participants in our study agreed that anyone could commit suicide ( 88.8% ) and that suicide is preventable ( 86.3% ) . after adjusting for age and sex , multivariate regression analysis revealed that maternal care approached a statistically significant association with the right to suicide attitude . under the same conditions , maternal care was shown to be significantly associated with the common occurrence attitude . no other significant relationships were observed between parental bonding and attitudes toward suicide.conclusionthis study suggests that a higher level of maternal care ensures that children think that suicide occurs less commonly . the promotion of best practices for suicide prevention among medical students is needed . child rearing support might be associated with suicide prevention .

copd is characterized by persistent airway obstruction , usually of a progressive nature and associated with an enhanced chronic inflammatory response in airways to noxious particles or gases.1 copd is associated with increased mortality and reduced life expectancy.2 of the 3.8 million mortalities caused by chronic respiratory system diseases in 2010 , 2.9 million were by copd.3 the first consensus report for the diagnosis , management , and prevention of copd was published in 2001 by the global initiative for chronic obstructive lung disease ( gold ) committee , and since then it has been updated several times . the major objectives of gold are to increase the awareness of copd and to help the millions of people who suffer from this disease and die prematurely from it or its complications by increasing the standards of care . the low adherence to guideline treatment recommendations has been reported in several studies.46 under or overprescription cause the inappropriate treatment of the disease.4,7 undertreatment of copd has also been reported in turkey.8 the bold study in adana , turkey showed that only 12.3% of the adults with copd used medication for their diseases in 2004.9 the chronic diseases and risk factors in turkey survey reported that 46.1% of the patients clinically diagnosed with copd used regular medication in 2011.10 to date , there has been no study on adherence to the gold guidelines for the treatment of copd among pulmonary physicians in turkey . at the time of the study , the patients with copd were still treated according to the gold 2010 guideline recommendations . therefore , stages and treatments in this study are also classified in accordance with the gold 2010.1 the aim of this study is to investigate the adherence rates to gold 2010 recommendations for stable copd treatment by pulmonary physicians with a national survey in outpatient clinics of turkish pulmonary units from different regions of the country . this multi - center , cross - sectional study was conducted with the participation of the pulmonary outpatient clinics of eleven centers ( university hospitals , research and training hospitals , state hospitals , and private hospitals ) from eight cities ( adana , ankara , diyarbakir , erzurum , istanbul , izmir , kocaeli , and samsun ) across turkey . sample size estimation has been calculated in order to have the representation of all gold stages , and sequential recruitment of 100 patients was anticipated per center . the study was approved by the institutional ethics committee of ankara university school of medicine ( november 21 , 2010 ; # 24,596 ) prior to its initiation and conducted in accordance with the latest version of declaration of helsinki and international conference of harmonization / good clinical practice guideline . the inclusion criteria included doctor s diagnosis of copd , age 40 years , available pulmonary function test ( pft ) results from within the past 2 years and copd treatment data from any time within 6 months of the test , and formal written consent . patients with a concomitant chronic disease that leads to obstructive ( ie , asthma ) or restrictive ( ie , disseminated bronchiectasis ) changes on pft , with pft results discordant to gold diagnostic criteria ( symptoms of copd , and postbronchodilator spirometric evaluation confirming forced expiratory volume in 1 second ( fev1)/forced vital capacity [ fvc ] < 70% , exclusion of other obstructive lung diseases),1 and those with acute copd exacerbations were excluded from the study analysis . in nonsmoker patients , biomass exposure is considered as one of the major risk factors especially in rural areas , and they were not excluded from the study if they meet the gold spirometric diagnostic criteria . patient recruitment has been performed according to the study inclusion and exclusion criteria in each study center . patient data were collected from hospital records on standardized forms and following parameters were recorded : any chronic diseases accompanying copd , age at the time of copd diagnosis , spirometry results ( fev1 and fev1/fvc ) and copd treatment recommended within 6 months of this result , and demographic information such as sex , age , location of residence , occupation , level of education , and health insurance status . overall , 1,125 patients have been recruited , and 719 patients meeting the gold diagnostic criteria ( fev1/fvc < 70% ) were included in this analysis . appropriateness of treatment was established in accordance with the 2010 gold guideline recommendations below : in stage i , add short - acting bronchodilator when needed.in stage ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama]).in stages iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 in stage i , add short - acting bronchodilator when needed . in stage ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama ] ) . in stages iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 all other treatment approaches ( under- or overtreatment ) were evaluated as inappropriate treatment . data are reported as mean and sd ; median , minimum , and maximum ; or frequency and percentage . monte carlo simulation was used for multigroup comparisons where chi - square test assumptions were not fulfilled . bonferroni - corrected mann whitney u - test was used for subgroup comparisons within numeric variables . seven - hundred and nineteen patients ( 614 males , 105 females ) with mean sd age of 62.99.7 years were included . six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% . of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv . mean sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv . there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) . of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity . the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) . overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group . appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss . the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii , adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) . of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations . the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) . of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics . the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss . the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) . of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics . the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated . inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) . the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) . only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients , guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment . while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd . the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively . the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 . of all the patients , 614 ( 89% ) received treatment containing ics or ics alone . the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage iv . of the 25 ( 3.5% ) patients who were not on any medication , one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy . of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy . long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients . seven - hundred and nineteen patients ( 614 males , 105 females ) with mean sd age of 62.99.7 years were included . six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% . of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv . mean sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv . there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) . of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity . the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) . overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group . appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss . the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii , adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) . of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations . the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) . of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics . the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss . the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) . of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics . the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated . inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) . the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) . only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients , guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment . while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd . the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively . the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 . of all the patients , 614 ( 89% ) received treatment containing ics or ics alone . the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage iv . of the 25 ( 3.5% ) patients who were not on any medication , one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy . of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy . long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients . this is the first large - scale real - life study in turkey that reflects the clinical practice of pulmonary specialists through an observational analysis of their adherence to treatment recommendations of gold 2010 guideline . the survey was carried out in eleven pulmonary outpatient clinics spread over the national territory . our findings show that only 59.5% of overall patients were treated in accordance with the guideline recommendations . while inappropriate treatment regimens were more frequently prescribed for stages i ( 93.8% ) and ii ( 85.3% ) , overtreatment in early stages and high rate of ics administration ( 89% ) in all stages of disease resulted in improper therapeutic approach according to gold 2010 recommendations . in more severe disease stages , guideline - adherent therapeutic approaches were detected by wide use of ics addition to one or more long - acting bronchodilators . most of the provider s guideline adherence studies have reported the current practices of general practitioners . a few recent studies have reported similar rates of adherence to guideline recommendations for copd patients ( 37.9%54.7% ) between pulmonologists in other countries.4,5 stage i patients were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss , while guidelines recommend short - acting bronchodilators as initial therapy . in a study conducted among the pulmonary specialists in italy , corrado and rossi reported that 87.7% of the stage i patients received regular treatment containing long - acting bronchodilators , ics alone , or other treatment combinations of ics plus long - acting bronchodilators.4 in a retrospective database study in the united states , fitch et al reported that only 1% of the stage i patients received ics added to laba and lama , while 6% received ics plus single long - acting bronchodilators , and 76% did not receive any medication in stage i.11 shariff et al reported that 7.7% of the patients in stage i were overtreated and received inappropriate treatment according to the guidelines , in another study on guideline adherence among practitioners and pulmonary specialists.5 in our study , only 14.7% of the patients in stage ii received appropriate therapy , and this was mainly due to overtreatment which was combined long - acting bronchodilators and ics in 77.3% of the patients . other reasons for nonadherent therapeutic approaches were single use of ics in 3.4% of the patients and lack of treatment in 3.8% of the patients . other studies found 26%49.2% adherence to gold recommendations in this stage . overtreatment was 67.3% in one study,4 and lack of copd treatment has been reported in 54% of the patients in another report for stage ii.11 severe and very severe copd patients were treated by the combined use of one or more long - acting bronchodilators and ics which was compliant to gold recommendations . this finding can be explained by the features of this group of patients who are more symptomatic and have more frequent exacerbations . similar studies on guideline adherence in different countries also reported frequent use of dual and triple therapies in stages iii and iv.4,5,11 the ics was frequently used at all stages ( 43.8% in stage i , 81.5% in stage ii , 89.3% in stage iii , and 87.4% in stage iv ) mostly as a part of combination therapy . this study demonstrates that the pulmonary specialists in turkey have a high tendency to use ics for the treatment of patients with copd . in a study by de miguel - diez et al in spain , 34.5% of the patients were prescribed ics , alone or in combination , with a rate of inappropriate use of 18.2%.12 asche et al reported that 33% of the patients received combination treatment containing laba , lama , and/or ics consistent with the current gold guidelines.13 pulmonary specialists in our study mostly preferred to prescribe combination treatments containing ics . this might be related to a number of factors : inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment.concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist.reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance.preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available.increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment . concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist . reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance . preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available . increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . in patients treated with regular bronchodilators ( alone or a part of a combination therapy ) , the most frequently used drug was laba ( n=613 ) , followed by lama ( n = 575 ) , t ( n=197 ) . among these results , t use was prescribed for 27.4% of stable copd patients which was remarkably higher than the finding of corrado and rossi that reported 2.2% of patients were using xanthines.4 gold remark on t is that it is effective in copd but , due to its potential toxicity , inhaled bronchodilators are preferred when available.1 in our study , this high rate of t prescription in addition to other inhaled bronchodilators may be explained by the choice of an oral bronchodilator drug even if it has potential toxicity and drug interactions in older patients . another finding of this study was that the use of long - acting bronchodilators such as laba and lama alone remains considerably low , and they have been mostly preferred in stages i , ii , and iii . the most commonly used bronchodilator in monotherapy was lama ( 4.2% ) , and this might be due to its single - dose use and duration of action of more than 24 hours.14,15 this study has several limitations . individual patient data were retrospectively collected through patient records : number of acute exacerbations in the previous year , complete information on comorbidities , adherence to nonpharmacologic interventions were lacking . gold guidelines were updated in 2011 to incorporate clinical presentation , including dyspnea as measured by copd assessment test and the modified british medical research council . the updated version of the guidelines incorporates clinical presentation with spirometry findings ( gold 2011).16 this study was undertaken between december 2010 and november 2011 . study protocol had been designed to evaluate gold 2010 guideline adherence . due to lack of knowledge on dyspnea level ( medical research council scale ) and exacerbation frequency during the last year even this new gold classification ( updated in 2011 ) has been changed by considering sypmtoms and exacerbations in addition to spirometric staging , we have not enough data as to the exact superiorities over the old gold classification . a very recent analysis showed that gold 2011 shifted the overall copd severity distribution to more severe categories . there were nearly three times more copd patients in stage d than in former stage iv ( p<0.05 ) . the predictive capacity for survival up to 10 years was significant for both systems ( p<0.01 ) and gold 2007 and 2011 did not differ significantly . in this study , the percent predicted fev1 thresholds of 85% , 55% , and 35% were found better to stage copd severity for mortality , which are similar to the ones used previously in the gold 2010 classification . increasing intensity of treatment of patients with copd according to their gold 2011 reclassification is not known to improve health outcomes.17 the classification recommended by the gold committee since 2011 does not seem to be adopted universally throughout europe . even if there is a real improvement over the previous classification due to taking into account clinical criteria , quality of life , and exacerbations , criticisms have arisen concerning the choice of certain pathways and therapeutic recommendations not based on prospective studies with a high level of evidence.18 at the national level , we do not have any published data on the adoption of the new gold 2011 classification . according to the previously mentioned obstacles of the new classification , and lack of the knowledge about the frequency of new gold 2011 classification use , instead of the gold 2010 . we believe this data on the guideline adherence among pulmonologists may still reflect the current attitude toward the routine approach of the diagnosis and therapies of copd in turkey , and also is concordant with the real life situation around the world . turkish thoracic society has published an updated report of copd prevention , diagnosis and treatment in 2014,19 and included in the global alliance against chronic respiratory disease ( gard ) with the ministry of health.20 the improvement of adherence to national and international copd guidelines may be achieved by continuing medical education supported by national thoracic societies . increased awareness of guidelines may influence the standardized care of copd patients , and will have prognostic influences . promotion of the compliance to evidence - based medicine by health authorities may have also positive effects on the attitude change in the real - life applications . this study showed that the pulmonary specialists in turkey have low rates of adherence to gold guidelines for treatment of stable copd patients . major improper approaches were overtreatment in early stages and excessive use of inhaler corticosteroids in all stages of disease . new strategies are needed to achieve a standardized approach for the treatment of copd , and thus to improve adherence to guideline recommendations , with particular focus on treatment indications and overtreatment in turkey .

backgroundlow adherence to global initiative for chronic obstructive lung disease ( gold ) guideline recommendations has been reported worldwide . there has been no study on the adherence to gold guidelines for copd treatment in turkey.objectivesto investigate the rates of adherence to gold 2010 guidelines for copd treatment among pulmonologists.designa multi - center , cross - sectional , observational study was carried out in eleven pulmonary outpatient clinics across turkey . adherence to gold was evaluated through hospital records . demographic and clinical data were recorded.resultsstudy included 719 patients ( mean age : 62.99.7 years ; males 85.4% ) of whom 16 was classified as gold stage i , 238 as ii , 346 as iii , and 119 as iv , and only 59.5% received appropriate treatment . rates of guideline adherence varied across gold stages ( i , 6.3% ; ii , 14.7% ; iii , 84.4% ; and iv , 84% ) . causes of inappropriate therapies were overtreatment ( stage i , 100% and stage ii , 91.1% ) , undertreatment ( stage iii , 3.3% and stage iv , 10.9% ) and lack of treatment ( stage ii , 3.8% ; stage iii , 2.3% ; and stage iv , 5.9% ) . the most preferred regimen ( 43.4% ) was long - acting 2-agonist - inhaled corticosteroid - long - acting muscarinic antagonist . overall , 614 patients ( 89% ) received treatment containing inhaled corticosteroid.conclusionpulmonologists in turkey have low rates of adherence to gold guidelines in copd treatment . inappropriateness of therapies was due to overtreatment in early stages and excessive use of inhaled corticosteroid ( ics ) in all disease stages .

amphotericin b ( amb ) , an amphoteric polyene macrolide , has been considered one of the main options of antifungal therapy for serious and life - threatening mycotic infections for over 50 years despite the introduction of newer antifungal medications such as the azoles ( e.g. , voriconazole ) and echinocandins ( e.g. , caspofungin ) , which have better safety profiles ( 1 , 2 ) . this may be due to the fact that azoles and echinocandins are costly , ineffective against certain pathogenic fungi ( e.g. , candida krusei and candida parapsilosis ) , and not readily available in developing countries such as iran ( 3 ) . many acute and chronic adverse reactions have been associated with amb , such as infusion - related reactions ( e.g. , fever , chills , and hypotension ) , normocytic normochromic anemia , cardiac toxicity ( e.g. , ventricular tachycardia and hypertension ) , hepatic toxicity ( e.g. , increase in liver enzymes and bilirubin ) , neurologic toxicity ( e.g. , confusion , delirium , tremor , and seizure ) , and nephrotoxicity ( 4 , 5 ) . major features of amb - induced nephrotoxicity include an increased serum creatinine level , a decreased glomerular filtration rate ( gfr ) , urinary potassium wasting , hypokalemia , urinary magnesium wasting , and hypomagnesemia ( 6 ) . although it is a common event in clinical settings , different aspects of amb nephrotoxicity have not been studied well or in detail in our population . the purpose of this investigation was to specifically assess the frequency , time onset , and possible associated factors of amb nephrotoxicity in hospitalized patients in hematology - oncology wards in the southwest of iran . a cross - sectional , observational study was performed over a period of 9 months from august 2015 to april 2016 in 2 hematology - oncology and 1 hematopoietic stem cell transplantation wards of namazi hospital , which is affiliated to shiraz university of medical sciences , shiraz , iran . the medical ethics committee of the hospital approved the study , and all patients signed and approved a written informed consent form . the inclusion criteria for recruiting patients were as follows : 1 ) age 15 years or older ; 2 ) no documented history of acute kidney injury defined by an increase in serum creatinine 0.3 mg / dl within 48 hours , or an increase in serum creatinine by 1.5 times the baseline in the previous 7 days , or a urine volume < 0.5 ml / kg / h for 6 hours ( 7 ) ; 3 ) no documented history of chronic kidney disease ( clearance creatinine below 60 ml / min/1.73 m calculated by the simplified modification of diet in renal disease equation ) ; 4 ) no documented history of peritoneal or hemodialysis for > 3 months ( 8) ; 5 ) no documented history of having received amb by any administration route in the previous 14 days ; and 6 ) having received any formulation of amb intravenously for at least 1 week . they included age , sex , weight , amb dose , duration and indication , duration of amb infusion , type of co - administered medications that may exacerbate or attenuate amb nephrotoxicity , length of hospital stay , and mortality . serum urea and creatinine were monitored daily during the course of the amb treatment . according to routine ward practice , serum potassium and magnesium were checked daily and once weekly for patients , respectively . because most cases of amb nephrotoxicity occur during the first 2 weeks of treatment ( 9 , 10 ) , urine urea , creatinine , sodium , potassium , and magnesium levels were determined at days 0 , 3 , 5 , 7 , 10 , and 14 of the amb treatment . serum as well as urine urea , creatinine , and the aforementioned electrolytes were determined using an auto - analyzer ( shanghai xunda medical instrument , shanghai , china ) . the creatinine levels in the serum and urine samples were established using the modified jaffe colorimetric reaction . amb nephrotoxicity was defined as an estimated clcr 50% ( calculated by the cockcroft - gault formula ) or an increase in serum creatinine ( double the baseline value ) ( 11 ) . either the fractional excretion of sodium > 2% or the fractional excretion of urea > 50% ( in cases of diuretic co - administration ) was considered as acute tubular necrosis ( atn ) ( 12 ) . serum level potassium and magnesium below 3 meq / l and 1.2 meq / l were defined as hypokalemia and hypomagnesemia , respectively ( 13 ) . a urine potassium - to - creatinine ratio above 13 meq / g was considered renal potassium wasting ( 14 ) . the time onset of nephrotoxicity , hypokalemia , and hypomagnesemia after the initiation of amb was also determined . daily dose reductions , every other day dosing , discontinuation , or performing dialysis as probable measures of managing amb nephrotoxicity were recorded . continuous data were expressed as either mean standard deviation ( sd ) or mean standard error ( se ) . probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test . the fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 . the parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests , respectively . using the stepwise method , the logistic regression analysis with the odds ratio ( or ) and 95% confidence interval ( ci ) were employed to determine the associated factors of amb nephrotoxicity . in the first step , the possible association of each independent variable , namely age , gender , amb cumulative dose , duration of amb infusion , baseline gfr value , mean daily oral / intravenous sodium supplementation , and the co - administration of aminoglycosides , calcineurin inhibitors , vancomycin , acyclovir , loop diuretics , corticosteroids , and sodium bicarbonate , with amb nephrotoxicity ( as the dependent variable ) was assessed separately by univariate analysis . those with p values of less than 0.4 statistical significance in all analyses was defined by p values < 0.05 , except for the first step of the logistic regression analysis ( p values < 0.4 ) . continuous data were expressed as either mean standard deviation ( sd ) or mean standard error ( se ) . probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test . the fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 . the parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests , respectively . using the stepwise method , the logistic regression analysis with the odds ratio ( or ) and 95% confidence interval ( ci ) were employed to determine the associated factors of amb nephrotoxicity . in the first step , the possible association of each independent variable , namely age , gender , amb cumulative dose , duration of amb infusion , baseline gfr value , mean daily oral / intravenous sodium supplementation , and the co - administration of aminoglycosides , calcineurin inhibitors , vancomycin , acyclovir , loop diuretics , corticosteroids , and sodium bicarbonate , with amb nephrotoxicity ( as the dependent variable ) was assessed separately by univariate analysis . those with p values of less than 0.4 were then considered together for the multivariate logistic regression analysis . statistical significance in all analyses was defined by p values < 0.05 , except for the first step of the logistic regression analysis ( p values < 0.4 ) . during the 9-month study period , 90 patients who were scheduled to receive amb were screened . a flowchart detailing the reasons patients dropped out of the study is presented in figure 1 . the most common admission diagnosis of the patients was acute myeloid leukemia ( 60% ) followed by acute lymphoid leukemia ( 20% ) , hodgkin disease ( 5% ) , and aplastic anemia ( 5% ) . prophylaxis of aspergillosis ( 52.5% ) , treatment of aspergillosis ( 17.5% ) , and treatment of mucormycosis ( 12.5% ) were the three most frequent indications of amb administration ( table 1 ) . the mean sd daily doses of conventional and liposomal amb were 28.79 10.84 mg and 212.78 99.33 mg , respectively . the mean sd cumulative dose of conventional amb was 223.79 125.53 mg and 2367.15 2137.89 mg for liposomal amb . all courses of either conventional or liposomal amb infusions were administered within 2 - 6 hours . potential nephrotoxic medications , namely acyclovir , corticosteroids , calcineurin inhibitors ( cyclosporine ) , vancomycin , loop diuretics ( furosemide ) , and aminoglycosides ( amikacin ) , were given to 38 ( 95% ) , 28 ( 70% ) , 21 ( 52.5% ) , 19 ( 47.5% ) , 5 ( 12.5% ) , and 1 ( 2.5% ) patients , respectively . in contrast , spironolactone and sodium bicarbonate as potential nephroprotective agents were administered to 7 ( 17.5% ) and 4 ( 10% ) individuals , respectively . no patient received cisplatin , ifosfamide , cyclophosphamide , dopamine , and mannitol during the study period . the mean sd daily oral / intravenous sodium supplementation administered in conjunction with the amb treatment was 124.75 62.15 meq . during the study , 11 ( 27.5% ) and 21 ( 52.5% ) patients developed amb nephrotoxicity and atn , respectively . the mean sd onset of amb nephrotoxicity was 6.73 2.36 days . in accordance with the univariate analysis , amb indication ( p = 0.259 ) , vancomycin co - administration ( p = 0.388 ) , duration of liposomal amb infusion ( p = 0.141 ) , and amount of oral / intravenous sodium supplementation ( p = 0.375 ) based on this model , none of these variables were significantly associated with amb nephrotoxicity ( table 2 ) . amb administration was discontinued permanently in 2 individuals who developed nephrotoxicity , and they underwent emergent hemodialysis . there was no statistically significant difference in the duration of hospitalization between the patients with and without amb nephrotoxicity ( 27.72 4.81 and 30.33 13.57 days , respectively ; p = 0.541 ) . the mortality rate was also comparable between the patients with ( 54.54% ) and without ( 48.27% ) amb nephrotoxicity ( p = 0.723 ) . the mean sd onset of hypokalemia during amb administration was 5.06 3.35 days . only one documented episode of hypomagnesemia was detected in the study population during the amb treatment . considering the fact that serum magnesium levels were not measured routinely for most of the patients , determining the rate of renal magnesium wasting was not feasible . the co - administration of neither loop ( p = 0.642 ) nor potassium - sparing diuretics ( p = 0.211 ) was significantly associated with hypokalemia . the mean se daily amounts of intravenous potassium and magnesium administered during the course of the amb treatment were 52.43 12.13 and 3.51 2.04 meq , respectively . the incidence ( 27.5% ) and time onset ( 5.06 3.35 days ) of amb nephrotoxicity in the present study were within the ranges reported in the literature . some degree of increase in serum creatinine has been detected within 2 weeks in up to 80% of patients who received amb ( 9 , 10 ) . in one of the most prominent studies in this regard , a 9-year retrospective analysis demonstrated that 138 ( 28% ) of 494 adult in - patients experienced some type of nephrotoxicity during amb treatment ( 10 ) . in a 1-year prospective observational study by tavakoli - ardakani et al . ( 15 ) in the hematology - oncology and stem cell transplantation wards at taleghani hospital in tehran , 9 ( 25.71% ) out of 35 patients developed an increase in serum creatinine and bun during the course of amb treatment . this rate was reported to be 27.8% at another referral hematology - oncology and stem cell transplantation center in tehran ( 16 ) . in the adult infectious diseases ward at imam khomeini hospital in tehran , khalili et al . ( 17 ) demonstrated that 10 ( 76.92% ) of 13 individuals receiving amb alone developed acute kidney injury ( aki ) ; however , the incidence of aki among the patients given amb along with ceftriaxone and/or vancomycin was 86.68% . the time onset of amb nephrotoxicity was not generally reported in the aforementioned iranian studies . the wide variations in the incidence of amb nephrotoxicity noted above could be due to different study methodologies , clinical settings , relevant risk factors , and definitions . notably , 62.5% of the individuals in our study were given liposomal amb , which has a considerably less nephrotoxic formulation . this could partially justify the relatively lower rate of amb nephrotoxicity in our cohort than in similar studies in iran . in the present study , amb nephrotoxicity in 45.45% of the affected patients resolved spontaneously without any intervention . only 2 patients in our cohort required emergency hemodialysis due to the severity and persistency of amb nephrotoxicity . it has been reported that amb nephrotoxicity is predominantly reversible within a few months after discontinuation of treatment ( 18 ) ; however , about 15% of affected patients may require renal replacement therapy such as dialysis ( 19 ) . in the shariati hematology - oncology and stem cell transplantation wards , hayatshahi et al . ( 16 ) showed that amb nephrotoxicity led to a dose reduction of this agent in 3.7% of cases . in terms of clinical outcomes , mortality and duration of hospitalization were comparable between patients with and without amb nephrotoxicity in the present population . in contrast , bates et al.s ( 20 ) study of a large population ( 707 adults ) in the united states reported that acute renal failure due to amb increased the mean length of hospital stay by 8.2 days ( p < 0.0001 ) and increased the total cost of treatment by $ 29,823 . the mortality rate was also much higher ( 54% vs. 16% ; p = 0.001 ) . differences in the confounding factors ( e.g. , the severity of the underlying disease ) , the type of amb formulation used ( conventional vs. lipid - based ) , and sample size could partially account for these disparities . we considered p values greater than 0.05 ( 0.4 ) in the univariate analysis to select all possible variables linked to the dependent variable . among the different demographic , clinical , and paraclinical features , amb indication , vancomycin co - administration , duration of liposomal amb infusion , and amount of oral / intravenous sodium supplementation nevertheless , none of these variables were significantly associated with amb nephrotoxicity in the multivariate logistic regression model . several large - scale retrospective and prospective studies have identified males , an average daily dose of amb above 35 mg , a cumulative dose of amb greater than 2 - 5 g , dehydration , co - administered diuretics and nephrotoxic agents ( e.g. , aminoglycosides , cyclosporine , foscarnet , cisplatin , and ifosfamide ) or corticosteroids , and baseline renal dysfunction as potential risk factors for amb nephrotoxicity ( 21 - 23 ) . inadequate statistical power resulting from the relatively small sample size and the relatively low incidence of amb nephrotoxicity due to the administration of liposomal amb appear to be the main reasons for our findings in this regard . it has been suggested that conventional amb should preferably not be administered to patients with two or more of the aforementioned risk factors of amb nephrotoxicity ( 10 , 24 ) . notwithstanding , with the introduction of considerably less nephrotoxic formulations of amb into the market , this suggestion does not seem to make sense in clinical practice . during the course of the amb treatment in the current study , hypokalemia and renal potassium wasting developed in 45% and 27.5% of the patients , respectively . in contrast , hypomagnesemia was detected in only 1 patient ( 2.5% ) . the incidence of amb - induced hypokalemia in our cohort was lower than that reported in the literature ( 75% - 90% ) ( 25 ) . this may be due to the fact that 62.5% of the patients in the present study received liposomal amb . several publications have estimated the frequency of amb - induced hypomagnesemia to be between 15% and 100% depending on the dose and formulation of amb ( 26 ) . apart from the type of amb formulation and the presence of relevant risk factors , such as the co - administration of loop and potassium - sparing diuretics , the low rate of hypomagnesemia in our population could have been because the patients serum magnesium levels were measured only once a week during the amb treatment in the studied wards . severe hypokalemia and hypomagnesemia due to amb can cause metabolic complications , rhabdomyolysis , and life - threatening arrhythmias ( 25 ) ; however , these adverse events were not observed in our cohort at least during the course of the amb treatment . in conclusion , nearly one - third ( 27.5% ) of our cohort developed nephrotoxicity within the first week of amb treatment . amb nephrotoxicity resolved spontaneously in about half ( 45.45% ) of the affected patients without any intervention . mortality and the duration of hospitalization were comparable between patients with and without amb nephrotoxicity . no studied demographic , clinical , and paraclinical features of the study population were significantly associated with amb nephrotoxicity . among the studied electrolyte abnormalities , hypokalemia and renal potassium wasting were the most notable , affecting about one - half and one - third of amb recipients , respectively . the co - administration of either loop or potassium - sparing diuretics did not significantly affect electrolyte abnormalities during the course of the amb treatment . close monitoring of renal function indexes , including serum creatinine , bun , serum potassium , and magnesium , during amb treatment is highly recommended . additionally , implementing approved prophylactic measures , such as saline loading ( 150 meq / day ) before and/or during amb infusion , and exploiting lipid - based formulations of amb ( especially liposomal ) , if available and affordable , should be considered to minimize the possibility of amb nephrotoxicity .

backgroundnephrotoxicity is the most clinically significant adverse reaction of amphotericin b. different aspects of amphotericin b ( amb ) nephrotoxicity have not been studied well in our population.objectivesthe purpose of this study was to assess the frequency , time onset , and possible associated factors of amb nephrotoxicity in hospitalized patients in hematology - oncology wards in the southwest of iran.patients and methodsa cross - sectional , observational study was performed over a period of 9 months at 2 hematology - oncology and 1 hematopoietic stem cell transplantation wards at namazi hospital . patients aged 15 years or older with no documented history of acute kidney injury or chronic kidney disease who were scheduled to receive formulations of amb intravenously for at least 1 week were included . the required demographic and clinical data of the patients were recorded . urine urea , creatinine , sodium , potassium , and magnesium levels were measured at days 0 , 3 , 5 , 7 , 10 , and 14 of the amb treatment . amb nephrotoxicity based on serum creatinine increase , renal potassium wasting , hypokalemia , and hypomagnesemia were determined.resultsamong the 40 patients recruited for the study , 11 ( 27.5% ) patients developed amb nephrotoxicity with a mean standard deviation onset of 6.73 2.36 days . in 5 patients , amb nephrotoxicity resolved spontaneously without any intervention . according to the multivariate logistic regression model , none of the studied demographic , clinical , and paraclinical variables were significantly associated with amb nephrotoxicity . the duration of hospitalization ( p = 0.541 ) and the mortality rate ( p = 0.723 ) were comparable between the patients with and without amb nephrotoxicity . hypokalemia and renal potassium wasting were identified in 45% and 27.5% of the patients during amb treatment , respectively.conclusionsnearly one - third ( 27.5% ) of our cohort developed nephrotoxicity within the first week of amb treatment . hypokalemia and renal potassium wasting were more notable , affecting about one - half and one - third of the amb recipients , respectively .

o - glycans have been found to play an important role in protein stability and tertiary structure , stabilize protein conformation , modulate the activity of enzymes ( e.g. by reversible attachment of o - linked glcnac to cytoplasmic and nuclear proteins ) and signaling molecules , are essential for a number of recognition processes and are sorting determinants guiding the modified protein in the cell from the place of biosynthesis to its target location [ 13 ] . unlike the n - glycans they may be linked to several different amino acid residues ( very often serine or threonine , but also hydroxylysine , tyrosine or hydroxyproline ) of the protein and their occurrence can not be predicted easily by a common consensus sequence . extended galnac residues linked to serine or threonine residues ( mucin - type glycans ) are the most frequent and best investigated o - glycans in higher eukaryotes . eight core subtype formations showing cell - tissue specific patterns related to their function have been described ( for a review see ) . also glcnac--ser / thr without any elongation , a typical feature for nuclear and cytoskeletal proteins existing in a dynamic equilibrium with phosphorylation , and fuc--ser / thr as well as glc--ser , primarily found in epidermal growth factor domains , have been found in eukaryotes . a linkage via mannose has been found to be the main o - glycan type in yeasts , but also some mammalian tissues carry this structural feature , which seems to be relevant in some muscular dystrophies , while o - xylosylation is the starting point for the biosynthesis of chondroitin and heparan sulfates , which have roles in development and morphogenesis . the o - glycans of nematodes are rather complex with methylation as further modification [ 9 , 10 ] . xenopus eggs have o - glycans with up to 10 monosaccharide constituents , often highly fucosylated , with a linkage to the protein via galnac [ 11 , 12 ] . even parasites ( helminths and t. cruzi ) display small galnac linked o - glycans . in plants o - glycans occur as arabinogalactans linked to hydroxyproline or serine ( gal--hyp / ser ) , or hydroxyproline is glycosylated with short arabinofuranosides ( ara--hyp , ara--hyp ) . here we present for the first time an overview on o - glycans of some species of the mollusk branch of the evolutionary tree . the glycosylation potential of snails is important in general , because some of them are intermediate hosts of pathogens ( biomphalaria glabrata and lymnaea stagnalis ) or a pest in agriculture . a broad knowledge of their glycosylation abilities may show targets for pest control in the near future . in the course of the years , n - glycan patterns of several snail species have been presented [ 1417 ] . methylated mannose and galactose residues were found to be frequent constituents . in the present study we determine the main o - glycan structures in snails and characterize a common core - trisaccharide . cepaea hortensis , planorbarius corneus , arion lusitanicus , limax maximus and helix pomatia were collected by the authors under the supervision of dr . manfred pintar ( department of integrative biology and biodiversity research , institute of zoology , university of natural resources and life sciences , vienna ) in the vicinity of vienna . all chemicals purchased were of the highest quality available from sigma or fluka . while water living snails could be dissected right away , land living snails were extensively washed to remove the extraneous mucous components . snails were dissected as previously described and proteins were purified according to with minor modifications : 1 g of wet tissue was homogenised in 10 ml of chaps - based lysis buffer ( 0.5 % ( w / v ) chaps , 150 mm nacl , 20 mm tris / hcl , 2.5 mm sodium pyrophosphate , 1 mm ethylene - glycol - bis(2-aminoethylether)-n , n , n,n-tetraacetic acid and 1 mm edta , ph 7.5 ) and incubated for 72 h at 4 c . snail proteins were then reduced by the addition of 500 l of 10 mm dithiothreitol and incubation at 56 c for 45 min . then 500 l of 55 mm iodoacetamide were added and incubated for 30 min at room temperature in the dark . the aqueous samples were precipitated with a 4-fold amount of pre - chilled acetone at 20 c for at least 4 h and then centrifuged at 32000 g for 60 min . the pellets were resuspended in 0.1 m sodium citrate buffer , ph 4.6 , and incubated with 2 units of -glucosidase from rice [ ec 3.2.1.20 ] at 37 c for 16 h. sugars and polysaccharides were eluted from cation exchange chromatography ( ag 50 w - x2 , biorad , ca ) with 2 % of acetic acid , while proteins were eluted by 1.0 m ammonium acetate , ph 9.0 . the protein fraction was again precipitated by acetone followed by a similar precipitation by methanol . the dry sample was dissolved in 0.1 m naoh containing 0.8 m nabh3 and incubated at 37 c for 68 h. the sample was carefully neutralised by the addition of 2 m acetic acid , dried in a vacuum centrifuge and washed at least twice with 30 % ( v / v ) methanol . the glycans were directly extracted by solid phase extraction on a porous graphitized carbon cartridge ( supelclean envi - carb , 100 mg bed weight , 1 ml column volume or 500 mg bed weight , 6 ml column volume , sigma - aldrich , germany ) according to by using ammonium formate buffer / acetonitrile for elution and then on a reversed - phase c18 cartridge ( strata , c18-e , 55 m , 50 mg bed volume , 1 ml column volume , phenomenex , germany ) using 5 % ( v / v ) acetic acid as loading solvent and 25 % ( v / v ) isopropanol in 5 % ( v / v ) acetic acid for elution . hplc separation was carried out on a porous graphitized carbon ( pgc ) column ( hypersil - keystone , hypercarb 5 150 3 mm , thermo scientific , austria ) at a flow rate of 0.6 ml / min using 0.3 % ( v / v ) formic acid adjusted to ph 3.1 with ammonium as solvent a and 95 % ( v / v ) acetonitrile in solvent a as solvent b. the elution protocol consisted of 2 min at 100 % ( v / v ) of solvent a followed by a gradient of 28 min till 25 % ( v / v ) of solvent b and a cleaning step of 4 min at 60 % ( v / v ) of solvent b. each fraction was tested by esi - ms for its glycan content . single glycan structures were pooled and lyophilised . for monosaccharide analysis alditol acetates and for linkage analysis gc analysis was carried out on a vf 5 ms capillary column ( 60 m , 0.25 mm inner diameter ; 0.1 mm film thickness ; varian , darmstadt , germany ) and electron impact ( ei ) mass spectrometry was performed in the positive ion mode using a polaris q instrument ( thermoquest - analytical systems ) . for analysis of small sample amounts with esi - ms an in - gel release method for glycans bound to coomassie reduced oligosaccharides were analyzed by pgc - lc - esi - ms on a hypercarb column ( 0.32 150 mm , thermo fisher scientific , austria ) coupled to an ultimate 3000 ( dionex ) capillary hplc and a q - tof ultima ms ( waters ) as described previously [ 21 , 26 ] . the interpretation of the ms data was performed with the help of the software tool glycoworkbench . arion lusitanicus proteins were isolated and separated from contaminants through several purification steps in order to obtain sufficient amounts ( 12 g ) of pure glycans for further gc - ms analysis ( total monosaccharide and linkage analysis ) . the preparation of o - glycans was carried out via -elimination followed by further separation steps . as a final procedure the glycans were separated on a pgc ( porous graphitized carbon ) hplc - column by collecting 1-minute fractions which were screened by esi - ms to gain clean single structures . in the course of the -elimination releasing the glycan from the protein backbone , sodium borohydride was used . in contrast , for reduction of monosaccharides before gc - ms analysis sodium borodeuteride was employed , which allowed further the discrimination between the sugar released by -elimination and those linked to other sugars previously . in fig . 1 monosaccharide constituents of the core trisaccharide representing the main structure found in a. lusitanicus , with a pseudomolecular mass ( [ m+h ] ) of m / z 576.3 da , were determined by gc - ms of the corresponding alditol acetates and identified on the basis of retention times in gc and their characteristic electron impact ( ei ) mass spectra . the results revealed the presence of a galnac - residue which was h - reduced , while all the other sugars were h reduced . therefore , galnac can be considered as the protein bound sugar . the high amount of 4-o - me - gal indicated that this residue is the one which is mainly involved in the elongation of the glycan . minor compounds were identified as contaminations by n - glycans ( glcnac and man ) or storage glycans ( glc and gal ) . the lc - esi - ms / ms fragmentation pattern confirmed the occurrence of two methylated hexoses linked to the amino sugar ( fig . 1monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination . alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms . peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced)fig 2lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . registered ions represent proton adducts [ m+h ] monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination . alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms . peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced ) lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . registered ions represent proton adducts [ m+h ] moreover , gc - ms analysis of partially methylated alditol acetates was carried out for further confirmation of the compounds and also for linkage determination . methylated sugar standards purified by preparative gc were used to elucidate the type of methylated hexose ( man or gal ) based on retention time . the selected ion chromatogram of the monosaccharide derivatives obtained from the o - glycan core structure identified 2,3,4,6-tetra - o - methyl - galacitol at 20.66 min and 1,4,5-tri - o - methyl galnac - ol at 33.33 min ( fig . 3a ) which could be verified by ei - ms data ( figs . 3b and c ) . to determine the position of the naturally occurring methyl group on the galactose this allows in the ei - ms fragmentation pattern the discrimination between the naturally occurring and introduced methylgroups in the fragments by a mass difference of 3 da . that way the occurrence of terminal 4-o - methylated galactose 4 ) . combining these data , the main o - glycan structure of a. lusitanicus was elucidated as a trisaccharide containing two terminal 4-o - methylated galactoses which are 3- and 6-linked to an inner galnac residue ( fig . 5).fig 3linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol . characteristic primary selected secondary fragment ions are assignedfig 4ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide . the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of glycoworkbench . this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) . square = galnac , circles = gal , me = methyl group linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol . characteristic primary selected secondary fragment ions are assigned ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide . the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of glycoworkbench . this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) . square = galnac , circles = gal , me = methyl group a small amount of an isoform with the same molecular weight , but slightly later retention time in the pgc ( porous graphitized carbon ) selected ion chromatogram ( fig . 6c ) , was identified analogously by gc - ms and ei - ms as trisaccharide containing a galnac residue substituted by one 4-o - methylated galactose and one 3-o - methylated mannose ( data not shown).fig 6selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) . square = amino sugar , circle = hexose , me = methyl group selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) . square = amino sugar , circle = hexose , me = methyl group besides the trisaccharide core several other glycan species were found during purification on pgc - column . a glycan consisting of the methylated core trisaccharide and one further hexose ( m / z 738.3 [ m+h ] ) was found . this additional hexose was an unmethylated gal residue linked alternatively to one of the two arms of the trisaccharide explaining the two isoforms detected by pgc selected ion chromatogram ( fig . the fourth structure ( m / z 914.4 [ m+h ] ) which was obtained in sufficient amounts for gc - ms consisted of the core trisaccharide elongated by an unmethylated hexose on one arm and a methylated hexose on the other one ( fig . the presence of significant amounts of man and 3-o - me - man indicated that these two residues were responsible for the elongation . gc - ms analysis using different derivatisation protocols requested a high amount of material and high quality of purification . especially for snail tissues this was a challenge and therefore this method could not be carried out for a large number of samples in due time . an in - gel -elimination after sds - page followed by a short clean - up ( pgc - cartridge ) procedure was sufficient . the shortened protocol also ensured that no weakly bound modifications , such as sulfate or sialic acids for example , got lost . combining our data on snail monosaccharide analysis , data from previous gc - ms methylation analysis and the fragmentation results of lc - esi - ms / ms analysis of selected snails we could determine the main structures of snail o - glycans and classify them according to their modification of the established core structure into six groups : ( i ) glycans smaller than the core , missing one or both methyl groups or missing one hexose ; ( ii ) the trisaccharide core containing galnac and two 4-o - methylated hexose residues ; ( iii ) the core with additional methylated hexoses ; ( iv ) the core with one or two additional unmethylated hexoses and sometimes one more methylated hexose ; ( v ) glycans containing fucose ; ( vi ) glycans containing one hexnac and up to six unmethylated hexoses . for an overview on the structures see table 1.table 1overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans . compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex)glycan compositiongroupobserved mass [ m+h]calculated mass [ m+h]arion lusitanicusachatina fulicacepaea hortensisplanorbarius corenusbiomphalaria glabratahelix pomatialimax maximusclea helenahexhexnac(i ) - glycans smaller than the core386.2386.1707000000mehexhexnac400.2400.1850310200hex2hexnac548.2548.22traces000traces000hexmehexhexnac562.3562.2390012182600(mehex)2hexnac(ii ) - core576.3576.2554692612292910089(mehex)3hexnac(iii ) - core with additional methyl hexoses752.3752.325212302700(mehex)4hexnac928.4928.39traces0000000(mehex)5hexnac1104.41104.46traces0000000hex(mehex)2hexnac(iv ) core with additional hexoses and methyl hexoses738.3738.30141617164512011hex2(mehex)2hexnac900.4900.36203720100hex(mehex)3hexnac914.4914.3725505400(mehex)2hexnacdhex(v ) - fucosylated structures722.3722.31220traces1traces00(mehex)3hexnacdhex898.4898.3860000000(mehex)4hexnacdhex1074.41074.44traces0000000hex3hexnac(vi ) hexnac and hexoses710.3710.2700061traces00hex4hexnac872.3872.32000460000hex5hexnac1034.31034.3800030000hex6hexnac1197.01196.43000traces0000 overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans . compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex ) analyzing snails in detail , it was obvious that the o - glycan trisaccharide core is an important decoration of snail proteins in all investigated species . in five of the analyzed snails ( a. lusitanicus , a. fulica , h. pomatia , l. maximus , c. helena ) this glycan was the most abundant type of o - glycan . b. glabrata and c. hortensis displayed as their main glycan the trisaccharide elongated by one or two unmethylated hexoses , respectively . only p. corneus was an exception possessing a large amount of hex4hexnac glycan . whereas the addition of one hexose to the core was relatively frequent , larger structures were minor compounds or were even not present in several species . the only detected amino sugar was galnac which provided in all cases the connection to the protein . gal and man were present in unmethylated or mono - methylated ( 3-o - me - gal , 4-o - me - gal , 3-o - me - man ) versions . fucose , which had been identified as such in previous works [ 23 , 28 ] , occurred only in low amounts and was detected linked to galnac as well as to terminal sugar residues by ms / ms fragmentation . during the last decades our knowledge on glycosylation processes - structure of the glycans and the corresponding biochemical pathways including the responsible enzymes - increased enormously , especially for glycans of mammalian origin . the glycosylation capabilities of other species were only under investigation if their glycans were for any reason connected with human life ( e.g. some recognition processes of pathogens or allergy on food or plant glycans ) or if they were potent candidates for cell culture systems for the expression of therapeutics ( some insect , yeast and plant cells ) . in the meantime more and more invertebrate systems are investigated because of their potential usefulness for biotechnological reasons or to get some deeper insights into the processing pathways of unusual glycan structures . previous studies on various invertebrates showed that glycosylation has a lot more varieties than we expected as long as only mammals were investigated , but the data are scattered . still the main focus is laid on n - glycans but the interest in o - glycans is growing . a number of difficulties arose during snail glycan analysis due to the tough tissue , which was not easy to homogenize and the rigid mucus , which had to be discarded diligently . the removal of storage carbohydrates , which might influence monosaccharide analysis and the separation of n- and o - glycans were other struggles to resolve . while the first was done by -glucosidase digestion from rice , the latter was an even more difficult task . n - glycosidases ( pngase a and f ) did not remove the n - glycans completely and even mild -elimination conditions seemed to be able to release , at least in part , residual n - glycans which resulted in n - glycan impurities in o - glycan fractions . furthermore some n - glycans of snails and digested storage glycans are so small ( 45 sugar residues [ 16 , 17 ] ) that they may co - migrate on hplc with o - glycans . therefore only a limited number of glycans could be prepared in terms of sufficient amount and purity for permethylation followed by maldi - tof - ms and gc - ms linkage analysis . however , in combination with lc - esi - ms of native glycans which is capable of a high throughput of samples the o - glycan pattern of 8 snail species ( land snails with shell : achatina fulica , cepaea hortensis and helix pomatia ; slugs : arion lusitanicus and limax maximus ; water snails : biomphalaria glabrata , clea helena , and planorbarius corneus ) could be identified . four monosaccharide constituents galnac , gal , man and fuc are the building blocks of the structures . the only further modification is a methylation of the hexoses , resulting in 3-o - me - gal , 4-o - me - gal and 3-o - me - man , which has already been shown for snail n - glycans [ 1417 , 23 ] . each o - glycan contains only one amino sugar , which is the protein linked galnac . the linkage amino acid was not determined in detail , but due to the easy release by -elimination the common galnac - ser / thr motif is very likely . the connection to other amino acids , for example tyrosine , is usually more stable . in snail o - glycans the inner galnac is frequently elongated by two 4-o - me - gal residues in ( 13)- and ( 16)-linkage . elongations of this trisaccharide core by one or two hexoses with or without methyl group appeared in most snail species , whereas further elongation or fucosylation are rare events . insects , one neighbor of mollusks in the phylogenetic tree , display also internal galnac elongated by one gal in their o - glycan structures , but further elongations and/or branching are frequently done by another amino sugar ( galnac or glcnac ) . the main o - glycans of toxocara canis are trisaccharides consisting of galnac , gal or 4-o - gal and 2-o - fuc . so far nothing is known about biological functions of snail glycans . for sure these glycans are similarly involved in recognition processes as they are in all other species , but especially the significance of the high degree of methylation of hexoses of n- as well as of o - glycans is waiting for elucidation . here we enlarge the current knowledge on glycosylation abilities of lower animals by presenting the first o - glycan analysis of snails , representatives of the mollusk phylum .

the glycosylation abilities of snails deserve attention , because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites . in analogy to many other host - pathogen relations , the glycosylation of snail proteins may likewise contribute to these host - parasite interactions . here we present an overview on the o - glycan structures of 8 different snails ( land and water snails , with or without shell ) : arion lusitanicus , achatina fulica , biomphalaria glabrata , cepaea hortensis , clea helena , helix pomatia , limax maximus and planorbarius corneus . the o - glycans were released from the purified snail proteins by -elimination . further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and for the main structures by gas chromatography / mass spectrometry . snail o - glycans are built from the four monosaccharide constituents : n - acetylgalactosamine , galactose , mannose and fucose . an additional modification is a methylation of the hexoses . the common trisaccharide core structure was determined in arion lusitanicus to be n - acetylgalactosamine linked to the protein elongated by two 4-o - methylated galactose residues . further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present . the typical snail o - glycan structures are different to those so far described . similar to snail n - glycan structures they display methylated hexose residues .