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to assess the extent of promoter reuse in bacteria we looked for groups of bacterial genes per genome that share highly similar sequences upstream of their transcriptional start site , but do not have obvious flanking paralogous coding sequences . more specifically , we extracted in silico the dna region between positions -150 and -50 relative to the start of translation for all genes in a genome ( including plasmids ) , except when this overlapped with the coding region or promoter region of a flanking gene . in escherichia coli the majority of the transcriptional start sites were shown to be between 20 and 40 nucleotides upstream of the translational start site , so most of our upstream sequence fragments will not contain the important -10 ( pribnow ) box , but should include the -35 element . using blast we then searched for sequence pairs that matched with 80% or more nucleotide identity over at least 50 nucleotides , to select for highly similar regions rather than for short conserved dna elements . sequence pairs that in addition showed a high nucleotide identity in their adjacent coding sequences were assumed to be paralogs and excluded because for this study we were interested in the independent mobility of promoters , not duplicated regions ( for details see the materials and methods in our nar paper ) . we analyzed all available complete prokaryotic genomes ( 1,362 ; july 2011 ) and even with our strict selection criteria found over 4,000 families of highly similar sequences upstream of apparently unrelated coding sequences . the majority of these families actually consist of pairs that on average share 92% nucleotide identity , meaning that at least 46 out of 50 base pairs were conserved , but we also found pairs that were completely identical over 100 base pairs . whether this level of high identity is the result of a strong selective pressure , or indicative of recent duplication events remains to be investigated . in fact , some of these sequences likely are not promoters but have a different function that causes their conservation . looking for known elements in our pmp set we actually found 42 trnas , 83 resembled other rna families like the regulatory riboswitches , and interestingly 210 were known insertion sequences . the > 4000 families that our study uncovered represent only a small sample of a large pool of repeated dna in promoter regions , a conservative reference of promoter reuse in prokaryotes . we anticipate many relevant examples of the phenomenon remain undetected because of our strict criteria . for example , filtering out paralogous genes also removes mobile promoters that extend into the coding region , like reported cases of correia elements that overlap with an orf . in addition , our initial extraction of promoter sequences is sensitive to wrongly annotated translational start sites , which is a known issue with genome annotation pipelines . more surprising even than the large number of promoter pairs sharing high nucleotide identity within one bacterial genome is that about 6% are shared between distantly related species . clustering these based on sequence similarity resulted in 62 distinct groups , of which four are present in species that are related only by belonging to the same phylum ( table 1 ) . as expected , inter - taxon transfers seem to decrease with phylogenetic distance and at the domain level , i.e. , between archaea and bacteria , no transfer events were observed . some non - coding sequence elements like trnas are very well conserved over large evolutionary distances , but if highly similar sequences are found only in small number of distantly related species horizontal gene transfer is a more likely scenario . the large majority of the pmps are located on a chromosome , but for one group of pmps all members are in fact on plasmids . these plasmids are associated with multiple - drug resistance in pathogenic salmonella and are frequently transferred between bacterial species . although the genetic code for translating dna to protein is extremely well conserved between species as distant as escherichia coli and homo sapiens , transcriptional cis - regulatory elements are much more variable and their activity can differ even between strains of the same species . it can therefore be expected that the 62 homologous pmps are not primarily transcription factor binding sites , but rather have other ( regulatory ) functions causing their high conservation . indeed , two of the pmps that are shared between families of bacteria are known s - adenosylmethionine ( sam ) binding riboswitches . the other 60 pmps however did not resemble any of the rna families included in the rfam database , so their function at present remains uncovered . we conclude that we have uncovered a large number of putative mobile promoter families , present in numerous bacterial genomes . these may be involved in rapid adaptive processes via transcriptional rewiring , or include post - transcriptional regulatory functions . the ways these pmps move within and between genomes is still unknown , but due to the large number of families , this may include diverse mobilization mechanisms . finally , although transcription regulation in eukaryotes is more complex than in bacteria , it seems obvious that also in eukaryotes promoter reuse offers a mechanism for rapid adaptation of gene expression . it would therefore be very interesting to extent our analysis to this domain , especially now more genomes and transcriptomes are becoming available that greatly facilitate the mapping of the core promoters .
obstructive sleep apnea ( osa ) is a chronic condition that affects 3 - 7% of the population . despite increasing public awareness over the last 20 years , osa is still underdiagnosed and untreated . the negative health consequences of untreated sleep apnea continue to be elucidated , but currently include significant morbidity - related entities such as cardiovascular disease , hypertension , and abnormal glucose metabolism . more readily available and cost - effective methods of diagnosing osa could have significant impact on public health and reduce health - care expenditures . factors known to increase risk of osa include neck size , age , male sex , obesity , family history , and craniofacial anatomy . upper airway anatomy is involved in the pathophysiology of osa . decreased cross - sectional area and increased compliance of the upper airway contribute to osa . neck circumference measurement reflects neck tissue mass and serves as a surrogate measure of neck mass . hypopnea index ( ahi ) than did the measure of body mass index ( bmi ) . neck circumference corrected for by height is more useful . in 1990 , katz et al . published an article entitled do patients with obstructive sleep apnea have thick necks ? our study restates the question to read , do patients with thick necks have obstructive sleep apnea ? we hypothesized that a simple and objective test using proportionate neck size ( subject spanning their own neck with their own hands ) rather than measured circumference would simplify the screening and increase the recognition and diagnosis of osa . this simple objective maneuver may eliminate the need to rely on first- and third - person subjective reporting of snoring quality , possible apnea , sleep quality , and sleepiness that is currently heavily relied upon for suspicion of osa . we evaluated a simple neck grasp maneuver as a predictor of osa in a general sleep clinic . the institutional review board at bassett medical center approved this retrospective chart review and waived the need for informed consent from patients . a retrospective review of charts was performed to obtain the following data : esap status , age , gender , bmi , and polysomnogram / home sleep test ( psg / hst ) result . fifty consecutive adult patients ( aged > 18 years ) requiring psg for clinical evaluation were assessed with the easy sleep apnea predictor ( esap ) test [ figure 1 ] at the time of the consultation . the patients were instructed to place their thumbs together at the anterior neck and to wrap their fingers around their neck until they met in the posterior . a positive esap test was defined as the inability to easily ( without excess squeezing and choking ) encircle the neck completely [ figure 2 ] . a negative esap test was defined as the ability to easily encompass their hands around the neck [ figure 3 ] . the result of the esap test was recorded in the medical record as positive or negative . clinical testing with psg or hst level 3 was dictated by the consulting clinician 's clinical determination and insurance coverage . psg was performed and scored according to current the american academy of sleep medicine ( aasm ) guidelines . in medicare patients ( n = 7 ) , the hypopnea definition of 30% reduction in flow with 4% desaturation was applied ; all others were scored using the 30% reduction with either a 3% desaturation or an arousal . hst ( n = 1 ) was performed and scored by manufacturer 's setting and reviewed and interpreted by a board certified sleep physician . for the purpose of the main study analysis , osa was defined as an ahi of 5 . subanalyses were conducted using ahi cutoffs of ahi of > 10 and ahi of > 15 . the easy sleep apnea predictor is performed by touching the thumbs in the anterior of the neck and encircling the neck with the digits in the posterior easy sleep apnea predictor positive is the inability to easily encircle the neck so that the patient 's digits meet on the posterior neck easy sleep apnea predictor negative is the ability to encircle the neck without excessive choking with digits touching at the posterior neck demographic variables were compared between esap positive and esap negative patients using chi - square ( for gender ) and the t - test ( for bmi , age , and ahi ) . using ahi as the gold standard for osa diagnosis , the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of esap to predict osa were calculated . sensitivity , specificity , ppv and npv of obesity classes ( bmi 30 and bmi 35 ) to predict osa were also calculated . demographic variables were compared between esap positive and esap negative patients using chi - square ( for gender ) and the t - test ( for bmi , age , and ahi ) . using ahi as the gold standard for osa diagnosis , the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of esap to predict osa were calculated . sensitivity , specificity , ppv and npv of obesity classes ( bmi 30 and bmi 35 ) to predict osa were also calculated . two of the 47 subjects had hst rather than psg by insurance requirement [ table 1 ] . the mean age was 51.6 years ( sd 14.4 , range 29 - 81 ) . the mean bmi was 38.8 ( sd 9.9 , range 20.4 - 69.5 ) . in our population , characteristics and demographics of the study population esap did differentiate a statically significant difference in the population for ahi ( p < 0.001 ) and bmi ( p both ahi and bmi were significantly greater on average for esap positive subjects as compared to esap negative subjects . between the esap positive and negative groups there is no statistical difference for gender ( p = 0.49 ) or age ( p = 0.66 ) [ table 2 ] . a comparison of esap negative and positive patients for bmi , ahi , age , and gender the sensitivity and specificity of esap were 68.3% and 100% , respectively , with regard to an ahi of 5 . the positive predictive power was 100% and the negative predictive power was 31.6% for an ahi of 5 . using bmi to predict ahi of 5 revealed weaker prediction . the ppvs for bmi 30 and bmi 35 were 92% and 90% , respectively , at an ahi of 5 . the ppvs of esap remained similar when stratified by bmi > 30 and bmi > 35 for three levels of ahi ( 5 , > 10 , and > 15 ) [ table 3 ] . the field of sleep medicine is working to improve the diagnosis and treatment of osa in a cost - effective manner ; it is estimated that 80% of the people with osa are undiagnosed . incorporating novel methods will be required to successfully meet these demands . over the past decade , many screening algorithms and questionnaires have been developed such as stop - bang and the berlin questionnaire . obesity and a large neck have long been established as risk factors for osa . to our knowledge , this is the first time that this well - recognized fact has been leveraged in a simple neck grasp maneuver , as a diagnostic tool for osa . our retrospective pilot data support our hypothesis that a positive esap , the inability to fit hands around the neck , strongly correlates with osa in a general sleep clinic population and this correlation is superior to bmi alone . esap 's usefulness is consistent with the known fact that neck size predicts osa and that proportionate data , corrected for body size , are more valuable than absolute measurements . esap is more easily applicable than linear measure , and it does not require the adjustments for gender required with linear measure . first , we recognize that our threshold for presence of osa was low ( ahi of 5 ) . however , in a symptomatic sleep clinic population , this is arguably an appropriate set point for consideration of treatment . when the threshold for osa was increased to ahi of > 15 , our data showed that the ppv of esap to remain clinically useful at 86% . we did not evaluate esap combined with any other parameter or test such as snoring , observed apnea , presences of hypertension , or epworth sleepiness scale . this raises the second point of note that the high pretest probability of this preselected referral population improved esap predictive power . this does not reduce the value of the esap test when applied to this population . the final point of comment on the 100% ppv pertains to an issue that needs to be clarified for the entire field of sleep medicine , not just our esap validity ; that is the definition of sleep disordered breathing events , namely hypopneas . our retrospective the more recent 2014 definition lowers the threshold for diagnosing osa and hence increases the ppv of esap . this does not stand to negate esap positive value , but needs to be recognized . collop ; we are not attempting to bring any additional clarity to the definitions other than to say that the esap data stands in the midst of this change in terminology . the most obvious and important limitation of the esap test is the rate of false negatives , npv of 31.6% . the lack of value of a negative esap needs recognition when the esap is applied . the standard testing ( psg or hst ) is still required if clinically indicated in an esap negative patient . other factors , such as craniofacial anatomy and upper airway anatomy , contribute to osa regardless of neck size and esap negativity . that point understood , the positive esap remains valuable at identifying the 80% of patients with undiagnosed osa , the majority of whom are obese and likely esap positive . the equally objective measure of bmi was inferior [ table 2 ] to esap over a range of ahi . esap is arguably the simplest of all objective tests , requiring only seconds of clinical time and no device . in unique populations , such as commercial drivers , this objective screen might prove invaluable to highway safety ; however , to date this population has not be evaluated . first , the general economic burden to society of untreated osa remains high despite three decades of recognition of the syndrome . second , the health - care expenditure for untreated osa is estimated at $ 3.4 billion in the us . the initial step in addressing this economic problem is improving diagnosis , which esap appears every effective in the sleep clinic population . lastly , esap may result in a cost reduction in the area of diagnostic testing to identify osa by reducing reliance on psg . as we hypothesized , esap positive ( inability to span neck ) was predictive of osa . esap shows promise for ease of clinical use to predict the presence of osa in a general sleep clinic population . further research to evaluate this simple stand - alone diagnostic maneuver is warranted to confirm the high predictive value in this and other populations . the process of disease recognition of osa remains a challenge and esap may prove to be the easiest and least expensive tool available . the authors have no conflicts of interest including financial support , or involvement with organizations that have a vested financial interest . the authors have no conflicts of interest including financial support , or involvement with organizations that have a vested financial interest .
oral disease is considered globally one of the most common public health problems with important socioeconomic implications . among oral diseases , poor oral health associated with dental caries and periodontal disease may lead to loss of teeth , and can result in a number of debilitating diseases such as cardiovascular disease and adverse pregnancy outcomes . in recent years , a substantial amount of research was conducted among saudi women to investigate their health and health risk - related behaviors . this favorably growing interest to collect data on women 's health was notably paralleled by quite a reluctance to investigate their oral health and oral hygiene practices . most of oral health research in this part of the world was conducted among children or among the general population , but young women received little attention . in this era of globalization and social networking , oral health may be impacted by the changing trends in social habits of women such as smoking and oral hygiene habits . a previous study reported an alarmingly high prevalence of dental disease as indicated by a high mean number of decayed , missing and filled teeth ( dmft ) among a sample of young women in the age range of 1825 years in the western region of saudi arabia . al madinah al munawarah ( the enlighted city in arabic ) is the second holiest site in islam after makkah , and is situated in the hijaz region of western saudi arabia . its 2 million population is multiracial ; a characteristic that has significantly impacted their social habits including diet , oral hygiene , and other health behaviors . a substantial proportion of al madinah women , regardless of their nationality , attend the dental college and hospital of taibah university to receive free dental treatment services . we conducted this study among a sample of young women to target those who are in an important phase of life and to address a pivotal period of any woman 's life , the age of founding families and child - bearing . with the obvious deficiency in current research on women 's oral health , the aims of this study were to estimate the prevalence of jaw findings as detected in panoramic radiographs and to investigate the significance of dmft components as predictors for the radiographic findings in a sample of young women attending the taibah university dental college & hospital ( tudch ) in al madinah al munawarah . this descriptive retrospective study was approved by the taibah university college of dentistry research ethics committee a waiver of informed consent was approved being a retrospective study , and confidentiality of the data was guaranteed . the study involved retrieving electronic files stored on the clinical software system used at tudch [ cs r4 clinical + practice management software ( carestream dental ltd , rochester , ny ) ] during the period of 20132014 . extracted data included patient 's age and the panoramic radiographs taken during the standard screening visit for all patients visiting tudch , unless otherwise necessitated . the panoramic radiographs were taken utilizing a digital imaging system [ cs 9000 select 3d extraoral digital imaging system sm749 ( rochester ny ) ] . presence of a recent , high quality , clear panoramic radiograph was also an inclusion criterion . patients with radiographs that were unclear or had distortion , overlapping , or positioning errors , were excluded from the study . taking into consideration that 1668 patients in the age range 1825 attended tucdh during the period 20132014 , sample size determination was calculated using special software ( epi info software , center for disease control ) based on 8090% power of the study and an expected frequency of 50% radiographic jaw findings and a confidence limit of 5% , the required sample size was 190 . data on dmft of patients were retrieved from dental charts that existed in the clinical records to study the associations with radiographic findings . author sk , a radiologist with experience in interpretation of pathologies and abnormalities affecting the jaw bones and teeth performed the radiographic interpretation . screened findings included alveolar bone loss ( localized or generalized ) , radiolucent periapical bony lesions , retained roots , supernumerary teeth , and impacted teeth . all working conditions were similar and standardized each time during panoramic interpretation in the work station . this scoring system is described as follows : pai : 1 = normal periapical structures , 2 = small changes in bone structure , 3 = changes in bone structure with some mineral loss , 4 = periodontitis with well - defined radiolucent area , and 5 = severe periodontitis with exacerbating features . a score greater than 2 ( pai , 3 ) was considered to be an indication of periapical lesion . with regards to alveolar bone loss , we calibrated the measurements using the linear measurement tool of cs9000 software to estimate the alveolar bone height on panoramic radiographs using specific anatomic landmarks as a reference ; namely the cementoenamel junction ( cej ) and alveolar crest . bone loss was noted when the distance from the cej to the alveolar crest exceeded 2 mm . alveolar bone loss in panoramic radiograph was considered either localized if 30% or less of sites were affected , or generalized if more than 30% of sites were affected . descriptive statistics in the form of frequency distributions were used to describe the study sample and prevalence of radiographic lesions . multiple linear regression was used to explore the significance of tooth caries , missing teeth , filled teeth , and the final dmft score as predictor variables for the occurrence of various radiographic lesions . data were analyzed using the statistical analysis software [ spss version 21 ( ibm corp . ) ] . descriptive statistics in the form of frequency distributions were used to describe the study sample and prevalence of radiographic lesions . multiple linear regression was used to explore the significance of tooth caries , missing teeth , filled teeth , and the final dmft score as predictor variables for the occurrence of various radiographic lesions . data were analyzed using the statistical analysis software [ spss version 21 ( ibm corp . ) ] . panoramic radiographs revealed a wide variety of jaw lesions and abnormalities including retained roots , alveolar bone loss , periapical lesions , supernumerary teeth , and impacted teeth [ figure 1 ] . jaw findings in number and percentage are shown in table 1 . a percentage of 53.6% had periapical lesions , and alveolar bone loss affected 17% of the study sample . a panoramic radiograph of a 20-year - old female patient showing neglected mouth with multiple carious teeth , retained roots , and periapical lesions periapical lesions , retained roots , alveolar bone loss , supernumerary teeth , and impacted teeth among the study sample tables 26 display the details of regression models for the prediction of number of periapical lesions and level of alveolar bone loss . based on the two regression models in this study , we can predict in a similar population the number of periapical lesions by regression analysis with dependent variable : number of periapical lesions . predictor variables : age of the patient , carious teeth , missing teeth , and filled teeth coefficients and significance of their contribution to the model . dependent variable : number of periapical lesions regression analysis with dependent variable : level of alveolar bone loss . predictor variables : age of the patient , carious teeth , missing teeth , and filled teeth coefficients and significance of their contribution to the model . dependent variable : level of alveolar bone loss correlation matrix of predictor variables for the two regression models no . pa lesions = 2.942 0.106 ( age of patient in years ) + 0.069 ( no . of carious teeth ) + 0.039 ( no . of missing teeth ) + 0.002 ( no . of filled teeth ) . the level of alveolar bone loss by level of alveolar bone loss= 0.765 + 0.044 ( age of patient in years ) 0.019 ( no . of carious teeth ) + 0.091 ( no . of missing teeth ) 0.006 ( no . of filled teeth ) . tables 3 and 5 show coefficients of the two models , and table 6 shows the correlation matrix of the predictor variables and the dependent variables in the two models . this retrospective study aimed at utilizing panoramic radiographs and dental charts available in electronic clinical records to estimate the prevalence of pathologic jaw findings , as well as aimed at investigating the significance of dmft components as predictors for the radiographic findings in a sample of young women . more than 50% of the study sample had periapical lesions , approximately quarter of the sample had retained roots , and slightly less than 20% had alveolar bone loss that was in most of the cases generalized in nature . other developmental nonpathologic findings included supernumerary teeth and impacted teeth that occurred in 6.4% and 33.7% of the sample , respectively . a previous study that was done in our center among the same age group found an alarmingly high dmft index of approximately 9 . this prompted us to study their panoramic radiographs to document and report radiographic findings , if any . panoramic radiographs are routinely taken for all adult patients on their first screening visit to tudch . the use of panoramic radiographs as a routine screening tool has been criticized for their cost and radiation exposure to patients . panoramic radiographs that were taken for our patients were based on digital rather than plain film radiography . using the digital technique the radiation dose is reduced by up to 80% , and the technique is cost - effective as well . an added feature of digital radiographs is the ability to manipulate radiographs by zooming in and changing contrast , which is a useful tool in assessing bony defects . we assessed the presence of radiolucent periapical lesions radiographically because it is considered as an indicator for the presence of apical periodontitis ( ap ) , which is an inflammation of the periodontium at the tooth apex due to dental pulp infection and tooth caries . we used the pai scoring system for identification of periapical lesions in panoramic images as advocated by several studies . more than half of our sample had periapical lesions ( pai > 2 ) . while periapical lesions are not always the result of pulpal necrosis , a high prevalence among a young age group indicates poor oral health . furthermore , almost one in every four had retained roots , confirming the general trend of poor oral health . a recent study conducted in malawi found a prevalence of 23.5% of periapical lesions in a sample of pregnant women . studies that were done in western countries among the same age group , on the other hand , have found completely different results with excellent oral and dental health , which , as the researchers suggest , may reflect the use of fluoride and high levels of oral health education . taking into consideration the critical age range of our sample , the child bearing age , such patients may be susceptible to an increased risk of shorter pregnancy duration and intrauterine growth restriction as a result of such a high prevalence of periapical lesions . another bone pathology in the form of alveolar bone loss affected 17% of the study sample , with most of the alveolar bone loss cases ( 11.6% ) having a generalized pattern . on the other hand , alveolar bone loss is a hallmark of periodontitis , which is multifactorial in nature . because records of patients with any systemic diseases were not included in the study , one could exclude the assumption that the detected alveolar bone loss was indirectly related to a systemic illness rather than local factors such as dental plaque . therefore , an association between oral hygiene status and alveolar bone loss could not be established . nevertheless , it is well - established that women who are in the child bearing age and who have periodontal disease are probably susceptible to adverse pregnancy outcomes . it was shown that periodontal disease is associated with preeclampsia , preterm birth , and low birth weight . mothers with periodontitis are six times more likely to have low birth weight children than mothers without periodontitis . furthermore , pregnancy itself has its own adverse impact on oral health due to hormonal changes . perhaps pathologic alveolar bone loss itself can be considered as a predictor for many pathologies that may appear in a population of young females , however , the absence of full scale radiographic data for any given population , poses another limiting factor . regression analysis in this study indicated that patient age and number of carious teeth are statistically significant predictors of the number of periapical lesions . on the other hand , patient age , number of carious teeth , and number of missing teeth were statistically significant predictors for alveolar bone loss . the older our patients were the higher was the incidence of alveolar bone loss , and the lower is the incidence of periapical lesions . this can be explained by the fact that periapical bone lesions do not persist over the years and affected teeth tend to be either lost by extraction as a result of pain and loss of function , or treated endodontically , if restorable . dmft was not used in the two regression models for periapical bone lesions and alveolar bone loss because of problems of multicollinearity of predictor variables ( i.e. , number of carious teeth , missing teeth and filled teeth ) . however , after the elimination of dmft as a predictor , multicollinearity was not a problem . based on the values of r , regression models could explain 9.6% of the variance in number of periapical lesions , and they could explain 16.2% of variance in the level of alveolar bone loss . however , both models were extremely significant for the predictors of the number of periapical bone lesions and the level of alveolar bone loss ( p = 0.0009 and p = 0.0001 , respectively ) . this study is considered the first study that provides an insight into the radiographic jaw findings of young females who reside in al madina al munawarah with interesting results . hopefully , more studies are to follow focusing on females in different age groups and various ethnic backgrounds . another strength of this study is that it highlighted the use of prediction of bone pathology ( periapical lesions and alveolar bone loss ) in case panoramic radiographs were not available . this becomes even more important in pregnant women who may oppose radiographic examination due to cultural misbeliefs . a recent study conducted among a sample of pregnant women in saudi arabia reported that approximately 50% of the study sample believed that dental radiographs affect the fetus adversely . however , the sample size was calculated to produce 85% power , which can be judged to be sufficient . in our previous study , we included 419 patient records , however , we found only 190 patients who had panoramic radiographs saved in the r4 system . this discrepancy resulted as the patient record system was converted to the electronic r4 system with unexpected loss of some radiographic data . another limitation of the study emanates from the fact that it was a retrospective study that utilized only the data that are stored in the electronic patients ' records , hence , the risk factors that may be associated with the current findings were not investigated . the possible risk factors , though , could be one or more of the following poor oral hygiene , poor dietary habits , socioeconomic status , and genetic factors . previous studies have reported that young females in saudi arabia are likely to neglect oral hygiene habits and follow poor dietary habits . economic rush has been considered an important factor in changing dietary and oral health habits of the saudis . however , one should be careful in interpreting the results of this study since al madinah is a cosmopolitan city and its residents come from almost all over the muslim world . further studies are needed to investigate the racial and genetic susceptibility of these patients to dental and periodontal disease . the findings of this study should alert local authorities such as the ministry of health to implement preventive oral health programs that target women , particularly young age groups . these preventive programs can be implemented as a joint collaboration between ministry of health and ministry of higher education to address female university students . the need for such programs becomes particularly obvious when assessing the current status of primary care centers affiliated to the local ministry of health . the dental sector of primary care centers faces important challenges that makes oral health care fall short of patient expectations . lack of advanced dental treatment and high cost of dental treatment in private clinics are two important factors that should motivate the local government to implement preventive programs . within the same context , organized screening programs in the form of national mass screening campaigns targeting oral diseases , can be integrated in the health system to seek out undiagnosed patients or patients with difficulties in accessing the healthcare system . another important aspect that can improve oral health care is to implement policies that target cultural attitudes that could hinder providing this type of care . one of the cultural attitudes that may be considered controversial is the difficulty , with which women can get transportation to access dental services in saudi arabia . albarakati ( 2009 ) investigated reasons for failed dental appointments among a sample of saudi women . she found that 68% relied on a family member for transportation to their dental appointment , and another 76.2% reported that transportation difficulty is a relevant factor in missing the appointment , some of them attributed this to appointment conflicting with their husbands ' work . however , lately , people have started to use smartphone applications to get transportation in various areas of kingdom of saudi arabia . in particular , women are the main recipients of these services which could replace the reliance on family members for transportation . a high prevalence of pathologic radiographic jaw findings were detected in a sample of young female patients in al madina al munawarah , indicating poor oral health . risk factors contributing to these findings are urgently needed to be addressed to design policies that counteract this problem . along with the number of missing teeth , they were also significant predictors for alveolar bone loss . in the case where obtaining radiographs is difficult or unavailable , regression models from this study can be utilized to predict the presence of intrabony lesions in patients ( after verification ) . it is anticipated that a research group that is fully dedicated to investigating all aspects of oral health of women in al madinah to be formed officially in the near future , with a vision to change women 's attitudes and perceptions towards oral health as a major factor in determining quality of life and general systemic health .
beekeeper profit for colony i : iv=[(1v)(hiv+riv)(ci+cv ) ] where iv is profit for colony i with varroa infestation v , hiv is honey revenue , riv is pollination rental revenues , ci is operation and maintenance costs , cv is a targeted varroa treatment cost , is the deterioration from a lack of effective varroa treatment , v(0,1 ) is the varroa load , and (0,1 ) is the rate of treatment resistance in the varroa mite population . (0,1 ) is introduced only when there is evidence of treatment resistance developing in the varroa mite population . honey price is consistent with the statistics canada average honey price for 2015 ( statistics canada [ stats can ] 2015 ) and can also be derived from calculating the $ /lb for honey from the 2014 data in page and darrach 2016 , where the total value of honey was cdn$201 , 620 for 81,556 pounds of honey . this price does not account for the dramatic fall in prices in the fall 2016 ( http://www.cbc.ca/news/canada/calgary/canadian-beekeepers-face-plummeting-prices-1.3746822 , accessed 28 february 2017 ) , and so we also calculate the economic impact of this lower honey price of $ 1.13/lb . quantity of honey produced by a focused honey - producing colony is derived from a 2011 study in alberta , where the average colony produced 143 lbs of honey ( laate 2013 ) , and from our canadian honey bee producer data and discussions collected during the beeipm project . our 12 cooperating producers managed a mixture of honey - producing and commercial pollinating colonies , with an average honey output of just over 110 lbs per colony . some of the high yield honey producers yielded well over 200 pounds . in this case study , our fully productive focused honey - producing colony yields 200 pounds , whereas our commercial pollinator yields 100 pounds . the pollination rental fee received from a canola grower for a healthy colony in alberta is cdn$150 ( canadian honey council [ chc ] 2016 ) . the total production cost to keep a colony in alberta this figure does not explicitly include supplemental targeted treatment costs for pathogens and diseases , which we estimate at $ 13cdn as an average annual supplemental treatment cost for a number of different varroa treatment options including labor ( apiguard [ thymol ] , apilife var [ thymol + essential oils ] , thymovar [ thymol ] , apivar [ amitraz ] , checkmite+ [ coumaphos ] , apistan [ fluvalinate , formic acid fumigation , oxalic acid ] , and hopguard [ potassium salt of hop beta acids ] ; brushy mountain bee farms [ bmbf ] 2016 , dadant 2016 , mannlake 2016 ) . for innately resistant mas colonies , there is no treatment necessary at lower varroa levels of 2% , and at higher loads of 20% , mas colonies are assumed to require half the number of treatments ( $ 6.50 ) . varroa levels in our field experiments are measured as a percent of varroa infestation per colony ( number of mites per 100 bees ) . varroa levels and economic thresholds vary by season and region , with spring thresholds for treatment in the canadian prairies at 1% ( currie 2008 ) . this threshold indicates the level of varroa infestation above which an untreated colony will deteriorate and suffer economic consequences . in our case study , both the lower varroa level ( 2% ) and higher varroa level ( 20% ) are above the treatment threshold at the time of monitoring and treatment . in our colony profit calculations , mas and non - mas colonies untreated mas colonies are more resistant and thus able to reduce varroa levels and mitigate harm more effectively than untreated non - mas colonies , which are taken into account in each colony s deterioration calculations . when we calculate apiary profit , we assume that our beekeeper has chosen to replace some of the non - mas colonies with mas colonies that have already reduced their varroa load to 2% and do not require treatment , and are thus , not susceptible to treatment resistance . the deterioration parameter , , reflects a given colony s ability to mitigate its varroa challenge in the absence of effective treatment . the greater the varroa infestation in a colony , the greater the economic consequences when a colony is not treated effectively . currie and gatien ( 2006 ) showed that honey production alone can fall by as much as 76% per colony owing to a lack of varroa treatment when lower varroa levels of only 2% were identified in the spring . their results also showed that at significantly higher varroa levels of 21% , honey output fell by over 4000% per untreated colony . our deterioration variable in the case study transforms the varroa - infestation level into a loss of colony productivity , which includes increased morbidity , decreased brood production , decreased population growth , and decreased honey production . for our case study , we parameterize the deterioration variable to capture just a portion of this potential loss given varroa levels similar to currie and gatien s experiments . untreated non - mas colonies in our case study experience a 15% decrease in total productivity ( = 7.5 ) at 2% varroa levels and a total productivity decrease of 40% ( = 2 ) when challenged with varroa levels of 20% . innately resistant untreated mas colonies experience no productivity loss at lower varroa levels and a 10% productivity loss ( = 0.5 ) when higher varroa levels of 20% are identified at the time of monitoring . the economic impact of colony mortality is calculated separately from this productivity loss . as significant untreated varroa leads to mortality ( fries et al 2006 , currie and gatien 2006 ) , in our case study , both non - mas and mas colonies with untreated varroa loads of 20% do not survive the season and must be replaced . resistance in varroa mites to acaracide treatments is becoming more common with increasing concern about the associated colony health and beekeeper costs ( spreafico et al . we add a treatment resistance variable , ix , that generates some deterioration of the colony s productivity when treatment is not fully effective ( whenixequals 1 , we have full resistance to the treatment in the varroa mite population ) . as some treatment resistance develops , the treated colony deteriorates , eventually becoming equal to the no treatment case when we have full resistance . we allow treatment resistance to be 0% , 25% , 50% , and 75% in our case study . in the case study , we assume that with the development of treatment resistance in the varroa mite population , the treated non - mas colonies are unable to survive when treatment resistance reaches 50% , when they are challenged with a 2% varroa load , and when treatment resistance reaches 25% with a higher 20% varroa load . for mas colonies , as no treatment is necessary at lower 2% varroa levels , mas colonies survive regardless of treatment resistance , and with higher 20% varroa levels , treated mas colonies are considered unable to survive when treatment resistance reaches 75% . when a colony with higher varroa is not treated effectively , that colony is unlikely to survive the season and will generate reduced profit according to the deteriorated levels of productivity and then will be replaced . the cost of replacing the colony is assumed to be the market cost of purchasing a nucleus colony or a package of bees ( 1 kg of workers and a mated queen ) , which can range from $ 180$230 ( laate 2013 , urban bee supplies [ ubs ] 2017 , valley beekeeping supply [ vbs ] 2017 ) . for our calculations , the cost of replacement is $ 180 . experimental data show colonies that are bred for hygienic behavior exhibit lower levels of varroa ( spivak and reuter 2001 ) and greater survival ( guarna et al . 2016 ) , ultimately requiring less or no targeted varroa treatment depending on levels of infestation . our mas colonies are left untreated under lower varroa pressure , and with higher varroa infestation , we apply half of the typical targeted varroa treatment in a given year ( one treatment at $ 6.50 per colony ) . beekeepers would likely apply a reduced number of treatments in a given year as opposed to half a dose per treatment . beekeeper profit for colony i : iv=[(1v)(hiv+riv)(ci+cv ) ] where iv is profit for colony i with varroa infestation v , hiv is honey revenue , riv is pollination rental revenues , ci is operation and maintenance costs , cv is a targeted varroa treatment cost , is the deterioration from a lack of effective varroa treatment , v(0,1 ) is the varroa load , and (0,1 ) is the rate of treatment resistance in the varroa mite population . (0,1 ) is introduced only when there is evidence of treatment resistance developing in the varroa mite population . honey price is consistent with the statistics canada average honey price for 2015 ( statistics canada [ stats can ] 2015 ) and can also be derived from calculating the $ /lb for honey from the 2014 data in page and darrach 2016 , where the total value of honey was cdn$201 , 620 for 81,556 pounds of honey . this price does not account for the dramatic fall in prices in the fall 2016 ( http://www.cbc.ca/news/canada/calgary/canadian-beekeepers-face-plummeting-prices-1.3746822 , accessed 28 february 2017 ) , and so we also calculate the economic impact of this lower honey price of $ 1.13/lb . quantity of honey produced by a focused honey - producing colony is derived from a 2011 study in alberta , where the average colony produced 143 lbs of honey ( laate 2013 ) , and from our canadian honey bee producer data and discussions collected during the beeipm project . our 12 cooperating producers managed a mixture of honey - producing and commercial pollinating colonies , with an average honey output of just over 110 lbs per colony . some of the high yield honey producers yielded well over 200 pounds . in this case study , our fully productive focused honey - producing colony yields 200 pounds , whereas our commercial pollinator yields 100 pounds . the pollination rental fee received from a canola grower for a healthy colony in alberta is cdn$150 ( canadian honey council [ chc ] 2016 ) . the total production cost to keep a colony in alberta this figure does not explicitly include supplemental targeted treatment costs for pathogens and diseases , which we estimate at $ 13cdn as an average annual supplemental treatment cost for a number of different varroa treatment options including labor ( apiguard [ thymol ] , apilife var [ thymol + essential oils ] , thymovar [ thymol ] , apivar [ amitraz ] , checkmite+ [ coumaphos ] , apistan [ fluvalinate , formic acid fumigation , oxalic acid ] , and hopguard [ potassium salt of hop beta acids ] ; brushy mountain bee farms [ bmbf ] 2016 , dadant 2016 , mannlake 2016 ) . for innately resistant mas colonies , there is no treatment necessary at lower varroa levels of 2% , and at higher loads of 20% , mas colonies are assumed to require half the number of treatments ( $ 6.50 ) . varroa levels in our field experiments are measured as a percent of varroa infestation per colony ( number of mites per 100 bees ) . varroa levels and economic thresholds vary by season and region , with spring thresholds for treatment in the canadian prairies at 1% ( currie 2008 ) . this threshold indicates the level of varroa infestation above which an untreated colony will deteriorate and suffer economic consequences . in our case study , both the lower varroa level ( 2% ) and higher varroa level ( 20% ) are above the treatment threshold at the time of monitoring and treatment . in our colony profit calculations , mas and non - mas colonies untreated mas colonies are more resistant and thus able to reduce varroa levels and mitigate harm more effectively than untreated non - mas colonies , which are taken into account in each colony s deterioration calculations . when we calculate apiary profit , we assume that our beekeeper has chosen to replace some of the non - mas colonies with mas colonies that have already reduced their varroa load to 2% and do not require treatment , and are thus , not susceptible to treatment resistance . the deterioration parameter , , reflects a given colony s ability to mitigate its varroa challenge in the absence of effective treatment . the greater the varroa infestation in a colony , the greater the economic consequences when a colony is not treated effectively . currie and gatien ( 2006 ) showed that honey production alone can fall by as much as 76% per colony owing to a lack of varroa treatment when lower varroa levels of only 2% were identified in the spring . their results also showed that at significantly higher varroa levels of 21% , honey output fell by over 4000% per untreated colony . our deterioration variable in the case study transforms the varroa - infestation level into a loss of colony productivity , which includes increased morbidity , decreased brood production , decreased population growth , and decreased honey production . for our case study , we parameterize the deterioration variable to capture just a portion of this potential loss given varroa levels similar to currie and gatien s experiments . untreated non - mas colonies in our case study experience a 15% decrease in total productivity ( = 7.5 ) at 2% varroa levels and a total productivity decrease of 40% ( = 2 ) when challenged with varroa levels of 20% . innately resistant untreated mas colonies experience no productivity loss at lower varroa levels and a 10% productivity loss ( = 0.5 ) when higher varroa levels of 20% are identified at the time of monitoring . the economic impact of colony mortality is calculated separately from this productivity loss . as significant untreated varroa leads to mortality ( fries et al 2006 , currie and gatien 2006 ) , in our case study , both non - mas and mas colonies with untreated varroa loads of 20% do not survive the season and must be replaced . resistance in varroa mites to acaracide treatments is becoming more common with increasing concern about the associated colony health and beekeeper costs ( spreafico et al . we add a treatment resistance variable , ix , that generates some deterioration of the colony s productivity when treatment is not fully effective ( whenixequals 1 , we have full resistance to the treatment in the varroa mite population ) . as some treatment resistance develops , the treated colony deteriorates , eventually becoming equal to the no treatment case when we have full resistance . we allow treatment resistance to be 0% , 25% , 50% , and 75% in our case study . in the case study , we assume that with the development of treatment resistance in the varroa mite population , the treated non - mas colonies are unable to survive when treatment resistance reaches 50% , when they are challenged with a 2% varroa load , and when treatment resistance reaches 25% with a higher 20% varroa load . for mas colonies , as no treatment is necessary at lower 2% varroa levels , mas colonies survive regardless of treatment resistance , and with higher 20% varroa levels , treated mas colonies are considered unable to survive when treatment resistance reaches 75% . when a colony with higher varroa is not treated effectively , that colony is unlikely to survive the season and will generate reduced profit according to the deteriorated levels of productivity and then will be replaced . the cost of replacing the colony is assumed to be the market cost of purchasing a nucleus colony or a package of bees ( 1 kg of workers and a mated queen ) , which can range from $ 180$230 ( laate 2013 , urban bee supplies [ ubs ] 2017 , valley beekeeping supply [ vbs ] 2017 ) . for our calculations , the cost of replacement is $ 180 . experimental data show colonies that are bred for hygienic behavior exhibit lower levels of varroa ( spivak and reuter 2001 ) and greater survival ( guarna et al . 2016 ) , ultimately requiring less or no targeted varroa treatment depending on levels of infestation . our mas colonies are left untreated under lower varroa pressure , and with higher varroa infestation , we apply half of the typical targeted varroa treatment in a given year ( one treatment at $ 6.50 per colony ) . beekeepers would likely apply a reduced number of treatments in a given year as opposed to half a dose per treatment . we present colony profit results as an average of a focused honey - producing colony s profit and a commercial pollinating colony s profit , except in the case of honey price variability which has differential effects on our two types of colony . apiary profit is calculated based on the profit of 40 colonies , with a varying number of resistant mas colonies replacing at - risk non - mas colonies . effective varroa treatment has an average net profit benefit for non - mas honey and pollinating colonies of 9.7% when varroa levels are 2% ( fig . 3 ) . when faced with higher varroa loads , untreated colonies do not survive the season and as a result , these colony profits subtract the cost of colony replacement , resulting in a net profit benefit of treatment increasing to 128% when varroa levels are 20% . honey and pollinating mas colonies challenged with a 2% varroa load show an average net profit benefit of 4.8% when compared with treated non - mas colonies ( owing to a lack of treatment cost for mas colonies ) , and an even smaller net profit increase of 1.7% when faced with a 20% varroa load for which mas colonies do require treatment . when our beekeeper chooses not to treat his colonies for varroa , we see an average net profit increase of 8.9% for the untreated honey and pollinating mas colonies challenged with a 2% varroa level compared with untreated non - mas colonies . when these honey and pollinating colonies are faced with a 20% varroa load , untreated mas colonies show an average net profit benefit of 96% compared with untreated non - mas colonies . 3honey and pollinating colony average profits for treated and untreated varroa infestations of 2% and 20% with non - mas and mas colonies . * honey and pollinating colony average profits for treated and untreated varroa infestations of 2% and 20% with non - mas and mas colonies . figure 4 shows the net profit effect at an apiary level for a beekeeper who increases the number of resistant mas colonies with 2% varroa levels in his yard . the beekeeper first replaces his non - mas colonies that are challenged with 20% varroa levels , and then once all these high - risk colonies are replaced , the non - mas colonies with 2% varroa are replaced . an apiary of 40 untreated mas colonies generates over 210% more profit than an apiary with 40 untreated non - mas colonies . the net profit gain from adopting mas colonies and replacing untreated non - mas colonies with high varroa loads decreases after the number of mas colonies reaches half the apiary ( 20 colonies ) . once the beekeeper replaces his weakest non - mas colonies that are challenged with 20% varroa and are unable to survive without treatment , the element of colony survival is taken out of the equation , as the remaining non - mas colonies with lower 2% varroa loads and the mas colonies are all able to mitigate mite loads and survive the season . 4forty - colony apiary profit with gradual adoption of mas colonies replacing weakest ( highest varroa loads ) existing non - mas colonies . untreated colonies with high varroa loads do not survive the season and are replaced.table 1distribution of colonies adopted in a 40-colony apiary2% varroa20% varroacommercial pollinatorhoney producercommercial pollinatorhoney producer0 mas colonies10 non - mas10 non - mas10 non - mas10 non - mas10 mas colonies15 ( 10 non - mas + 5 mas)15 ( 10 non - mas + 5 mas)5 non - mas5 non - mas20 mas colonies20 ( 10 non - mas + 10 mas)20 ( 10 non - mas + 10 mas)0040 mas colonies20 mas20 mas00 forty - colony apiary profit with gradual adoption of mas colonies replacing weakest ( highest varroa loads ) existing non - mas colonies . distribution of colonies adopted in a 40-colony apiary when there is 25% treatment resistance within a honey or pollinating colony s varroa population in our case study , we see an average net profit gain of 8.4% with varroa loads of 2% and a 102% average net profit gain under varroa loads of 20% for treated mas colonies compared with treated non - mas colonies ( fig . 5 ) . at 50% treatment resistance , the average net profit gain for a treated mas colony reaches 99.3% at varroa loads of 2% and 112.6% at varroa loads of 20% , when compared with a treated non - mas colony . when there is 75% treatment resistance within a colony , there is a 77.5% average net profit gain for treated honey and pollinating mas colonies compared with treated non - mas colonies . at an apiary level with 50% treatment resistance overall , fig . 6 shows a net profit gain of 145% from replacing 40 treated non - mas colonies with 40 innately resistant mas colonies . 5average honey and pollinating colony profit for treated and untreated varroa infestations of 2% and 20% , with varying levels of treatment resistance within the varroa mite population for mas and non - mas colonies . non - mas colonies are now unable to survive when treatment resistance reaches 50% when faced with a 2% varroa load , and when treatment resistance reaches 25% with a higher 20% varroa load . for mas colonies , as no treatment is necessary at lower varroa levels , mas colonies survive regardless of treatment resistance , and with higher varroa levels , treated mas colonies are considered unable to survive when treatment resistance reaches 75%.fig . 6a 40-colony apiary ( half honey producers and half commercial pollinators ) with gradual mas colony adoption when treatment resistance develops in the varroa mite population at 50% . in this simulation with 50% treatment resistance , all treated and untreated non - mas colonies with 2% and 20% varroa loads are replaced . average honey and pollinating colony profit for treated and untreated varroa infestations of 2% and 20% , with varying levels of treatment resistance within the varroa mite population for mas and non - mas colonies . non - mas colonies are now unable to survive when treatment resistance reaches 50% when faced with a 2% varroa load , and when treatment resistance reaches 25% with a higher 20% varroa load . for mas colonies , as no treatment is necessary at lower varroa levels , mas colonies survive regardless of treatment resistance , and with higher varroa levels , treated mas colonies are considered unable to survive when treatment resistance reaches 75% . a 40-colony apiary ( half honey producers and half commercial pollinators ) with gradual mas colony adoption when treatment resistance develops in the varroa mite population at 50% . in this simulation with 50% treatment resistance , all treated and untreated non - mas colonies with 2% and 20% varroa loads are replaced . when we take into account the recent drop in honey prices in canada from $ 2.50/lb to $ 1.13/lb , both the commercial pollinating colonies and the focused honey producers experience significant drops in profit . at the lower honey price , we see negative profits for all focused honey - producing colonies at all varroa levels , with the highest profit of $3.40 generated by an untreated non - mas colony subjected to a 2% varroa load ( fig . 7a ) . given the lower honey price , with a 2% varroa level , there is no longer an economic incentive to treat colonies or to invest in mas colonies . however , at a 20% varroa level , a focused honey - producing non - mas colony still generates a net profit gain from treatment of over 90% , highlighting the economic benefit of treatment at high levels of infestation . as well , an untreated focused honey - producing mas colony with a 20% varroa level generates 97% more profit than an untreated non - mas colony , pointing to the value of innate resistance as a risk mitigator in the face of ineffective treatments and market fluctuations . for commercial pollinating colonies , we see marginally higher profits with a similar pattern : 30% net decrease in profit from treating a non - mas colony at lower varroa - infestation levels ; and a 116% net profit increase from adopting an untreated commercial pollinating mas colony compared with an untreated non - mas pollinator at higher varroa levels ( fig . 7(a , b ) colony profit for mas and non - mas focused honey - producing colonies and commercial pollinating colonies with variable honey prices and treatment decisions as well as low and high varroa - infestation levels . ( a , b ) colony profit for mas and non - mas focused honey - producing colonies and commercial pollinating colonies with variable honey prices and treatment decisions as well as low and high varroa - infestation levels . marker - assisted selection testing could provide beekeepers with a valuable tool for improving the accuracy and efficiency of their efforts to breed stronger , more resistant colonies in the face of varroa or other pathogen challenges while also reducing their vulnerability to treatment resistance . this case study showed that given that there is a 10128% net profit benefit from treatment for varroa infestations of 2% and 20% , mas honey bee colonies that are innately resistant to varroa will add value to a beekeeping operation of between 9% and over 96% when there is a lack of treatment . innate resistance could play an important role as a risk mitigation tool when treatment resistance threatens to develop in a varroa mite population , increasing net colony profits by between 8% and 112% depending on the level of treatment resistance . we see profit gains of over 200% for our beekeeper in the case study from replacing weaker non - mas colonies that are not able to effectively manage their higher varroa loads with stronger more resistant mas colonies . with a greater number of stronger colonies that have an innate resistance to varroa , treatment becomes less critical , as these colonies are better able to survive without effective acaracide or other treatment . when a colony is faced with a varroa load of 20% and honey prices fall , both treatment and mas adoption are still able to mitigate the negative effects of varroa and minimize the decrease in profits . however , when a focused honey producer or a commercial pollinating colony is challenged with a 2% varroa load and honey prices fall to less than half of their previous level , it is no longer profitable to treat these colonies or to adopt mas colonies . as treatment becomes less profitable under these conditions , however , there is a negative externality as more untreated colonies will populate a beekeeper s apiary , which will spread the varroa infestation to other colonies and potentially result in greater than anticipated deterioration and mortality . fries and camazine ( 2001 ) suggest that selecting for healthier , more disease - resistant colonies in an apiary reduces the risk of intercolony transmission of pathogens , whereas others have shown that healthier colonies are also assumed to resist or absorb the impact of negative elements more effectively than weaker colonies ( cornman et al . there are hidden economic consequences resulting from a lack of treatment that need to be evaluated carefully by a beekeeper . as well , the greater the number of strong , resistant colonies in bee apiaries , the lower the operating costs for beekeepers and the larger supply of pollinator strength colonies available for crop pollination . in the long run , as colony health and strength increase , we are likely to see greater average honey production and higher pollination rental grades and rates for beekeepers . as well , when mas testing comes to market and is adopted by an increasing number of diagnostic labs ( most university protein mass spectrometry core labs can offer these types of analyses with little effort by just adding in honey bee proteins ) , the cost per test will fall , further increasing mas colony profits for beekeepers . our parameterization of the deterioration variable likely captures only a fraction of the productivity impact from a lack of effective treatment , which would mean potentially even greater profit gains from adopting mas colonies . the assumption built into our case study that mas colonies require some treatment when faced with higher varroa loads may further underestimate the economic impact of mas when treatment resistance develops in the varroa mite population . on - going research may reveal that mas colonies have enough varroa resistance that they do not require any treatment and thus are not at all susceptible to the development of treatment resistance . as well , our simulation assumes that non - mas colonies challenged with low varroa loads are able to survive through the season . however , some nonresistant colonies may be unable to withstand even low levels of varroa above the treatment threshold and need replacing , resulting in even greater profit differentials between mas and non - mas colonies . this case study also focuses on the isolated economic impact of adopting queens with the hygienic behavior ( hb ) trait identified through mas in one beekeeper s apiary . as current and future research progresses , mas breeding could be shown to have much broader implications for the honey bee industry , namely , that a ) multiple traits can be identified simultaneously at no increased cost , resulting in greater economic , social , and colony health benefits for the beekeeper . for example , increased revenues from higher honey production alongside decreased mortality from varroa mites and less aggression in the bees ; b ) screening of many colonies at one time becomes possible with mas ; c)mas enables screening breeder queens for markers developed in other countries , such as screening new zealand queens for canadian winterability ; and finally d ) mas screening could provide a valuable insurance policy for beekeepers who are faced with mitigating the risk of impending treatment failure for a number of pathogens , diseases , and pests . marker - assisted selection breeding tools have the potential to increase colony profit and reduce mortality , which could contribute positively to the continued viability of the apiculture industry globally , particularly in the face of increased varroa infestations and the risk of treatment resistance . more research into the effects of hygienic behavior and more specific varroa - sensitive hygiene ( vsh ) would allow us to better predict the impact of the hb or vsh trait on highly infected colonies . as well , further scientific inquiry into the genomics of honey bees and the correlations between trait identification , expression , treatment resistance , and the marginal benefit to the beekeeper is necessary . finally , expanding the selective breeding queen supply sector in canada and globally through knowledge translation to industry about selective breeding tools is essential for optimal adoption .
astrocytes , a major glial cell type in the central nervous system , have emerged as powerful regulators of brain function where they maintain ion , metabolic , and neurochemical homeostasis . astrocytes form organized networks that communicate through gap junctions formed by hemichannel proteins called connexins , potentially creating a functional syncytium.1 the close structural and functional relationship between the presynaptic bouton and the postsynapse , both engulfed by astrocyte processes , has been named the tripartite synapse.2 thus , by extending processes that wrap around brain capillaries and synapses , astrocytes control neurovascular coupling , as well as synaptic transmission.3,4 increasing evidence has specified their important functions in the nervous system , regulating blood brain barrier permeability , cellular responses to brain injury , formation and elimination of synapses , synaptic maintenance and plasticity , as well as axon myelination in the adult brain , among others.58 table 1 shows a summary of the core functions of astrocytes in relation to neuronal function . though most of this evidence has been obtained in vitro , encouraging additional evidence comes from in vivo models , as recently revealed by measures of astrocytic ca dynamics with high - resolution ca imaging in living mice.9 the nature of astrocyte - to - neuron interactions are mediated by direct cell - to - cell contact as well as by a complex array of astrocyte - derived molecules , ie , the astrocyte secretome , that varies according to neuronal activity and astrocyte activation and thus according to physiological or pathological conditions.3,10 it has been recently proposed and in part documented that the different cell types in the central nervous system could communicate by means of extracellular vesicles , among which small extracellular vesicles , also named exosomes , have received intense attention.11 however , the involvement of exosomes in astrocyte - to - neuron communication has not been explored in depth . thus , in this brief commentary , we will discuss the new exciting possibilities related to the interaction of astrocyte - derived exosomes with neurons in the nervous system , focusing on the content of micrornas ( mirnas ) present in these vesicles . cells release a variety of extracellular vesicles that differ in their subcellular origin and , possibly , in their biology . among them , exosomes are defined by their endosomal origin , storage in multivesicular bodies , and characteristic size of ~30120 nm . by fusion of multivesicular bodies with the plasma membrane , exosomes are released by exocytosis from most eukaryotic cells.32 these vesicles carry a large diversity of molecules , such as rna species , proteins , and lipids , that can modify the physiology of nearby or distant target cells . the current knowledge on the mechanisms involved in the biogenesis and secretion of exosomes , as well as their possible functional interaction with target cells , has been described in several excellent recent reviews.3235 most of this knowledge relies on the isolation of exosomes from extracellular fluids by differential ultracentrifugation and further characterization by size and presence of protein markers.36 however , a major problem has more recently been identified : this isolated exosome - like small extracellular vesicle fraction contains a range of small vesicle subpopulations consisting of vesicles of larger size ( > 150 nm ) or of nonendosomal origin , while in most exosome studies , their endosomal origin has not been demonstrated.37 thus , the identification of specific protein markers and general protein markers among vesicle subpopulations as well as the implementation of additional purification steps leading to homogeneous preparations are under active research . in this commentary , we will focus on the available literature in the field that uses mostly small extracellular vesicle preparations obtained by ultracentrifugation that are enriched in exosomes , as revealed by the current available experimental tools . despite the large amount of evidence about exosomes derived from cells of the nervous system,38 specific research about astrocyte - derived exosomes is scarce . some of the pioneers evidences for the presence of exosomes in astrocyte cultures came from milligan s and agnati s group in 2007 and 2010 , respectively . they found the presence of classical exosome markers in a fraction obtained after the well - accepted differential centrifugation protocol.39,40 three years later , it was shown that astrocyte - derived exosomes harbor synapsin-1 , a glycoprotein that was released from these extracellular vesicles following an increase in extracellular potassium levels . although they did not show a direct effect of these exosomes on neurons , it was suggested that released synapsin-1 could have a positive effect on neurite outgrowth and neuronal survival.41 soon after , it was reported that astrocytes exposed to -amyloid both in vitro and in vivo release exosomes that trigger apoptosis in other astrocytes , acting in an autocrine fashion.42 using a different strategy , astrocytes may use extracellular vesicles , probably including exosomes , to transport the excitatory amino acid transporters eaat-1 and eaat-2 , essential components in the maintenance of glutamate homeostasis . interestingly , this extracellular vesicle fraction is able to actively take up aspartate , suggesting that in addition to their potential role in cell cell communication , exosomes may participate in glutamate clearance.43 interestingly , members of the heat shock protein 70 family as well as astrocyte - specific glycolitic enzyme aldolase c have also been found in this fraction , raising the question about the potential transfer of these proteins with functional consequences to neurons.39,44 accordingly , it was recently shown that astrocytes release exosomes containing nonpathogenic prion protein that can be transferred to neurons , inducing protection after exposure to a series of stressors such as hypoxia , hypoglycemia , and ischemia.45 thus , astrocyte - derived exosomes are capable of transferring proteins and lipids to target cells to modify their function.42 strikingly , astrocyte - derived exosomes may have far - reaching consequences in vivo : they facilitate the outgrowth of metastatic cells in the brain by inhibiting the expression of the tumor suppressor phosphatase and tensin homolog ( pten ) through a functional transfer of mir-19a.46 nevertheless , as far as we know , there is no functional evidence of exosome - mediated transfer of astrocytic mirnas to neurons . after the first evidences were provided by valadi et al , the functional transfer of exosomal components such as proteins and nucleic acids to elicit responses on target cells has been reported in several studies47,48 and has been summarized recently.35 next , we will focus here on the possible transfer of mirnas from astrocytes to neurons by means of exosomes . exosomes as well as other extracellular vesicles contain mrnas and a plethora of noncoding rnas , among which mirnas stand out for their capacity of reprogramming protein expression in recipient cells . a long precursor mirna molecule is transcribed in the nucleus , and after several sequential processing steps by rnase complexes , it is transported as pre - mirna to the cytoplasm.4951 here , the pre - mirna hairpin is cleaved by the rnase dicer producing the ~22 nucleotides , double - stranded mature form of the mirna.52 the mature form is loaded onto the rna - induced silencing complex ( risc ) , where one of the strands ( ie , the passenger strand ) is degraded after recruiting argonaute proteins while the other , called the leading strand , is held inside the mature complex carrying argonaute 2 ( ago2).53 after this processing , a short sequence of 68 nucleotides or seed sequence of the mirna is capable of targeting the 3 utr region of mrnas to inhibit translation by two mechanisms : ( 1 ) the direct degradation of the mrna or ( 2 ) translational repression by reducing the recognition capacity of ribosomes to mrna.54 as each mirna harbors target - recognition motifs in as many as hundreds of mrnas , they are able to control complex biochemical processes in a coordinated manner and thus , disease and health states . it is important to consider that genes coding for mirnas tend to form clusters , defined as groups of genes that are separated by no more than 1 mb between them , in such a way that three to six mirna genes are usually found in one cluster . all of these genes may be transcribed as polycistrons and , surprisingly , have preference for targeting functionally related genes.55,56 for example , one of the most studied mammalian clusters is mir-17 - 92 , which is composed of six members : mir-17 , mir-19b , mir-20a , mir-92 , mir-18a , and mir-19a . all of them , except mir-18a , are demonstrated to downregulate pten , inducing axonal elongation.57 interestingly , all of these mirnas have been previously described in astrocytes , and mir-19a is contained in exosomes.46 in addition , mir-26a , although located outside this cluster , also favors neurite / axonal elongation by targeting pten as well as glycogen synthase kinase-3 ( gsk-3 ) ( see below).58,59 the loading of mirnas into exosomes leads to a specific enrichment of some mirnas in them in comparison to their originating cells , mediated by sorting mechanisms that include : ( 1 ) the presence of specific mirna sequences ( including uridylated mirnas and exomotifs ) , ( 2 ) mirna binding to lipid raft - like regions in the cytoplasmatic face of the multivesicular body limiting membrane , and ( 3 ) interactions with proteins such as sumoylated heterogeneous nuclear ribonucleoproteins or ago2.60,61 the participation of ago2 in mirna loading is further demonstrated because its kras mek erk pathway - dependent phosphorylation decreases the sorting of ago2 and its associated mirnas to exosomes.62,63 although mirnas are selectively retained or released by different cell types,48,6466 the mechanisms underlying selective packaging of mirnas into astrocyte exosomes needs to be further explored . over the past few years several studies have provided a deeper insight into the mirna species that are expressed or enriched in astrocytes , or even unique compared to other cns cell types ( eg , neurons , oligodendrocytes , and microglia).6772 we will summarize some of the reported mirnas and their functional relevance in different cellular contexts . some of the mirnas found in astrocytes may be crucial to determine a glial fate ( as opposed to a neuronal one ) from progenitor cells.73 mir-146a , for example , may prevent astrocytes from expressing neuronal proteins,67 and provided that certain conditions are met , mir-34a can promote astrogliogenesis.74 mir-9 and mir-124 provide another example of a mirna in regulating cellular differentiation fate . if the mir-124 binding site in the 3utr of the histone methyltransferase enhancer of zeste homolog 2 ( ezh2 ) is manipulated as to prevent the action of this mirna , astrocytic differentiation is enhanced ( and neuronal differentiation diminished).75 consistent with these results , overexpression of mir-124a or mir-9 in neural precursor cells promotes neurogenesis and reduces gliogenesis.76 studies performed in different brain regions of humans show that many mirnas are common to all astrocytes , regardless of the brain region analyzed , whereas others are unique to certain areas ( eg , mir-129 and mir-181a were not found in interlaminar astrocytes , but were detected in astrocytes from deep cortical layers ) , suggesting their involvement in the regulation of factors specific to those regions . in line with the previously mentioned results , a low expression of mirnas involved with inflammatory responses was observed in fetal compared to adult white - matter astrocytes . a differential expression was also observed between adult interlaminar astrocytes compared to those found at the fetal germinal matrix , as the latter contains mirnas involved in neurogenesis with antiapoptotic properties ( eg , mir-210 , mir-129 , and mir-214).77 in various pathological contexts , astrocyte mirnas have been shown to increase or decrease their levels , as summarized in table 2 . the highly expressed mir-29 family in astrocytes is transported with functional consequences in exosomes.82 when the expression of mir-29b increases by exposure to pathological conditions in the brain and in cultured astrocytes , its derived exosomes reduce neuronal viability by targeting the platelet - derived growth factor ( pdgf - b).78 surprisingly , transfecting a mimic of mir-29a into cultured astrocytes protects them from oxygen - glucose deprivation.79 as the targets of mir-29 are both pro- and antiapoptotic , this apparently controversial effect can be explained by different mrnas being overexpressed and thus targeted under diverse physiological or pathological conditions.70 another frequently addressed neuropathological phenomenon is astrogliosis . for example , after spinal cord injury , the activation of astrocytes involving the overexpression of astrocyte - specific genes such as the glial fibrillary acid protein favors the repair and limits the inflammation of the damaged zone.80 the concomitant increase of mir-21 , however , attenuates the beneficial astrocytic response to injury and limits axon numbers in the scar zone . although the targets of mir-21 explaining this effect are not clear , inhibiting mir-21 facilitates recovery and increases axon density.81 the expression of mir-146 is upregulated in tissue obtained from patients with intractable epilepsy , especially in areas of noticeable gliosis . this mirna regulates inflammation : in cultured astrocytes , its upregulation reduces the interleukin-1 ( il-1 ) mediated increase of interleukin-6 ( il-6 ) and cyclooxygenase 2 ( cox-2 ) , two molecules associated with inflammatory gliosis.82 in tissue obtained from multiple sclerosis patients , most of the mirnas induced by the lesions are present in astrocytes , with a subset of these being upregulated by cytokines ( eg , mir-23a , mir-146a , and mir-155 ) . some of these mirnas ( mir-155 , mir-326 , and mir-34a ) regulate the expression of cd47 , leading to the release of macrophages and promoting myelin phagocytosis.83 a differential expression of astrocytic mirnas has been observed after oxygen - glucose deprivation , and this expression profile changed with time after deprivation , as was the case with mir-29b and mir-21 , upregulated after 12 hours , or of mir-30b and mir-107 , upregulated after 6 and 8 hours after oxygen - glucose deprivation , respectively.84 the levels of mirnas in astrocytes also change after exposure to various stressors that increase oxidative stress such as a-42,85 ammonia,69 and transfection with human heme oxygenase 1 , an enzyme induced by oxidative stress that is overexpressed in a number of cns disorders.68 in line with these observations , exposing astrocytes to cytokines and pro - inflammatory molecules ( eg , interleukins , tumor necrosis factors , and interferon gamma ) has a profound impact on the expression of mirnas.73,82,83,8588 though still speculative , the prospect that some of the abovementioned mirnas may be found in exosomes circulating in body fluids and thus amenable to be used as biomarkers , or the possibility of engineering exosomes with sequences that either mimic or antagonize these mirnas to target astrocytes under pathological conditions remains an exciting topic of exploration . an extensive rna sequencing profile of astrocyte - derived exosomes under different physiological or pathological conditions is a must to predict the main functional pathways that exosomal mirnas modify on target cells . on the next part of this commentary , we will focus on the possible transfer of mir-26a within exosomes based on the following arguments : ( 1 ) it is highly expressed in astrocytes when compared with neurons,72 ( 2 ) we have found mir-26a to be present in astrocyte - derived exosomes ( unpublished data ) , ( 3 ) this mirna is dysregulated in several cns diseases ( see below ) , and ( 4 ) mir-26a can be sorted to exosomes and transported by these vesicles in the plasma , serum , whole blood , urine , or secreted in vitro by human umbilical vein endothelial cells.9094 altered levels of mir-26 have been reported for some of the most prevalent disorders of the cns . in human postmortem tissue , this mirna can be upregulated or down - regulated , depending on the brain region , at different stages of alzheimer s disease ( ad)9599 as well as in biofluids ( eg , blood , serum , and cerebrospinal fluid ) obtained from ad patients.100,101 increased levels of mir-26b have been observed in the substantia nigra of patients with parkinson s disease ( pd),102,103 as well as in the blood of pd patients who underwent treatment compared with those who did not.104 levels of mir-26a are enhanced in the relapsing phase of patients with relapsing - remitting multiple sclerosis compared to the remitting phase and to healthy controls.105 patients diagnosed with major depression showed lower blood levels of mir-26a / b,101 while treatment with the antidepressant escitalopram increased its blood levels.106 patients diagnosed with schizophrenia showed lower blood levels of mir-26a / b,101 while the precursor and mature mir-26b was upregulated in postmortem tissue from the superior temporal gyrus and the dorsolateral prefrontal cortex107 and downregulated in postmortem tissue from the prefrontal cortex.108 during neurogenesis , an increase in the level of some mir-26 family members has been reported,109111 in contrast to pre - mir-26,72 which remains at constant levels.110 in zebrafish , mir-26b targets the mrna of the c - terminal domain of the small phosphatases ( ctdsp2 ) gene . as the sequence for mir-26b is localized in one of the introns of ctdsp2 , this is a case where a gene is transcribed simultaneously with the mirna that regulates its expression . for neuronal differentiation to occur , mir-26b has to inhibit ctdsp2 translation , therefore allowing for genes containing the repressor element 1 ( re-1 ; otherwise inhibited by ctspd2 ) sequence to be expressed . mir-26a is also able to repress the expression of ctdsp2 and shows a upregulation similar to mir-26b during progression of neurogenesis.110,112 the predicted mrna targets of a mirna are commonly validated by tools such as quantitative real - time polymerase chain reaction ( qrt - pcr ) , western blots , or luciferase reporter assays . the first two do not exclude that the decrease of a given mrna or protein is secondarily associated with the downregulation of the mirna target sequence . however , the luciferase reporter assay provides direct evidence of mrna targets , because the expression of the luciferase reporter 3-utr construct will decrease in the presence of a regulatory mirna , which is able to interact with the introduced 3-utr sequence.113 figure 1 contains the proposal of the present commentary , in which astrocyte - derived exosomes carrying mir-26a will influence neuronal function and brain physiology through validated and predicted targets . one of the validated targets of mir-26 members in neurons and other cell types under physiological / pathological conditions is gsk-3.59,114116 gsk-3 is a cytosolic protein that takes part in the canonical wnt pathway , and it is capable of forming a complex with -catenin and two other proteins : axin and adenomatous polyposis coli.117 in the absence of wnt signaling , -catenin is phosphorylated by gsk-3 and degraded.118 however , under wnt signaling , gsk-3 is inhibited , allowing the stabilization of -catenin and its translocation into the nucleus to activate the expression of wnt target genes.119,120 another target of mir-26 detected in neurons is the phosphatase pten , a negative regulator of the phosphoinositide 3-kinase ( pi3k)/akt pathway.58 pi3k / akt inhibits the previously mentioned target of mir-26a gsk-3 , by phosphorylation of a serine residue.121 inhibition of pten increases neurite outgrowth both in vitro and in vivo,122125 and these changes resemble those observed under mir-26a regulation , with the concomitant decrease in pten levels and increase in akt protein levels.58,126 other targets validated by luciferase reporter assays include wnt5a,127,128 a ligand of the noncanonical , -catenin independent pathway,129 and brain - derived neurotrophic factor ( bdnf),130 a growth factor that controls a wide range of mechanisms in the cns , including plasticity and synaptic maturation.131 in hippocampal neurons , the exogenous addition of mir-26a mimics induces an increment in axonal length;111 consistently , electroporation of an inhibitor of mir-26a prevents axonal regeneration in vivo after sciatic nerve crush.59 neurite outgrowth is also enhanced by the addition of mir-26a in dorsal root ganglia and in primary cortical neurons.58,126 in mature primary hippocampal neurons ( ie , 14div ) , transfection with a mir-26a - expressing plasmid or a mir-26a sponge did not affect spine area or density , but mir-26a did inhibit the spine enlargement induced after 90 minutes of chemical long term potentiation ( ltp ) . reduction in the levels of mir-26a is necessary for maintenance of ltp but not for its induction , and the effects of mir-26a on both , spine remodeling and ltp , was mediated by the mir-26a targeting of ribosomal s6 kinase 3 ( rsk3).132 this evidence suggests that this mirna could have a differential effect in two different neuronal compartments : the dendritic and the axonal arborization . outside the cns , other targets of mir-26a have been validated : plasminogen activator inhibitor 1 rna - binding protein ( serbp1),133 phosphatidylinositol-4-phosphate 3-kinase c2 domain - containing alpha polypeptide ( pik3c2),134 and mothers against decapentaplegic homolog 1 and 4 ( smad1 and smad4).135137 cyclooxgenase-2 ( cox-2),138 metadherin ( mtdh),139,140 cyclin - dependent kinases regulatory subunit 2 ( csk2),141 enhancer of zeste homolog 2 ( ezh2),139,142,143 estrogen receptor alpha ( er),144 pleiomorphic adenoma gene 1 ( plag1),145 empty spiracles homeobox 2 ( emx2),146 high mobility group at - hook 1 ( hmga1),147 cyclin - dependent kinase 6 ( cdk6 ) , cyclin e1,148 and cyclins d2 and e2.149 further experiments will be needed to study if mir-26a is also regulating some of these targets in the cns . last , it is worth mentioning that in silico studies of predicted targets for mir-26a include mrnas that express subunits for receptors involved in synaptic transmission , such as gabar , ampar , and nmdar . these need to be validated in the cns and the role of exosomes derived from astrocytes on neuronal physiology promises to be an exciting area of study . such a mechanism of communication suggests a new level of complexity in the processing of information in the cns . mirnas stand out as key regulators of cellular processes , due to their capacity to inhibit hundreds of different mrna targets . we have focused on mir-26a in this review , as it targets mrnas that may impact neuronal function and morphology , and its dysregulation has been implicated in many neurological disorders , such as depression and ad . as such , it may be an interesting therapeutic target and biomarker for pathologies of the cns . this and other mirnas packaged in exosomes of astrocytic origin may open the doors to a better understanding of how astrocytes impact on neuronal functions , and it may also provide us with new tools to compensate for cellular malfunctions under pathological conditions .
there is considerable evidence suggesting that cancer patients suffer from substantial and long - term psychological distress associated with different forms of cancer and its medical treatment . cancers and any type of treatment may lead alteration in patients appearance . this issues can affect the quality of life ( qol ) , especially may reduce self - confidence , decrease self - esteem , having a problem with social interest , and finally may result in social withdrawal . studies have shown that psychological stress has a strong impact on accelerating the growth of various types of malignant tumors . hence , psychosocial management of adjustment problems experienced by people with cancer seems to be an obvious requirement for a more effective treatment of the disease . recently , indicators of medical outcome for cancer patients have expanded to include qol along with the survival rate , and accordingly , comprehensive patient care has focused on psychological as well as physical aspects . cancer and it 's treatment has a substantial impact on mental and social health and consequently , on qol of patients . patient 's psychological state and qol should be considered in the light of factors such as the stage of disease and treatment process . furthermore , it is important to recognize the relationship between psychological state / qol and individual factors such as personality and coping style . coping style , the individual cognitive response to stressors acknowledged by folkman in the 1980s , might function as a mediating variable controlling stress burden . on the other hand , coping style could conceivably be the cause or result of a patient 's psychological state / qol . there are three general styles of coping with stress : problem - focused coping style , emotion - focused coping style , and avoidance style . given the high costs that cancer imposes on patients and society , and considering the relationship between coping style and qol in cancer patients , we can learn more effective coping styles for cancer patients to improve their qol so that their psychological and even physical complications are reduced . the problem which we studied can be formulated in the following questions : what styles of coping do patients with cancer adopt?what is the state of qol of patients with cancer ? what styles of coping do patients with cancer adopt ? what is the state of qol of patients with cancer ? eventually , in this study our primary aims were an evaluation of coping styles and qol in cancer patients , and our secondary aim was an investigation of the relationship between coping styles and qol in cancer patients . in this study , patients with cancer who were referred to health centers affiliated with kermanshah university of medical sciences ( both inpatient and outpatient ) were studied . inclusion criteria entail : over 18-year - old patients with diagnosed cancer that tended to participate in this study . exclusion criteria include : patients with any chronic physical or mental illness ( other than cancer ) . drug or psychotropic addiction were excluded , also patients who were in the end stage of cancer who could not communicate or interviews bothered them and patients who did not tend to collaborate . demographic data including : age , sex , marital status , salary , home location , education and cancer type was recorded via a self - structured questionnaire . type of treatments at the time of the study was considered and recorded . in the present study , endler and parker questionnaire was used to investigate patients stress coping styles and using world health organization 's quality of life questionnaire ( whoqol - bref ) , patient 's qol was evaluated . endler and parker questionnaire comprises 48 items on likert scale from never ( 1 ) to very high ( 5 ) . the inventory consists of three main scales : task - oriented coping ( toc ) , emotion - oriented coping ( eoc ) , and avoidance - oriented coping ( aoc ) , with the last one with two subscales : engagement in substitute activity and seeking for social relationships . total qol was measured using the iranian version of the whoqol - bref questionnaire which has been validated in our local iranian population . the whoqol - bref questionnaire consists of 26 questions and is based on a four - domain structure including : physical health activities of daily living , dependency on medical substances and medical aids , energy and fatigue , mobility , pain and discomfort , and sleep and restpsychological body image and appearance , negative feelings , positive feelings , self - esteem , spirituality / religion / personal beliefs , and thinking , learning , memory , and concentrationsocial and personal relationships , social support , and sexual activity andenvironmental , including financial resources , freedom , physical safety and security , health and social care . physical health activities of daily living , dependency on medical substances and medical aids , energy and fatigue , mobility , pain and discomfort , and sleep and rest psychological body image and appearance , negative feelings , positive feelings , self - esteem , spirituality / religion / personal beliefs , and thinking , learning , memory , and concentration social and personal relationships , social support , and sexual activity and environmental , including financial resources , freedom , physical safety and security , health and social care . the scores ranged from 24 to 120 for the total qol and 735 for the physical health , 630 for the psychological health , 315 for the social relationships , and 840 for the environment domains . chicago , usa ) using descriptive statistics ; correlation test was used to determine the correlation between qol and various copying styles . chi - square test was used to compare ratios for instance percentages ; student 's t - test was used to compare the mean value of two quantitative group data and analysis of variance to compare averages between more than two groups . in the present study , 150 patients with cancer ( 71 females and 79 males ) were investigated . 83 patients were in the young group ( 2039 years ) , 60 in the middle - aged group ( 4059 years ) and seven in the elderly group ( above 60 years ) . n = 120 ) were single and 20% ( n = 30 ) were married . in the case of salary , 13.5% were in the high - salary group , 59.0% with medium - salary , and 23.7% had low salary . the most observed cancer type was breast cancer , and the least was uterus cancer [ table 2 ] . our study showed 17.3% of cancer patients were in stage 1 , 50.0% were in stage 2 , 23.7% in stage 3 , and 4.5% in stage 4 . relationship between gender , marital status , location , education , and salary with quality of life mean value of quality of life in different type of cancer in patients about 44.2% of patients were undergone radiotherapy ; 73.7% were undergone chemotherapy , and 68.6% were undergone surgery operations . there was no relationship between the type of treatment and copying style . according to oncologist prescription , 4.5% of patients who were in stage 4 of cancer and were considered end stage just have received palliative cares and other patients have received both of curative and palliative cares . as illustrated in table 1 , mean value of qol was significantly higher in men than in women ( p = 0.006 ) , it was also significantly higher in single patients than in married ones ( p = 0.003 ) . furthermore , there was a significant relationship between qol and salary ; it was higher in patients with high income ( p = 0.017 ) . age had a significant negative correlation with qol ( p = 0.034 , r = 0.174 ) . furthermore , higher education showed a significant positive association with qol ( p = 0.014 ) . there was a significant relationship between qol and cancer type ( p = 0.00 ) . testis cancer group had the highest mean value of qol while patients with uterus cancer showed the lowest mean value of qol [ table 2 ] , ( p = 0.000 ) . comparing different therapeutic methods , the surgery caused a significant difference in qol of patients while such difference was not observed in the other methods including chemotherapy and radiotherapy [ table 3 ] . regarding the impact of stage of the disease on the qol of patients , the only significant difference was observed in patients with stage 3 of cancer with patients in stages 1 and 2 , as illustrated in table 4 . difference in quality of life - based on therapeutic method quality of life in patients in different stages of disease statistical analysis showed copying type was significantly associated with cancer type ( p = 0.028 ) , [ figure 1 ] . as it is depicted in the graph , patients with leukemia , ovary , uterus , lung , and colon cancer used problem - based copying type more than other copying types , while in breast and skin cancer , employment of problem and emotion - based copying types was more than avoidant copying style . association between cancer type and copying type in cancer patients there was not any significant association between copying type and income ( p = 0.33 ) , but patients with medium salary used problem - based copying type more than other patients [ figure 2 ] . association between salary and copying style in cancer patients the results also showed that qol of cancer patients have a positive correlation with avoidant coping style ( p = 0.038 , r : 0.170 ) , while there was a significant negative association between emotion - focused coping styles and qol ( p = 0.000 , r : 0.378 ) , [ table 5 ] . both in women and men , no significant correlation was found between qol and problem - focused and avoidant coping styles , but qol and emotion - focused coping style had a significant negative relationship ( women : p = 0.000 , r : 0.532 , men : p = 0.027 , r : 0.249 ) [ table 5 ] . correlation between quality of life ( in general , in different genders , and different age groups ) and different copying styles in young adults ( 2039 years ) , a significant negative relationship was observed between qol and both problem - focused coping style ( p = 0.012 , r : 0.275 ) and emotion - focused coping style ( p = 0.000 , r : 0.423 ) . the relationship between qol and avoidance coping style was not significant . in the middle - aged group ( 4059 years ) qol was significantly associated with problem - focused coping style ( p = 0.012 , r : 0.925 ) , also a significant positive relationship was found with avoidant coping style ( p = 0.008 , r : 0.341 ) . a significant negative relation with emotion - focused coping style also was observed ( p < 0.01 , r : 0.423 ) . in the elderly group ( over 60-year - old , 7 cases ) , qol and problem - focused style the correlations of qol with emotion - focused and avoidant coping styles were not significant ( p > 0.05 ) , [ table 5 ] . it also showed in single patients and those with a higher salary ; qol was significantly higher than that in other patients . arthritis , perceived health - related quality of life ( hrqol ) were similarly found higher qol in male than female patients . chang et al . in a cross - sectional study , investigated gender impacts on hrqol and related factors of postoperative lung - cancer patients . they also showed male participants had better physical , role , emotional , and cognitive functioning , so they had higher qol . however , they showed married patients had significantly higher qol , which is not in accordance with our findings . demonstrated income is positively associated with qol and in another study it has been reported that qol of cancer patients get worse with age . one of the contributing factors of lower qol in cancer patients can be a high prevalence of anxiety in these patients . . reported a weak significant negative correlation between anxiety score and total qol among prostate cancer patients . furthermore , in another study it has been demonstrated that stress can be negatively correlated with qol . we used the endler and parker and whoqol - bref questionnaires to assess coping styles and qol among cancer patients , showing that the emotion - focused coping style , in particular , were related to qol , so that patients tending to use this coping style more probably experienced lower qol than others . young adults ( 2039 years ) with problem - focused coping style and emotion - focused coping style showed significantly lower qol . in the middle - aged group ( 4059 years ) , qol was significantly higher in those with avoidance and problem - focused coping style , and lower in patients with emotion - focused coping style . moreover , in the elderly group ( over 60 years ) , the only positive correlation between qol and problem - focused coping style was significant . the results of this study showed there was a significant negative association between qol and emotion - focused coping style in both women and men . however , there was no significant association between avoidance and problem - focused coping styles with qol in both sexes . in a study , on gender differences in stress and coping styles , matud reported compared to men , women used more emotional and avoidant coping styles , and their coping style was more emotion - focused . in a review article on cancer patients , baider and bengel revealed women initiate and communicate emotions while men try to avoid or withdraw from feelings and emotional demands . several studies have shown in chronic diseases ; patients used positive and problem - focused coping styles less than other copying styles . in a study , on 78 elderly patients ( over 70 years ) and 105 younger patients ( 4060 years ) with carcinoma of the oral cavity and pharynx , reported younger patients often use more active coping styles while older patients used religious coping styles in all assessments . in both groups , avoidant coping style was associated with more depressive symptoms . a study on 131 cancer patients in india has shown that the level of qol in a patient with cancer were increased by chemotherapy treatment . as we have mentioned above , in young adults we observed those with problem - focused coping style and emotion - focused coping style showed significantly lower qol . we did not find plenty of relevant references in literature , but according to eslami et al . study on congenital heart disease patients , the active problem - solving coping style was associated with never married marital status , parenthood , unemployment , higher level of anxiety / somatic symptoms , lower level of depressive symptoms , and better social support . from this study , we can interpret young adults with problem - solving copying style may experience some of these negative situations which can be a contributor to lower qol among them . similar to our findings , a study in japan that aimed to assess coping styles in 85 patients with gastrointestinal cancer showed the higher the score of eoc style , the bigger the decline in qol subscales . under the same conditions , the tendency to demonstrate eoc style ( i.e. , whether or not to respond emotionally ) could in part establish qol . a substitute view might be that the lower the qol value becomes , the more emotionally a patient is possibly to react . therefore , it would appear more likely that a person with a trait of adopting a particular coping style would tend to experience a decline in qol level as a result , rather than that a person would select a special coping style as a response to a preexisting low qol . in a nutshell , in very stressful conditions , a person who had a negative coping style originally would have a more negative coping style , although a person who usually had a positive coping style was likely to take on a negative coping style . studied the relationships between coping style assessed using the coping inventory for stressful situations and personality characteristic assessed using the munich personality test , and reported that the toc style was associated with extraversion and frustration tolerance neuroticism , esoteric tendency and isolation tendency , and aoc style was linked to extraversion . this recommends that people who tend to implement a more eoc style might be likely to assess their circumstances negatively rather than affirmatively . with regard to coping style among cancer patients , greer et al . have reported a significant correlation between recurrence rate after 15 years and coping style assessed using mental adjustment to cancer and that between life expectancy and coping style . in addition , according to studies by ho et al . using mini - mental adjustment to cancer , dunkel - schetter et al . using ways of coping - cancer version , and akechi et al . using the japanese version of mac , coping style could influence the psychological state in cancer patients . therefore , the psychological aspect of qol depends on the way in which individuals identify their disease state , treatment conditions , and associated circumstances and on how they deal with this . hence , in the care of cancer patients , even if it is not probable to change a patient 's coping style predominantly , we could optimize care by focusing on their individual coping style . as a result , we could not only discriminate whether or not the patient is in a risk group but also deliver and plan efficient and specific care by recognizing the patient 's coping style . moreover , such a psycho - educational approach might enable patients to acquire a more appropriate coping style . this study had some limitations including : lack of patient 's tendency to participate in the study . in general , emotion - focused coping style was associated with lower qol in cancer patients . the results recommended that focusing on a patient 's coping style , predominantly on an eoc style , is an important and that patients possibly to adopt a more eoc style should be given enough attention , particularly before discharge .
to date , according to the hugo database ( www.genenames.org ) , 21 lysine demethylases have been identified in the human genome , most of which target histones in a residueselective manner.5 nineteen demethylases belong to the jumonji domaincontaining dioxygenase family , while lsd1 and lsd2 ( kdm1b ) are the only members of the flavindependent amine oxidase family , which require flavin adenine dinucleotide for their enzymatic activity ( fig . biochemical and cellbased analyses revealed that h3k4me was the endogenous substrate of lsd1 ( fig . 1b).7 , 8 up to three methyl groups form cohesive bonds with h3k4 , which are generally recognized as an active chromatin signature in cells . monomethylated h3k4 is a hallmark of enhancer regions , while dimethylated and trimethylated h3k4 are enriched in active promoters.4 because a lone pair of electrons in the nitrogen atom in the methylated lysine is required for catalytic activity , lsd1 is capable of demethylating monomethylated and dimethylated lysine but not trimethylated lysine.9 in addition to the catalytic amine oxidase domain , lsd1 contains two unique structural domains that are closely associated with its molecular function ( fig . the tower domain contains two antiparallel helices forming a protruding structure , which serves as a platform for sant ( swi3 , ada2 , ncor and tfiiib ) domain proteins , such as corest and metastasisassociated protein , mta.10 association of lsd1 with these proteins facilitates demethylation activity.11 , 12 because these binding partners form hdaccontaining repressor complexes , lsd1mediated h3k4 demethylation is linked to the transcriptionally repressive chromatin structure.12 , 13 basic characteristics of lysinespecific demethylase1 ( lsd1 ) . lsd1 is involved in the repression of promoter and enhancer activities through the regulation of h3k4me , while activating transcription through h3k9 demethylation in cooperation with the androgen receptor ( ar ) . lysinespecific demethylase1 has also been implicated in transcriptional activation through h3k9 demethylation activity but in limited circumstances . upon stimulation by androgen receptor ( ar ) agonist , lsd1 facilitates the demethylation of h3k9 and , thus , augments armediated transcriptional activation in prostate cancer cells ( fig . 1b).14 a recent study by laurent et al . demonstrated that a splice variant of lsd1 containing an extra four amino acids , which is selectively expressed in the neural cell lineage , mediates h3k9 demethylation possibly through an indirect mechanism.15 , 16 structural models have revealed a highly selective recognition of h3k4 by lsd1.17 , 18 , 19 mechanistic insight into lsd1mediated h3k9 demethylation awaits further investigation . increased expression of lsd1 has been reported in various types of cancer . in particular , many types of hematopoietic and lymphatic neoplasm , including acute myeloid leukemia ( aml ) , acute lymphoblastic leukemia , myelodysplastic syndromes , t cell nonhodgkin lymphoma and hodgkin lymphoma , exhibit lsd1 overexpression.20 , 21 in addition , solid tumors in bladder , liver ( hepatocellular carcinoma ) , colon , prostate and lung ( small cell cancer ) show elevated levels of lsd1 mrna and/or protein.22 , 23 , 24 although the overexpression of lsd1 may be a shared feature across cancer types , the regulatory mechanism of lsd1 gene transcription , both in normal and oncogenic contexts , is poorly understood . a recent study proposed that increased protein stability might also contribute to the gainoffunction of lsd1 . a deubiquitinase , usp28 , has been experimentally shown to protect lsd1 from proteasomal degradation through direct deubiquitination.25 in breast cancer cells , the depletion of usp28 resulted in a reduced level of lsd1 protein and elevated levels of h3k4me at lsd1target genes , which was accompanied by the loss of stem cell properties . importantly , the protein levels of lsd1 and usp28 were positively correlated in human breast tumors . this evidence strongly indicates the involvement of lsd1 in shaping the epigenomic landscape in cancer . lysinespecific demethylase1 is essential for embryonic development in mice . when the lsd1 gene was conventionally deleted , no viable embryo could be found after e7.5.26 , 27 moreover , conditional deletion of lsd1 in the pituitary , hematopoietic system and adipose tissue led to severe dysplastic phenotypes , suggesting the requirement of lsd1 for stem cell maintenance and/or differentiation.26 , 28 , 29 lsd1ko embryonic stem ( es ) cells have been generated by several groups , exhibiting somewhat different phenotypic outcomes . 27 report that lsd1deleted mouse es cells exhibited impaired growth , with an increased rate of apoptosis and the failure of embryoid body formation . in contrast , foster et al.,30 using a gene trap method , demonstrated that lsd1 deletion did not cause any defect in proliferation , while showing an increased apoptosis rate when embryoid body formation was induced . interestingly , a chipseq analysis of lsd1bound sites in mouse es cells revealed that the vast majority of active enhancers and promoters were occupied by lsd1.31 however , lsd1 was not required for the maintenance of stemness . instead , lsd1 was essential for h3k4 demethylation and the silencing of es cellenriched genes upon differentiation , a process called enhancer decommissioning.31 moreover , in human es cells , a reduced level of lsd1 has been linked to impaired cell cycle progression and aberrant expression of developmentally regulated genes.32 the difference in phenotypic outcomes may reflect the different methods used for gene manipulation , cell line differences or species variation . in neural stem cells ( nsc ) in mice , lsd1 is required for the maintenance of proliferative capacity through interaction with an nsc maintenance factor , tlx.33 in contrast , in human fetal nsc , lsd1 mediates neuronal differentiation by repressing a notchtarget gene , heyl.34 overall , these data indicate the importance of lsd1 function both in embryonic and somatic stem cells . important roles for lsd1 in both normal hematopoiesis and leukemogenesis have been characterized ( fig . lsd1 was identified as a binding partner of growth factor independence ( gfi)1 and ( gfi)1b , transcription factors ( tf ) , which are involved in multiple steps of hematopoiesis.35 rnaimediated depletion of lsd1 resulted in the compromised differentiation in erythroid and megakaryocytic cells . mechanistically , lsd1 occupies the promoter region of gfi1btarget genes , and represses their expression most likely through h3k4 demethylation . another transcriptional regulator of hematopoiesis , tal1 , has also been shown to bind lsd1.36 differentiation stagedependent interaction of these proteins is essential for the timely expression of genes associated with the erythroid lineage . a later study , using genetic approaches in mice , revealed that lsd1 was required for early and late differentiation processes in the hematopoietic lineage.28 both prenatal and postnatal deletion of lsd1 resulted in a dramatic reduction of mature blood cells accompanied by a fatally severe anemia . specifically , lsd1deficient mice lacked mature myeloid progenitor cells , but the colony forming potential of hematopoietic stem cells was reserved . in addition , numbers of terminally differentiated granulocytes and erythrocytes were reduced , while their precursors were accumulated . transcriptomic and epigenomic data indicates that lsd1 represses stem and progenitor cellassociated genes through h3k4 demethylation at their promoter and enhancer regions . these studies indicate that lsd1 is important for hematopoietic differentiation , especially in the erythroid lineage gfi1 and 1b , growth factor independence1 and 1b ; tal1 , tcell acute lymphocytic leukemia 1 ; mll , myeloid / lymphoid or mixedlineage leukemia ; rar , retinoic acid receptor . the increased expression of lsd1 in different types of human hematopoietic neoplasm indicates its possible involvement in leukemogenesis . this prediction has been shown to be true , most prominently in the case of aml . in acute promyelocytic leukemia , which harbors the pmlrara gene fusion , treatment with alltransretinoic acid ( atra ) efficiently induces cellular differentiation and growth arrest , but this therapeutic effect has not been achieved in other types of aml.37 schenk et al.38 demonstrated that inhibition of lsd1 activity in combination with atra exposure promoted the differentiation of aml cells with different genetic backgrounds . upon lsd1 inhibition and atra treatment , the expression of genes associated with myeloid differentiation was upregulated with a concomitant increase of h3k4me2 levels at these genes . lsd1 inhibitor exerts synergistic effects with other anticancer agents , such as arac or an inhibitor of h3k27 methyltransferase , on the induction of aml cell death , indicating the multifaceted function of lsd1.39 moreover , harris et al . report the contribution of lsd1 in maintaining stem cell properties in a subtype of aml harboring an mll gene translocation.40 increased expression of lsd1 was detected in mllmutant leukemia cells , especially in cells expressing the mllaf9 fusion protein , which acts as an oncogenic transcriptional regulator . interestingly , lsd1 and mllaf9 cooperatively promoted the expression of these genes , although mll itself is a h3k4 methyltransferase normally counteracting lsd1 to dynamically remodel h3k4 methylation status . these findings indicate a distinct mode of epigenetic regulation in leukemia cells with specific genetic backgrounds . direct evidence that the increased expression of lsd1 can support malignant transformation of hsc has been reported.21 among the four reported lsd1 splice variants , the transgenic expression of the shortest , and perhaps the most wellknown , isoform induced lymphocyte hyperplasia in mice , and when exposed to irradiation , the mice developed tlymphoblastic leukemia ( tlbl ) . lsd1 is a key epigenetic effector downstream of notch signaling , which is frequently activated in lymphoid malignancies.41 , 42 considering that lsd1 is often overexpressed in human tlbl,21 lsd1 may be a strong driver of epigenetic disruption that paves the way to leukemogenesis . lysinespecific demethylase1 is a key epigenetic regulator of the cellular state ; therefore , it is plausible that it also contributes to the environmental adaptation of cancer cells . indeed , a number of reports have shown that lsd1 is critically involved in the regulation of the epithelialtomesenchymal transition ( emt ) . emt confers mesenchymal cell properties on tumor cells , including the cell motility that is required for invasion and metastasis.43 , 44 emt is also associated with the acquisition of cancer stem celllike properties , such as selfrenewal and colony forming capacities.43 emt involves highly ordered transcriptional regulation , in which several master tf , including snail family proteins , repress epithelial marker genes and activate mesenchymal markers.44 , 45 two groups independently demonstrated that lsd1 physically associates with snail1 in breast cancer cells.46 , 47 lsd1 is recruited to the ecadherin gene promoter in a snail1dependent manner , and represses its expression via h3k4 demethylation ( fig . 3 ) . interestingly , an inhibitor of lsd1 enzymatic activity abolished the lsd1/snail1 interaction , leading to impaired cell motility.46 the expression of lsd1 was highly correlated with that of snail1 in human breast tumor specimens , indicating the cooperativity of these proteins during tumor development.46 the lsd1/snail1 complex has also been shown to enhance bone marrow homing activity in aml cells , indicating its conserved regulatory role in cell motility across different cell types.48 moreover , the expression of lsd1 was increased during transforming growth factor ( tgf)induced emt of noncancerous hepatocytes.49 this emt process was accompanied by an increase of gross h3k4 methylation and a decrease of h3k9 methylation , which was reversed by lsd1 depletion . although the mechanism for this is not clear , the data indicate that lsd1 is a major determinant of genomescale epigenetic reprogramming during emt . wang et al.12 show that lsd1 cooperates with the nurd complex to repress a set of genes associated with tgf signaling , which , in turn , inhibits cell migration ( fig . the loss of lsd1 enhanced the metastatic behavior of breast cancer cells transplanted into mice . it has also been reported that lsd1 represses the expression of snail1 and other emtassociated genes.50 another snail family tf , slug / snai2 , also binds to lsd1.51 this protein complex colocalized at and transcriptionally repressed lineagespecific genes to maintain an undifferentiated state in breast cancer cells.52 lysinespecific demethylase1 ( lsd1 ) regulates cell motility and emt in cancer cells . demethylation activity of lsd1 exerts opposite effects on cell motility and epithelialtomesenchymal transition ( emt ) depending on interacting partners . these lines of evidence suggest that lsd1 is a pivotal regulator of the phenotypic plasticity of cancer cells . it can either promote or inhibit emt and cell motility , presumably depending on the genetic background of the cells and/or environmental cues that can influence the behavior of lsd1 . it is also important to note that remodeling of h3k4me status , either local or global , is intimately associated with the progression of emt . cancer cells undergo a rewiring of their energy metabolism pathways , a process known as metabolic reprogramming , in order to adapt to their microenvironment and to support their proliferative potential.53 a hallmark of cancer cell metabolism is glycolysisshifted energy production rather than mitochondrial respiration . such an energy strategy enables not only survival under hypoxic conditions but also efficient production of macromolecules , including lipids and nucleotides , which serve as building blocks for cell division.54 although the expression of metabolic genes is dramatically remodeled in cancer cells , the underlying epigenetic mechanism is poorly understood.55 , 56 we have previously demonstrated that lsd1 is an integrative regulator of the glycolytic shift in hepatocellular carcinoma ( hcc ) cells ( fig . 4).57 mechanistically , lsd1 represses mitochondrial respirationassociated genes such as ppargc1a , acadm and ehhadh through h3k4 demethylation at their promoter regions . moreover , lsd1 promotes the expression of most of the glycolytic genes , including glut1 , hk2 and pkm2 , by facilitating hypoxiainducible factor1 ( hif1)mediated transcriptional activation . interestingly , lsd1 was required to sequester hif1 from proteasomal degradation , and , thus , contributes to the stabilization of hif1 protein under hypoxia . consistent with this gene regulatory function , loss of lsd1 resulted in increased oxidative phosphorylation ( oxphos ) capacity as well as reduced glucose uptake and glycolytic activity . using an hcc xenograft in mice we also observed a significant correlation between lsd1 and glut1 expression in human hcc specimens . highly similar results were observed in human esophageal cancer ( ec ) , in which lsd1 expression levels were significantly correlated with glucose uptake as detected by fluorodeoxyglucosepositron emission tomography.58 in ec cells , lsd1 was essential for the maintenance of glycolytic gene expression . intriguingly , the reduction of glycolytic activity by lsd1 inhibition was accompanied by compromised motility rather than a proliferative defect or cell death , indicating the requirement of metabolic adaptation for cell migration . moreover , an lsd1dependent glycolytic shift has also been demonstrated in pancreatic cancer cells.59 these results suggest a conserved role of lsd1 in the control of metabolic reprogramming across different types of cancer . , lsd1 suppresses mitochondrial respiration but does not influence glycolytic activity in adipose cells,60 suggesting that cancerspecific conditions , such as oncogenic signaling and/or the tumor microenvironment , contribute to selective gene regulation by lsd1 . in addition , it is not clear how h3k4 demethylation by lsd1 can be triggered by specific signaling and tf during metabolic reprogramming . lysinespecific demethylase1 ( lsd1 ) as an integrative regulator of the glycolytic shift in cancer cells . lsd1 promotes the glycolytic shift by directly suppressing mitochondrial respiration and by activating glycolysis via hypoxiainducible factor1 ( hif1 ) . although not much is known about the biological function of lsd2 , some reports have described its role in the regulation of cellular metabolism . in hepatic cells , lsd2 represses the expression of genes associated with lipid metabolism and transport directly through the demethylation of h3k4 at their enhancers.61 lsd2depleted cells exhibited an increased rate of fatty acid uptake and an accumulation of large lipid metabolites , such as cholesterol and phospholipids . the growth of lsd2depleted cells was markedly impaired by fatty acid exposure , indicating that lsd2 protects the cell from lipotoxic damage . a recent study has shown that lsd2 represses the expression of glycolytic genes.62 mir215 , a microrna whose level is increased in gliomainitiating cells , represses the expression of lsd2 , leading to reduced expression of glycolytic genes . of note , the low level of lsd2 expression and the high level of mir215 expression coexisted in glioblastoma patients . these findings indicate that lsd1 and lsd2 have nonredundant roles in regulating energy metabolism and in the development of cancer . because both lsd1 and lsd2 show relatively ubiquitous expression patterns across cell and tissue types , these proteins may work either cooperatively or competitively in certain circumstances . lysinespecific demethylase1 plays a pivotal role in various biological processes , including the maintenance of stemness , cell motility , emt and glycolysisshifted metabolism , all of which are typically associated with oncogenesis . indeed , the increased expression of lsd1 in many types of cancer is consistent with the hypothesis that lsd1 gainoffunction leads to aberrant epigenomic regulation . because lsd1 regulates the h3k4me status of key genes both in normal and cancer cells , it is important for future studies to elucidate whether the overexpression of lsd1 causes a redistribution of h3k4me marks in cancer . it is also tempting to examine whether somatic mutation and/or sequence variation of lsd1 could contribute to aberrant epigenome formation in cancer . however , it is mostly unclear how lsd1 and specific h3k4 methyltransferases counteract to establish a certain h3k4me equilibrium . because multiple h3k4 methyltransferases exist , lsd1 may exert different impacts on the epigenetic plasticity and stability , depending on the coworking methyltransferase . monoamine oxidase inhibitors have a potent inhibitory effect on lsd1 demethylase activity.63 moreover , recently developed lsd1 inhibitors with increased potency and selectivity exert marked anticarcinogenic effects.39 , 40 , 64 , 65 because lsd1 participates in diverse biological processes depending on the cellular context and partner proteins , lsd1 inhibition in combination with the perturbation of other pathways and molecules might confer selective effects against desired target cells .
controlling increasing antimicrobial resistance in clinically important bacteria is a key challenge for clinicians and scientists , and there is an urgent need to prevent the emergence and subsequent spread of resistant isolates . enterococcus faecium is a primary example of one such troublesome pathogen ; while innately resistant to many classes of antibiotics , this bacterium has demonstrated a remarkable capacity to evolve new antimicrobial resistances . in fact , e. faecium has been highlighted by the infectious diseases society of america as one of the key problem bacteria , or eskape ( enterococcus faecium , staphylococcus aureus , klebsiella pneumoniae , acinetobacter baumannii , pseudomonas aeruginosa , and enterobacter species ) pathogens , requiring new therapies ( 1 ) , and there has been a steadily increasing prevalence of e. faecium - related nosocomial infections ( 2 ) . millions of dollars are spent each year by health care systems trying to contain antibiotic - resistant bacteria and prevent cross - transmission . nosocomial outbreaks of vancomycin - resistant enterococci ( vre ) are thought to occur when a patient already carrying vre in his / her bowel sheds vre , which are then transmitted by health care workers or via the environment to other patients ( 3 ) . this model predicts that interventions based on screening and isolation of vre - colonized patients , improved hand hygiene , and enhanced hospital cleaning will limit cross - transmission ( 4 ) . hospital based on modified use of antibiotics have also been associated with reducing vre transmission ( 5 ) . at the austin hospital in melbourne , australia ( an 800-bed tertiary referral institution ) , hand hygiene schemes have resulted in a significant reduction in the incidence of methicillin - resistant s. aureus ( mrsa ) ; however , the incidence of vre has continued to rise ( 6 ) . a similar story has unfolded in other regions , with vre now being responsible for over 30% of e. faecium bacteremias in some european countries ( 7 ) and for outbreaks in other european countries , asia , and the united states ( 811 ) . in the united kingdom , concern about increasing vre incidence led to mandatory reporting of rates of vre bacteremia , and more than 500 cases per year have been reported since 2004 ( http://www.hpa.org.uk/ ; accessed , 3 december 2012 ) . clinically significant vre strains were first reported from europe and the united kingdom in 1986 and in the united states soon after ( 1214 ) . glycopeptide resistance in enterococci is mediated predominantly by mobile gene clusters that confer resistance to vancomycin and teicoplanin ( vana genotype ) or vancomycin only ( vanb genotype ) ( 15 ) . in the united states , vana - containing vre dominate , in europe , a mix of vana and vanb - containing vre is found , while in australia , vanb - containing vre dominate ( 1618 ) . nonenterococcal species ( predominately anaerobes ) may carry the vanb resistance determinant as part of transposon tn1549 ( 19 ) in the human bowel , and in australia , while carriage of vancomycin - resistant e. faecium ( vrefm ) in the general community is rare , up to 50% of healthy adults have detectable vanb in their fecal samples as determined by pcr ( 20 , 21 ) . transmission of a tn1549-like element from one human fecal anaerobe , clostridium symbiosum mlg101 , to enterococcus faecium and enterococcus faecalis in the digestive tracts of mice has been demonstrated , suggesting that the same process may occur in the human bowel ( 23 ) . recently , the first complete genome sequences of e. faecium have been reported ( 24 , 25 ) , and draft genomes of additional e. faecium isolates have been studied to explore antibiotic resistance mechanisms and phylogenomic relationships ( 2629 ) . hospital - associated ampicillin - resistant e. faecium isolates , which are represented by three dominant sequence types ( st17 , st18 , and st78 ; st203 is an st78 single - locus variant [ slv ] ) , are genetically distinct and replace the normal reservoir of ampicillin - susceptible e. faecium strains in hospitalized patients ( 24 , 25 , 30 ) . these three sequence types have been collectively referred to as clonal complex 17 ( cc17 ) ; however , the growing realization of the significant impact of recombination on the evolution of e. faecium suggests that the cc designation may not be accurate ( 31 , 32 ) because of the significant genetic variation that has been revealed among these hospital - associated isolates ( 33 ) . because of the failure of intense infection control interventions to curtail the rise of vre , we applied genome sequencing and detailed comparative genomic analysis to a collection of e. faecium isolates to better understand the relationship between vancomycin - susceptible e. faecium ( vsefm ) and vrefm isolates and to test the hypothesis that de novo generation of vrefm is a frequent occurrence that is undermining infection control efforts . in this study , we sequenced and compared the genomes of 61 e. faecium isolates . fifty - six were bloodstream isolates from unique patients over a 12-year period in a single institute in melbourne , australia ( 32 vrefm and 24 vsefm isolates ) , 3 were rectal - screening isolates from royal perth hospital in western australia ( all vrefm ) , and 2 were clinical isolates from royal darwin hospital , darwin , northern territory , australia ( 1 vrefm and 1 vsefm isolate ) . relevant epidemiological information and a summary of sequencing results are presented in dataset s1 in the supplemental material . the aus0004 ( st17 ) ( 24 ) and aus0085 ( st203 ) ( margaret m. c. lam , torsten seemann , nicholas j. tobias , honglei chen , volker haring , robert j. moore , susan ballard , m. lindsay grayson , paul d. r. johnson , benjamin p. howden , and timothy p. stinear , submitted for publication ) genome sequences were used as primary references , and two publicly available international e. faecium blood culture isolates were included for comparison : the st18 do ( tx16 ) strain isolated from texas in 1998 ( 25 ) and the st203 strain 1,231,502 from the broad institute collection ( 26 ) . in addition , five anaerobic bacteria previously isolated from human feces and demonstrated to carry tn1549 within the vanb operon were also subjected to genome sequencing to allow comparison of the vanb transposon sequences from these anaerobes and the vrefm isolates . the details and sequencing results for these isolates are summarized in dataset s2 in the supplemental material . to address the question of whether patients develop vrefm within their bowel or acquire vrefm from an exterior source , we first conducted a detailed analysis of the characteristics of the tn1549 transposon among the vanb - positive isolates . in our recent comparison of the st203 vrefm ( aus0085 ) and st17 vre ( aus0004 ) whole genomes ( margaret m. c. lam , torsten seemann , nicholas j. tobias , honglei chen , volker haring , robert j. moore , susan ballard , m. lindsay grayson , paul d. r. johnson , benjamin p. howden , and timothy p. stinear , submitted for publication ) , we identified a number of tn1549 features that would permit identification of unique transposon acquisition events , including transposon sequence type , insertion site , insertion direction , and coupling sequence composition ( fig . 1 ) . analysis of the 36 vrefm isolates and the anaerobic bacteria in this study found that 15 vrefm isolates harbored a larger 57-kb version of tn1549 than the other isolates ( fig . 2 ) . the tn1549 phylogeny , inferred by comparing the 118 variable nucleotides within this element among all isolates ( fig . 3a ) , revealed two dominant populations of tn1549 , those represented by the sequences from the short , 33-kb element from aus0069 and those represented by the 57-kb element from aus0085 . overall , there were five different transposon sequence groups among the 36 vrefm isolates ( based on a difference of three or more single nucleotide polymorphisms [ snps ] ) ( fig . notably , tn1549 sequences identified in the anaerobic bacteria were similar to those from vrefm isolates . remarkably , the tn1549 sequence from clostridium sp . strain mlg055 , isolated from the feces of a dialysis patient in melbourne , australia ( 20 ) , was identical to tn1549 from a number of vrefm isolates from bloodstream infections , indicating that transfer of this element among the bowel microbiota may occur . characteristics used to identify unique tn1549 transmission events in vanb - containing e. faecium isolates , including a unique transposon sequence ( 1 ) , an insertion site ( 2 ) , the insertion orientation ( 3 ) , and the flanking coupling sequence ( 4 ) . flanking imperfect left and right inverted repeat ( ir - l and ir - r ) insertion sites are highlighted in orange and blue , respectively . the vanb locus contains vancomycin resistance genes ( green ) , which are conserved in all vanb sequence types , as well as other genes , which leads to variation in total transposon size , as indicated . dna - dna comparison of tn1549 sequences ( 33 kb , short version [ s ] ) from aus0004 and aus0085 ( 57 kb , long version [ l ] ) , visualized with the artemis comparison tool , showing the location of the vanb locus in both elements ( orange shading ) and the putative novel conjugation locus ( pink shading ) in the long version of tn1549 . percentages of gc content ( 500-bp sliding window ) are shown above , followed by heatmap visualizations of results of read mapping normalized to both aus0004 and aus0085 for a representative selection of vrefm and vanb - positive bowel anaerobes . note that changes in read mapping density across tn1549 are explained by sequencing biases , as these regions correlate with significant changes in gc content . ( a ) snp - based phylogeny of tn1549 elements in e. faecium and anaerobes ( red dots indicate anaerobes ) . the color scale represents the tn1549 sequence type ( based on snp analysis ) as well as the transposon insertion site in the e. faecium chromosome , relative to the aus0004 chromosome sequence . the coupling sequence is the 5- to 8-bp sequence introduced from the donor during transposition ( < > refers to the site of insertion , as shown in figure 1 ; * indicates nucleotide deletion ) . isolates shaded in pink contain the shorter ( 33-kb ) version of tn1549 , while those in blue contain the longer ( 57-kb ) version of tn1549 . ( b ) overview of the seven unique transposon insertion sites for the 36 vrefm clones mapped to the aus0004 chromosome , as well as the transposon sequence type . the color scheme follows that described in panel a. outter numbers indicate numbers of isolates with insertions at that position . ( c ) maximum - likelihood phylogeny for e. faecium based on snps in the core genome , with major multilocus sequence type groups highlighted . isolates from other institutions are highlighted in yellow and include five isolates from other institutions in australia ( rph isolates from perth ; isolates 928 and 755 from darwin ) and two isolates from the united states sequenced by others ( do , 1 - 231 - 502 ) . colored bars indicate transposon sequences and insertion sites as defined for panel a. gray triangles indicate predicted independent vrefm clones based on tn1549 characteristics and core genome phylogeny . the node represented by the black circle shows the estimated divergence date for these st17 and st252 isolates , as inferred by path - o - gen . the chromosomal insertion site of all tn1549-like elements for the 36 vrefm isolates was investigated , revealing a restricted repertoire of insertions , indicating that tn1549 insertion is not random ( fig . seven different chromosomal insertion sites were identified , yet transposons of the same sequence were consistently inserted at the same chromosomal location ( fig . 3a and b ) . 3a ) all integrated within efau004_00592 , while transposons represented by isolate 85 ( blue in fig . 3a ) all integrated within the signal peptidase i gene ( efau004_00734 ) . in conjunction with the core genome phylogeny ( see below ) , we then used tn1549 sequence type , site of insertion , insertion orientation , and coupling sequence to distinguish inherited from independent tn1549 acquisitions among e. faecium isolates ( fig . 1 and 3 ) . to understand the population structure of the e. faecium strains included in this study , a core genome e. faecium phylogeny was constructed using 14,988 snps identified among the 63 isolates ( excluding insertions and deletions ) . split decomposition analysis produced a reticulate phylogeny , suggesting that recombination contributes to the phylogenetic signal ( data not shown ) . because recombination would perturb the phylogeny , we employed an iterative algorithm that searched for a high density of snps and excluded chromosome regions likely acquired by recombination ( fig . 4 ) . this analysis predicted 297 recombination events , spanning 1.3 mb ( 44% of the chromosome ) and accounting for 12,849 snps ( 85.7% of all nucleotide variation ) . the remaining 2,139 snps were aligned among all isolates , and a tree was inferred using maximum likelihood based on these vertically inherited base substitutions alone ( fig . areas of predicted recombination in the e. faecium chromosome are highlighted , based on snp density . mlst alleles are indicated by triangles . only purk and adk are in regions without predicted recombination . two isolates ( 69 and 71 , circled ) are part of the st252 complex by mlst but cluster with the st203 isolates based on core genome snp analysis . regions in red are recombinant in multiple isolates in the collection , while regions in blue are unique to one strain . isolates generally clustered within their previously defined multilocus sequence types ( mlsts ) ; however , there were also some clear exceptions highlighting the impact of recombination events on mlsts . for example , isolates 69 ( slv of st252 ) and 71 ( st252 ) clustered among the st203 isolates based on the nonrecombinant core genome analysis ( fig . 4 ) . as expected , the non - cc17 isolates ( isolates 10 , 92 , and 101 ) were more distantly related to all other isolates . significantly , cc17 vsefm isolates were closely related to , and in some cases essentially indistinguishable from , cc17 vrefm isolates at this core genome level , suggesting that the cc17 vsefm and cc17 vrefm strains were part of the same circulating population rather than that they evolved independently . thus , the evolution of vrefm strains from colonizing vsefm strains , and not just the clonal spread of vrefm strains , explains the epidemiology of invasive vrefm disease observed in this study , suggesting that the epidemiology of vsefm in hospitals is a key factor driving the emergence of clonally related vrefm . in addition to examining the evolutionary relationship between the isolates based on a core genome , we also assessed an e. faecium pan genome which included plasmids , prophages , and other genomic elements ( dataset s3 in the supplemental material and fig . the pan genome included more than 5 mb of sequence bearing > 5,000 genes and demonstrated that even among isolates that were found to be very closely related based on their core genomes , variability in gene content was detected . for example , isolates 73 and rph3 had closely related core genomes ; however , isolate 73 harbored a unique region ( efpan04301 to efpan04389 ) that likely represented a plasmid with a large number of hypothetical proteins and plasmid - associated genes . similarly , isolates 72 and 78 displayed closely related core genomes , with isolate 72 harboring tn1549 and isolate 78 lacking this element but possessing a unique prophage ( efpan02546 to efpan 02583 ) . blue bar indicates the 5.4-mb e. faecium pan genome , including 5,000 annotated genes , detected among genomes analyzed in this study . a heat map shows the presence ( black ) and absence ( white ) of the 5,000 genes . dataset s3 in the supplemental material summarizes the variable regions and pan - genome contents for all isolates and includes efpan locus tags for all predicted coding sequences . in some cases , for example , the st203 cluster that included isolates 149 , 86 , 85 , 81 , 77 , 93 , and 96 was isolated over a 12-month period from patients in the same hospital ( fig . 5 ) suggests that they may have been transmitted directly between patients , except for isolates 86 and 96 , where differences in gene content , possibly due to plasmid acquisition , were detected . for example , isolate 86 contained the genes efpan04820 to efpan04855 , encoding a number of hypothetical proteins and a type iv secretion system , while isolate 96 harbored a unique region ( efpan04913 to efpan04927 ) . in contrast , we were able to combine the detailed transposon analysis with the core genome phylogeny to define independent episodes of vrefm evolution ( fig . at least 18 distinct episodes of vrefm evolution of the total 36 vrefm isolates were detected , indicating that de novo evolution of vrefm strains is an important source of vrefm infection at our hospital . for example , the st17 isolates 4 and 21 are closely related at the core genome level , but their transposon sequence and insertion sites are different . furthermore , the dominant red - orange and blue - aqua tn1549 types are dispersed throughout the e. faecium core genome phylogeny and can be explained by instances of independent tn1549 acquisition ( fig . four vrefm bloodstream isolates were sequenced from our hospital , and at least three of these indicate the independent evolution of vrefm strains . in the 33-month period from march 2007 to november 2009 , we had an outbreak of clinical vre infection at our institution ( 6 ) . based on mlst analysis , it appeared that we had a clonal outbreak of vrefm due to the introduction of a new clone of st203 vrefm . however , analysis of the st203 vrefm genome data from these isolates demonstrates that at least 9 unique clones of st203 vrefm have emerged ( out of the 22 sequenced ) . these unique st203 vrefm isolates highlight the polyclonal nature of vrefm at our hospital , even within a single mlst cluster that was first detected as late as 2007 . in order to determine if these observations were unusual and restricted to our hospital , we included genome sequences from geographically dispersed e. faecium isolates for comparison . the more recent isolates from other australian cities were closely related to st203 and st252 isolates from our institution ( highlighted in yellow in fig . the older st18 vrefm isolate from the united states ( do strain , 1998 ) was more distantly related to the majority of e. faecium isolates in this study . the recent vre isolate 1 - 231 - 502 ( 2008 ) was very similar to st203 isolates from our institution , suggesting global dissemination of this e. faecium clone . the collection of e. faecium isolates from a single hospital over a 12-year period allowed us to analyze the evolutionary changes and mutation rate in the isolate collection . for this analysis , we used only st17 and st252 isolates from our institution , because they were isolated throughout the 12-year study period . 6 ) and allowed estimation of a core genome mutation rate of 1.5 10 substitutions per site per year or ~5 snps per genome per year ( excluding snps introduced via recombination ) . this analysis suggested that the st17/st252 isolates from the collection arose from a common ancestor that existed in the late 1970s . estimate of the divergence date for st17 and st252 isolates by branch - to - tip analysis using path - o - gen and branch lengths inferred by maximum - likelihood analysis ( http://tree.bio.ed.ac.uk/software/pathogen/ ) . using large - scale comparative genomics , we have demonstrated that hospital vanb vrefm epidemiology is far more complex than previously recognized and is significantly driven by de novo vrefm generation rather than simply nosocomial transmission of extant vrefm strains . we found that many distinct clones of vanb vrefm were responsible for bloodstream infection isolates from patients at our institution . in fact , at least 18 of 36 vrefm isolates sequenced in this study were due to unique vrefm evolution events . this includes a time period where a new sequence type of e. faecium ( st203 ) emerged in our institution and caused what we thought was a clonal outbreak ( 6 ) . these findings indicate that unique vrefm evolution events are common , presumably occurring in the human bowel , and taken together suggest that control strategies based on screening for vrefm and isolation of colonized patients might not be completely effective . we identified a number of tn1549 features that could be utilized to determine independent vrefm acquisition events ( fig . 1 ) , demonstrating the power of genome sequencing to uncover complex relationships between bacteria . significantly , the transposon sequences from anaerobic bacteria isolated from human feces were very similar and , in one case , identical to those from vrefm isolates associated with bloodstream infections in humans , suggesting that anaerobic bacteria might form an important component of the tn1549 reservoir . if the transposon pool is restricted and the vsefm population is highly clonal , as occurs in hospitalized patients ( 30 ) , even unique in vivo generation episodes of vrefm may appear like clonal spread of preformed vre . this issue was highlighted by detailed analysis of three st203 isolates , 149 , 86 , and 96 . based on core genome phylogeny and transposon analysis , these isolates appeared clonal ; however , analysis of the pan - genome components of these isolates ( fig . 5 ) demonstrated additional genome diversity , in some cases driven by acquisition of plasmids . while it is possible that individual genome variability evolved in a clonally disseminated preformed vrefm strain , it is also possible that clonally dispersed ( but not identical ) vsefm isolates if this is the case , then even more than 18 of the 36 vrefm isolates sequenced in this study may have evolved independently . while we focused our investigation on a single institution with the primary aim of understanding the persistent vrefm outbreak that we had noted , we included isolates from other australian cities and the united states . sequences from these isolates were very closely related to vrefm isolates from our institution in melbourne , australia , including the united states isolate ( 1,231,502 ) . hospital - hospital transfer of colonized patients may explain this observation in australia ; however , such movements are infrequent between darwin and melbourne . in our view , the best model which explains our observations is silent circulation internationally of closely related vsefm isolates , with local tn1549 transposition events , which then lead to the appearance of new clinical cases of vre colonization and infection in hospitals . the e. faecium genome is known to have high rates of recombination , especially compared to other important hospital pathogens , such as staphylococcus aureus ( 25 , 34 , 35 ) . here we applied whole - genome sequencing and snp clustering analysis to infer a phylogeny and predict regions of recombination within the 63 e. faecium genomes used in this study ( fig . 3 and 4 ) . a total of 1.3 mb ( 44% ) of the chromosome was affected by recombination . this includes five of the seven mlst loci ( fig . 4 ) , indicating that mlst analysis of e. faecium based on the currently selected alleles may give misleading results and must be interpreted with care . we also defined an e. faecium pan genome , which includes 1,872 core genes as well as 3,128 accessory genes identified within the 63 e. faecium isolates in this study ( fig . this indicates significant plasticity within the e. faecium genome , with large accessory elements potentially impacting the clinical behavior of these isolates , as previously suggested ( 25 , 33 ) . clonal complex 17 e. faecium emerged as an important hospital - associated pathogen in the 1980s ( 36 ) . by analyzing the st17/st252 isolates from this study , we found a mutation rate in the core genome of 5 snps per genome per year and predicted that the common ancestor strain for e. faecium at our institution existed around 1978 ( 95% confidence interval [ ci ] , 1973 to 1983 ) , in keeping with the appearance of cc17 e. faecium and vrefm in the 1980s . our findings have implications for the control of vrefm in hospitals , where generation of new vrefm clones is a frequent event . because a high proportion of hospital patients carry nonenterococcal vanb - containing organisms in their bowels ( 19 , 20 , 22 , 37 ) , we suggest that specific vsefm clones should also be targets for control in the hospital environment . it remains to be determined if hospital - associated vsefm is acquired nosocomially by patients , as suggested in european studies , or if in fact these isolates of vsefm are present in the community in australia ( 30 , 38 ) . a complete understanding of vse epidemiology and the biology of the tn1549 element will be critical to improving strategies to curtail the vre epidemic . these data dictate that alternative strategies directed at identifying and isolating patients colonized with high - risk vsefm clones , identifying patients who harbor vanb - containing tn1549 and who are likely at increased risk of in vivo generation , and identifying and modifying the selective pressures promoting in vivo evolution are needed to prevent the ongoing emergence of vrefm as a public health threat . isolates used in this study were vancomycin - susceptible and -resistant enterococcus faecium strains recovered between 1998 and 2009 from blood culture specimens at austin health , a tertiary referral hospital in melbourne , australia . additionally , five recent clinical and screening isolates of vsefm and vrefm from other cities in australia ( darwin and perth ) were included for comparison , as were five previously identified and characterized human bowel anaerobes containing tn1549 within their vanb operon that were isolated in melbourne , australia ( 19 , 20 ) . all vrefm isolates were found to display the vanb genotype , as determined by pcr ( 17 ) . enterococcus faecium isolates were previously analyzed by mlst and were found to belong predominately to clonal complex 17 ( cc17 ) ( 6 ) . epidemiological data , including date and location of isolation , were recorded . a summary of isolates and epidemiological data is provided in dataset s1 ( e. faecium ) and dataset s2 ( anaerobes ) in the supplemental material . genomic dna was extracted , and all isolates were subjected to whole - genome shotgun sequencing using an illumina hiseq-2000 sequencing system and 100-bp - paired - end truseq chemistry or ion torrent single - end 100-bp or 200-bp sequencing chemistry ( 39 ) ( datasets s1 and s2 ) . a read mapping approach was used to align the sequences from these isolates with the recently completed e. faecium st203 genome , aus0085 ( margaret m. c. lam , torsten seemann , nicholas j. tobias , honglei chen , volker haring , robert j. moore , susan ballard , m. lindsay grayson , paul d. r. johnson , benjamin p. howden , and timothy p. stinear , submitted for publication ) , using shrimp v2.0 ( 40 ) . single nucleotide polymorphisms ( snps ) in the e. faecium core genome were identified using nesoni v0.93 , which aligns the read of each genome to that of the reference to construct a tally of putative differences at each position ( http://www.bioinformatics.net.au ) . in order to exclude regions of suspected recombination from the phylogenomic analysis , regions with high frequencies of polymorphic nucleotides were removed from the alignment , as previously described ( 41 , 42 ) , and a phylogeny was inferred using maximum likelihood with raxml ( 43 ) . the temporal signals in the sequence data were analyzed using path - o - gen ( http://tree.bio.ed.ac.uk/software/pathogen/ ) . snps likely introduced via recombination were identified using the method described in croucher et al . , an e. faecium pan genome was defined after de novo assembly using velvet v1.2.04 ( 44 ) and alignment of contigs to the aus0085 genome using mummer ( 45 ) . unaligned contigs were appended to the aus0085 genome to construct a pan genome , which was annotated using prokka ( http://bioinformatics.net.au/ ) . the proportion of the length of each annotated gene covered by reads was assessed for each isolate and a map summarizing all variable genes and their distribution in each strain produced . variable nucleotide positions in tn1549 for all vrefm isolates and anaerobes were also identified , and a tn1549 phylogeny was inferred using the neighbor - joining method with uncorrected p distances , as implemented in splitstree v4.12.3 ( 46 ) . in addition , the chromosomal tn1549 insertion site was determined by aligning the contigs that spanned the left- and right - hand ends of tn1549 ( obtained from de novo assembly of each vrefm clone , as described above ) to the aus0004 reference genome . other features of the tn1549 insertion that were determined included the orientation of insertion within the chromosome and identification of the 5- to 8-bp coupling sequence . separate clones of vrefm ( unique vrefm generation events ) were defined as taxa with distinct core genome phylogenies , or if they possessed specific tn1549 characteristics , they were defined by including the variation in their transposon sequence , the insertion site , the insertion orientation , or the coupling sequence . the sequence reads for all isolates have been submitted to genbank under bioproject identifier prjna205886 . isolates used in this study were vancomycin - susceptible and -resistant enterococcus faecium strains recovered between 1998 and 2009 from blood culture specimens at austin health , a tertiary referral hospital in melbourne , australia . additionally , five recent clinical and screening isolates of vsefm and vrefm from other cities in australia ( darwin and perth ) were included for comparison , as were five previously identified and characterized human bowel anaerobes containing tn1549 within their vanb operon that were isolated in melbourne , australia ( 19 , 20 ) . all vrefm isolates were found to display the vanb genotype , as determined by pcr ( 17 ) . enterococcus faecium isolates were previously analyzed by mlst and were found to belong predominately to clonal complex 17 ( cc17 ) ( 6 ) . epidemiological data , including date and location of isolation , were recorded . a summary of isolates and epidemiological data is provided in dataset s1 ( e. faecium ) and dataset s2 ( anaerobes ) in the supplemental material . genomic dna was extracted , and all isolates were subjected to whole - genome shotgun sequencing using an illumina hiseq-2000 sequencing system and 100-bp - paired - end truseq chemistry or ion torrent single - end 100-bp or 200-bp sequencing chemistry ( 39 ) ( datasets s1 and s2 ) . a read mapping approach was used to align the sequences from these isolates with the recently completed e. faecium st203 genome , aus0085 ( margaret m. c. lam , torsten seemann , nicholas j. tobias , honglei chen , volker haring , robert j. moore , susan ballard , m. lindsay grayson , paul d. r. johnson , benjamin p. howden , and timothy p. stinear , submitted for publication ) , using shrimp v2.0 ( 40 ) . single nucleotide polymorphisms ( snps ) in the e. faecium core genome were identified using nesoni v0.93 , which aligns the read of each genome to that of the reference to construct a tally of putative differences at each position ( http://www.bioinformatics.net.au ) . in order to exclude regions of suspected recombination from the phylogenomic analysis , regions with high frequencies of polymorphic nucleotides were removed from the alignment , as previously described ( 41 , 42 ) , and a phylogeny was inferred using maximum likelihood with raxml ( 43 ) . the temporal signals in the sequence data were analyzed using path - o - gen ( http://tree.bio.ed.ac.uk/software/pathogen/ ) . snps likely introduced via recombination were identified using the method described in croucher et al . ( 41 ) . to investigate the total e. faecium gene content , an e. faecium pan genome was defined after de novo assembly using velvet v1.2.04 ( 44 ) and alignment of contigs to the aus0085 genome using mummer ( 45 ) . unaligned contigs were appended to the aus0085 genome to construct a pan genome , which was annotated using prokka ( http://bioinformatics.net.au/ ) . the proportion of the length of each annotated gene covered by reads was assessed for each isolate and a map summarizing all variable genes and their distribution in each strain produced . variable nucleotide positions in tn1549 for all vrefm isolates and anaerobes were also identified , and a tn1549 phylogeny was inferred using the neighbor - joining method with uncorrected p distances , as implemented in splitstree v4.12.3 ( 46 ) . in addition , the chromosomal tn1549 insertion site was determined by aligning the contigs that spanned the left- and right - hand ends of tn1549 ( obtained from de novo assembly of each vrefm clone , as described above ) to the aus0004 reference genome . other features of the tn1549 insertion that were determined included the orientation of insertion within the chromosome and identification of the 5- to 8-bp coupling sequence . separate clones of vrefm ( unique vrefm generation events ) were defined as taxa with distinct core genome phylogenies , or if they possessed specific tn1549 characteristics , they were defined by including the variation in their transposon sequence , the insertion site , the insertion orientation , or the coupling sequence . the sequence reads for all isolates have been submitted to genbank under bioproject identifier prjna205886 . download dataset s1 , xls file , 0.1 mb anaerobic bacteria metadata . epidemiological data and sequencing results for anaerobic bacteria included in this study . download dataset s2 , xls file , 0.1 mb enterococcus faecium pan - genome contents of all isolates . summary of the e. faecium accessory elements used to define the pan genome and presence or absence of these elements in each strain .
genomics studies often start with the organization of a dataset retrieved from public databases , such as the ensembl.1 the ensembl database is currently in release 71 ( april 2013 ) and covers genomes from vertebrates and other eukaryotic species , and therefore holding a large amount of genomic data . ensembl provides well characterized gene and gene family annotations together with well defined gene homologous ( orthologs and paralogs ) relationships , which are very accurate and a rich source of information for evolutionary and comparative genomics . it is very important , therefore , to develop faster , easier , and more user friendly methods in order to make them available to the broad community of biologists enabling this important resource to be exhaustively used . at present , the process of creating a dataset is very tedious and time consuming , particularly if the user must search and download every sequence individually , making it almost impossible to use such important and valuable resource . to overcome such hurdles , users currently have a few available tools for downloading data from ensembl , namely biomart,2 the application programming interfaces ( apis ) , and the retrieve - ensembl - seq3 program . the biomart application , however , can be limiting considering the number of species that can be obtained per query , which is at most five.3 the apis are not useful to the common biologist , as high programming skills are required to maximize its use . although the retrieve - ensembl - seq program is more user - friendly , its dataset preparation is cumbersome with some limitations regarding the taxon level , where no option exists to choose the desired species , an option very much needed for comparative genomics . in addition , users have to select multiple options from web - based non - intuitive menus making the overall process tedious , particularly when building a large dataset , thus diminishing the overall purpose of fast and large - scale data preparation . in order to address this problem , we have developed a much more user friendly application for biologists , which has the advantage of allowing all types of genomics features to be downloaded ( table 1 ) . this includes complete genes , coding - sequences ( cds ) , peptide , exon , intron , 5 and 3 utr , upstream , downstream , as well as both extended regions , 5 end and 3 end , as per user defined lengths in base - pairs ( bp ) based on a given relationship ( table 2 ) for a given gene i d . hence , it becomes more efficient to download ensembl defined genomic features for as many gene ids and as many species as per user requirement . following the download , the user often needs to format the descriptions of sequences , such as the word limits and the use of special characters4 for the requirements of the downstream analyses . we further provided the user with the option of naming the downloaded sequences in one of the given four easer - specific options : 1 ) species name with gene symbol ; 2 ) ensembl i d ; 3 ) ensembl i d with species name and gene symbol ; and 4 ) species name abbreviated and gene symbol ( fig . we present easer ( easer.py ) , a simple python ( http://www.python.org/ ) program for the retrieval of large amounts of sequence data from the ensembl database . in the case of coding - sequences , the script provides an easy , fast , and effortless way for downloading the homologous sequences from any given number of available species selected by the user . the user can select all the desired options at once or one by one from interactive mode , and in a matter of seconds or a few minutes the sequences are saved in the user personal computer in fasta file format . the perl ( http://www.perl.org/ ) script enamer.pl can be used independently for renaming the ncbi and ensembl specific sequence descriptions in any selected format from the available options provided interactively . our application provides the user the access to an alternative and oversimplified way of working , focused in the personal computer environment . the use of this api ensures that a constant up - to - date access to sequences is gained . quick start : just typing ( easer.py s ensg00000108511 a 1 ) will help the user to download the coding sequences ( orthologs one - to - one ) for all species available . to view a list of available options , the user types the option -h ( easer.py-h ) in the terminal ( fig . for instance , option -s enables the user to specify a single ensembl i d ( -s ensg00000108511 ) or an input file containing multiple ensembl ids ( -s ensemblids.txt ) organized in one single column for a set of genes . other options include option -r for the ensembl homology relationship choice ( see table 2 ) , option -r for the ensembl database release number , option -a for the species selection ( if omitted , the user will be prompted for the species selection ) , option -o for output file name ( by default ensembl ids are used as file names ) , and option -c for sequences renaming . depending on the user requirement , option -a can be used to select all species ( -a 1 ) or select a group of species specified in a file and provided by the user ( -a mammals.txt ) . in this case , the possibility to provide a file with the required group of species is given to the user ( e.g. , for mammals group , see file mammals.txt provided with easer archive ) . similarly , it is possible to create a file with desired species from the complete species list ( see interactive mode ) . therefore , the user has freedom to choose any species or taxon regarding the diverse research interests . by default the script downloads coding - sequences , thus the user must specify option -d in order to obtain the desired feature ( see table 1 ) . for example , by typing the single command ( easer.py s ensg00000108511 -a 1 -r 67 -o mydata.fas -d e ) all one to one orthologs for exons from all the available species in the release 67 of the ensembl database will download for the given ensembl gene i d , and save in mydata.fas output file . if multiple ensembl ids are specified in a file ( e.g. , -s ensemblids.txt ) , the results are saved separately in different output files where the naming is done in accordance to the output file name given and the position of the ensembl i d in input file following its top - down order ( e.g. , mydata1.fas is meant for download results from first ensembl i d in ensemblids.txt ) . since the pycogent5 library ( currently in version 1.5.3 ) does not provide automated updates of the organisms list , our script fulfills this gap by providing an option ( -m ) which prompts for the species scientific and common names to be added to the list , thus adding the new species to the initial species list . in the case where no options are given in the command - line , the easer.py script enters the interactive mode and a set of successive questions is posed to the user in order to obtain the necessary options for data retrieval . when the option -a is not selected in command - line , the script enters this mode to present a list of available species , from which the user can select their option by choosing the corresponding species numbers separated by commas . quick start : just typing ( easer.py s ensg00000108511 a 1 ) will help the user to download the coding sequences ( orthologs one - to - one ) for all species available . to view a list of available options , the user types the option -h ( easer.py-h ) in the terminal ( fig . for instance , option -s enables the user to specify a single ensembl i d ( -s ensg00000108511 ) or an input file containing multiple ensembl ids ( -s ensemblids.txt ) organized in one single column for a set of genes . other options include option -r for the ensembl homology relationship choice ( see table 2 ) , option -r for the ensembl database release number , option -a for the species selection ( if omitted , the user will be prompted for the species selection ) , option -o for output file name ( by default ensembl ids are used as file names ) , and option -c for sequences renaming . depending on the user requirement , option -a can be used to select all species ( -a 1 ) or select a group of species specified in a file and provided by the user ( -a mammals.txt ) . in this case , the possibility to provide a file with the required group of species is given to the user ( e.g. , for mammals group , see file mammals.txt provided with easer archive ) . similarly , it is possible to create a file with desired species from the complete species list ( see interactive mode ) . therefore , the user has freedom to choose any species or taxon regarding the diverse research interests . by default the script downloads coding - sequences , thus the user must specify option -d in order to obtain the desired feature ( see table 1 ) . for example , by typing the single command ( easer.py s ensg00000108511 -a 1 -r 67 -o mydata.fas -d e ) all one to one orthologs for exons from all the available species in the release 67 of the ensembl database will download for the given ensembl gene i d , and save in mydata.fas output file . if multiple ensembl ids are specified in a file ( e.g. , -s ensemblids.txt ) , the results are saved separately in different output files where the naming is done in accordance to the output file name given and the position of the ensembl i d in input file following its top - down order ( e.g. , mydata1.fas is meant for download results from first ensembl i d in ensemblids.txt ) . since the pycogent5 library ( currently in version 1.5.3 ) does not provide automated updates of the organisms list , our script fulfills this gap by providing an option ( -m ) which prompts for the species scientific and common names to be added to the list , thus adding the new species to the initial species list . in the case where no options are given in the command - line , the easer.py script enters the interactive mode and a set of successive questions is posed to the user in order to obtain the necessary options for data retrieval . when the option -a is not selected in command - line , the script enters this mode to present a list of available species , from which the user can select their option by choosing the corresponding species numbers separated by commas . we provide the user an easy access to the retrieval of biological sequence features from the ensembl database for comparative genomics and evolutionary studies . the desired genomic features ( table 1 and 2 ) can be easily downloaded for the corresponding ensembl i d and the multi - sequence file can be prepared given the user requirements . this set of sequences can further be renamed and readily used for downstream analyses , thereby saving lots of time and energy . our script is a valid tool that interacts with well - defined ensembl features and an easy - to - use alternative to the currently available options . since it runs on the user s personal computer , the easer program is a more effective and practical way of generating a sequences dataset from the ensembl database directly to a fasta file . given the easer is command - line driven , the efficiency is greatly improved in building appropriate datasets . if the user has multiple ensembl ids to use as query , these can be provided in a file and thus avoid running the script several times for the same options . the user can even start several instances of this script simultaneously in several terminal windows , choosing options wisely for every case and make the download of data instantly for every ensembl i d or set of ids . our script has been successfully used by us and is cited in several manuscripts under review and published from our group.6 easer is open for further improvements , which can be performed in the near future regarding the user s needs . easer is currently in version 1.7.0 and is freely available under gnu general public license upon request or on the web at http://easer.sourceforge.net/. this script is implemented in python ( http://www.python.org ) and is based in the pycogent5 library , for which installation instructions can be found in quick installation at http://www.pycogent.org . presently pycogent5 library supports linux , windows 64-bit and macos , enabling the use of easer in any of these systems . easer was developed for unix / linux environment . in order to start using the script , and provided that 1 ) python and 2 ) pycogent5 library are already installed in the user s system , the user must give it execution permissions and it will be ready to run from its current location directory . the user can also place the scripts in a binaries directory , enabling the use of easer from any working directory .
mccune - albright syndrome ( mas ) is a rare disease presenting with the classical triad of polyostotic fibrous dysplasia ( fd ) , skin hyperpigmentation ( caf - au - lait spots ) and endocrine dysfunction , usually seen as precocious puberty in females . the mosaicism of mutations in the gnas1 gene results in hyperactive gs protein which causes activation of hormonal receptors and bone dysplasia [ 13 ] . there is increased osteoclastic activity , which can be detected by the elevation of biochemical markers of bone turnover , and normal bone marrow architecture is progressively replaced by abnormal fibrous tissue [ 4 , 5 ] . bisphosphonates , antiresorption agents , largely used in the treatment of osteoporosis have been tried as an option for improvement of fd symptoms in mas [ 69 ] . most patients are treated with intravenous pamidronate , in a hospital regimen , or as outpatients in clinic settings . alendronate ( aln ) is a second - generation bisphosphonate that has , as an advantage , oral administration . it has been used for treatment of several disorders related to increased bone resorption ; however , it is rarely used by the pediatric population . since we could not find reports on the use of oral aln in children with fd of mas , although we are aware of an ongoing , as yet unpublished , study with children , we present the case of a girl with mas treated during a one - year period with oral aln . a 10.5-year - old white girl was admitted with complaints of severe weekly headache , diplopia , and nasal obstruction . she was in outpatient followup since she was 20 months old when it was noticed caf - au - lait spots . by the age of 3 years she presented with left exophthalmos , and a severe form of fd of skull , which leads to the diagnosis of mas . she had already undergone left optical nerve surgical decompression at 5 years of age and a second procedure was under consideration . at 6 and 10 years of age she was submitted to two turbinectomy procedures ; however , she still had complaints of nasal obstruction . when she was 7 years old she was treated twice with intravenous pamidronate , 1 mg / kg / d during 3 days in a 6 month interval , and had presented vomiting and headache during the infusion . in - between these two treatments she received 10 mg / day of oral aln for 30 days which was well tolerated . at 9 years of age she was born to nonconsanguineous parents ( wt : 3000 g ; ht : 48 cm ) . her father and two uncles had caf - au - lait spots , and her mother had high blood pressure and anxiety disorder . physical examination showed severe craniofacial deformity , left proptosis and large caf - au - lait spots with irregular borders in the left scalp and sacral region . her weight was 36 kg ( + 1 sd ) and height was 136.2 cm ( 0 sd ) ; body mass index was normal ( bmi : 19.73 kg / m ; 75 centile ) ; she had a bp of 90/60 mmhg and b3 p2 tanner stage . she was very upset with the bone pain and impaired quality of life , especially regarding school absenteeism , and a limited trial with oral aln was prescribed . two and a half months before the aln trial she had undergone screening for endocrinopathies ( ft4 : 1.0 mg / dl ( rv : 0.81.9 ) , tsh : 1.35 mui / ml ( rv : 0.45.0 ) , prl : 2.2 ng / ml ( rv : 1.925 ) , cortisol : 13.9 ng / dl ( rv : 5.025)rv : reference value ) and renal phosphate wasting , which was absent . serum calcium , phosphorus , 25oh vitamin d and pth levels were normal ( table 1 ) . bone scintigraphy revealed typical pattern of polyostotic fd with intense uptake in facial bones , right femur , right arm and ribs . dexa showed normal total body bone mineral density ( bmd : 0.907 g / cm ; + 0.09 sd ) . computerized tomography of skull showed fibrous dysplasia lesions without signs of increased intracranial pressure . biochemical markers of bone turnover , alkaline phosphatase and 24-hour urinary hydroxyproline levels were elevated . blood count , electrocardiogram and serum liver enzymes were normal . na : not available ; rv : reference value ; ca : calcium ; p : phosphorus ; alp : serum alkaline phosphatase . after the patient and her mother signed an informed consent form , aln ( 70 mg tablets with 200 ml of water ) was administered once a week , initially under medical supervision . she was advised to be fasting and remain upright for at least 30 minutes following ingestion of the drug . after two weeks , the patient continued medication at home , with no difficulties . aln treatment was interrupted for 15 weeks ( between 8 and 29 weeks of followup ) due to a complaint of muscle pain and dysphagia during the postoperative period of her third turbinectomy . she also presented mild self - limited dyspepsia with no need for treatment interruption . throughout her followup visits improvement was also evaluated through a numeric pain scale where the perception of pain varied from 0 to 10 . pain perception went from an intensity of 8 to 5 following 7 months of treatment . after 10 months of treatment the pain intensity was 3 and after 12 months of treatment she felt no pain . growth velocity was of 6 cm / y associated with proportional advancement of bone age during the 2 year study ( ca : 11 y3 m and ba : 11 y ) . sclerotic bands in the left wrist metaphyses were observed on plain x - ray , after treatment . improvement during aln treatment was also attested by the lowering of urinary hydroxiproline levels during followup and near complete normalization of alkaline phosphatase ( table 1 ) . she was treated with aln for a year and the benefits lasted during the year following the end of treatment . at her last visit she was 14 years old ; she was clinically stable and felt no pain . the primary and likely only effect of bisphosphonate treatment observed in mas patients is the reduction or complete resolution of bone pain . reduction of bone fractures , no harm to linear growth or altered fracture healing and better quality of life have been described [ 6 , 10 , 11 ] . until now , the majority of pediatric patients received parenteral pamidronate [ 6 , 1116 ] ; however , there is no consensus on the effectiveness of this treatment [ 7 , 17 ] . chapurlat et al . found that almost 50% of patients had radiological improvement of lesions as well as increase in cortical thickness and filling of lytic lesions . improvement of bone pain may be explained by the antiosteoclastic action of the drug , with reduction of cytokines and other pain - related substances released by the lesions . furthermore , improvement of bone mineral density may occur due to inhibition of bone resorption leading to improvement of surgical outcome in mas patients treated with bisphosphonates , yet a controversial issue [ 4 , 14 ] . aln is a biphosphonate which is largely used in the treatment of osteoporosis in adults and osteogenesis imperfecta in children [ 10 , 19 ] . it was related to satisfactory improvement of bone pain and less affected sites in the scintigraphy scan in some reports [ 8 , 20 ] . most common adverse effects are upper gastrointestinal , mainly , esophageal irritation , which can be prevented with special care during the administration . headache , diarrhea , intestinal constipation and atrial fibrillation may also occur [ 2123 ] . recently there were reports of esophageal cancer with oral bisphosphonate use in adult patients . sclerotic bands , observed in wrist metaphyses on plain x - ray , are often described in the literature and appear to have no clinical implications . there is also some concern about longer - term possible side effects of bisphosphonates on future pregnancies of treated females . instead of classical daily administration , the new therapeutic regimens such as 70 mg / weekly dose given to adults has also been used in children with osteogenesis imperfecta ( according to the weight ) with good tolerance . the 70 mg / weekly dose administered to this girl was based on treatment protocols that have been used in pediatric patients with different diagnoses [ 10 , 19 , 26 ] . these weekly regimens may improve compliance and reduce the incidence of adverse effects . nonetheless , the decision to initiate bisphosphonates and the adequate time to cease their use is controversial . there are no well - defined criteria for precise indication and response to treatment . different parameters for treatment suspension in children with osteoporosis have been reported : bmd z score sd ; reduction of 50% in fracture incidence ; resolution of bone pain ( the most useful indicator of treatment effect ) or occurrence of adverse effects [ 19 , 21 , 27 ] . there is no consensus on the changes in the markers of bone turnover during bisphosphonate therapy . alkaline phosphatase ( bone formation marker ) and urinary hydroxyproline ( bone resorption marker ) are indicators of severity and activity of osseous lesions in mas . lowering of the biochemical markers of bone resorption is more pronounced than the changes in formation markers after bisphosphonate treatment [ 21 , 22 , 2729 ] . there are reports of significant reduction in alkaline phosphatase , osteocalcin , urinary hydroxyproline and urinary ntx during bisphosphonate use . however , reference values for some of these biochemical markers are not well established in the pediatric population mainly because their increment could be a reflection of the normal bone growth and remodeling processes . although we noticed a trend towards normality in our patient biochemical markers it is not possible to foresee if there will be a sustained beneficial effect to treatment . there are reports of improvement in bmd persisting for 2 years after interruption of intravenous bisphosphonate treatment in children with osteogenesis imperfecta ; biochemical markers of bone turnover were normal after 69 months of treatment . until standard criteria for treatment interruption have been established we believe the endpoint should be clinical improvement . improvement of bone pain and of daily activities including reduction in school absenteeism , showing an increase in quality of life were the criteria which defined interruption of treatment in the present paper . we considered the outcome to have been favorable although it is still not possible to predict its endurance or reproducibility . we concluded that weekly aln regimen was a good option for improving symptoms of fd of mas in this girl , due to ease of administration and lack of severe adverse effects reported in the patient . we also believe there is an urgent need for ongoing studies addressing the many unanswered questions regarding the use of bisphosphonates in pediatric patients , in order to safely use this medication .
prostate cancer ( pca ) is one of the most commonly diagnosed neoplasms in elderly men . in western europe and the usa , it is the most frequent malignant neoplasm in men and accounts for 30% of all newly diagnoses cases [ 1 , 2 ] . the precancerous lesion of pca is considered a high grade of intraepithelial neoplastic growth ( also known as intraepithelial anaplasia of high degree pin among patients subjected to biopsy due to cancer suspicion ranges from 1.5% to around 16% [ 610 ] . in meta analysis of a number of studies , epstein and bostwick calculated the mean risk of diagnosing cancer during second biopsy in patients in whom hg pin was diagnosed on first biopsy to be 18.1% and 30% , respectively [ 11 , 12 , 13 ] . atypical small acinar proliferation ( asap ) can be considered to be an element in the progression of changes in cell morphology between healthy tissue of the prostate and pca . in most clinical studies , asap is diagnosed in 0.52% of patients subjected to biopsy of the prostate due to the suspicion of cancer [ 14 , 15 ] . diagnosis of asap is proposed , when it is not possible to find certain significant changes in the morphology of cells to diagnose pca [ 16 , 17 ] . the frequency of diagnosing pca in the second biopsy in patients with asap in the first biopsy ranges from 1770% ( mean 40.2% ) [ 18 , 19 ] . as a result of the lower estimate of risk of pca diagnosis in subsequent biopsies in patients with hg pin in the first biopsy and normal levels of psa , in comparison to men with benign changes and the associated increased levels of psa , the european association of urology guidelines of 2010 excluded hg pin , without elevated levels of psa , from the indications for subsequent prostate biopsy . only extensive hg pin can be a reason to re biopsy , because the risk of subsequent pca is increased . concurrently , due to the opinion that at least some cases of asap diagnosis in biopsy specimens are actually undiagnosed cancers , the indication to second biopsy in such patients has been preserved . some studies stand at least partially in contradiction to the opinion that patients with asap have a worse prognosis in comparison to hg pin . a study by schoenfield ( 2007 ) found that the risk of cancer development in patients with hg pin was 33% while it was only 25% in asap . the practice of undeserved exclusion of hg pin in the prognosis of cancer development occurs more and more frequently but it should be evaluated . therefore , the aim of this prospective study was to determine the incidence of pca , intraepithelial anaplasia of high grade ( hg pin ) , atypical small acinar proliferation ( asap ) , and other pathological states in men subjected to subsequent and repeat prostate biopsy due to suspicion of pca , and to assess whether there is any difference in risk of cancer development depending on type of changes identified in the first biopsy ( asap versus hg pin ) . another aim was to asses if asap or hg pin change the value of psa . this prospective study included 1010 men who were evaluated from april 2006 to september 2007 , after approval of the local ethical committee . the study 's inclusion criteria included the suspicion of pca on the basis of elevated values of prostate specific antigen ( psa , threshold set at 4 ng / ml ) , changes in digital rectal examination ( dre ) , and/or presence of changes on transrectal ultrasound ( trus ) . patients who were qualified to join the study were referred to two urology departments where , with quinolone chemoprophylaxis and local anesthesia , 1012 core biopsy using a tru cut cook 18 g needle under trus control was performed . in general , ten specimens were collected , but the protocol was sometimes expanded to include additional biopsies from suspicious areas of the prostate as seen in trus imaging . if the results of the biopsy specimen revealed pca then the patient was qualified for further treatment or observation depending on the severity of the disease . if the results revealed asap or extensive hg pin then the patient entered the outpatient observation program and a second biopsy was proposed three months later . the same protocol was applied for those with benign changes or prostatitis in the first biopsy with accompanying rising or persistent elevation of psa levels . wilk test was used to evaluate the compliance of the parameters with the normal distribution . the analyzed parameters did not have a normal distribution , which is why the non parametric mann whitney u test was used to compare the sum of the rank in two samples , which were presented using the median ( me ) , upper ( q1 ) and lower quartiles ( q3 ) , the range of minimums and maximums , as well as mean and standard deviation . among 1,010 men who were subjected to first time biopsy , pca was diagnosed in 336 patients ( 33.27% ) . the presence of asap and hg pin lesion were diagnosed in 159 men ( 15.47% ) ( table 1 ) . the mean value of psa in the studied men was 19.17 ng / ml ( median 9.16 ng / ml ) . the highest psa levels were found in the group of patients with pca ( mean 34.93 ng / ml , median 13.5 ng / ml ) and the lowest in the group of patients with low grade prostatic intraepithelial neoplasia ( lg pin ) ( mean 9.3 ng / ml , median 6.90 ng / ml ) . results of second prostate biopsy comparing the mean values of psa in patients with asap and hg pin in relation to patients with benign prostatic hyperplasia ( bph ) , a higher mean value was observed . a similar correlation is observed when comparing medians with only the case of co occurrence of asap and hg pin ; it was found that the value was equal to the median of patients with bph . the differences in psa levels between these lesions ( asap and hg pin ) were not statistically significant from each other ( p = 0.6139 ) , similar to the levels of psa in the group of patients with asap in comparison to those with asap occurring together with hg pin ( p = 0.1684 ) and hg pin in comparison to asap occurring together with hg pin ( p = 0.4737 ) . there was , however , a statistically significant difference between the values of psa in the group of patients with asap in comparison to those with bph ( p = 0.005 ) as well as in patients with hg pin in comparison to bph ( p = 0.02 ) ( figures 1 and 2 ) . mean values of psa in the group of patients with asap and hg - pin in relation to patients with bph . median psa in the group of patients with asap and hg - pin in relation to patients with bph . the most common diagnosis made in the course of second biopsy was bph ( 32 patients 32.65% ) and pca was diagnosed in 16 ( 16.33% ) patients in whom it was not previously found . in the second biopsy , 17.34% ) was diagnosed more frequently than hg pin ( 14 patients 14.28% ) ( table 2 ) . results of second prostate biopsy pca was diagnosed in six of 19 patients subjected to the second biopsy ( 31.57% ) in whom first biopsy revealed the presence of hg pin , in four of 40 ( 10% ) in whom bph was diagnosed , and in four of 18 ( 22.22% ) with asap or changes of lg pin type together with asap , and two of five ( 40% ) in whom first biopsy revealed coexistence of asap and hg pin ( table 3 ) . results of first biopsy in patients in whom pca was diagnosed in second biopsy there were no statistically significant differences in the incidence of pca diagnosis in repeat biopsy between patients with the diagnosis of asap and hg pin in first biopsy ( p = 0.2929 ) . also , there was no statistically significant difference in the incidence of diagnosing cancer in repeat biopsies after diagnosing asap coexisting together with hg pin in first biopsy ( p = 0.3512 ) in comparison to asap and hg pin occurring independently . among 1,010 men who were subjected to first time biopsy , pca was diagnosed in 336 patients ( 33.27% ) . the presence of asap and hg pin lesion were diagnosed in 159 men ( 15.47% ) ( table 1 ) . the mean value of psa in the studied men was 19.17 ng / ml ( median 9.16 ng / ml ) . the highest psa levels were found in the group of patients with pca ( mean 34.93 ng / ml , median 13.5 ng / ml ) and the lowest in the group of patients with low grade prostatic intraepithelial neoplasia ( lg pin ) ( mean 9.3 ng / ml , median 6.90 ng / ml ) . results of second prostate biopsy comparing the mean values of psa in patients with asap and hg pin in relation to patients with benign prostatic hyperplasia ( bph ) , a higher mean value was observed . a similar correlation is observed when comparing medians with only the case of co occurrence of asap and hg pin ; it was found that the value was equal to the median of patients with bph . the differences in psa levels between these lesions ( asap and hg pin ) were not statistically significant from each other ( p = 0.6139 ) , similar to the levels of psa in the group of patients with asap in comparison to those with asap occurring together with hg pin ( p = 0.1684 ) and hg pin in comparison to asap occurring together with hg pin ( p = 0.4737 ) . there was , however , a statistically significant difference between the values of psa in the group of patients with asap in comparison to those with bph ( p = 0.005 ) as well as in patients with hg pin in comparison to bph ( p = 0.02 ) ( figures 1 and 2 ) . mean values of psa in the group of patients with asap and hg - pin in relation to patients with bph . median psa in the group of patients with asap and hg - pin in relation to patients with bph . the most common diagnosis made in the course of second biopsy was bph ( 32 patients 32.65% ) and pca was diagnosed in 16 ( 16.33% ) patients in whom it was not previously found . in the second biopsy , 17.34% ) was diagnosed more frequently than hg pin ( 14 patients 14.28% ) ( table 2 ) . results of second prostate biopsy pca was diagnosed in six of 19 patients subjected to the second biopsy ( 31.57% ) in whom first biopsy revealed the presence of hg pin , in four of 40 ( 10% ) in whom bph was diagnosed , and in four of 18 ( 22.22% ) with asap or changes of lg pin type together with asap , and two of five ( 40% ) in whom first biopsy revealed coexistence of asap and hg pin ( table 3 ) . results of first biopsy in patients in whom pca was diagnosed in second biopsy there were no statistically significant differences in the incidence of pca diagnosis in repeat biopsy between patients with the diagnosis of asap and hg pin in first biopsy ( p = 0.2929 ) . also , there was no statistically significant difference in the incidence of diagnosing cancer in repeat biopsies after diagnosing asap coexisting together with hg pin in first biopsy ( p = 0.3512 ) in comparison to asap and hg pin occurring independently . currently , the only recognized precancerous lesion for pca is considered to be hg pin . the incidence of hg pin among patients subjected to biopsy due to suspicion of cancer ranges from 1.5% to about 16.5% with an average around 6% [ 23 , 24 ] . asap is a condition associated with the occurrence of lesions of atypical proliferation in the prostatic glandular epithelium , which is likely to be associated with the development of under appreciation of pca . asap has been recognized in the results of prostate biopsy since the end of the 90s . in many studies its frequency varies and is about 0.52% ( up to 6% is some studies ) [ 25 , 26 ] . the correct classification of the changes observed in histopathological preparation is extremely difficult , but more and more authors classify changes found in the asap as an under diagnosed cancer . braussi , among such patients , post operatively confirmed the presence of pca in case of patients , and almost all of them with clinical significance . therefore it is to be discussed , whether asap is still a valid term , or if it should be classified as a cancer , and radical treatment should be proposed . in most studies , asap or hg pin alone does not cause a rise in psa levels , and it is believed that only cancer and acute inflammation can account for such a rise . however , in some works , especially the older ones , opposing opinions can be found [ 28 , 29 ] . in our study , among patients in whom asap and extensive hg pin were diagnosed , median values of psa were statistically higher in comparison to healthy men ( those with bph ) ( p = 0.005 for the group with asap and p = 0.02 for the group with hg pin ) . such a difference can , however , suggest that at least a portion of the patients with asap and hg pin , already in the moments of the first biopsy , have already developed an undiagnosed pca , which caused the elevated values of psa . unfortunately , it is impossible to fully assess the impact of all the histopathological changes of the prostate on the level of psa , as well as to exclude the lack of possible impact of accidentally missing the cancer . in fact , such a correlation could only be established after examining the whole prostate on autopsy and correlating its results with levels of psa . that is why it is impossible to definitively exclude that a coexisting undiagnosed pca could cause the observed elevation of psa level in men with precancerous lesions . in recently published studies it is observed that somewhat less frequent diagnoses of pca in second biopsy occur in patients with hg pin in comparison to studies from previous years . it seems that one of the causes may be the increased number of specimens collected during the first biopsy , and , therefore , increased probability of cancer diagnosis already in the first biopsy . currently , only a few centers still perform sextant biopsies and the suggested biopsy standard has become a 10 or 16core collection . herawi proposed an explanation of this fact , and outlined that if the first and second biopsies were sextant , then the risk of pca diagnosis was 14.1% for each biopsy . if the first sextant biopsy diagnosed hg pin and the second if , however , both the first and subsequent biopsies were performed using eight cores then the chance of diagnosing cancer in both groups fell to 14.6% . he claims that the decrease in diagnostic ability in the last group is probably caused by the greater frequency of diagnosing cancer in the first biopsy and he proposes not subjecting patients to subsequent biopsies in such cases . pin is diagnosed on first biopsy is ambiguous . in earlier studies published before the era of asap ( studies published up to the end of the nineties ) however , this assessment was conducted on small groups of men ( 150200 patients ) . among them , analyzing only the studies that included a large number of patients , the mean risk of pca development among patients with hg pin was 24.1% ( range : 21.7% to 28.9% ) [ 3133 ] . in more recent studies , the estimated risk is currently estimated at 18.1% ( range : 16.7% to 19.6% ) [ 34 , 35 , 36 ] . in the analyzed study , a repeat biopsy was performed in 19 patients with asap , and in four of them ( 21.05% ) pca was diagnosed . the proportion of patients with hg a biopsy was performed in 22 patients , and pca was found in six of them ( 27.27% ) . in all of the cases , hg the difference between the risk of pca diagnosis after the diagnosis of asap or hg pin in the first biopsy is statistically insignificant and also stands in contradiction to the majority of presently published studies , in which the assessed risk of pca development in the second biopsy after asap being discovered in the first is significantly ( sometimes even two fold ) greater than after hg pin in the first . in our work , risk is comparable for both diseases and the slightly more frequent finding of pca after hg pin in the first biopsy does not clearly establish a worse prognosis in these patients . it should be worth mentioning that , if the results of this analyzed study were compared to older data from before the era of asap , the risk of pca development after hg pin in the first biopsy would be similar to that presented in the literature . very few studies confirm our results and from recent works , only schoenfield presents a risk of pca development that is similar to our study , indicating it as 33% for hg pin and only 25% for asap . in light of these results it seems that hg pin can not be clearly rejected as a precancerous lesion and , as other authors propose , performing subsequent prostate biopsies should not be abandoned , especially when hg such patients have a similar risk of pca diagnosis in the second biopsy as those with asap in the first and at a much higher risk than those with benign changes found in the first biopsy ( the presence of pca was confirmed in our study in four of 50 patients with bph it seems that patients with both histopathologic entities asap and extensive hg pin should be subjected to an urgent second prostate biopsy . it is very likely that these are patients in whom , at the moment of the first biopsy , the pca had already developed , but was not explicitly confirmed . this is also why there seems to be no reason to subject these patients to different treatment protocols as they both should be subjected to an urgent second biopsy in around 46 weeks following the initial procedure . a precancerous lesion diagnosed upon biopsy causes a statistically significant increase in the values of psa in relation to bph , likewise in the case of asap as well as extensive hg pin . the estimate of risk of pca diagnosis in patients with asap and those with extensive hg pin in the first biopsy is comparable , which is why there is no reason for different treatment of patients with the above mentioned diagnoses . both should be subjected to an urgent second biopsy in around 46 weeks following the initial procedure .
the etude epidemiologique de femmes de la mutuelle gnrale de l'education nationale ( e3n ) prospective cohort was initiated in france in 1990 to investigate risk factors for cancer in women ( 10 ) . the cohort included 98,995 women living in france , aged 4065 years at baseline , who were covered by the mutuelle gnrale de l'education nationale , a national health insurance plan for teachers and coworkers . all women signed an informed consent form , in compliance with the rules of the french national commission for computed data and individual freedom ( commission national informatique et liberts ) from which approval was obtained . women completed baseline and biennial self - administered questionnaires with demographic and anthropometric characteristics , reproductive history , health status , lifetime use of hormonal treatment , parental diabetes , and smoking status . follow - up questionnaires updated information , especially on medication use and menopausal status , and recorded occurrence of major health events , among which was diabetes . a first set of potential cases of diabetes included women who had self - reported either diabetes , a diet to manage diabetes , use of diabetes drugs , or a hospitalization for diabetes in at least one of the eight questionnaires up until july 2005 . a total of 4,289 self - reported potential cases were thus identified . among them , 2,315 cases were validated when women were identified from a drug reimbursement file provided by the health insurance plan as having been reimbursed for a diabetes drug between 1 january 2004 ( date when the file became available ) and 30 june 2007 ( end point in the present study ) . among the 1,974 women without diabetes drug reimbursement , women alive and with an accurate address ( n = 1,735 ) were mailed a questionnaire specifically designed to validate diabetes . it included questions on the circumstances of diagnosis ( date of diagnosis , symptoms , and biological data including fasting or random glucose concentrations at diagnosis ) , the current therapy ( prescription of diet and/or physical activity and list of diabetes drugs ) , and the monitoring of diabetes ( last values of fasting glucose and a1c levels ) . among the 1,480 women who completed this questionnaire ( 84% response rate ) , 342 potential cases were confirmed when glucose at diagnosis was reported to comply with world health organization recommendations ( fasting glucose 7.0 mmol / l or random glucose 11.1 mmol / l ) and/or when women reported taking diabetes drugs and/or when their last values of fasting glucose or a1c levels were reported to be 7.0 mmol / l and/or 7% , respectively . in this first set of potential cases , a total of 2,657 diabetes cases were thus validated . among the 1,632 nonvalidated cases , a second set of women with potential cases of diabetes was identified exclusively from the drug reimbursement file ( n = 1,216 ) without a prior report of diabetes in any of the eight study questionnaires . we mailed the diabetes - specific questionnaire detailed above to 1,139 of them and 734 women completed it . we considered as noncases women who reported being nondiabetic and who had been reimbursed for diabetes drugs only once before 30 june 2007 ( n = 233 ) ; we validated as diabetic cases women who confirmed diabetes in the diabetes - specific questionnaire ( n = 458 ) as well as those who did not answer the diabetes - specific questionnaire but who received reimbursement for diabetes drugs at least twice ( n = 381 ) . other potential cases were considered as nonvalidated ( n = 144 ) . altogether , 3,496 diabetes cases were validated up until 30 june 2007 . although this procedure did not systematically allow differentiation between type 1 and type 2 diabetes , very few incident cases of type 1 diabetes were expected considering the age range . they could report their exact birth weight , and we subsequently categorized it as low ( < 2,500 g ) , medium ( 2,5004,000 g ) , or high ( > 4,000 g ) . women were also asked whether they had been told they had a low , medium , or high birth weight as a newborn ( we used this information when birth weight was missing , n = 40,817 ) and whether their birth was premature . , women provided information on their body silhouette ( 11 ) at different ages ( 8 years , menarche , 2025 years , and 3540 years ) . a four - level categorical variable was used with values from 1 ( leanest silhouette ) to 4 ( the four largest silhouettes were grouped together because of small numbers ) , except at age 3540 years , at which categories were ranked from 2 to 5 . silhouette 3 seemed to be the best choice for a common reference category at all ages , given the corresponding number of subjects at each age . bmi was computed at each follow - up ( eight biannual questionnaires ) from self - reported weight and height . we excluded women who declared themselves diabetic on the first questionnaire ( n = 715 ) as well as those with nonvalidated incident diabetes or with an unknown date of diagnosis ( n = 1,968 ) , those with no follow - up after baseline ( n = 2,714 ) , and those with missing baseline bmi ( n = 2,145 ) , leaving 91,453 women for analysis . women contributed person - time until the date of diagnosis of diabetes , date of last completed questionnaire if the 2005 questionnaire was not completed , or 30 june 2007 , whichever occurred first . we used cox proportional hazards regression models with age as the time scale to estimate the hazards ratios ( hrs ) for diabetes and 95% ci associated with birth weight categories or silhouettes at different ages in separate models . in a more global analysis of weight history over the life course , subjects were categorized according to birth category ( low , medium , or high ) , silhouette at 8 years ( 1 , 2 , or 3 ) , and baseline bmi ( overweight or not ) in an 18-level class variable . the hr for each category of this variable was estimated in reference to women with birth weight in the medium class , silhouette 2 at 8 years , and baseline bmi < 25 kg / m . this analysis was restricted to subjects without missing values for any of the three variables . to respect the proportional hazards assumption , analyses were performed according to 5-year interval birth cohorts using the strata option of sas phreg procedure ( sas 9.1.3 ; sas institute , cary , nc ) . we controlled for potential confounders by adjusting models for education level , baseline physical activity , prematurity , parental history of diabetes , high cholesterol level , age at menarche , parity , and ever use of oral contraceptive pills . data on smoking , menopausal status , use of menopause hormonal therapy , hypertension , and bmi were also considered as potential confounders and analyzed as time - dependent variables . we replaced missing values by the modal value ( all were categorical variables ) when data were missing in fewer than 5% of women or else by a missing category . we performed sensitivity analyses that also included all nonvalidated incident diabetes as diabetes cases and all prevalent cases of diabetes . a first set of potential cases of diabetes included women who had self - reported either diabetes , a diet to manage diabetes , use of diabetes drugs , or a hospitalization for diabetes in at least one of the eight questionnaires up until july 2005 . a total of 4,289 self - reported potential cases were thus identified . among them , 2,315 cases were validated when women were identified from a drug reimbursement file provided by the health insurance plan as having been reimbursed for a diabetes drug between 1 january 2004 ( date when the file became available ) and 30 june 2007 ( end point in the present study ) . among the 1,974 women without diabetes drug reimbursement , women alive and with an accurate address ( n = 1,735 ) were mailed a questionnaire specifically designed to validate diabetes . it included questions on the circumstances of diagnosis ( date of diagnosis , symptoms , and biological data including fasting or random glucose concentrations at diagnosis ) , the current therapy ( prescription of diet and/or physical activity and list of diabetes drugs ) , and the monitoring of diabetes ( last values of fasting glucose and a1c levels ) . among the 1,480 women who completed this questionnaire ( 84% response rate ) , 342 potential cases were confirmed when glucose at diagnosis was reported to comply with world health organization recommendations ( fasting glucose 7.0 mmol / l or random glucose 11.1 mmol / l ) and/or when women reported taking diabetes drugs and/or when their last values of fasting glucose or a1c levels were reported to be 7.0 mmol / l and/or 7% , respectively . in this first set of potential cases , a total of 2,657 diabetes cases a second set of women with potential cases of diabetes was identified exclusively from the drug reimbursement file ( n = 1,216 ) without a prior report of diabetes in any of the eight study questionnaires . we mailed the diabetes - specific questionnaire detailed above to 1,139 of them and 734 women completed it . we considered as noncases women who reported being nondiabetic and who had been reimbursed for diabetes drugs only once before 30 june 2007 ( n = 233 ) ; we validated as diabetic cases women who confirmed diabetes in the diabetes - specific questionnaire ( n = 458 ) as well as those who did not answer the diabetes - specific questionnaire but who received reimbursement for diabetes drugs at least twice ( n = 381 ) . other potential cases were considered as nonvalidated ( n = 144 ) . altogether , 3,496 diabetes cases were validated up until 30 june 2007 . although this procedure did not systematically allow differentiation between type 1 and type 2 diabetes , very few incident cases of type 1 diabetes were expected considering the age range . they could report their exact birth weight , and we subsequently categorized it as low ( < 2,500 g ) , medium ( 2,5004,000 g ) , or high ( > 4,000 g ) . women were also asked whether they had been told they had a low , medium , or high birth weight as a newborn ( we used this information when birth weight was missing , n = 40,817 ) and whether their birth was premature . , women provided information on their body silhouette ( 11 ) at different ages ( 8 years , menarche , 2025 years , and 3540 years ) . a four - level categorical variable was used with values from 1 ( leanest silhouette ) to 4 ( the four largest silhouettes were grouped together because of small numbers ) , except at age 3540 years , at which categories were ranked from 2 to 5 . silhouette 3 seemed to be the best choice for a common reference category at all ages , given the corresponding number of subjects at each age . bmi was computed at each follow - up ( eight biannual questionnaires ) from self - reported weight and height . they could report their exact birth weight , and we subsequently categorized it as low ( < 2,500 g ) , medium ( 2,5004,000 g ) , or high ( > 4,000 g ) . women were also asked whether they had been told they had a low , medium , or high birth weight as a newborn ( we used this information when birth weight was missing , n = 40,817 ) and whether their birth was premature . 1 ( available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-1304/dc1 ) . at baseline , women provided information on their body silhouette ( 11 ) at different ages ( 8 years , menarche , 2025 years , and 3540 years ) . a four - level categorical variable was used with values from 1 ( leanest silhouette ) to 4 ( the four largest silhouettes were grouped together because of small numbers ) , except at age 3540 years , at which categories were ranked from 2 to 5 . silhouette 3 seemed to be the best choice for a common reference category at all ages , given the corresponding number of subjects at each age . bmi was computed at each follow - up ( eight biannual questionnaires ) from self - reported weight and height . we excluded women who declared themselves diabetic on the first questionnaire ( n = 715 ) as well as those with nonvalidated incident diabetes or with an unknown date of diagnosis ( n = 1,968 ) , those with no follow - up after baseline ( n = 2,714 ) , and those with missing baseline bmi ( n = 2,145 ) , leaving 91,453 women for analysis . women contributed person - time until the date of diagnosis of diabetes , date of last completed questionnaire if the 2005 questionnaire was not completed , or 30 june 2007 , whichever occurred first . we used cox proportional hazards regression models with age as the time scale to estimate the hazards ratios ( hrs ) for diabetes and 95% ci associated with birth weight categories or silhouettes at different ages in separate models . in a more global analysis of weight history over the life course , subjects were categorized according to birth category ( low , medium , or high ) , silhouette at 8 years ( 1 , 2 , or 3 ) , and baseline bmi ( overweight or not ) in an 18-level class variable . the hr for each category of this variable was estimated in reference to women with birth weight in the medium class , silhouette 2 at 8 years , and baseline bmi < 25 kg / m . this analysis was restricted to subjects without missing values for any of the three variables . to respect the proportional hazards assumption , analyses were performed according to 5-year interval birth cohorts using the strata option of sas phreg procedure ( sas 9.1.3 ; sas institute , cary , nc ) . we controlled for potential confounders by adjusting models for education level , baseline physical activity , prematurity , parental history of diabetes , high cholesterol level , age at menarche , parity , and ever use of oral contraceptive pills . data on smoking , menopausal status , use of menopause hormonal therapy , hypertension , and bmi were also considered as potential confounders and analyzed as time - dependent variables . we replaced missing values by the modal value ( all were categorical variables ) when data were missing in fewer than 5% of women or else by a missing category . we performed sensitivity analyses that also included all nonvalidated incident diabetes as diabetes cases and all prevalent cases of diabetes . among the 91,453 women studied , 2,534 cases of incident diabetes were ascertained during a median follow - up of 15.1 years . characteristics of the study population according to diabetes status at the end of the follow - up , are presented in table 1 . the relationship between body weight or silhouette at different ages and diabetes risk was investigated ( table 2 ) . characteristics of the e3n study population , according to diabetes status at the end of the 17-year follow - up data are means sd or % . * birth weight in grams categorized as low ( < 2,500 g ) or women 's self - classification as newborn of low birth weight ( when birth weight in grams was missing ) . hrs ( 95% ci ) for incident diabetes in relation to body silhouette history in the e3n cohort ( 19902007 ) * model 2 : includes physical activity ( < 34/3447/4762/ 62 met - h / week ) , education ( 9/1011/1214/1516/17 years ) , prematurity ( no / yes ) , family history of diabetes ( none / only one parent / both parents ) , smoking ( never / former / current smoker , time - dependent variable ) , high cholesterol level ( no / yes ) , hypertension ( no / yes , time - dependent variable ) , menopausal status ( no / yes , time - dependent variable ) , hormone replacement therapy ( never / ever , time - dependent variable ) , oral contraceptive pills ( never / ever ) , parity and age at first child ( nulliparous / first child at < 30 years , 12 children / first child at < 30 years , 3 + children / first child at 30 years ) , and age at menarche ( 12/13/14 years old ) , according to birth cohort ( 1,9251,930/1,9301,935/1,9351,940/1,9401,945/1,9451,950 ) . model 3 : confounders as above and further adjustment for adult bmi as a time - dependent variable . after adjustment for confounders , the risk of diabetes decreased with increasing birth weight ( ptrend < 0.001 ) . this result remained statistically significant after additional adjustment for adult bmi during follow - up . at 8 years , both a lean and a large silhouette were associated with an increased risk of incident diabetes . after controlling for potential confounders . body silhouette at menarche was inversely associated with the risk of incident diabetes ( ptrend < 0.001 ) , and the association was enhanced after full adjustment . body silhouette at age 2025 years was positively associated with the risk of diabetes ( ptrend < 0.001 ) , even after adjustment for potential confounders . however , the association reversed after additional adjustment for adult bmi during follow - up , with a decrease in risk among women with a large silhouette and an increased risk in lean women ( ptrend < 0.001 ) . a significantly increased risk of diabetes was associated with the larger silhouettes at age 3540 years . after controlling for potential confounders and additionally for adult bmi during follow - up , we found that hrs were of a lower magnitude , but the relationship remained statistically significant . adjustment for birth weight did not substantially modify the above findings ( data not tabulated ) . interactions between birth weight and body silhouettes were also tested in the model adjusted for confounders , but none was statistically significant . missing values on silhouettes were associated with a significantly increased diabetes risk , and they were more frequent both among overweight or obese women ( p < 0.001 for birth weight and each body silhouette , except at age 2025 years , p = 0.012 ) and among older women ( p < 0.001 for birth weight and each body silhouette ) independently . because self - reported birth weight and body silhouette may be influenced by baseline body weight , we investigated a potential interaction between these variables and baseline bmi . a statistically significant interaction was found for birth weight or body silhouettes ( all p < 0.001 ) . however , analyses stratified on baseline overweight status ( supplementary table 1 , available in an online appendix ) displayed associations between diabetes risk and birth weight or body silhouette until early adulthood similar to those presented in table 2 . only the positive association between body silhouette at 3540 years and new - onset diabetes in each subgroup was no longer significant after adjustment for bmi during follow - up ( all p > 0.2 ) . in a more global analysis of weight history over the life course ( table 3 ) , as compared with nonoverweight women with medium birth weight and silhouette 2 at 8 years , women who were lean in childhood were at higher risk for new - onset diabetes . the highest risk for new - onset diabetes was found for overweight women who had low birth weight and were lean in childhood . finally , normal - weight women who reported a larger silhouette in childhood , tended to have a lower risk of diabetes . the only situation for which a large silhouette in childhood was not associated with a lower risk for diabetes compared with a thinner silhouette was in overweight women with a high birth weight . fully adjusted hrs ( 95% ci ) for incident diabetes in relation to body shape history over the life course , from birth to middle age , in the e3n cohort ( 19902007 ) * adjusted for physical activity ( < 34/3447/4762/2 met - h / week ) , education ( 9/1011/1214/1516/17 years ) , prematurity ( no / yes ) , family history of diabetes ( none / only one parent / both parents ) , smoking ( never / former / current smoker , time - dependent variable ) , high cholesterol level ( no / yes ) , hypertension ( no / yes , time - dependent variable ) , menopausal status ( no / yes , time - dependent variable ) , hormone replacement therapy ( never / ever , time - dependent variable ) , oral contraceptive pills ( never / ever ) , parity and age at first child ( nulliparous / first child at < 30 years , 12 children / first child at < 30 years , 3 + children / first child at 30 years or more ) , age at menarche ( 12/13/14 years old ) , and adult bmi as a time - dependent variable , according to birth cohort ( 1,9251,930/1,9301,935/1,9351,940/1,9401,945/1,9451,950 ) . when women with validated and nonvalidated cases of incident diabetes were included in the analysis ( n = 3,867 ) , the results remained similar to those presented above ( data not tabulated ) . in a sensitivity analysis , we also included women with prevalent diabetes , and estimates were consistent with the main results presented ( data not shown ) . in this large cohort study of french women , we described a complex relationship between body shape throughout life and adult - onset diabetes . low birth weight and thinness from childhood to early adulthood increased the risk of diabetes , independently of adult bmi . the association between body silhouette from childhood to adulthood and diabetes risk was not modified by birth weight . compared with having a medium silhouette both as a child and as a middle - aged adult , an evolution from a lean to a large silhouette conferred the highest risk , whereas evolution from medium to lean conferred the lowest risk . early adulthood ( 2025 years ) seemed to be a critical period when the relationship between body silhouette and diabetes reversed . because there is no birth weight registry for the whole french population , we used self - reported birth weight , as in other large studies ( 1214 ) . potential nondifferential misclassification may occur and reduce the association ; thus , the actual association between low birth weight and diabetes might be even stronger than that reported here . a recent systematic review suggested that the association between birth weight and type 2 diabetes decreased linearly ( 15 ) , except in native north american populations , in which there is a high prevalence of maternal diabetes . although we lacked information on the duration of their mothers ' gestation , adjustment for having been a premature baby did not modify our results . this finding is consistent with previous studies that have shown , using precise information on the length of gestation , that the relationship between small birth weight and incidence of type 2 diabetes was not explained by prematurity . a recent study showed that a genetic locus previously identified as a marker of type 2 diabetes could also influence birth weight , suggesting that the association between low birth weight and type 2 diabetes could be genetically mediated ( 16 ) . in addition to low birth weight , thinness in childhood and adolescence was associated with later diabetes risk . they speculated that the association between low weight gain between 6 months and 1 year and the risk of later type 2 diabetes may be due to impaired development of the endocrine pancreas , because islet development continues from late gestation until adolescence ( 17,18 ) . however , high bmi in childhood or adolescence has also been associated with impaired glucose tolerance ( 19 ) and even type 2 diabetes ( 20 ) in early adulthood . a rapid increase in bmi during childhood was also found to be related to higher plasma insulin levels ( 21 ) . in our study , few women reported either of the two largest silhouettes ( silhouettes 7 or 8) at 8 years or at menarche ( n = 94 and 177 , respectively ) , and we may have lacked the power to detect associations with a larger silhouette in early life . however , a lean silhouette in childhood was not associated with the risk of diabetes in overweight adult women with large birth weight . as pointed out by eriksson et al . ( 8) , the pathway leading to diabetes seems to be different in subjects with low and high birth weight . it is also noteworthy that the adjustment for adult bmi , by inclusion as a time - dependent variable , reinforced the associations between thinness in childhood and adolescence and diabetes risk . in our study , after we controlled for bmi in middle - age , the body silhouette at age 2025 years was inversely associated with the risk of incident diabetes in middle age . this is the first cohort indicating that the relationship between body silhouette and risk of later - onset diabetes could reverse so late in life . our results are consistent with previous observations that increases in bmi from childhood to adulthood are an important risk factor for adult - onset diabetes ( 1 ) . although increased adiposity is the strongest risk factor for type 2 diabetes ( 8,14 ) , our results add evidence that increased adiposity is particularly detrimental for those who have experienced slow growth in utero but also throughout childhood . in our cohort , the adjustment for bmi in middle age increases the risk associated with body silhouette in childhood but not with birth weight . differences between studies for the latter point may be related to the period in life when overweight developed . in our sample of french women born between 1925 and 1950 , it occurred more often in early adulthood . a strength of our study is the large number of participants and the large number of women with incident cases of diabetes from whom we were able to obtain data on body silhouette from childhood to middle age . the exclusion of women with nonvalidated cases of diabetes limited misclassification errors , whereas sensitivity analyses suggested the absence of a major selection bias . analyses were replicated with all potential cases of diabetes , and the results were similar . a limitation is the use of recalled body silhouette to estimate corpulence over the life course . however , a validation study to determine the accuracy of reported anthropometric measurements and perceived body silhouettes showed a correlation of 0.78 between bmi measured by technicians and the current self - reported silhouette , with no significant differences between self - reported and technician - measured mean bmi ( 22 ) . body shape has been evaluated by recall in other studies and found to be reliable ( 23,24 ) , providing a good ranking of past corpulence and changes in corpulence over time . ( 23 ) reported that the bmi percentile at menarche was well correlated with recalled body size at this period , after a recall time ranging from 23 to 33 years . the ability to recall events around menarche appeared to be good in that study , probably because the occurrence of the first menstrual period is usually a disrupting event . ( 23 ) also described systematic recall biases , with the thinnest girls overestimating their body size , whereas normal and heavier girls underestimated it . such a pattern was also observed among adult women in the validation study conducted in our population ( 22 ) . however , this type of bias would tend to underestimate any association between body silhouette and incident diabetes . in addition , our results were robust when we stratified analyses by overweight status at baseline ( supplemental table 1 ) . finally , an increased risk of incident diabetes was observed in the missing value category for women with all body silhouette measurements , a result that could be partially explained by the lower response rate among overweight or obese women and among older women . the prevalence of diabetes assessed in french women , aged 6069 years , was 8% in 2005 ( 25 ) . in our cohort the prevalence of diabetes in women of similar age at the end of the follow - up was lower ( 3% ) . this difference may be explained in part by a low prevalence of obesity in our cohort ( 3% at inclusion ) . our results could thus be very different in a younger cohort , born from an increasing number of overweight mothers with glucose intolerance , and experiencing overweight throughout childhood . in summary , among women born in 19251950 , both low birth weight and thinness in childhood and early adulthood were found to be associated with the risk of diabetes in middle age , independent of adult bmi . weight gain prevention programs need to be implemented to prevent diabetes in young adult women , especially among those who were lean in childhood .
biological nanostructures can induce the perception of the entire range of a rainbow s color , but often only a reduced range of colors is produced in biological iridescence ; there are often sharp discontinuities between target areas that display iridescence and those that do not , and perceived colors can change dramatically with viewing angle ( not just gradually along a rainbow ) . diffraction gratings ( where light is diffracted by a periodic structure , usually of ridges ) are one way in which biological iridescence can be produced . differences in the degree of perfection in diffraction gratings that generate iridescence might impact the visual system of insect pollinators . for definition of the optical effects of different surfaces , the spectral reflection of the range of hues produced by target epoxy disks ( varying in iridescence production and pigment ) was measured using optical spectroscopy in the 300-to-700-nm wavelength range ( near - uv to infrared ) . light from a xenon light source was reflected off the target disks at a 45 incidence angle , and the reflected and scattered light was analyzed at angles varying from 45 to 90 in 5 steps . this was repeated in 10 steps from a position of 90 to one of 0 across the diffraction grating . the spectral reflectance curves measured at each angle can be used to determine the relative excitation values in each of the bees color receptors ( uv , blue , and green ) , and , using the methods specified by chittka , these excitation values were used to calculate hexagon color loci under lab conditions ( as detailed in ) for all target flowers used in this study ( figure 1 ) . figure 1a shows the color loci of two disks that contain the same blue pigment but have different types of diffraction grating , calculated in bee hexagon color space . artificial gratings were cast from a commercial diffraction grating film , whereas floral diffraction gratings were cast from the iridescent tepal of the queen of the night tulip cultivar . both the two different types of diffraction grating ( artificial perfect and floral imperfect ) produce scattering within bee color space ; however , the pattern of this scattering differs between the two types of grating . the distribution of colors produced by the grating is considerably wider than that produced by the imperfect floral grating . the underlying pigment also impacts the perceived color , with two disks with identical perfect diffraction gratings but different underlying pigment displaying discrete scattering in color space ( figure 1b ) . further analysis of the individual receptor signals suggests that the interaction between underlying pigment and overlying iridescence may have a range of effects ; for example , the stimuli whose color loci are displayed in figure 1b , when analyzed for individual receptor contrast , show a much wider spread in the uv channel for the blue pigment disk than for the red pigment disk ( figure s1 and tables s1 and s2 ) . iridescent objects show a broader spread of loci within bee color space , and detectability depends in part on the amount of contrast that is presented to a visual system over time [ 14 , 15 ] . the analysis of the individual receptor signals ( supplemental information ) shows that there is a diversity in the median and range of signals shown by different disk types . although blue artificial iridescent disks show the greatest range of uv receptor signals , it is actually non - iridescent disks that show the highest green receptor signal ( which is of interest because this signals determines target detectability , in addition to color contrast ) . an explanation for this is suggested by figure 1b , showing a spread of color loci in the non - iridescent disks , due to specular reflection or gloss ( gloss measurements , as measured in , are included in figure s2 ) . so that the impact of iridescence on flower detection and identification by bees could be determined it has previously been shown that the color contrast of an object with its background can impact its salience ( detectability ) . however , all investigations into this topic have used pigment colors . iridescence , including that generated by plant structures , can be extremely colorful and produce highly contrasting bands of distinct hues . this could provide a high contrast with any surrounding background , as well as between the different hues produced by the iridescence , and make the object highly detectable , which would be a distinct advantage for any floral billboard . additionally , the color - shifting qualities of iridescence itself could also increase flower detectability as an animal moves toward it . target detectability depends in part on the amount of contrast that is presented to a visual system over time [ 14 , 15 ] . with an iridescent target , the hue of color produced is dependent on the angle from which it is observed . for a mobile forager searching for suitable targets , an iridescent object could therefore produce more visual change per unit time than one where the color is produced by pigment and is therefore homogenous . this would increase detectability and could have significant effects on the behavioral ecology of foragers , to the advantage of both flower and visitor . although studies have explored the effectiveness of iridescence as a sexual signal [ 19 , 20 ] , none has yet analyzed the detectability of iridescent targets compared to conventional visual cues that do not change appearance with viewing perspective . using the method described in spaethe et al . , we determined the extent to which the addition of diffraction - grating generated iridescence ( either perfect iridescence derived from an artificial grating or imperfect iridescence derived from a flower ) to an object influenced its detectability . the contrast of the chemical ( pigment ) color of an object with that of the background has been shown to determine its detectability , and in both plant and animal systems structural color is usually combined with pigments . in this study , targets with different pigment colors ( ultramarine blue and quinacridone red ) were used in combination with iridescent , imperfect floral - derived iridescent , or non - iridescent disks ( equivalent , but lacking diffraction grating , artificial or floral surfaces were used to make non - iridescent disks ) . bees were pre - trained to locate 3-cm disks of a single color randomly located in a flight arena at set distances from each other . during the test phase , the time taken by an individual bee to locate 1-cm disks of the same color was recorded ( disk 1 to disk 2 and disk 2 to disk 3 ) . this was repeated with an independent set of ten bees for both red and blue disks with the different iridescence options . we find that the presence of iridescence reduced the travel time between disks ( f1,88 = 33.349 , p < 0.001 ; figure 2 ) . both the underlying pigment color and the type of overlying iridescence affected the travel time ( f2,88 = 13.325 , p < 0.001 ) . disks than they do to find the equivalent blue disks , suggesting that when overlying iridescence is equal , the underlying pigment color contributed to disk detectability in a similar way to that previously described in the literature ( tukey s honest significant difference [ hsd ] between red perfect and blue , p < 0.001 ; figure 2 ) . perfect red ones ( tukey s hsd , p < 0.001 ; figure 2 ) and were equally fast between blue disks with perfect iridescence and red disks with imperfect floral iridescence ( tukey s hsd , p = 0.780 ; figure 2 ) . for flowers , where a high level of detectability is important , producing pigment colors with strong contrast to the background increases detectability . flowers that produced both pigment colors with a high degree of contrast to the background and iridescence would have the greatest color - dependent salience . furthermore , this increase in detectability is achieved to an equivalent or even higher degree by imperfect our findings show that red targets with floral iridescence are located as quickly by bees as are blue flowers with producing iridescence could therefore be of benefit in situations , including flowering , where a high level of detectability is important . this might apply to plants that flower in relative isolation and would benefit from a greater likelihood of being spotted by a pollinator , to flowers in highly competitive situations where any increase in detectability would be an advantage , and to plants with very short - lived flowers that depend on high detectability in a narrow time window . on this point , it is interesting to note that the iridescent flower of hibiscus trionum only blooms for 23 hr before wilting . a more detectable flower will have the potential to attract pollinators from a greater distance and would also benefit foraging insects because flowers that require minimal search time will minimize time and energy expended on searching . optimal foraging theory predicts that search time is a key variable in determining the value of a target food source [ 21 , 22 ] . so the more detectable a flower , the more valuable it becomes as a resource . this would promote flower constancy in visiting pollinators and thus the effective transfer of pollen between members of the same plant species . these observations lead to the quandary as to why so many animals subject to high levels of predation , such as beetles , have non - sexually selected iridescence if it increases their detectability by visual predators [ 2 , 23 ] . the answer to this may lie in aposematism [ 23 , 24 ] , but the visual effect resulting from the shift in hues might come with an additional effect it may also obscure object identity . most flowers are not iridescent , and those that are do not display the bright , intense iridescence that , for example , fruits are capable of producing [ 5 , 6 , 18 ] in certain situations , where camouflage is important , this corruption of identity by disruptive coloration could be a significant advantage [ 4 , 25 ] . however , to a foraging pollinator , disruption of color identity would weaken search image formation and compromise the pollinator s ability to identify rewarding flowers . previous work has shown that foraging bumblebees can see iridescence independently of other visual cues , including underlying pigment color . although this is important in determining that iridescence can act as a cue in itself , iridescence does not usually occur independently in most natural systems , with both animal and plant iridescence frequently being enhanced by chemical color or requiring an underlying pigment . in flowers , we hypothesize that in the visual perception of an insect , iridescence could interact with pigment color . could the iridescence disrupt an insect s perception of pigment color and thus produce disruption of color identity ? to test whether this hypothesis could be correct when flowers have strongly distinct pigment colors , we trained bees with absolute conditioning to blue disk targets ( perfect iridescent , imperfect floral - derived iridescent , or non - iridescent disks ) . individual bees were then tested for 20 visits with both blue target disks and an equal number of red distractor disks . in this initial experiment , we find that perfect iridescence does not disrupt color identity when targets are perceptually far apart in color space ( median correct visits with iridescent disks = 19.5 [ range = 1420 , n = 10 ] , non - iridescent disks = 18 [ range = 1620 , n = 11 ] ; w = 59.5 , p = 0.764 ; figure 3a ) . these data are supported by the optical analysis shown in figure 1b , which shows that although both the red and blue iridescent disks display a wide scatter of perceived hues in bee color space , there is no overlap in bee color space of the perceptual color distances between the range of colors produced by the targets . therefore , we conclude that iridescence does not always perturb color identity . when this experiment was repeated with floral - derived iridescence , again the presence of iridescence did not disrupt color identity ( median correct visits with floral iridescent disks = 20 [ range = 1720 , n = 7 ] , non - iridescent disks = 20 [ range = 1820 , n = 7 ] ; w = 22.5 , p = 0.831 ; figure 3a ) . however , flowers within a bee s flight range are often much closer in pigment color than the distinct red and blue used here . for assessment of whether iridescence disrupts identity when targets are more similar in pigment hue , the experiment was repeated using disks that contained pigments much closer in bee color space ( purple , purple blue , and purple red ) . bees were trained to purple target disks ( perfect iridescent , floral - derived imperfect iridescent , or non - iridescent disks ) again using absolute conditioning . individual bees were then tested with target disks ( purple ) and an equal number of distractor disks ( purple blue and purple red ) . using these disks , with the spectral reflectance of the underlying pigments perfect iridescence disrupted target identity significantly ( median correct visits with artificial iridescent disks = 10 [ range = 914 , n = 12 ] , non - iridescent disks = 14 [ range = 1316 , n = 8 ] ; w = 6 , p = 0.001 ; figure 3b ) . however , this disruption does not occur if the iridescence is floral derived ( mean correct visits with imperfect floral iridescent disks = 14.63 0.66 [ se ] , n = 11 ; non - iridescent = 13.42 0.79 , n = 12 ; t20.66 = 1.18 , p = 0.252 ; figure 3b ) . these results are supported by the spectrometry measurements of the disks and the modeling of these data in bee color space ( figures 1c and 1d ) . unlike the red and blue disks , the three shades of purple are much closer in bee color space , such that when the disks have iridescence generated by a perfect diffraction grating , individual points ( representing positions in bee color space ) overlay and overlap between the different disks ( figure 1c , with photographs of the disks in figure s3 ) . however , when the iridescence is generated by an imperfect floral diffraction grating , the individual points do not overlap , except in the center of the hexagon , which corresponds to the specular reflection of the white light source from the surface of the disk ( figure 1d ) . the modeling also confirms that the underlying pigment and overlying iridescence interact , with the floral iridescence generating the same pattern of scatter in all three pigment disks and the contribution of the pigment meaning that the loci are scattered along lines that radiate separately from the center . that this second color - discrimination test was a more difficult perceptual task is also confirmed by the behavioral data for the non - iridescent disks , where the accuracy rate drops from the 90% accuracy shown with the non - iridescent red and blue disks to 72% in the purple disk discrimination . iridescence appears to have multiple impacts on an insect visual system both independently and through interaction with any underlying pigmentation . iridescence can both increase the detectability of an object and also alter the extent to which aspects of the object can be accurately identified . our behavioral data are supported by modeling spectral analysis of iridescent targets in bee color space . they also confirm that iridescence can produce a broader scatter within color space compared to non - iridescent objects and that this scatter has an impact both on how detectable an object is and on the extent to which it disrupts the identity of the underlying pigmentation color . although the pattern of scatter within color space may be key in determining the extent to which the iridescence impacts detectability and identity , analysis of the contrast of each color receptor showed that there may be multiple factors at work here , with color contrast and achromatic specular contrast contributing to different extents in different iridescence - pigment combinations . some optical effects can be dismissed , for example , the polarization of reflected light . although iridescence is known to impact the polarization of light reflected from a target , this is not a cue that bees use when identifying iridescent targets , and furthermore it is not a cue that bees can use when targets are presented to them horizontally [ 5 , 13 ] . more work is needed to determine the interactions of iridescence with other optical effects such as gloss . further spectral and behavioral studies of iridescence , particularly when compared with an analysis of which floral pigments are found in combination with floral iridescence , will be useful to unravel these factors and determine the mechanisms by which floral iridescence affects flower detection and identification . the iridescence produced by floral diffraction gratings may appear to the human eye to be subtle ( examples of floral iridescence are included in figure s3 ) ; however , in the production of iridescence , there appear to be two conflicting pressures : vivid iridescence to enhance detectability and reduced iridescence to maintain potential color constancy . the subtle floral iridescence therefore appears to be optically optimal for its role to advertise to pollinators while maintaining their ability to identify rewarding flowers . floral iridescence increases detectability while minimizing the color disruption that iridescence generated by an artificial diffraction grating produces . however , this is not to say that floral iridescence may not have any costs . the petal epidermal surface is multifunctional , and the shape and structure of this epidermis contributes to many biotic and abiotic interactions . for example , the presence of conical cells ( which would preclude the production of floral iridescence by scattering light ) might impact the temperature and wettability of the petal surface [ 28 , 29 ] and would also enhance the ability of a foraging insect pollinator to grip and handle a flower [ 30 , 31 ] . the flat , striated surface that produces iridescence in flowers has been shown to reduce the ability of insects to grip when it is found on leaves . therefore , there is a potential trade - off between floral surface structures that provide a secure grip for pollinators and those that enhance floral detectability . this trade - off may be another factor behind the relative rarity of floral iridescence and may also explain why that , when it does occur , it frequently occurs in defined regions of the petal in combination with spatially distinct conical epidermal cells . in all organisms that produce iridescence , there is potential for trade - off between increasing detectability and maintaining signal identity . in the case of floral - generated iridescence however , if iridescence is used as camouflage , the reverse could be the ideal , with increased disruption and reduced detectability . conceivably , either objective could be attained by modifying the structure responsible for the generation of the iridescence or by combining the diverse ways in which iridescence can be produced , each of which gives rise to different optical properties , each of which would differently impact visual detectability and disruption . in the case of floral - derived iridescence the apparently imperfect diffraction gratings of flowers generate a signal whose adaptive nature only emerges when one considers the color vision and behavior of the intended signal receivers , the pollinators of the flowers . target disks were measured using a zeiss mcs 230 diode array photometer , with illumination by a zeiss clx 111 xenon lamp . the illumination fiber optic was held at 45 to the normal by a zeiss gk 111 goniometer . the angle of the measurement fiber optic was held at angles ranging from 45 to 90 to the normal , in 5 increments . spectra were recorded in 1-nm steps from 300 to 700 nm and were recorded relative to a spectralon 99% white standard . both this white - standard calibration and a true - dark reference calibration ( where light was fully blocked from the detector ) were taken immediately prior to measurement of each disk , to minimize measurement error . each measurement was taken from a 2-mm diameter spot on the disk , randomly chosen from within a uniform region . for each disk , spot measurements were taken at each 5 increment , the disk was then rotated 10 , and measurements repeated from a position of 90 to one of 0 across the diffraction grating . each bee was tested on one disk type only ( i.e. , one combination of iridescence and pigment ) . bees were initially trained on the large 3-cm disks of the stated iridescence and pigment type , each of which provided a 30% sucrose solution reward in a cap in the center of the disk . three disks were positioned in an equilateral triangle pseudo - randomly orientated within the flight arena . a successful trial was one in which the bee found and drank from all three disks . once the bee had successfully completed 15 bouts of training , the disks were swapped for those of the same type but smaller size ( 1 cm ) . the time taken for the bee to locate and land on each disk type the data were analyzed with spss 19 using a repeated - measures general linear model . iridescence ( whether the disks presented were iridescent or not ) , and disk type ( whether the disks were blue with an artificial pattern , red with an artificial pattern , or red with a floral pattern ) were considered as between - subject factors . visit order ( the lengths of time to move between the first and second disk and between the second and third disk ) and experimental repeat ( the five consecutive repetitions on each individual ) were considered as within - subject factors . initial analyses suggested that third- and fourth - order interactions were non - significant ; therefore , within the model used , interactions were not considered between the two within - subject factors or between the two between - subject factors . post hoc tukey s hsd tests were conducted to explore differences between disk types . for determination of whether bees could distinguish between the target color and that of the distractor disks , absolute conditioning was used . in these experiments , a single trial consisted of releasing a marked bee from the colony into a flight arena containing an array of disks of the target color , each of which provided a 30% sucrose solution reward in a cap in the center of the disk . the bee was permitted to forage on disks of the target color until at least 20 visits had been made to different target disks and permitted to return to the colony . the array was then changed such that an equal number of target and distractor disks were presented . the trained bee was released again into the flight arena , and the first 20 disks that the bee landed on recorded . the correct number of choices made during the first 20 visits was compared between iridescent and non - iridescent pairings using either a welch s t test of the log - transformed data ( floral three - color test ) or a wilcoxon rank - sum test with continuity correction , using r 2.15.1 . conceptualization , h.m.w . , l.c . , and b.j.g . ; formal analysis , s.a.r . ; , l.c . , and b.j.g ; writing review & editing , h.m.w , s.a.r . ,
one of the objectives of periodontal therapy is the reduction of bacterial deposits and calculus on tooth surface . this objective can be achieved with hand scalers and curettes or ultrasonic scaling instruments . recent clinical studies do not indicate a difference between ultrasonic / sonic and manual debridement in the treatment of chronic periodontitis . complete removal of subgingival calculus with hand or ultrasonic instruments is impossible or rare even when a surgical approach is used . the efficacy of root planing procedures can be studied in two different ways . the tissue healing around the treated teeth can be evaluated , or the teeth may be extracted immediately after treatment in order to observe directly the cleanliness and surface characteristics of the root planed surfaces . a number of authors have used the stereomicroscope to evaluate the residual calculus after extraction of the root planed teeth . however , precise study of the root planed surface can be performed only by means of scanning electron microscope ( sem ) . initially , ultrasonic scalers were employed with apprehension because of suspected root surface damage . this concern was subsequently put to rest by studies such as that of ritz , et al . thus , the present study was conducted to comparatively evaluate the efficacy of manual , magnetostrictive and piezoelectric ultrasonic instruments . thirty periodontally compromised extracted human teeth with supragingival and subgingival calculus were divided into 3 experimental groups using a randomized rank selection programme . each tooth was positioned horizontally on a dental stone block of 1 " by 1 " . the teeth were to be instrumented with hand and ultrasonic instruments resembling clinical application . in group a all teeth were carefully scaled with a new universal hand curette ( hu friedy gracey after five vision curette ; hu friedy , chicago , usa ) in group b cavitron fsi - sli ultrasonic device with focused spray slimline inserts ( dentsply international inc . , york , pa , usa ) were used . in group c teeth were scaled with an ems piezoelectric ultrasonic device with prototype modified ps inserts . with the hand curette the working strokes ran from apical to coronal direction , parallel to the long axis of the tooth . 6a , hu - friedy , leimen , germany ) after instrumenting each tooth . the insert tips were parallel to the tooth axis and the working strokes ran perpendicular to the tooth axis . the application method for both ultrasonic devices was same . clinically appropriate force of application was ensured as only one operator duly trained in the set procedure carried out debridement of all teeth . each tooth was instrumented till the root surface was visually and tactilely clean and smooth as confirmed by a sharp cow horn explorer ( hu - friedy ) . the surfaces were analyzed by a precision profilometer ( form surtronic 3 + , rank taylor hobson , leicester , uk ) to measure the surface roughness ( ra value in m ) consecutively before and after the instrumentation . special care was taken to make the post experimental tracings in the same positions as at baseline . the samples were examined under scanning electron microscopy ( sem ) ( jeom jsm- 6380 la , analytical sem ) at magnifications ranging from 17x to 300x . the surfaces were examined for damage , scratches , gouges , cracks and any remnants of debris . the baseline and end point ra values as analyzed by the profilometer were compared in the intergroup using anova while intragroup comparison was done using tukey 's test . also , percentage reduction of ra value was done by anova and fischer 's test . the baseline and end point ra values as analyzed by the profilometer were compared in the intergroup using anova while intragroup comparison was done using tukey 's test . also , percentage reduction of ra value was done by anova and fischer 's test . all teeth had supragingival and subgingival calculus and were instrumented until no visible calculus could be assessed by the naked eye and felt by the explorer . the mean ra values ( m ) before and after instrumentation in groups a , b and c are presented in the form of a bar diagram ( figure 1 ) . after statistically analyzing the data , no significant difference was observed in the three experimental groups . mean ra values ( in m ) before and after instrumentation in all three groups the percent reduction of ra value was calculated by : though there was a decrease in the percentage reduction of ra values consecutively from group a to c ( figure 2 ) , it was not statistically significant . in other words , although the piezoelectric ultrasonic ( group c ) device with prototype inserts produced root surfaces as clean as with the curette or magnetostrictive ultrasonic device , the overall surface roughness was greater after the piezoelectric instrumentation than the other two instruments . percentage reduction of surface roughness ( ra ) values in group a , b and c the images acquired from the sem were used for descriptive analysis . the entire tooth sample as observed in all 3 groups showed cracks on the surface caused by dehydration procedures ( figures 3 - 5 ) . . a : sem view of group a sample at 300x magnification ; b : sem view of group a sample at 600x magnification scanning electron microscopy ( sem ) view of group c sample . a : sem view of group c sample at 300x magnification ; b : sem view of group c sample at 600x magnification the sem observation revealed that all the instruments managed to remove the calculus deposits quite effectively . remnants of calculus were seen at magnification 300x ( figures 3a , 4a , 5a ) and at 600x ( figures 3b , 4b , 5b ) . presence of smear layer was noted in all the three groups at high magnifications . the images acquired from the sem were used for descriptive analysis . the entire tooth sample as observed in all 3 groups showed cracks on the surface caused by dehydration procedures ( figures 3 - 5 ) . scanning electron microscopy ( sem ) view of group a sample . a : sem view of group a sample at 300x magnification ; b : sem view of group a sample at 600x magnification scanning electron microscopy ( sem ) view of group c sample . a : sem view of group c sample at 300x magnification ; b : sem view of group c sample at 600x magnification the sem observation revealed that all the instruments managed to remove the calculus deposits quite effectively . remnants of calculus were seen at magnification 300x ( figures 3a , 4a , 5a ) and at 600x ( figures 3b , 4b , 5b ) . presence of smear layer was noted in all the three groups at high magnifications . studies investigating the differences between manual , piezoelectric and magnetostrictive ultrasonic systems are inconclusive . the same has been corroborated by lea and walmsley 's exhaustive review of literature specifically with respect to powered instruments . it is worthwhile recapitulating that a large number of variables - vibration generation method , water flow rate , tip cross section and generator power , contact load , angle and duration , generator power , tip shape , instrumentation end point , vibration generation method , tip variability , water flow rate , tip cross - section and tip motion - associated with attempts to investigate such differences , make it practically impossible to reach a definite conclusion regarding the method of instrumentation that causes the least amount of root surface alteration . using both , stereomicroscope and sem evaluation , breininger , et al . evidently , some form of standardization is required to allow meaningful comparisons to be made between studies . further , a number of studies have shown that one session of closed root instrumentation does not achieve the goal of total elimination of all calculus deposits . other investigations in which flaps have been reflected to secure access and visibility before scaling and root planing have failed to secure calculus free root surfaces . it is worth mentioning at this juncture that even under optimal conditions in vitro it is not always possible to remove entire calculus from all root surfaces . the first is to apply instrumentation until the tooth surface is clean and clear of calculus as deemed by the operator . the second is to instrument for a controlled length of time or number of strokes . while the latter is often more controlled insofar as operating parameters such as load and contact angle are concerned , clinically it is less appropriate . employing the former method in the present study , teeth samples were instrumented until they were clean - clinically a more pertinent aim . one of the highlights of this study is the fact that periodontally diseased teeth were selected . results from such a work are more meaningful because that the sample mimics actual conditions in patients unlike works that are carried out on healthy teeth extracted originally for orthodontic reasons . moreover , through random allocation of teeth to their respective groups they were well matched as reflected by a lack of any visible differences among initial calculus in each group . this was an important aspect given that these teeth were diseased and affected by calculus . the teeth were then examined under a stereomicroscope ( magnification 10x ) ; remnants of calculus were seen . subsequent sem analysis showed that surfaces with smaller mean ra values exhibited less gouges and scratches than those with higher mean ra values . sem observations in our study indicate that the use of hand instruments resulted in a smooth surface than obtained by ultrasonic instruments . ( 2006 ) had also concluded that diamond coated sonic tips and ultrasonic universal tips produced similar roughness of surface which was higher than that produced by hand curettes . our findings corroborate those of some others regarding instrumentation with curettes and ultrasonic instruments . however , ewen and gwinnett ( 1977 ) did not find any difference while jones , et al . such variations in results can be attributed to methods of processing the specimens , magnifications used and techniques of instrumentation . we discovered a difference in the surface finish quality between the two ultrasonic instruments where we felt that the magnetostrictive instrument produced a better surface finish than the piezoelectric device . however , caution should be exercised in interpreting the results too strictly given that a limited number of surfaces were examined and the interpretations were purely subjective . the difference in ra values between the two ultrasonic instruments could also be due to a difference in the power output . thus , while medium power setting was used for both and the same operator conducted the procedure , there was no way of deducing if the two ultrasonic devices delivered similar power at the same settings . the power of the piezoelectric device could have been higher than that of the magnetostrictive device causing more root damage which was interpreted as a higher ra value . in a study published in 2006 the authors assessed the manual and ultrasonic root surface scaling at low , medium and high power settings ; roughness of the instrumented teeth was evaluated . the authors concluded that ultrasonic instrumentation at high power settings produces rougher root surfaces than ultrasonic instrumentation at lower power ; and that manual instrumentation with curettes produces lower roughness than ultrasonic instrumentation independent of power setting . notwithstanding these limitations , our results clearly indicate that all three instruments considerably reduced calculus on the root surfaces . both ultrasonic and manual instrumentation removed calculus quite effectively as seen by naked eye and sem at low magnification . though the ultrasonic methods produced greater disturbance on surface topography than hand instrumentation , given that the differences in surface roughness produced by the three different instruments were not significant , we suggest that ultrasonic scalers are operationally more viable since they ensure more patient comfort and cause less operator fatigue . within the limits of the present study , given that the manual , magnetostrictive and piezoelectric ultrasonic instruments produce the same surface roughness , it can be concluded that their efficacy for creating a biologically compatible surface of periodontally diseased teeth is similar . the authors thank the technical staff who ceded the surface roughness measuring instrument ( profilometer ) for the execution of this study at the mechanical engineering department , manipal institute of technology , manipal university , manipal ; and , department of metallurgy , national institute of technology , suratkhal , karnataka for the sem examination .

to address the roles of cytokines and t reg lymphocytes in immune - mediated inflammation , we have established a model in which a systemic autoimmune disease is caused by a monospecific t cell population that can be followed quantitatively in vivo . in this model , cd4 t cells from the do11.10 ( do11 ) t cell receptor transgenic mouse , specific for the ovalbumin ( ova)323 - 339 peptide , are transferred into a lymphopenic ( rag ) host expressing ova as a secreted , systemic antigen . the transferred t cells expand , develop into th1 effector cells , and cause a severe disease characterized by weight loss and skin inflammation ( 16 ) . the disease has many similarities to graft - versus - host disease ( gvhd ) , in which transferred t cells react against host antigens in an environment deficient in endogenous lymphocytes . since the signature cytokine of th1 cells is ifn- , we first asked if ifn- was responsible for the disease . to do this , we compared the pathogenic effects of wild - type ( wt ) do11 cells with those of do11 cells lacking either ifn- or the th1-specific transcription factor , t - bet ( 17 ) . ablating the th1 response did not ameliorate weight loss and , surprisingly , led to more severe inflammatory skin lesions ( fig . the relative severity apparent from the external appearance and histology was corroborated by scoring the lesions ( fig . cell numbers in lymphoid organs of recipient mice that received wt do11 cells were similar to recipients that were transferred with either ifn- or t - bet do11 cells ( unpublished data ) . ifn- has been suggested to play a role in the development and/or function of t reg lymphocytes ( 18 ) . to ask if the exacerbated disease in the absence of ifn- could reflect a failure of endogenous t reg lymphocytes development , we followed the do11 cells for generation of cd25foxp3 t reg lymphocytes . these assays showed no defect in the numbers of t reg lymphocytes in the absence of ifn- or t - bet ( fig . s1 , available at http://www.jem.org/cgi/content/full/jem.20061341/dc1 ) . role of ifn- and t - bet in the systemic immune reaction . cd4kj1 - 26cd25 cells were isolated from the lymph nodes and spleens of wt , ifn- , or t - bet do11 mice by cell sorting , and 10 cells were transferred into sova tg rag mice on day 0 . ( a ) body weight is plotted over time as the percentage of the starting weight before t cell transfer . data are pooled from six to eight mice per group ( * , p < 0.05 compared with wt do11 cells ) . ( b ) skin of lower abdomen is shown on day 23 after transfer for one representative mouse in each group . stain of skin sections is shown on the left , and macroscopic appearance of the skin is shown in the middle . histological scores were obtained as described in materials and methods and averaged from three to four mice per group sd . we have begun to identify which cell populations infiltrate the skin in sova transgenic ( tg ) rag recipients by isolating cells from skin and flow cytometry . s2(available at http://www.jem.org/cgi/content/full/jem.20061341/dc1 ) shows that the skin is infiltrated by cd3kj1 - 26 t cells and gr-1cd11b granulocytes . this is not the case in normal naive do11 rag mice or normal sova tg rag mice that have not received do11 cells . it is possible that the inflammatory disease is caused by the cytokine il-17 , and production of il-17 is increased in the absence of ifn- ( 19 , 20 ) . because the entire reaction in this model involves one t cell population , one can easily follow the profiles of cytokines produced by these cells . intracellular cytokine stains showed that early during the course of the disease ( 5 d after initiation of the reaction ) , there were populations of activated t cells in lymphoid organs that produced either ifn- or il-17 or both cytokines . over time , the il-17 producers decreased in number ( fig . strikingly , in the absence of ifn- or t - bet , the number of il-17producing t cells did not decline in lymphoid organs or in the skin ( fig . only very few il-4producing do11 cells could be found before ( day 510 ) or after ( day 30 ) development of skin disease ( fig . the correlation between increased skin inflammation and il-17 production in the absence of a th1 response suggested that the inflammation was caused primarily by il-17producing t cells . in fact , an anti il-17 antibody was able to reduce the skin inflammation when do11 t - bet t cells were transferred into sova tg rag recipients , as indicated by the severity of alopecia macroscopically and microscopically ( fig . these results support a reciprocal relationship between th1 responses and il-17 production and indicate that il-17 is an important cytokine in tissue ( i.e. , skin ) inflammation . ( a ) 10 purified cd4 do11 rag cells were transferred into sova tg rag mice on day 0 . peripheral lymph nodes were harvested at the indicated time points after transfer and stained for intracellular cytokines . representative plots are shown from one experiment out of four with two to three mice per group . ( b ) cd4kj1 - 26cd25 cells were isolated from lymph nodes and spleens from wt , ifn- , or t - bet do11 mice by cell sorting , and 10 were transferred into sova tg rag mice on day 0 . peripheral lymph nodes and skin were harvested on day 10 after transfer , and the percentage of kj1 - 26cd4 cells that express intracellular ifn- , il-17 , and il-4 was measured by flow cytometric analysis . ( c ) the percentage of cells producing intracellular il-17 was determined as in b at the indicated time points . each bar represents three to four mice from two representative experiments ( * , p < 0.05 ) . ( d ) 10 purified cd4cd25 do11 t - bet cells were transferred into sova tg rag mice on day 0 . every 3 d. alopecia was scored on day 16 as described in materials and methods . macroscopic appearance of the skin of the lower abdomen is shown on day 18 from one representative mouse in each group . histological scoring was done as described in materials and methods , and depicted numbers represent averages of six to seven mice per group sd . because of the potential beneficial effect of blocking il-2 signaling in immunological reactions , we have been interested in the types of pathologic immune responses that are influenced by il-2 we have previously shown that in the absence of il-2 , i.e. , when il-2 do11 cells are transferred into sova tg rag il-2 recipients , the mice develop a severe , progressive skin disease with massive accumulation of do11 cells in lymphoid organs and skin . these mice fail to recover from the disease and this is associated with lack of generation of regulatory t cells ( 16 ) . to address the role of il-17 in this reaction , we assayed t cells for il-17 production in vivo in the absence of il-2 . surprisingly , large numbers of il-17producing t cells accumulated in the skin in the il-2deficient setting ( fig . 3 ) . in fact , skin infiltration was dominated by il-17producing cells , demonstrating that efficient generation of tissue - infiltrating il-17producing cells occurs in the absence of il-2 . because classical th1 responses are dependent on il-2 ( 21 ) , our findings raise the possibility that il-17 is an important mediator of the inflammatory disease that develops in knockout mice lacking il-2 production or signaling ( 22 ) . cd4-purified do11 rag wt cells were transferred into sova tg rag wt mice ( wt ) , or do11 rag il-2 cells were transferred into sova tg rag il-2 mice ( il2ko ) . ear skin was harvested on day 26 after transfer , and cells were isolated as described in materials and methods . ( a ) the total number of cd4kj1 - 26 cells recovered from ear skin and ( b ) the percentage of cytokine - producing kj1 - 26cd4il-17 cells was determined by intracellular staining . ( c ) the total number of il-17producing kj1 - 26 cd4 cells was calculated ( * , p < 0.05 ; * * , p < 0.01 ) . it has been shown recently that t reg lymphocytes promote il-17 production in vitro , in the presence of inflammatory cytokines such as il-6 ( 13 ) . if this happens in vivo , then t reg lymphocytes may not be able to control reactions that are dominated by il-17 . to address this directly , we asked if ova - specific t reg lymphocytes could prevent the systemic inflammatory syndrome , and how the production of pathogenic il-17 is affected by the t reg lymphocytes . t reg lymphocytes specific for a defined antigen can be generated experimentally by exposing developing cd4 t cells specific for the antigen to the cognate protein expressed in the thymus . we have shown previously that in crosses of do11 mice with rip - mova - transgenics , cd25foxp3 t reg lymphocytes develop , because the antigen is expressed in the thymus and t cells encounter the antigen during maturation ( 23 ) . cd25cd4 do11 t cells were purified from do11rip- mova tg mice and cotransferred with wt do11 cells expressing a different allelic marker ( thy1.1 ) into lymphopenic sova tg recipients . b ) that are characteristic of the inflammatory disease . because we were able to distinguish the t reg lymphocytes from the effector cells , we could determine the effects of the ova - specific transferred t reg lymphocytes on the response patterns of the effector t cells of the same specificity . the most striking effect of t reg lymphocytes was a profound inhibition of t cell accumulation , which was detectable as early as 5 d after transfer ( fig . 4 c ) . t reg lymphocytes almost completely inhibited the production of ifn- but , surprisingly , enhanced the frequency of il-17producing cells among the remaining do11 cells ( fig . 4 c ) . however , even though the percentage of il-17producing cells was increased within the surviving effector cell population , the total number of cytokine - secreting cells was markedly decreased . importantly , when t reg lymphocytes were cotransferred with effector cells at a lower ratio , there was no longer an increase in the percentage of il-17producing cells ( unpublished data ) and the total number of il-17producing cells was reduced ( fig . 4 d ) . these data indicate that strong immune responses to a systemic antigen can be prevented when t reg lymphocytes are present at the time of the initial activation , and this protective effect of t reg lymphocytes is dominant over their il-17promoting ability . it remains to be determined how t reg lymphocytes inhibit the accumulation of effector cells . a plausible explanation could be competition for il-2 or other growth factors , or , alternatively , t reg lymphocytes may inhibit il-2 production and lead to apoptosis of early activated cells . cd4kj1 - 26cd25 cells were purified from lymph nodes and spleens of do11 wt thy1.1 mice , and 10 cells were transferred into sova tg rag mice on day 0 . cd4kj1 - 26cd25 cells were isolated from lymph nodes and spleen of do11rip - mova tg mice ( thy1.1 ) , and 10 cells were cotransferred in a separate injection on the same day . ( a ) body weight is plotted over time as the percentage of the starting weight before t cell transfer . alopecia was scored as described in materials and methods ( * , p < 0.05 compared with recipients that did not receive t reg lymphocytes ) . ( b ) h&e stain of skin sections is shown on day 23 after transfer from one representative mouse in each group . histological scoring was done as described in materials and methods , and depicted numbers represent averages of three to four mice per group sd . ( c ) cells were harvested from peripheral lymph nodes on day 5 or 9 . the total number of isolated cd4kj1 - 26 cells on day 5 and 9 ( left ) , the percentage of cytokine - producing effector cells by intracellular staining on day 9 ( middle ) , and total cell numbers of cytokine - producing effector cells on day 9 ( right ) are shown . all plots are gated on cd4kj1 - 26thy1.1 ( * , p < 0.05 compared with recipients that did not receive t reg lymphocytes ) . ( d ) 0.3 10 cd4kj1 - 26cd25 cells from do11 wt thy1.1 mice were transferred into sova tg rag mice on day 0 , either alone or with cd4kj1 - 26cd25 from do11rip - mova tg mice ( thy1.1 ) at a ratio of 1:1 or 1:5 ( t reg lymphocytes to effector cells ) . the total number of isolated cd4kj1 - 26 cells ( left ) and the total cell numbers of cytokine - producing effector cells ( right ) are shown . all plots are gated on cd4kj1 - 26thy1.1 ( * , p < 0.05 compared with recipients that did not receive t reg lymphocytes ) . whether or not t reg lymphocytes can also have a protective effect if they are generated or introduced late in the course of disease is obviously of clinical relevance . to address this question we transferred t reg lymphocytes into sova tg rag recipients 6 d after the transfer of naive do11 cells . this late administration of t reg lymphocytes did not prevent the systemic disease ( unpublished data ) . these studies have explored the roles of cytokines and t reg lymphocytes in a mouse model of a systemic inflammatory disease . this model resembles systemic gvhd , the common feature of both being antigen recognition by transferred t cells in the context of lymphopenia ( 24 ) . the strengths of the experimental system we have used are that the pathologic immune response is quantifiable because it is caused by t cells of a single specificity reacting to their target antigen , the disease that develops has both systemic abnormalities and local tissue inflammation , and the disease is controlled by endogenously generated t reg lymphocytes . we show that the cytokine il-17 is the mediator of tissue inflammation in this disease ( figs . 1 and 2 ) . paradoxically , skin inflammation becomes worse in the absence of th1 cytokines , presumably because of a concomitant increase in il-17 production . worsening of disease in the presence of defective th1 response has also been noted previously in polyclonal models of gvhd ( 25 , 26 ) . the cross - regulation of th1 responses and il-17 has been demonstrated in vitro ( 19 , 20 ) , and our results suggest that this is also true in vivo , but the lineage relationships between th1 cells and il-17producing cells remain to be defined . massive accumulation of il-17producing cells in peripheral tissues is independent of il-2 and also occurs in the absence of t reg lymphocytes . in fact , tissue - infiltrating il-17producing cells are increased in the absence of il-2 , and thus in the absence of t reg lymphocytes . this is emphasized by the observation that early after transfer of do11 rag cells , which do not contain t reg lymphocytes and at a time point in the disease course when no t reg lymphocytes have developed yet ( 16 ) , strong differentiation into th17 has occurred . our results demonstrate conclusively that the development of th17 cells is , in fact , increased in the absence of t reg lymphocytes . our studies also show that t reg lymphocytes are able to completely prevent the inflammatory disease ( 1315 , 27 ) . the potent disease - preventing effect of t reg lymphocytes is likely because of the ability of these cells to block initial activation and , as a consequence , antigen - induced t cell expansion after recognition of the systemic antigen . the inability of t reg lymphocytes to ameliorate the disease when given late in the course is consistent with the finding that the principal effect of these cells is to inhibit the initial t cell proliferation and generation of pathogenic effector cells . this result raises the possibility that t reg lymphocytes therapy may be of limited benefit in established immunological diseases . however , it is possible that the propagation of such diseases is dependent on continuous recruitment of new t cells and their differentiation into pathogenic effectors . if this is indeed the case , t reg lymphocytes may reduce disease severity by inhibiting the continued development of effector cells from newly arising autoreactive precursors . it is also possible that t reg lymphocytes may have little effect on established inflammatory lesions that are caused by local il-17 secretion , because t reg lymphocytes do not inhibit , and appear to enhance , production of this inflammatory cytokine . thus , protective or pathologic effects of inflammatory cytokines and the therapeutic effects of t reg lymphocytes vary depending on the location , timing , and context . transgenic mice expressing the do11.10 tcr ( do11 ) , specific for the chicken ova peptide ( ova323339 ) in the context of the mhc class ii molecule i - a , were obtained from dr . some experiments were performed using do11 cells that had been crossed onto a rag2 background . l. glimcher ( harvard medical school , boston , ma ) , and ifn- mice on a balb / c background were obtained from the jackson laboratories and crossed with the do11 tcr tg mice . soluble ova transgenic mice ( sova tg ) on a balb / c background , expressing a soluble form of ova in the serum have been described ( 28 ) . r. locksley ( university of california , san francisco , san francisco , ca ) . scoring for alopecia was done as follows : < 1 cm = 1 point , < 2 cm = 2 points , > 2 cm = 3 points , 50% of body surface = 4 points , complete alopecia = 5 points . all mice were bred and maintained in our pathogen - free facility in accordance with the guidelines of the laboratory animal resource center of the university of california , san francisco . all experiments were conducted with the approval of the committee on animal research of the university of california , san francisco . lymph node cells , splenocytes , and skin were stained with the clonotypic antibody kj1 - 26 ( caltag laboratories , burlingame , ca ) , anti - cd4 ( gk1.5 , h129.19 , and rm4 - 5 ) , anti - cd25 ( pc61 , 7ad ) , anti - cd62l ( mel-14 ) , anti - thy1.1 ( ox-7 ) , anti - thy1.2 ( 30-h12 ) , anti - cd3 ( 145 - 2c11 ) , anti - gr-1 ( rb6 - 8c5 ) , anti - cd11b ( m1/70 ) , and anti - foxp3 ( fjk-16s ; ebioscience ) . antibodies were used as fitc , pe , pe - cy7 , pe - cy5.5 , allophycocyanin , or peridin chlorophyll protein conjugates . flow cytometric analyses were done on a facscalibur with cellquest software ( both from becton dickinson ) . propidium iodide ( sigma - aldrich ) was used to exclude dead cells in flow cytometry of skin samples . for intracellular cytokine staining , do11 t cells recovered from peripheral lymph nodes , spleen , or skin of transfer recipients were restimulated on mitomycin c treated balb / c splenocytes for 14 h in the presence of 1 g / ml ova peptide . brefeldin a ( epicentre ) was added ( 10 g / ml ) for the last 2 h of stimulation . cells were stained for intracellular cytokines il-17 , ifn- , il-4 and analyzed by flow cytometry gating on cd4kj1 - 26 cells . anti il-17 antibody ( m210 ) was provided by amgen or obtained from r&d systems ( clone 50104 ) . a total of 250 g ( r&d systems ) or 500 g ( amgen ) statistical analysis was done using two - tailed t test assuming equal variance between the compared groups ( * * , p < 0.01 ; * , p < 0.05 ) . for isolation of t cells from ear skin , the ear was cut off and split into ventral and dorsal half with forceps . the ear halves were minced with a razor blade and incubated at 37c for 45 min in digestion cocktail containing 0.3% trypsin from bovine pancreas , collagenase xi ( 4,100 u / ml ) , hyaluronidase ( 260 u / ml ) , dnase ( 0.1 mg / ml ) diluted in rpmi , containing 10 mm hepes and 5% fcs ( all from sigma - aldrich ) . digested ears were minced , and isolated cells were washed twice with ice - cold medium containing 10 mm edta and filtered through a 40-m filter . sections were scored blinded using a histological score for dermal inflammation ( grade 1 = less than 15% necrotic cells , infiltration less than 20% of dermis ; grade 2 = 1520% necrotic cells , infiltration 2033% of dermis ; grade 3 = 2550% necrotic cells , infiltration 3350% of dermis , grade 4 = necrosis in greater than 50% of dermis , infiltration of more than 50% of dermis ) and epidermal changes ( grade 1 = less than 25% infiltrating cells , 12 cell thickening ; grade 2 = 2550% infiltrating cells , 34 cell thickening ; grade 3 = 5075% infiltrating cells , 46 cell thickening , grade 4 = 75100% infiltrating cells , more than 6 cell thickening ) ( 29 ) . s1 demonstrates that peripheral foxp3-expressing t reg lymphocytes develop from naive cd25 wt , ifn- , and t - bet do11 cells when transferred into sova tg rag . s2 shows that the dermatitis of sova tg rag recipients is characterized by accumulation of transferred do11 t cells and gr-1cd11b granulocytes in the skin . transgenic mice expressing the do11.10 tcr ( do11 ) , specific for the chicken ova peptide ( ova323339 ) in the context of the mhc class ii molecule i - a , were obtained from dr . some experiments were performed using do11 cells that had been crossed onto a rag2 background . l. glimcher ( harvard medical school , boston , ma ) , and ifn- mice on a balb / c background were obtained from the jackson laboratories and crossed with the do11 tcr tg mice . soluble ova transgenic mice ( sova tg ) on a balb / c background , expressing a soluble form of ova in the serum have been described ( 28 ) . r. locksley ( university of california , san francisco , san francisco , ca ) . scoring for alopecia was done as follows : < 1 cm = 1 point , < 2 cm = 2 points , > 2 cm = 3 points , 50% of body surface = 4 points , complete alopecia = 5 points . all mice were bred and maintained in our pathogen - free facility in accordance with the guidelines of the laboratory animal resource center of the university of california , san francisco . all experiments were conducted with the approval of the committee on animal research of the university of california , san francisco . lymph node cells , splenocytes , and skin were stained with the clonotypic antibody kj1 - 26 ( caltag laboratories , burlingame , ca ) , anti - cd4 ( gk1.5 , h129.19 , and rm4 - 5 ) , anti - cd25 ( pc61 , 7ad ) , anti - cd62l ( mel-14 ) , anti - thy1.1 ( ox-7 ) , anti - thy1.2 ( 30-h12 ) , anti - cd3 ( 145 - 2c11 ) , anti - gr-1 ( rb6 - 8c5 ) , anti - cd11b ( m1/70 ) , and anti - foxp3 ( fjk-16s ; ebioscience ) . antibodies were used as fitc , pe , pe - cy7 , pe - cy5.5 , allophycocyanin , or peridin chlorophyll protein conjugates . flow cytometric analyses were done on a facscalibur with cellquest software ( both from becton dickinson ) . propidium iodide ( sigma - aldrich ) was used to exclude dead cells in flow cytometry of skin samples . for intracellular cytokine staining , do11 t cells recovered from peripheral lymph nodes , spleen , or skin of transfer recipients were restimulated on mitomycin c treated balb / c splenocytes for 14 h in the presence of 1 g / ml ova peptide . brefeldin a ( epicentre ) was added ( 10 g / ml ) for the last 2 h of stimulation . cells were stained for intracellular cytokines il-17 , ifn- , il-4 and analyzed by flow cytometry gating on cd4kj1 - 26 cells . il-17 antibody ( m210 ) was provided by amgen or obtained from r&d systems ( clone 50104 ) . a total of 250 g ( r&d systems ) or 500 g ( amgen ) were injected i.p . statistical analysis was done using two - tailed t test assuming equal variance between the compared groups ( * * , p < 0.01 ; * , p < 0.05 ) . for isolation of t cells from ear skin , the ear was cut off and split into ventral and dorsal half with forceps . the ear halves were minced with a razor blade and incubated at 37c for 45 min in digestion cocktail containing 0.3% trypsin from bovine pancreas , collagenase xi ( 4,100 u / ml ) , hyaluronidase ( 260 u / ml ) , dnase ( 0.1 mg / ml ) diluted in rpmi , containing 10 mm hepes and 5% fcs ( all from sigma - aldrich ) . digested ears were minced , and isolated cells were washed twice with ice - cold medium containing 10 mm edta and filtered through a 40-m filter . sections were scored blinded using a histological score for dermal inflammation ( grade 1 = less than 15% necrotic cells , infiltration less than 20% of dermis ; grade 2 = 1520% necrotic cells , infiltration 2033% of dermis ; grade 3 = 2550% necrotic cells , infiltration 3350% of dermis , grade 4 = necrosis in greater than 50% of dermis , infiltration of more than 50% of dermis ) and epidermal changes ( grade 1 = less than 25% infiltrating cells , 12 cell thickening ; grade 2 = 2550% infiltrating cells , 34 cell thickening ; grade 3 = 5075% infiltrating cells , 46 cell thickening , grade 4 = 75100% infiltrating cells , more than 6 cell thickening ) ( 29 ) . s1 demonstrates that peripheral foxp3-expressing t reg lymphocytes develop from naive cd25 wt , ifn- , and t - bet do11 cells when transferred into sova tg rag . s2 shows that the dermatitis of sova tg rag recipients is characterized by accumulation of transferred do11 t cells and gr-1cd11b granulocytes in the skin .
in line with the global increase in prediabetes and diabetes , a significant rise in the proportion of diabetes during pregnancy has been reported . women with diabetes are regarded as a high - risk group with respect to maternal , fetal , and neonatal outcomes . according to the medical birth registry in norway reporting for the year 2008 , diabetes type i ( t1 dm ) and type ii ( t2 dm ) were diagnosed in 0.4% and 0.3% , respectively , of the pregnancies in 2009 . gestational diabetes mellitus ( gdm ) was recorded in 1.4% of pregnancies , a number that has doubled during the last decade . in norway , antenatal care for diabetes pregnancies is delivered in hospital outpatient clinics by multidisciplinary diabetes teams , in line with national guidelines for diabetes in pregnancy . in this team , in addition , the midwife provides information and advice related to the consequences of living with diabetes and the expressed needs of the woman . several studies point to the fact that midwifery consultations are perceived as valuable for the women [ 4 , 5 ] , but there is limited knowledge on how these consultations are carried out and performed in an antenatal diabetes care setting . there are numerous studies on the risk for perinatal outcomes related to pregnancy and diabetes from a medical perspective . however , fewer studies have been conducted on women 's perceptions of diabetes and pregnancy . the research on diabetes in pregnancy could be divided into two groups : research of prepregnancy conditions in women with t1 dm and t2 dm , and research on gdm . women with t1 dm have been reported to be more stressed and anxious compared to women in normal pregnancies . during pregnancy , berg stresses the importance of the provider acting in a manner that promotes the woman 's mastery of her condition instead of acting as a controller . women have reported the consultations with the professionals to be overshadowed by the diabetes diagnosis and several studies have revealed a lack of communicative topics related to normal pregnancy and maternity developmental tasks including focus on emotional needs [ 5 , 10 , 11 ] . women with t1 dm have also been reported to have a large need of access to reliable information during the whole childbearing period , not only in pregnancy . according to sjgren , pregnant women receiving a gdm diagnosis show great concerns about the baby 's health as well as their own . in their study , women with gdm recalled having been being more worried during pregnancy compared to women without this pregnancy complication . persson et al . describe women with gdm as experiencing a chaotic situation , one of being out of balance . they face different challenges to women who already have a diabetes diagnosis when entering pregnancy as they have to become confident with blood sugar monitoring procedures as well as adapting to dietary restrictions . some of these women need to start administering insulin . according to griffiths et al . , it is important to provide information in a timely manner once the diagnosis is set , as well as to tailor health advice to the actual woman 's situation . regardless of the type of diabetes , women with diabetes in pregnancy are likely to have a higher risks of preeclampsia , a baby large for gestational age , shoulder dystocia , and cesarean section compared to women with normal pregnancies [ 16 , 17 ] . there are a few studies of providers ' perspectives of providing care for women with diabetes during pregnancy . berg and dahlberg , reporting on midwives working in hospital settings , describe them as struggling to balance the medical and normal perspectives , here the view of childbearing process as a normal life which also has to be supported , in their provision of midwifery care for women at high risk , including women with t1 dm . in community primary health care settings , persson et al . found midwives counseling women with a gdm diagnosis as experiencing moral and ethical challenges in exerting control and providing the women with support as well as promoting lifestyle changes . linell and bredmar discuss the moral implications of midwifery consultations in primary health care / community - based settings in talking about lifestyle - focused topics such as smoking , drinking , and sexually transmitted diseases and risk for malformations of the fetus . midwifery consultations for women with diabetes could therefore be regarded as complex consultations , during which the midwife encounters all types of women , regardless of parity and diagnosis , in a vulnerable period of their life . from the research , it is clear that women in diabetic pregnancies seem to have an extended need for emotional , appraisal , and informational support during their childbearing period including pregnancy [ 10 , 21 ] . the professional midwife has an explicit responsibility to enable the pregnant women to be involved and participate in decisions of different kinds . at the same time , the pregnant women must have the opportunity to express any concerns she has about the pregnancy , motherhood , and any other issues , concerns about life and living . these midwife - woman encounters could be considered as social processes where two experts meet , the woman as an expert on her life and living , and the midwife , as the professional expert in childbearing - related issues . health care personnel have a special responsibility for ensuring that patients set their own agenda and as far as possible understand what is offered from a professional perspective . previous research about women with diabetic pregnancies examines either the perceptions and experiences of the women or the providers ' perspectives , mainly through qualitative interviews . less focus has been directed to the conversations taking place between midwives and women in consultations and practice . findings from our previous study suggest that pregnant women to a lesser degree , compared to the midwives , initiated topics with concerns in the conversations . this indicates the relevance of describing consultations as cocreated communicatively by the parties . to improve clinical practice , we first need to illuminate and examine the taken - for - granted practice and behaviors in order to reflect on and challenge the provision of midwifery care . to gain such knowledge is important for midwives as well as other health care providers , who have a responsibility for promoting health and wellbeing in childbearing women . therefore , the aim of this study was to explore verbal communicative patterns in midwifery consultations , focusing on sequences where mothers initiated topics and concerns in the conversation . what topics and concerns were addressed / identified ? which variations in verbal communication were identified ? the study has an exploratory and interpretive design , based on the assumption that consultations between the midwife and the pregnant woman are socially constructed , situated , and relational phenomena . the midwife and pregnant woman contribute verbally and nonverbally to the understanding of the consultation situation [ 2528 ] . this understanding of what happens during the consultation is produced and reproduced through social processes , with the language employed playing a significant role . this study was conducted using a theme - oriented discursive approach , as described by roberts and sarangi , which means that we took a close look at how language constructs professional practice . we regard communication as not context - neutral ; rather , ways of talking are formed and take place in a discourse space within a larger context , such as , in this case , societal expectations of midwifery consultations . five antenatal diabetes clinics at hospitals in urban areas of norway were initially contacted about participation in the study , one of which declined . the diabetes team an obstetrician , an endocrinologist , and a midwife met weekly on a particular day at the clinics . the pregnant women were scheduled to meet the team every 14 days and sometimes weekly . all consultation rooms were equipped with a desk , a computer and chairs , a scale , and equipment for measuring blood pressure . some rooms had an examination bench where the midwife could conduct a physical examination of the pregnant woman . some consultation rooms were also equipped with an ultrasound machine and a cardiotocograph ( ctg ) machine . we invited 40 pregnant women to participate , asking them for permission to audio - record one of their consultations in the second or third trimester . the reason for selecting women at this stage of pregnancy was that we assumed that the midwife - mother relationship was well established by then . we also asked the women to take part in a postconsultation interview , which is not presented in this study . the inclusion criteria were having a diabetic condition and no other medical complication and being fluent in speaking and understanding norwegian . the secretary at the clinic sent letters of request to women enrolled at the clinics during april and june 2008 . midwives working at the diabetes clinics were invited to participate by letter of request , in addition to receiving oral information about the study . none of the midwives declined ; only ten of the pregnant women agreed to participate . five were primiparous , two were expecting their second and third baby , and one woman was pregnant with her fourth baby . all had postsecondary education and were employed outside their home , although two were on part - time sick leave . a pilot study was carried out by the first author ( c. f. risa ) prior to the main study , using informal conversations with midwives ( and in some clinics also with doctors ) and observing them in their daily work . this revealed that different tasks as well as time scheduled for the midwifery consultations varied between the clinics , which informed the author 's understanding of the context surrounding the consultations . to capture the verbal conversation , a voice recorder was placed on the desk , and a microphone was fastened to the midwife 's collar . since the consultation rooms were small and the researcher did not want to interfere with the consultation , she then left the room and was not present during the consultation . our data consists of ten audio - recorded consultations conducted at one of the scheduled appointments with participants in their second as well as third trimester ( gestational week 2636 ) . this study was conducted in accordance with the declaration of helsinki and was approved by the norwegian ethical committee ( no . 013/08 ) . midwives were instructed to stop the recording if needed or at the women 's request . all participants were informed that they could withdraw from the study at any time without stating a reason or explanation why . the voice recordings were transcribed without disclosing the identity of the speaker and were kept from unauthorized parties . in section 3 , descriptions of the participants ' medical situation as well as circumstances in care have been limited to secure confidentiality . the audio recordings were transcribed verbatim by the first author . the transcriptions were read and compared to the recordings several times , to both check for accuracy and get a sense of the whole and a sense of what to look for . results from a previous study in the project ( authors ) have revealed that the midwives dominated the discursive space ; that is , they did most of the talking and also initiated most topics . therefore , to understand and describe in detail the language constructs in midwifery consultations , we closely examined the text , especially those sequences where the pregnant women initiated topics and concerns in the conversation . in those sequences , we identified features of direct and indirect ways of talking as well as ambiguities , which in the theme - oriented approach are referred to as the focal themes of the analysis . this impression led us to take a closer look at goffman and his notion of frames , footings , and face - saving strategies in interactions . as we understand goffman , the term frame refers to how participants make use or sense of events as they construct those events . , a footing is the ways in which we position ourselves in relation to other persons by managing the production or reception of an utterance [ 34 , page 129 ] . we understand a change in footing as utterances where the participants shift their frame of conversation and therefore the relations . face - preserving strategies are described as strategies used to determine the degree to which it is possible to be implicit / indirect or explicit / direct in a conversation . brown and levinson , in developing goffman 's ideas further , named face - preserving strategies indirect politeness strategies can be used while disagreeing with more powerful persons . a strong request could , for instance , be softened or mitigated by use of phrases such as i think , you could , or you would . in this paper , we refer to these strategies as ambiguous ways of talking . in the analysis , we also investigate metaphors and choice of words , as well as paralinguistic features , such as pauses and accentuations in speech . in the quotes , pauses are marked with an ellipsis ( ) and their length in seconds is given in brackets , for example , [ silence 6 seconds ] . in our analysis , we have categorized instances where , during the consultation , the pregnant women initiated concerns into different frames , namely , ( i ) the professional expert 's frame , and ( ii ) the shared experts ' frame . in this section , the professional expert 's frame describes patterns of conversation mainly derived from the professional 's point of departure . the women used implicit and explicit strategies to express their concerns and the midwives responded in an explicit way . the topics addressed by the women were concerns about delivery time and mode and breastfeeding , which we here will present in detail . the first communicative variation within the professional expert 's frame is procedural talk , as seen in the following conversation on delivery - related issues , including sensations of labor onset . the second variation is called persuasion talk , here shown in a conversation where women 's breastfeeding was in focus , especially previous breastfeeding experience . a steering talk took place when a mother signaled a need to talk about her former birth experience . , we join the conversation as the woman raises her concern about the coming childbirth as an implicit question , a think - aloud question , or ambivalent statement , , the midwife talks in an objective and distanced manner while the woman acts as a listener . we interpret the woman 's laugh as a way to alleviate a situation which she perceives as threatening that is , as a face - saving strategy . however , the midwife steers the consultation forward ( line 20 ) and initiates a shift in footing by introducing a new topic , the baby 's kicks . the baby 's health is drawn into the conversation and keeps the conversation in a question - answer pattern , a typical feature of the professional expert 's frame . w : i really do n't know , i 'm thinking if i 'm not going to feel contractions as a vague pain in the back ? m : if you get contractions it is more than cramps in your back , then , sure it is . m : but since you are a diabetic , you will never be going longer maybe i 'm going to shut this door here since you are a diabetic you will never be going longer than m : if you haven't started labor by yourself by the due date , you will get started . m : but since you were previously delivered by cesarean , we have to use some other method to start you up . m : hm , well those who tried both , think when the birth starts spontaneously , it is better than to be started , they think that it 's something with the body that has decided for the baby to come out . m : but in your case , we have your health to take into consideration , we will never let it go past the due date . m : if you haven't started by the due date you will automatically be started . w : since you had an operation in your uterus , you have a uterus that is not quite so able as if you had n't been operated and so we have to use other methods to start you . one does n't need to talk so much about it just now , but so you got some ideas anyway . m : so , then it becomes exiting as you know that before the [ due date ] you will have the baby or at the latest on the [ due date ] . as they talk , it seems that the midwife misses the woman 's concern , as in line 10 the woman says , i 'm hoping to let go this time . we do not know if the mother is referring to hope to let go of a cesarean section or a vaginal birth . in answer the pregnant woman has had previous breastfeeding experience , which she is willing to share with the midwife . from the conversation , it appears that the midwife does not pick up on , or shows little interest in , the woman 's preferences . the woman 's pauses and hmmm , which we interpret as a noncommittal agreement , suggest that she does not agree with what the midwife says . such face - saving strategies are used when disagreeing with a more powerful person . in line 9 , we interpret the woman 's response i think as an attempt to soften her disagreement while still holding on to her own experience . [ the previous topic was former birth experiences . ] w : yeah but do n't know how it 's going to work out with breastfeeding but that [ abrupt speech ] m : yes , there are some that only breastfeed . ] yeah , but the first time is history , you have to forget ok ? then m : but you can think in a way , i would like to try and see , because it would save you a lot of work . w : i think it was much easier to give by bottle [ bottle feed ] . m : but think of everything you will have to boil , keep in order , and prepare , while you are out , wherever you go , it 's like [ laughs ] so if you get this working so it is , and if you do n't have all meals it saves a lot of hassle w : [ continues to talk about former experiences . ] i remember awfully well how much of a struggle it was ; i had to get up and latch and pump and m : no it 's not like that this is another way of keeping the talk in a certain frame but here the midwife seems to negotiate with the woman about why she has to keep strict control of her blood sugar level . in the following excerpt , the mother signals a need to talk about her former birth experiences . the use of the word things ( line 5 ) directs the focus to the pregnant woman 's physical health ( by things is meant physical measurements , e.g. , blood pressure , etc . ) and the completing of the maternity record . the footing is the talk - aloud sequence ( let 's see we are going to see how it is with your things ) to lead the woman 's attention back to the expert 's frame . the midwife uses it to cut the conversation short in order to keep to her schedule . in line 6 , it seems that the woman has already come to understand that there is no time to talk today and she modifies her initial request as a way to save the midwife 's face . in line 10 , the midwife calls up a different frame , by saying just for a chat , which may call up a different situation , where the woman may be encouraged talk about her situation with the midwife on a more equal footing . the last let 's see is understood as a way of verbally signaling a change in footing to keep up with the things , that is , her tasks that have to be done . m : hmm , it was a vacuum and a forceps delivery but you can say , it was the first time and probably a bit of a case of your rupture then , hmm , it probably is , w : yeah , but i 'm thinking , if i 'm going to have a birth i need to go through this whole birth process thoroughly to know what happened . m : yes , there is for sure a possibility of that , yeah . when are you scheduled for the next appointment , is it about 2 weeks ? w : yes , i do n't know , it is usually every 2 weeks . m : yes , it becomes that , does n't it , you know , so let 's see we are going to see how it is with your things . m : a bit so it is time for that , it is as you know , it is so awfully hectic these days you are here , is n't it , but i was thinking we could arrange a pure midwifery consultation for you [ is interrupted by a colleague who is asking a question from the doorway ] m : so we could arrange that and go through and have a decent talk , but you should then visit [ name ] because it 's she who 's usually here those days [ confidential tone ] . w : it 's actually the same m : actually the same , ok , then we try to find a time before then and quite simply book you in for a day , other than the diabetes control day , just for a chat , but of course we 'll check your blood pressure and all that but m : i think we 'll do that , wo n't we , so we 'll have a lot of time so it would n't be like on top of everything else . so i can go out and look at your urine let 's see [ proceeds with the blood pressure measurement ] . the conversations that took place in this frame were composed of midwives reasoning from a medical viewpoint but at the same time taking account of the women 's understanding and experiences . however , the midwives were more prone to communicate in an ambiguous way in their responses and explanations . the following topics were introduced into the conversation by the women : ( i ) diabetes management and fetal weight , ( ii ) early signs of labor , and ( iii ) labor pain . in the first communicative variation , the second variation is called contextualization talk , during which the midwife seems to reveal the woman 's perceptions and expectations and transforms the information into contextual knowledge . finally , the way in which the conversational dynamics can change during a conversation is illustrated by the switch talk , where the midwife introduces an episode of a shared experts ' frame into an otherwise professional expert 's frame . in this sequence , we quote part of a conversation where , early in the consultation , a first - time mother is being greeted by the midwife who acknowledges the mother 's belly . the mother 's response is made impersonal and objective by the midwife 's words , we should n't expect the opposite , should we ? the midwife seems to encourage the woman to voice any concerns by accentuating the pronoun you . this is to signify that she wants to hear about the woman herself and , briefly , shift the focus away from the baby . over the next few sentences , the mother and midwife take a conversational journey with a lot of back and forth , assisted by the midwife 's listening , comments , and questions as well as the use of continuers such as hmm , yes , and uhhm . these continuers seem to signal and call up a listening frame , i want to hear more , keep on talking . in this particular sequence , it takes about 36 lines before the mother shares her concerns about the baby 's weight as a consequence of her blood sugar fluctuations . note the hesitations and unfinished sentences which may indicate that she is approaching a sensitive topic . however , the possible resistance interpreted in this sequence could also be due to the fact that the mother did not really want to talk about this issue with the midwife . it 's been a long time since i last saw you , anyway it 's been almost the whole summer , i wonder when we saw each other [ looking in the maternity records ] well it was in the last week of june well , it 's 2 months now a lot has happened to you , your belly w : yeah , it has grown . m : we should n't expect the opposite , should we , but how are you now ? w : in general very well , i feel the body is very heavy , i do that . w : i keep on gaining w : i , and i have some nights that are n't so good . w : the less sleep i have [ the more ] it affects my blood sugar ; it changes when i ca n't sleep m : yes stress may well make it rise , maybe ? m : do you sleep badly when your husband is n't home , or ? w : more like when i am more active the sleep becomes poor ; more contractions w : yeah , i sleep much worse . w : no , you know i have to watch my blood sugar at least every third hour m : [ surprised tone ] you have ? w : i haven't slept through one night since [ continues talking about diabetes management and tests . ] m : when you think , what are you concerned about ? what 's in your head ? w : [ silence 3 seconds . ] blood sugar . [ silence 3 seconds . ] i do n't like the fluctuations ; i find it [ sic ] very distressing . and i know too , i think i sort of know how much this kid weighs ; does he stress enormously those hours it [ the blood sugar ] goes high in the following contextualization talk , a first - time mother - to - be is asking about the hospital routines during the early stages of labor . the midwife leaves her sentences unfinished , and there are delays and hesitations which result in an ambiguous way of talking ; this is contrasted by the more direct and fact - oriented sequences in the expert 's frame . the ambiguous talk , with its unfinished sentences , in this sequence may indicate the sensitive nature of diabetic pregnancies . pregnant diabetic women are likely to have more interventions than women with normal pregnancies and may not have natural onset of labor . however , the midwife provides a context for the explanation that is understandable to the woman . w : is it something special that i as a diabetic need to think of when i think it [ the birth ] has started ? any routines , like i have to call earlier or something or can i wait as long as others ? m : as long as you feel fine and your blood sugar 's fine and as long as you in a way have control over it and eat and it does n't lie too high or low , it must lie between 4 and 8 w : i see . m : and you feel fine and feel [ fetal ] movements then it 's normal for you as for others ; then it 's really good . m : you do recognize kicks and movements like that while you also have contractions , m : hope you will start by yourself so w : i hope so but you never know . m : no , one can never know , one can hope because having the birth started is as if w : yeah , that is probably what it is . m : it 's often more strenuous so it is often better when you start at home . m : you can hang around at home and maybe you have moved into the new house ? later on in the consultation , the woman inadvertently introduces her concerns in response to the midwife 's direct question . in line 21 , the midwife 's utterance may be interpreted as indirect advice regarding what the woman should expect during birth . the woman seems to embrace the advice , as we interpret her responses of i see , and m : exactly , when you were attending the antenatal class did you get any more thoughts or were you thinking , was it something new that was revealed or something you w : yes , it was some kind of scary . it did n't sound quite good with that epidural in my spine or near to , then m : yes , it 's not in the spine , it is w : i did n't like it and i hope to give birth as naturally as possible but i should maybe be thinking of having a bit of , hmm , pain relief because i ca n't bear to have it as if w : i 'm not against it but haven't i do n't have to take it if i do n't need to , that 's what i think . m : and if it stops and they see you are struggling and are very strained w : m : yes , sometimes it is , when one can relax a little , your body becomes [ making an exhalation ] and then it can , all of a sudden , just open up when the bodily resistance in a way has gone . m : so then they may suggest , but no - one would do anything until you w : no no , it 's not that i don't[interrupted / overlapping speech ] w : no , and so i will choose the safest way . this excerpt serve as an example of how , during the talk , the midwife shifts her footing by interposing an episode of a shared experts ' frame before returning to the expert 's frame . the incomplete sentence in line 13 is metaphorically seen as a key which opens up for the woman 's own ideas of her situation . in line 24 , the conversation is then continued on a question and answer basis in keeping with the expert 's frame . m : then i 'm going to take this one [ the ctg probe ] away and then it 's smart to , while you are at home , you write down questions if you have something you wonder about , ok ? w : but i 'm on the internet m : hmm , it is n't always you find the best , hmm , answers on the internet you know , we do n't know , you ca n't guarantee the quality of what 's been said on the internet w : but it 's usually when things tense up as m : it 's maybe , maybe that you really do n't know how this will turn out ? : i have always been like that ; i ca n't really take on worrying myself . m : that is absolutely smart just about that [ unstrapping the ctg from the woman 's belly ] . w : i think a little about i 'm becoming a multipara [ she was pregnant with her second child ] when it comes to birthing . m : well , you are a second parous woman , but you are not multiparous in your body ; in your birth canal you are like a primipara . m : yes , since no baby has passed through your birth canal , you are regarded as a primipara . since you haven't been at the beginning of a birth . m : you are perfectly right ; you are a primipara in your body and birth canal . m : here you see acceleration and there an acceleration [ looking on the ctg printout ] and it 's having variations , great ups and downs , perfectly excellent as far as i can see anyway . the two patterns that emerged from the analysis reflect some strategies employed by midwives in responding to women 's concerns , in an professional expert 's frame or a shared experts ' frame , using either a direct way of talking or an indirect way of talking . in this section , we discuss the findings within each frame . the first excerpt in the expert 's frame , procedural talk , describes a communication pattern that could be characterized by a transmission model of communication . it shows the midwife talking in a general and objective way while informing the woman about routines and procedures . in the second excerpt , persuasion talk , we suggest that the midwife takes a health promotive role . she argues from a professional stance and does not take the woman 's own experience into account . it may seem that the midwife is expecting the woman to comply with recommendations within the breastfeeding discourse , as it is expected that women breastfeed their children , no matter what . similar patterns of relating to each other have been reported in a study of midwifery antenatal consultations in a primary health care setting . the authors found five different patterns , three of which were regarded as basic patterns , named , in that study , the respectful gardener and her developing plants , the propagandist teacher , and the steering inspector . our finding of the expert 's frame may be in line with the pattern of the steering inspector where the midwife either missed or disregarded the woman 's concerns and actual situation , and instead imposed her own norms and expected compliance with those norms . the last excerpt from an expert 's frame is a further example of steering talk , where the surrounding context is revealed in the conversation , as the midwife seems to explain the circumstances of midwifery consultations to the woman . we can ask if the midwife , with her choice of words and metaphors , is implicitly saying that talk is not the main agenda at these consultations . she refers to talk as something that happens in addition to the actual purpose of the consultation , the checkup . pure may be interpreted to mean that the current consultation is contaminated with medical issues and does not represent a proper midwifery consultation . the pattern that the consultation takes may have consequences for how women perceive these consultations and comply with the expected behaviors and roles within the frame . we suspect that these women , some of them due to their background , have a history of encounters with health care professionals and may come to their consultation with a traditional provider - patient communication model in mind , which means the professional asks questions and the patient takes on a passive role , merely answering the questions . as these consultations have a strong focus on monitoring tasks to enable early detection of complications , we suggest that there may be a risk that the woman comes to perceive herself as an object , a container for the unborn baby . on the other hand , on the basis of previous research we understand that women highly regard the monitoring and surveillance aspects of the regular biomedical assessments of the unborn baby 's health [ 11 , 38 ] . because midwives are officially recognized as professional experts in childbearing issues , they should reflect on how they use and exercise their power in the consultation , especially as they seem to set the tone in the consultation , regarding which topics are given relevance in the consultation . on the other hand , it does seem that time limitations may necessitate a more tightly managed , expert consultation with focus on monitoring physical parameters at the expense of attention to emotional needs . as we have come to understand , as a consequence of the large increase in women with diabetes , primarily gdm , the diabetes team has to counsel more women within the same time frame and resources ( personal communication ) . it may be that this expert 's approach is a strategy to handle the workload and that without it ; the individual consultation would delay the other team members ' time schedule . the steering talk sequence could possibly indicate such a conflict , as the midwife seems to explain and justify her need to keep up with her institutional demands at the expense of meeting the woman 's need to talk . , midwives who do to not work in accordance with their ideal or philosophy may experience less work satisfaction as well as a variety of negative emotions , such as frustration , anxiety , and anger . we also found that the frequent use of the pronoun we by the midwife in the procedural talk could be interpreted in line with the midwife 's face work strategies . the recommendations do not stem from her ; she is replicating the institution 's voice . the communication pattern in the shared experts ' frame has a more two - way , dialogic approach where the midwife sets a listening context , as well as providing the woman with an opportunity to tell her story . it also seems that the point of departure for the conversation is the woman 's actual situation ; from there , the conversation continues , with an interweaving between the woman 's personal knowledge and the midwife 's professional and personal knowledge . from a pedagogical perspective , we can understand this in terms of how general information is transformed and contextualized to the individual pregnant woman , to be used in the forthcoming birth . the midwife 's listening attitude , with use of continuers such as ahh and mmm , seems to encourage the woman to open up about her concerns , rather than simply ask open questions . this is interesting because in contemporary health care , open questions are regarded as important tools in facilitating the expression of mothers ' ( and patients ' ) views , symptoms , and concerns . , report that patients seldom explicitly reveal concerns , and that instead , they offer clues and statements from their life situation , which might be associated with an emotion . the provider often misses these cues , so a listening attitude from the professional may lead to a direct expression of the patient 's emotional concerns . consequently , we suggest that it is insufficient to routinely ask open questions and lay the responsibility on the woman to voice her concerns explicitly and directly . in the example of the switch talk , the woman is being advised to collect and write down her questions and ask them at the next consultation . the beginning of this excerpt may mirror this consultation as an arena for information transfer , where knowledge is constructed by the parties and where both parties get to know something new . ambiguous talk was found in the shared experts ' frame , and it was characterized by pauses and unfinished sentences , which suggest communicative caution . according to linell and bredmar , this is a way of informing the pregnant woman while aiming not to cause anxiety , a face - saving strategy in line in goffmanian terms . similar patterns of ambiguous talk have been identified in studies where counseling touched lifestyle issues which may have moral implications , such as in the human immunodeficiency virus ( hiv)/acquired immunodeficiency syndrome ( aids ) counseling context and also in nurse - diabetic counseling encounters . in this study , ambiguous ways of talking were revealed in topics regarding some general sources of uncertainty related to childbirth : the when , how , and outcome of the birth , as well as pain in birthing . ambiguous ways of talking may be a strategy for the midwife to handle information of a delicate nature , since aggregated to large groups , women in diabetic pregnancies are more prone to have more inductions , instrumental deliveries , and deliveries by cesarean section compared to women in normal pregnancies and therefore may to a lesser degree come to experience normal birthing . the ambiguous way of talking , by being implicit and tentative , seems to function as an invitation from the midwife to the pregnant mother to have an input . similar findings have been reported in a study of family therapy , where ambiguous ways of talking were denoted as reciprocal editing of the ongoing talk . we found it challenging to demarcate the sequences in ongoing conversations , as the conversations had an evolving character ; what one said influenced what the other said in a spiral fashion . therefore , some of the illustrated excerpts are long and still have to be regarded as this study is based on transcriptions of voice recordings from consultations and some methodological considerations apply . we fully agree that transcripts are not an objective representation of a conversation ; they are regarded as a theoretical contextual construction of reality . the analytical perspective we used in this study is based on a linguistic sociological approach ; a different analytical focus may well have led to different findings . we have taken into consideration the fact that transcripts can never reproduce all aspects of speech or the exchange between the individuals nor illuminate the whole of the participants ' intended meanings , experiences , or intentions . in order to get close to the data , the first author collected as well as transcribed the audiotapes into texts which were read and revised several times . a further consideration is that what is heard in the consultations depends on the hearer . the hearer in this case , the first author , is a nurse - midwife experienced in antenatal consultations in primary health care settings . this background was an advantage in the process of understanding the midwives ' ways of working as well as their choice of vocabulary and phrases . the first authors discussed and reflected on the interpretations during the process . due to the limited extent of this study , we have not presented the original norwegian versions of the excerpts . this could have given the reader some information on the structure and semantics of the original language and may have allowed readers to make their own interpretations . the advantage of slowing down an activity or talk by analyzing it at a microlevel is that it provides an opportunity to identify how participants process talk through a conversation . in our study , this led to an understanding of the complexity of midwifery consultations as well as raising questions as to other contextual situational factors which may have influenced the communication at a microlevel , such as the time available , the initial and subsequent contact with the actual midwife , and the incidence of the knock on the door individuals invited to participate in research do not need to state their reason for declining to take part , so we do not know the reasons for the low participation rate in this study . voice - recording a consultation could possibly be regarded as intrusive and this may explain the low number of participants . since communication is more than just verbal speech , the use of a video recorder may have captured some aspects of the interaction that were missed in the voice recording . however , the data collected were rich in detail and served the purpose of addressing the research questions in these consultations . in a larger material , further categories and patterns may have emerged . one methodological question that needs to be raised is this : would the same interpretations as ours , the professional expert 's and shared experts ' frames , have evolved in consultations with other pregnant women in a different context ? if so , would the patterns have been equally , more , or less expert - oriented ? the findings may not be transferable to all antenatal diabetes consultations in norway or other countries that may have different health care . we suggest that the findings may provide insight into midwifery consultations beyond the particular settings and may also be useful for other health care professionals . different ways of communicating create different prerequisites for the mother 's perspective to unfold as well as different opportunities for the parties to share each other 's perspectives . from a professional perspective , providing adequate responses as well as creating space for a narrative approach with a listening attitude can be important for the mother 's concerns and experiences to be revealed in the consultations . professionals need to consider and discuss which behavior can best reach women , especially mothers in vulnerable situations such as high - risk pregnancies , and in which roles they can best assist them in their need to know and understand . it is also important to address the premises of , and obstacles to , providing midwifery in a midwifery setting . the findings in this study may serve as a basis for further research aimed at broadening our understanding of the communicational dynamics in antenatal diabetes consultations .
the advent of digital fluorescence microscopy and the development of fluorescent proteins allows for unprecedented observation of molecular behavior during complex cellular processes . because visualization of fluorescent probes is possible in live cells , not only can the cellular location of proteins be studied , but in vivo molecular and cellular protein dynamics can be observed . a number of sophisticated techniques , including fluorescence recovery after photobleaching , fluorescence resonance energy transfer , and fluorescence correlation spectroscopy have been developed to further utilize fluorescence technology . although fluorescently labeled proteins can localize to cellular components with a high degree of specificity , the spatial resolution of fluorescence microscopy is limited by the diffraction of light from a fluorophore and by the brightness of the fluorophore against background fluorescence . the spatial resolution of fluorescence microscopy is inherently limited by the wavelength of visible light , with a maximum achievable lateral resolution of ~200 nm and longitudinal resolution of ~500 nm ( the abbe limit ) for commonly used oil - immersion objective lenses having a numerical aperture of 1.4.15,28 in addition , conventional fluorescence microscope images contain information from out - of - focus planes both above and below the focal plane , which further complicates the analysis.1 while confocal fluorescence microscopy can reduce this contribution from out - of - focus fluorescence , it suffers from increased noise as well as from object distortion in the plane of focus.27 background noise reduces the maximum achievable resolution of spatially separated fluorophores , especially in the case of dim fluorescent signals . thus , although fluorescent markers can precisely localize cellular proteins and structures , difficulties in resolving individual same - color fluorophores through standard digital fluorescence microscopy hinders accurate quantitative analysis of feature locations . this is particularly true for live - cell imaging of proteins fused to green fluorescent protein ( gfp ) . new optical methods are being developed that break through the abbe limit5,6,12,13 for 3d imaging , but practical considerations so far limit resolution to ~100 nm . the ability to validate theoretical spatial models for the location of cellular components at resolutions beyond the abbe limit would increase the utility of fluorescence microscopy , allowing for improved quantitative analysis and for modeling of dynamic cellular processes . theoretical models can be developed via qualitative analysis of fluorescence images , complementary experimental methods such as electron microscopy , or computer modeling . in previous work , we have applied the model - convolution method to validate a variety of theoretical models,3,4,10,11,25 and found that the model - convolution approach provides a seamless and objective method to directly compare theoretical models to experimental results . in addition , we have found that simulated images generated through the model - convolution method can be used to evaluate the reliability of experimental measurement methods for a particular application , such as in evaluating the accuracy of gaussian fitting to track beads or evaluate filament curvature distributions.2,4 a typical solution used to reduce blur and out - of - focus fluorescence and thereby improve spatial resolution in conventional fluorescence microscopy is image deconvolution.21 the spreading of light by diffraction through a microscope lens relative to the focal plane of the point light source is termed the point spread function ( psf ) , and can be either experimentally measured or theoretically calculated for a given microscope . computational deconvolution methods use the experimentally measured or theoretically predicted psf to deblur fluorescence images . deconvolution methods work by estimating and then removing the contribution of light both from out - of - focus fluorescence and from in - plane spreading of light due to diffraction . although image deconvolution can be successful in improving image detail and contrast , as well as in reducing background haze,21 it is unclear whether this method is always appropriate for quantitative spatial characterization in images containing same - color multiple fluorescent protein copies ( > 2 copies ) . specifically , the presence of noise in the data makes it difficult to exactly reconstruct the underlying fluorophore distribution , so that deconvolution can only produce the distribution in a statistical sense , often via least - squares minimization.21 in addition , the interpretation of deconvolved images can be problematic , as the computational deconvolution operation is performed without regard to known physical or molecular information about the system being studied . we illustrate a potential pitfall of deconvolution with an example that involves detection of the positions of kinetochores between spindle poles within the budding yeast mitotic spindle . a simulated distribution was made that included 32 green fluorophores ( representing 32 kinetochores ) and 2 red fluorophores ( representing two spindle pole bodies ) , located along a ~1500 nm spindle axis . this is the fluorophore distribution predicted by electron microscope reconstructions of a typical budding yeast mitotic spindle22,31 ( figs . a simulated fluorescence image of the mitotic spindle was generated by convolving this fluorophore distribution with the wide - field microscope psf and then by adding a level of background noise to the image that is typical of budding yeast cells expressing proteins fused to gfp ( fig . this simulated image was deconvolved using standard constrained iterative deconvolution software ( deltavision , fig . resolution of the two widely spaced red fluorophores at the spindle poles is clearly successful in the deconvolved image . thus , experimental deconvolution is useful in this instance for contributing to accurate measurement of spindle pole locations ( through gaussian fitting or similar ) , in assessing the relative brightness of the poles , and in confirming that there are indeed two fluorescent objects present in the image.figure 1image deconvolution can not always resolve individual fluorophore locations . ( a ) a typical yeast mitotic spindle experimental fluorescence image ( kinetochore - associated fluorescence , green ; spindle pole body fluorescence , red ) . ( b ) theoretical point - source fluorophores ( 32 green points , representing individual kinetochores , and 2 red points , representing the spindle pole bodies ) along a 1500 nm length . the bright green pixels indicate the presence of multiple fluorophores within the pixel area . for simplicity , it was assumed that there are no fluorophores in out - of - focus focal planes . ( c ) point - source fluorophores in ( a ) are convolved with the microscope psf and noise is added . ( d ) the image in ( b ) has been deconvolved using the identical psf . the image deconvolution process can not resolve the individual point - source fluorophores and tends to generate fluorescent clusters in the periphery which are artifacts of deconvolving noise image deconvolution can not always resolve individual fluorophore locations . ( a ) a typical yeast mitotic spindle experimental fluorescence image ( kinetochore - associated fluorescence , green ; spindle pole body fluorescence , red ) . ( b ) theoretical point - source fluorophores ( 32 green points , representing individual kinetochores , and 2 red points , representing the spindle pole bodies ) along a 1500 nm length . the bright green pixels indicate the presence of multiple fluorophores within the pixel area . for simplicity , it was assumed that there are no fluorophores in out - of - focus focal planes . ( c ) point - source fluorophores in ( a ) are convolved with the microscope psf and noise is added . ( d ) the image in ( b ) has been deconvolved using the identical psf . the image deconvolution process can not resolve the individual point - source fluorophores and tends to generate fluorescent clusters in the periphery which are artifacts of deconvolving noise in contrast , image deconvolution did not yield an accurate distribution of the kinetochores given the typical level of noise . here , the deconvolution operation identified artificial clusters of green fluorescence as the underlying distribution of fluorescent kinetochores , both in the spindle as well as outside of the spindle ( fig . 1d ) . in this case , the gaussian white noise in the background of the raw image results in the kinetochore example demonstrates two undesirable outcomes from deconvolution : ( 1 ) fluorophores are misplaced ( inaccurate localization of green spots ) , and ( 2 ) noise is identified as signal ( deconvolution of green background noise to produce artificial fluorescent clusters ) . in the absence of noise , deconvolution is a simple linear operation that yields the exact fluorophore distribution in a single step . noise is produced by photon statistical variance , which is defined as the square - root of the average integrated number of photons captured per detector picture element ( pixel ) , and by detector read - out noise . for example , if the average number of photons per pixel in the background is 100 , then the background photon noise is 10 photon counts per pixel . similary , typical detector readout noise for cooled ccd cameras is equivalent to about 10 photon counts per pixel . in this case , the total noise per pixel for background fluorescence is the square root of ( 10 + 10 ) = ~14 photon counts ( via the addition of variances ) . in addition , the signal itself also has photon noise , such that peak signal intensity fluctuates between image frames as the square root of the average number of photons . all of these noise sources confound the problem of separating meaningful signal from background signal . this uncertainty makes quantitative characterization of fluorescent protein locations via image deconvolution particularly problematic in images with a low signal - to - noise ratio ( snr ) , i.e. , when the fluorescent probe markers are dim as compared to the image background noise . to illustrate the effect of background noise on the accuracy of experimental deconvolution , we convolved a single fluorophore with a theoretical psf , and then deconvolved the resulting image using a standard wiener filter - based deconvolution algorithm , using the identical theoretical psf ( fig . , we were able to achieve varying levels of success in identifying the simulated fluorophore after image deconvolution . here , we define success as a result in which the brightest pixel in the deconvolved image is in the location of the original simulated fluorophore.figure 2high noise levels limit the utility of the image deconvolution method . ( a1 ) a simulated point - source fluorophore has been convolved with a theoretical psf ( no background noise ) to produce a 32 32 image having a single signal in the center of the field . ( a2 ) subsequent image deconvolution ( by wiener filtering - based deconvolution using the matlab image processing toolbox ) precisely resolves the spreading of light due to the psf , and correctly identifies the fluorophore location to be at the center . ( b1 ) a simulated point - source fluorophore has been convolved with a theoretical psf , but noise has been added to the image such that the snr = 8 . ( b2 ) in this case , subsequent image deconvolution is able to correctly resolve the fluorophore location . ( b3 ) in another image with snr = 8 , image deconvolution is not able to separate the fluorophore from background noise and misidentifies the location of the point source . ( c ) the ability of wiener - filter - based deconvolution to separate fluorophores from background noise decreases substantially with decreasing snr . the quantitative relationship between the failure rate and the snr depends upon the specifics of the problem , but generally failure rate increases with decreasing snr high noise levels limit the utility of the image deconvolution method . ( a1 ) a simulated point - source fluorophore has been convolved with a theoretical psf ( no background noise ) to produce a 32 32 image having a single signal in the center of the field . ( a2 ) subsequent image deconvolution ( by wiener filtering - based deconvolution using the matlab image processing toolbox ) precisely resolves the spreading of light due to the psf , and correctly identifies the fluorophore location to be at the center . ( b1 ) a simulated point - source fluorophore has been convolved with a theoretical psf , but noise has been added to the image such that the snr = 8 . ( b2 ) in this case , subsequent image deconvolution is able to correctly resolve the fluorophore location . ( b3 ) in another image with snr = 8 , image deconvolution is not able to separate the fluorophore from background noise and misidentifies the location of the point source . ( c ) the ability of wiener - filter - based deconvolution to separate fluorophores from background noise decreases substantially with decreasing snr . the quantitative relationship between the failure rate and the snr depends upon the specifics of the problem , but generally failure rate increases with decreasing snr if the snr was large ( > 15 ) , the deconvolution algorithm was nearly always able to separate signal from noise ( fig . in contrast , if the snr was set equal to 8 , it was just as likely for the deconvolution algorithm to successfully identify a fluorophore as it was for it to ignore the signal ( figs . as the snr approaches 1 , the deconvolution algorithm essentially picks the fluorophore location at random , and so yields no new information . in the yeast mitotic spindle example ( fig . 1 ) , although there are many fluorophores concentrated in a small area , experimental snrs are ~3 . in this case , the deconvolution algorithm is likely to confuse background noise with fluorophore signals , making quantitative spatial analysis of deconvolved images highly uncertain . we consider here an alternate method to deconvolution which we term model - convolution . this method enables digital fluorescence microscopy data to be interpreted in the context of a model with a measurable degree of confidence , when deconvolution is not successful because fluorophores are closely spaced and noise is significant ( which is common for many live - cell imaging applications ) . with model - convolution , we use other structural information ( from high - resolution electron micrographs , for example ) to hypothesize an underlying distribution of photon counts from proteins or structures labeled with fluorophores.11 this 3d distribution is then convolved with the 3d distribution of the microscope psf.8,9,25 the background fluorescence and noise typical of our microscope images is then added to obtain a simulated fluorescence image ( figs . 1b and 1c).25 to convolve a model for an underlying distribution of photon counts with the 3d distribution of the microscope psf , the microscope psf must first be defined . we have found that a reliable technique for defining a 3d psf is to estimate the psf experimentally by imaging sub - resolution fluorescent beads at different microscope focal planes.25,26 these images are then convolved with a 3-d model to account for the spreading of light through the microscope lens due to diffraction , as described below . when defining a hypothesized spatial model for the underlying distribution of photon counts , the resolution of the model can , however , the spatial model must ultimately be translated into a resolution that is consistent with the experimental imaging apparatus . for this reason , although the fine grid array used for modeling can be arbitrarily small , it is most accurate to define its dimensions such that it can be directly re - binned into the pixel size of the microscope used for imaging . for example , if the microscope pixel size is 66 66 nm , then an appropriate fine grid bin size may be 22 22 nm , so that nine fine grid elements can then be summed to correspond to the experimental pixel size.26 once a hypothesized model has been defined and the corresponding fine grid array is re - binned such that each element of the final model grid is representative of an experimental microscope pixel size , then the model distribution is directly convolved with the experimentally measured 3d psf . here , the correct z - slice psf is centered on each non - zero element in the binned model grid , and then the psf is then convolved with the binned model grid . this can be accomplished through the use of commercially available software packages ( e.g. , matlab ) , which have built - in convolution commands . after convolving the binned model grid with the experimentally measured psf , then the final step in creating a model - convolved image is to add in the typical experimental noise and background fluorescence levels . the experimental background fluorescence and noise are established by measuring both the mean and the standard deviation of experimental fluorescence in an experimental image area that is far from the experimental signal . then , this measured background fluorescence and noise can be applied to each pixel of the binned model grid , as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{bin}}_{\text{final } } = { \text{bin}}_{\text{convolved } } ( { \text{signal } } - { \text{bkrd ) } } + { \text{bkrd } } + { \text{rand}}*{\text{noise } } $ $ \end{document}where binfinal is the final model - convolved fluorescence intensity , calculated for each pixel of the image , binconvolved is the binned grid element value after convolution with the psf , signal is the average measured experimental signal , bkrd is the average measured experimental background , and noise is the standard deviation of the background signal . here , rand represents a gaussian random number with mean = 0 and standard deviation = 1 . quantitative statistical comparison of simulated to raw experimental microscope images then allows for quantitative evaluation of the theoretical fluorophore distribution . previously , quantitative statistical comparison of simulated data to experimental data has been completed by calculating the sum - of - squares error for many individual simulations as compared to the overall average simulation curve , and then by calculating a p - value based on how the experimental sum - of - squares error compares to the simulation results ( for detailed description of statistical methods , see gardner et al.8 and sprague et al.25 ) . by directly creating simulated fluorescence images from theoretical fluorophore arrangements , artifacts in deconvolution algorithms generated as a result of difficulties in distinguishing background noise from true signal are eliminated . furthermore , in contrast to computational deconvolution , the final model - convolution simulated image is generated by incorporating known physical and molecular information about the system , thus simplifying interpretation of the results . although deconvolution software iteratively seeks a fluorophore distribution that minimizes the sum - of - squares error , there may be other distributions with only slightly worse sum - of - squares error than the best - fit the advantage of the model - convolution method is that it naturally incorporates the known physical and molecular constraints of the system ( i.e. , number of kinetochores , in the yeast mitosis case , etc . ) . in situations with a limited number of fluorescent proteins and/or with multi - colored fluorescent proteins , 2d - gaussian fitting and centroid tracking have been used effectively to achieve a spatial resolution beyond the abbe limit.18,29 in this case , the model - convolution approach is helpful as a complement to standard tracking algorithms to establish the limits of detectability and accuracy . validation of tracking algorithms is not routinely performed , but could be done readily with model - convolution.3,4 the model - convolution method is most useful as a technique for quantitative analysis and hypothesis testing with digital fluorescence microscopy . cellular objects that are more closely spaced than half the wavelength of light used for imaging can not be visually resolved as separate objects using single channel fluorescence microscopy . however , model - convolution can be used to accurately estimate the information content in fluorescence images . clustering patterns of multiple fluorophores , changes in fluorophore locations over time and with genetic mutations , and recovery patterns of photobleached fluorophores can all be quantitatively analyzed using the model - convolution method as described above . in our yeast mitotic spindle analysis , data from electron microscopy23,31 was used to program into a computer a three - dimensional geometric representation of a mitotic spindle . at metaphase in yeast , sister kinetochores stretch out their intervening centromere chromatin by pulling forces toward opposite poles generated by kinetochore attachment to a single microtubule . the 16 pairs of sister kinetochores , each tagged with cse4-gfp , thus become centered on average near the spindle equator , although each sister kinetochore oscillates poleward and away from its pole , coupled to kinetochore microtubule plus - end depolymerization and polymerization at the kinetochore attachment sites . on average , spindle pole - to - pole length , measured via labeling with spc29-cfp , is about 1500 nm in the yeast metaphase spindle , and each kinetochore microtubule is ~300 nm , such that sister kinetochore pairs are separated by ~900 nm . theoretical numbers of kinetochore - associated fluorophores as well as the size and general organization of the mitotic spindle could be deduced from the three - dimensional ultrastructural analysis . once the spindle geometry was defined based on electron microscopy , a computer simulation was run to test various models for the dynamics of fluorescently labeled proteins at kinetochores and the polymerization / depolymerization dynamics of kinetochore microtubules.9,25 in this way , a specific model for regulation of kinetochore microtubule polymerization / depolymerization was developed . this model relied on the regulation of microtubule dynamics via the physical separation of sister kinetochores . thus , although individual sister kinetochore spacing can not be resolved experimentally , the model - convolution method could be used to test models for the regulation of kinetochore microtubule dynamics via simulation of results from multiple experiments targeting both tubulin and kinetochore - associated proteins . in addition , statistical comparison of the model against the results of multiple experiments provided for quantitative validation of the model . thus , a model for the dynamics of individual proteins in the spindle was developed via model - convolution , despite the inability to experimentally resolve individual fluorescent proteins via light microscopy . the model - convolution method can also be used to design experiments for quantifying the physical separation distance between same - color fluorescent protein markers . as shown in fig . 3a , simulated yeast mitotic spindle images for various sister kinetochore spacings can be generated via model - convolution ( fig . these images are similar to those expected for a mutant phenotype that modified kinetochore microtubule dynamics , resulting in altered sister kinetochore spacing . by quantitatively comparing the spacing of kinetochore - associated fluorescence clusters in simulated images , it is possible to predict the number of independent experimental images that would be required to make statistical conclusions about more subtle differences in sister kinetochore spacing between mutant and wild - type cells ( fig . for example , in order to spatially resolve differences in fluorescent cluster spacing of ~12 nm with a statistical significance of p < 0.01 , ~50 different spindle experimental images would be required . specifically , model - convolution simulations predict that if the distance between fluorescence cluster centroids is calculated for each unique spindle , and then this sampling of distances is statistically evaluated against the null hypothesis that the separation distance is equal to 0 nm , it would require 50 independent spindles to resolve a 12 nm centroid spacing with a statistical significance of p < 0.01.figure 3the model - convolution method as applied to experimental design . ( a ) simulated images can be generated via model - convolution with varying mean distances between fluorescent protein clusters . in the upper images , mean distance between fluorescent protein clusters is ~600 nm , shown as raw simulated fluorophores and after model - convolution . in the lower images , mean distance between fluorescent protein clusters is ~800 nm . in this example , the difference in spacing is large enough to be distinguished readily upon visual examination . ( b ) for more subtle differences in fluorescent protein separation differences , statistical comparison of fluorescent protein localization is required . by generating simulated images of the yeast spindle example via model - convolution and performing a statistical comparison of these images , it is possible to predict the number of images required to achieve more subtle experimental spatial resolution . for example , to resolve a difference in fluorescent cluster spatial localization of ~12 nm , ~50 experimental images would be required the model - convolution method as applied to experimental design . ( a ) simulated images can be generated via model - convolution with varying mean distances between fluorescent protein clusters . in the upper images , mean distance between fluorescent protein clusters is ~600 nm , shown as raw simulated fluorophores and after model - convolution . in the lower images , , the difference in spacing is large enough to be distinguished readily upon visual examination . ( b ) for more subtle differences in fluorescent protein separation differences , statistical comparison of fluorescent protein localization is required . by generating simulated images of the yeast spindle example via model - convolution and performing a statistical comparison of these images , it is possible to predict the number of images required to achieve more subtle experimental spatial resolution . for example , to resolve a difference in fluorescent cluster spatial localization of ~12 nm , ~50 experimental images would be required similar methods to the model - convolution technique have been employed in a variety of applications , including fluorescent speckling on microtubules,30 ca sparks in skeletal muscle,17 measurement of three - dimensional diffusion rates,19 and the sizing of small fluorescently labeled particles.7 in each of these cases , simulation of the fluorescence imaging process was employed to allow for testing of a theoretical model . method similar to the model - deconvolution method was successfully employed by littlefield and fowler20 to measure thin filament lengths of myofibrils . here , the authors detected length ranges within 40100 nm by utilizing information from electron microscopy about the ultrastructural organization of actin and myosin filaments within the sarcomere repeat . the model - convolution method can also be used to aid in the interpretation of fluorescence images . as shown in fig . 4a , two - dimensional fluorescence images can lead to misinterpretation of three - dimensional structures , such as branched actin filaments.14 from the simulated image , it is apparent that long side branches from the mother filament are nearly imperceptible , except at their base , when they project at various angles out of the focal plane . assuming these filaments have the diameter of a single actin filament ( 7 nm ) , a theoretical structure in three dimensions is constructed and then model - convolution is used to generate a simulated fluorescence image ( fig . this allows one to assess the detectability of side branches as a function of branch angle and length.figure 4the model - convolution method as compared to the image deconvolution process . in the image deconvolution process , an experimental image is deblurred using the theoretical microscope psf . with the model - convolution method , a theoretical fluorophore distribution is convolved with the microscope psf and noise , and a simulated image is generated . thus , the model - convolution method is essentially the inverse of the image deconvolution process . ( a ) the example shown is a computational model of arp2/3-mediated actin filament branching in three dimensions based on experimental observations by ichetovkin et al.14 the model results in a branched actin filament structure stemming from an initial nucleation site ( 1blue arrow ) and leading to a series of branches off the main filament . the model - convolution method is applied to create a theoretical microscope image at the focal plane of the main filament . branches that are close to the focal plane of the microscope ( 2orange arrow ) are clearly visible in the simulated fluorescence image . branches that project out of the focal plane ( 3red arrow , and 4white arrow ) are less visible in the simulated image , indicating that the branching complexity and branch length distribution of the actin filament could be misinterpreted from experimental fluorescence images . scale bar , 1000 nm . ( b ) the model - convolution approach to estimating microtubule curvature . a simulated microtubule is constructed with a known analytical function ( sine function on a 2 nm pixel grid ) , showing the true underlying relation of the curvature to the outer diameter . this simulated microtubule has curvature that would be at the high extreme of observed curvatures in living cells.24 the model - convolution operation is performed , and the resulting image is binned to the pixel size associated with a high na lens and ccd detector ( 50 nm pixel size ) . the convolved image appears more highly curved than the underlying filament , and the digitization on the camera makes quantitative analysis of curvature prone to errors . scale bar , 250 nm the model - convolution method as compared to the image deconvolution process . in the image deconvolution process , an experimental image is deblurred using the theoretical microscope psf . with the model - convolution method , a theoretical fluorophore distribution is convolved with the microscope psf and noise , and a simulated image is generated . thus , the model - convolution method is essentially the inverse of the image deconvolution process . ( a ) the example shown is a computational model of arp2/3-mediated actin filament branching in three dimensions based on experimental observations by ichetovkin et al.14 the model results in a branched actin filament structure stemming from an initial nucleation site ( 1blue arrow ) and leading to a series of branches off the main filament . the model - convolution method is applied to create a theoretical microscope image at the focal plane of the main filament . branches that are close to the focal plane of the microscope ( 2orange arrow ) are clearly visible in the simulated fluorescence image . branches that project out of the focal plane ( 3red arrow , and 4white arrow ) are less visible in the simulated image , indicating that the branching complexity and branch length distribution of the actin filament could be misinterpreted from experimental fluorescence images . scale bar , 1000 nm . a simulated microtubule is constructed with a known analytical function ( sine function on a 2 nm pixel grid ) , showing the true underlying relation of the curvature to the outer diameter . this simulated microtubule has curvature that would be at the high extreme of observed curvatures in living cells.24 the model - convolution operation is performed , and the resulting image is binned to the pixel size associated with a high na lens and ccd detector ( 50 nm pixel size ) . the convolved image appears more highly curved than the underlying filament , and the digitization on the camera makes quantitative analysis of curvature prone to errors . scale bar , 250 nm as another example , consider the analysis of microtubule bending in living cells . in this example , fluorescently tagged microtubules are ~25 nm in outer diameter , but fluorescent microscope images or images simulated via microtubule model - convolution yield a virtual microtubule image that is ~250 nm in diameter . the apparent 10-fold increase in filament thickness can lead to the perception that microtubule is more curved and under more mechanical stress than is actually the case . in addition , the presence of noise and the digitization inherent in ccd cameras can lead to substantial errors when quantitatively estimating the local curvature of a filament from a digital fluorescence image ( fig . in similar work by janson and dogterom , microtubule shape was digitized by convolution of microtubule image intensity profile with one period of a sine function to mimic the shadow - cast appearance of a microtubule16 imaged via differential interference contrast microscopy . this digitized data was then smoothed and used to analyze shape fluctuations on elongating microtubules . computational modeling is an emerging tool that can be used to better understand and predict complex cellular processes . by converting numerical data generated from computational models into simulated fluorescence images that can be directly compared to experimental images , the model - convolution method provides for a strong link between sophisticated simulation methods and experimental results . in this way , the results of computational simulations can be better understood and interpreted , regardless of the model complexity . in general , model - convolution facilitates the interpretation of the digital fluorescence microscopy data now being collected in large quantities .
alzheimer s disease ( ad ) is the most prevalent neurodegenerative condition that leads to dementia . along with memory loss and cognitive impairment , patients with ad also suffer from dysfunctions of homeostatic and autonomic mechanisms , including mood regulation , pain perception , and sleep wake cycles.1 such noncognitive abnormalities are clinically referred to as behavioral and psychological symptoms of dementia ( bpsd ) . sleep - related bpsd affects up to 40% of ad patients prior to their clinical diagnosis of overt dementia.2 sleep is essential for brain function , such as memory consolidation , and it has been suggested that disturbed sleep exacerbates cognitive decline or ad progression.2,3 although the connection between the etiology of sleep disorders and ad is poorly understood , previous findings have suggested a robust connection and a possible bidirectional link between sleep distortion and ad pathogenesis.4,5 they showed that chronic sleep deprivation increased chronic accumulation of -amyloid ( a ) in the brain.5 moreover , cerebral a retention may lead to wakefulness and altered sleep patterns.5 these results stress the importance of examining brain structures pertinent to regulation of sleep , which could help to pinpoint important loci in the progression of ad . findings in postmortem studies have located detectable ad - related biochemical changes in the brain stem , which contains neuronal networks that play key roles in the regulation of sleep.6 in addition , our previous in vivo structural imaging study using magnetic resonance imaging ( mri ) showed significant morphological deformations in the upper posterior regions of the brain stem in ad patients.7 in this regard , brain stem structural abnormalities might be important neurobiological mechanisms underlying sleep disturbances associated with ad . the aim of this study was to explore the relationships between brain stem morphological changes and sleep disturbances in patients with ad using mri - based automated subcortical segmentation methods . we hypothesized that sleep disturbances would be correlated with deformations in posterior brain stem morphology in ad patients . a total of 84 subjects ( 44 with ad and 40 healthy elderly controls ) participated in this study . they were recruited from the geriatric psychiatry brain imaging database , which holds brain scans of outpatients and inpatients at the department of geriatric psychiatry , saint vincent hospital , the catholic university of korea . ad subjects met the national institute of neurological and communicative disorders and stroke / alzheimer disease and related disorders association criteria for probable ad,8 and scored 1 or greater on the clinical dementia rating scale.9 participants who suffered from other neurological or psychiatric conditions , including other forms of dementia , or who were taking any psychotropic medications were excluded . we measured cognitive functions using the korean version of the consortium to establish a registry for alzheimer s disease.10 severity of sleep disturbance was measured using the sleep - disturbance domain of the neuropsychiatric inventory ( npi ) scale . patients who had a score of 1 or more on the scale were characterized as having sleep disturbance . to minimize the influence of other psychiatric conditions on sleep disturbances , we included only participants who scored zero in the other eleven domains of the npi scale ( delusions , hallucinations , agitation or aggression , depression , anxiety , euphoria , apathy , disinhibition , irritability , motor disturbance , and eating behavior ) . ethical and safety approval for the study was obtained from the local institutional review board of the catholic university of korea . mri scans of all participants were obtained using a 3 t whole - body scanner equipped with an 8-channel phased - array head coil ( verio ; siemens ag , munich , germany ) . the scanning parameters of the t1-weighted three - dimensional magnetization - prepared rapid gradient - echo sequences were as follows : echo time 2.5 ms , repetition time 1,900 ms , inversion time 900 ms , flip angle 91 , field of view 250250 mm , matrix 256256 mm , and voxel size 1.01.01.0 mm . the three - dimensional mri images were registered and processed within the bayesian appearance model framework as designed in an earlier study by the oxford centre for functional mri of the brain ( fmrib).11 during registration , mri images were transformed to the mni152 standard space by affine transformations on the basis of df=12 . different subcortical structures , such as the brain stem , were located based on shape models and voxel intensities through the application of a subcortical mask and the fmrib integrated registration and segmentation tool ( first ) . absolute volumes of the subcortical structures were calculated with consideration of the transformations performed during the initial registration steps . brain stem volumes ( bvs ) were normalized to the total intracranial volume ( ticv ) , measured using the sienax software.12 normalized bv ( nbv ) was defined as nbv = mean ticv bv / ticv . first analyzes the volume and shape of each subcortical structure using a deformable surface - mesh model . although the segmentation process of first is largely automated , two important aspects of the mesh model must be inspected in its application : vertex correspondence and surface alignment . first , the number of vertices for each structure must have a fixed value , so that corresponding vertices can be compared across individuals and between groups . such vertex correspondence is crucial for the first methodology , because group differences in the spatial location of each vertex are directly used for the examination of localized shape differences . second , even when the vertices retain correspondence , the surfaces of the mesh reside in the native image space , and thus are arbitrarily oriented or positioned . therefore , surface alignment must be standardized before investigating any group differences . the mean surface from first models was used as the target to which surfaces from individual participants were aligned . pose was removed by minimizing the sum - of - squares difference between the corresponding vertices of a participant s surface and the mean surface . correlation analyses of the vertices were performed using f - statistics with the statistical significance threshold set at a p - value of less than 0.05 ( false - discovery rate [ fdr ] ) to resolve the issue of multiple comparisons . the duvernoy brain stem atlas was incorporated as a reference in determining the approximate anatomical location of our statistical maps.13 a total of 84 subjects ( 44 with ad and 40 healthy elderly controls ) participated in this study . they were recruited from the geriatric psychiatry brain imaging database , which holds brain scans of outpatients and inpatients at the department of geriatric psychiatry , saint vincent hospital , the catholic university of korea . ad subjects met the national institute of neurological and communicative disorders and stroke / alzheimer disease and related disorders association criteria for probable ad,8 and scored 1 or greater on the clinical dementia rating scale.9 participants who suffered from other neurological or psychiatric conditions , including other forms of dementia , or who were taking any psychotropic medications were excluded . we measured cognitive functions using the korean version of the consortium to establish a registry for alzheimer s disease.10 severity of sleep disturbance was measured using the sleep - disturbance domain of the neuropsychiatric inventory ( npi ) scale . patients who had a score of 1 or more on the scale were characterized as having sleep disturbance . to minimize the influence of other psychiatric conditions on sleep disturbances , we included only participants who scored zero in the other eleven domains of the npi scale ( delusions , hallucinations , agitation or aggression , depression , anxiety , euphoria , apathy , disinhibition , irritability , motor disturbance , and eating behavior ) . ethical and safety approval for the study was obtained from the local institutional review board of the catholic university of korea . mri scans of all participants were obtained using a 3 t whole - body scanner equipped with an 8-channel phased - array head coil ( verio ; siemens ag , munich , germany ) . the scanning parameters of the t1-weighted three - dimensional magnetization - prepared rapid gradient - echo sequences were as follows : echo time 2.5 ms , repetition time 1,900 ms , inversion time 900 ms , flip angle 91 , field of view 250250 mm , matrix 256256 mm , and voxel size 1.01.01.0 mm . the three - dimensional mri images were registered and processed within the bayesian appearance model framework as designed in an earlier study by the oxford centre for functional mri of the brain ( fmrib).11 during registration , mri images were transformed to the mni152 standard space by affine transformations on the basis of df=12 . different subcortical structures , such as the brain stem , were located based on shape models and voxel intensities through the application of a subcortical mask and the fmrib integrated registration and segmentation tool ( first ) . absolute volumes of the subcortical structures were calculated with consideration of the transformations performed during the initial registration steps . brain stem volumes ( bvs ) were normalized to the total intracranial volume ( ticv ) , measured using the sienax software.12 normalized bv ( nbv ) was defined as nbv = mean ticv bv / ticv . first analyzes the volume and shape of each subcortical structure using a deformable surface - mesh model . although the segmentation process of first is largely automated , two important aspects of the mesh model must be inspected in its application : vertex correspondence and surface alignment . first , the number of vertices for each structure must have a fixed value , so that corresponding vertices can be compared across individuals and between groups . such vertex correspondence is crucial for the first methodology , because group differences in the spatial location of each vertex are directly used for the examination of localized shape differences . second , even when the vertices retain correspondence , the surfaces of the mesh reside in the native image space , and thus are arbitrarily oriented or positioned . the mean surface from first models was used as the target to which surfaces from individual participants were aligned . pose was removed by minimizing the sum - of - squares difference between the corresponding vertices of a participant s surface and the mean surface . correlation analyses of the vertices were performed using f - statistics with the statistical significance threshold set at a p - value of less than 0.05 ( false - discovery rate [ fdr ] ) to resolve the issue of multiple comparisons . the duvernoy brain stem atlas was incorporated as a reference in determining the approximate anatomical location of our statistical maps.13 table 1 shows the baseline demographic data of our participants . of the 44 ad subjects with sleep disturbances , 26 ( 59% ) had circadian rhythm disturbances , 14 ( 32% ) had insomnia , and four ( 9% ) had hypersomnia . the data from surface - based vertex analyses revealed significant structural alterations in the left posterior lateral region of the ad patients brain stems that correlated with their npi sleep - subscale scores ( fdr - corrected p<0.05 , figure 1a ) . in addition , significant group differences between the ad group and the control group were observed in posterior brain stem morphology ( fdr - corrected p<0.05 , figure 1b ) . furthermore , the normalized bvs of the ad patients correlated with their npi sleep - subscale scores ( r=0.33 , p=0.024 , figure 2 ) . to the best of our knowledge , this is the first in vivo study on brain stem morphological changes associated with sleep disturbances in ad . recently , a growing body of evidence has suggested a potential causal relationship between sleep disturbances and ad.14 daytime drowsiness was shown to be an accurate predictor of ad within a time span of 24 years.14,15 also , improved sleep was linked with delayed onset of ad , while reduction in sleep was correlated with lower cellular tolerance of oxidation stress in the brain.16 a shared genetic risk background , such as variations in the apoe and maoa genes , has also been implicated in disrupted sleep and ad.17,18 in addition , research with mouse models has proposed that disturbed sleep and ad pathology could affect each other by affecting the accumulation of a , which is widely accepted as a major contributor to altered sleep patterns and ad pathogenesis.5 additional lines of evidence indicate that decline in brain stem function holds important implications in the potential connection between sleep and ad . the brain stem contains networks of aminergic and cholinergic nuclei that are critical for sleep regulation . brain stem aminergic and cholinergic neurons , such as the dorsal raphe ( serotonin ) , locus coeruleus ( noradrenaline ) , ventral tegmental area ( dopamine ) , and laterodorsal tegmentum ( acetylcholine ) in particular , have been strongly implicated in sleep quality.1 specific disruption or transection of these neurons is directly related to fragmented sleep with rapid eye - movement sleep deficits and even a near - constant wakefulness state.19 slow - wave sleep , during which memory consolidation occurs , is also governed by the brain stem structures , is commonly fragmented in ad patients , and disruption of this process could potentially be a contributing factor to the cognitive decline in ad.20 the results of our study identified significant structural changes in the brain stem that correlated with the severity of sleep disturbances in ad patients . these results are consistent with those from previous studies that identified neuropathological changes and morphological alterations in the brain stems of ad patients.6,7 although preliminary , our findings lend support to the involvement of the brain stem in both sleep and ad . first , the sample size was small , and control subjects with sleep disturbances were not included in the study . therefore , our study design was insufficient to arrive at robust conclusions about the relationship between ad and sleep . second , our analyses relied on the underlying assumption that morphological changes in the brain are measures of neuronal loss caused by intracellular neurofibrillary tangles of hyperphosphorylated tau and extracellular a plaques , which are hallmark biochemical symptoms of ad.6 therefore , a direct link between such biochemical alterations and sleep disturbances may require additional in vivo analyses utilizing amyloid or tau positron - emission tomography.21,22 finally , the different kinds of sleep - related bpsd were not distinguished in the npi - sleep subscale . in future studies , objective and specific measurements of sleep disturbances , such as polysomnography , might be helpful to differentiate specific types of sleep disturbances and their associations with brain stem morphology in ad . in summary , this study identified morphological alterations in the brain stems of ad patients that significantly correlated with disturbances in their sleep . sleep complications commonly occur in ad patients , starting in preclinical stages , and the connection between ad and sleep disorders has become a praised topic in the field.4 although further research and data are needed , our results suggest that brain stem alterations might be at the core of underlying neurobiological mechanisms of sleep disturbances associated with ad .

the world health organization attributes hypertension , or high blood pressure , as the leading cause of cardiovascular mortality . an elevated arterial pressure is probably the most important public health problem in developed countries . it is common , asymptomatic readily detectable and easily treatable , and if untreated , often leads to complications . although our understanding of the pathophysiology of elevated arterial pressure has increased , in 90 to 95% of cases the etiology is still largely unknown . as a consequence , in most cases hypertension is treated non - specifically , resulting in the large number of minor side effects and relatively high non - compliance rate . the ratio of hypertension frequency in women versus men increases from 0.6 to 0.7 at age 30 to 1.1 to 1.2 at age 65 . it is a professional responsibility of a dental clinician to inform the patient of their hypertensive state and to offer medical advice , including epropriate referrals . there are no recognized oral manifestations of hypertension but antihypertensive drugs can often cause side - effects , such as xerostomia , gingival overgrowth , salivary gland swelling or pain , lichenoid drug reactions , erythema multiforme , taste sense alteration , and parasthesia . studies have indicated that , afflictions such as heart disease , diabetes , stroke , hypertension , multiple sclerosis , and hiv / aids often can be discovered during a routine visit to the dentist . the mouth is an ideal breeding area for bacteria and those affected by periodontal disease are at increased risk , for potentially fatal bacteria entering the bloodstream via infected oral tissue . those suffering from periodontitis are highly susceptible to major health issues such as premature births and low birth - weight babies , cancer , anorexia , vascular and heart disease including that caused by the introduction of germs that attack the heart 's mitral valve . periodontal disease in an expectant mother has been identified as one of the signs that an infant will be born prematurely or at a low birth weight . recent studies have shown that the inflammatory effects of periodontal disease help to promote blood clot formation in arteries . this clinical study was aimed an investigation the effect or oral manifestations in hypertensive patients . 0the study sample consisted of 465 known hypertensive patients , between the age group of 20 - 80 years of which 250 patients were males and 215 were females . about 105 were between the age group of 1 - 40 years , 136 in 41 - 50 years , 110 in 51 - 60 years , and 114 in 60 or above of age , all patients were from different socio - economic status . patients were examined clinically using a mouth mirror , probe , williams 's periodontal probe to check the periodontal pockets , russell 's periodontal index was used to estimate the gingival and periodontal health and further histopathological examination for mucosal changes was also carried out [ figure 1 ] . all the patients included in the study were known hypertensive patients and who were under the medication of the antihypertensive drugs . gingival enlargements in hypertensive patients oral changes mainly observed were gingivitis , periodontitis , lichenoid reactions , and hypo salivation and facial nerve paralysis . gingivitis and periodontitis were confirmed from patients russell 's periodontal index , and hypo salivation was noted by asking questions to the patients regarding symptoms [ figures 2 and 3 ] . from the data recorded it was evident that majority of the hypertensive patients examined during the course of study belonged to the age group of 41 - 50 years . around 79.14% of the patients under study presented with russell 's periodontal index score ranging from 2- 4.9 . 85.38% of patients presented with gingival bleeding on probing and were characterized by redness of the marginal gingiva . 4.5% of the total patients presented with lichenoid reactions and they were characterized by linear striations occurring on the buccal mucosa . 1.2% of patients presented with facial nerve paralysis and 16.9% patients presented with gingival enlargement characterized by firm nodular gingival overgrowth seen on both buccal and facial and lingual or palatal aspects of the marginal gingival [ figure 4 ] . oral manifestations observed in the clinical observed patients the risk factors for old men and women to develop hypertension were 90% , among those who survived to ages 65 to 89 years . systolic bp elevations were found in 87% of hypertensive men and 93% of hypertensive women , as they were examined for their hypertension during this study . hypertension ( htn ) or high blood pressure , sometimes arterial hypertension , is a chronic medical condition in which the blood pressure in the arteries is elevated . this requires the heart to work harder than normal to circulate blood through the blood vessels . patients showed the russell 's periodontal index score of 0.3 - 0.9 . in a study by mailboridin et al wherein the russell 's index was show to be 2.0 - 4.9 with 79.14% of the patients presented with the symptoms of periodontitis , was similar to the results of the this present study . mailboridin et al studied the micro lympho hemocirculatory bed and leucocytogram of gingival tissue by the light microscopy in patients with chronic periodontitis having normal and high arterial blood pressure . in most cases of arterial hypertension the gingival mucous was characterized by widening of lymphatic vessels and interstitial spaces . in cases of arterial hypertension combination with inflammatory reaction the tendency for widening of lymphatic vessels and interstitial spaces persisted compared with cases of normal blood pressure . it testifies to high probability of lymphogenic generalization of inflammation . besides , in cases of inflammatory gingival pathology in arterial hypertension the absolute neutrophil number was significantly higher showing for more acute inflammatory process and greater volume of tissue involvement . thus , concluding that the increased periodontitis in hypertensive patients could probably attributed as one of the manifestation of hypertension . this hypo salivation was related to the sustained increase in both systolic as well as diastolic blood pressure and also in patients who were under antihypertensive medication especially with diuretics . the results of this study , 16.99% ( 79 ) patients who were under medication with diuretics presented hypo salivation . these results were similar to the studies of tenovuo et al and glick et al . lichen planus like lesions or lichenoid reactions are white lesions characterized by linear striations occurring on the buccal mucosa . these are sometimes seen in hypertensive patients as a manifestation secondary to the use of the drug or medication . the most common drugs causing this side effect are the ace inhibitor drugs especially the captopril . they are characterized by acanthosis , basal cell degeneration , hyperparakeratosis , numerous chronic inflammatory cell infiltrate throughout the connective tissue especially the plasma cells and histiocytes . the results were similar to the studies of mandys et al , but this study results did not correlate with the studies of christensen et al as their investigation did not support the hypothesis of relationship between lichen planus and hypertension . facial nerve paralysis in hypertension is because of edema or hemorrhage in the facial canal , but the exact etiology is unknown . usually facial nerve paralysis is seen in patients with malignant hypertension . it is characterized by the sustained increase in systolic blood pressure 200 mm hg and/or sustained increase in diastolic blood pressure 120 mm hg . but it is more significant in patients with increased diastolic blood pressure rather than increase in systolic blood pressure . facial nerve paralysis was assessed clinically by paralysis of facial musculature , drooling of saliva from the corner of the mouth , loss of wrinkles on the forehead with expressionless appearance , inability to blow , inability to close the eyes and continuous rolling of tears . the results of this present study were similar to the study results of margabanthu et al , ellis et al . gingival enlargement is also one of the most common clinical finding in patients with ypertension taking anti - hypertensive medication especially calcium channel blockers . gingival enlargements appear clinically as firm nodules of gingival overgrowth seen on either buccal or facial aspects and lingual or palatal aspects of the marginal gingiva . the results of this study showed that 79 patients with gingival enlargement who were under either amlodipine or nifedipine therapy . these results were not significant to the studies of lawrence et al , and lafzi et al and barclay et al . but the finding failed to assess whether the periodontitis is due to the hypertension per se or not . there was some group of people showing increased incidence of dental caries , this may be correlated with the hypo salivation in patients who were under anti - hypertensive therapy . this study concludes the need to do further studies on the relation between hypertension and periodontitis , the relation between hypertension and facial nerve paralysis . an affirmative response to at least one of the five following questions about symptoms was shown to correlate with a decrease in saliva : does your mouth usually feel dry?does your mouth feel dry when eating a meal?do you have difficulty swallowing dry foods?do you sip liquids to aid in swallowing dry foods?is the amount of saliva in your mouth too little most of the time , or do nt you notice it ? does your mouth usually feel dry ? does your mouth feel dry when eating a meal ? do you sip liquids to aid in swallowing dry foods ? is the amount of saliva in your mouth too little most of the time , or do nt you notice it ? xerostomia , also known as dry mouth , is marked by a significant reduction in the secretion of saliva erythema multiforme is a skin condition of unknown cause , possibly mediated by deposition of immune complex ( mostly igm ) in the superficial microvasculature of the skin and oral mucous membrane that usually follows an infection or drug exposure . it is a common disorder , with peak incidence in the second and third decades of life . aids a severe immunological disorder caused by the retrovirus hiv , resulting in a defect in cell - mediated immune response that is manifested by increased susceptibility to opportunistic infections and to certain rare cancers , especially kaposi 's sarcoma . it is transmitted primarily by exposure to contaminated body fluids , especially blood and semen . multiple sclerosis a chronic autoimmune disease of the central nervous system in which gradual destruction of myelin occursin patches throughout the brain or spinal cord or both , interfering with the nerve pathways and causing muscular weakness , loss of coordination , and speech and visual disturbances . hypo salivation abnormally reduced salivation facial nerve paralysis is a common problem that involves the paralysis of any structures innervated by the facial nerve . the pathway of the facial nerve is long and relatively convoluted , and so there are a number of causes that may result in facial nerve paralysis . the most common is bell 's palsy , an idiopathic disease that may only be diagnosed by exclusion . lichen planus a dermatologic disease of unknown etiology that also occurs in the mouth , on the tongue , or on the lips as smooth lacy networks of white lines or , less commonly , as white patches that may become ulcerative . acanthosis any thickening of the prickle - cell layer of the epidermis ; associated with many skin diseases . xerostomia , also known as dry mouth , is marked by a significant reduction in the secretion of saliva erythema multiforme is a skin condition of unknown cause , possibly mediated by deposition of immune complex ( mostly igm ) in the superficial microvasculature of the skin and oral mucous membrane that usually follows an infection or drug exposure . it is a common disorder , with peak incidence in the second and third decades of life . aids a severe immunological disorder caused by the retrovirus hiv , resulting in a defect in cell - mediated immune response that is manifested by increased susceptibility to opportunistic infections and to certain rare cancers , especially kaposi 's sarcoma . it is transmitted primarily by exposure to contaminated body fluids , especially blood and semen . multiple sclerosis a chronic autoimmune disease of the central nervous system in which gradual destruction of myelin occursin patches throughout the brain or spinal cord or both , interfering with the nerve pathways and causing muscular weakness , loss of coordination , and speech and visual disturbances . hypo salivation abnormally reduced salivation facial nerve paralysis is a common problem that involves the paralysis of any structures innervated by the facial nerve . the pathway of the facial nerve is long and relatively convoluted , and so there are a number of causes that may result in facial nerve paralysis . the most common is bell 's palsy , an idiopathic disease that may only be diagnosed by exclusion . lichen planus a dermatologic disease of unknown etiology that also occurs in the mouth , on the tongue , or on the lips as smooth lacy networks of white lines or , less commonly , as white patches that may become ulcerative . acanthosis any thickening of the prickle - cell layer of the epidermis ; associated with many skin diseases .
traumatic brain injury ( tbi ) is a leading cause of morbidity and mortality in children , resulting in numerous physical , behavioral and cognitive deficits.1 ) cognitive deficits may involve problems with attention , memory and executive functions that worsen with increasing tbi severity.24 ) attention deficit hyperactivity disorder ( adhd ) that develops after injury without evidence of preinjury adhd is often referred to as secondary adhd.5 ) methylphenidate ( mph ) , a well - accepted treatment for adhd , may be associated with behavioral adverse effects.6 ) amongst these reactions , psychotic symptoms are the most terrifiying ones for families . although firstly reported in the early 1970s , the terminology of psychotic symptoms due to mph use has not been well described . the first report of stimulant - induced psychosis and/or mania symptoms in children included three cases and used the definition of methylphenidate hallucinosis.7 ) in these cases , hallucinations were the prominent presenting symptoms . in later reports , the term toxicosis has been used to distinguish transient psychotic symptoms associated with stimulant use.8 ) toxicosis term specifically indicates that the psychotic - like or mania - like symptoms resolve following discontinuation of stimulant . when the adverse reaction symptoms continue or recur after discontinuation , a rediagnosis of bipolar disorder or schizophrenia may be considered.8 ) the efficacy and tolerability of stimulants in children with a history of tbi have not been completely identified . in this case report , the emergence of manic symptoms with mph use in an adolescent with tbi will be summarized . a male patient aged 17 years was admitted to the child and adolescent psychiatry clinic with the complaints of attention difficulties on schoolwork and forgetfullness . patient s medical history revealed that he had a car accident 3 months ago which included a tbi . upon admission to emergency room , his neurologic assessment was marked with upper and lower muscular weakness , memory deficits and frank mutism . this clinic picture resolved in 2 months ; however , he has been speaking slowly since then . before the accident , he was reported to have a succesfull academic life in both elementary and secondary school . there was no known history of psychiatric disorders in both parents and extended family members . after the accident , he had difficulty on sustaining attention on school work and forgetfulness on daily duties . i study hard and try to do my best but i am far from my previous academic performance . i ca nt focus , ca nt sustain my attention and i do simple mistakes . deficits in cerebellar tests , slow speech and difficulty at walking on hills were evident in his neuorologic examination . his magnetic resonance imaging ( mri ) , which was performed 10 days after the injury , revealed postinjury findings on the right frontal lob , lef temporal lobe and corpus callosum ( fig . sleep and awake electroencephalography ( eeg ) of the patient was also obtained on the 7th day after injury . a diagnosis of major neurocognitive disorder due to tbi was made and osmotic release oral system ( oros ) mph was administered with the dose of 18 mg / day for attention problems . after one week , patient was admitted to the clinic with the complaints of talking to himself , delusional thoughts , irritability and sleeplessness . his psychiatric interview revealed elevated mood , incongruent affect and delusional thoughts about his mission to save the world . the suspect of a medication induced adverse reaction , mph was discontinued and all of the symptoms resolved in 3 days . almost one fifth of children who sustain a tbi are under the risk of attention problems after injury.9 ) children with severe tbi are shown to have a greater risk for developing postinjury attention problems.10 ) the consequences of attention problems may extend beyond school functioning and can have negative impacts on a child s social relationships , emotional well - being , self esteem and quality of life.11 ) despite the high burden of attention problems in children and adolescents after tbi , only a limited number of controlled studies , all with small sample size and short duration , have been conducted to date . in an early study by williams et al.,12 ) no signifficant effect of mph was reported in 10 children with mild - to - severe head trauma . mahalick et al.13 ) studied 14 children with tbis of varying severity and mph treatment resulted in significantly greater scores on the study measures of attention when compared with placebo . a chart review on 10 children with tbi has also shown positive findings on attention and behavior.14 ) a recent placebo controlled study , in which only five children with tbi have completed the study procedures , reported a small but significant effect on attention and hyperactivity with psychostimulants.1 ) of note , none of the mentioned studies focused on the possible adverse effects of mph and treatment associated mania or psychosis was not reported . the risk of stimulant - induced psychosis and/or mania symptoms in children as estimated as 1 in 400.8 ) in a significant proportion of cases , toxicosis symptoms develop at the theraupatic doses of stimulants.15 ) presenting symptoms usually emerge shorty after the start of the treatment or with a dose increase . in the great majority of such reports , behavioral symptoms resolve within a week after discontinuation.16 ) in accordance with the literature , manic symptoms started within the first week of mph treatment , and rapidly subsided with discontinuation in our case . it has been shown that stimulant induced toxicosis symptoms are highly similar to those of bipolar disorder or schizophrenia.8 ) symptoms may include euphoria , grandiosity , paranoid delusions , confusion and auditory hallucinations.8,16 ) bizarre behaviors , hallucinations involving visual and/or tactile sensations of insects , snakes , or worms were also reported , especially in younger children.17 ) our case also presented with grandiose delusions with religious content and socialy disinhibited behaviors which are commonly reported in manic episodes . in the differential diagnosis , bipolar disorder manic episode might be a possibility . however , the short duration of symptoms and the direct relationship with mph use do not fit the diagnostic and statistical manual of mental disorders 5th edition ( dsm - v ) criteria.18 ) moroever , mph - induced mania symptoms must be considered as a different clinical entity than the highly hereditery bipolar disorder . acute onset of clinical picture , absence of mood symptoms in the prior psychiatric history and the lack of the family history in the present case are against a bipolar disorder diagnosis.17 ) in light of the available research and the suggested terminology , toxicosis term appears to be the best description of the case . although not completely identified , the emergence of psychotic symptoms may be related with the mechanism of action of mph . mph mainly works via the reuptake inhibition of dopamine , and less prominently noradrenaline , in the striatal regions . effects on selected frontal and temporal regions were also observed with mph use.19 ) increased dopaminergic and/or noraderengic transmission with mph , especially in higher doses , may be associated with psychotic / manic symptoms.8 ) although stimulant - induced toxicosis appears to be an idiosyncratic reaction , some risk factors have been proposed . the use of high doses , premorbid adhd symptoms and mental retardation were suggested to be related with an increased risk.20 ) in the present case , the disrupted brain regions , as shown by the mri findings , might give rise to a vulnerability for this behavioral adverse reaction . right frontal , left temporal regions and corpus callosum , which were injuried in our case , are among the shown regions for the neurobiology of bipolar disorder in children and adolescents.21,22 ) deficits in executive functions , visual - motor responses and working memory were reported in children with traumatic lesions on corpus callosum.23 ) the previous literature on mania symptoms after tbi is limited . some studies have shown that multifocal lesions , mainly in temporal poles , are associated with mania.24 ) both left and right temporal lesions were found to be linked with mania symptoms.25,26 ) a previous review on 66 adult tbi patients have shown the incidence of mania as 9% within 12 months of follow - up.27 ) there is evidence that severe tbi and male gender are associated with an increased risk of mania.28,29 ) the relationship between age and mania has not been specifically documented , however , younger age has been found as a risk factor for psychiatric disorders after tbi.30,31 ) in our case , diffuse disruptions in fronto - temporal grey matter might have resulted with an increased vulnerability to manic symptoms . since these vulnerable regions are also among the shown target regions of mph , an additive effect of mph might have occured . in other words , mph use might have led to either irreguler or overt activity in the brain regions which were already disrupted with the head trauma . eventually , the additive effects of head trauma and mph use might have resulted in the emergence of manic symptoms in our case , which did not have any premorbid psychiatric history . this case highlights the fact that therapeutic dose of mph may cause mania - like symptoms in children with tbi . close monitarization and slow dose titration are crucial when considering mph in children with tbi .
pantothenate - kinase - associated neurodegeneration ( pkan ) is an autosomal recessive neurodegenerative disorder that is characterized by progressive extrapyramidal signs , visual loss , and cognitive impairment . pkan is caused by mutations in the pantothenate kinase gene ( pank2 ) , which is located on chromosome 20p13 and encodes pantothenate kinase , the key regulatory enzyme in coenzyme - a biosynthesis . in this report we describe a case of atypical pkan with a novel pank2 mutation , presenting with a 10-year history of postural tremor involving both hands . upon neurological examination , brain mri revealed a typical " eye - of - the - tiger " sign . a mutation analysis revealed three pank2 mutations : two in exon 3 ( asp 378gly and leu385cysfsx13 ) and one in exon 4 ( arg440pro ) . parkinsonism is not an unusual presenting symptom in patients with atypical pkan , and so it is important for physicians to consider pkan in the differential diagnosis of patients presenting with young - onset parkinsonism . pantothenate - kinase - associated neurodegeneration ( pkan ) , formerly called hallervorden - spatz syndrome , is an autosomal recessive neurodegenerative disorder characterized by progressive extrapyramidal signs , visual loss , and cognitive impairment.1 pkan is caused by mutations in pank2 , which is a gene located on chromosome 20p13 and encodes pantothenate kinase , the key regulatory enzyme in coenzyme - a biosynthesis.2 pkan belongs to a group of neurodegenerative diseases that includes neurodegeneration with brain iron accumulation ( nbia ) , infantile neuroaxonal dystrophy , aceruloplasminemia , and neuroferritinopathy . iron accumulation in the globus pallidus can be frequently detected by mri in a characteristic pattern referred to as the " eye - of - the - tiger " sign.3 two main phenotypes of pkan have been reported : classical and atypical.1 patients with classical pkan typically present in the first decade of life with severe extrapyramidal signs , progressing relentlessly and leading to independent walking disability within 10 - 15 years after onset . however , atypical pkan cases show later onset , less severe extrapyramidal signs , and slower progression . here we describe a patient with atypical pkan presenting with adult - onset , slowly progressive parkinsonism due to a novel pank2 mutation . a 45-year - old man was admitted to our hospital because of a 10-year history of tremor involving both hands . there was no history of perinatal complications and he met normal developmental milestones . from the age of 35 years , the patient began experiencing bilateral postural hand tremor . symptoms worsened gradually over several years . at the age of 40 years he was unable to continue working as a hairdresser due to the hand tremors . he had three brothers and two sisters ; the oldest brother also had mild hand tremors that did not prevent him performing the activities of daily living independently . upon neurological examination , pursuit eye movements were unremarkable . outstretching his arm elicited a postural hand tremor with a moderate amplitude and a frequency of 5 - 6 hz . tongue tremor , which seemed to be grossly synchronous with hand tremor , was also observed . on walking , his gait velocity was slow with decreased arm swing on his right side . complete blood count , biochemical screening , erythrocyte sedimentation rate , c - reactive protein , and urine were normal . the serologic test for human immunodeficiency virus and assays for anti - nuclear antibodies were negative . brain mri revealed bilateral and symmetric hypointensity with medial hyperintensity in the globus pallidus on fluid attenuation inversion recovery imaging . this feature was in accordance with the " eye - of - the - tiger " sign ( fig . 2 ) , in which pcr sequencing was utilized to identify pank2 mutations , revealed three mutations : two in exon 3 ( asp378gly and leu385cysfsx13 ) and one in exon 4 ( arg440pro ) of the gene . an a - to - g change at nucleotide 1133 results in the substitution of aspartate for glycine at amino acid 378 ( d378 g ) , an insertion of threonine at nucleotides 1154 - 1155 results in a frameshift at amino acid 385 ( l385fs ) , and a g - to - c change at nucleotide 1319 results in the substitution of arginine for proline ( r440p ) . there was no overt response of the patient 's parkinsonism to the introduction of l - dopa . however , his hand tremor improved partially after treatment with pramipexole dihydrochloride ( 0.5 mg three times daily ) and clonazepam ( 0.25 mg twice daily ) . phenotypically , the present case is compatible with atypical pkan in terms of adult onset , its slowly progressive nature , and its parkinsonian presentation . several cases of nbia that presented with adult - onset parkinsonism have been reported in the literature , and in their series of patients with nbia , thomas et al.4 reported that four out of ten patients with a pank2 mutation initially presented with parkinsonism . regarding the phenomenology of pkan - associated movement disorders , patients with an early onset predominantly presented with dystonia , whereas adult - onset patients exhibited parkinsonism as the initial symptom . although parkinsonism has frequently been observed in early - onset pkan patients , other neurologic abnormalities of dystonia , pyramidal signs , and cognitive impairment were generally combined.5,6 accordingly , pure parkinsonism is not an unusual presenting symptom of atypical pkan.4 the genetic analysis revealed two pank2 mutations in exon 3 ( asp378gly and leu385cysfsx13 ) and one in exon 4 ( arg440pro ) . of those , d378 g is an already established mutation found in pkan.7 however , l385fs and r440p are novel mutations that have not been reported previously . insertion of threonine at nucleotides 1154 - 1155 in exon 3 results in a frameshift mutation with a premature termination of translation at amino acid 385 . a novel substitution at nucleotide 1319 in exon 4 replaces arginine in position 440 with proline . arginine provides a positive hydrophilic charge with basic side chains , while proline is a cyclic amino acid that has a neutral hydrophobic charge . therefore , it may be speculated that the arginine - to - proline substitution results in loss of the functional properties of the pank2 protein . regarding the association between pank2 mutation and clinical phenotype , two loss - of - function alleles and residual enzymatic activity of pank2 are known to significantly determine the age of onset of the disease , but have a much weaker influence on the rate of disease progression.6 similarly , thomas et al.4 suggested that patients with atypical pkan were more likely to have mutations resulting in amino- acid changes . although we were unable to determine the enzymatic activity of pank2 in our case , this compound heterozygous mutation may have been responsible for the adult - onset and delayed progressive nature of the disease . mutations on both alleles of pank2 are identified in about 50% of patients with clinical features of pkan , and in more than 98% of patients with radiographic features of the disease.7 as mentioned previously , parkinsonism is not an unusual presenting symptom in patients with atypical pkan , and it is important for physicians to consider pkan in the differential diagnosis of patients presenting with early - onset parkinsonism .
the main applications include ligand - based in silico ( virtual ) drug screening , adme - tox ( adsorption , distribution , metabolism , excretion , and toxicity ) property prediction , physical molecular property prediction ( 1-octanol - water partition coefficient , solubility ) , and measurement of the diversity of chemical compounds in a library . the molecular similarity measure generally assigns a one - dimensional ( 1d ) and/or two - dimensional ( 2d ) descriptor that is , molecular fingerprints based on substructure , molecular mass , number of rotatable bonds , number of hydrogen donors / acceptors of the compound , and so forth to compounds so that the similarities of the compounds can be evaluated [ 15 ] . many methods have been proposed for the similarity search of chemical compounds , such as the comparison of overlapping substructures in the form of daylight fingerprints ( daylight chemical information systems inc . , aliso viejo , ca , usa ) , the chemically advanced template search ( cats ) descriptor method developed by pickett , and the burden - cas - university of texas ( bcut ) descriptor method . one of the most widely used methods is to compare the existence of fragment structures ; this is the technique employed by the macss key , which was developed by molecular design limited ( mdl , santa clara , ca , usa ) . each element of the feature vector of the molecule represents the existence of a particular fragment structure in the molecule ( dictionary based fingerprinting ) . a rather large example of a dictionary used for this fingerprinting technique is the software program dragon developed by talete srl ( geographical information ) , which consists of more than 3200 molecular descriptors . the affinity fingerprint approach is a new type of similarity search method based on a multiprotein / multicompound affinity matrix [ 821 ] . in this method , each element of the feature vector of the molecule represents the binding affinity of the molecule with a particular protein . usually , the binding affinity is measured by calculation using a protein - compound docking program . there are various applications for molecular similarity , and thus many types of similarity measures are needed . most of the conventional molecular descriptors aim scaffold hopping ( lead hopping ) to find a compound with a different scaffold from the known active compound . however , in some cases , we want to find similar compounds with similar scaffolds . for example , in lead generation , we want to find a series of similar compounds with the same or similar scaffold instead of performing an actual synthesis . , a comparison of the indices of molecular topologies is much faster than a substructure search . a topological index , which is any of several numerical parameters of a molecular graph , is also widely used . the wiener index , hosoya index , and randic 's molecular connectivity index , are graph invariants and conventional topological indices . these topological indices show correlation to the physical or chemical properties of molecules , although these indices do not recognize atom types and they can be quite difficult to calculate . in the current study , we proposed a new similarity measure for identifying topologically similar compounds based on their molecular topologies and evaluated this method by applying it to a ligand - based drug screening test . first , the all - atom model compound structures are converted to united atom models , in which all hydrogen atoms are omitted and the atomic charge of the hydrogen atom is added to the atomic charge of the connected heavy atom . in this method , the adjacency matrix e and the distance matrix d are used , and figure 1 shows an example of these two matrices for a simple graph . the topology of the compound can be represented by an edge - adjacency matrix e [ 4 , 5 ] : ( 1)eab=1 , when the ath and the bth atoms are connectedeab=0 , otherwise , where eab is the a - b element of matrix e. the value of eab could be the bond order between the ath and the bth atoms ( the value of eab could be 1.5 for an aromatic bond ) . just as in the bcut method , the diagonal part ( eaa ) is replaced by the converted atomic charge qc : ( 2)qc=11+exp ( cqa ) , where qa is an atomic partial charge and c is a coefficient . in this study , c was set to 1.0 . the qc value is > 0 for any qa value . the a - b matrix element of the pseudodistance matrix d represents the minimum path length between the ath and bth atoms : ( 3)dab = ln ( 1+dab ) , where dab is a shortest path length between the ath and the bth atoms . we also tried dab = dab ( shortest - path topological distance matrix [ 4 , 5 ] ) and dab = dab and found that dab = ln ( 1 + dab ) gave the best result among these definitions . let i and n be the ith eigenvalue of the matrix e or d and the number of atoms of the united - atom model of the compound . we define the eigenvalue histogram g( ) as follows : ( 4)g()=i=1nexp ( c(i)2 ) . here the distance s(a , b ) between molecules a and b is defined based on the eigenvalue histogram of molecule a(ga( ) ) and that of molecule b(gb( ) ) as follows : ( 5)s(a , b)=\|ga()gb()\|d. in the current in silico drug screening , candidate hit compounds are selected using the following method . let s(a , b ) and s(a , b ) be the distance between a and b based on the adjacency matrix e and that based on the distance matrix d. these two distances give the consensus distance s(a , b ) with the weight parameter : ( 6)s(a , b)=se(a , b)+(1)sd(a , b ) . compounds that are close to the known active compounds are selected as the candidate hit compounds . the distance from the kth compound to the average position of the active compounds ( distk ) is defined as ( 7)distk=i=1ms(ak , ci)2/m , where ak , ci , and m are the kth compound , ith active compounds , and the total number of the active compounds . when the number of active compounds is one , distk = s. we call distk the molecular - graph ( mg ) distance and we call this screening procedure the molecular - graph distance ( mgd ) method . the eigenvalues ( i ) of s and s of each compound of the compound library are stored in a database file a priori . for a query compound , the eigenvalues of s and s must be calculated , which costs less than 1 second . the database search is conducted only to perform the calculations in ( 4)(7 ) and thus is quite fast . for the drug screening test , our target proteins were the macrophage migration inhibitory factor ( mif ) , cyclooxygenase-2 ( cox-2 ) , human immunodeficiency virus protease-1 ( hiv ) , thermolysin ( thr ) , glutathione s - transferase ( gst ) , the histamine h1 receptor , the adrenaline beta receptor , the serotonin receptor , and the dopamine d2 receptor . for validation of the present method , we used the same set of compounds as used in our previous study . namely , the compound set consisted of 12 inhibitors of mif , 28 inhibitors of thr , 15 inhibitors of cox-2 , 20 inhibitors of hiv , 12 inhibitors of gst , 10 antagonists of the histamine h1 receptor , 12 agonists and 13 antagonists of the adrenaline beta receptor , 8 agonists and 9 antagonists of the serotonin receptor , and 6 agonists and 15 antagonists of the dopamine d2 receptor as the active compounds , along with 11050 potentially negative compounds from the random compound library of the coelacanth chemical corporation ( east windsor , nj , usa ) . typically , only one hit compound could be found out of 10 randomly selected compounds ; we therefore expected that there would be no more than a few , if any , hit compounds among these 11212 compounds . the 160 active compounds are listed in the supplemental materials available online at doi:10.1155/2009/231780 . the size distribution of compounds was as follows : percentage of compounds with 0 ~ 19 atoms , 0.1% ; with 20 ~ 29 atoms , 1.2% ; with 30 ~ 39 atoms , 1.6% ; with 40 ~ 49 atoms , 9.3% ; with 50 ~ 59 atoms , 22.5% ; with 60 ~ 69 atoms , 37.9% ; with 70 ~ 79 atoms , 20.5% ; and with more than 80 atoms , 7.0% . the atomic charge of each ligand was determined by the gasteiger method [ 28 , 29 ] . to calculate the gasteiger charge , the three - dimensional ( 3d ) coordinates of the 11050 random compounds above were generated to add the hydrogen atoms by the concord program ( tripos , st . louis , mo , usa ) from 2d sybyl sd files provided by the coelacanth chemical corporation . the 3d coordinates of the active compounds ( inhibitors , substrates , agonists , and antagonists ) were generated by chem3d ( cambridge software , cambridge , ma , usa ) . to evaluate the efficiency of this method , the leave - one - out cross - validation test was applied ; namely , the active compounds of each target protein were selected one by one as the known active compounds for this software and the other unknown active compounds were discovered by the software . the test dataset consists of these active compounds and the other approximately 10 potential inactive compounds ( decoy set ) . a total of 160 (= total 160 active compounds ) database enrichment curves were calculated for these 9 target proteins and 11212 compounds and the results were averaged . the surface area ( q or area under curve : auc ) under the total database enrichment curve ( f ) is a measure of the database enrichment : ( 8)q=0100f(x)dx , where x and f(x ) are the percentages of compounds that are selected from the total compound library and the database enrichment curve , respectively . a higher q value corresponds to better database enrichment , and the q value is always greater than zero and less than 100 . for the random screening , q = 50 . first , the dependence of the hit ratio was examined in the mgd method . the average q values and the hit ratio of the 160 trials with various values are summarized in table 1 . the coefficients c for matrices e and d were optimized for every to maximize the hit ratio . when = 0 or = 1 , the hit ratio and the q values were lower than those in the other cases . this result showed that the combination of matrices d and e is more effective than the single usage of either d or e. the optimized coefficients were used in the following study . the average eigenvalues of d and e were 3.21 10 and 0.505 for the decoy set , respectively . the histograms g( ) of ( 4 ) were close to single gaussian distributions . we show the distributions of g( ) of h1 antagonist diphenhydramine and cox-2 inhibitor indomethacin in the supplementary data . for diphenhydramine , the average g( ) values of s and s the deviations of g( ) values of s and s were 75.75 and 9.712 , respectively . for indomethacin , the average g( ) values of s and s were 1.46 10 and 1.009 10 , respectively . the deviations of g( ) values of s and s were 75.88 and 9.739 , respectively . the average values and the standard deviations for the values are summarized in table 1 . figure 2 shows a score distribution with = 0.25 using diphenhydramine as the template ( see figure 4 ) . the frequency was normalized ; the surface area under the curve was set to 1 . figure 3 shows the average database enrichment results of the 160 trial screening tests by the leave - one - out cross - validation test . the mgd method worked well and showed good enrichment . in this calculation , was set to 0.25 . within the first 1% , 5% , and 10% of the database , 36.5% , 52.8% , and 60.0% of the active compounds were found by the similarity measure defined by ( 7 ) , respectively . the average q value by the mgd method using ( 7 ) was 82.53 . this result is worse than the result by the machine - learning docking score index method reported previously . namely , about 70% of the active compounds were found within the first 1% of the database and the average q value was 98.5 . ten histamine h1 receptor antagonists were included in the compound library ( see figure 4 ) . figure 5 shows the known active compound ( template ) and the best ranked molecules , when = 0 , 0.25 and 1 . the distk values and z - scores (= ( score average score)/standard deviation ) of the three top - ranked compounds are summarized in table 1 . these z - scores show that the score distribution is slightly different from the gaussian distribution . in the gaussian distribution , the number of compounds with a z - score > 3 is 0.1% of the database ( 10 compounds in this case ) . the z - scores of the top - ranked compounds were only 2 , in this case . = 0.25 , the other h1 receptor antagonist , chlorpheniramine , was found as the third compound . the other compounds ( a1 and a2 ) are not very similar to the template . for = 0 , compound b2 , which is not an h1 antagonist , is similar to the template . figure 3 shows that 36.5% of the active compounds were found within the first 1% of the compounds of the whole library . our previous study showed that 12.4% , 43.4% , and 67.5% of the active compounds were found within the first 1% of the database by the docking score index ( dsi ) , factor - selection dsi ( fs - dsi ) , and machine - learning dsi ( ml - dsi ) methods , respectively , when 180 proteins were used to calculate the affinity fingerprint . the three - dimensional ( 3d ) shape and charge distribution of a compound govern the protein - compound binding energy . the dsi , fs - dsi , and ml - dsi methods utilize the 3d shape and charge distribution of the compound through the affinity fingerprint . on the other hand , the 2d structure of the compound does not govern the protein - compound binding energy . thus , the current similarity measure was not better than the previously developed screening methods for in silico drug screening , when it was used as a single measure to describe the molecular similarity . however , the mgd method did have an advantage in terms of its computational speed . the mgd method can search 10 000 000 compounds within 1 hour on a xeon 3 ghz computer , which is 1000 times faster than the msm - dsi method . as shown in figure 4 , some compounds were very similar to each other by human - eye inspection . the main reason for this similarity was likely that these compounds were generated from a progenitor compound by lead optimization . diphenhydramine , chlorpheniramine , homochlorcyclizine , cetirizine , and clemastine have a diphenyl - like group . in promethazine , olopatadine , mequitazine , and cyprohrptadine , the conformations of two phenyl groups are fixed . most of these antagonists are structurally similar , which should be the reason why the current similarity measure was effective for the in silico drug screening . in other words , this method is not suitable for scaffold hopping ( lead hopping ) . for scaffold if all matrix elements of the off - diagonal part are zero , the eigenvalues are equal to the values of the diagonal part . the off - diagonal part shifts the eigenvalues from the values of the diagonal part . in ( 1 ) , the bond information close to the ith atom can give major perturbation on the ith diagonal value ( atomic charge of the ith atom ) . thus the matrix e represents the short - range information of the molecular topology . on the other hand , in ( 3 ) , the i - j matrix element of the matrix d becomes large when the ith atom is far from the jth atom on the molecular topology . thus the matrix d can represent the long - range information of the molecular topology . the information of the matrix e and that of d are independent of each other . for each compound in the test database of 10 compounds , the correlation coefficient of the values of s and s was only 0.08 , indicating that there was no correlation between these values . thus , the compounds in the compound library are widely distributed in the ( s , s ) two - dimensional ( 2d ) space , and the mgd method selects the compounds around the query compound from the compound library in the 2d space . we developed a similarity measure for chemical compounds that is based on the molecular topology , the atomic charge , and the minimum path length between atoms . the difference and overlap between these two histograms define the distance between the two compounds . this similarity measure was applied to ligand - based in silico drug screening . in this calculation , compounds whose molecular topology structures are similar to the given active compounds were selected by using this similarity measure .
hybrid organic inorganic halides have been of interest since the start of the 20th century ; however , the first report of a perovskite - structured hybrid halide appears to have been by d. weber in 1978 . in the same journal volume , he reported both ch3nh3pbx3 ( x = cl , br , i ) and the ch3nh3snbr1xix solid solution . in the subsequent decades , these materials were studied in the context of their solid - state chemistry and physics , with the first solar cell reported in 2009 . the resulting explosion of research effort and success in the photovoltaic applications of these materials has been the subject of many review papers and commentaries . hundreds of materials have been tried and tested for use as light absorbing layers in solar cells , so one question has been frequently posed : what makes hybrid halide perovskites special ? the question is difficult to answer with certainty as our understanding of the physical properties of these materials , including how the solar cells operate , continues to evolve . one of the unique features of this class of material is their large dielectric constants ( 0 > 20 ) , compared to conventional semiconductors ( 0 < 20 ) , which include a rotational component associated with molecular dipole relaxation . the aim of this feature article is to step back and recount the fundamental physical chemistry underpinning the performance and potential limitations of hybrid perovskite materials . the work discussed here is primarily from our research group ; however , many others have contributed to the computational studies in the area . simulations on the electronic structure , alloy formation , and lattice defects have been the subject of recent review papers . we previously produced a gentle introduction to the fundamental chemistry of hybrid perovskites , which is not duplicated here . instead , we first discuss the principles of chemical bonding in these systems , followed by approaches to tune the electronic structure , and finally outline ten outstanding challenges in the field . schematic of the perovskite crystal structure with respect to the a , b , and x lattice sites . the redox chemistry of the component ions can be used to influence the valence and conduction band energies and orbital composition , and hence the stability of electrons and holes in the material . note that for larger molecular a sites layered perovskites are formed . beyond halide perovskites , a wider range of stoichiometries and superstructures are known , e.g. , the ruddlesden popper , aurivillius , and dion jacobson phases . the chemical bonding in hybrid perovskites with abx3 stoichiometry ( shown in figure 1 ) can be separated into three distinct components . it should be noted that these materials are organic inorganic but not organometallic following the iupac definition as there is no direct bond between a metal and carbon atom . in the context of metal organic frameworks , they are considered to be io materials due to the combination of a three - dimensional inorganic network with a zero - dimensional ( molecular ) organic component . the bonding within the bx3 anionic framework is unambiguously heteropolar ( mixed ionic / covalent interactions ) . the formal oxidation states of pb(+2 ) and i(1 ) , resulting from the chemical composition , are a good approximation of the chemical species here . , the quantification of partial charges remains ill - defined due to the collective nature of the periodic electronic wave function . the born effective charges in halide perovskites are large ( the value for pb can exceed 4 ) , consistent with high ionicity . the lattice energy ( defined with respect to the ions held infinitely far apart ) and electrostatic site potentials are listed for a range of perovskite stoichiometries in table 1 . in comparison to the three types of oxide ( group vi anion ) perovskite , for the halide ( group vii anion ) perovskite the electrostatic stabilization is notably reduced . the lattice energy is just 29.71 ev per abx3 cell , with an electrostatic potential on the anion site ca . due to this weaker potential alone , lower ionization potentials ( workfunctions ) are expected for halide perovskites compared to , for example , metal oxides . a second consequence is that lattice vacancies are facile to form and do not result in deep ionization levels . in contrast , for rocksalt - structured metal halides , the halide is in an octahedral coordination environment with a large confining electrostatic potential . in such cases , a halide vacancy will trap electrons with ionization levels deep in the band gap : an f - center . for ch3nh3pbi3 , the formal electronic configurations of pb 6s6p and i 5p are apparent from the electronic band structure , where the upper valence band is formed from the i p orbitals and the lower conduction band is formed from the unoccupied pb p orbitals . there is an admixture of pb s in the valence band , but here the cationic lone pair electrons are stereochemically inactive , at least in the equilibrium structural configuration . polar instabilities of the pb(ii ) ion are common in ferroelectric and multiferroic oxide perovskites . strong hybridization ( orbital overlap ) along the octahedral framework results in light electron ( 0.15 me ) and hole ( 0.12 me ) effective masses a fraction of the free electron mass . these light carrier masses provide the means for high - mobility band transport in high - quality materials . the high atomic numbers of lead and iodine suggest that relativistic effects are important for an accurate determination of the electronic structure . many - body electron electron interactions have been shown to be important . these factors combine to make high - quality electronic structure studies , such as relativistic gw theory , computationally and methodologically challenging . while density functional theory calculations can now be performed routinely on system sizes of up to 100 s of atoms , for relativistic quasi - particle self - consistent gw theory ch3nh3pbi3 represents the most complex system studied to date . note that this approach is superior to non - self - consistent g0w0 methods but still neglects electron the potentials are aligned to a common vacuum level at 0 v. the hybrid halide perovskites are of type i ii vii3 . reprinted with permission from ref ( 17 ) . ( in contrast to several erroneous statements in the published literature , ch3nh3 is not a free radical . ) the large permanent electric dipole ( 2.29 d with respect to the center of charge of the ion ) results in an estimated electrostatic point dipole dipole interaction energy of 25 mev . for two static dipoles , the interaction tails off as ( 1/r ) ; however , the screening effect of two freely rotating dipoles shortens this keesom force ( one component of the van der waals interaction ) to ( 1/r ) . as the dipole dipole interaction energy is comparable to available thermal energy at room temperature , we expect a complex ferroelectric behavior . monte carlo simulations have shown that for a fixed lattice a striped antiferroelectric alignment of dipoles is favored at low temperatures , which become increasingly disordered and finally paraelectric at high temperatures . at room temperature , there is significant local structure which can be linked with regions of high and low electrostatic potential . even for a single - crystal film the topology of the electrostatic potential resembles a bulk heterojunction more familiar to organic photovoltaics . larger polar domain structures have recently been observed from piezoelectric force microscopy , which may be associated with effects from local chemical and lattice strain . the orientation of molecular dipoles is involved in the unusual dielectric response of ch3nh3pbi3 . at high ( optical ) frequencies , there exists the standard electronic response of the system to an applied electric field . at lower ( thz ) frequencies , an additional vibrational response from lattice phonons gives rise to the static dielectric constant of 25 ( previously computed from density functional perturbation theory ) . the molecular response occurs at even lower frequencies ( ghz regime ) , which can be associated with rotational order . toward audio frequencies a colossal permittivity emerges , which can be linked to ionic and/or electronic conductivity : the maxwell wagner effect . schematic of the ordering of molecular dipoles in the presence of an external electric field , as well as the four regimes in the dielectric response from lowest frequency ( electronic excitations ) to highest frequency ( space charges and electronic or ionic conductivity ) . each process will have a characteristic relaxation time and can combine to give a complex temporal response to an external perturbation . the dominant bonding between the molecule ( a site ) and framework is electrostatic in nature . ch3nh3 is a positively charged ion inside a negatively charged cage , so there is a strong electrostatic potential ( 8 v ; table 1 ) holding the molecule at its lattice site . an additional electrostatic contribution to the chemical bonding between the molecular dipole and the pbi6 octahedra is the charge there is also the effect of primary polarization . given the appreciable polarizability of the i ions ( ca . 7 10 cm ) , an induced dipole interaction is expected ( the so - called debye force ) . due to these interactions , a correlation is expected between molecular orientation and octahedral deformation in molecular dynamic simulations ; more in - depth studies are ongoing . the molecular dipole framework interaction has also been discussed in terms of hydrogen bonds ; however , both interactions are electrostatic in nature and are difficult to distinguish between . the significant mobility of the cations ( including hydrogen atoms ) at room temperature does suggest that a dipole interaction is a more appropriate and general description . the van der waals interaction collectively describes the intermolecular ( keesom force ) and molecule it should be noted , however , that the term van der waals is sometimes used synonymously with there are now many flavors of dispersion - corrected exchange - correlation functional , which aim to recover a description of the london force ( secondary polarization ) associated with dynamic correlation . by taking a first - generation generalized - gradient functional ( e.g. , pbe ) which overestimates equilibrium bond lengths by 12% , the addition of a weakly attractive r potential will result in better agreement with experimental structures . it does not require that these interactions are london dispersion in nature . our approach has been to employ a functional optimized for solids ( e.g. , pbesol or hse06 ) , which quantitatively describes structural parameters of dense materials without system - specific parametrization . in addition to improved lattice parameters , pbesol also describes the vibrational properties of solid - state systems more accurately . from this discussion , it is clear that a variety of interactions give rise to the properties of the hybrid perovskites important to their photovoltaic performance . in particular , the combination of the light carrier effective masses provided by the metal halide framework and the strong dielectric screening including the molecule framework and intermolecular interactions favors free carrier generation over excitons ( bound electron it is possible to chemically substitute on all of the perovskite lattice sites , and appropriate examples of each can be found in the literature . it is important to recognize the chemical distinction between the three approaches . in the limit of a small perturbation , the physical response to a hydrostatic volume change can be described by the band gap deformation potential1which for ch3nh3pbi3 is positive ( vr = 2.45 ev ) . as the fundamental band gap is determined at the boundary of the brillouin zone ( r for the pseudocubic structure ) , the out - of - phase band - edge states are stabilized as the lattice expands . temperature - dependent photoluminescence indicates a decrease in band gap with decreasing temperature ( lattice contraction ) from 1.61 ev at 300 k to 1.55 ev at 150 k , which is at the onset of a phase change . the chemical effects of substitution will generally exceed this physical volume effect , as discussed below . calculated natural band offsets of ch3nh3pbi3 and related materials based on density functional calculations ( with quasi - particle corrections ) . interfacial or surface electric dipoles ( or quadrupoles ) are not considered here . adapted with permission from ref ( 17 ) . the a site of ch3nh3pbi3 does not directly contribute to the frontier electronic structure , but it can have an indirect influence by changing the crystal structure ( figure 3 ) . following eq 1 , lower band gap values should be observed for smaller molecular cations . the replacement of ch3nh3 by nh4 in the perovskite lattice reduces the band gap by 0.3 ev . the smallest possible counterion is a proton ( h ) ; hpbi3 has a theoretical cubic perovskite lattice parameter of 6.05 and an associated band gap of less than 0.3 ev . the limitation of this logic ( hydrostatic deformation ) is the relationship between the size of the ion and the local structure . for example , in reality both nh4pbi3 and hpbi3 adopt alternative chain or layer structures due to a mismatch in ionic radius . the geometric constraints for the formation of a stable perovskite lattice are summed up in the radius ratio rules , which have been recently extended to hybrid perovskites . an a - site ion too small for the bx3 framework results in an instability of the octahedral networks with respect to tilting , which can change the electronic properties ( e.g. , a transition from an antiferroelectric to ferroelectric phase ) . ch3nh3pbi3 has a goldschmidt tolerance factor of 0.91 ( unstable with respect to tilting ) . nh4pbi3 has a tolerance factor of 0.76 , and an alternative nonperovskite structure is favored . more asymmetric molecular ions ( e.g. , formamidinium , nh2chnh2 or fa ) can also result in built - in structural distortions due to their deviation from spherical symmetry . the chemical and physical strains associated with the molecular substitution can not be neglected when considering band gap engineering . the choice of a - site ions that are too large for the bx3 framework can result in layered perovskite structures ( e.g. , ruddlesden popper type an1a2bnx3n+1 phases ) . the quantum confinement associated with these layered structures has itself attracted significant interest . substitution on the b site can be used to directly alter the conduction band . isovalent substitution of pb for sn has been successfully reported ; however , sn(ii ) is less chemically stable in an octahedral environment . oxidation to sn(iv ) results in the low performance and high carrier concentrations found for the sn halide perovskites . the stability of ge(ii ) is further reduced , owing to its lower binding energy 4s electrons and is unlikely to result in a candidate photovoltaic material . the b site is substituted by two aliovalent ions ( one higher and one lower oxidation state)2an example here would be the substitution of pb(ii ) by bi(iii ) and tl(i ) , which is likely to reduce the electronic band gap due to the lower binding energy of the bi 6p orbitals and the fluctuations in electrostatic potential caused by the combination of monovalent and trivalent ions . an advantage of this approach is that controlled substitutions beyond the 1:1 stoichiometry could be used to influence the n - type ( excess bi ) or p - type ( excess tl ) carrier concentrations . for ch3nh3pbi3 , the anion ( x site ) dictates the valence band energy . the observed band gap changes upon halide substitution are influenced by the electronic states of the anion ; i.e. , from cl to br to i the valence band composition changes from 3p to 4p to 5p with a monotonic decrease in electron binding energy ( lower ionization potential ) . the valence band energy varies by as much as 0.6 ev between the methylammonium chloride and iodide perovskites . this holds true for other choices of the molecular ion : the substitution of br by i in fapbx3 decreases the optical band gap from 2.23 to 1.48 ev . the successful incorporation of the tetrafluoroborate polyanion into the perovskite structure has been recently reported . we have shown , however , that both bf4 and pf6 do not hybridize significantly with pb , which results in an increase in the band gaps and a decrease in the band widths . such substations , if stable structures were formed , could be exploited to produce a novel high - k dielectric with potential applications in transistors or memristors . in addition to their application in photovoltaics , hybrid halide perovskites display a rich physical chemistry . we have discussed the salient features of their chemical bonding and routes to tuning the properties beyond the widely studied methylammonium lead iodide . hybrid halide perovskites still pose many fundamental challenges relating to their physical chemistry and chemical physics . ten issues of current interest include:1.local structure the average crystal structure inferred from standard x - ray diffraction experiments is likely to be far from the local structure of the perovskite framework.2.dynamic disorder knowledge is required of the time scales associated with molecular motion and how this changes from single crystals to thin films and with the method of preparation.3.lattice point defects there have been reports of n - type , p - type , and intrinsic semiconducting samples of ch3nh3pbi3 . what causes this behavior , and how can the semiconductivity be controlled?4.ionic conductivity many perovskite materials support vacancy - mediated ion diffusion . is iodine , methylammonium , or hydrogen mass transport contributing to low - frequency impedance spectra?5.surface and interfaces the chemical nature of extended defects is poorly understood , in particular the interface between the perovskite and the hole transport layer.6.ferroelectricity simulations demonstrate short - range ferroelectric order at room temperature ; however , external electric fields and internal strains will change this behavior.7.grain boundaries and domain walls the perovskite microstructure may provide alternative pathways for conductivity and electron hole separation or recombination . what is their form and abundance?8.increased stability the long - term air instability of these materials is in part associated with the volatility of the molecular components . the development of alternative ions without labile protons would be advantageous.9.pb-free compositions a major goal remains to identify a ( stable ) pb - free material that maintains the same exceptional performance as ch3nh3pbi3 in solar cells . the difficulty is in maintaining a small band gap with lighter metals.10.device models there are standard electron transport models for p n junction devices and extensions to bulk heterojunctions ; however , there is no band transport model that encompasses the complex behavior of the hybrid perovskites including current voltage hysteresis . local structure the average crystal structure inferred from standard x - ray diffraction experiments is likely to be far from the local structure of the perovskite framework . dynamic disorder knowledge is required of the time scales associated with molecular motion and how this changes from single crystals to thin films and with the method of preparation . lattice point defects there have been reports of n - type , p - type , and intrinsic semiconducting samples of ch3nh3pbi3 . ionic conductivity many perovskite materials support vacancy - mediated ion diffusion . is iodine , methylammonium , or hydrogen mass transport contributing to low - frequency impedance spectra ? surface and interfaces the chemical nature of extended defects is poorly understood , in particular the interface between the perovskite and the hole transport layer . ferroelectricity simulations demonstrate short - range ferroelectric order at room temperature ; however , external electric fields and internal strains will change this behavior . grain boundaries and domain walls the perovskite microstructure may provide alternative pathways for conductivity and electron hole separation or recombination . what is their form and abundance ? increased stability the long - term air instability of these materials is in part associated with the volatility of the molecular components . pb - free compositions a major goal remains to identify a ( stable ) pb - free material that maintains the same exceptional performance as ch3nh3pbi3 in solar cells . n junction devices and extensions to bulk heterojunctions ; however , there is no band transport model that encompasses the complex behavior of the hybrid perovskites including current voltage hysteresis .
paragonimiasis is a food - borne illness of the lung caused by trematodes of the genus paragonimus . humans can become infected with this lung fluke after consuming raw or undercooked freshwater crustaceans . before infecting mammalian hosts , paragonimus species require a snail as the first intermediate host , and a freshwater crab or crayfish as the second intermediate host . in mammalian definitive hosts , the infective metacercariae excyst in the duodenum and migrate to the lungs , causing pulmonary paragonimiasis , with respiratory symptoms ( e.g. coughing ) that mimic tuberculosis ( toscano et al . , 1995 ) . ectopic paragonimiasis occurs when flukes migrate internally , causing damage to muscles and organs , including the brain ( cerebral paragonimiasis ) ( blair , 2014 ) . infect more people globally than any other foodborne trematode , and infections cause an estimated 196,710 disability adjusted life - years ( frst et al . , 2012 ) . is best known from asia and latin america ( frst et al . , 2012 ) . however , two known species , paragonimus africanus and p. uterobilaterlis , infect humans in africa ( blair , 2014 ) . paragonimus uterobilateralis is considered the principal causative agent of paragonimiasis in nigeria ( aka et al . , 2008 ) . prior to the biafran war in nigeria ( 19671970 ) , paragonimiasis was known only from a handful of cases ( nnochiri , 1968 ; nwokolo , 1964 ) . during the war , food shortages and limited access to cooking facilities led to increased consumption of inadequately cooked or raw crab , and cases of paragonimiasis increased dramatically ( nwokolo , 1972 ) . human infections nearly disappeared again after the war , until recent surveys revealed unexpected high prevalence ( up to 13.2% ) in communities in the southeast part of nigeria ( aka et al . , 2008 ) . the epidemiology of paragonimus sp . in nigeria differs from that in cameroon , where the disease has a longer history of endemicity due to the cultural practice of eating raw crabs in some areas ( world health organization , 1995 ) . the african civet ( viverra civetta ) is considered the natural host of p. uterobilateralis in nigeria ( voelker and sachs , 1974 ) , with the swamp mongoose ( atilax paludinosus ) and domestic dog ( canis familiaris ) harboring the parasite in cameroon and liberia , respectively ( voelker and vogel , 1965 ) . paragonimus africanus has a broader host range , infecting the mongoose ( crossarchus obscurus ) , palm civet ( nandinia binotata ) , drill monkey ( mandrillus leucophaeus ) , potto ( perodicticus potto ) , and domestic dog ( c. familiaris ) in cameroon ( voelker and vogel , 1965 ; sachs and voelker , 1975 ) . the intermediate and definitive hosts of p. africanus range throughout the contiguous forest of southeastern nigeria bordering cameroon ( kingdon , 2005 ; abraham and akpan , 2011 ) , suggesting that p. africanus could be more widely distributed than is currently appreciated . dna directly from eggs in feces to identify it to species and compare it to parasites reported in human populations . finally , we examined clinical observational data prior to and following treatment of the study population with anthelminthic drugs . we use this information to assess the presence of paragonimus sp . in red - capped mangabeys in nigeria , the role of these primates as potential hosts , and the clinical effects of the parasite on monkeys . between may and august 2012 , we collected fecal samples and recorded coughing opportunistically from a group of 49 [ 23 adults / sub - adults ( 3 yo ) and 12 juveniles ( <3 yo ) ] individually identifiable red - capped mangabeys that lived in a 1-ha open topped forest enclosure within the natural home range of the species ( fig . 1 ) . the population was provisioned daily , but also had access to wild foods within the enclosure . the animals had access to water ad libitum , from a natural stream that ran through the enclosure . they were exposed to natural predators ( e.g. snakes and birds of prey ) and parasites . all animals were rescued from the bushmeat and pet trades in nigeria as young juveniles , or were captive - born . thirteen individuals were moved from a sancutary to the open - topped enclosure in 2004 as part of the rehabilitation and release program of the centre for education , research and conservation of primates and nature ( cercopan ) , and the remaining 36 individuals were born in the enclosure . for 30 days in late may and june 2012 , we collected triplicate fecal samples from each individual . then , in late june 2012 , the population was treated for paragonimus sp . via orally administered praziquantel ( approximately 20 mg / kg for three consecutive days ) . we collected subsequent triplicate fecal samples from each individual over 30 days post - treatment , for a total of 294 samples ( 147 pre - treatment and 147 post - treatment ) . over the same time periods ( 30 days pre - treatment and 30 days post - treatment ) , we recorded all observed instances of coughing between 6:00 and 17:00 daily . the institutional animal care and use committee at university of wisconsin , madison approved all research activities ( protocol v1490 ) . fecal samples were collected from known individuals immediately following defecation , stored temporarily in plastic bags , and fixed within 2 h of collection . we removed two aliquots from each sample for preservation of gastrointestinal parasite eggs and dna separately . one aliquot was fixed in 10% formalin for microscopic analysis , and the other in rnalater nucleic acid stabilizing solution for genetic analysis . samples were transported to the university of wisconsin , madison following all applicable import , export , and international air transport association regulations . one gram of formalin - preserved feces was concentrated by sedimentation and examined microscopically at x10 and x40 magnification ( greiner and mcintosh , 2009 ) . we calculated prevalence ( percent of individuals infected ) as number of individuals shedding eggs divided by the total number of individuals examined , and we approximated mean and median intensity of infection ( number of eggs per gram ( epg ) of a particular parasite species in the feces of a single infected host ) ( bush et al . we calculated pre- and post - treatment intensity by taking the average epg of triplicate samples for each individual . we compared paragonimus sp . prevalence and intensity to host characteristics and rates of coughing using fisher 's exact test , mann whitney test , and spearman rank correlation . we then compared parasite intensity and coughing rates pre- and post - treatment using paired wilcoxon rank sum test . for all pre- and post - treatment comparisons , we used one - tailed tests under the directional hypotheses that coughing frequency would be positively associated with parasite infection . we extracted dna from 150 mg of the fecal sample with the highest egg count ( 1,067 epg ) using the zymo zr fecal dna miniprep kit ( zymo research corporation , irvine , ca , usa ) , following the manufacturer 's protocols . pcr and nucleotide sequencing were performed on the internal transcribed spacer 2 region ( its2 ) using primers 3s ( 5-cggtggatcactcggctcgt-3 ) and a28 ( 5-cctggttagtttc ttttcctccgc-3 ) , previously used to amplify p. africanus ( nkouawa et al . , 2009 ) . pcr was performed using phusion high - fidelity pcr mastermix ( new england biolabs , ipswich , ma ) , and cycled in a biorad cfx96 platform ( bio - rad laboratories , hercules , ca , usa ) with the following cycling parameters : 98 c for 30 min ; 40 cycles of 98 c for 10 s , 55 c for 30 s , 72 c for 90 s ; and a final extension at 72 c for 10 min . amplicons were electrophoresed on an agarose gel stained with ethidium bromide and then purified using the zymoclean gel dna recovery kit ( zymo research corporation , irvine , ca , usa ) . amplicons were sequenced on abi 3730xl dna analyzers ( applied biosystems , grand island , ny , usa ) at the university of wisconsin madison biotechnology center dna sequencing facility . sequences were aligned to published paragonimus sequences using clustal w ( thompson et al . , 1994 ) . we recovered parasite eggs that were consistent with p. africanus ( 87.3 7.9 x 45.4 4.5 ) in 43% of the population . our dna sequence ( gen bank accession number kr780065 ) was 100% identical to a p. africanus sequence from a human in kumba , cameroon and 99% identical to another from bulutu , cameroon ( nkouawa et al . , 2009 ) . mean intensity prior to treatment was 96.70 epg ( 95% ci : 29.73163.67 ; median = 32.67 ; range = 11067 ) . we found no association between parasitism ( prevalence or intensity ) and host sex or age . we recovered paragonimus sp . from animals transferred to the enclosure and those born in the enclosure . we observed 516 coughs in 88% ( n = 43 ) of individuals . frequency of coughing was over two times higher in paragonimus - positive individuals ( = 11.42 ) than in paragonimus - negative individuals ( = 4.03 ; w = 192 ; p < .02 ; fig . eggs shed in feces ( rs = .64 , p < .001 ; fig . de - worming led to a significant reduction in epg ( = 6.13 ; p < .0001 ; fig . 3c ) and a corresponding reduction of coughing to baseline levels ( = 4.95 ; p < .01 ; fig . we provide the first molecular evidence of paragonimus sp . in african wildlife , and the first report of paragonimus sp . recent surveys revealing re - emergence of paragonimus sp . in human populations in southeast nigeria suggest a sylvatic cycle in which the parasite is maintained in crab - eating wildlife reservoirs ( blair , 2014 ) . indeed , c. torquatus in gabon eat crabs as a normal part of the diet ( cooke , 2014 ) . our results demonstrate that c. torquatus can be a host for p. africanus , and infection is associated with respiratory illness . however , given limited sampling and dna sequencing , we can not exclude the possibility that this population hosts other species within the genus paragonimus . together with observations from voelker and sachs ( 1977 ) and sachs and voelker ( 1980 ) , these results suggest that wild primates in nigeria may help maintain paragonimus sp . perhaps contributing to human disease . for example , there was no sequence information available for p. uterobilateralis on genbank as of august 7 , 2015 , and only two sequences from p. africanus were available ( nkouawa et al . , 2009 ) . our p. africanus sequences were between 99% and 100% identical to sequences from humans in cameroon , demonstrating only limited intraspecific variation in the its2 region of the parasite . paragonimus africanus therefore appears to be genetically homogeneous across nigeria and cameroon , albeit based on a limited number of samples and only one genetic locus . paragonimus mexicanus , previously considered the sole etiological agent of paragonimiasis in the americas , is now believed to include cryptic species ( lpez - caballero et al . , 2013 ) . significantly , we found that coughing was more frequent in infected individuals , and that animals with more intense infections coughed more frequently . furthermore , treatment with praziquantel in our study population led to reduced parasite burden and a corresponding reduction of coughing to baseline levels in infected monkeys . determining whether coughing is communicative or physiological is problematic in studies of primate behavior ( hauser , 2000 ) . our results suggest that coughing may actually indicate respiratory disease , such as infection with lung flukes . this observation not only complicates interpretations of primate behavior , but it also suggests a method for clinical assessment of wild primates for paragonimiasis and similar respiratory pathogens . interestingly , hunters in this area report using primate skulls and feces to treat cough ( friant et al . , 2015 ) . in interviews , hunters made reference to seeing monkeys in the area cough as justification for these traditional remedies ( s.friant , unpub.data ) , indicating an intriguing link between parasitism , clinical disease , local beliefs , and primate conservation . knowledge of sylvatic reservoirs will be critical for improved understanding and control of re - emerging paragonimiasis in nigeria . given the difficulties associated with eradicating multi - host pathogens , as well as their high potential for emergence and re - emergence , control of paragonimiasis will require not only sustained behavior change away from raw or undercooked crab consumption and improved education , but also surveillance of potential wildlife hosts . in the case of primates , observations of coughing should be considered suspicious for paragonimiasis .
this is a method in which one factor is varied while all other factors are fixed under certain conditions ( logothetis and wynn , 1989 ) . however , this method does not take all factors into account at the same time . rsm is a set of statistical techniques for designing experiments , creating models , evaluating the impacts of factors , and exploring optimal conditions for desirable responses ( myers et al . , 2009 ) . regarding experimental designs in rsm , central composite designs ( ccd ; box and wilson , 1951 ) have been used most frequently . a ccd is a three- or five - level design that can fit a second - order polynomial model to data within a cubic or spherical experimental region . for a second - order model to be a good predictive model , it should satisfy some criteria that the p - value of the model 0.05 , the pvalue of the lack of fit > 0.1 , and the adjusted r - square 0.8 ( myers et al . , 2009 ) . if the model fitted to the data does not meet these criteria , modeling and optimization results might not be accurate . however , in reality , it is observed that the models that do not satisfy the above criteria are used in the analyses of response surface experiments . this seems to be because researchers have little knowledge of what to do in such a situation . rheem and rheem ( 2012 ) improved a second - order model with a significant lack of fit by adding cubic terms to it . however , cases can exist where a third - order model still falls short of such criteria for a good predictive model . in these cases , there arises a need to use a fullest model that has no lack of fit . when a spherical ccd is used as an experimental design , fullest models with no lack of fit exist , but they are not unique . however , among them , a balanced model is unique . this article proposes such a model , which is called a fullest balanced model , and how to use it . for the last ten years ( from 2007 to 2016 ) , sixteen articles using a ccd in rsm were published in the korean journal for food science of animal resources . the number of such articles published each year during that period is shown in fig . , we found that three independent variables ( three factors ) were most frequently used in such articles ( fig . 2 ) . thus , a dataset with three factors , which is in park et al . ( 2014 ) published in the korean journal for food science of animal resources , will be re - analyzed for the illustration of the method suggested in this article . how to use a fullest balanced model will be explained through re - analysis of a dataset described in the article entitled application of response surface methodology ( rsm ) for optimization of anti - obesity effect in fermented milk by lactobacillus plantarum q180 authored by park et al . three factors were used in an experiment to model three responses . among them , the third response , which was anti - adipogenetic activity ( % ) , had the poorest fit of a second - order model . factors ( x variables ) in this experiment and their coded and actual levels are given in table 1 . table 1.response and factorsresponse = yactual factorcoded factoractual factor level at the coded factor level of1.681791011.68179anti - adipogenetic activity ( % ) skim milk powder ( % ) x18.3189101111.682incubation temp . ( c)x231.95534374042.045incubation time ( h)x312.8412030.54148.159 the dataset to be re - analyzed is shown in table 2 . in this dataset , the experimental design is the ccd for three factors with an axial value of 1.68179 and three center runs . using this design , to the data , we can fit a second - order model , a third - order model , and a fullest balanced model . table 2.experimental design in coded levels and responsesstandard orderdesign pointx1x2x3y1111119.17221112.393311113.73441115.945511110.29661114.027711112.28881115.58991.681790026.7810101.68179002.57111101.68179013.91121201.6817905.761313001.6817930.041414001.6817910.11151500018.44161500016.45171500015.00 data were analyzed using sas software . how to use a fullest balanced model will be explained through re - analysis of a dataset described in the article entitled application of response surface methodology ( rsm ) for optimization of anti - obesity effect in fermented milk by lactobacillus plantarum q180 authored by park et al . three factors were used in an experiment to model three responses . among them , the third response , which was anti - adipogenetic activity ( % ) , had the poorest fit of a second - order model . factors ( x variables ) in this experiment and their coded and actual levels are given in table 1 . table 1.response and factorsresponse = yactual factorcoded factoractual factor level at the coded factor level of1.681791011.68179anti - adipogenetic activity ( % ) skim milk powder ( % ) x18.3189101111.682incubation temp . ( c)x231.95534374042.045incubation time ( h)x312.8412030.54148.159 the dataset to be re - analyzed is shown in table 2 . in this dataset , the experimental design is the ccd for three factors with an axial value of 1.68179 and three center runs . using this design , to the data , we can fit a second - order model , a third - order model , and a fullest balanced model . first , the second - order polynomial regression model containing 3 linear , 3 quadratic , and 3 interaction terms was fitted to the data by using rsreg procedure of sas / stat . results of analysis of variance for the second - order model are shown in table 3 . in table 3 , the p - value of the model = 0.0642 > 0.05 , the p - value of the lack of fit = 0.0526 < 0.1 , and the adjusted r - square = 0.5654 < 0.8 ; none of the three criteria are satisfied . since this second - order model has a poor fit , next we will fit to the data a third - order model that consists of linear , quadratic , cubic , and two - way and three - way interaction terms , anticipating a possible improvement in modeling . table 4 shows the results of analysis of variance for this third - order model . table 3.analysis of variance for the second - order modelmodel terms : x1 , x2 , x3 ; x1 , x2 , x3 ; x1x2 , x1x3 , x2x3sourcedegrees of freedomsum of squaresmean squaref - valuep - valuemodel91187.5291131.94773.310.0642error7278.827739.8325 - -total161466.3568 - --root mse = 6.3113r - square = 0.8099adjusted r - square = 0.5654test of lack of fitsourcedegrees of freedomsum of squaresmean squaref - valuep - valuelack of fit5272.862354.572518.30.0526pure error25.96542.9827 - - in table 4 , the p - value of the model = 0.2627 > 0.05 , the p - value of the lack of fit = 0.0230 < 0.1 , and the adjusted r - square = 0.5221 < 0.8 ; none of the three criteria are satisfied . this third - order model is worse than the previous second - order model , let alone better . now , the lack - of - fit part has 1 degree of freedom , which means that we can add one more term to the model . for the model to be balanced , this additional term needs to contain all of x1 , x2 , and x3 . then , since the latest term in the model is x1x2x3 , the next term to enter the model should be x1x2x3 . now , we add this term to the model , expecting a possible improvement in modeling . results of analysis of variance for this fullest balanced model are given in table 5 . table 4.analysis of variance for the third - order modelmodel terms : x1 , x2 , x3 ; x1 , x2 , x3 ; x1x2 , x1x3 , x2x3 ; x1 , x2 , x3 ; x1x2x3sourcedegrees of freedomsum of squaresmean squaref - valuep - valuemodel131334.9522102.68862.340.2627error3131.404543.8015 - -total161466.3568 - --root mse = 6.6183r - square = 0.9104adjusted r - square = 0.5221test of lack of fitsourcedegrees of freedomsum of squaresmean squaref - valuep - valuelack of fit1125.4391125.439142.060.0230pure error25.96542.9827 - - in table 5 , the p - value of the model = 0.0281 < 0.05 , and the adjusted r - square = 0.9675 > 0.8 ; two criteria are satisfied . the lack - of - fit part has 0 degree of freedom , which means that this model has no lack of fit . and finally , we have obtained the improved model that will be used for optimization . letting denote the predicted value of y , we specify this model as = b0 + b1x1 + b2x2 + b3x3 + b11x1 + b22x2 + b33x3 + b12x1x2 + b13x1x3 + b23x2x3 + b111x1 + b222x2 + b333x3 + b123x1x2x3 + b112233x1x2x3 where the coefficients b1 , b2 , , b112233 are given in table 6 , which says that x1x2x3 is the most significant term among the model terms . table 5.analysis of variance for the fullest balanced modelmodel terms : x1 , x2 , x3 ; x1 , x2 , x3 ; x1x2 , x1x3 , x2x3 ; x1 , x2 , x3 ; x1x2x3 ; x1x2x3sourcedegrees of freedomsum of squaresmean squaref - valuep - valuemodel141460.3914104.313734.970.0281error25.96542.9827 - -total161466.3568 - --root mse = 1.7271r - square = 0.9959adjusted r - square = 0.9675test of lack of fitsourcedegrees of freedomsum of squaresmean squaref - valuep - valuelack of fit00 ... pure error25.96542.9827 - - table 6.coefficient estimates in the fullest balanced modeltermparameter estimatestandard errort - valuep - valueinterceptb0 = 16.630000.9971116.680.0036x1b1 = 4.965531.024654.850.0400x2b2 = 4.125121.024654.030.0565x3b3 = 0.858381.024650.840.4903x1b11 = 1.599830.557402.870.1030x2b22 = 2.402400.557404.310.0498x3b33 = 1.218000.557402.190.1605x1x2b12 = 2.672500.610604.380.0484x1x3b13 = 1.042500.610601.710.2299x2x3b23 = 1.087500.610601.780.2169x1b111 = 1.329470.518892.560.1245x2b222 = 2.315120.518894.460.0467x3b333 = 2.398380.518894.620.0438x1x2x3b123 = 0.770000.610601.260.3345x1x2x3b112233 = 6.273260.967356.490.0230 according to park et al . thus , through a search on a grid ( oh et al . , 1995 ) , we maximized the model with the coefficients in table 5 . in this experiment , the bounds are 1.682 xj 1.682 for j = 1 , 2 , 3 . in the ccd in table 2 , every design point is under the constraint x1 + x2 + x3 ( 1 ) + ( 1 ) + ( 1 ) = 3 , which makes the design region spherical with the radius 3=1.732 . thus , satisfying these bounds and constraint , we conducted a search on a grid using the sas data step programming . here , a search for the maximum on a grid was performed by calculating the function over a grid of the values of x1 , x2 , and x3 with an increment of 0.01 under the bounds 1.682 xj 1.682 for j = 1 , 2 , 3 and the constraint x1 + x2 + x3 3 , and then sorting the calculated function values in desc - ending order . the optimum point at which is maximized was found this way and presented in table 7 . ( c)incubation time ( h)anti - adipogenetic activity ( % ) 0.420.031.681.731969.5837.0912.8632.6492 in park et al . their optimum conditions for this maximum were skim milk powder = 8.4677% , incubation temperature = 65.3815c , and incubation time = 12.8412 h. these maximum and optimum conditions are different from our optimization results . our predicted maximum was 32.6492% , which was greater than their predicted maximum 31% . ( 1995 ) , for any two of the three factors , a three - dimensional ( 3d ) response surface plot was drawn with the vertical axis representing the predicted response and two horizontal axes representing the coded levels of two explanatory factors . in each 3d plot , the factor not represented by the two horizontal axes is fixed at its optimum level . two - dimensional contour plots of response surfaces were also drawn with two axes indicating two coded factors . in each contour plot , the factor not represented by the two axes is fixed at its optimum level . first , the second - order polynomial regression model containing 3 linear , 3 quadratic , and 3 interaction terms was fitted to the data by using rsreg procedure of sas / stat . results of analysis of variance for the second - order model are shown in table 3 . in table 3 , the p - value of the model = 0.0642 > 0.05 , the p - value of the lack of fit = 0.0526 < 0.1 , and the adjusted r - square = 0.5654 < 0.8 ; none of the three criteria are satisfied . since this second - order model has a poor fit , next we will fit to the data a third - order model that consists of linear , quadratic , cubic , and two - way and three - way interaction terms , anticipating a possible improvement in modeling . table 4 shows the results of analysis of variance for this third - order model . table 3.analysis of variance for the second - order modelmodel terms : x1 , x2 , x3 ; x1 , x2 , x3 ; x1x2 , x1x3 , x2x3sourcedegrees of freedomsum of squaresmean squaref - valuep - valuemodel91187.5291131.94773.310.0642error7278.827739.8325 - -total161466.3568 - --root mse = 6.3113r - square = 0.8099adjusted r - square = 0.5654test of lack of fitsourcedegrees of freedomsum of squaresmean squaref - valuep - valuelack of fit5272.862354.572518.30.0526pure error25.96542.9827 - - in table 4 , the p - value of the model = 0.2627 > 0.05 , the p - value of the lack of fit = 0.0230 < 0.1 , and the adjusted r - square = 0.5221 < 0.8 ; none of the three criteria are satisfied . this third - order model is worse than the previous second - order model , let alone better . now , the lack - of - fit part has 1 degree of freedom , which means that we can add one more term to the model . for the model to be balanced , this additional term needs to contain all of x1 , x2 , and x3 . then , since the latest term in the model is x1x2x3 , the next term to enter the model should be x1x2x3 . now , we add this term to the model , expecting a possible improvement in modeling . results of analysis of variance for this fullest balanced model are given in table 5 . table 4.analysis of variance for the third - order modelmodel terms : x1 , x2 , x3 ; x1 , x2 , x3 ; x1x2 , x1x3 , x2x3 ; x1 , x2 , x3 ; x1x2x3sourcedegrees of freedomsum of squaresmean squaref - valuep - valuemodel131334.9522102.68862.340.2627error3131.404543.8015 - -total161466.3568 - --root mse = 6.6183r - square = 0.9104adjusted r - square = 0.5221test of lack of fitsourcedegrees of freedomsum of squaresmean squaref - valuep - valuelack of fit1125.4391125.439142.060.0230pure error25.96542.9827 - - in table 5 , the p - value of the model = 0.0281 < 0.05 , and the adjusted r - square = 0.9675 > 0.8 ; two criteria are satisfied . the lack - of - fit part has 0 degree of freedom , which means that this model has no lack of fit . and finally , we have obtained the improved model that will be used for optimization . letting denote the predicted value of y , we specify this model as = b0 + b1x1 + b2x2 + b3x3 + b11x1 + b22x2 + b33x3 + b12x1x2 + b13x1x3 + b23x2x3 + b111x1 + b222x2 + b333x3 + b123x1x2x3 + b112233x1x2x3 where the coefficients b1 , b2 , , b112233 are given in table 6 , which says that x1x2x3 is the most significant term among the model terms . table 5.analysis of variance for the fullest balanced modelmodel terms : x1 , x2 , x3 ; x1 , x2 , x3 ; x1x2 , x1x3 , x2x3 ; x1 , x2 , x3 ; x1x2x3 ; x1x2x3sourcedegrees of freedomsum of squaresmean squaref - valuep - valuemodel141460.3914104.313734.970.0281error25.96542.9827 - -total161466.3568 - --root mse = 1.7271r - square = 0.9959adjusted r - square = 0.9675test of lack of fitsourcedegrees of freedomsum of squaresmean squaref - valuep - valuelack of fit00 ... pure error25.96542.9827 - - table 6.coefficient estimates in the fullest balanced modeltermparameter estimatestandard errort - valuep - valueinterceptb0 = 16.630000.9971116.680.0036x1b1 = 4.965531.024654.850.0400x2b2 = 4.125121.024654.030.0565x3b3 = 0.858381.024650.840.4903x1b11 = 1.599830.557402.870.1030x2b22 = 2.402400.557404.310.0498x3b33 = 1.218000.557402.190.1605x1x2b12 = 2.672500.610604.380.0484x1x3b13 = 1.042500.610601.710.2299x2x3b23 = 1.087500.610601.780.2169x1b111 = 1.329470.518892.560.1245x2b222 = 2.315120.518894.460.0467x3b333 = 2.398380.518894.620.0438x1x2x3b123 = 0.770000.610601.260.3345x1x2x3b112233 = 6.273260.967356.490.0230 thus , through a search on a grid ( oh et al . , 1995 ) , we maximized the model with the coefficients in table 5 . in this experiment , the bounds are 1.682 xj 1.682 for j = 1 , 2 , 3 . in the ccd in table 2 , every design point is under the constraint x1 + x2 + x3 ( 1 ) + ( 1 ) + ( 1 ) = 3 , which makes the design region spherical with the radius 3=1.732 . thus , satisfying these bounds and constraint , we conducted a search on a grid using the sas data step programming . here , a search for the maximum on a grid was performed by calculating the function over a grid of the values of x1 , x2 , and x3 with an increment of 0.01 under the bounds 1.682 xj 1.682 for j = 1 , 2 , 3 and the constraint x1 + x2 + x3 3 , and then sorting the calculated function values in desc - ending order . the optimum point at which is maximized was found this way and presented in table 7 . ( c)incubation time ( h)anti - adipogenetic activity ( % ) 0.420.031.681.731969.5837.0912.8632.6492 in park et al . their optimum conditions for this maximum were skim milk powder = 8.4677% , incubation temperature = 65.3815c , and incubation time = 12.8412 h. these maximum and optimum conditions are different from our optimization results . our predicted maximum was 32.6492% , which was greater than their predicted maximum 31% . ( 1995 ) , for any two of the three factors , a three - dimensional ( 3d ) response surface plot was drawn with the vertical axis representing the predicted response and two horizontal axes representing the coded levels of two explanatory factors . in each 3d plot , the factor not represented by the two horizontal axes is fixed at its optimum level . two - dimensional contour plots of response surfaces were also drawn with two axes indicating two coded factors . in each contour plot , the factor not represented by the two axes is fixed at its optimum level .
pemphigus vulgaris ( pv ) is an autoimmune disease characterized by blisters and erosion on skin and mucous membrane due to acantholysis . it is caused by autoantibodies against desmosomal caderins desmoglein 3 ( dsg 3 ) which are cell adhesion molecules . it affects men and woman equally and usually occurs in the fifth and sixth decade . the mainstay of treatment remains to be corticosteroids with or without adjuvant therapies which may include immunosuppressive , anti - inflammatory and immunomodulatory procedures . due to the variation in severity , varied response to the conventional treatment protocols and severe side effects , now dermatologists all over the world have ventured into new modalities of adjuvant therapies , like intravenous immunoglobulins ( ivig ) , therapeutic plasma exchange ( tpe ) and rituximab to grapple the increasing number of patients with severe pv who show little or no response with conventional steroid treatment . we herewith report a case of severe pv , who was resistant to corticosteroids pulse therapy , cyclophosphamide and was successfully treated with tpe , an option hardly being considered in indian scenario . a 65-year - old male presented to the emergency with multiple blisters and erosions all over his body including oral mucosa , scalp with superadded maggot infection since last 6 months . he had previous episodes of such lesions which subsided within 3 - 4 months after treatment with steroids and some unknown generic medications from outside last 1 year . the patient again developed lesions all over the body over the period of 6 month which gradually worsened and was brought to our tertiary hospital . on physical examination , he was febrile and had numerous flaccid blisters and erosions covering more than 85% of body surface area with some of them covered with slough and maggots [ figure 1 ] . nikolsky sign was strongly positive and the diagnosis of pv was confirmed by a skin biopsy [ figure 2 ] and tzanck smear . he was put on intravenous ( iv ) dexamethasone pulse ( 120 mg ) along with supportive care such as fluids , fresh frozen plasma ( ffp ) and dressings on admission for three consecutive days , once routine blood investigation were normal and blood and urine cultures were sterile . intravenous antibiotics ( piperacillin / tazobactam and teicoplanin ) were given according to the pus culture and sensitivity . adjuvant immunosuppressors were held back because of infection . due to extensive lesions and high susceptibility to further infections the patient there was still oozing from skin ulcerations and hemorrhagic excoriation with peeling of skin was present . he was maintained on 1 mg / kg of oral methyl prednisolone after the pulse but there was no improvement in the skin lesions and general condition of the patient worsened further with hypoproteinemia and developed pleural effusion on day 7 . blood culture showed enterobacter and pus culture from erosions show staphylococcus aureus and proteus mirabilis . the erosions still persisted and did not show any sign of re - epithelialization , perilesional nikolsky sign was still positive and he continued to develop new blisters . as he was in sepsis with persistent high grade fever , albumin levels fell to 2.1 mg , it was considered to hold the pulse therapy of iv methyl prednisolone and cyclophosphamide . keeping this in mind , tpe was performed using a single volume plasma exchange with intermittent cell separator ( haemonetics mcs plus , kit 980/790 ) machines based on centrifugation by femoral access using a 12-french double lumen dialysis catheter . it was scheduled preferably on alternate - day intervals for five sessions over a period of 10 days . replacement of plasma removed during the procedure was done with isotonic sterile saline , to make up one - half of the volume and with 4% purified human albumin and ffp to complete it . a careful monitoring of hemodynamic parameters was done and complications during or following procedure were rapidly recognized and reverted by rationale interventions . indications for tpe , number of cycles and sessions , duration of each session , volume of plasma exchanged and patient tolerance to the procedure were systematically recorded . 10 ml of 10% calcium gluconate was administered through the procedure to avoid citrate toxicity and hemogram , serum electrolytes , total protein , albumin were monitored daily . extensive blisters and non - healing erosions on face and neck histopathology of a skin biopsy from erosion showing an acantholytic cleft separates the upper part of the epidermis from the basal keratinocytes ( h and e , 40 ) the amount of plasma to be exchanged must be determined in relation to the estimated plasma volume ( epv ) . a simple means of estimating the epv can be done from the patient 's weight and hematocrit using the formula : epv= [ 0.065 wt ( kg ) ] [ 1-hct ] . a total of 12.75 l of plasma was removed in five sessions in icu . after the third session of tpe , nikolsky sign became negative and no new lesions appeared . the lesions showed 60 - 70% re - epithelization and 90% healing after the last sessions [ figure 3 ] . the oral lesions also healed completely though little erosions on back , anterior aspect of thigh and buttocks persisted . serum antibody levels before and after tpe sessions could not be done as the patient could not afford it . healing of erosions over the head and neck after five sessions of tpe in order to prevent the rebound antibody synthesis , the patient was given pulse therapy of iv methyl prednisolone ( 1 gm / day ) and cyclophosphamide ( 500 mg / day ) for three consecutive days . at the end of pulse therapy , there was further improvement in the lesions and the patient was clinically stable . to achieve further improvement as evident by previous , plan was to given one more tpe cycles for three sessions but due to cost restraint it was not done and patient was discharged on maintained dose of monthly iv dexamethosone pulse along with daily oral prednisolone ( 60 mg / day ) . tpe is an extracorporeal blood purification technique in which the plasma is separated from blood , discarded in total , and replaced with a substitution fluid such as albumin or with plasma collected from healthy donors . this is generally performed to remove high - molecular - weight substances such as pathogenic auto antibodies , immune complexes , cryoglobulins and toxins that have accumulated in the plasma . usually it is combined with immunosuppressive treatment , such as ivig , prednisone and azathioprine , to avoid rebound effect and to maintain the improvement . it is used for various autoimmune diseases like goodpasture syndrome , myasthenia gravis , guillain - barre syndrome , systemic lupus erythematosus , pv and bullous pemphigoid etc . the first therapeutic implications of tpe in pv were described by cotterill . since then , it has been described as an effective adjuvant therapy in pv patients in controlling disease activity by reducing serum levels of autoantibodies . even though , there is no standardized protocol for the number of cycles and frequency of sessions , it is commonly believed that four or five sessions of plasma exchanges over a period of 7 to 10 days constitutes an adequate short - term therapy , to remove 90% of the total initial body immunoglobulin . tpe is relatively safe and the risk of infections associated with it is mainly due to the steroids and immunosuppressives given along with it . the overall incidence of adverse reaction reported in the literature range for 1.6% to 2.5% with severe reaction occurring in 0.5 - 3.1% . studied the role of tpe as steroid sparing therapy in seven patients and found 86% response rate with three sessions of tpe per week along with monthly iv dexamethasone pulse , without any major adverse effects . we could safely practice tpe in our patient despite evidence of sepsis and a similar observation was made by saraceno et al . in a 63-year - old male who developed nocardia asteroides infection when on conventional therapy and responded well to tpe . while immuno - adsorption is superior to plasma exchange in terms of efficacy and safety but the high cost of the adsorbers is the major limiting factor . it is a useful intervention in patients with pv who are not responding to standard therapy or who require unacceptably high doses of steroids or immunosuppressant . while the cost of four injections of rituximab or one cycle of ivig could be approximately 1.2 to 1.5 lakh rupees in india , five to six sessions of tpe will cost less than half of the same with better results . the use of tpe has changed in recent years , given advances in medical technology that have allowed a wider clinical application in the critical care setting . therapeutic plasma exchange improves outcome in patients of pemphigus vulgaris who experience the exacerbations in spite of treatment with steroids and oral immunosuppressives . it may considered as treatment options especially in developing countries like india as it is comparatively less costly and as equally effective .
in 2001 , an estimated 70 million americans had some form of arthritis or other rheumatic conditions , a major increase over the previous estimate of 43 million cases reported in 1997 . in 1992 , economy about $ 64.8 billion dollars ( about 24% was due to direct medical costs , 76% due to indirect costs from lost wages ) . the cost of treating arthritis and other musculoskeletal conditions in 1992 was $ 149.4 billion , which was about 2.5% of the gross national product [ 2 , 3 ] . because arthritis conditions do not usually cause death , quality of life ( qol ) is a better indicator of the disease impact than mortality rates in a nonterminal disease like arthritis . in addition to the quality of life , arthritis can also negatively impact the well - being and satisfaction with life in people with severe forms of the disease [ 4 , 5 ] . debilitating nature of the disease , mainly due to chronic physical pain , can affect all aspects of a patient 's life , including the physical , behavioral , social , and psychological . consequently , an overall evaluation of health status in arthritis is viewed to be important for broadening the focus of care and improving patient outcomes beyond survival . the adverse impact of arthritis on mental health of a patient has been documented frequently in the literature , particularly in the case of rheumatoid arthritis ( ra ) [ 68 ] . rates of depression are noticeably higher among patients with chronic pain such as rheumatoid arthritis . for persons of 65 years of age or older , depression is often correlated with a decline in physical health . according to an expert estimate , about 90 percent of patients with arthritis and other rheumatic diseases experience depression . depression is often assumed to be a normal response to aging , physical pain , or other life events and is frequently overlooked as a clinical diagnosis in older people . because depression can make an independent and additive contribution to disability accompanying arthritis , early recognition of depressive symptoms is essential for an effective management of the disease . maintenance of good health status that is free from the burden of mental illness is crucial for producing positive health outcomes in patients affected by arthritis . accordingly , the primary objective of this study was to comprehensively measure the health status of older adults with arthritis and to determine the prevalence of depressive symptomatology in this population . researchers in health services area have used short - form 36 ( sf-36 ) , a health status survey , and center for epidemiological scale for depression ( ces - d ) , a screening tool for depression , extensively for many years . health related quality of life ( hrqol ) , an outcome that is measured by sf-36 , represents multiple dimensions of health that cover physical and social activities , well - being , and overall evaluation of health . the sf-36 is suitable for self - administration , computerized administration , or administration by a trained interviewer in person or by telephone to persons aged 14 and older . the reliability of the scale has been previously established to meet or exceed the minimum standards recommended for measures used in group comparison . even though sf-36 has been used routinely in the assessment of health in patient populations , with the exception of a few studies [ 15 , 16 ] , its utility as an instrument that screens for depression remains largely unexplored . one of the eight domains of sf-36 , the 5-item mental health ( mh ) subscale , has been shown to be useful as a first stage screen for depression . using the standard ( 0100 ) scoring , a cut - point of 52 on the mh scale is often cited as best in several studies using receiver operating characteristic ( roc ) analysis to screen for major depressive disorders [ 11 , 17 , 18 ] . yet , to a large part , the use of sf-36 in the past has been mostly confined to the assessment of health status . the 20-item ces - d , the instrument originally developed for national institute of mental health studies , is judged among the best screening instruments for symptoms of depression in older adults [ 20 , 21 ] . its reliability and validity to detect both clinical and nonclinical symptoms of depressed mood are established for a wide range of study populations [ 12 , 22 ] , including older adults [ 13 , 23 , 24 ] . concurrent use of sf-36 and ces - d has been reported in the literature before , but seldom with a goal to examine the utility of sf-36 as a depression screener , when a more established ces - d is also used . it can be argued that the usefulness of sf-36 would substantially increase if it could also serve as a screening tool for depression , obviating the need for a separate depression instrument . therefore , a secondary purpose of this research was to determine whether sf-36 could be a good tool for the measurement of depression and to assess its performance relative to that of an established instrument like ces - d . a cross - sectional , web - based , survey research methodology with random sampling techniques was employed . households across 44 states . to be eligible , the respondents had to be 65 years of age or older , have arthritis , and have no clinical diagnosis of depression . sf-36 and ces - d surveys were coadministered through webtv technology . in august 2001 , proprietary random digit dialing ( rdd ) survey procedures , developed by a new york - based commercial market research firm , were employed for screening , subject recruitment , and survey administration . the research firm employs rdd sampling for selecting telephone numbers of eligible households and invites them to join a panel . the participants are then sent a webtv set - top box and given free internet access in exchange for completing surveys approximately once a week . a sample of about 40,000 10-digit randomly selected numbers constituted our sampling frame and was representative of the entire usa . following the initial screening achieved through internet and/or telephone contacts to establish the participant 's age and medical condition , a sample of 550 randomly selected elderly arthritis - affected individuals agreed to participate in the study . protocols reviewed and approved by the institutional review board were adopted to ensure voluntary participation and confidentiality of survey responses . both sf-36 and ces - d tests were administered together , and a 2-week deadline was imposed for survey completion . participants were instructed so as to the purpose of coadministration of the surveys and asked to complete both questionnaires in one session . it was estimated through pilot - testing in a small group of older adults that the surveys took about 30 minutes to complete . in order to ensure uniformity in survey administration across the entire sample , participants were instructed to respond to the sf-36 survey first , followed by the ces - d questionnaire . reminder e - mails were sent a week following the initial online dispatch of the survey instruments . the sample contained individuals with a range of background education and reported themselves to be in a good to excellent physical health . the 36 items in sf-36 measure eight health concepts ( or constructs ) and health transition ( physical function , role function , bodily pain , vitality , social functioning , role - emotional , mental health , general health , and health transition ) . the instrument also generates two summary measures of health , namely , physical component score ( pcs ) and mental component score ( mcs ) , which together represent all the eight sf-36 domains . the scale items were scored using likert 's method of summated ratings , where multi - item scale score is computed by simply summing the scores assigned to item responses and by transforming scores to a 0100 range . in addition to eight domain scores , summary physical and mental component scores ( pcs and mcs ) were also computed . cutoff scores of < 52 on the mental health ( mh ) subscale were used to identify older adults at risk for depression . in order to facilitate easy data interpretation , the publishers of sf-36 survey tools recommend the use of normative data obtained from studies conducted in general populations , which are then used as benchmarks against which to compare observed scores and changes in scores . this comparison is called norm - based interpretation of sf-36 data and enables ready interpretation of the burden of disease . for example , the lower the observed score for a patient from the norm is , the greater the burden of disease is . useful published norms are available with adjustments for age , gender , and medical conditions . the 20-item ces - d scale is designed to measure the depressive mood in the general population . the respondents are asked to rate the frequency , over the past week , of 20 symptoms of depression by choosing one of four response options ranging from rarely or none of the time to most or all of the time . scores range from 0 to 60 , where a score greater than 60 indicates impairment . in addition to the two instruments , data on respondents ' sociodemographics , medication use , and arthritis - related care were also gathered . the data analysis was carried out with the aid of spss ( version 11.0 ) statistical software package . descriptive univariate statistics were used to analyze crucial study variables and bivariate associations among different health status domains , and ces - d score was determined using pearson 's correlations , using a priori significance level of 0.05 . survey responses were retrieved electronically following the 2-week deadline and stored in a spreadsheet format . the surveys were then appropriately coded according to the scoring guidelines recommended for sf-36 and ces - d . incomplete surveys or questionnaires with missing values for more than half of the items on each subscale of the sf-36 questionnaire were discarded . similarly , for the ces - d survey , those who failed to answer at least 17 of the 20 items were discarded . a response rate of 87% ( n = 480 ) was achieved following elimination of incomplete and/or unusable surveys . a majority of the older adults were white ( 95.5% ) , female ( 52.9% ) , married ( 70.9% ) , and belonged to the age group of 6574 years ( 57.3% ) . about 42.3% of the sample ( n = 203 ) reported having osteoarthritis , about 16.5% ( n = 79 ) had rheumatoid arthritis , 12.7% osteoporosis ( n = 61 ) , and about 29% ( n = 137 ) had diagnosis of other rheumatic conditions . table 2 provides information on health status scores of arthritic individuals on the eight sf-36 domains . table 3 describes group differences on summary component scores between male and female respondents and between two age groups . however , the difference was statistically significant ( p < 0.05 ) between the two groups for only mcs scores . respondents aged 75 and over exhibited poorer physical and mental component scores than the respondents in the younger age group . the potential range of the ces - d scale is 060 ; the range for our sample was 057 . table 4 presents differences in ces - d scores on gender and age variables . according to the student t - test analysis , respondents aged 75 and over appeared to be significantly more depressed ( p < 0.05 ) than the rest of the sample . using cesd cutoff score of > 16 , 16% ( n = 76 ) of the respondents were found to be at risk for clinical depression , as opposed to only 4% ( n = 19 ) of the subjects who scored below the cut point ( < 52 ) on the mh subscale of sf-36 . as expected , a strong , negative correlation ( r = 0.68 , p < 0.05 ) was found between the ces - d total scores and mh subscale of sf-36 . further , analysis of variance ( anova ) was performed to examine overall group differences on sf-36 variables and between different demographic subgroups to identify variables where differences originated ( table 5 ) . data showed significant group differences on pcs component scores among subgroups based on marital status , education , and personal income ( p < 0.05 ) . anova showed that income and education contributed more to between - group differences than differences based on other sociodemographic factors . significant differences on mental health status scores were obtained only for different levels of educational qualification ( p < 0.05 ) . we performed a series of partial correlations between depression and health status scores to investigate whether age was the factor in contributing to the relationship between health and depression . based on the cutpoint defined for ces - d , we identified 16% of the older people as the group that is very likely at risk for a disorder requiring further medical attention . it must be noted here that these individuals probably had diagnosable psychiatric disorders and depression , even though diagnosis of depression per se might not be ruled out . the prevalence rates found in this study of 16% is similar to the rates found in the previous studies [ 26 , 27 ] . the number we have witnessed here is significant and calls for primary care providers to be more vigilant when caring for elderly patients with arthritis , as depression seems to be largely undiagnosed in this group . according to one report , the prevalence of significant depressive symptoms in medically ill elderly and residents of long term - care residents may be as high as 50% . the numbers we obtained in our study are not as alarming but do stress the need for early detection and diagnosis of depression and prompt treatment . therefore , from health services perspective , primary care can be an appropriate setting for arthritis care so as to minimize unnecessary utilization of health care resources when arthritis is concomitant with depression in elderly individuals . one good primary care strategy might be to identify individuals having arthritis who exceed the threshold on the ces - d scale and provide them with appropriate care so as to minimize mental health services use and pharmacotherapy necessary to manage care . lower mh and mcs scores for both men and women aged 75 years and over showed that older adults in this group were more likely to report symptoms of depression than people in younger age groups . further , older men and women with arthritis were more likely to report symptoms of depression than older men and women with no chronic condition , as evidenced by the comparison of our study population with the benchmarks obtained from the general elderly u.s . population . it appears that the relationship between physical health and functioning and depressive symptoms may have occurred as a result of association between items representing somatic symptoms on the ces - d scale and physical illness . it is not known if depression in our sample was caused by the physical illness alone , older age , or both . even though depression can be a reaction to physical illness , it can also cause physical impairment due to what can be regarded as a cyclical relationship between the two . given the nature of these complex interrelationships , it is often possible to overlook cases of depression in older people who might tend to attribute their depressive symptoms to physical illness alone . available evidence seems to indicate that that physical illness and depression might be intricately linked . according to one study , for patients who were managed under a comprehensive depression management program comprising antidepressant medications and multiple sessions of psychotherapy , improved primary care treatment of depression for the elderly not only reduced depressive symptoms but also reduced arthritis pain and improved quality of life as well . the special program developed in this study for patients with arthritis and concurrent depression produced significant improvement over the usual care group in functional impairment , general self - assessed health status , and quality of life . improvement in both arthritis pain and depression among patients in the depression management program was seen at the same time , suggesting a strong relationship between the two outcomes . because of the cross - sectional nature of our data , it is impossible to draw inferences concerning the direction of causality with regard to physical illness and depression in our study . from a clinical perspective , the best approach at treatment seems to be one that combines depression screening and a systematic , ongoing assessment of pain in patients with arthritis in order to maximize functional status and quality of life , especially when high comorbidity of arthritis and late - life depression exist . the mental health subscale ( mh ) of the sf-36 and its cut - offs seem to perform somewhat satisfactorily in the detection of depression . however , the fact that mh scale identified fewer cases of depressive mood than a more reliable and valid ces - d raises questions about the effectiveness of sf-36 as an independent depression screener . one reason for the underestimation of depression in our sample by the sf-36 scale ( compared to an established ces - d ) may be that it contains fewer items that measure symptoms of depression . therefore , underrepresentation of depression items in the instrument at this time makes it less preferable for the assessment of depression along with health status . it can be argued that sf-36 was not designed with a purpose to measure depression , even though the instrument , when originally conceived and developed , embodied many of the concepts represented by the well - known geriatric depression instruments available at the time , including ces - d . we did not perform roc analyses in this study to assess true positive ( sensitivity ) and false positive rates ( specificity ) of the full range of mh and its cutoffs in detecting depression . doing so would have perhaps provided a more definite word on the utility of sf-36 in depression measurement . in absence of such efforts , however , caution should be exercised while using sf-36 as a sole screener for depression . at best , mh cutpoint scores should serve as first stage screen for further testing and not the final word on the diagnosis of depression . more research is warranted to explore possibilities for sf-36 to be used as a tool in the identification and treatment of depression . results from this study can also help guide future research on predictors and risk factors of depression concurrent to a physical illness and the value of early detection and intervention in arthritis management . we conclude that older individuals having arthritis are at a moderate risk of clinical depression based on their mental health status . from health services perspective , a significant decline in health in all the domains of arthritis - affected older persons , particularly in mental and social functioning domains , when compared to the general population should be an area of concern . early recognition by primary care specialists of depressive symptomatology and its prompt treatment is essential to lessen the public health burden arising from the comorbidity with arthritis . more evidence is needed to support the use of sf-36 as a depression screener as it appears to perform inadequately compared to an established depression scale in the identification of population at risk for depression .
small rna - seq raw data has been deposited in the ddbj sequence read archive ( dra ) under accession numbers dra003930 and dra004107 ( cc-124 replicate # 1 , drx040414 ; cc-124 replicate # 2 , ccdrx040415 ; gluc1( ) replicate # 1 , drx040416 ; gluc1( ) replicate # 2 , drx040417 ; ago31 replicate#1 , drr045098 ; ago31 replicate#2 drr045099 ; dus161 replicate # 1 , drx043778 ; and dus161 replicate # 2 , drx043779 ) . we would like to thank the functional genomics facility , nibb core research facilities for technical support . this work was supported by nibb collaborative research program 15103 ( to t.y . ) , jsps grant - in - aid for young scientists ( b ) 16k18480 ( to t.y . ) , and a grant from the national science foundation ( to h.c . ) .
laparoscopic ventral hernia repair has grown in popularity since it was first reported in the early 1990s . lower recurrence rates , fewer complications , and shorter hospital stays have led some to believe that it sets the new standard of care for ventral hernia repair . controversy exists regarding the optimal method to fix the prosthetic mesh to the anterior abdominal wall . currently , the 2 most popular methods of mesh fixation are via transabdominal sutures and laparoscopic tacks . sutures pass through all layers of the fascia and muscle of the anterior abdominal wall , while tacks secure the mesh to the innermost millimeters of the peritoneal cavity . most controversy in laparoscopic repair centers on the tensile strength of the mesh fixation method . postoperative pain produced by the securing methods is another consideration in deciding between sutures and tacks . this study compares these 2 methods and examines the consequential pain that occurs after each type of fixation . from 2004 through 2006 , patients undergoing laparoscopic ventral hernia repair by 8 different surgeons at the mount sinai medical center were prospectively enrolled in the study . the patients were sorted into 2 groups : those undergoing hernia repairs primarily with transabdominal sutures ( sutures group ) and those undergoing hernia repairs primarily with tacks ( tacks group ) . patients in the sutures group had repairs with transabdominal sutures placed circumferentially approximately 2 cm to 3 cm apart . these patients typically had 10 to 20 sutures placed , depending on the size of hernia . patients in the tacks group included those with hernias completely repaired with only tacks and repairs that may have involved 4 stay sutures with the rest of the mesh secured to the anterior abdominal wall with tacks . choice of repair was made by surgeon preference , including type of mesh and type of tacks . telephone follow - up was used to determine verbal pain scores at 1 week , 1 month , and 2 to 3 months postoperatively ( 0=pain free , 10=excruciating pain / worse pain ever ) . in addition , patients were asked regarding time to return to work and need for narcotic pain medications . informed consent was obtained , and this study was approved by the institutional review board . we needed to enroll 50 patients into the study to detect a 50% difference in pain scores ( power 80% , level of significance p=0.05 ) . no significant difference was found between the groups in terms of proportion of patients with recurrent hernias , multiple hernia defects , and total defect size . the type of mesh used was surgeon dependant and was variable across both groups . demographics and hernia characteristics table 2 shows the operative and postoperative characteristics of the 2 groups . both groups were similar in operative time . the tacks group had a longer length of postoperative hospital stay ( 2.4 vs 1.7 days ) ; however , this difference was not statistically significant . operative and postoperative characteristics differences between groups were determined by two tailed student 's t - tests . no difference was reported in mean pain scores between the 2 groups at 1 week , 1 month , and 3 months ( p>0.05 ) . on a scale of 0 to 10 , patients from both groups had moderate pain 1 week after the operation . pain scores in both groups decreased at 1 month and were minimal by 2 to 3 months . in addition , use of narcotic pain medications during the postoperative period was similar in both groups ( table 3 ) . mean postoperative pain scores at 1 week , 1 month , and 2 to 3 months after laparoscopic ventral hernia repair . no difference in pain score existed between groups ( p>0.05 , 2-tailed student t test ) . many proponents of the use of transabdominal sutures cite lower recurrence rates due to higher tensile holding strengths of sutures in comparison to tacks . other authors argue that the use of tacks reduces surgical time considerably while maintaining similar recurrence rates . these authors also argue that the use of tacks significantly reduces postoperative pain . to date , most studies of mesh fixation during laparoscopic ventral hernia repair focus on the risk of recurrence . however , this is the only study that compares postoperative pain after hernia repair with sutures versus tacks . in addition , numerous sutures are typically needed around the perimeter of the hernia defect . because mesh overlap on normal muscular fascia is usually aimed for around 3 cm to 5 cm , we found no difference in postoperative pain in patients undergoing hernia repair with sutures or tacks . both groups had moderate pain at one week and minimal pain on further follow - up . it is possible that early pain caused by multiple tacks penetrating the parietal peritoneum is equivalent to the pain caused by transfascial sutures . in the long - term , cobb et al has also proposed that intercostal nerves may become entrapped within the transabdominal sutures causing chronic , persistent neuropathic pain . series of repairs using transfascial sutures report persistent pain and discomfort in 1% to 6% of patients . most authors feel oral anti - inflammatory medications or injections of a local anesthetic can alleviate the symptoms in the majority of cases . others have reported re - explorations for persistent pain , finding immediate relief after the release of a suture from the site of symptoms . none of the patients in our study had persistent pain severe enough to undergo local anesthetic injection or reoperation . the reports of persistent cases of pain seem to be isolated at one particular suture site , supporting the nerve entrapment theory . pain from muscle ischemia would seem to be more generalized at all of the suture sites . our data suggest that both methods of mesh fixation are generally not different in terms of their resultant postoperative pain . however , because our study only included 50 patients , occasional episodes of chronic persistent pain due to nerve entrapment are certainly possible if more patients were followed . our findings are somewhat consistent with those of leblanc et al , whose study noted that patients in the earlier half of their series had more pain . these patients had fewer sutures used , suggesting the use of these sutures was unrelated to postoperative pain . though the use of laparoscopic tackers may seem to be simpler and faster , we did not find a significant difference in operative time between the 2 fixation methods . this is contrary to the general opinion that the use of tacks reduces surgical time . operative time during laparoscopic ventral hernia repair significantly involves extensive adhesiolysis and dissection of peritoneal contents from the anterior abdominal wall . conceivably , surgeons may misinterpret the amount of time spent on the different phases of the operation and focus on time spent on mesh fixation . in our study , we did not specifically look at operative time during different components of the operation . . therefore , the assumption that repair with transabdominal sutures takes longer than tack repair remains largely unproven . however , large differences should be found . considering that most anecdotal evidence suggests a large difference in pain experience in addition , although data were prospectively recorded , the patients in this study were not randomized to treatment arms . the type of repair was based on surgeon preference , as each had his or her own strong feeling regarding the best method of fixation . larger controlled trials may be necessary to optimally determine which method contributes to the most pain . patients undergoing laparoscopic ventral hernia repair with primarily transabdominal sutures or tacks experience similar overall postoperative pain . symptoms are moderate by the end of the first postoperative week and mild by 1 month . occasional episodes of chronic , persistent suture site pain are possible and have been reported . postoperative pain should be a minor factor when deciding between repair with sutures or tacks in laparoscopic ventral hernia repair .
tyrosinase is a kind of oxidase enzyme that involves in reactions of melanin synthesis . in food industry , the inhibition of tyrosinase is very important , because this enzyme catalyzes the oxidation of phenolic compounds found in fruits and vegetables into quinones , which contribute in undesirable color and taste of fruits and vegetables . arbutin , kojic acid , and hydroquinones have been reported to have tyrosinase inhibitory activity . also , they have been used in cosmetic industry as whitening composition ( 1 , 2 ) . the genus teucrium consists of more than 340 species widely distributed around the world ( south - west of asia , north of africa , and south and north - east of iran . ) . the main compounds reported from the genus teucrium include terpenoids and flavonoids ( 3 , 4 ) . it has been long used in iranian traditional medicine to treat stomach disorders , malabsorption , grippe , cold , and was reputable for having hypoglycemic , anti - hyperlipi - demia ( 3 ) , diuretic , analgesic , antipyretic , anti - spasmodic , anti - inflammatory and anti - hypertensive properties . all species of this genus have been showed considerable hypoglycemic and anti - hyperlipidemia properties ( 5 ) . a literature review shows that there are a large number of phytochemical studies on the genus teucrium including t. polium . different parts of plants from the genus teucrium have been reported to have monoterpenes ( 6 ) , sesquiterpenes ( 7 ) , polyphenols and flavonoids such as apigenin and rutin ( 8 , 9 ) and some fatty acids and steroids such as -sitosterol and stigmasterol . gnaphalodes and its traditional applications , that are similar to those of t. polium , we aimed to characterize its components and primarily evaluate their antioxidant and anti - tyrosinase properties . in this study , we reported the isolation and structure elucidation of the main constituents of t. polium var . gnaphalodes , and antioxidant ( frap and dpph test ) and anti - tyrosinase ( mushroom tyrosinase inhibition assay ) activities of the main compounds . preparative hplc - dad was performed on a knauer liquid chromatograph system consisting of a quaternary pump ( smartline pump 1000 ) . detection was carried out using uv / vis diode array detector ( smartline dad 2800 ) , and data were processed using ez chrom elite software . the fractions were subjected to reverse - phase hplc using a gradient method of 20 - 100 % methanol in water as the eluent including 0.05 % trifluoroacetic acid . the h - nmr spectra of the isolated compounds were recorded at 30 c on a varian 600 h - nmr spectrometer . the h - nmr chemical shifts were referenced to the solvent peaks at h 2.50 ppm . gnaphalodes were collected in october from north of iran , firoozkouh , alborz mountains , 2200 meters height . the plant material was identified by mohammad reza joharchi . a voucher specimen ( no . 11377 ) has been deposited at the herbarium of school of pharmacy , mashhad university of medical sciences . aerial parts have been dried in room temperature and then have been finely powdered by a miller . the powdered aerial parts ( 250 g ) were extracted in 500 ml of methanol at room temperature for three times each for 24 hr by maceration method . after concentration of extracts with a rotary evaporator and completion of drying of them with a freeze dryer , the obtained extract ( 37 g ) has been conserved in the refrigerator . 10 g of the dried extract was loaded on silica gel column chromatography ( 5 50 cm , normal phase ) . the column has then been eluted by hexane and then gradual adding of ethyl acetate and methanol to increase mobile phase polarity . the obtained fractions ( 200 ml each ) were compared by tlc and those giving similar spots were combined . , b and c were subjected to more purification via hplc apparatus ( c-18 reversed phase with methanol : water solvent system ) to obtain pure compound of 1 ( 21 mg ) , 2 ( 9.3 mg ) , 3 ( 101.4 mg ) and 4 ( 10.4 mg ) . purification of fractions was carried out using an ace 5 c18 ( 5 m , 250 21.2 mm ) at a flow rate 9 ml / min and linear gradient conditions of 20 % - 100 % meoh ( 0.05 % tfa ) within 20 min , followed by an isocratic condition of meoh ( 0.05 % tfa ) for 5 min . dpph is a stable radical that is used in a popular method for screening free radical - scavenging ability of compounds or antioxidant activity of plant extracts ( 10 ) . free radical scavenging activity was evaluated by measuring the scavenging activity of the compounds in the solution of 2 , 2-diphenyl-1-picrylhydrazyl ( dpph briefly , a 0.3 mm solution of dpph in ethanol was prepared . an aliquot ( 50 l ) of samples ( at four different concentrations ( g / ml ) were added to 150 l of the dpph solution in each well of a 96-well plate . for blank , the decrease in absorbance was measured at 515 nm after 30 min of incubation at 37 c using the biotek micro plate reader ( synergy h4 , usa ) . all tests were performed in triplicate ( 11 ) and the data presented as mean of the three values . when a solution of dpph is mixed with that of a substance , this gives rise to the reduced form diphenylpicrylhydrazyl with the loss of this violet color . the ic50 values were calculated as the concentration of extracts causing a 50% inhibition of dpph radical . frap reagent was prepared by adding 10 ml of acetate buffer 300 mm , ph 3.6 ( 3.1 g sodium acetate trihydrate ) , to 1.0 ml of ferric chloride hexahydrate 20 mm ( dissolved in distilled water ) and 1.0 ml of 2,4,6-tri-(2-pyridyl)-s - triozine ( tptz ) 10 mm ( dissolved in hcl 40 mm ) . in a well of a 96-well plate , an aliquot ( 10 l ) of sample ( at five different concentrations ( g / ml ) was added to 190 l of the frap solution . after 30 min of incubation at 37 c , absorbance of the reaction mixture was measured at 593 nm using biotek micro plate reader ( synergy h4 , usa ) . all tests were carried out in triplicate . melanin , which is secreted by melanocyte cells , is the major pigment for color of human skin . tyrosinase , a copper - containing monooxygenase , is a key enzyme that catalyzes melanin synthesis in melanocytes . inhibiting this enzyme may be the least invasive procedure for maintaining skin whiteness ( 10 ) . the l - dopa oxidation activity of tyrosinase was measured by spectrophotometry as described previously with some modifications . briefly , 160 l of 5 m l - dopa ( in 100 m sodium phosphate buffer ph 6.8 ) and 20 l of the same buffer with and without the test sample were placed in the wells of a 96 micro plate , and then 20 l of mushroom tyrosinase ( 200 units / ml ) were mixed into each well at 37c over 30 min . the amount of dopachrome produced in the reaction mixture was measured at 475 using the micro plate reader . kojic acid was used as positive control of tyrosinase inhibitor . the 50 % inhibition of tyrosinase activity ( ic50 ) preparative hplc - dad was performed on a knauer liquid chromatograph system consisting of a quaternary pump ( smartline pump 1000 ) . detection was carried out using uv / vis diode array detector ( smartline dad 2800 ) , and data were processed using ez chrom elite software . the fractions were subjected to reverse - phase hplc using a gradient method of 20 - 100 % methanol in water as the eluent including 0.05 % trifluoroacetic acid . the h - nmr spectra of the isolated compounds were recorded at 30 c on a varian 600 h - nmr spectrometer . the h - nmr chemical shifts were referenced to the solvent peaks at h 2.50 ppm . gnaphalodes were collected in october from north of iran , firoozkouh , alborz mountains , 2200 meters height . the plant material was identified by mohammad reza joharchi . a voucher specimen ( no . 11377 ) has been deposited at the herbarium of school of pharmacy , mashhad university of medical sciences . aerial parts have been dried in room temperature and then have been finely powdered by a miller . the powdered aerial parts ( 250 g ) were extracted in 500 ml of methanol at room temperature for three times each for 24 hr by maceration method . after concentration of extracts with a rotary evaporator and completion of drying of them with a freeze dryer , the obtained extract ( 37 g ) has been conserved in the refrigerator . 10 g of the dried extract was loaded on silica gel column chromatography ( 5 50 cm , normal phase ) . the column has then been eluted by hexane and then gradual adding of ethyl acetate and methanol to increase mobile phase polarity . the obtained fractions ( 200 ml each ) were compared by tlc and those giving similar spots were combined . then fractions a , b and c were subjected to more purification via hplc apparatus ( c-18 reversed phase with methanol : water solvent system ) to obtain pure compound of 1 ( 21 mg ) , 2 ( 9.3 mg ) , 3 ( 101.4 mg ) and 4 ( 10.4 mg ) . purification of fractions was carried out using an ace 5 c18 ( 5 m , 250 21.2 mm ) at a flow rate 9 ml / min and linear gradient conditions of 20 % - 100 % meoh ( 0.05 % tfa ) within 20 min , followed by an isocratic condition of meoh ( 0.05 % tfa ) for 5 min . dpph is a stable radical that is used in a popular method for screening free radical - scavenging ability of compounds or antioxidant activity of plant extracts ( 10 ) . free radical scavenging activity was evaluated by measuring the scavenging activity of the compounds in the solution of 2 , 2-diphenyl-1-picrylhydrazyl ( dpph briefly , a 0.3 mm solution of dpph in ethanol was prepared . an aliquot ( 50 l ) of samples ( at four different concentrations ( g / ml ) were added to 150 l of the dpph solution in each well of a 96-well plate . for blank , the decrease in absorbance was measured at 515 nm after 30 min of incubation at 37 c using the biotek micro plate reader ( synergy h4 , usa ) . all tests were performed in triplicate ( 11 ) and the data presented as mean of the three values . when a solution of dpph is mixed with that of a substance , this gives rise to the reduced form diphenylpicrylhydrazyl with the loss of this violet color . the ic50 values were calculated as the concentration of extracts causing a 50% inhibition of dpph radical . frap reagent was prepared by adding 10 ml of acetate buffer 300 mm , ph 3.6 ( 3.1 g sodium acetate trihydrate ) , to 1.0 ml of ferric chloride hexahydrate 20 mm ( dissolved in distilled water ) and 1.0 ml of 2,4,6-tri-(2-pyridyl)-s - triozine ( tptz ) 10 mm ( dissolved in hcl 40 mm ) . in a well of a 96-well plate , an aliquot ( 10 l ) of sample ( at five different concentrations ( g / ml ) was added to 190 l of the frap solution . after 30 min of incubation at 37 c , absorbance of the reaction mixture was measured at 593 nm using biotek micro plate reader ( synergy h4 , usa ) . dpph is a stable radical that is used in a popular method for screening free radical - scavenging ability of compounds or antioxidant activity of plant extracts ( 10 ) . free radical scavenging activity was evaluated by measuring the scavenging activity of the compounds in the solution of 2 , 2-diphenyl-1-picrylhydrazyl ( dpph briefly , a 0.3 mm solution of dpph in ethanol was prepared . an aliquot ( 50 l ) of samples ( at four different concentrations ( g / ml ) were added to 150 l of the dpph solution in each well of a 96-well plate . for blank , the decrease in absorbance was measured at 515 nm after 30 min of incubation at 37 c using the biotek micro plate reader ( synergy h4 , usa ) . all tests were performed in triplicate ( 11 ) and the data presented as mean of the three values . when a solution of dpph is mixed with that of a substance , this gives rise to the reduced form diphenylpicrylhydrazyl with the loss of this violet color . the ic50 values were calculated as the concentration of extracts causing a 50% inhibition of dpph radical . frap reagent was prepared by adding 10 ml of acetate buffer 300 mm , ph 3.6 ( 3.1 g sodium acetate trihydrate ) , to 1.0 ml of ferric chloride hexahydrate 20 mm ( dissolved in distilled water ) and 1.0 ml of 2,4,6-tri-(2-pyridyl)-s - triozine ( tptz ) 10 mm ( dissolved in hcl 40 mm ) . in a well of a 96-well plate , an aliquot ( 10 l ) of sample ( at five different concentrations ( g / ml ) was added to 190 l of the frap solution . after 30 min of incubation at 37 c , absorbance of the reaction mixture was measured at 593 nm using biotek micro plate reader ( synergy h4 , usa ) . melanin , which is secreted by melanocyte cells , is the major pigment for color of human skin . tyrosinase , a copper - containing monooxygenase , is a key enzyme that catalyzes melanin synthesis in melanocytes . inhibiting this enzyme may be the least invasive procedure for maintaining skin whiteness ( 10 ) . the l - dopa oxidation activity of tyrosinase was measured by spectrophotometry as described previously with some modifications . briefly , 160 l of 5 m l - dopa ( in 100 m sodium phosphate buffer ph 6.8 ) and 20 l of the same buffer with and without the test sample were placed in the wells of a 96 micro plate , and then 20 l of mushroom tyrosinase ( 200 units / ml ) were mixed into each well at 37c over 30 min . the amount of dopachrome produced in the reaction mixture was measured at 475 using the micro plate reader . the 50 % inhibition of tyrosinase activity ( ic50 ) was calculated by the use of prism graph pad software . normal - phase column chromatography of the methanol extract of aerial parts , followed by semi - preparative hplc , afforded four pure known compounds including isorhoifolin ( 1 ) , jaranol ( 2 ) , poliumoside ( 3 ) and verbascoside ( 4 ) . jaranol showed the highest tyrosinase inhibitory activity . on the other hand , poliumoside was the best antioxidant among the tested compounds . it was clear from the h and c nmr data ( table 1 ) that compound 1 consisted of a flavone nucleus and two sugar moieties . h - nmr and c - nmr data of compounds 1 and 2 ( 600 mhz , dmso - d6 ) the h - nmr spectrum revealed the presence of glucose and rhamnose . the glycoside structure and its connectivity were confirmed by a hmbc experiment , which showed long - range correlations between the signals of h-1 and c-7 and h-1 and c-6 . the -configuration of the anomeric glucose unit was assigned on the basis of its typical coupling constant , j = 7.8 hz . the spectrum showed four aromatic protons appearing as two doublets at 7.93 with ( j= 8.8 hz ) and 6.94 with ( j= 8.8 hz ) due to ortho - coupled protons , that were assigned to h-2 , 6 and h-3 , 5 , respectively ( figure 1 ) . also a singlet at 6.83 ppm corresponding to h-3 , two protons broad singlet at 6.75 and 6.43 ppm , assigned to h-8 and h-6 , respectively . from these data and by comparing them with the published data ( 12 ) , compound 1 was identified as isorhoifolin . the h and c nmr data of 2 showed a bi - methoxylated flavone structure for compound 2 ( table 1 ) . the aromatic part of the h nmr spectra of compound 2 suggested the presence of a flavone structure . typical h nmr signals of two methoxy groups at 3.72 and 3.91 correlated in the hmbc spectrum to the c nmr signals at 132.3 ( c-3 ) and 159.0 ( c-7 ) , respectively , suggesting that methoxyl groups were located at c-3 and c-7 . the spectrum also showed four aromatic protons appearing as two doublets at 7.94 with ( j = 8 hz ) and 6.91 with ( j = 8 hz ) due to ortho - coupled protons , that were assigned to h-2 , 6 and h-3 , 5 , respectively ( figure 1 ) . the meta position protons of ring a were also appeared as two singlets in 6.82 and 6.90 ppm . the other h and c nmr data of compound 2 were in agreement with those of jaranol ( syn . h and c - nmr data indicated the presence of three sugar moieties , due to the presence of signals at c 102.8 with h 4.35 ( d , j = 7.9 hz ) for -glucose moiety and at c 101.6 with h 5.01 ( d , j = 1 ) and c 100.9 with h 4.47 ( d , j = 1 ) for rhamnose moieties . the h nmr spectral data also revealed the presence of trans - olefinic protons attached to tri - substituted benzene ring along with the carbonyl carbon ( trans - caffeoyl moiety ) . the presence of trans - caffeoyl moiety was indicated from the signals at h 7.44 ( h-3 , j = 15.7 hz ) and h 6.17 ( h-2 , j = 15.7 hz ) for trans double bond and h 7.01 ( h-5 ) , 6.73 ( h-8 ) and 6.95 ( h-9 ) for the tri - substituted benzene ring , which was supported by the c nmr spectrum . from the above mentioned data and by comparing these data with the published data ( 15 - 17 ) , compound 3 was identified as poliumoside . the h - nmr and c - nmr spectral data of 4 were very similar to those of 3 ( table 2 ) except for the loss of one of rhamnose moieties . therefore , compound 4 was assumed to have a disaccharide structure . by comparing these data with the published data ( 18 ) , the nmr data of the compounds 1 - 4 has been summarized in tables 1 and 2 . the structure assignments of four compounds were also supported by other 2d nmr experiments including hsqc and hh - cosy . h - nmr and c - nmr data of compounds 3 and 4 ( 600 mhz- dmso - d6 ) as shown in table 3 , poliumoside ( 3 ) possessed the highest antioxidant capacity in dpph test with an ic50 of 4.23 g / ml . in frap assay , it should be pointed out , however , all four compounds , but not jaranol , showed antioxidant activity comparable to the positive control -tocopherol . jaranol exhibited the lowest activity among the tested compounds due to the blockage of its two active hydroxyl groups with methyl groups . in contrast , jaranol showed the most tyrosinase inhibitory activity with an ic50 value of 0.04 mm , comparable with the ic50 of kojic acid as positive control ( table 4 ) . gnaphalodes possessed antioxidant compounds with anti - tyrosinase activity , particularly jaranol as potent tyrosinase inhibitor . antioxidant activities of compounds 1 - 4 using frap and dpph radical - scavenging activity mushroom anti - tyrosinase activity of compounds 1 - 4 during last decade , much interest has been att - racted to natural and synthetic phenylpropanoids for medicinal use as antioxidant , uv screens , anticancer , antiviral , anti - inflammatory , wound healing , and antibacterial agents . they are of great interest for cosmetic and perfume industries as active natural ingredients ( 19 ) . phenylethanoid glycosides ( or phenylpropanoid glycosides ) such as verbascoside and poliumoside are structurally characterized with a hydroxyl - phenylethyl moiety attached to a - glucopyranose through glycosidic linkage . the core structures are often inundated with substituents such as aromatic acids like cinnamic and caffeic acids , and various sugars such as rhamnose , xylose and arabinose attached to the glucose residue through ester or glycosidic linkages . several pharmacological studies have shown that these compounds possess a broad spectrum of biological activities including antibacterial , antitumor , antiviral , anti - inflammatory , antioxidant and tyrosinase inhibitory actions ( 20 ) . studies have also demonstrated that both the number and position of the phenolic hydroxyls play an important role in the antioxidative activity of phenylpropanoid glycosides ( 21 ) . the length of methylene - chain connected to the benzene ring and the type of phenylpropanoid acids and sugar connected to the aglycone moiety , are major factors affecting the overall activity ( 20 , 22 , 23 ) . overproduced free radicals can often lead to oxidative stress that may result in oxidative injury and diseases such as cardiovascular diseases , retinal ischemia and neurodegenerative diseases ( 20 ) . together with searching new antioxidants , there has been increasing interest in replacing synthetic antioxidants with natural ones for safety concern ( 24 ) . verbascoside [ first isolated from verbascum sinuatum ( scrophulariaceae ) in 1963 ( 25 ) ] showed considerable antibacterial activities against of s. aureus strains with the minimum inhibitory concentration ( mic ) values ranging from 64 poliumoside , has also significantly attenuated glutamate - induced neurotoxicity at concentrations ranging from 0.1 to 10 m . these compounds , having a caffeoyl moiety , showed stronger neuroprotective activity than those of unsubstituted phenylethanoid glycosides ( 27 ) . the role of melanin is to protect the skin against uv light damage by absorbing uv sunlight and removing reactive oxygen species . many tyrosinase inhibitors find applications in cosmetic products for whitening and depigmentation after sunburn and in the treatment of dermatological disorders related to melanin hyperpigmentation . due to the harmful effects of commercial skin whitening , natural compounds have been noticed . among them , phenolic compounds seem to be potent agents ( 29 ) . flavonoids are a large class of natural phenolics with various biological activities that occur in many fruits and vegetables ( 30 - 34 ) . the studies showed that the presence of the 3 , 3 and 4-hydroxyl group in flavonoids was essential for high anti - tyrosinase activity , whereas , the presence of a methoxyl group at the c-4 and c-7 position tended to reduce the anti - tyrosinase activity . since flavones showed higher antityrosinase activities than flavanones , it was concluded that the presence of the c2c3 double bond is also essential for tyrosinase inhibitory ability ( 35 ) . the mechanism of tyrosinase inhibition of flavonoids might also be due to chelating with copper in the active center of tyrosinase enzyme ( 30 ) . the inhibitory activity of phenylethanoid glycosides could be attributed to the presence of ortho - hydroxyls on the phenolic rings , which give them a property to chelate with metals ( 29 ) . no phytochemical study has been conducted on this variety of t. polium . in this study , we have isolated and characterized four compounds including two phenylpropanoid glysosides ( verbascoside and poliumoside ) and two flavonoids ( jaranol and isorhoifolin ) from this plant for the first time . jaranol showed the highest tyrosinase inhibitory activity . on the other hand , poliumoside was the best antioxidant among the tested compounds tyrosinase inhibitors of this plant , particularly jaranol , have a potential of application like other tyrosinase inhibitors in cosmetic and food industries .
the story of sarcopenia has a rather recent beginning , although a search of the terms muscle wasting and sarcopenia in the online database pubmed buttresses its growing importance over the last years ( fig . involutional changes of the musculature were described as early as 1931 by macdonald critchley , then junior neurologist at king s college hospital in london , who wrote that the entire musculature tends with advancing age to undergo involutional changes , which are manifested as wasting . he went on saying that probably the chief cause of this change is to be demonstrated in the general process of senile atrophy , which shows itself in the muscles and elsewhere . later on , in the 1970s , nathan shock published a series of articles on age - related physiologic functions using data from the first large - scale longitudinal study in this field . altogether , it evolved that no decline in structure and function is more dramatic than the decline in muscle mass that develops as we age . irwin rosenberg realized that if this phenomenon was to be taken seriously , a name was required , and at a meeting in albuquerque , new mexico , in 1988 , he suggested to use the term sarcopenia . following the recommendation by morley , the term took hold over these last 20 years . 1number of pubmed entries retrieved after entering the search term muscle wasting or sarcopenia . assessed on 23 october 2012 from www.pubmed.gov number of pubmed entries retrieved after entering the search term the name sarcopenia is derived from greek sarx ( flesh ) and penia ( loss ) , literally meaning poverty of flesh . sarcopenia is one of the four main reasons for loss of muscle mass , the others being anorexia , dehydration , and cachexia [ 5 , 6 ] . it is difficult to estimate the prevalence of sarcopenia ( table 1 ) , mostly because of practical difficulties in assessing muscle mass . many different methodologies have been used over the last 20 years , and new techniques are still being introduced . on average , it is estimated that 513 % of elderly people aged 6070 years are affected by sarcopenia , and the numbers increase to 1150 % for those aged 80 or above . in line with these data , other sources estimate that 840 % of elderly people above the age of 60 years are affected by sarcopenia . sarcopenia may lead to frailty , but not all patients with sarcopenia are frail . in essence , sarcopenia is about twice as common as frailty .table 1prevalence of sarcopenia cohort ( country)n ( % female)agesarcopenia definition ( assessment method)sarcopenia prevalencereferencechs ( usa)5,036 ( 56.4 % ) > 65 yearscategories of skeletal mass index , defined as muscle mass normalized for height ( bia)moderate sarcopenia , m : 70.7 % , f : 41.9 % ; severe sarcopenia , m : 17.1 % , f : 10.7 % epidos ( france)1,458 ( 100 % ) all > 70 years ; mean 80.3 3.8 yearsappendicular skeletal muscle mass < 2 sd below the mean of a young female reference group ( dexa)9.5 % inchianti ( italy)1,030 ( 54.5)range : 20102calf muscle cross - sectional area more than 2 sd below population mean ( ct scan)m : 20 % at 65 years , 70 % at 85 years ; f : 5 % at 65 years , 15 % at 85 yearsnhanes iii ( usa)14,818>18 years ; 30 % > 60 yearsskeletal mass index was defined as muscle mass / body mass x 100 ; sarcopenia class i defined as skeletal muscle mass 12 sd , sarcopenia class ii defined as skeletal muscle mass > 2 sd from the mean of young subjects ( bia)in subjects aged > 60 years : sarcopenia class i , m : 45 % , f : 59 % ; sarcopenia class ii : m : 7 % , f : 10 % nmehs ( usa)808 ( 47.3 % ) m : 73.6 5.8 years ; f : 73.7 6.1 yearsappendicular skeletal muscle mass < 2 sd below the mean of a young reference population ( substudy of dexa)<70 years , m : 13.516.9 % , f : 23.124.1 % ; 7074 years , m : 18.319.8 % , f : 33.335.1 % ; 7580 years , m : 26.736.4 % , f : 35.335.9 % ; > 80 years , m : 52.657.6 % , f : 43.260.0 % bia bioelectrical impedance assessment , chs cardiovascular health study , ct computed tomography , dexa dual - energy x - ray absorptiometry , epidos european patient information and documentation systems , nhanes national health and nutrition examination survey , nmehs new mexico elder health study , sd standard deviation prevalence of sarcopenia bia bioelectrical impedance assessment , chs cardiovascular health study , ct computed tomography , dexa dual - energy x - ray absorptiometry , epidos european patient information and documentation systems , nhanes national health and nutrition examination survey , nmehs new mexico elder health study , sd standard deviation the broadness in the range of sarcopenia prevalence is partly due to the heterogenecity of study populations , but also due to the different techniques used to assess muscle mass . dual - energy x - ray absorptiometry ( dexa ) is currently considered the gold standard . the name is derived from the fact that two x - ray beams are used with different energy levels of minimal intensity . other methods used to measure muscle mass include bioelectrical impedance , computed tomography , magnetic resonance imaging , urinary excretion of creatinine , anthropometric assessments , and neutron activation assessments . depending on the actual technique used in different studies and on the cutoff values chosen , the prevalence of muscle mass may vary considerably ( table 1 ) . physical performance can be analyzed using simple and easy - to - do tests such as the short physical performance battery test , usual gait speed , the timed get - up - and - go test , or the stair climb power test . a combination of serum markers , diagnostic imaging , and functional tests of muscle function would constitute an ideal biomarker panel . a recent consensus statement acknowledges that the list of potential serum markers of sarcopenia is quite long . it embraces markers of inflammation ( e.g. , c - reactive protein , interleukin-6 , and tumor necrosis factor- ) , clinical parameters , urinary creatinine , hormones ( e.g. , dehydroepiandrosterone sulfate , testosterone , insulin - like growth factor-1 , and vitamin d ) , products of oxidative damage , or antioxidants . since all the aforementioned markers are rather indirect measures of muscle loss , novel serum markers directly associated with changes in skeletal muscle mass and function are also promising . the one mentioned in the consensus statement is procollagen type iii n - terminal peptide ( p3np ) . another interesting marker in this regard is c - terminal agrin fragment , a degeneration product of the neuromuscular junction [ 16 , 17 ] . using assessments of physical performance it became clear that aging is associated with changes not only in muscle mass but also in muscle composition , contractile , and material properties of muscle as well as in the function of tendons . therefore , recent consensus definitions include not only changes in muscle mass , but also changes in muscle function like exercise performance . in aging muscle , there is a loss of motor units via denervation . these denervated motor units are recruited by surviving motor units , which puts an increased burden of work on them . altogether , there is a net conversion of fast type ii muscle fibers into slow type i fibers with resulting loss in muscle power necessary for activities of daily living such as rising from a chair or climbing steps . these effects in contrast to cachexia do not lead to a net loss in body weight , but to a significant reduction in muscle strength . indeed , in healthy volunteers , the maximal velocity during cycle ergometry decreased by 18 % from the third age decade ( 2029 years ) to the sixth ( 5059 years ) . another 20 % were lost between the seventh ( 6069 years ) and the ninth age decade ( 8089 years ) . the loss of maximal oxygen consumption ( peak vo2 ) with increasing age has also been attributed to reduced muscle mass and cardiac output . our group has recently demonstrated that 19.5 % of a prospectively enrolled cohort of 200 patients with clinically stable chronic heart failure fulfilled the criteria of sarcopenia . among these patients , muscle wasting remained an independent predictor of reduced peak vo2 after adjusting for age , gender , new york heart association class , hemoglobin value , left ventricular ejection fraction , 6-min walk distance , and the number of co - morbidities . importantly , the risk of falls and consequently of fractures in elderly subjects is closely related to reduced muscle mass as well : another study found a 2.3-fold increase in the risk of falls after multivariable adjustment among patients in the lower third of handgrip strength as compared to the upper third . likewise , another large - scale study in more than 2,100 elderly subjects found that a low walking speed is an independent risk factor of falls . using the above knowledge , it does not come as a surprise that the likelihood of having a physical disability is higher in elderly subjects who present with height - adjusted appendicular muscle mass 2 standard deviations below the mean of young adult as compared to those with normal muscle mass . having said that , the difficulty in making a correct diagnosis of sarcopenia is easily understood . a consensus definition formulated by experts from a vast array of different medical fields recently suggested to diagnose sarcopenia when two criteria are fulfilled : ( 1 ) a low muscle mass and ( 2 ) a low gait speed . normal muscle mass is defined using data derived from young subjects aged 1839 years from the third nhanes population , and the requirement for a diagnosis of sarcopenia is the presence of a muscle mass 2 standard deviations below the mean of this reference population . a low gait speed is defined as a walking speed below 0.8 m / s in the 4-m walking test . the european working group on sarcopenia in older people suggested diagnosing sarcopenia when at least two of three criteria apply : ( 1 ) low muscle mass , ( 2 ) low muscle strength , and/or ( 3 ) low physical performance . cutoff points are defined in a similar manner as by the consensus group mentioned before , namely 2 standard deviations below the mean reference value for muscle mass and muscle strength of a reference population and a gait speed 0.8 m / s . a more recent consensus document defines sarcopenia with limited mobility as present in a person with muscle loss whose walking speed is equal to or less than 1 m / s or who walks less than 400 m during a 6-min walk , and who has a lean appendicular mass corrected for height squared of 2 standard deviations or more below the mean of healthy persons between 20 and 30 years of age of the same ethnic group . however , these criteria remain cumbersome in daily clinical practice , and easily applicable tests such as handgrip strength testing or one of the biomarkers mentioned above may help to identify patients in need of a more thorough examination . no consensus has been reached so far as to whether the term sarcopenia should be limited to older persons above 60 years of age or whether it should be used in adults of any age , particularly also in patients with chronic disease . many different approaches have been pursued , but exercise and physical activity are important considerations for both sarcopenia prophylaxis [ 29 , 30 ] and sarcopenia management . progressive resistance training , performed two three times per week by older people , has been shown to improve gait speed , timed get - up - and - go , climbing stairs , and overall muscle strength . indeed , a recent study in patients with heart failure has shown that daily exercise using a cycle ergometer reduces the expression of ligases from the ubiquitin - proteasome pathway . current recommendations state that protein should be consumed at a rate of 0.8 g / kg / day , but about 40 % of persons above the age of 70 years do meet this amount . additional calorie intake of 360 cal per day together with resistance exercise training has been shown to increase leg muscle strength in nursing home residents after 10 weeks . supplementation of essential amino acids has been shown to improve handgrip strength and 6-min walking distance in elderly subjects after 3 months . other therapeutic approaches include the use of testosterone , estrogens , growth hormones , vitamin d , and angiotensin - converting enzyme inhibitors [ 5 , 30 , 37 ] . in addition , animal studies have recently reported beneficial effects of soluble activin receptor type iib ( actriib ) treatment and myostatin inhibition . the first step in the sarcopenia journey is to create more awareness of this clinical problem , both by the general public and by healthcare professionals . in this respect , however , sarcopenia is a phenomenon that is present not only in healthy elderly subjects but also in those with chronic illnesses , such as chronic heart or renal failure . more prospective studies are required to understand the prevalence , incidence , phenotype , and the clinical impact of sarcopenia . furthermore , the discussion continues whether sarcopenia , i.e. , loss of muscle mass , should be separated from dynopenia , i.e. , loss of power . also , as enthusiasts try to make sarcopenia all encompassing by adding a functional definition , the definition lines between frailty and sarcopenia are becoming blurred . there is a need for a consensus decision regarding the use of these terms .
about 80% of the rural population in sub - saharan africa depends on traditional herbal remedies for primary health care and veterinary use . in many developing countries antibiotic resistance , adverse drug reactions , and the high costs of antimicrobials natural products of higher plants may be a source of new antimicrobial agents with possibly novel mechanisms of action [ 3 , 4 ] . contain fatty acids , lupeol , and betulin ; saponins and resins are usually found in the seeds and crotepoxide and crotomacrine in fruits . roots contain chalcone and secondary metabolites such as 3-acetoxy taraxer-14-en-28-oic acid , trachyloban-19-oic acid , trachyloban-18-oic acid , neoclerodan-5,10-en-19,6;20,12-diolide , 3,19-dihydroxytrachylobane , and 3,18,19-trihydroxytrachylobane . several hydroalcoholic c. macrostachyus stem bark extracts have been tested against a clinical strain of neisseria gonorrhoeae with the minimum inhibitory concentration ( mic ) of 125250 mg / ml . there are both negative and positive reports on the antibacterial activity of methanol extracts from c. macrostachyus leaves [ 5 , 10 ] . contrasting results could be attributed to the locality of plant species , parts used , time of collection , storage conditions , and methods of analysis . in the present study we tested the antimicrobial activity of methanol , ethyl acetate , and isobutanol extracts from c. macrostachyus stem bark against human pathogenic e. coli , salmonella typhi , klebsiella pneumoniae , enterobacter aerogenes , listeria monocytogenes , and candida albicans . the ethyl acetate extract was submitted for further purification using column chromatography fractionation and the purified compound was identified by nmr spectroscopy . agar well diffusion method was used to determine the zones of inhibition and the mic values . c. macrostachyus stem bark was collected from baraton community in nandi district of kenya from the nature preserve of the university of eastern africa , baraton , kenya . sample of plant was deposited in the herbarium at the department of biological sciences , university of eastern africa , baraton , and the national museums of kenya for identification . the air - dried bark was powdered using a mechanized hand grinder . the powdered material ( 500 g ) the extract was separated from the solvent by a rotary evaporator ( rotavapor r300 ) below 40c . thin layer chromatography was used to detect the presence of polar compounds . in order to isolate pure compounds from the ethyl acetate extract silica gel column chromatography the column was washed with hexane and toluene and the sample eluted and collected with ethyl acetate . each plant extract was then dissolved in dimethyl sulfoxide ( dmso , rankem , india ) to the concentration of 500 mg / ml to determine the zones of inhibition . for the minimum inhibitory concentration measurements the stock solution ( 500 mg / ml ) was serially diluted to give the concentrations of 250 mg / ml , 125 mg / ml , 62.5 mg / ml , 31.25 mg / ml , 15.63 mg / ml , 7.81 mg / ml , 3.90 mg / ml , and 1.95 mg / ml . the crystallized ethyl acetate extract was dissolved in deuterated chloroform ( cdcl3 ) and transferred into a 5 mm nmr tube . subsequent nmr analysis was carried out at rt on bruker avance av400 spectrometer with 5 mm automatic tuning and matching ( atm ) multinuclear broadband observe ( bbo ) probe including h and c experiments . the following bacterial strains were used for the study : escherichia coli atcc 25922 , salmonella typhi atcc 2202 , klebsiella pneumoniae atcc 1583380 , enterobacter aerogenes the microbial freeze - dried strains were stored in the deposit of the university of baraton and regenerated in brain heart infusion broth ( himedia laboratories pvt . , ltd . , the organisms were then cultured overnight on meller - hinton agar ( himedia laboratories pvt . , ltd . , mumbai , india ) . for the antimicrobial experiments one colony of each microbial strain was inoculated from the agar into 5 ml of tryptone soya broth ( tsb ) ( himedia laboratories pvt . , the microbial strains were cultured overnight , bacterial strains at 37c , and c. albicans at 30c . for the antimicrobial experiment the densities of the cultures were adjusted to the a625 nm value of 0.1 against a tsb control using a spectrophotometer . antimicrobial activity of the extracts was analyzed using agar well diffusion assay according to the technique described by taye et al . . several plates of meller - hinton agar were made and labeled according to the extract and by the concentration , including a negative dmso control and the following positive antibiotic controls : for s. typhi , e. coli , k. pneumoniae , and e. aerogenes antibiotics used were amoxicillin and ciprofloxacin . for l. monocytogenes antibiotic controls were ampicillin and benzylpenicillin and for c. albicans antibiotic controls were clotrimazole and cefotaxime . the selection of antibiotics and antifungal agents was based on the general knowledge of the typical susceptibilities of the chosen indicator microorganisms , and the expected sensitivities were confirmed during the actual experiments . , mumbai , india ) ( 2 ml ) was introduced into tubes and sterilized by autoclaving . 200 l of organism was mixed with 2 ml of the soft agar and the mixture was aseptically transferred to the surface of the solid meller - hinton agar plate and allowed to solidify for 40 minutes . in addition to control wells three sample wells were drilled in the extract concentration - labeled agar plate using a sterilized 6 mm cork borer , giving 3 replicates per plate . the extract ( 75 l ) was transferred to each of the 3 wells per plate . the zones of inhibition ( mm ) were recorded from measurements of the clear zones around the agar wells . for each solvent extract and each organism , three different experiments were conducted , giving nine readings per extract for each organism . the ethyl acetate extract induced the zone of inhibition between 10.1 0.6 mm and 16.0 1.2 mm against s. typhi , e. coli , k. pneumoniae , e. aerogenes , and l. monocytogenes with slight antimicrobial activity against c. albicans ( zone of inhibition : 5.6 1.0 mm ) . the isobutanol extract showed activity against s. typhi , e. coli , k. pneumoniae , and e. aerogenes ( zone of inhibition between 8.3 1.5 mm and 13.7 1.4 mm ) but did not show any activity against l. monocytogenes and c. albicans . the methanol extract was active against e. coli , k. pneumoniae , e. aerogenes , and c. albicans with the zone of inhibition between 9.0 1.1 mm and 14.9 1.3 mm . the methanol extract was not active against l. monocytogenes and had very low activity against s. typhi ( zone of inhibition : 2.3 1.87 mm ) . all the extracts had quite equal activity against e. aerogenes ( zone of inhibition within 10.1 0.610.8 1.2 mm ) . against s. typhi and l. monocytogenes the ethyl acetate extract was the most active ( zone of inhibition between 16.0 1.2 mm and 11.7 1.3 mm , resp . ) . against e. coli the isobutanol extract possessed the highest activity ( zone of inhibition : 12.2 1.6 mm ) . against k. pneumoniae and c. albicans the methanol extract was the most active ( zones of inhibition : 14.9 1.3 mm and 12.0 1.4 mm , resp . ) . for the methanol and isobutanol extracts the mics were 250500 mg / ml ( results not shown ) . the methanol extract against e. coli had mic of 250 mg / ml and against k. pneumoniae , e. aerogenes , and c. albicans mic was 500 mg / ml , although the methanol extract induced the narrowest zone of inhibition against e. coli . isobutanol extract had mic of 250 mg / ml against both e. coli and s. typhi and 500 mg / ml against k. pneumoniae and e. aerogenes . for the ethyl acetate extract the mics were 125250 mg / ml against all the studied pathogens except against c. albicans ( mic of 500 mg / ml ) . lupeol had the lowest mic for k. pneumoniae ( 125 mg / ml ) and the highest mic against c. albicans ( 500 mg / ml ) . against e. coli , s. typhi , e. aerogenes , and l. monocytogenes mic was 250 mg / ml ( table 2 ) . the chemical shifts of the carbon atoms were compared to the literature information ( table 3 ) . the chemical shifts showed that the purified compound was triterpene lupeol ( figure 1 ) . when testing methanol extracts of c. macrostachyus leaves and roots wagate and colleagues found mics between 15.6 and 250 mg / ml against three bacterial organisms , e. coli , bacillus cereus , and pseudomonas aeruginosa . in the present study the extracts from c. macrostachyus stem bark induced growth inhibition against all the studied pathogens including four gram - negative and one gram - positive bacteria with mic values of 125250 mg / ml . the results achieved in the present study are in accordance with previous results carried out by suffredini et al . were constantly high and some extracts induced inhibition which almost reached the antibiotic level , when measured as zone of inhibition . our results show that between the bacterial strains there is variation in susceptibility to extracts . the antimicrobial effect of the extract depends on the bacterial strain and the extraction solvent . the broadest effect against all the studied bacteria in a dose - dependent manner was achieved by the ethyl acetate extract . however , against e. coli the isobutanol extract and against k. pneumoniae and c. albicans the methanol extract had even higher activity . the main components of c. macrostachyus stem bark are lupeol , betulin , and fatty acids . because of their solubility properties one could conclude that the isobutanol and methanol extracts contained mixtures of these compounds . lupeol isolated from other plants has been reported to inhibit the growth of several types of bacteria , fungi , and viral species [ 1420 ] . in the present study lupeol was isolated from c. macrostachyus stem bark by ethyl acetate extraction followed by silica gel column chromatography . , it was the first time when lupeol was extracted from the stem bark of c. macrostachyus and showed broad antimicrobial activity against several important human pathogens e. coli , s. typhi , k. pneumoniae , and c. albicans with the novel finding against e. aerogenes and l. monocytogenes . the present study gives scientific evidence for the use of the extracts and especially lupeol from c. macrostachyus stem bark as antimicrobial candidates against several human pathogens . they could be utilized as preventive agents in disinfectants or submitted to further process for drug or dietary supplement development . the provision of safe and effective traditional medicines could offer increasing access to health care . various studies have established that herbal medicines can be developed as safe , effective , and less costly alternatives to the current medicines to treat certain bacterial infections .
in september 1992 , 36 990 residents of takayama city , gifu , japan , aged 35 years who were not hospitalized were eligible to participate in the takayama study . a total of 31 552 residents ( 85.3% ) participated in the baseline survey and completed a selfadministered questionnaire including an ffq . the details of this populationbased cohort study have been described elsewhere.14 anthropometric characteristics , sociodemographic status , medical history , physical activity , smoking status , alcohol consumption , and regular diet were asked about in the baseline questionnaire . smokers were defined as people who had smoked a total of at least 20 packs of cigarettes in their life . we asked former and current smokers how long they had smoked . to assess physical activity both at work and at leisure , participants were asked the average time they spent on the following listed activities during the past year : strenuous sports , vigorous work , and moderate sports or work . the number of hours per week spent engaging in each activity was multiplied by the corresponding energy expenditure , expressed as metabolic equivalent of task ( met ) , and the product was taken as the physical activity score expressed as meth / week . the details of this approach including its validity are described elsewhere.15 diet , including meat intake , was assessed using a semiquantitative ffq . data were collected on the average frequency of consumption and the usual serving size for 169 food items and dishes during the past year . food items for red meat products included beef steak , pork steak , pork cutlet , grilled meat , grilled offal , and liver . ham , sausage , bacon , and yakibuta ( chinese style roasted pork ) were defined as processed meat . these items and some other dishes including meat products used as cooking ingredients were accounted for to obtain the estimates for meat intake . total meat was defined as the sum of any kind of meat , including red meat , poultry , and processed meat . each intake of nutrients was estimated using the japanese standard table of food composition ( 5th revised and enlarged edition ) , published by the japan science and technology agency.16 both the validity and reproducibility of the questionnaire were previously reported to be reliable.17 spearman correlation coefficients between the ffq and 12day diet records kept over a 1year period in men and women were 0.18 and 0.62 for total meat , 0.21 and 0.54 for red meat , and 0.58 and 0.69 for processed meat , respectively . corresponding values were 0.44 and 0.53 for total energy , 0.78 and 0.73 for calcium , 0.55 and 0.36 for vitamin d , 0.63 and 0.60 for dietary fiber , and 0.72 and 0.64 for alcohol , respectively . participants were followed until the end of march 2008 . at the baseline , 753 who were diagnosed with colorectal cancer before the baseline , and/or reported a positive history of any cancer and 535 who had a history of colorectal adenoma were excluded ( there was some overlap ) . we also gained some information on histological identification using colonoscopy from two main hospitals in takayama city.8 , 18 the causes of cancer were coded according to the international classification of diseases and health related problems , 10th revision ( icd10 ) . colorectal cancer was defined as the sum of code c18 ( colon cancer ) and code c19 and c20 ( rectal cancer ) . migration data during the study period were obtained from the city residential registers or family registers . the endpoint of followup was determined by the earliest occurrence of one of the following events : a diagnosis of colorectal cancer , emigration from the study area , death , or the end of the study period . during the study period , 1767 persons ( 5.8% ) moved away from the study area . for participants with who moved away on an unknown date ( n = 238 ) , their last date of residence that we could confirm was assigned as the endpoint of followup . colorectal cancer developed in 429 men and 343 women , and 115 men and 107 women died of colorectal cancer . the mortalitytoincidence ratio for colorectal cancer was 0.29 , and patients who were ascertained by death certificateonly registration were 6.2% , indicating satisfactory completeness of cancer information in this cohort . the values of the nutrients and foods consumed were controlled for the total energy intake using the residual method developed by willett.19 participants were categorized into quartile groups ( q1 , q2 , q3 , or q4 ) according to the distribution of their energyadjusted intake of total meat , red meat , and processed meat . relative risks and 95% confidence intervals ( cis ) for colorectal cancer were estimated for the quartile groups of each category of meat intake using a cox proportional hazard model . the reference group was set as the lowest quartile ( q1 ) of each meat intake . the following covariates were included as potential confounders in the models : for men , age ( years , continuous ) , height ( quartiles ) , body mass index ( quartiles ) , physical activity score ( continuous ) , smoking status ( never , past , current smoker who had smoked for 30 years , current smoker who had smoked for 31 years ) , years of education ( 8 , 911 , 1214 , 15 years ) , history of aspirin use ( yes , no ) , alcohol consumption ( g / day ) , and energyadjusted intake of dietary fiber ( g / day ) , calcium ( mg / day ) , and vitamin d ( g / day ) ; for women , age , height , body mass index , physical activity score , smoking status ( never , past , current smoker ) , years of education , history of aspirin use , alcohol consumption , menopausal status ( premenopausal , postmenopausal ) , and energyadjusted intake of dietary fiber , calcium , and vitamin d. indicator terms were specifically created for missing data of categorical covariates . tests for linear trend were conducted in the cox model by treating meat intake as a continuous variable . all analyses were conducted using the sas program , version 9.4 ( sas institute , cary , nc ) . in september 1992 , 36 990 residents of takayama city , gifu , japan , aged 35 years who were not hospitalized were eligible to participate in the takayama study . a total of 31 552 residents ( 85.3% ) participated in the baseline survey and completed a selfadministered questionnaire including an ffq . the details of this populationbased cohort study have been described elsewhere.14 anthropometric characteristics , sociodemographic status , medical history , physical activity , smoking status , alcohol consumption , and regular diet were asked about in the baseline questionnaire . smokers were defined as people who had smoked a total of at least 20 packs of cigarettes in their life . we asked former and current smokers how long they had smoked . to assess physical activity both at work and at leisure , participants were asked the average time they spent on the following listed activities during the past year : strenuous sports , vigorous work , and moderate sports or work . the number of hours per week spent engaging in each activity was multiplied by the corresponding energy expenditure , expressed as metabolic equivalent of task ( met ) , and the product was taken as the physical activity score expressed as meth / week . data were collected on the average frequency of consumption and the usual serving size for 169 food items and dishes during the past year . food items for red meat products included beef steak , pork steak , pork cutlet , grilled meat , grilled offal , and liver . ham , sausage , bacon , and yakibuta ( chinese style roasted pork ) were defined as processed meat . these items and some other dishes including meat products used as cooking ingredients were accounted for to obtain the estimates for meat intake . total meat was defined as the sum of any kind of meat , including red meat , poultry , and processed meat . each intake of nutrients was estimated using the japanese standard table of food composition ( 5th revised and enlarged edition ) , published by the japan science and technology agency.16 both the validity and reproducibility of the questionnaire were previously reported to be reliable.17 spearman correlation coefficients between the ffq and 12day diet records kept over a 1year period in men and women were 0.18 and 0.62 for total meat , 0.21 and 0.54 for red meat , and 0.58 and 0.69 for processed meat , respectively . corresponding values were 0.44 and 0.53 for total energy , 0.78 and 0.73 for calcium , 0.55 and 0.36 for vitamin d , 0.63 and 0.60 for dietary fiber , and 0.72 and 0.64 for alcohol , respectively . participants were followed until the end of march 2008 . at the baseline , 753 who were diagnosed with colorectal cancer before the baseline , and/or reported a positive history of any cancer and 535 who had a history of colorectal adenoma were excluded ( there was some overlap ) . we also gained some information on histological identification using colonoscopy from two main hospitals in takayama city.8 , 18 the causes of cancer were coded according to the international classification of diseases and health related problems , 10th revision ( icd10 ) . colorectal cancer was defined as the sum of code c18 ( colon cancer ) and code c19 and c20 ( rectal cancer ) . migration data during the study period were obtained from the city residential registers or family registers . the endpoint of followup was determined by the earliest occurrence of one of the following events : a diagnosis of colorectal cancer , emigration from the study area , death , or the end of the study period . during the study period , 1767 persons ( 5.8% ) moved away from the study area . for participants with who moved away on an unknown date ( n = 238 ) , their last date of residence that we could confirm was assigned as the endpoint of followup . colorectal cancer developed in 429 men and 343 women , and 115 men and 107 women died of colorectal cancer . the mortalitytoincidence ratio for colorectal cancer was 0.29 , and patients who were ascertained by death certificateonly registration were 6.2% , indicating satisfactory completeness of cancer information in this cohort . the values of the nutrients and foods consumed were controlled for the total energy intake using the residual method developed by willett.19 participants were categorized into quartile groups ( q1 , q2 , q3 , or q4 ) according to the distribution of their energyadjusted intake of total meat , red meat , and processed meat . relative risks and 95% confidence intervals ( cis ) for colorectal cancer were estimated for the quartile groups of each category of meat intake using a cox proportional hazard model . the reference group was set as the lowest quartile ( q1 ) of each meat intake . the following covariates were included as potential confounders in the models : for men , age ( years , continuous ) , height ( quartiles ) , body mass index ( quartiles ) , physical activity score ( continuous ) , smoking status ( never , past , current smoker who had smoked for 30 years , current smoker who had smoked for 31 years ) , years of education ( 8 , 911 , 1214 , 15 years ) , history of aspirin use ( yes , no ) , alcohol consumption ( g / day ) , and energyadjusted intake of dietary fiber ( g / day ) , calcium ( mg / day ) , and vitamin d ( g / day ) ; for women , age , height , body mass index , physical activity score , smoking status ( never , past , current smoker ) , years of education , history of aspirin use , alcohol consumption , menopausal status ( premenopausal , postmenopausal ) , and energyadjusted intake of dietary fiber , calcium , and vitamin d. indicator terms were specifically created for missing data of categorical covariates . tests for linear trend were conducted in the cox model by treating meat intake as a continuous variable . all analyses were conducted using the sas program , version 9.4 ( sas institute , cary , nc ) . the characteristics of participants are shown in table 1 as the mean ( standard deviation ) or the percentage of each category , according to the quartile groups of energyadjusted total meat intake for each gender . male and female participants in the higher quartile of total meat intake were younger and taller , and had attended school for a longer period at baseline . men and women among the lowest quartile of total meat intake had a higher level of alcohol consumption . men in the lowest quartile and women in the highest quartile of total meat intake had a higher physical activity score . quartile groups according to the energyadjusted intakes of total meat by willet method . in table 2 , after adjustments for multiple confounders , a significantly increased relative risk of colorectal cancer was observed in the highest intake group ( q4 ) of total meat and red meat in men ; the estimated hazard ratios ( hrs ) were 1.36 ( 95% ci : 1.03 , 1.79 ) for total meat , and 1.44 ( 95% ci : 1.10 , 1.89 ) for red meat . the linear trends in these associations were statistically significant . a higher intake of total meat was significantly associated with a higher risk of colon cancer . participants in the highest group of red meat intake had a significantly increased risk of rectal cancer . in addition , participants in the highest group of processed meat intake had a significantly increased risk of colon cancer . in women , there was no significant association between colorectal cancer and total meat , red meat , or processed meat ( table 3 ) . associations between meat consumption and colorectal cancer incidence among men estimated hazard ratio after adjustments for age , height ( quartiles ) , body mass index ( quartiles ) , physical activity score , smoking status ( never , past , current smoker for 30 years or less , current smoker for 31 years or more ) , education years ( 8 , 911 , 1214 , 15 years ) , history of aspirin use ( yes , no ) , alcohol consumption ( g / day ) , and the intakes ( quartiles ) of total fiber , calcium , and vitamin d. meat consumption was adjusted for total energy intake by willet method . associations between meat consumption and colorectal cancer incidence among women estimated hazard ratio after adjustments for age , height ( quartiles ) , body mass index ( quartiles ) , physical activity score , smoking status ( never , past , current smoker ) , education years ( 8 , 911 , 1214 , 15 years ) , history of aspirin use ( yes , no ) , alcohol consumption ( g / day ) , menopausal status ( premenopausal , postmenopausal ) , the intakes ( quartiles ) of total fiber , calcium , and vitamin d. meat consumption was adjusted for total energy intake by willet method . to eliminate those who might have had colorectal cancer but who had not yet been diagnosed at baseline , we excluded 63 patients who were diagnosed with colorectal cancer in the first 2 years of followup and then reanalyzed the data . although none of the results were substantially altered , the positive association between meat consumption and colon cancer in men seemed to be slightly strengthened ( table 2 ) . additionally , we observed a significant association between higher intake of red meat and increased risk of colon cancer ( p for linear trend = 0.047 ) . when the association between seafood ( fish and shellfish ) consumption and colorectal cancer was evaluated similarly , the estimated hrs ( 95% ci ) for colorectal cancer in the q2 , q3 , and q4 vs the q1 of seafood consumption were , respectively , 1.36 ( 0.99 , 1.87 ) , 1.16 ( 0.80 , 1.67 ) , and 1.29 ( 0.86 , 1.94 ) for men , and 0.88 ( 0.62 , 1.26 ) , 1.17 ( 0.80 , 1.72 ) , and 1.48 ( 0.96 , 2.27 ) for women . after the addition of cancer registry data and the extension of followup to our previous report,8 this study revealed significant positive associations of total and red meat consumption with colorectal cancer risk in men . higher intake of processed meat was associated with a higher risk of colon cancer in men . several supporting mechanisms have been proposed for the carcinogenic effects of meat.20 when meat is cooked at high temperatures by panfrying , grilling , or barbecuing , heterocyclic aromatic amines and polycyclic aromatic hydrocarbons ( pahs ) , which are carcinogenic and mutagenic , are formed.21 , 22 meat processing such as curing and smoking also produces pahs and another carcinogenic nnitroso compounds ( nocs).22 , 23 in the digestive tract , heme iron from red meat mediates the formation of nocs,24 , 25 and the high fat content of red meat could also be a candidate for promoting tumorigenesis by enhancing the production of secondary bile acids by gut bacteria.26 in our study however , dietary intake of iron , animal fat , or saturated fat overall were not directly associated with colorectal cancer risk ( data not shown ) . the japanese population is reported to consume a much lower amount of meat compared to western populations.27 furthermore , dietary habits , lifestyle , and genetic background in japan are also different from those in western countries . in a 2014 metaanalysis of six japanese cohort studies and eight japanese casecontrol studies on meat consumption and colorectal cancer incidence or mortality , pham et al . reported that colorectal cancer risk had a statistically significant positive association with the consumption of red and processed meat , but not with total meat consumption.7 however , individual prospective studies have failed to demonstrate a clear association between meat consumption and colorectal cancer . three japanese cohort studies on meat consumption and colorectal cancer mortality revealed no significant association.11 , 12 , 13 three other japanese cohort studies have estimated the incidence of colorectal cancer.8 , 9 , 10 firstly , the miyagi cohort study revealed no significant association with total , red , or processed meat consumption.10 secondly , the japan public health centerbased prospective study reported that the hr of colon cancer for the highest versus lowest quintiles of red meat intake was 1.48 ( 95% ci : 1.01 , 2.17 , p for linear trend = 0.03 ) in women . also , in men , the corresponding hr for total meat intake was 1.44 ( 95% ci : 1.06 , 1.98 , p for linear trend = 0.07).9 thirdly , using updated data of the takayama study , here we found significant positive associations of total and red meat consumption with colorectal cancer risk in men , in addition to an increased risk of colon cancer among men with high consumption of processed meat as observed previously . in women , there was no significant association of colorectal cancer with the intake of total , red , or processed meat , although the link between meat and colorectal cancer was not modified by sex . the effects of heme iron in red meat , which may be involved in carcinogenesis , might be weakened in women because of blood loss by menstruation , although the associations between meat consumption and colorectal cancer were not substantially different between premenopausal and postmenopausal women in our study ( data not shown ) . also , the lower intake of meat in women than men might have made it difficult to detect any association with colorectal cancer ( mean in men and women : 74.3 and 59.0 g / day for total meat , 41.0 and 30.1 g / day for red meat , and 13.3 and 11.0 g / day for processed meat , respectively ) . we conducted a metaanalysis of these six prospective studies and found that the pooled rrs of colorectal cancer in the highest category of consumption compared with the lowest category were recalculated as 1.22 ( 95% ci : 1.07 , 1.39 ) for red meat , 1.09 ( 95% ci : 0.95 , 1.26 ) for processed meat , and 1.07 ( 95% ci : 0.91 , 1.26 ) for total meat , respectively , among all subjects . the increased risk of higher consumption of red meat was stronger in men ( 1.31 [ 95% ci : 1.10 , 1.56 ] ) than women ( 1.06 [ 95% ci : 0.81 , 1.39 ] ) , and more prominent for colon cancer ( 1.29 [ 95% ci : 1.10 , 1.52 ] ) than rectal cancer ( 1.06 [ 95% ci : 0.81 , 1.40 ] ) . thus , the present study reinforced the positive association between red meat consumption and colorectal cancer in japan , and also provided possible evidence for the difference in sex or subsites . in addition , the pooled rr did not show a significant association with processed meat , which has more robust evidence of carcinogenicity based on the international classification , but high consumption of processed meat is suggested to be associated with an increased risk of colon or colorectal cancer as observed in the takayama study . the strengths of our study include the prospective design , which minimalized recall bias , as well as the robust representation of the general population , good participation rate , long followup duration , and information on several confounders . the ffq was designed to measure an individual 's relative intake of foods and nutrients , rather than absolute values . some of values for dietary intakes we presented in the table may have been overestimated by the ffq . in addition , the validity of the ffq for total and red meat was low in men , which might have affected the results with colorectal cancer risk in this study . however , the observed association of total and red meat with colorectal cancer might have been underestimated because a misclassification on dietary intake would likely have occurred nondifferentially between participants with colorectal cancer and those without colorectal cancer . the exposure evaluation was performed only at baseline and changes in lifestyle patterns during the followup period were unknown . although underlying diseases or preclinical signs may have affected lifestyle at baseline , the exclusion of cases during the first 2 years of followup did not substantially change the results . despite considering several lifestyle and reproductive factors in the analyses , we could not fully exclude the possibility of residual confounders . in conclusion , this prospective study in japan demonstrated increased risk of colorectal cancer among men with higher intake of total and red meat . higher intake of processed meat was associated with a higher risk of colon cancer in men . these results suggest that the intake of red and processed meat increases the risk of colorectal or colon cancer especially among men . in accordance with the assessment of the iarc , abstaining from overeating meat the authors have no conflict of interest . abbreviationsciconfidence intervalsffqfood frequency questionnaireiarcthe international agency for research on cancermetmetabolic equivalentnocnnitroso compoundpahpolycyclic aromatic hydrocarbon food frequency questionnaire the international agency for research on cancer polycyclic aromatic hydrocarbon
a 40-year - old male patient was admitted to our hospital with amnesia , nervousness , personality changes , hostile attitudes , aggressive behaviors , hallucinations and illusions with a history of one year . his symptoms started 3 years ago with complaints of unwillingness to move and loss of appetite . he was consulted by a psychiatrist and put on antipsychotic drugs with no benefit . in the following year , urinary and fecal incontinence and speech disorders began . necrotic unhealed and deeply seated wounds were detected on his left knee and big toe of his right foot . on neurological examination , he had dysarthria and his comprehension ability was restricted to single commands . on cranial nerve examination , right and left pupil size were 2 and 3 mm , respectively with bilateral decrease in light reflexes . blood analyses including complete blood count , hepatic , renal , thyroid function tests , fasting blood glucose , electrolytes , erythrocyte sedimentation rate , c - reactive protein were within normal limits . markers of vasculitis were negative . among serum serological tests , treponema pallidum hemagglutination assay ( tpha ) was positive at 1/2560 dilution . venereal diseases research laboratories ( vdrl ) and rapid plasma reagin ( rpr ) tests were also positive . cranial magnetic resonance ( mr ) images were evaluated as global cerebral and cerebellar atrophy and sequela of trauma in subcortical white matter of the right parietal lobe without contrast enhancement ( figure 1 ) . glucose was 67 mg / dl , protein 36 mg / dl , pandy test positivity and leukocyte 3/mm . in serologic examination of csf , fluorescent treponemal antibody absorption ( fta- abs ) and vdrl was positive at 1/2 and tpha at 1/10240 dilutions . the patient was diagnosed as neurosyphilis and after consultation with the department of infectious diseases , intravenous crystalized penicilline g ( 24 million units / day ) was administered for 21 days . he was diagnosed as organic brain syndrome and offered risperidone ( 3 mg / d ) , haloperidol ( 10 mg / d ) and carbamazepine ( 800 mg / d ) therapy . control csf tests performed 6 months later were reported as tpha positivity at 1/64 dilution , vdrl positivity and rpr 27.54 s / co . during his follow - up , we did not observe any improvement in his psychiatric symptoms , cognitive functions , urinary and fecal incontinence . flair - weighted axial section ( a ) , t2-weighted sagittal section ( b ) and t1-weighted coronal section ( c ) of cranial mr images show diffuse cerebral , cerebellar atrophy and ventricular enlargement secondary to atrophy . it can be encountered in almost all psychiatric disorders including dementia , personality changes , mania , depression , psychosis and delirium . in our patient , psychiatric manifestations started with introversion followed by gradually worsening cognitive decline , psychotic symptoms , dysarthria , urinary and fecal incontinence . his medical history did not reveal any clinical and dermatological complaints and signs peculiar to primary and secondary stages of syphilis . differential diagnosis of the patient involved treatable causes of cognitive dysfunction and psychiatric disorders , primary degenerative dementia and vascular disease . primary dementia was discarded because of the patient age and development of cognitive decline within a short time ( 1 - 2 years ) and severe course . mri was negative for cerebrovascular disease , space - occupying mass lesion and vascular dementia . normal blood tests results , negative hepatitis and hiv serology ruled out metabolic and infectious causes of dementia . clinical findings and positive serologic results were compatible with general paresis , one of the forms of neurosyphilis . tertiary syphilis is a form of progressive dementia , also termed as general paresis , paretic neurosyphilis or dementia paralytica . generally it develops 10 - 25 years after onset of the infection . in early phases of the disease , neurological examination may be normal or there are some pathological findings such as dysarthria , argyl robertson pupil defects , hypotonia , intentional tremor and reflex abnormalities . literature reports indicate that outcome of patients with general paresis is poor and death occurred within nearly 2.5 years before the era of penicillin treatment . treatment response should be examined with csf analyses . after a treatment period , quantitative evaluation of blood serology is performed at 3-month - intervals and a decrease in the level of positivity in serologic tests can be observed . follow - up with neurological examination and csf analysis can be stopped after the patient is cured , clinical stabilization is achieved and csf serologic tests return to normal limits . treatment response is less frequently obtained in patients with general paresis when compared with cases with meningitis and meningovascular syphilis . indeed , irreversible neuronal damage is seen in general paresis , while the others reflect types of cns inflammation . as the case presented here is in the advanced stage of neurosyphilis , no improvement was observed in cognitive functions , psychotic manifestations and symptoms of dysarthria , urinary and fecal incontinence despite penicillin treatment . nowadays , it should be remembered that syphilis in early stages can be overlooked , left untreated and can lead to irreversible manifestations , especially in developing countries .
between march 1999 and december 2002 , tae for nonvariceal upper gastrointestinal bleeding was performed in 93 patients at our institution . the endoscopic approach had failed or was discarded because of its inability to control the bleeding in participating patients . prospective collection of data about clinical records and medical imagings was carried out for all patients . among the 93 patients , nbca was used as the primary embolic material for tae in 32 patients and they were included in this study . there were 28 men and four women , with an age range of 33 - 84 years ( mean age , 59.1 years ) . the causes of bleeding included : benign ulcers ( n = 20 ) , gastric cancers ( n = 7 ) , pancreatitis ( n = 2 ) , duodenal cancer ( n = 1 ) , duodenal tuberculosis ( n = 1 ) and esophageal candidiasis ( n = 1 ) . the embolized arteries were : duodenal branch of gastroduodenal arteries ( n = 11 ) , left gastric arteries ( n = 9 ) , right gastric arteries ( n = 5 ) , right gastroepiploic arteries ( n = 2 ) , esophageal arteries ( n = 2 ) , antral branch of gastroduodenal artery ( n = 1 ) , dorsal pancreatic artery ( n = 1 ) and the transverse pancreatic artery ( n = 1 ) . the angiographic findings were extravasation of contrast media in 27 patients and pseudoaneurysms in five patients . written informed consent for transcatheter embolization was obtained from each patient or a family member . n - butyl cyanoacrylate ( histoacryl , braun , melsungen , germany ) was used as an embolic agent in all participating patients . after a common femoral artery puncture , a standard 5-f angiographic catheter was used to access the sites of suspected bleeding and it was also used as an introducing catheter . diagnostic angiography was performed to identify the bleeding site ; next a 3-f microcatheter ( microferret ; cook , bloomington , in ) was advanced , to as close as possible , to the bleeding point . the indications for using nbca as an embolic material were inability to adequately reach the bleeding site and effective wedging of the microcatheter into the bleeding artery to restrict pericatheter flow ( figs . 1 , 2 ) . for the embolotherapy , nbca was mixed with iodized oil ( lipiodol , andre guerbet , aulnay - sous - bois , france ) in ratios varying from 1:1 to 1:3 ; the iodized oil supplied radiopacity to the mixture and delayed the polymerization time ( 5 ) . prior to injection of the nbca mixture , the microcatheter was flushed with 5% dextrose solution to prevent premature polymerization of the mixture in contact with residual blood or saline . the end point for the injection was extravasation of the mixture from the bleeding site or filling of the pseudoaneurysm with or without appearance of anastomotic channels ( fig . 3 ) . only a small amount of nbca mixture ( 0.2 - 0.6 ml ) was necessary to achieve target embolization . immediately after the injection , the microcatheter was removed to prevent adherence of the catheter tip to the vessel wall and discarded without flushing . then the inner lumen of the guiding catheter was aspirated and post - embolic angiography was performed . the angiographic and clinical success rate , recurrent bleeding rate , procedure related complications and clinical outcomes were evaluated . angiographic success was defined as when post - embolic angiography demonstrated cessation of extravasation with no backbleeding from collateral flow or non - opacification of pseudoaneurysm at the end of the procedure . clinical success was defined as clinical improvement with complete cessation of bleeding after embolization without the need for emergent surgery or other interventional procedures . cessation of bleeding was defined by clearing of the nasogastric aspirate by 24 hours after embolization or absence of bleeding on endoscopic examination as well as stabilization of the hemoglobin level within 48 hours of the embolization procedure . patients who met one of the following criteria were considered to have a coagulopathy : prothrombin ratio greater than 1.5 , partial thromboplastin time greater than 45 seconds , or platelet count of less than 80,000/l ( 2 , 6 , 7 ) . between march 1999 and december 2002 , tae for nonvariceal upper gastrointestinal bleeding was performed in 93 patients at our institution . the endoscopic approach had failed or was discarded because of its inability to control the bleeding in participating patients . prospective collection of data about clinical records and medical imagings was carried out for all patients . among the 93 patients , nbca was used as the primary embolic material for tae in 32 patients and they were included in this study . there were 28 men and four women , with an age range of 33 - 84 years ( mean age , 59.1 years ) . the causes of bleeding included : benign ulcers ( n = 20 ) , gastric cancers ( n = 7 ) , pancreatitis ( n = 2 ) , duodenal cancer ( n = 1 ) , duodenal tuberculosis ( n = 1 ) and esophageal candidiasis ( n = 1 ) . the embolized arteries were : duodenal branch of gastroduodenal arteries ( n = 11 ) , left gastric arteries ( n = 9 ) , right gastric arteries ( n = 5 ) , right gastroepiploic arteries ( n = 2 ) , esophageal arteries ( n = 2 ) , antral branch of gastroduodenal artery ( n = 1 ) , dorsal pancreatic artery ( n = 1 ) and the transverse pancreatic artery ( n = 1 ) . the angiographic findings were extravasation of contrast media in 27 patients and pseudoaneurysms in five patients . written informed consent for transcatheter embolization was obtained from each patient or a family member . n - butyl cyanoacrylate ( histoacryl , braun , melsungen , germany ) was used as an embolic agent in all participating patients . after a common femoral artery puncture , a standard 5-f angiographic catheter was used to access the sites of suspected bleeding and it was also used as an introducing catheter . diagnostic angiography was performed to identify the bleeding site ; next a 3-f microcatheter ( microferret ; cook , bloomington , in ) was advanced , to as close as possible , to the bleeding point . the indications for using nbca as an embolic material were inability to adequately reach the bleeding site and effective wedging of the microcatheter into the bleeding artery to restrict pericatheter flow ( figs . 1 , 2 ) . for the embolotherapy , nbca was mixed with iodized oil ( lipiodol , andre guerbet , aulnay - sous - bois , france ) in ratios varying from 1:1 to 1:3 ; the iodized oil supplied radiopacity to the mixture and delayed the polymerization time ( 5 ) . prior to injection of the nbca mixture , the microcatheter was flushed with 5% dextrose solution to prevent premature polymerization of the mixture in contact with residual blood or saline . the end point for the injection was extravasation of the mixture from the bleeding site or filling of the pseudoaneurysm with or without appearance of anastomotic channels ( fig . 3 ) . only a small amount of nbca mixture ( 0.2 - 0.6 ml ) was necessary to achieve target embolization . immediately after the injection , the microcatheter was removed to prevent adherence of the catheter tip to the vessel wall and discarded without flushing . then the inner lumen of the guiding catheter was aspirated and post - embolic angiography was performed . the angiographic and clinical success rate , recurrent bleeding rate , procedure related complications and clinical outcomes were evaluated . angiographic success was defined as when post - embolic angiography demonstrated cessation of extravasation with no backbleeding from collateral flow or non - opacification of pseudoaneurysm at the end of the procedure . clinical success was defined as clinical improvement with complete cessation of bleeding after embolization without the need for emergent surgery or other interventional procedures . cessation of bleeding was defined by clearing of the nasogastric aspirate by 24 hours after embolization or absence of bleeding on endoscopic examination as well as stabilization of the hemoglobin level within 48 hours of the embolization procedure . patients who met one of the following criteria were considered to have a coagulopathy : prothrombin ratio greater than 1.5 , partial thromboplastin time greater than 45 seconds , or platelet count of less than 80,000/l ( 2 , 6 , 7 ) . in all study patients , it was possible to deliver the nbca mixture to the bleeding site ; the angiographic success rate was 100% ( 32/32 ) . the nbca mixture was the only embolic material used to achieve angiographic success in 29 patients . other embolic agents were used in combination with nbca mixture in three patients , microcoils in two cases and gelatin sponge pieces in one case . microcoils were used in combination with nbca for proximal blockade of pseudoaneurysms in two cases during our early experience . backbleeding from collateral flow after nbca embolization occurred in one patient with massive gastric cancer bleeding and additional embolization of collateral flow from short gastric artery with gelatin sponge pieces could effectively stop the backbleeding . however , a second tae successfully stopped the rebleeding in two patients and emergency surgery was performed in one patient . in two patients with a second tace , the causes of the bleeding were large duodenal ulcers in both . a repeat angiography revealed that the previously embolized vessels were not recanalized , and that other collateral vessels were responsible for the rebleeding at the same ulcer lesions that were previously embolized . there was no evidence of symptomatic bowel infarction or other major complications directly related to nbca embolization in any of the patients . follow up endoscopic evaluations were performed in 20 patients between 1 - 105 days ( mean 16 days ) after the embolization and postembolization bowel ischemia was not observed in any of the patients . coagulopathy was present at the time of tae in 18 patients ( 56% ) and the clinical success rate in this group of patients was 83% ( 15/18 ) . since rosch et al . ( 8) introduced the technique of tae as an alternative to surgery for the control of upper gastrointestinal bleeding in 1972 , tae has gained widespread acceptance for first - line treatment of acute nonvariceal upper gastrointestinal bleeding resistant to endoscopic therapy . tae is generally preferred over surgery , especially in high - risk patients ( 3 , 6 , 9 , 10 ) . the efficacy of tae in initial hemostasis of acute nonvariceal gastrointestinal bleeding has been reported to be 52 - 90% ; however , relatively high rates of rebleeding have also been reported ( 3 , 6 , 7 , 10 - 12 ) . a variety of embolic agents such as gelatin sponge pieces , microcoils , polyvinyl alcohol particles and autologous blood clots have been used in tae for gastrointestinal bleeding . some authors have reported the use of liquid adhesives for transcatheter embolization of gastrointestinal bleeding ( 11 , 13 - 15 ) . however , most interventional radiologists are reluctant to use this technique because of the potential risk of complicated ischemic injury and the difficulty with handling liquid adhesives . liquid adhesives or glue have been used as embolic agents for nearly three decades , but experience with liquid adhesives outside of neurological interventions has been generally limited ( 16 ) . n - butyl cyanoacrylate is a well known liquid adhesive used in the vascular system ; the us food and drug administration recently approved its use for cerebral arteriovenous malformations in 2000 . a localized vascular occlusion mimicking a surgical ligation can be achieved within seconds as the nbca polymerizes on exposure to the anions in blood ( 17 ) . unlike temporary embolic agents , vascular occlusion using nbca is permanent and can not be removed by reversal of vessel flow . however , its lack of radiopacity and its rapid polymerization time make it difficult to use . the addition of iodized oil to nbca can opacify the agent and polymerization time can be tempered with an increased ratio of iodized oil to nbca . reported that the mixture of nbca and iodized oil at a mixing ratio of 1:3 - 1:4 provided optimal embolic material with a polymerization time of 7.5 - 11.5 seconds with excellent contrast definition reported in their experimental study in 1982 ( 18 ) . however , the in vivo polymerization time for nbca appears to be more rapid than the in vitro time because of the higher body temperature compared to room temperature and a greater presence of anions intravascularly ( 19 ) . pollak and white suggested that the estimated in vivo polymerization time for the nbca to iodized oil mixtures of between 1:1 and 1:4 were 1 - 4 seconds , with a linear relationship between time and mixture ratio ( 16 ) . in the present study , we mixed nbca with iodized oil in ratios varying from 1:1 to 1:3 , and we determined the appropriate amount and dilution of nbca depending on the size and shape of the vessel and its distribution after test injection with contrast material to the target of embolization . however , we used the 1:3 mixing ratio in most cases because the 1:3 ratio was thought to provide a useful polymerization time for embolization in most patients with gi bleeding . the test injection took only one or two minutes and did not cause a significant time delay during emergency procedures . among the variety of embolic agents , coils superselective catheterization and stable catheter positioning are required for safe release and ideal coil embolization . however , sometimes it is difficult to selectively place the catheter close to and beyond the pseudoaneurysm in small or tortuous vessels ( 20 ) . furthermore , when there are many efferent arteries originating from the pseudoaneurysm , or many collateral vessels coming from the adjacent arteries , the procedure becomes time consuming and technically difficult to carry out with coils . although catheterization may be possible , coils may not be delivered because of the tortuosity of vessels ( 20 ) . however , because of its low viscosity , nbca can be injected through a microcatheter into small arteries and collateral circulation , which are difficult to embolize with a coil . even though it is impossible to advance the microcatheter to the bleeding point , when the microcatheter is tightly engaged in the bleeding artery , it is possible to deliver nbca selectively to the bleeding site without embolizing innocent vessels . in the upper gastrointestinal tract there are rich anastomoses between adjacent arteries ; this had made backbleeding a problem with embolization procedures . for effective hemostasis , embolization of both feeding arteries and potential collateral channels is required . however , gelatin sponge pieces or polyvinyl alcohol particles have a chance to embolize only the feeding artery and sometimes post - embolic angiography reveals backbleeding from collateral channels that are usually more complex and difficult to access ( 16 ) . superselection of those collateral feeders is very time consuming and can occasionally result in failed catheterization and embolization . in this series , backbleeding from collateral channels did not occur except in one case of massive gastric cancer bleeding . this suggests that nbca can effectively embolize all potential collateral channels together with the main feeder vessels with only a single injection . in cases of life threatening bleeding , the choice of nbca as an embolization material can be lifesaving by minimizing the risk of backbleeding , which can occur with the use of other embolic materials . toyoda et al . reported that the time for tae , using nbca , was significantly faster than for tae procedures that do not use nbca . this is important particularly in cases of massive bleeding that require urgent hemostasis ( 11 ) . coagulopathy has been associated with clinical failure after tae ( 2 , 6 , 7 ) . reported that embolization was 2.9 times more likely to fail in patients with coagulopathy , and that death from bleeding after embolization was 9.6 times more likely to occur in this group ( 6 ) . also reported that embolization in patients with a coagulopathy at the time of intervention was 2.8 times more likely to fail , and the odds of dying were 3.4 times greater for patients with a coagulopathy ( 2 ) . in cases of coil or polyvinyl alcohol particle embolization , occlusion of vessels or pseudoaneurysms is dependent on thrombosis rather than the embolic material itself and the coagulation state of the patient is important for the ultimate success of the embolization procedure . however , nbca does not depend on the coagulation process for its therapeutic effect and therefore can be used effectively in patients with a coagulopathy . eighteen of 32 patients ( 56% ) in our study had a diagnosis of a coagulopathy prior to arrival to the angiographic suite ; the angiographic success rate in this group of patients was 100% and the clinical success rate was 83% in spite of their coagulopathy . considering that angiographic success rate of tae in patients with coagulopathy has been previously reported to be less than 46% , nbca appears to provide a more successful approach in patients with a coagulopathy ( 2 , 6 ) . transcatheter embolization with nbca has the potential risk of complications including : embolization of the proximal segment without adequate embolization at the bleeding site due to rapid polymerization , distal embolization of untargeted sites due to too rapid injection of the nbca mixture or an excessively prolonged polymerization time ( 21 ) and reflux of glue into another innocent vessel by overinjection . complications related to nbca embolization can be minimized by careful attention to the specific vascular anatomy and the information obtained from test injections with contrast material . in this series , none of these patients developed symptomatic bowel infarction or mesenteric ischemia after nbca embolization . ( 13 ) who similarly had no cases of organ infarction . in order to carry out a precise and safe embolization and to reduce the possibility of complications with the use of nbca as an embolic material , we recommend the following : 1 ) pay careful attention to the specific vascular anatomy and the information obtained from the test injections with contrast media ; 2 ) use the wedged catheter technique with no pericatheter flow . it allows the interventional radiologists to precisely control the extent of glue embolization ; 3 ) perform a simulated injection with contrast media in the same condition before the actual nbca injection with precise volume calculation . the single use of a microcatheter , without flushing after nbca injection , is highly recommended for precise embolization with the wedged technique because overinjection of nbca can occur during flushing of the microcatheter . the intraluminal volume of microcathter is usually around 0.3 ml and this should be considered for precise volume estimation . for example , if initially 0.8 ml of nbca mixture was drawn up in a 1-ml syringe and 0.1 ml was left in the syringe after injection , then 0.4 ml was injected into the embolized vessels and 0.3 ml was left in the discarded microcatheter . although most interventional radiologists are familiar with the use of coils and particles such as gelatin sponge pieces and polyvinyl alcohol , few have experience with liquid adhesives such as nbca ( 16 ) . nbca is difficult to use initially , however after becoming familiar with its properties in the animal laboratory , it can be used safely for the care of patients . in conclusion , tae with nbca is a highly effective and safe treatment modality for upper gastrointestinal bleeding , especially in cases when it is technically difficult to advance the microcatheter to the bleeding site and in patients with a coagulopathy .
addressing the various needs and concerns faced by down 's syndrome ( ds ) subjects require the education of their caregivers by medical and dental professionals alike . thus providing dental health care services for individuals with special health care needs continue to be an important aspect in these patients . other than the periodontal disease ds individuals are characterized by poor oral seal , poor tongue control , hypersalivation , cracked lips , angular cheilitis , fissured tongue , submucous cleft and cleft palates , reduction in salivary flow rate , bruxism , midline deviation , developmental disturbances of the midfacial complex as well as the occlusal relationships . ds individuals usually present with poor oral hygiene , which is manifested as marginal gingival inflammation , acute necrotizing gingivitis , suppuration or abscess formation and advanced chronic periodontitis including furcation involvement , increased tooth mobility , and loss of teeth . ds individuals have a prevalence of 60 - 90% increased severity of periodontal disease compared with normal age - matched controls and subjects with other mental disabilities of similar age . gingival inflammation is a characteristic feature seen in ds subjects where it persists predominantly in the lower dental arch and mixed dentition . aggregatibacter actinomycetemcomitans ( aac ) , a key periodontal pathogen commonly found in aggressive periodontitis patients is often seen in individuals with ds . porphyromonas gingivalis ( pg ) also have a strong correlation to severe periodontal disease and have an increased prevalence with age , thus playing an important role in the onset and progression of periodontal disease in ds individuals . agholme et al . in 1999 in his 7 years observational study found that the prevalence of bone loss increased from 35% to 74% among 33 ds individuals , however , the severity and progression of the disease was not as rapid as reported in the literature . though , the majority of ds children had poor oral hygiene the incidence of earlier onset periodontal disease could not be demonstrated in these subjects . simultaneously , there was only moderate relationship between dental plaque and periodontal disease severity in ds individuals thus , rapid and severe periodontal destruction in those ds individual affected could not be explained by poor oral hygiene alone . the current study hypothesis shows that , presence of elevated levels of aac and pg , predisposes ds subjects to severe periodontal destruction . hence , the study was designed to correlate the presence of aac and pg with the severity of periodontal disease in ds subjects when compared to systemically healthy controls with periodontal disease . twenty - nine ds subjects were considered as part of the experimental group and 30 age - matched systemically healthy individuals were included as controls in this study . ds subjects were recruited from opportunity school , vepery , chennai , tamil nadu and ds association of tamil nadu , adyar , chennai , tamil nadu , while systemically healthy age - matched controls with gingivitis and periodontitis were recruited from the patients attending the department of periodontology , s.r.m . dental college , ramapuram , chennai . the institutional ethical committee approval was obtained for the study and informed consent was obtained from the heads of both the above - mentioned special schools and amongst the control subjects . group i ( ds subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth 3 mm , no clinical loss of attachment , gingival index score 2.0group ii ( ds subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth of 4 mm and clinical attachment loss ( cal ) 3 mmgroup iii ( control subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth was 3 mm , no clinical loss of attachment and gingival index score 2.0group iv ( control subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth 4 mm and cal 3 mm . group i ( ds subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth 3 mm , no clinical loss of attachment , gingival index score 2.0 group ii ( ds subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth of 4 mm and clinical attachment loss ( cal ) 3 mm group iii ( control subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth was 3 mm , no clinical loss of attachment and gingival index score 2.0 group iv ( control subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth 4 mm and cal 3 mm . the experimental group ( group i and group ii ) included 29 ds subjects and the control group ( group iii and group iv ) included 30 systemically healthy subjects . of the 29 ds subjects , 11 subjects had periodontitis and 18 had gingivitis . while , of the 30 control subjects , 15 had periodontitis and 15 had gingivitis according to community periodontal index for treatment needs ( cpitn ) scores . subjects with mental retardation , endocrinal disease , coronary heart disease , smokers , pan chewers and other systemic diseases were excluded from the study . the presence or absence of calculus , bleeding on probing , shallow and deep pockets were assessed using cpitn index . subgingival pooled plaque samples were collected from the mesial and buccal sites of teeth with the deepest pocket by means of a sterile paper point ( no . 35 , diadent , almere , the netherlands ) as shown in figure 1 . the samples were stored at 80c and processed at the central research facility , sri ramachandra medical college and research institute , porur , chennai . the samples were then analyzed for quantification of aac and pg using real time polymerase chain reaction ( rtpcr ) . the processing reagent , pcr reagents and master mix kit were obtained from applied biosystems , warrington , uk . species - specific primers ( inqaba biotech industries ( pty ) ltd . ) were used to detect the presence of the aac and pg [ table 1 ] . the expected product lengths were 404 bp for pg and ( 593 bp ) for aac and pg . a pair of ubiquitous primers product length ( 602 bp ) , which matched most bacterial 16s rrna genes at the same position was used as a positive control for the pcr reaction . collected plaque samples were stored in eppendorf tubes containing ethanol solution ( 99% ) at 80c and centrifuged at 7000 rpm for 5 min . mixture of 1 g of lysosome powder was mixed with tris hydrochloride ( 1 ml ) , ethylenediaminetetraacetic acid 200 l , triton 600 l and sterile water milli q was added to make the solution up to 40 ml and then vortexed for 5 min . 0.8 l of this solution was added to each sample and incubated for 30 min at 37c . 20 ml of sodium dodecyl sulfate was added to the sample , vortexed again and incubated for 30 min at 37c . to this , equal volume of phenol chloroform isoamyl was added and centrifuged at 10,000 rpm for 10 min and the supernatant was extracted and added to new eppendorf tubes . 1 ml chloroform isoamyl alcohol was added to the new tubes and centrifuged at 10000 rpm for 10 min . a volume of 1 ml sodium acetate and 1 ml ethyl alcohol was added and centrifuged under 10,000 rpm for 10 min . the supernatant obtained was discarded and 500 l of ethanol was added to the remaining of the sample and centrifuged at 10,000 rpm for 5 min . the supernatant was discarded and the pellets that remained at the bottom of the tubes were dried for 2 h. a volume of 30 l of sterile water was added to it , which was then frozen and sent for pcr analysis . commercially available sybr green master mix ( applied biosystems sybr green master mix , life technologies 5791 van allen way carlsbad clifornia 92008 united states of america ) 5 l was added along with 0.5 l of forward primer and reverse primer each , 2.0 l of template dna and 2.0 l of sterile water . 10 l of the prepared mix was dropped in micro wells and subjected to pcr analysis , while amplification was done using values obtained from cycle threshold ( ct ) equation . mean values were compared among different study groups by using independent t - test with one - way anova kruskal - wallis followed by post - hoc ( tukey hsd ) test . the statistical software spss version 17.0.0 ( spss manufacturer ibm corporation , 1 new orchard road , armonk , new york 10504 - 1722 , united states ) was used for the analysis of the data . twenty - nine ds subjects were considered as part of the experimental group and 30 age - matched systemically healthy individuals were included as controls in this study . ds subjects were recruited from opportunity school , vepery , chennai , tamil nadu and ds association of tamil nadu , adyar , chennai , tamil nadu , while systemically healthy age - matched controls with gingivitis and periodontitis were recruited from the patients attending the department of periodontology , s.r.m . dental college , ramapuram , chennai . the institutional ethical committee approval was obtained for the study and informed consent was obtained from the heads of both the above - mentioned special schools and amongst the control subjects . group i ( ds subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth 3 mm , no clinical loss of attachment , gingival index score 2.0group ii ( ds subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth of 4 mm and clinical attachment loss ( cal ) 3 mmgroup iii ( control subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth was 3 mm , no clinical loss of attachment and gingival index score 2.0group iv ( control subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth 4 mm and cal 3 mm . group i ( ds subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth 3 mm , no clinical loss of attachment , gingival index score 2.0 group ii ( ds subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth of 4 mm and clinical attachment loss ( cal ) 3 mm group iii ( control subjects with gingivitis ) : plaque - associated gingivitis with positive bleeding on probing , sulcus depth was 3 mm , no clinical loss of attachment and gingival index score 2.0 group iv ( control subjects with periodontitis ) : patients diagnosed with chronic periodontitis with probing depth 4 mm and cal 3 mm . the experimental group ( group i and group ii ) included 29 ds subjects and the control group ( group iii and group iv ) included 30 systemically healthy subjects . of the 29 ds subjects , 11 subjects had periodontitis and 18 had gingivitis . while , of the 30 control subjects , 15 had periodontitis and 15 had gingivitis according to community periodontal index for treatment needs ( cpitn ) scores . subjects with mental retardation , endocrinal disease , coronary heart disease , smokers , pan chewers and other systemic diseases were excluded from the study . oral hygiene status was assessed using simplified oral hygiene index . the presence or absence of calculus , bleeding on probing , subgingival pooled plaque samples were collected from the mesial and buccal sites of teeth with the deepest pocket by means of a sterile paper point ( no . 35 , the samples were stored at 80c and processed at the central research facility , sri ramachandra medical college and research institute , porur , chennai . the samples were then analyzed for quantification of aac and pg using real time polymerase chain reaction ( rtpcr ) . the processing reagent , pcr reagents and master mix kit were obtained from applied biosystems , warrington , uk . species - specific primers ( inqaba biotech industries ( pty ) ltd . ) were used to detect the presence of the aac and pg [ table 1 ] . the expected product lengths were 404 bp for pg and ( 593 bp ) for aac and pg . a pair of ubiquitous primers product length ( 602 bp ) , which matched most bacterial 16s rrna genes at the same position was used as a positive control for the pcr reaction . collected plaque samples were stored in eppendorf tubes containing ethanol solution ( 99% ) at 80c and centrifuged at 7000 rpm for 5 min . the supernatant was discarded . mixture of 1 g of lysosome powder was mixed with tris hydrochloride ( 1 ml ) , ethylenediaminetetraacetic acid 200 l , triton 600 l and sterile water milli q was added to make the solution up to 40 ml and then vortexed for 5 min . 0.8 l of this solution was added to each sample and incubated for 30 min at 37c . 20 ml of sodium dodecyl sulfate was added to the sample , vortexed again and incubated for 30 min at 37c . to this , equal volume of phenol chloroform isoamyl was added and centrifuged at 10,000 rpm for 10 min and the supernatant was extracted and added to new eppendorf tubes . 1 ml chloroform isoamyl alcohol was added to the new tubes and centrifuged at 10000 rpm for 10 min . a volume of 1 ml sodium acetate and 1 ml ethyl alcohol was added and centrifuged under 10,000 rpm for 10 min . the supernatant obtained was discarded and 500 l of ethanol was added to the remaining of the sample and centrifuged at 10,000 rpm for 5 min . the supernatant was discarded and the pellets that remained at the bottom of the tubes were dried for 2 h. a volume of 30 l of sterile water was added to it , which was then frozen and sent for pcr analysis . commercially available sybr green master mix ( applied biosystems sybr green master mix , life technologies 5791 van allen way carlsbad clifornia 92008 united states of america ) 5 l was added along with 0.5 l of forward primer and reverse primer each , 2.0 l of template dna and 2.0 l of sterile water . 10 l of the prepared mix was dropped in micro wells and subjected to pcr analysis , while amplification was done using values obtained from cycle threshold ( ct ) equation . mean values were compared among different study groups by using independent t - test with one - way anova kruskal - wallis followed by post - hoc ( tukey hsd ) test . the statistical software spss version 17.0.0 ( spss manufacturer ibm corporation , 1 new orchard road , armonk , new york 10504 - 1722 , united states ) was used for the analysis of the data . the samples are subjected to rtpcr and the results showed increased level of pg and aac in ds subjects in gingivitis and periodontitis group compared to systemically healthy subjects [ graphs 1 and 2 ] . polymerase chain reaction amplification plot for aggregatibacter actinomycetemcomitans real time polymerase chain reaction amplification plot for porphyromonas gingivalis the results revealed a statistical significant increase in the level of aac in both gingivitis and periodontitis group in ds subjects with the p = 0.008 and 0.001 , respectively compared with the control gingivitis and periodontitis groups as shown in table 2 . similarly a statistically significant level of pg were found in both ds gingivitis and periodontitis with the p = 0.001 and 0.001 respectively when compared to control gingivitis and periodontitis groups as shown in tables 3 and 4 and represented as bar diagrams [ figures 2 and 3 ] . the inter group comparison of ct values of aac and pg by one way anova revealed a statistical significance in the level of aac in ds subjects when compared to healthy controls with the p = 0.001 . independent t test for aac among ds and systemically healthy subjects group statistics independent t test for pg among ds and systemically healthy subjects group statistics comparison of aggregatibacter actinomycetemcomitans in down 's syndrome and control subjects with gingivitis and periodontitis comparison of porphyromonas gingivalis in down 's syndrome and control subjects with gingivitis and periodontitis in this study , pg levels were elevated in ds subjects with gingivitis and periodontitis compared with the healthy controls . however the levels of pg in ds gingivitis group were most significant when compared to all other groups in the present study as shown in tables 5 and 6 . the post - hoc test for multiple comparisons of aac among the four groups showed a statistically significant levels of aac in ds gingivitis group when compared to control gingivitis and control periodontitis groups with p = 0.011 and 0.003 respectively , similarly , the test revealed a statistically significant levels of aac in ds periodontitis group when compared to control gingivitis and control periodontitis groups with the p = 0.047 and 0.017 , respectively as shown in table 7 . intergroup comparisons for the levels of aac using anova between ds and systemically healthy subjects intergroup comparisons for the levels of pg using anova between ds and systemically healthy subjects multiple comparisons using post - hoc test for the levels of aac between ds and systemically healthy subjects with gingivitis and periodontitis the post - hoc test for multiple comparisons of pg among the four groups showed a statistically significant levels of pg in ds gingivitis group when compared to control gingivitis and control periodontitis groups with p = 0.001 and 0.001 respectively , similarly the test revealed a statistically significant levels of pg in ds periodontitis group when compared to control gingivitis and control periodontitis groups with the p = 0.001 and 0.001 , respectively as shown in table 8 . multiple comparisons using post - hoc test for the levels of pg between ds and systemically subjects with gingivitis and periodontitis the samples are subjected to rtpcr and the results showed increased level of pg and aac in ds subjects in gingivitis and periodontitis group compared to systemically healthy subjects [ graphs 1 and 2 ] . polymerase chain reaction amplification plot for aggregatibacter actinomycetemcomitans real time polymerase chain reaction amplification plot for porphyromonas gingivalis the results revealed a statistical significant increase in the level of aac in both gingivitis and periodontitis group in ds subjects with the p = 0.008 and 0.001 , respectively compared with the control gingivitis and periodontitis groups as shown in table 2 . similarly a statistically significant level of pg were found in both ds gingivitis and periodontitis with the p = 0.001 and 0.001 respectively when compared to control gingivitis and periodontitis groups as shown in tables 3 and 4 and represented as bar diagrams [ figures 2 and 3 ] . the inter group comparison of ct values of aac and pg by one way anova revealed a statistical significance in the level of aac in ds subjects when compared to healthy controls with the p = 0.001 . independent t test for aac among ds and systemically healthy subjects group statistics independent t test for pg among ds and systemically healthy subjects group statistics comparison of aggregatibacter actinomycetemcomitans in down 's syndrome and control subjects with gingivitis and periodontitis comparison of porphyromonas gingivalis in down 's syndrome and control subjects with gingivitis and periodontitis in this study , pg levels were elevated in ds subjects with gingivitis and periodontitis compared with the healthy controls . however the levels of pg in ds gingivitis group were most significant when compared to all other groups in the present study as shown in tables 5 and 6 . the post - hoc test for multiple comparisons of aac among the four groups showed a statistically significant levels of aac in ds gingivitis group when compared to control gingivitis and control periodontitis groups with p = 0.011 and 0.003 respectively , similarly , the test revealed a statistically significant levels of aac in ds periodontitis group when compared to control gingivitis and control periodontitis groups with the p = 0.047 and 0.017 , respectively as shown in table 7 . intergroup comparisons for the levels of aac using anova between ds and systemically healthy subjects intergroup comparisons for the levels of pg using anova between ds and systemically healthy subjects multiple comparisons using post - hoc test for the levels of aac between ds and systemically healthy subjects with gingivitis and periodontitis the post - hoc test for multiple comparisons of pg among the four groups showed a statistically significant levels of pg in ds gingivitis group when compared to control gingivitis and control periodontitis groups with p = 0.001 and 0.001 respectively , similarly the test revealed a statistically significant levels of pg in ds periodontitis group when compared to control gingivitis and control periodontitis groups with the p = 0.001 and 0.001 , respectively as shown in table 8 . multiple comparisons using post - hoc test for the levels of pg between ds and systemically subjects with gingivitis and periodontitis down 's syndrome individuals usually present with poor oral hygiene which manifests as marginal gingival inflammation , acute necrotizing gingivitis , suppuration or abscess formation and advanced chronic periodontitis . the severity of periodontal disease among ds individuals has improved in the recent years owing to better dental care and awareness . sakellari et al . evaluated the severity of periodontal disease in ds individuals and compared with healthy individuals or cerebral palsy patients and concluded that periodontal inflammation and treatment needs were significantly higher in ds individuals . oral health in individuals with ds were similar to that of early onset periodontitis and lower incisors reportedly exhibited early signs of alveolar bone loss in approximately 35% of ds adolescents . as the amount of plaque and calculus alone can not explain the severity of periodontal disease in ds individuals , abnormal capillary morphology , disorders in connective tissue and anatomical aspects of teeth might also contribute to the pathology . alteration in immunological response may also play a role in the progression of the disease process in ds subjects . other immune cell defects like decline in t - cell functioning after the first 10 years of life , decrease in t - cell counts and other immune defects have been reported increasing plasma levels of interleukin-6 and defective neutrophil migration . it has been proposed that when compared to ds children living at home , institutionalized ds children live in conditions of relative stress hence explaining the difference in severity of periodontal disease . in this study , the gingivitis group in ds subjects showed a statistically significant increase in the levels of both aac ( p < 0.008 ) and pg ( p < 0.001 ) when compared with healthy controls , however pg was more significantly increased when compared to aac . the results of this study is in accordance with amano et al . who stated that increase in pg levels in ds gingivitis subjects played an important role in the initiation of gingival inflammation and plaque maturation . in the periodontitis group , increased levels of aac ( p < 0.001 ) and pg ( p < the results were similar to the study done by amano et al . and agholme et al . the reason for the prevalence of these organisms in ds could be due to early colonization of these organisms in their childhood . multiple comparisons within the four groups using post - hoc test showed that the ct levels of aac and pg were statistically significant in ds gingivitis and ds periodontitis groups . the results of this study correlated with the study done by sakellari et al . however agholme et al . and amano et al . in their studies on ds subjects without controls had shown elevated levels of aac and pg respectively . elevated levels of pg in ds subjects resulting in early onset periodontitis could be due to the more susceptible host for the causative microbial agents including pg with type ii fimbrea . in the present study both pg and aac were elevated in ds subjects with gingivitis and periodontitis compared to healthy controls , further there is a highly significant increase in the level of pg than aac in ds gingivitis group . the probable explanation for the above results can be due to the fact that institutionalized ds subjects are exposed to periodic oral prophylaxis programs limiting the progression of gingivitis to periodontitis . this statement is further validated by yoshihara et al . , where the author evaluated the effect of periodic preventive care on the progression of periodontal disease in young adults with ds and concluded that institutionalized ds subjects have less periodontal destruction and high prevalence of gingival inflammation with pg being the predominant microflora . this study is one of the first studies to evaluate and quantify specific periodontal pathogens in ds subjects among the indian subpopulation comparing the results with healthy controls . the limitations of the current study could be that it is a single centered trial , with uneven distribution of samples in the ds and healthy control groups and nonevaluation of posttreatment changes in microflora . within the limits of the study , it can be concluded that the presence of aac and pg with a probable contributory compromised immune response exists in ds subjects , predisposing them to gingival inflammation and possible early onset periodontitis . further studies evaluating multiple periodontal pathogens and assessing the immune cell functions could widen our understanding on the etiopathogenesis of periodontal diseases in ds subjects . the present study showed a significant presence of potent periodontal pathogens aac and pg in ds subjects compared to healthy controls .
beta - hemolytic streptococcal isolates from humans can be subdivided into large - colony and small - colony ( < 0.5 mm in diameter ) formers ( 1 ) . the small colony - forming -hemolytic strains with lancefield group a , c , f , or g antigens belong to the anginosus or previously termed ' streptococcus milleri ' species group . members of the anginosus group ( streptococcus anginosus , streptococcus constellatus , and streptococcus intermedius ) are considered part of the viridans group streptococci ( vgs ) , the majority of which display -hemolytic or non - hemolytic reactions . the vgs also include streptococcus mitis , streptococcus oralis , streptococcus mutans , streptococcus salivarius , streptococcus sanguis , and streptococcus bovis ( 1 - 3 ) . although small - colony - forming -hemolytic vgs are found as commensals whose pathogenic abilities appear to be much more subtle than those of the pyogenic streptococci , they may also participate in various infections such as subacute bacterial endocarditis , catheter - related and neutropenia - related bloodstream infections , purulent abdominal , hepatobiliary , brain , and dental infections ( 3 - 5 ) . in addition , they play a significant role as a reservoir of antimicrobial resistance genes , transferring different resistance traits to more pathogenic organisms such as streptococcus pneumoniae and streptococcus pyogenes ( 6 ) . beta - lactam agents are the treatment of choice for vgs infections , but increasing resistance to penicillin has recently been reported in many parts of the world ( 7 ) . macrolides and related drugs have been suggested as alternatives , but recent studies have shown that macrolide resistance may also be a problem ( 7 ) . moreover , clinical and laboratory standards institute ( clsi ) recommended that vgs isolated from normally sterile body sites should be tested for penicillin susceptibility using the minimum inhibitory concentration ( mic ) method , and interpretive criteria for the vgs should be used ( 2 ) . however , laboratory personnel have difficulty in differentiating -hemolytic vgs from pyogenic streptococci based on colony size . although many recent studies have elucidated susceptibilities of vgs isolated from clinical samples ( mainly from blood cultures ) to various anti- microbial agents , such studies on -hemolytic vgs from the various specimens are scarce . the objectives of the present study were to determine the incidence and patterns of antimicrobial resistance among -hemolytic vgs isolated from various clinical specimens in a korean hospital and to clarify the macrolide resistance phenotypes and genotypes of erythromycin - resistant isolates . one hundred and three isolates of -hemolytic vgs isolated from various clinical specimens were collected between january 2003 and december 2004 at wonju christian hospital in south korea . isolates were identified by standard methods : streptex latex agglutination assay ( murex biotech limited , dartford , england ) and the api rapid id32 strep system ( biomrieux , marcy - l'etoile , france ) . these were stored in thioglycollate broth plus 20% glycerol at -70 until studied . susceptibility to penicillin g , erythromycin , clindamycin , ceftriaxone , chloramphenicol , tetracycline ( sigma chemical co , st . louis , mo , u.s.a . ) , and vancomycin ( daewoong lilly , korea ) was determined by an agar dilution method ( 2 ) , and mics were interpreted using the clsi criteria ( 2 ) . macrolide resistance phenotypes of erythromycin - resistant isolates were determined using a double - disk test with erythromycin ( 15 g ) and clindamycin ( 2 g ) disks on mueller - hinton agar plates containing 5% sheep blood . genomic dna was extracted with an easy - dna kit ( invitrogen , carlsbad , ca , u.s.a . ) , according to the manufacturer 's instructions . the presence of erm and mef class genes was determined by pcr amplification using previously described primers specific for erm(a ) , erm(b ) , erm(c ) , erm(tr ) , and erm(a ) ( 8) . to confirm that pcr reactions were amplifying the target sequences for specific primers , pcr products were sequenced ( macrogen inc . , of the 103 -hemolytic vgs , nine ( 8.7% ) , forty ( 38.8% ) , and fifty - three ( 51.5% ) isolates were agglutinated with group c , f , and g antisera , respectively . one isolate of -hemolytic s. intermedius was nongroupable . using the agar dilution method , the overall resistance ( intermediate and resistance ) rates of -hemolytic vgs were measured at 47.5% to tetracycline , 3.9% to chloramphenicol , 9.7% to erythromycin and 6.8% to clindamycin , whereas all isolates were susceptible to penicillin g , ceftriaxone , and vancomycin . one s. intermedius isolated from peritoneal fluid was susceptible to tetracycline , erythromycin , and clindamycin . resistance rates to tetracycline , erythromycin , and clindamycin of group f and g isolates were 37.5% vs. 52.8% , 17.5% vs. 5.7% , and 15.0% vs. 3.8% , respectively . all of group c -hemolytic vgs isolates were susceptible to erythromycin , clindamycin , and chloramphenicol , although tetracycline resistance rate ( 66.7% ) of group c was higher than other serogroups ( table 1 ) . among the -hemolytic vgs , 68 ( 66.0% ) and the remaining isolates included gemella haemolysans ( six isolates ) , s. intermedius ( one isolate ) , s. oralis ( one isolate ) , s. salivarius ( one isolate ) , and streptococcus alactolyticus ( one isolate ) . the serogroup proportions of s. anginosus were g ( 63.2% ) , f ( 33.8% ) , and c ( 2.9% ) , in decreasing order , whereas those of s. constellatus were f ( 44.0% ) , g ( 36.0% ) , and c ( 20.0% ) . among the s. anginosus isolates , group g was frequently isolated from the lower respiratory tract specimens such as bronchial washing and sputum ( 55.8% ) , and wound cultures ( 37.2% ) , whereas group f was somewhat prevalent in genitourinary tract specimens ( 52.2% ) . as contrasted with s. anginosus , the specimen distribution of s. constellatus by serogroups had little or no difference . of the nine erythromycin - resistant s. anginosus strains , six and three were group f and g , respectively . among ten erythromycin - resistant isolates , six isolates expressed a constitutive mlsb ( cmlsb ) phenotype , and each of the two isolates expressed an m phenotype and an inducible mlsb ( imlsb ) phenotype , respectively . all cmlsb and m phenotypes carried the erm(b ) and mef(a ) genes , respectively . of the two imlsb phenotypes , one isolate with the cmlsb phenotype carried both of erm(b ) and erm(tr ) genes ( table 2 ) . although most strains of s. anginosus group have been known to be non - hemolytic or -hemolytic , it is estimated that 25% are -hemolytic ( 9 ) . ( 10 ) reported that a majority ( 84% ) of the s. milleri strains possessed lancefield group antigen ( 3 a , 27 c , 41 f , and 5 g ) , whereas 16% were nongroupable . in this study , however , group g was the most commonly isolated serogroup among hemolytic vgs , and only one strain was nongroupable . many authors ( 1 , 3 ) have reported that : s. anginosus strains may possess lancefield group a , c , f , or g antigens ; s. constellatus strains are more -hemolytic than s. anginosus or s. intermedius , and possess mainly lancefield group f antigens ; and that isolates of s. intermedius strains are rarely -hemolytic . some authors ( 1 , 5 ) have documented that there tends to be an association between the clinical specimen sources and three species of anginosus group organisms . in addition , -hemolytic group c strains of s. constellatus constitute two subspecies : s. constellatus subsp . constellatus , which is isolated from a relatively broad clinical background , and s. constellatus subsp . s. anginosus isolates are commonly isolated from urogenital and gastrointestinal sources , and strains of s. constellatus have been isolated relatively frequently from the respiratory tract and many other sources , while s. intermedius strains are often isolated among the polymicrobial flora of deep - seated abscesses , notably in the liver and brain . identification to the species level among -hemolytic vgs should not be ignored because -hemolytic vgs differ from large - colony - forming -hemolytic streptococci in clinical significance and interpretive criteria of susceptibility . additionally , anginosus group organisms recovered from abscess or wound specimens , even when other organisms are present , are likely to be pathogens rather than contaminants . careful evaluation of the significance of -hemolytic vgs according to specimen types is needed , and a clinical laboratory setting for the precise identification of -hemolytic vgs should be a priority . until the 1980s , vgs were considered uniformly susceptible to -lactam antibiotics , but resistance had spread rapidly in the 1990s . vgs that are not susceptible to penicillin also showed reduced susceptibility to ceftriaxone , erythromycin , and clindamycin . the resistance rates to penicillin , clindamycin , erythromycin , and ceftriaxone of vgs isolated from blood cultures in our previous study ( 8) were 57.6% , 33.9% , 17.9% , and 9% , respectively . in this study , however , any of -hemolytic vgs isolates did not show resistance to either penicillin or to ceftriaxone . in our previous study , the overall resistance rates of -hemolytic streptococci were found to be 80.0% to tetracycline , 22.8% to chloramphenicol , 20.2% to erythromycin and 19.1% to clindamycin , whereas all isolates were susceptible to penicillin g , ceftriaxone and vancomycin ( 11 ) . these results suggested that susceptibilities of -hemolytic vgs to -lactams are similar to pyogenic -hemolytic streptococci rather than non--hemolytic vgs . the rate of clindamycin resistance in vgs has been determined mainly by the distribution of mlsb resistance phenotypes . in this study , all of erythromycin - susceptible isolates were susceptible to clindamycin , and the cmlsb phenotype was the most frequently observed mechanism among erythromycin - resistant isolates . however , the numbers of erythromycin - resistant strains were too small to draw a conclusion . as for the non--hemolytic vgs , the distribution of mlsb phenotypes of -hemolytic vgs may also vary with many factors such as geography , specimen sources , species identified , and so on ( 8 , 12 ) . we think that susceptibility testing of -lactams for treatment of -hemolytic vgs is not necessary for clinical purpose , and may not be done routinely in the hospitals , because , as with vancomycin , resistant strains have not been detected in this study . the resistance rates to erythromycin and clindamycin of -hemolytic vgs seemed to be relatively lower than non--hemolytic vgs in our hospital , although cmlsb phenotype - carrying erm(b ) was dominant in -hemolytic vgs . further epidemiologic studies are needed to confirm whether or not our susceptibility data on -hemolytic vgs are restricted to our area .
a 28-year - old male visited our department with complaint of visual disturbance , hyperemia , and epiphora in his right eye , which had developed about four weeks earlier . after symptoms developed , he visited a general ophthalmologist and was prescribed 3% acyclovir ointment ( 5 times / day ) , 1% topical prednisolone acetate ( 8 times / day ) , 0.5% topical levofloxacin ( 8 times / day ) , and oral acyclovir 400 mg ( 1 time / day ) to treat herpetic stromal keratitis . his medical and familial history was unremarkable . on initial examination , his best - corrected visual acuity ( bcva ) was 20/50 in the right eye and 20/20 in the left eye . intraocular pressure was in normal range in both eyes . slit lamp examination of the right eye showed conjunctival hyperemia , localized edema of the central and inferior half of cornea with epithelial microcysts , and moderate anterior chamber ( 2 + ) inflammation . a focal mid - stromal scar at the temporal cornea was observed , which had not been in attention ( fig . 1 ) . suspecting herpetic stromal keratitis , his medication was increased to 1% topical prednisolone acetate every one hour and oral acyclovir 400 mg five times a day while maintaining the other medications . two weeks after the initial visit , his visual acuity decreased to 20/80 and corneal edema with microcysts were still observed . oral prednisolone ( 30 mg once a day ) was prescribed in addition to the other medications . after five days , anterior chamber inflammation was improv ed and his visual acuity recovered to 20/20 , but subtle corneal edema and microcysts still remained ( fig . , he maintained 20/20 vision and his ophthalmic examination showed no significant interval changes . on tapering the medication , after three months , a previously unobserved intraocular foreign body was incidentally identified in the inferior angle of anterior chamber ( fig . thorough history taking revealed that the patient had ocular trauma with a mechanical pencil 12 years earlier . under the operating microscope , the foreign body was surgically removed via corneal incision , which measured about 0.5 1.5 mm in size ( fig . the foreign body was freely movable in the chamber and there was no evidence of fibrosis or granulation reaction at the adjacent tissue . elemental analysis of the foreign body using energy dispersive x - ray spectrometer ( bruker quantax 200 ; bruker axs , berlin , germany ) and scanning electron microscope ( philips xl 30s feg ; philips , eindhoven , the netherlands ) revealed that it consisted mainly of elementary carbon , which is compatible with the component of graphite pencil lead ( fig . two weeks after the surgery , the patient 's bcva was 20/15 , and remaining corneal edema and microcysts were completely resolved ( fig . specular microscopy showed that endothelial cell density was reduced following the event , but the percentage of hexagonal cells increased after removal of the foreign body ( fig . there was no inflammatory reaction during six years of follow - up in one case , while endophthalmitis developed two days after trauma in the other case . the authors mentioned the possibility of infectious endophthalmitis or sterile endophthalmitis caused by elemental aluminum released by the retained graphite fragment . there are also cases of retained graphite in the conjunctiva simulating melanoma , and intracorneal graphite particles , which were well tolerated . in this case , the foreign body which had penetrated 12 years earlier , did not show any cellular adhesion or pigment precipitation on its surface , thus a graphite foreign body seems to be well tolerated in the anterior chamber without inducing any foreign body reactions or inciting ocular inflammation . moreover , given the response to surgical removal of the foreign body , the patient 's manifestations , such as corneal edema and inflammatory response on initial visit , should be attributed to mechanical irritation of the adjacent tissues including the iris and corneal endothelium . based on the absence of ocular symptoms for 12 years , we may assume that the foreign body had remained in a place where it did not contact the corneal endothelium . a certain event such as minor trauma could have dislodged the foreign body from the original place , and resulted in corneal edema by irritating the corneal endothelium . when a young patient presents with unilateral corneal stromal edema and anterior chamber inflammation , differential diagnosis should include glaucomatocyclitic crisis , herpes stromal keratitis , and intraocular foreign body . in this case , although the patient had a history of ocular trauma , it was 12 years earlier , and no evidence of intraocular foreign body except an old focal corneal scar was observed at the first visit . he also revealed normal intraocular pressure , and was therefore treated with anti - virals and anti - inflammatory agents to treat herpetic stromal keratitis . the proper diagnosis and treatment were delayed until the foreign body was detected through the course of follow - up examination . the anti - inflammatory effect of medication or spontaneous interaction with adjacent tissue of the foreign body would explain the moderate response to steroid treatment during follow - up . persistent corneal problems indicate that there had been intermittent mechanical contact of the foreign body with the corneal endothelium . complete resolution of corneal edema and epithelial microcysts was achieved only after surgical removal of the graphite foreign body from the anterior chamber . careful history taking and meticulous slit lamp examination would have led to the proper management earlier . this case showed that localized corneal stromal edema or endothelial dysfunction can be caused by an intraocular foreign body , even with history of trauma that occurred much earlier . a physician should always arouse suspicion and perform a thorough examination , including gonioscopy , in such patients .
the effects of electromagnetic fields ( emfs ) on simple systems , such as colloids , particles in solution , and on living matter , have been investigated for many years [ 13 ] . while high - frequency emfs produce microvibrations in the molecules that have been interpreted as resonance phenomena , elfs induce slight and more modest actions [ 46 ] . despite the large number of studies regarding the effects of emfs , there is no consensus on the effects of pulsed emfs on different biological targets . it is possible that emfs with different characteristics , such as different waveforms , frequencies , and intensities , may have different or even opposing effects on cells . interest on the effects of emfs has greatly increased in recent years , especially in terms of their possible effects , their potential therapeutic and diagnostic applications . however , a comprehensive understanding is lacking about which frequency has the most positive effects and causes the least damage to living matter , or even to simple molecules . it is impossible to investigate the entire range of elf emfs : systematic exploration would require a limitless number of tests and could be impractical in terms of workload . however , it is possible to compare the effects of a specific emf to a comprehensive reference emf with parameters that change with time ; such a reference emf would involve combinations of different parameters . at physics department of the university of siena in italy , a new system has been pioneered , termed the therapeutic application of musically modulated electromagnetic field or tammef system , in which a large spectrum of low frequency emfs are generated . the frequency , wave shape , and wave induction of the emf varies using the tammef system because the emf is generated by a musical piece . this apparatus has been used in our institution for several years for therapeutic purposes , that is , to treat patients affected by several diseases , and the efficiency and effectiveness of the analgesic and anti - inflammatory effects of tammef have been demonstrated [ 710 ] . many published studies have examined the effects of emfs on cell proliferation , cell cycle regulation , cellular differentiation , metabolism , and other physiological characteristics . although it is unlikely that elf emfs themselves have genotoxic effects , it is possible that some cellular processes are altered upon exposure to emf . in this study , we investigated and compared the in vitro effects of 100 hz elf emf and the new tammef system on cellular metabolism . the study was performed on peripheral blood mononuclear cells ( pbmcs ) from 20 healthy volunteers ; these cells were used frequently in these studies because they are well characterized and relatively easy to obtain [ 1215 ] . specifically , we evaluated total cellular protein content and the activity of some key enzymes involved in nucleotide metabolism after exposure to emfs . we chose to examine nucleotide metabolism because nucleotides are precursors of dna and rna , and the relationship of changes in nucleotide metabolism with some pathologies and drug interactions has been studied extensively [ 1620 ] . the enzymes ecto-5-nucleotidase ( ec 3.1.3.5 ) ( ecto-5-nt ) , adenosine deaminase ( ec 3.5.4.4 ) ( ada ) , and adenosine kinase ( ec 2.7.1.20 ) ( adk ) play important roles in intracellular adenosine homeostasis , while adenylate kinase ( ec 2.7.4.3 ) ( ak ) regulates the cellular electrical charge [ 2124 ] . a schematic drawing of the instruments used to generate elf emfs and tammefs are shown in scheme 1 . the emf apparatus consisted of an audiotape player that sends a monochannel - microphone signal to 2 low - frequency amplifiers , a and m , both with adjustable gain . the elf field was modulated at a frequency of 100 hz and was generated by an opposing pair of heteronomous polar expansions that made up the characteristic spatial conformation of the setup . the polar expansions consisted of the sides of two iron - silicon nuclei ( 3 4 cm ) joined by a ferromagnetic arc . the coils ( 4.5 cm long ) consisted of 900 turns of copper wire ( 0.7 mm in diameter ) . the alternating current power supply was regulated to maintain the effective value of the oscillation amplitude at ~10 gauss at the midpoint of the interpolar distance ( 30 cm ) . the tammef apparatus was similar to the elf apparatus ; however , the audiotape contained a different signal , that is , a musical passage , which produced a suitably modulated emf with varying frequencies and varying waveforms . we generally used a piece of classical music ( i.e. , rachmaninov 's piano concerto number 2 in c minor op.18 for piano and orchestra ) to generate a microphone signal , with the current from the amplifier going to a loudspeaker that played the musical passage , so that the piece can be contemporary listened during the therapy ( in fact , it seems that the listening of the musical piece add a positive psychological effect to the emf therapy ) . the emf generated in this way had characteristics that varied with time according to the musical signal ; by simply changing the music , we could produce fields with varying frequencies , intensities , and wave shapes . a 75-cm flask containing the biological sample ( about 70 10 cells ) was positioned between the polar expansions in order to be directly in the generated emf . the resulting functional modifications in the cellular microenvironment depended not only on the particular microenvironment or the emf but also on changes in the field due to the responses of the cells . figure 2 shows the position of the flask containing pbmcs during the 30-minute exposure to the emf . the research protocol was approved by our local ethics committee , and each blood donor provided informed written consent . blood was donated by 20 healthy subjects , 10 males and 10 females , aged 2545 years old . fresh aliquots of na - citrate blood ( 30 ml ) were added to histopaque 1077 ( sigma - aldrich , st . louis , mo , usa ) 1 : 1 and centrifuged ( 30 minutes , 750 g , 4c , according to the protocol developed in our laboratory ) . the pbmc layer was removed and washed with a lyses mixture consisting of 155 mm nh4cl , 10 mm khco3 , and 0.1 edta for the lyses of the residual erythrocytes . we obtained five samples of pbmcs , from pools of 4 individuals , to diminish the effects of individual variation . the cells in each sample were counted using a brker chamber , divided equally into 4 aliquots of about 70 10 cells ( one for each condition ; described hereafter ) then transferred to 75-cm flasks containing dmem and 10% fbs . cells were incubated in a controlled atmosphere ( 5% co2 , 95% air ) , with controlled humidity and temperature conditions ( 37c ) for 2448 hours , until treatment . prior and at the end of the treatment with emfs , cell survival was monitored using the alamarblue assay ( invitrogen , carlsbad , calif , usa ) , which utilizes a water - soluble fluorimetric indicator based on a redox fluorescence indicator read at 590 nm . cells that were not removed from the incubator ( termed t0 cells , as these cells represent the pretreatment controls ) and cells that were removed from the incubator but not exposed to emfs ( termed control cells ) served as the two reference controls . after treatment ( or after 30 minutes for the t0 and control cells ) , the cells were submerged in an ultrasound bath for 10 seconds , ultracentrifuged for 60 minutes at 50,000 g , and aliquots of the supernatants were assayed for enzymatic activity [ 25 , 26 ] . to test for ada activity : reactions contained 0.1 mm adenosine , 50 mm tris - hcl ( ph 7.4 ) , and supernatant ( 1530 g protein ) . to test for ecto-5-nt activity : reactions contained 0.1 mm amp , 5 mm mgcl2 , 50 mm tris - hcl ( ph 7.4 ) , and supernatant ( 1530 g protein ) . samples were incubated for 15 minutes at 37c , and the reactions were stopped by adding 0.2 n hclo4 ; after centrifugation at 12,000 rpm for 10 minutes at 4c , supernatants were neutralized with 0.2 n koh , centrifuged to remove potassium perchlorate , and subjected to capillary electrophoresis analysis for quantification of the products . a multiple wavelength biofocus 3000 capillary electrophoresis system ( bio - rad , hercules , calif , usa ) was used with an uncoated silica capillary ( 40 cm 50 m i d ) . the separation was performed with 20 mm sodium borate buffer ( ph 10 ) and followed by uv detector at 254 nm . reaction products were identified on the basis of their retention times compared to standards analyzed under the same conditions . to test for adk activity : the reactions contained 50 mm tris - hcl ( ph 7.5 ) , 0.018 mm [ c]ado ( 58 mci / mmole ) , 1 mm atp , 0.5 mm mgcl2 , 0.005 mm erythro-9-(2-hydroxy-3-nonyl)-adenine ( ehna ) , 0.1 mm adenosine 5-(,-methylene)-diphosphate ( amp - cp ) , and supernatant ( 4560 g protein ) . ehna and amp - cp inhibit adenosine deaminase and 5nucleotidase , respectively , enzymes from pbmc that are involved in purine metabolism and that can alter assay results by consuming the assay substrate . the reaction mixtures were incubated for 10 minutes at 37c , then stopped by heating at 100c for 1 minute . after centrifugation at 12,000 rpm for 5 minutes , 0.05 ml of cleared supernatant was injected into the hplc apparatus to separate the labeled products . we used a vista 5500 instrument ( varian , palo alto , calif , usa ) , equipped with a uv detector at 254 nm and a supelcosil c18 column 5 m ( 250 4.6 mm ) ; the products were eluted with 92% 10 mm kh2po4 ( ph 5.5 ) buffer and 8% ethanol at a flow rate of 1.5 ml / min . the labeled amp was eluted from the chromatographic column , collected , added to scintillation liquid , and radioactivity was determined using a 1500 tri - carb beta - counter ( hewlett - packard , palo alto , calif , usa ) . to test for ak activity : the reactions contained 50 mm mops ( ph 7.4 ) , 3 mm mgcl2 , 0.15 m kcl , 3 mm atp , 0.3 mm phosphoenolpyruvate ( pep ) , 0.22 m nadh , 0.6 iu pyruvate kinase ( pk ) , and 0.6 iu lactate dehydrogenase ( ldh ) and suitable aliquots of supernatant ( 2030 g proteins ) . ak activity was determined using a spectrophotometric method by following the decrement at 340 nm for 15 minutes at 37c . the activities of all enzymes were expressed as moles of product formed in 1 h/10 cells . total protein was determined in samples using the bradford assay with bovine serum albumin as a standard . one - way analysis of variance ( anova ) or nonparametric kruskal - wallis analysis was used to determine the significance of differences among the groups . statistical analysis was performed using prism 4.00 for windows ( graphpad software , san diego , calif , usa ) . most ( 99% ) of cells survived treatment with emfs , which was comparable to survival under control conditions . table 1 shows the total protein content and ecto-5-nt , ada , adk , and ak enzymatic activities of pooled human pbmcs for t0 and control cells as well as for cells treated with elf emfs and tammefs . the protein content increased after both emf treatments compared to t0 and control cells , but the increase was not significant . however , ak activity was significantly higher ( p < .05 ) after elf emf treatment , with an increase of about 89% compared to t0 cells and 102% compared to control cells . in contrast , ak activity was slightly reduced ( 0.2% ) after tammef treatment compared to t0 cells . the values shown in table 1 are also depicted in figures 3 and 4 as histograms in order to better illustrate the differences between the groups . in the present study , we evaluated the influence of elf emfs ( at 100 hz ) and tammefs ( with variable frequency ) on the cellular metabolism of pbmcs . the total protein content and the activities of enzymes involved in purine metabolism were determined and compared in cells treated with elf emfs and tammefs , as well as in control cells ( t0 and control ) . the small increase in the protein content of cells after treatment with either type of emf can be attributed to accelerated cellular metabolism , as demonstrated previously by others . it has also been shown previously that exposure to elf emfs can affect cell proliferation , cell cycle regulation , and differentiation , although the specific effects on health have not been determined . the mechanisms underlying the interactions of emfs on living cells and organisms are yet unknown ; until now , reports have presented largely theoretical rather than experimental observations . the enzymes ecto-5-nt , adk , and ada showed no significant changes in activity compared to controls regardless of emf treatment . other enzymes , such as glycolytic enzymes in human erythrocytes treated with 50 hz emf , have also been reported to be unchanged following emf exposure . in contrast , cells treated with elf emfs in our study showed an increase in ak activity ; this enzyme is involved in nucleotide metabolism , which regulates the homeostasis of adenylic nucleotides , thus contributing to the cellular energy charge . a study of the effects of elf ( 75 hz ) emfs on ak activity in bovine retinal rods reported a sharp decrease of its activity ( approximately 55% ) relative to nonexposed control cells : this effect was observed only for ak bound to the retinal membrane , for when the ak was extracted from the membranes using triton x-100 , the enzymatic activity was no longer affected much by elf emfs . based on these observations , the authors suggest that effects of the emf are influenced by the organization and structure of the cellular membrane . in our experiments , ak activity was higher in pbmcs treated with elf emfs compared to t0 and control cells . this could indicate that elf emfs s affect the stability of the cell 's electrical charge , and we hypothesize that the tendency of ak activity to increase upon exposure to elf emfs is a direct consequence of cellular mechanisms that work to restore intracellular equilibrium . magnetic field variations induce emfs that act on the electrical charges in the cell , such as ions , free radicals , and dipolar structures . these charges are affected by the induced electromotive forces within the constraints of the cellular environment , possibly modifying cellular function . other investigators who have studied the effects of elfs on the cell 's bioenergetic system [ 31 , 32 ] have also reported increases in the activity of enzymes involved in cellular respiration , such as activation of cytocrome c oxidase . exposure of pmbcs to tammefs does not increase ak activity ; rather , there is a small decrease compared to the control . thus , tammef does not interfere with the cell 's energetic and metabolic state and may in fact be useful for stimulating and maintaining the homeostatic response . the data presented here hints at the potential for using tammefs therapeutically . further research , with a larger sample size , is needed for further investigations , toward the structural and molecular sites of action , in native or stimulated cells , as in the presence of optimal concentration of phytohemagglutinin ( pha ) .
kidney transplantation is the treatment of choice for most patients with stage five chronic kidney disease ( ckd ) . the risk of death is less than half of that for dialysis patients regardless of the immunosuppression protocol used . furthermore , most recipients acknowledge improved quality of life . the united network for organ sharing ( unos ) has over 80,000 patients on the kidney transplant waiting list , many of whom are highly sensitized . data obtained from the unos ( 20012008 ) showed that the rates of transplantation for living donor ( ld ) and deceased donor ( dd ) by panel reactive antibody ( pra ) status are less than 16% per year for patients with pras of 10% to 80% , and less than 8% for patients with pras more than 80% . thus , sensitized patients with any level of pra are difficult to transplant and have longer waiting times on the transplant list literature review demonstrates 1-year allograft survival between 69% and 96% for desensitizieted patients . the rejection risk for all patients in the first year post transplant is less than 12% based on the 2009 usrds database . highly sensitized transplant recipients , regardless of the desensitization protocol used , are at increased risk for amr . both desensitization and amr are managed with the similar therapeutic arsenal ; however protocols are center - specific and there are no consensus guidelines . the two desensitization protocols for which clinical efficacy has been demonstrated are high - dose ivig or low - dose ivig with either plasmapheresis ( pp ) or immunoadsorption [ 6 , 7 ] . additionally , some transplant centers may add intravenous steroids , rabbit antithymocyte globulin ( ratg ) , or rituximab . as mentioned above , these modalities are variably effective in decreasing reactive antibody levels [ 911 ] . there is concern that the role of plasma cells in mediating humoral rejection is not adequately addressed . since plasma cells do not express cd20 , they are not depleted by rituximab 's ability to deplete cd20 positive b - cell line members as detailed in ( figure 1 ) . there is one variant of amr in which over 30% of infiltrating cells are mature plasma cells , and once diagnosed graft survival is generally less than one year post diagnosis . hence , it is of importance to target this cell lineage in desensitization and amr treatment strategies . reservations were expressed in the literature that plasma cells were unaffected by current desensitization protocols . the group conducted a study where the spleens of patients receiving desensitization were histologically compared to control spleens for their levels of different b - cell line members . the study showed that levels of nave b cells ( cd20 + and cd79 + ) , memory b cells ( cd27 + ) , and plasma cells ( cd138 + ) in the spleens of patients desensitized with pp and low - dose ivig did not differ significantly from control spleens . it was also noted that despite the addition of rituximab to the pp and ivig protocol , the amount of memory b cells and plasma cells were still comparable to controls . combination therapy in the study ( pp , low - dose ivig , rituximab , and ratg ) did show a small reduction of memory b cells , but plasma cell levels were still on par with controls . this study confirmed the reservations expressed in the literature that plasma cells were unaffected by current desensitization protocols [ 9 , 13 ] . bortezomib ( velcade , millennium pharmaceuticals , cambridge , ma ) depletes plasma cells via proteasome inhibition . in 2008 , investigators at the university of cincinnati published their experience of six patients with amr and donor - specific antibodies ( dsa ) elevation post transplantation who had reversal of amr with a single cycle of bortezomib . several other transplant centers have since utilized bortezomib for treatment of amr with varying success [ 1420 ] . herein , we review the current body of literature regarding using bortezomib in pretransplant desensitization and treatment of amr . the food and drug administration ( fda ) approved this drug as treatment for multiple myeloma , a plasma cell dyscrasia , which remains its only approved indication . a phase 3 multicenter trial published in the new england journal of medicine in 2008 demonstrated marked improvement in outcomes for newly diagnosed myeloma patients who received bortezomib in addition to the treatment standard of melphalan and prednisone . this 682-patient randomized trial demonstrated the superiority , along with safety and efficacy of bortezomib . now this drug is first - line treatment in patients with newly diagnosed myeloma who can not receive immediate autologous stem cell transplantation . the drug has been used off label in the transplant setting since 2005 both to reduce dsas in highly sensitized patients and as an adjunct therapy for amr . bortezomib ( c19h25bn4o4 ) has a central boron atom which binds the catalytic site of the 26s proteasome with high affinity and specificity . present in all cells , the proteasome degrades ubiquitinylated , abnormal , and misfolded proteins ; thus regulating protein expression and function . simply put , proteasome inhibition during mitosis inhibits the degradation of cell - cycle regulatory proteins resulting in cell - cycle death via apoptosis . one such regulatory protein is nfkb , which has an important role in controlling cell cycle progression , loading of class i mhc molecules , cell adhesion , and activation of cytokines . bortezomib 's interference with these two molecules leads to the accumulation and aggregation of unfolded proteins and eventual plasma cell apoptosis . both in vitro and in vivo ( murine and human ) studies have noted that this drug has a propensity to cause apoptosis of cd138 + plasma cells [ 16 , 24 ] . bortezomib also exerts numerous indirect effects on circulating b cells and th cells ; for example , it may lead to blockade of t - cell cycling leading to apoptosis of th cells and reduction of bone marrow interleukin-6 can decrease b - cells numbers . the pharmacokinetics of bortezomib can be characterized by rapid and wide distribution , a prolonged elimination half life , and hepatic cytochrome p-450 ( cyp ) isoenzyme metabolism . after a rapid distribution half life of approximately 10 minutes , peak plasma bortezomib concentrations range 60 to 120 ng / ml following repeated doses of 1 to 1.3 mg / m . l / h after the first dose and from 15 to 32 l / h following repeated doses . a high volume of distribution ( 500 to 1800 l / m ) in patients with multiple myeloma is suggestive of extensive distribution to peripheral tissues . in vitro studies indicate bortezomib metabolism to occur primarily via hepatic oxidation by cyp3a4 , cyp2c19 , cpy1a2 , and to a lesser extent cyp2d6 and cyp2c9 , to inactive metabolites . while inhibitors and inducers of these isoenzymes are commonly seen especially in organ transplant populations , clinically significant interactions with bortezomib and enzyme inducers concomitant administration of ketoconazole has been shown to increase systemic bortezomib exposure by 35% with corresponding increased proteasome inhibitory activity , although side effects were similar to those not receiving ketoconazole . on the other hand , ascorbic acid ( vitamin c ) has been shown to interfere with bortezomib 's proteasome inhibitory activity through alternative mechanisms . following intravenous administration , maximum percent inhibition of 20s proteasome is observed after 5 minutes , reaching a mean of 7084% inhibition . with decreases in drug concentration , 20s proteasome inhibition is reversed with mean inhibition declining to 2248% at 48 hours and returning to baseline at 72 hours post administration . adverse effects are reported in phase ii and phase iii studies from multiple myeloma and mantle cell lymphoma populations . the main adverse effect of this drug is neurotoxicity which manifests as a dose - related peripheral sensory neuropathy that may occur in about 30% of treated patients . this neuropathy can often be severe but is reversible with discontinuation of the drug . severe events ( national cancer institute common toxicity criteria grade 3 event ) also noted with bortezomib therapy include thrombocytopenia ( 28% ) and neutropenia ( 11% ) which are usually managed with standard approaches . thrombocytopenia due to bortezomib has been observed to occur in patients primarily with low baseline platelet levels and resolves upon drug discontinuation . other commonly reported side effects include nausea ( 55% ) , diarrhea ( 44% ) , and fatigue ( 12% ) ; the gastrointestinal disturbances are usually mild and managed easily with standard approaches . in the largest series of kidney transplant recipients to date , walsh et al . report a similar pattern of adverse effects , including low - grade gastrointestinal side effects , mild to moderate anemia , neutropenia , and thrombocytopenia , and primarily mild cases of peripheral neuropathy in patients undergoing treatment for desensitization and humoral rejection . dosing of bortezomib is similar regardless of the route of application , it does not require renal or hepatic dosing adjustments , and the drug is no longer detectable within 30 minutes of injection . to our knowledge , this drug has never consistently been used in nonkidney transplant protocols . there are several published case reports and case series detailing bortezomib 's application in kidney transplantation . the first published data is from university of cincinnati by idica et al . in 2008 . thirteen highly sensitized patients received this drug and all had reduction in the normalized mean fluorescent units ( mfi ) of the donor - specific antibodies ; ten of whom ( 77% ) had significant decrease in dsa . . reported on 11 patients with a posttransplant anti - hla antibody titer greater than 1000 mfi , but without acute rejection . the use of bortezomib with plasmapheresis was successful in decreasing antibody levels to under 1000 mfi within a median time of 24 days from treatment initiation in all but two patients . overall the study suggests that bortezomib can be used to decrease dsa levels with minimal toxicity . four of 11 patients had reappearance of anti - hla antibodies despite initial effective reduction with one cycle of bortezomib . the authors suggested that certain patients may need more than one cycle of treatment to decrease dsa levels . with clinically stable patients , the study 's findings neither argues for nor against bortezomib 's ability to affect the clinical course of graft rejection , but the study does point to the possible role that bortezomib can play in decreasing dsa levels that are implicated in amr . in the case , the group used two cycles of bortezomib for pre - transplant desensitization for two highly sensitized kidney recipients . the first cycle of bortezomib was given alone followed by a cycle of bortezomib with dexamethasone as dexamethasone has been shown to enhance treatment efficacy in multiple myeloma patients . in the two patients , pra decreased from 87% to 80% in patient 1 and 37% to 13% in patient 2 . despite the mild decrease in pra levels , bortezomib therapy led to more than 50% decrease in the levels of anti - hla antibodies triggering c4d deposition on single antigen luminex beads as measured in mfi after 6 months of followup . this suggests that bortezomib may have a role in decreasing complement fixation especially as c4d is one of the histological markers leading to the diagnosis of amr . yet , bortezomib 's mild effect on pra for pre - transplant desensitization may suggest the need for adjunct modalities that target antibodies such as pp and ivig as well as further exploration of bortezomib in transplant desensitization . in the first study to use bortezomib as an antirejection modality , 6 kidney transplant recipients with amr and acute cellular rejection ( acr ) refractory to plasmapheresis , ivig , and/or ratg , and/or rituximab were treated with bortezomib . bortezomib therapy led to prompt rejection reversal ( within days to weeks ) and in all the cases , there was improved renal function and reduction in dsa levels . recurrent rejection episodes in 2 patients were suppressed for up to 5 months ; furthermore , the anti - hla antibody with the highest levels ( immunodominant dsa ) were decreased by more than 50% within 14 days and remained suppressed for up to 5 months . as the first positive study of incorporating bortezomib as a suppressor of dsa in the treatment of amr , others groups have emulated this strategy . perry et al . analyzed the effects of ratg , ivig , rituximab , and bortezomib on enriched populations of bone marrow derived plasma cells and indicated that only bortezomib was successful in causing plasma cell apoptosis and completely blocking anti - hla igg secretion against all specificities in vitro compared to controls . in comparing the bone marrow of 2 positive cross - match kidney recipients at the time of amr and at 1 week after treatment with bortezomib for amr , in vivo data showed a decrease in the percentage of bone marrow plasma cells , a decrease in antibody production ( including dsa ) , and a decrease in the number of plasma cell allospecificities in the bone marrow aspirates obtained after bortezomib treatment . with resolution of amr and normal kidney function at one year post transplant , the study suggests that bortezomib can target plasma cells implicated in amr . in the study by walsh et al . , two patients undergoing acute amr with high dsa and positive cd4 staining on biopsy two weeks after kidney transplantation were treated with a multiday regimen consisting of pp paired with methylprednisolone and bortezomib along with a single dose of rituximab . the theoretical rationale behind this protocol was that bortezomib would target plasma cells , rituximab would target plasma cell production from the pool of memory b - cells , and plasmapheresis would remove antibody levels creating a demand for increased antibody production to potentiate the effect of bortezomib 's proteasome inhibition . in addition , plasmapheresis was used to remove pre - existing antibodies that may have been circulating for weeks and thus measure antibody levels that would correlate with the magnitude of the antibody producing plasma cell population . by nearly 14 days after treatment , dsa levels had dropped significantly as well as repeat biopsy showed faint peritubular capillary c4d labeling and decreased glomerular c4d deposition . for patient 1 , dsa remained below detectable thresholds for 6 months following bortezomib treatment . for patient 2 , dsa remained below detectable thresholds for 2 months before a rebound in dsa titers were observed . a repeat cycle of pp , rituximab , and bortezomib treatment was given showing undetectable dsa within the first week of retreatment . the group concluded that bortezomib - based regimens may be beneficial in rapid dsa elimination in the setting of acute amr as an adjunct to commonly used modalities such as pp . in the study by sberro - soussan et al . , the group came to an opposite conclusion of bortezomib efficacy in four patients at least 1 year out from renal transplantation . in these four patients experiencing subacute amr , no significant decrease in dsa intensity as measured by mfi occurred despite use of pp , ivig , rituximab , and bortezomib in 150 days of followup . in addition , bortezomib 's activity on long - lived plasma cells as measured by observing any decrease in antiviral antibodies ( antihepatitis b surface antigen ) and total igg was not demonstrated as neither decreased significantly with a cycle of bortezomib . the group postulated that lack of activity against dsa may have been secondary to a long period of dsa stability following transplant as bortezomib was administered nearly 1 year or more after transplantation of these patients . they concluded that a single cycle of bortezomib does not seem to exert an effect on any long - lived antibody levels ( further than 1 year post - transplant ) whether the long - lived antibodies be dsa in sensitized kidney transplant recipients , anti - viral antibodies , or total immunoglobulins . yet , the group postulated that bortezomib may be more effective in more short - lived , intensely producing plasma cells as previously noted in the literature . finally , as this is the only study to use bortezomib as solo immunotherapy , steroids may be critical for a synergistic , proapoptotic effect in normal plasma cells . bortezomib 's ability to target antibody producing plasma cell has generated interest in its use for pre - transplant desensitization and treatment of amr . this drug may provide a promising insight into the management of patients undergoing kidney transplantation especially considering the large numbers of highly sensitized patients on the kidney transplant waiting list . new therapeutic strategies targeting reduction in dsa as well as managing amr can provide opportunities for these patients . although limited experience with bortezomib may seem to show promise in the realm of transplant recipients desensitization and treatment of amr , there is also experience that may suggest otherwise . specifically , the use of bortezomib with other accepted modalities for desensitization ( pp , ivig , and rituximab ) may make it difficult to tease out bortezomib 's role in transplant desensitization and treatment of amr as highlighted in the limited reported instances of its use . bortezomib 's role in transplant desensitization may be better elucidated as more clinical data and well - designed clinical trials become available .
demyelinating disorders like multiple sclerosis ( ms ) is a common cause of neurological morbidity and mortality among the general population . rare variants of ms like balo concentric sclerosis ( bcs ) are classically considered to have a rapid progression , poor outcome , and poor response to treatment . newer diagnostic modalities like magnetic resonance spectroscopy ( mrs ) have helped in early ante - mortem diagnosis and have also shed light on the pathogenesis of the disorder , which has until now remained largely obscure . a 26-year - old male reported in august 2009 , with complaints of acute - onset of loss of speech , deviation of face toward the right , nasal regurgitation of fluids , and altered behavior in the form of irrelevant and persistent smiling or crying , for the last 13 days . a day before there was no history of fever , headache , blurring of vision , diplopia , tinnitus , vertigo , seizures , unconsciousness , weakness of limbs , or bladder bowel involvement . there was no previous history of preceding infection or vaccination few days or weeks prior to the onset of neurological symptoms . during the examination , the patient was conscious , oriented with stable vitals , but aphasic ( transient , motor ) , and left upper motor neuron type facial nerve palsy with ninth and tenth cranial nerve paralysis , in the form of an absent gag reflex . neck rigidity and kernig 's sign were absent ; bilateral fundii , pupillary reflex , visual acuity , and color vision were normal with no ophthalmoparesis . the sensory and motor system and deep tendon reflexes were normal , the plantars were bilaterally downgoing , the romberg sign was negative , and coordination was normal . there was no history of diabetes , hypertension , palpitation , breathlessness , photosensitivity , or joint pains in in past . human immunodeficiency virus ( hiv ) , anti - nuclear antibody , anti - dsdna , rheumatoid factor , and anti - phospholipid antibody were negative . differential diagnosis included confluent acute demyelinating encephalomyelitis ( adem ) and confluent lesions of ms . cerebrospinal fluid ( csf ) analysis showed a total white blood cell ( wbc ) count of 06 cells ( all lymphocytes ) , sugar 36 mg% , protein 97.5 mg% , and adenosine deaminase 7.2 iu . it was negative for oligoclonal bands or igg . t2-weighted mri sequences [ figure 1 ] and fluid - attenuated inversion recovery ( flair ) sequence images showed bilateral asymmetric hyperintense confluent lesions in the periventricular and centrum semiovale location , suggestive of a demyelinating pathology [ figure 2 ] . axial t2wi and gadolinium - enhanced scans showed characteristic concentric and laminated lesions [ figures . 3 and 4 ] . magnetic resonance spectroscopy obtained as a single voxel image at 135 ms , at the time of presentation , showed a non - specific choline peak , suggestive of a demyelinating pathology [ figure 5 ] . sagittal t2wi showing confluent hyperintense lesions in the periventricular location axial fluid - attenuated inversion recovery image showing hyperintense white matter lesions in the periventricular lesions contrast - enhanced gadolinium scan showing no enhancement . the concentric laminated pattern of the lesions can be seen axial t2wi and fluid - attenuated inversion recovery showing concentric laminated hyperintense lesions characteristic of concentric sclerosis magnetic resonance spectroscopy single voxel at 135 ms showing a non - specific choline peak the patient was put on oral prednisolone based on clinical suspicion of an acute demyelinating pathology . the decision to continue with this we started oral prednisolone 1 mg / kg for four weeks , and then tapered it over six weeks . the patient 's speech , swallowing , and facial deviation returned to almost normal during the eight days of hospital stay . the patient was normal at the three- , six- , and twelve - month follow - up , with no relapse . magnetic resonance imaging after 12 months showed reduction in size of the demyelinating plaques . on follow - up scans balo concentric sclerosis is considered a rare acute demyelinating disease of the central nervous system , of unknown etiology . some consider it as a discrete disorder and some consider it to be a variant of ms . it is more common in young males , presenting as focal neurological deficits like , hemiplegia , hemianesthesia , or coordination defects . involvement of the cerebellum , spinal cord , and optic chiasm has also been reported . the alternating lamellae of demyelination and normal myelination with perivascular lymphocytic infiltrates and absence of intrathecal igg synthesis suggests that it is a different disease entity from ms . several physiochemical , anatomical , and cytotoxic theories have been proposed , to explain the genesis of these typical lesions , the latest being the theory of tissue preconditioning , by stadelmann et al . brain biopsy is the gold standard , but with the advent of mri , many more cases are now being diagnosed ante mortem . earlier it was considered as a disease with poor prognosis , but recent cases and our case show that in a single monophasic event , complete response to therapy and survival is possible . steroids , plasma exchange , and immunosuppressive therapy have been tried , with variable responses .
orthodontic root resorption ( orr ) is one of the most difficult procedure - related adverse events to predict in cases of orthodontic tooth movement ( otm ) , and may cause permanent loss of the dental structure at the root apex . in an epidemiological study by kaley and phillips,1 all patients who underwent comprehensive orthodontic treatment presented root shortening , and 3% of the patients had severe root resorption ( shortening by more than one - quarter of the root length ) with root shortening of the maxillary central incisors . previous studies have linked the severity of root resorption to various factors , including the type of orthodontic appliance,23 magnitude of the applied force,456 duration of force application , type of tooth movement , local and systemic diseases , patient age , genetic factors related to root anomalies , previous trauma , and ethnicity . orthodontists encounter orr when teeth ( especially maxillary anterior teeth ) are shifted by a reciprocating movement ( " jiggling " ) during orthodontic treatment ; the generated forces move the roots of the teeth mesiodistally or buccolingually . jiggling movements are thought to induce orr.78 however , the influence of jiggling forces during otm are not fully understood . therefore , this study focused on the relationship between orr and jiggling . in an in vivo experiment , we investigated the protein expression levels of interleukin ( il)-6 , cytokine - induced neutrophil chemoattractant 1 ( cinc-1 ; an il-8-related protein in rodents ) , receptor activator of nuclear factor b ligand ( rankl ) , and osteoprotegerin ( opg ) during experimental jiggling tooth movement in a rat model . the animal experimental protocol used in this study was approved by the ethics committee for animal experiments at the nihon university school of dentistry at matsudo ( chiba , japan ) ( approval number : ap13md003 ) . in total , 50 8-week - old male wistar rats ( body weight , 350 10 g ; sankyo labo service , tokyo , japan ) were used for the experiments . the animals were anesthetized with pentobarbital sodium ( 40 mg / kg of body weight ) for the application of orthodontic devices . experimental tooth movement was induced using the method described by hayashi et al.9 with a quad helix - type device ( diameter : 0.012 inches [ 0.3048 mm ] , stainless steel wire ; tomy international , inc . , tokyo , japan ) ligated to a maxillary first molar cleat with a 0.008-inches ( 0.2032 mm ) stainless steel ligature wire ( tomy international , inc . ) the maxillary first molar was palatally or buccally moved by the appliance with a force of 10 g or 50 g ( figure 1 ) . the rats were randomly assigned to four groups : the control group ( n = 5 ) with no appliance ; the optimal force ( of ) group ( n = 15 ) , treated with 10 g of compression ( palatal side of the root ) ; the heavy force ( hf ) group ( n = 15 ) , treated with 50 g of compression ( palatal side of the root ) ; and the jiggling force ( jf ) group ( n = 15 ) , treated with 10 g of compression from day 0 to day 7 ( palatal side of the root ) , 10 g of tension from day 7 to day 14 ( buccal side of the root ) , 10 g of compression day 14 to day 21 ( palatal side of the root ) ( figure 2 ) . the following in vivo experiments were performed , as described by nakano et al.10 each sample was sliced continuously into 4-m sections perpendicular to the long axis of the through center of pulp at distal palatal ( dp ) root of the maxillary first molar in the frontal cross - section , and prepared for hematoxylin and eosin ( h&e ) and immunohistochemical staining.9 the periodontal tissue was observed in the buccal and palatal portions of the dp root of the left first upper molar . detailed observations were made in the a area , a section of the root 300 m in height and 225 m in width in the direction from the top of the alveolar bone surface on the palatal side ( box a ) ; and in the b area , a section 300 m in height and 225 m in width that accounted for less than 150 m of the root in the direction of the bifurcation on the buccal side ( box b ) , on the pressure / tension side during tooth movement ( figure 3).11 in both areas , positive cells were counted manually . the a area was on the compression side for 21 days ( days 021 ) in the of and hf groups ; and in the jf group , on the compression side for the first and final 7 days ( days 07 and 1521 ) and on the tension side during the remaining 7 days ( days 814 ) . conversely , the b area was on the tension side for 21 days ( days 021 ) in the of and hf groups , versus on the tension side during the first and final 7 days ( days 07 and 1521 ) and on the compression side during the remaining seven days ( days 814 ) in the jf group . frontal serial sections were generated to include 15 consecutive slides adjacent to the center of the line connecting the dp root and distal buccal root . the mean values number of cell counts from 15 slides at the rats were used to calculate the ratio of positive cells to all cells . the tissue sections were deparaffinized , and endogenous peroxidase activity was quenched via incubation in 3% h2o2 in methanol for 30 minutes at room temperature . after washing in tris - buffered saline ( tbs ) , the sections were incubated with polyclonal anti - goat cathepsin k ( santa cruz biotechnology , inc . , dallas , tx , usa ; working dilution , 1:100 ) , polyclonal anti - goat matrix metalloproteinase ( mmp)-9 ( santa cruz biotechnology ; working dilution , 1:50 ) , polyclonal anti - goat il-6 ( santa cruz biotechnology ; working dilution , 1:100 ) , monoclonal anti - rat cinc-1 ( american research products , inc . , belmont , ma , usa ; working dilution , 1:50 ) , polyclonal anti - rabbit rankl ( santa cruz biotechnology ; working dilution , 1:100 ) , and polyclonal anti - goat opg ( santa cruz biotechnology ; working dilution , 1:100 ) for 18 hours at 4. cathepsin k , mmp-9 , il-6 , cinc-1 , rankl , and opg were stained using the histofine simple stain max po kit ( nichirei , co. , tokyo , japan ) according to the manufacturer 's protocol . the sections were rinsed with tbs and the final color reactions were performed using a 3,3'-diaminobenzidine tetrahydrochloride substrate reagent and aminoethyl carbazole . several sections were incubated with either nonimmune rabbit immunoglobulin g or 0.01 m phosphate - buffered saline instead of the primary antibody , and negative reactivity was observed . the kruskal - wallis , and steel - dwass tests were used to compare the means of the groups , with values of p < 0.05 and p < 0.01 considered to indicate significant differences from the corresponding control . the animal experimental protocol used in this study was approved by the ethics committee for animal experiments at the nihon university school of dentistry at matsudo ( chiba , japan ) ( approval number : ap13md003 ) . in total , 50 8-week - old male wistar rats ( body weight , 350 10 g ; sankyo labo service , tokyo , japan ) were used for the experiments . the animals were anesthetized with pentobarbital sodium ( 40 mg / kg of body weight ) for the application of orthodontic devices . experimental tooth movement was induced using the method described by hayashi et al.9 with a quad helix - type device ( diameter : 0.012 inches [ 0.3048 mm ] , stainless steel wire ; tomy international , inc . , tokyo , japan ) ligated to a maxillary first molar cleat with a 0.008-inches ( 0.2032 mm ) stainless steel ligature wire ( tomy international , inc . ) the maxillary first molar was palatally or buccally moved by the appliance with a force of 10 g or 50 g ( figure 1 ) . the rats were randomly assigned to four groups : the control group ( n = 5 ) with no appliance ; the optimal force ( of ) group ( n = 15 ) , treated with 10 g of compression ( palatal side of the root ) ; the heavy force ( hf ) group ( n = 15 ) , treated with 50 g of compression ( palatal side of the root ) ; and the jiggling force ( jf ) group ( n = 15 ) , treated with 10 g of compression from day 0 to day 7 ( palatal side of the root ) , 10 g of tension from day 7 to day 14 ( buccal side of the root ) , 10 g of compression day 14 to day 21 ( palatal side of the root ) ( figure 2 ) . the following in vivo experiments were performed , as described by nakano et al.10 each sample was sliced continuously into 4-m sections perpendicular to the long axis of the through center of pulp at distal palatal ( dp ) root of the maxillary first molar in the frontal cross - section , and prepared for hematoxylin and eosin ( h&e ) and immunohistochemical staining.9 the periodontal tissue was observed in the buccal and palatal portions of the dp root of the left first upper molar . detailed observations were made in the a area , a section of the root 300 m in height and 225 m in width in the direction from the top of the alveolar bone surface on the palatal side ( box a ) ; and in the b area , a section 300 m in height and 225 m in width that accounted for less than 150 m of the root in the direction of the bifurcation on the buccal side ( box b ) , on the pressure / tension side during tooth movement ( figure 3).11 in both areas , positive cells were counted manually . the a area was on the compression side for 21 days ( days 021 ) in the of and hf groups ; and in the jf group , on the compression side for the first and final 7 days ( days 07 and 1521 ) and on the tension side during the remaining 7 days ( days 814 ) . conversely , the b area was on the tension side for 21 days ( days 021 ) in the of and hf groups , versus on the tension side during the first and final 7 days ( days 07 and 1521 ) and on the compression side during the remaining seven days ( days 814 ) in the jf group . frontal serial sections were generated to include 15 consecutive slides adjacent to the center of the line connecting the dp root and distal buccal root . the mean values number of cell counts from 15 slides at the rats were used to calculate the ratio of positive cells to all cells . the tissue sections were deparaffinized , and endogenous peroxidase activity was quenched via incubation in 3% h2o2 in methanol for 30 minutes at room temperature . after washing in tris - buffered saline ( tbs ) , the sections were incubated with polyclonal anti - goat cathepsin k ( santa cruz biotechnology , inc . , dallas , tx , usa ; working dilution , 1:100 ) , polyclonal anti - goat matrix metalloproteinase ( mmp)-9 ( santa cruz biotechnology ; working dilution , 1:50 ) , polyclonal anti - goat il-6 ( santa cruz biotechnology ; working dilution , 1:100 ) , monoclonal anti - rat cinc-1 ( american research products , inc . , belmont , ma , usa ; working dilution , 1:50 ) , polyclonal anti - rabbit rankl ( santa cruz biotechnology ; working dilution , 1:100 ) , and polyclonal anti - goat opg ( santa cruz biotechnology ; working dilution , 1:100 ) for 18 hours at 4. cathepsin k , mmp-9 , il-6 , cinc-1 , rankl , and opg were stained using the histofine simple stain max po kit ( nichirei , co. , tokyo , japan ) according to the manufacturer 's protocol . the sections were rinsed with tbs and the final color reactions were performed using a 3,3'-diaminobenzidine tetrahydrochloride substrate reagent and aminoethyl carbazole . for the immunohistochemical controls , several sections were incubated with either nonimmune rabbit immunoglobulin g or 0.01 m phosphate - buffered saline instead of the primary antibody , and negative reactivity was observed . the kruskal - wallis , and steel - dwass tests were used to compare the means of the groups , with values of p < 0.05 and p < 0.01 considered to indicate significant differences from the corresponding control . the body weights of the rats in each force group showed no change during the experimental period . no significant difference in body weight was observed during the experimental period between the force groups ( data not shown ) . regarding areas a and b , in the control group ( applied orthodontic force ; 0 g ) on days 7 , 14 , and 21 after tooth movement , the rats specimens were composed of relatively dense connective tissue fibers and fibroblasts that ran regularly in a horizontal direction from the root cementum towards the alveolar bone . blood capillaries were mainly recognized near the alveolar bone in the periodontal ligament ( pdl ) , and the root surfaces were relatively smooth ( figure 4a4f ) . in the a and b areas in the of group ( applied orthodontic force ; 10 g ) , the arrangement of fibers and fibroblasts became coarse and irregular , and blood capillaries were compressed on days 7 and 14 . resorption lacunae with few multinucleated odontoclasts were observed on palatal root surfaces ( figure 4 g , 4h , 4j , and 4k ) . on day 21 , root resorption lacunae with a few multinucleated odontoclasts were observed on root surfaces ( figure 4i and 4l ) . in the a area in the hf group ( applied orthodontic force ; 50 g ) , root resorption lacunae with multinucleated odontoclasts were identified on root surfaces on day 7 after the application of orthodontic force ( figure 4 m ) . many resorption lacunae with multinucleated odontoclasts were observed on roots on day 14 ( figure 4n ) . on day 21 , root resorption lacunae with multinucleated odontoclasts were mostly observed on the root surface ( figure 4o ) . multinucleated odontoclasts on the palatal root surface and root resorption lacunae gradually increased from day 7 through day 21 . conversely , in the b area in the hf group ( applied orthodontic force ; 50 g ) , resorption lacunae with few multinucleated odontoclasts were observed on buccal root surfaces on days 14 and 21 ( figure 4p4r ) . in the a and b areas in the jf ( applied orthodontic force ; 10 g ) group , the arrangement of fibers and fibroblasts became coarse and irregular , and blood capillaries were compressed on day 7 , as in the of group ( applied orthodontic force ; 10 g ) . resorption lacunae with few multinucleated odontoclasts were observed on palatal root surfaces ( figure 4s and 4v ) . on day 14 , root resorption lacunae were increased compared to day 7 ( figure 4 t and 4w ) . on day 21 , many root resorption lacunae containing multinucleated odontoclasts were observed on the palatal roots ( figure 4u and 4x ) . in the control group , no resorption lacunae with cathepsin k - positive or mmp-9-positive multinucleated odontoclasts were observed on the surfaces of the roots in either the a area or b area during the experimental period ( figures 5 and 6a6f ) . in the of group , resorption lacunae with cathepsin k - positive and mmp-9-positive multinucleated odontoclasts were also not observed on the surfaces of roots in the a area or b area from day 7 to day 21 ( figures 5 and 6g6l ) . in the a area in the hf group , few root resorption lacunae with multinucleated cathepsin k - positive and mmp-9-positive odontoclasts were identified on root surfaces on day 7 ( figures 5 and 6 m ) . on days 14 and 21 , many root resorption lacunae with cathepsin k - positive and mmp-9-positive multinucleated odontoclasts were observed ( figures 5,6n , and 6o ) . in the b area , no root resorption lacunae with cathepsin k - positive or mmp-9-positive multinucleated odontoclasts were observed on day 7 ( figures 5,6p , and 6q ) . however , they were observed on days 14 and 21 ( figures 5,6q , and 6r ) . in the jf group on day 7 after tooth movement , no resorption lacunae with cathepsin k - positive or mmp-9-positive multinucleated odontoclasts were observed on root surfaces in the a or b areas ( figures 5 and 6s ) . on day 14 , a few cathepsin k - positive and mmp-9-positive odontoclasts were observed ( figures 5,6 t , and 6w ) . furthermore , on day 21 , many root resorption lacunae with cathepsin k - positive and mmp-9-positive odontoclasts were observed ( figures 5,6u , and 6x ) . in the control group in the a and b areas , il-6-positive , cinc-1-positive , and rankl - positive cells were rarely observed in pdl tissues during days 7 through 21 ( figures 7,8 , and 9a9f ) . in the of group , few il-6-positive , cinc-1-positive , and rankl - positive cells were observed in pdl tissues on root surfaces in the a area through day 21 ( figures 7,8 , and 9g9i ) . conversely , in the of group ( 10 g ) in the b area , they were rarely observed in pdl tissues during days 7 through 21 ( figures 7,8 , and 9j9l ) . in the hf group , few il-6-positive , cinc-1-positive , and rankl - positive cells were observed in pdl tissues on day 7 in the a areas of the roots , while increased numbers were observed on days 14 and 21 ( figures 7,8 , and 9m9o ) . conversely , in the b area , no il-6-positive , cinc-1-positive , or rankl - positive cell was observed in pdl tissues on day 7 , while a few cells were observed on days 14 and 21 ( figures 7,8 , and 9p9r ) . in the jf group , no il-6-positive , cinc-1-positive , or rankl - positive cell was observed on day 7 , although cells were observed on day 14 , and increased numbers were observed on day 21 in both the a and b areas of the root surfaces ( figures 7,8 , and 9s9x ) . in all groups , few opg - positive cells were observed in the pdl during days 7 through 21 ( figure 10 ) . in our quantitative evaluations , the numbers of cathepsin k - positive and mmp-9-positive odontoclasts were found to significantly increase in the hf and jf groups on day 21 versus the control group in both the a and b areas ( p < 0.01 ) . furthermore , their numbers were significantly increased in the jf group on day 21 compared with the hf group ( p < 0.01 ) . there was no significant difference in the a or b areas between the of group and the control group ( figure 11a and 11b ) . the numbers of il-6-positive , cinc-1-positive , and rankl - positive cells were found to increase significantly in the hf and jf groups on day 21 versus the control group in both the a and b areas . their numbers were greater in the jf group than in the hf group in both the a and b areas on day 21 ( p < 0.01 ) ( figure 11c11e ) . more significant differences were observed in the b area than in the a area ( figure 11c11e ) . the number of opg - positive cells was significantly ncreased in the of , hf and jf group on day 21 compared with the control group ; however , there was no significant difference between of , hf and jf group ( figure 11f ) . the body weights of the rats in each force group showed no change during the experimental period . no significant difference in body weight was observed during the experimental period between the force groups ( data not shown ) . regarding areas a and b , in the control group ( applied orthodontic force ; 0 g ) on days 7 , 14 , and 21 after tooth movement , the rats specimens were composed of relatively dense connective tissue fibers and fibroblasts that ran regularly in a horizontal direction from the root cementum towards the alveolar bone . blood capillaries were mainly recognized near the alveolar bone in the periodontal ligament ( pdl ) , and the root surfaces were relatively smooth ( figure 4a4f ) . in the a and b areas in the of group ( applied orthodontic force ; 10 g ) , the arrangement of fibers and fibroblasts became coarse and irregular , and blood capillaries were compressed on days 7 and 14 . resorption lacunae with few multinucleated odontoclasts were observed on palatal root surfaces ( figure 4 g , 4h , 4j , and 4k ) . on day 21 , root resorption lacunae with a few multinucleated odontoclasts were observed on root surfaces ( figure 4i and 4l ) . in the a area in the hf group ( applied orthodontic force ; 50 g ) , root resorption lacunae with multinucleated odontoclasts were identified on root surfaces on day 7 after the application of orthodontic force ( figure 4 m ) . many resorption lacunae with multinucleated odontoclasts were observed on roots on day 14 ( figure 4n ) . on day 21 , root resorption lacunae with multinucleated odontoclasts were mostly observed on the root surface ( figure 4o ) . multinucleated odontoclasts on the palatal root surface and root resorption lacunae gradually increased from day 7 through day 21 . conversely , in the b area in the hf group ( applied orthodontic force ; 50 g ) , resorption lacunae with few multinucleated odontoclasts were observed on buccal root surfaces on days 14 and 21 ( figure 4p4r ) . in the a and b areas in the jf ( applied orthodontic force ; 10 g ) group , the arrangement of fibers and fibroblasts became coarse and irregular , and blood capillaries were compressed on day 7 , as in the of group ( applied orthodontic force ; 10 g ) . resorption lacunae with few multinucleated odontoclasts were observed on palatal root surfaces ( figure 4s and 4v ) . on day 14 , root resorption lacunae were increased compared to day 7 ( figure 4 t and 4w ) . on day 21 , many root resorption lacunae containing multinucleated odontoclasts were observed on the palatal roots ( figure 4u and 4x ) . in the control group , no resorption lacunae with cathepsin k - positive or mmp-9-positive multinucleated odontoclasts were observed on the surfaces of the roots in either the a area or b area during the experimental period ( figures 5 and 6a6f ) . in the of group , resorption lacunae with cathepsin k - positive and mmp-9-positive multinucleated odontoclasts were also not observed on the surfaces of roots in the a area or b area from day 7 to day 21 ( figures 5 and 6g6l ) . in the a area in the hf group , few root resorption lacunae with multinucleated cathepsin k - positive and mmp-9-positive odontoclasts were identified on root surfaces on day 7 ( figures 5 and 6 m ) . on days 14 and 21 , many root resorption lacunae with cathepsin k - positive and mmp-9-positive multinucleated odontoclasts were observed ( figures 5,6n , and 6o ) . in the b area , no root resorption lacunae with cathepsin k - positive or mmp-9-positive multinucleated odontoclasts were observed on day 7 ( figures 5,6p , and 6q ) . however , they were observed on days 14 and 21 ( figures 5,6q , and 6r ) . in the jf group on day 7 after tooth movement , no resorption lacunae with cathepsin k - positive or mmp-9-positive multinucleated odontoclasts were observed on root surfaces in the a or b areas ( figures 5 and 6s ) . on day 14 , a few cathepsin k - positive and mmp-9-positive odontoclasts were observed ( figures 5,6 t , and 6w ) . furthermore , on day 21 , many root resorption lacunae with cathepsin k - positive and mmp-9-positive odontoclasts were observed ( figures 5,6u , and 6x ) . in the control group in the a and b areas , il-6-positive , cinc-1-positive , and rankl - positive cells were rarely observed in pdl tissues during days 7 through 21 ( figures 7,8 , and 9a9f ) . in the of group , few il-6-positive , cinc-1-positive , and rankl - positive cells were observed in pdl tissues on root surfaces in the a area through day 21 ( figures 7,8 , and 9g9i ) . conversely , in the of group ( 10 g ) in the b area , they were rarely observed in pdl tissues during days 7 through 21 ( figures 7,8 , and 9j9l ) . in the hf group , few il-6-positive , cinc-1-positive , and rankl - positive cells were observed in pdl tissues on day 7 in the a areas of the roots , while increased numbers were observed on days 14 and 21 ( figures 7,8 , and 9m9o ) . conversely , in the b area , no il-6-positive , cinc-1-positive , or rankl - positive cell was observed in pdl tissues on day 7 , while a few cells were observed on days 14 and 21 ( figures 7,8 , and 9p9r ) . in the jf group , no il-6-positive , cinc-1-positive , or rankl - positive cell was observed on day 7 , although cells were observed on day 14 , and increased numbers were observed on day 21 in both the a and b areas of the root surfaces ( figures 7,8 , and 9s9x ) . in all groups , few opg - positive cells were observed in the pdl during days 7 through 21 ( figure 10 ) . in our quantitative evaluations , the numbers of cathepsin k - positive and mmp-9-positive odontoclasts were found to significantly increase in the hf and jf groups on day 21 versus the control group in both the a and b areas ( p < 0.01 ) . furthermore , their numbers were significantly increased in the jf group on day 21 compared with the hf group ( p < 0.01 ) . there was no significant difference in the a or b areas between the of group and the control group ( figure 11a and 11b ) . the numbers of il-6-positive , cinc-1-positive , and rankl - positive cells were found to increase significantly in the hf and jf groups on day 21 versus the control group in both the a and b areas . their numbers were greater in the jf group than in the hf group in both the a and b areas on day 21 ( p < 0.01 ) ( figure 11c11e ) . more significant differences were observed in the b area than in the a area ( figure 11c11e ) . the number of opg - positive cells was significantly ncreased in the of , hf and jf group on day 21 compared with the control group ; however , there was no significant difference between of , hf and jf group ( figure 11f ) . all methods in this study , including the application of a 10-g light force , 50-g heavy force , and 10-g jiggling force , produced tooth movement over a period of 21 days in rats . gonzales et al.12 showed that the application of a 10-g light force produced significantly greater tooth movement with significantly less root resorption over 28 days versus a greater force in rats . the optimum force for moving rat upper molars may be even less than 10 g , as previously suggested.13 many investigators have reported that root resorption is aggravated by increased force magnitudes.613 for example , gameiro et al.14 demonstrated osteoclastic resorption of the roots on mesial surfaces of teeth subjected to a large orthodontic force ( 50 g ) . therefore , the present model represents a method for inducing efficient tooth movement and root resorption . furthermore , hayashi et al.9 established a method for achieving a jiggling movement in rats . we referred to this method and produced a force applications in opposing directions force by moving the roots of rat molars bucco - palatally once a week for 21 days in vivo . in the a area in this study , the h&e results in the of and hf groups were largely consistent with those of previous studies.10 the application of a jiggling force increased resorption lacunae in comparison with the hf group on day 21 ( figure 4 ) . conversely , in the b area , many resorption lacunae were observed in the jf group on days 14 and 21 ( figure 4 ) . chan and darendeliler6 quantified the extent of root resorption under compression ( 150 g ) and tension ( 150 g ) in human teeth undergoing otm using volumetry . the volume of root resorption was greater under compression than under tension or under both compression and tension . furthermore , there was greater root resorption under both compression and tension than under tension alone . interestingly , the number of resorption lacunae was significantly greater under the condition of a jiggling force of 10 g than under the condition of a continuous unidirectional force of 50 g during tooth movement in rats ( figure 5 ) . therefore , weaker jiggling forces may induce more root resorption than stronger continuous forces . conversely , eross et al.15 reported no significant difference in root resorption between a heavy continuous force ( 225 g ) and heavy jiggling force ( 225 g every 4 weeks ) in humans . the discrepancies between previous results and the present results may be due to differences between species and the magnitudes and intervals of the applied forces . orr occurs at the periphery of necrotic hyalinized tissue,16 and the pattern at the site of compression is related to the lesion.17 the pathogenesis of orr is associated with the removal of necrotic tissue from areas of the pdls compressed by orthodontic loads.1819 previous studies have also shown that orr is caused by the removal of necrotic hyalinized tissue.2021 tartrate - resistant acid phosphatase ( trap ) staining in a rat tooth movement model highlighted the involvement of trap - positive macrophages and multinucleated giant cells in the removal of hyalinized tissue.18 recently , ohashi et al.22 reported that immunoreactivity for trap was stronger in resorbed roots exposed to a jiggling force ( 10 g ) versus a heavy force ( 50 g ) on day 21 . therefore , the number of trap - positive odontoclasts was significantly increased in the jiggling force group compared with the hf group on day 21 . previous studies have reported that 2 to 4 weeks are required to remove hyalinized tissue.23 a jiggling movement leads to compression on both the buccal and lingual sides ; therefore , the formation of hyalinized tissue may extend over a wide area . hence , the application of forces in opposing directions before periodontal tissue repair induces the formation of hyalinized tissue , consequently aggravating orr . eross et al.15 concluded that jiggling forces applied alternately in different directions with a short interval of reactivation are critically important in inducing severe root resorption . cathepsin k - positive and mmp-9-positive odontoclasts were also increased in the a area in the jf group on day 21 compared with the hf and of groups , and many cathepsin k - positive and mmp-9-positive odontoclasts were observed in the jf group on days 14 and 21 in the b area ( figures 5,6 ) . osteoclasts and odontoclasts resorb mineralized tissues by lowering the ph in resorption lacunae , followed by tissue degradation via the secretion of proteolytic enzymes , which are classified as either cysteine proteinases , including the cathepsin family , or mmps . in particular , cathepsin k and mmp-9 are characteristic proteinases expressed in osteoclasts and odontoclasts.2425 tsuchiya et al.26 reported that cathepsin k and mmp-9 are expressed in odontoclasts under root resorption conditions during tooth movement in rats . taken together , these findings and the present results suggest that jiggling forces may induce odontoclast formation during otm . the relationship between orr and inflammatory cytokines has been reported in many studies using rat tooth movement models . heavy forces of 50 g induce orr via rankl / opg , il-6 , and il-8 production.272829 these findings support the results observed in the of and hf groups in this study . in our study , at a jiggling force of 10 g ( the optimal force ) , il-6-positive , cinc-1-positive , and rankl - positive pdl cells in the a areas were increased in the jf group on day 21 compared with the hf group ( 50 g ) and the of group ( 10 g ) . many il-6-positive , cinc-1-positive , and rankl - positive odontoclasts were also observed in the jf group on days 14 and 21 in the b areas ( figures 7,8,9 ) . in all groups , few opg - positive cells were observed in pdl tissues during days 7 through 21 ( figure 10 ) . considering the mechanism of enhancement of these cytokines by jiggling forces , in vitro studies applying compression and tension forces to pdl cells may provide clues . previous studies have reported that compression forces induce the production of inflammatory cytokines , such as il-6 , il-8 , and rankl in a magnitude - dependent manner in human pdl cells in vitro.272829 interestingly , tension forces also induce these cytokines in pdl cells . therefore , jiggling forces may increase cytokines more significantly in response to both compression and tension forces than in response to unidirectional forces . regarding differences between the a and b areas , the numbers of il-6-positive , cinc-1-positive , and rankl - positive cells were greater in the jf group in a than in the jf group in b on day 21 ( figure 11 ) . the pdl tissues in a were exposed to compression forces twice and tension forces once , whereas those in b were exposed to tension forces twice and compression forces once . garlet et al.30 demonstrated increased expression of rankl on both the compression and tension sides versus the controls , with significantly greater expression on the compression side than on the tension side . therefore , a jiggling force may increase cytokines more significantly in the a area than the b area . these results suggest that jiggling forces may induce orr via the production of inflammatory cytokines during otm and may be a risk factor for orr . orthodontists must seek to avoid the application of jiggling forces to teeth as much as possible to reduce the incidence of orr .
health claims are potentially powerful tools in consumer communication as they convey information on the health benefits of food or food components . however , health claims also have the potential to misdirect consumers towards food choices that may be against their best interests . thus , to help ensure correct health claims and prevent exaggerations , many countries have developed laws , guidelines , and codes of practice regarding health claims . in south korea , health / functional food ( hff ) regulations include controls on communications regarding health claims such as through labeling , presentation , and advertisement [ 2 - 5 ] . kfda introduced an evidence - based rating system created by world health organization ( who ) ; the system has four categories based on levels of evidence : convincing , probable , possible , and insufficient . the category assigned is determined by considering the type and quality of individual studies and the quantity , consistency , and relevance of the aggregate of studies . the us food and drug administration ( fda ) also uses an evidence - based ranking system for qualified health claims . however , certain consumer advocacy groups , such as consumer federation of america ( cfa ) and center for science in the public interest ( cspi ) , are opposed to qualified health claims . they argue that consumers can not differentiate between the various levels of scientific evidence and will likely be misled into purchasing items with health claims that have not yet passed " sufficient " scientific scrutiny . although the effects such claims have on consumer perceptions have not been extensively studied , a number of studies have tested consumers ' reactions to various nutrition and health messages [ 11 - 18 ] . to properly use hffs , consumers must be able to clearly understand the meaning of health messages behind a health claim . to address this issue , this study examined the effect of various qualified levels of health claims on consumer attitudes . our goals were to determine whether consumers can differentiate between levels of claims , and to clarify how a visual aid influences consumer understanding of the different claim levels . the study participants were 2,000 male and female adults aged 19 years or older living in korea . a direct - interview survey was conducted from august 1 , 2005 to september 1 , 2005 . measures of confidence in the information on health claims and expected health benefits of the product were used as indicators of whether the consumers could distinguish between the various levels of health claims . for this study , we used a hypothetical hff product , namely a supplement containing plant sterol ( fig . plant sterol was approved in korea in 2005 as a hff to help manage blood cholesterol . because plant sterol was not approved as a food ingredient before 2005 , the average korean consumer had no information about plant sterol and its health benefits at the time of our study . thus , we could exclude bias originating from previous information about the health benefits of plant sterol . the study was conducted in 13 geographically dispersed shopping malls in urban areas nationwide . at each site , a central interviewing facility was available where we could recruit participants , select participants with the required background characteristics , and observe and document the interview process . adults aged 19 and older , were recruited for a study about hff labeling and were randomly assigned to label conditions . we asked the participants two kinds of questions while showing them different cases for the hypothetical product . each case made a different level of health claim ( probable and possible ) and was presented with or without a visual aid ( table 1 ) . that is , participants were given product labels with probable and possible claims accompanied by different levels of information . we then asked them to compare the information level and their belief in the health benefits using a seven - point scale ( table 2 ; 1 = strongly disagree ; 7 = strongly agree ) . differences between the control and treatment groups were analyzed by student 's t - test . the study participants were 2,000 male and female adults aged 19 years or older living in korea . a direct - interview survey was conducted from august 1 , 2005 to september 1 , 2005 . measures of confidence in the information on health claims and expected health benefits of the product were used as indicators of whether the consumers could distinguish between the various levels of health claims . for this study , we used a hypothetical hff product , namely a supplement containing plant sterol ( fig . plant sterol was approved in korea in 2005 as a hff to help manage blood cholesterol . because plant sterol was not approved as a food ingredient before 2005 , the average korean consumer had no information about plant sterol and its health benefits at the time of our study . thus , we could exclude bias originating from previous information about the health benefits of plant sterol . the study was conducted in 13 geographically dispersed shopping malls in urban areas nationwide . at each site , a central interviewing facility was available where we could recruit participants , select participants with the required background characteristics , and observe and document the interview process . adults aged 19 and older , were recruited for a study about hff labeling and were randomly assigned to label conditions . we asked the participants two kinds of questions while showing them different cases for the hypothetical product . each case made a different level of health claim ( probable and possible ) and was presented with or without a visual aid ( table 1 ) . that is , participants were given product labels with probable and possible claims accompanied by different levels of information . we then asked them to compare the information level and their belief in the health benefits using a seven - point scale ( table 2 ; 1 = strongly disagree ; 7 = strongly agree ) . differences between the control and treatment groups were analyzed by student 's t - test . approximately 26% of the participants were male , and 74% were female . by age range , 21.3% were 20 - 29 years old , 31.5% were 30 - 39 years , and 27.0% were 40 - 49 years old . of the participants , 73.3% reported having eaten hffs , whereas 26.7% reported that they had never eaten such foods ( table 3 ) . when the participants were asked to read the label and then answer questions about their confidence in the claims about the product on a seven - point scale , the average scores were 4.17 and 4.07 for claims rated as probable and possible , respectively ( table 4 ) . scores were significantly higher for the probable claim than for the possible claim ( p < 0.05 ) . to confirm the effect of a visual aid , average scores with the visual aids were 5.37 for the probable level and 4.41 for the possible level . again , the score for the probable - level claim was significantly higher ( p < 0.05 ) . we then asked the participants to read two different claims , determine whether there was a difference in the strength of the scientific level behind those claims . as shown in table 5 , half of the respondents answered that there was more evidence for the higher - level claim . we investigated consumer confidence in the scientific evidence presented in labels with and without a visual aid ( table 6 ) . scores for confidence in labels with and without the visual aid were 5.27 and 4.43 , respectively , with the score for the label with a visual aid significantly higher than that for the label without a visual aid ( p < 0.01 ) . in this study , we investigated that consumers could differentiate between levels of claims , and clarify how a visual aid influences consumer understanding of the different claim levels . health claims communicate potential benefits to the consumer but may also create a bias in perception due to the way a claim is presented or to the beliefs of the individual reading it . with the kfda 's permission , health claims have been ranked at different levels based on the quality and quantity of scientific evidence . in order to understand the perception of consumers for scientific level of health claims , we interviewed 2,000 consumers with the questionnaire in the shopping malls . because of interviewing place , which was shopping area , most respondents were middle - aged women ( table 3 ) . when the participants were asked for their confidence with seven point scales , scores were significantly higher for the probable claim than for the possible claim ( p < 0.05 ) regardless of visual aids ( table 4 ) moreover , scores for confidence in labels with and without the visual aid were 5.27 and 4.43 , respectively , with the score for the label with a visual aid significantly higher than that for the label without a visual aid ( table 6 ) . a few studies of qualified health claims have been conducted in the us . using a mall - intercept data collection process , the us fda evaluated a range of product label formats and types of message language . visual- and text - based constructs tested the formats ' ability to convey the strength of scientific support . the results suggested that text alone did not convey the message of the degree of scientific support for qualified health claims . visual based formats generally helped consumers identify differences between levels of claims , yet not always in the correct manner . our study also shows that qualifying language for health claims can be specified to help consumers better understand the strength of scientific evidence . moreover , with a visual aid , the consumers perceived the levels of scientific evidence more clearly and had greater confidence in their meaning to health than in the case without a visual aid . this result is similar to that found in the us fda 's consumer survey for qualified health claims . although this result suggests that consumers react differently to several claim levels , it is not clear whether consumers actually understand the difference in the degree of scientific support for claims . food label information is just one source of marketing communications available to food manufacturers and consumers . consumers are more readily affected by advertisement than by labeling . according to reports by the us federal trade commission , none of the tested qualifying statements in advertisements was correctly interpreted by consumers as describing an associated weaker level of scientific support for the health claim . in experimental studies using self - reports , this method makes it possible to assess consumer understanding , but it is very different from a normal shopping experience . hence , it is probable that studies in which consumers are specifically asked to respond to health claims will overestimate the use of claim information relative to most real - life conditions . understanding consumer responses to health and nutrition information on product packages is critical when designing food - labeling regulations . the key message of this study is that further investigation is needed on how to effectively communicate novel , emerging nutrition information on product labels to consumers . the government 's goal is to permit the use of more , better , easily understood , and up - to - date scientific information on labels to communicate how food choice can affect the health of consumers . our results suggest that policymakers should try to enact regulations that will ensure that the exact meanings of health claims are presented on food labels to consumers . further research on the impacts and effectiveness of food advertisements and labeling is also needed with regard to health claims . qualitative studies such as focus group interviews may be helpful in identifying more specific disclaimers and effective ways of delivering health messages for food or food components .
successful endodontic therapy depends on thorough chemo - mechanical preparation as well as three - dimensional obturation that provides a complete sealing of the root canal system . during cleaning and shaping procedure instrumentation with various root canal instruments leaves an amorphous , granular , and irregular layer covering root dentin known as smear layer that contains inorganic and organic material . the first researchers to describe the smear layer on the surface of instrumented root canals were mccomb and smith . the smear layer may adversely affect the disinfection of dentin walls while blocking irrigants and sealants from entering dentinal tubules . in addition , it may increase post obturation microleakage , and may serve as a source of nutrients for some species of intra - canal microbiota . removal of smear layer after root canal instrumentation and before canal obturation improves the adaptation of root filling materials to the canal walls , resulting in a superior seal . so far the most commonly used method of smear layer removal has been the chemical method using chelating agents , with ethylenediaminetetraacetic acid ( edta ) being the most common agent used . the most advocated combination of 17% edta plus 5.25% naocl removes the smear layer completely in the coronal and middle thirds but is less effective in the apical third . for effective removal of smear layer apart from edta new irrigating agents such as qmix 2 in 1 , mtad , have been introduced as final irrigants . the aim of the present study is to evaluate the smear layer removal efficacy of different final irrigating solutions . they were decoronated to get the stable reference point and to standardize the root canal length of 14 mm . all specimen teeth were randomly divided into four groups as follows : group 1 : saline.group 2 : edta.group 3 : biopure mtad.group 4 : qmix 2 in 1 . patency of the root canal is established by passing a stainless steel number 15 k - file ( kendo , germany ) just beyond the apex of all canals . canals were prepared using protaper rotary system ( dentsply maillefer , ballaigues , switzerland ) . india ) irrigant was used between each subsequent file size in all experimental groups while saline was the sole irrigant in group 1 . to determine the effects of final irrigating solutions on the surface of root canals after instrumentation , the irrigating solution was delivered using a 30-gauge side - vented needle ( dentsply tulsa dental , tulsa , ok , usa ) passively placed to within the apical third of the root canals . nonpenetrating grooves were made in all specimen teeth at the cement - enamel junction ( cej ) and longitudinally on the buccal and lingual aspects . the teeth were then longitudinally split into two halves using chisel and mallet and the half containing the greater part of the apex was selected as the representative sample for each group and they were evaluated under scanning electronic microscope . coded samples were mounted on metallic stubs , gold sputtered and viewed under a scanning electron microscope ( sem ) . photographs at magnifications of 1000 were taken for each specimen in the apical third ( 4 mm from root apex ) . the images at 1000 magnification were then analyzed for the amount of smear layer present [ figures 1a , b and 2a , b ] . the amount of smear layer remaining on the surface of the root canal and dentinal tubules was scored according to a three score system developed by torabinejad et al . ( a ) scanning electron microscope picture of saline , ( b ) scanning electron microscope picture of ethylenediaminetetraacetic acid ( a ) scanning electron microscope picture of mtad , ( b ) scanning electron microscope picture of qmix 2 in 1 score 1 : no smear layer no smear layer was detected on the surface of the root canal , and all tubules were open.score 2 : moderate smear layer no smear layer on root canal walls but tubules contained debris.score 3 : heavy smear layer smear layer covered the root canal wall surface and the tubules . score 1 : no smear layer no smear layer was detected on the surface of the root canal , and all tubules were open . score 2 : moderate smear layer no smear layer on root canal walls but tubules contained debris . score 3 : heavy smear layer smear layer covered the root canal wall surface and the tubules . coded samples were mounted on metallic stubs , gold sputtered and viewed under a scanning electron microscope ( sem ) . photographs at magnifications of 1000 were taken for each specimen in the apical third ( 4 mm from root apex ) . the images at 1000 magnification were then analyzed for the amount of smear layer present [ figures 1a , b and 2a , b ] . the amount of smear layer remaining on the surface of the root canal and dentinal tubules was scored according to a three score system developed by torabinejad et al . ( a ) scanning electron microscope picture of saline , ( b ) scanning electron microscope picture of ethylenediaminetetraacetic acid ( a ) scanning electron microscope picture of mtad , ( b ) scanning electron microscope picture of qmix 2 in 1 score 1 : no smear layer no smear layer was detected on the surface of the root canal , and all tubules were open.score 2 : moderate smear layer no smear layer on root canal walls but tubules contained debris.score 3 : heavy smear layer smear layer covered the root canal wall surface and the tubules . score 1 : no smear layer no smear layer was detected on the surface of the root canal , and all tubules were open . score 2 : moderate smear layer no smear layer on root canal walls but tubules contained debris . score 3 : heavy smear layer smear layer covered the root canal wall surface and the tubules . statistical analysis was done using kruskal - wallis h test and mann - whitney u - test using software version spss 17.0 version ( spss inc . , the results showed that qmix 2 in 1 ( group 4 ) showed least smear layer scores ( 1.30 0.48 ) when compared to others . this was followed by mtad ( 2.00 0.00 ) , edta ( 2.40 0.52 ) , and saline showed highest smear layer scores ( 3.00 0.00 ) . during mechanical instrumentation smear layer formation occurs on the root canal dentinal wall occluding dentinal tubules , for efficient disinfection and sealing of the root canal system its removal is necessary . irrigation is an important step during and after instrumentation for effective removal of smear layer and also for lubrication of root canal system . naocl is the traditional irrigant which is used most commonly , however due to its inability to remove inorganic part of smear layer other irrigants were introduced . there are different final irrigating solutions which are used after instrumentation for effective smear layer removal . qmix 2 in 1 is a new root canal irrigating solution , and it contains a mixture of a bisbiguanide an antimicrobial agent , a polyaminocarboxylic acid , a calcium - chelating agent , saline , and a surfactant . eliot et al . evaluated the effectiveness of the three different formulations of qmix on the removal of canal wall smear layer and compared to a standard solution of 17% edta . sem analysis showed the effectiveness of all three qmix formulations were superior to edta in smear layer removal and exposure of dentinal tubules in the root canal system in single - rooted teeth . analyzed the precipitate formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a final rinse of chlorhexidine or qmix and concluded no precipitates or pca were detected with qmix and naocl in dentinal tubules . evaluated efficacy of a novel root canal irrigant , qmix , against enterococcus faecalis and its ability to remove smear layer was examined , using sem . it was concluded that qmix and naocl were superior to chx and mtad under laboratory conditions in killing e. faecalis and ability to remove smear layer by qmix was comparable to edta . this was also supported by studies done by ballal et al . , who showed that wetting of root canal dentin was better with qmix 2 in 1 due to its low surface tension . these studies correlate with our study ; qmix 2 in 1 showing better smear layer removal compared with other final irrigating solutions and there is a significant difference among all the groups . dai et al . , evaluated the smear and debris scores of qmix in the coronal third , middle third , and apical third of root canals using two versions of qmix 2 in 1 ( qmix i [ ph = 8 ] , qmix ii [ ph = 7.5 ] ) using sem . it was concluded that the two versions of the experimental antimicrobial ( qmix ) are as effective as 17% edta in removing canal wall smear layers from the entire root canal space in straight root canals after the initial use of naocl as the initial rinse . biopure mtad is a mixture of tetracycline isomer ( 3% doxycycline ) , 4.25% citric acid , detergent ( 0.5% polysorbate 80 ) . is used instead of its free - base doxycycline monohydrate , to increase the water solubility . it is considered to be clinically effective and a biocompatible endodontic irrigant . in this study mtad shown to be next to qmix 2 in 1 in removing smear layer , this may be because of its low surface tension ( 34.5 mj / m ) , these results correlate with the study conducted by paul et al . , who showed that mtad is better irrigating solution removing smear layer even in apical third . shenoy et al . , compared the smear layer removal efficacy of tublicid plus , mtad , edta and found that tublicid plus and mtad are better when compared to edta . within the limitations of this study , it was observed that among the final irrigating solutions used qmix 2 in 1 displayed effective smear layer removal when compared to edta , mtad and saline .
lumbosacral nerve root anomalies are the main causes of failed lumbar surgery and intraoperative neural injury . the most common ones are the adherent nerve root anomalies characterized by abnormal adherence of two adjacent roots . the prevalence of the disorder ranges between 1.2% and 17% , and it is as high as 6% in magnetic resonance imaging ( mri ) studies and 30% in cadaveric studies . some researchers have reported that radicular symptoms are more common in this disease owing to tension of the adherent nerve roots . combination of l5-s1 spondylolysis and adherent nerve root anomaly has been only rarely reported in the literature . in this paper , we aimed to discuss clinical , radiological signs and surgical therapy in a patient with a rare combination of lumbar disc herniation and spondylolysis with lumbosacral nerve root anomaly . this is a case report of a 49-year - old male with lumbosacral root anomaly accompanying l5/s1 lumbosacral spondylolysis and disc herniation . previous literature was reviewed and main findings were compared with literature data . our patient had occasional low back pain for 5 years and severe right leg pain for 1 week . bilateral and dynamic lumbosacral x - rays and three - dimensional computed tomography ( ct ) showed bilateral pars interarticularis defect and spondylolysis of l5 , and mri demonstrated l5/s1 disc herniation with right paramedian foraminal extension [ figures 1 and 2 ] . neurological examination was remarkable for hypoesthesia on a region corresponding to l5 and s1 dermatome , and loss of strength in extensor hallucis longus and tibialis anterior muscles . ( a and b ) preoperative axial and sagittal computed tomography images showing bilateral pars interarticular is defect ( a and b ) preoperative magnetic resonance imaging showing l5/s1 herniation and enlarged transverse diameter of the right root on axial sections l5 spinolaminar process was totally removed using the line of bilateral pars interarticularis , and l5/s1 posterior instrumentation was performed . whether the appearance thicker than normal is a disc or root anomaly is not differentiated ; it was noted that s1 root ascended with l5 root as a tense and single root when pushed medially [ figure 3 ] . lateral portion of the facet was partly removed and foraminotomy was performed followed by completion of bilateral l5/s1 discectomy and performance of posterior lumbar interbody fusion with spinolaminar process in the resulting space [ figure 4 ] . ( a and b ) perioperative view of right l5 and s1 roots emerging together ( a - c ) postoperative lumbar axial , coronal , and sagittal computed tomography showing intervertebral bone graft the visual analog scale of the patient , which was nine at the preoperative period , regressed to one . the patient mobilized at postoperative day 1 later and discharged at postoperative day 2 . during the review of the preoperative mri of the patient , it was noted that the root traversing the right l5/s1 level had a greater transverse diameter than that of the left root on axial cross - sections [ figure 2 ] . it has been reported that root anomalies may cause radiculopathy in the absence of mechanical compression such as in lumbar disc hernia or spinal stenosis . lumbosacral root anomalies are one of the main causes of neural injuries and surgical failure . it is particularly sensitive to retraction owing to tension on nerve roots and discectomy is more difficult to perform . clinical symptoms are more severe than expected in cases with root anomaly . in these patients , a prodromal claudication with or without radiculopathy should make one think of a diagnosis of root anomaly . a straight leg raising test is near normal . later , ct myelography has been used as the ideal imaging modality to define these anomalies . findings such as parallel , crescent fat , corner , or sagittal shoulder are present , especially on axial t1 and t2-weighted cross - sections . it should be always remembered during surgical exploration that a nerve root anomaly may be present . in the case of an unnoticed adherent nerve root the risk of nerve root injury is increased by excessive pulling of neural components by intraoperative manipulation . neural injury may also ensue , while the surgeon tries to find a piece of lumbar disc hernia during the operation . dural laceration , nerve root injury , or root avulsion can occur by excessive pulling an abnormal nerve root . this , in turn , may lead to persistent , or even worsened , leg pain , that is battered root syndrome . moreover , nerve root anomaly may be misperceived as a disc hernia and incised with resultant iatrogenic neural injury . pamir et al . in an article published in 1992 , reported that laminectomy , facetectomy , and pediculectomy are required , and a standard surgical procedure does not suffice to complete adequate decompression in such patients . in another report it was suggested that routine microdiscectomy would not suffice and foraminal decompression would be needed in the presence of root anomaly combined with disc herniation , as white et al . have reported , it is obvious that morbidity rate will increase due to excessive traction of neural components when an additional facetectomy is not performed , taghipour et al . operated all of 22 patients with root anomaly with additional laminectomy , medial facetectomy and pediculectomy . patients with lumbar spondylolysis and disc hernia with more severe clinical and neurological signs than expected should prompt physicians to consider coexisting lumbosacral root anomalies . in such circumstances , the diagnosis should be established with mri and meticulous preoperative assessments should be made when planning surgery . at perioperative period , the possibility of presence of a root anomaly should always be kept in mind to avoid neurological injuries .
the metabolic syndrome ( ms ) is a cluster of cardiometabolic alterations that include the presence of arterial hypertension , insulin resistance , dyslipidemia , cardiovascular disease ( cvd ) , and abdominal obesity [ 1 , 2 ] . ms presents a prothrombotic state as a result of endothelial dysfunction , the presence of a hypercoagulability state produced by an imbalance between coagulation factors and the proteins that regulate fibrinolysis and increased platelet reactivity [ 35 ] . in this latter regard , we have recently described that obese - diabetic rats with ms have an altered megakaryopoiesis that contributes to increased thrombosis . these alterations are due to an increased platelet turnover caused by a combination of accelerated death and an increased platelet production confirmed by the observation of an increased number of reticulated platelets ( the youngest , more immature , and more reactive platelets ) . importantly , all these alterations were associated with an increased thrombotic risk , analyzed in vivo by real time intravital microscopy , in wild - type obese - diabetic animals as well as in lean normoglycemic controls transplanted with bone marrow from obese - diabetic donors . moreover , we have also described that obese nondiabetic rats also show increased platelet counts and an increased mean platelet volume ( mpv ) which are associated with an increased thrombotic risk ( similar to that observed in obese - diabetic rats ) . in fact , we have shown that platelet number , mpv , and thrombotic risk are directly correlated with weight and that a reduction of peripheral insulin resistance can contribute to reduce thrombotic risk in obese subjects . these alterations might be a consequence of the low - grade chronic inflammatory state observed in obesity , as increased platelet size ( i.e. , mpv ) has been associated with the presence of low - grade inflammation , and several inflammatory proteins have been proven to influence megakaryocyte maturation and platelet formation . indeed , inflammation is receiving increased attention for its potential role in the pathogenesis of disorders ranging from insulin resistance and type 2 diabetes to fatty liver and cvd [ 9 , 10 ] . obesity is associated with a chronic inflammatory response characterized by abnormal adipokine production and the activation of several proinflammatory signalling pathways , resulting in the induction of several biological markers of inflammation . in obese patients , increased accumulation of macrophages is a hallmark of a proinflammatory state that links obesity with systemic inflammation . in addition , obesity is accompanied by other clinical complications ; these include fatty liver , cholesterol gallstones , sleep apnea , osteoarthritis , and polycystic ovary disease . adipose tissue has long been considered a nonfunctional storage pool of energy without any direct impact on organ function . however , it has recently been shown that adipose tissue is a secretory organ and a potent source of hormones , peptides , and cytokines involved in food intake regulation , glucose and lipid metabolism , inflammation , coagulation , and blood pressure control . moreover , it has also become an appealing stem cell source for cell therapy and tissue engineering . therefore , adipose tissue is now considered to be an active endocrine organ that secretes various humoral factors ( adipokines ) , capable of enhancing the release and production of proinflammatory cytokines in obesity , primarily through nonfat cells , likely contributing to the low - grade systemic inflammatory state found in ms - associated chronic pathologies ( e.g. , atherosclerosis ) . for instance , adiponectin is highly expressed in adipose tissue , and circulating adiponectin levels are decreased in patients with obesity , insulin resistance related to type 2 diabetes , and coronary heart disease . on the other hand , the changes presented by adipose tissue in the setting of ms favor the secretion of several molecular mediators capable of activating or suppressing a number of transcription factors ( ppars , peroxisome proliferator activated receptors ; c / ebps , ccaat - enhancer - binding proteins , among other ) that regulate different ms - related metabolic pathways [ 21 , 22 ] . the present paper reviews the principal molecular mechanisms involved in adipose tissue inflammation in the setting of ms and provides an in - depth description of the main genetic pathways involved in adipose tissue metabolism . the ms is characterized by a high amount of visceral fat , insulin resistance in skeletal muscle , and hypoadiponectinemia . ms subjects showed higher levels of blood pressure , waist circumference , and plasma triglycerides with a high risk of developing type 2 diabetes and cvd in the future [ 24 , 25 ] . the physiopathologic changes associated with ms are diverse including , among others , endothelial dysfunction which triggers atherogenic lesions development and enhanced coagulability [ 3 , 26 ] . we have reported that ms patients show higher levels of circulating svcam-1 and scd40l , but not se - selectin , as compared to non - ms patients likely indicating endothelial activation [ 27 , 28 ] . on the other hand , elevated plasma levels of plasminogen activator inhibitor-1 ( pai-1 ) , tissue factor , and fibrinogen detected in ms patients may contribute to the abnormally increased coagulability further enhancing the risk of cvd [ 29 , 30 ] . the metabolic changes related to obesity are largely attributable to the amount of intra - abdominal fat mass , rather than total body fat mass . the increased oxidative stress in accumulated fat is an important pathogenic mechanism of obesity - associated ms . the progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity . thus weight loss larger than 10% is associated with an increase in serum adiponectin and a decrease in hs - crp and plasma fibrinogen . the increase of intermuscular adipose tissue was primarily related to age , total body adiposity , and subclinical inflammation . functional failure of the adipose tissue results in changes in systemic energy delivery , impaired glucose consumption , and activation of self - regulatory mechanisms that extends adipose tissue influence to the whole homeostatic system , by the enhancement of adipokines secretion with the subsequent vascular - related effects [ 36 , 37 ] . adipose cell enlargement leads to a cellular proinflammatory state with reduced secretion of adiponectin and increased secretion of il-6 , il-8 , and monocyte chemotactic protein-1 ( mcp-1 ) , among others . data suggest that plasma adiponectin does not change with age , but levels are negatively associated with percent body fat , visceral fat , subcutaneous abdominal fat , insulin , and leptin levels [ 39 , 40 ] . there has been a paradigm shift from the traditional notion of adipose tissue merely as an energy storage site to one where adipose tissue plays an active role in energy homeostasis and other various processes [ 41 , 42 ] . adipose tissue plays a critical role in energy homeostasis , not only in storing triglycerides , but also responding to nutrient , neural , and hormonal signals and secreting adipokines that regulate feeding , thermogenesis , immunity , and neuroendocrine function . inflammation is increasingly known as a key process underlying metabolic diseases in obese subjects . in particular , adipose tissue - related production of proinflammatory molecules ( tnf- , il-1 , il-6 , il-8 , transforming growth factor- , and nerve growth factor ) as well as its acute - phase response ( plasminogen activator inhibitor-1 , haptoglobin , and serum amyloid a ) detected during obesity contributes to a low grade of systemic inflammation seen in chronic diseases associated with ms [ 4649 ] . in obese subjects , adiponectin levels are decreased , and the ability of adiponectin to inhibit the inflammatory processes is limited . low adiponectin levels are inversely related to high levels of c - reactive protein ( crp ) in patients with obesity , type 2 diabetes , and cvd [ 5052 ] . in fact , crp may promote the formation of intimal neovessels in vulnerable atherosclerotic plaques increasing the likelihood of rupture . in mice fed a high - fat diet , thus a high percentage ( > 50% ) of the total adipose tissue mrna transcripts found to be upregulated during diet - induced weight gain is inflammatory - related genes . in fact , overexpression of low - density lipoprotein receptor - related protein-1 and very low - density lipoprotein receptor in epicardial fat may play a key role in the alterations of lipid metabolism associated with type 2 diabetes mellitus . thus , 12/15- lipoxygenases ( alox ) and their lipid metabolites , involved in the oxidative metabolism of polyunsaturated fatty acids , act as upstream regulators of many of the cytokines involved in the adipose tissue - related inflammatory response contributing to the development of insulin resistance and diabetes . the gene expression and localization of alox isoforms have shown to be exclusively expressed in human visceral fat . resistin and tnf- are adipokines that have been implicated in insulin resistance in skeletal muscle by the addition of fatty acids to the diacylglycerol [ 57 , 58 ] . adipocytes are sensitive to the effects of tnf- , which , through its p55 and p75 tnf receptors , stimulates nuclear factor- ( nf- ) b , extracellular signal - regulated kinase , and p38 mitogen - activated protein kinases pi-3 kinase and junn - terminal kinase cascades . the correlation between insulin resistance , chronic inflammation , hypertension , endothelial dysfunction , and dyslipidemia could be due to the activation of nf-b . the transcription factor nf-b and the tnf- gene promoter were activated by hypoxia in adipocytes and fibroblasts . nf-b signaling represses e2f transcription factors eventually inhibiting adipogenesis and maintaining a chronic inflammatory condition . in contrast , hypoxia reduced adiponectin expression , detected d in adipocytes gene promoter . tnf- is chronically elevated in adipose tissues of obese rodents and humans . increased levels of tnf- are implicated in the induction of atherogenic adipokines , such as pai-1 and il-6 , and the inhibition of the antiatherogenic adipokine , adiponectin . obese individuals have increased tnf- gene expression , as shown by a study in which a 2.5-fold increases in mrna . also a strong positive correlation has been detected between tnf- mrna expression levels and the level of hyperinsulinemia ( an indirect measure of insulin resistance ) in fat tissue . adipokines exert potent modulatory actions on target tissues and cells involved in rheumatic disease and obesity - related diseases [ 66 , 67 ] . for establishing a relationship with the obesity , using ra as a model a complex adipokine - mediated interaction among white adipose tissue , cvd , and chronic inflammatory autoimmune diseases like ra has been described . in this regard , in ra adipocytes and their surrounding macrophages produce a range of adipokines that regulate systemic inflammation . in ra patients undergoing anti - tnf infliximab therapy because of severe disease , high - grade inflammation shows an independent and negative correlation with circulating adiponectin concentrations , whereas low adiponectin levels clustered with ms features such as dyslipidemia and high plasma glucose levels that have been reportedly to contribute to atherogenesis in ra . leptin in ra . in patients undergoing anti - tnf- therapy because of severe disease refractory to conventional therapy , there was a positive correlation between body mass index in patients with ra and leptin serum levels . in addition , these patients showed a significant correlation between leptin levels and vcam-1 [ 70 , 71 ] . this is of potential interest as biomarkers of endothelial dysfunction - endothelial cell activation are elevated in patients with ra and anti - tnf blockade improved endothelial dysfunction as well as decreased the levels of endothelial cell activation biomarkers . resistin in ra . in patients with ra in treatment with the anti - tnf- monoclonal antibody infliximab for severe disease refractory to conventional therapy , a positive correlation between markers of inflammation , in particular with c - reactive protein , and resistin levels was observed . tnf- blockade led to a rapid reduction in the levels of resistin in these patients . these results highlight a potential role of resistin in the inflammatory cascade in diseases like ra that are associated with chronic inflammatory burden . a complex adipokine - mediated interaction among white adipose tissue , cvd , and chronic inflammatory autoimmune diseases like ra has been described . in this regard , in ra adipocytes and their surrounding macrophages produce a range of adipokines that regulate systemic inflammation . in ra patients undergoing anti - tnf infliximab therapy because of severe disease , high - grade inflammation shows an independent and negative correlation with circulating adiponectin concentrations , whereas low adiponectin levels clustered with ms features such as dyslipidemia and high plasma glucose levels that have been reportedly to contribute to atherogenesis in ra . leptin in ra . in patients undergoing anti - tnf- therapy because of severe disease refractory to conventional therapy , there was a positive correlation between body mass index in patients with ra and leptin serum levels . in addition , these patients showed a significant correlation between leptin levels and vcam-1 [ 70 , 71 ] . this is of potential interest as biomarkers of endothelial dysfunction - endothelial cell activation are elevated in patients with ra and anti - tnf blockade improved endothelial dysfunction as well as decreased the levels of endothelial cell activation biomarkers . resistin in ra . in patients with ra in treatment with the anti - tnf- monoclonal antibody infliximab for severe disease refractory to conventional therapy , a positive correlation between markers of inflammation , in particular with c - reactive protein , and resistin levels was observed . tnf- blockade led to a rapid reduction in the levels of resistin in these patients . these results highlight a potential role of resistin in the inflammatory cascade in diseases like ra that are associated with chronic inflammatory burden . macrophage and lymphocyte infiltration in adipose tissue may greatly contribute to obesity - related metabolic dysfunction and chronic inflammation [ 75 , 76 ] . recent studies have demonstrated that over 90% of the adipokine release by adipose tissue , except for adiponectin and leptin , could be attributed to nonfat cells . the sequence of events in the inflammatory cascade within the adipose tissue comprises immune cells , first lymphocytes , and then macrophages [ 78 , 79 ] . t lymphocytes present in visceral adipose tissue contribute to the initiation and perpetuation of adipose tissue inflammation and the development of insulin resistance . thus it is observed that a large number of cd8 + effector t cell infiltrate adipose tissue promoting the recruitment and activation of macrophages in obese mice , while the number of cd4 + t cell and regulatory t is diminished ( figure 1 ) . white adipose tissue hypoxia and cd8 + t cell invasion are features of obesity in c57bl/6j mice and are potential contributors to their local and generalized inflammatory state . natural killer t ( ntk ) cells play a crucial role in the development of adipose tissue inflammation and glucose intolerance in diet - induced obesity . however , the deletion of nkt cells , in the absence of alterations in the cd8 + t cell population , is insufficient to protect against the development of the metabolic abnormalities of diet - induced obesity . macrophage can be characterized as m1-type ( involved in proinflammatory processes such as tnf- , il-6 , and il-12 ) or immunomodulatory and tissue remodeling ( m2 ) . the latter can secrete il-10 , which is an anti - inflammatory cytokine and partake mostly in the downregulation of proinflammatory cytokines [ 84 , 85 ] . infiltrated macrophages play the most prominent role , and this low grade inflammation is mediated by the activation and recruitment of macrophages into expanding adipose tissue [ 12 , 86 ] . in this context , it has been established that the diet - induced obesity leads to a shift in the activation state of adipose tissue macrophages from an m2-type to an m1 proinflammatory state that contributes to insulin resistance [ 87 , 88 ] . adiponectin is an abundantly expressed adipokine in adipose tissue and has multiple effects on glucose , metabolism of lipids and free fatty acids , cytokine secretion , and direct insulin sensitizing activity [ 89 , 90 ] . an increase of adiponectin concentrations or the maintenance of higher concentration may be negatively associated with cvd and diabetes , especially in patients with high glycaemic level and independent of adiposity and smoking status [ 91 , 92 ] . adiponectin has antiatherosclerotic as well as anti - inflammatory properties that may play an important role in preventing the progression of coronary artery disease [ 93 , 94 ] . in this context , adiponectin acts on cultured murine and human macrophages to promote a switch to an anti - inflammatory m2 phenotype . adiponectin can either act directly on macrophages to shift polarization or prime human monocyte differentiation into anti - inflammatory m2 macrophages [ 95 , 96 ] . possible pathways of action of adiponectin that leads to a shift in macrophages to an antiinflammatory phenotype include ( i ) adipor1 il-10 ho-1-dependent pathway to decrease tlr4 expression and dampen inflammatory cytokine expression in macrophages and ( ii ) adipor2 il-4 stat6-dependent signaling pathway that leads to a shift in macrophages to an m2 polarization [ 97 , 98 ] . genetic variations in adiponectin receptors ( adipor1 or adipor2 ) are unlikely to lead to a common genetic predisposition to insulin resistance or type 2 diabetes [ 99 , 100 ] . thus , an independent inverse correlation between plasma adiponectin levels and hs - crp may suggest that decrease of adiponectin contributes to the systemic and vascular inflammation commonly found in obesity . however , patients with advanced heart failure present increased adiponectin with reduced expression of adipor1 and adipor2 as well as reduced activation of amp kinase , a known downstream signaling molecule , suggesting a functional adiponectin resistance in advanced heart failure . also , high levels of adiponectin have been found in chronic inflammatory autoimmune diseases such as sle , type i diabetes , and rheumatoid arthritis [ 102104 ] . interactions of genetic factors such as single nucleotide polymorphisms ( snps ) in the adiponectin gene and environmental factors causing obesity result in hypoadiponectinaemia , which appears to play an important causal role in obesity - linked insulin resistance , type 2 diabetes , and the ms . in women with ms , visceral fat volume was negatively related to leptin and tended to be negatively related to adiponectin gene expression . thus obese subjects exhibit increased systemic oxidative stress , likely derived from the accumulated fat , being an early instigator of ms . a study of 2828 subjects showed that smoking , diabetes , and body mass index were highly associated with systemic oxidative stress and suggest an important role on obesity . lipid - rich diets are also capable of generating reactive oxygen species because they can alter oxygen metabolism . increase of free radicals together with low antioxidant capacity detected in obese adults indicate an elevated oxidative stress , which is , in concurrence with systemic inflammation , further potentiated in the case of patients with metabolic syndrome . an increased oxidative stress is also associated with adiponectin deficiency . increased oxidative stress has shown to inhibit preadipocyte differentiation as a result of reduced cell proliferation and an inhibition of g(1)s - phase transition through a transcriptional mechanism involving the inhibition of e2f recruitment and transactivation of its target promoters . abdominal adiposity and leptin are independent predictors of adiponectin gene expression , and in human adipocytes , adiponectin gene expression is strongly related to ib- mrna . the significant independent relationship between adiponectin gene expression and ib- mrna suggests that when adiponectin gene expression is high , there is a higher expression of ib- and the subsequent inhibition of nf - kb transcriptional activity with lower inflammation at the adipocyte level . adipokine zinc - alpha2-glycoprotein ( zag ) gene expression in adipose tissue is downregulated with increased adiposity and circulating insulin . zag mrna is positively correlated with adiponectin mrna ( zag enhances adiponectin production by human adipocytes ) . the action of zag is associated with downregulated lipogenic enzymes ( fas , acc1 , and dgat mrna ) and upregulated lipolytic enzyme ( hsl mrna ) expressions in adipose tissue . adiponectin gene transcription is stimulated by several transcription factors involved in adipogenesis such as ppars , foxo1 , c / ebps , and srebps and is suppressed by hypoxia , inflammation , and transcription repressors such as nfats and creb . proinflammatory cytokines such as tnf- , il-6 , and il-18 also negatively regulate adiponectin gene transcription by activating several pathways such as the jnk and erk1/2 pathways [ 115117 ] . changes in the life style , reduction of obesity , and food habits are fundamental in reducing the risk factors . however , there are key factors in ms regulation that depend on those transcription factors that , by responding and adapting to signals from the environment , are able to change the levels of relevant gene expression [ 119 , 120 ] . even , gene expression in the metabolic pathways ( apoptosis , lipid metabolism , and inflammation ) is directly related to the levels of igm antioxldl antibodies . changes in gene expression of adipose tissue suggest that carbohydrate modification can affect the risk of cvd and type 2 diabetes . the maturation of adipocytes is regulated by a series of transcription factors , mainly ppars and c / ebps , that in conjunction regulate the expression of hundreds of proteins that participate in the metabolism and storage of lipids and , as such , the secretion of adipocytes . meanwhile , the chronic inflammation in adipose tissue is evident from the differential expression of genes involved in inflammatory responses and activation of natural immunity ( figure 2 ) . ppars are transcriptions factors of a superfamily of nuclear receptors . the gene expression in the adipose tissue of people with ms seems to be affected by changes in tissue morphology or insulin sensitivity , where a diet high in saturated fatty acids produces a proinflammatory state via the repression of ppars . the double action of ppar- and ppar- increases the action of adiponectin and the expression of its receptors , which results in an improvement in obesity and a reduction of the inflammatory process . this class of compounds includes two drugs , pioglitazone and rosiglitazone , that are widely used to treat patients with diabetes . ppar- plays a fundamental role in adipogenesis , as a key regulator in the differentiation and function of adipocytes and the absorption of stored fatty acids [ 129131 ] . meanwhile , it has been suggested recently that ppar- is also involved as a key regulator of inflammatory and immune response . ppar- is required for maturation of alternatively activated macrophages whatever has a beneficial role in regulating nutrient homeostasis and suggests that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes . however , ppar- activation does not influence m2 marker expression in resting or m1 macrophages , indicating that only native monocytes can be primed by ppar- activation to an enhanced m2 phenotype . in addition , ppar- transcriptional signaling is required for maturation of an anti - inflammatory m2 phenotype , whereas ppar- controls the expression of alternative phenotype in kupffer cells of obese mice . apoe expression in adipocytes is regulated by factors involved in modulating systemic insulin sensitivity . increased plasma apoe levels adipocytes synthesize and secrete apoe , and its regulation by ppar- agonists and tnf- raises an issue regarding the potential significance of adipocyte - derived apoe . tnf- suppresses apoe gene expression in adipocytes , and ppar- agonist increases expression of apoe in adipose tissue . thus tnf- and ppar- agonists regulate apoe gene response via distinct apoe gene control elements . for ppar- , liver receptor x ( lxr ) is a key pathway for mediating stimulation of the adipocyte apoe gene . while that tnf- repression of adipocyte apoe gene expression required an intact nf-b binding site at 43 in the apoe promoter . on the other hand , the loss of function of ppar- due to dominant mutations brings about a resistance to insulin and the early onset of severe hypertension [ 142 , 143 ] . moreover , il-6 expression in subcutaneous adipose tissue was significantly associated with intermuscular adipose tissue ; il-6 messenger rna ( mrna ) was negatively associated with adiponectin and ppar- expression . also , loss of ppar- in immune cells impairs the ability of abscisic acid to improve insulin sensitivity by suppressing mcp-1 expression and macrophage infiltration into white adipose tissue . at least six members of this family have been isolated and characterized to date ( c / ebp--c / ebp- ) . c / ebps- , , and are tissue specific and highly expressed in adipose tissue . c / ebps and nuclear factor - y ( nf - y ) are critical for the regulation of the adiponectin expression in response to nutrients and in the course of adipocyte differentiation . even , the transcriptional activity of adiponectin gene during adipocyte differentiation is enhanced by the motif in a novel adiponectin enhancer region , via the recruitment of the c / ebps and sterol regulatory element - binding proteins ( srebps ) [ 148 , 149 ] . the adiponectin promoter was activated by both c / ebp- and c / ebp- , and the fold increase by c / ebp- was larger than that by c / ebp- . c / ebp- accesses the adiponectin promoter through two forkhead box protein o1 ( foxo1 ) binding sites and acts as a coactivator . further , sirt1 increases adiponectin transcription in adipocytes by activating foxo1 and enhancing foxo1 and c / ebp- interaction . thus low expression of sirt1 and foxo1 leads to impaired foxo1-c / ebp- complex formation , which contributes to the diminished adiponectin expression in obesity and type 2 diabetes . therefore , c / ebp- is a key transcription factor for full activation of human adiponectin gene transcription in mature adipocytes through interaction with response elements in the intronic enhancer . however , common allelic variants in cebp- and cebp- could influence abdominal obesity and related metabolic abnormalities associated with type 2 diabetes and cvd . meanwhile , activation of pi3 k induced proinflammatory gene expression through activating c / ebp- and c / ebp- but not nf-b , which may explain the proinflammatory effect of insulin in the insulin - resistant state . in the hyperinsulinaemic state , c / ebp- leads to the upregulation of ccl2 , an inflammation - related protein , which may initiate the process of atherosclerosis . moreover , adipose tissue glut4 regulates the expression of carbohydrate - responsive - element - binding protein ( chrebp ; also known as mlxipl ) , a transcriptional regulator of lipogenic and glycolytic genes . chrebp- expression in human adipose tissue predicts insulin sensitivity , indicating that it may be an effective target for treating diabetes . moreover , upregulation of glut4 gene transcription might be directly mediated by srebp-1c in adipose tissue . obesity triggers a chronic inflammatory state that promotes the production of proinflammatory factors contributing to the impairment of the pathogenesis of ms . the participation of leukocytes plays a critical role in the initiation and propagation of adipose tissue inflammation . most genetic modifications of adiponectin are due to oxidative stress generated during obesity . adiponectin and ppar- can directly act on macrophages to shift polarization or human monocyte differentiation into anti - inflammatory m2 macrophages . therefore the clarification of inflammatory processes in the adipose tissue during obesity appears to be essential for the understanding of ms . thus ms requires the development of new therapeutic strategies addressed to alter the main transcription genetic pathways that regulate adipose tissue metabolism .
quantitative compositional analysis of specific primary amines is applied in food science and agriculture to characterize samples for quality control . primary amines in soil samples provide information about the available sources of organic and bioorganic n in an ecosystem , a boon for management of agriculture , and a central aspect to researching systems that involve n or the nitrogen fixation cycle . primary amines and amino acids are also used to indicate reactions in food processing and to directly indicate nutritional value and quality of products [ 25 ] . a large majority of known bioactive molecules and neurotransmitters are primary amines , amino acids , or low molecular weight metabolites of these species , so primary amine analysis is of continually increasing interest for metabolomics , pharmaceuticals , and detection of hazardous agents in biowarfare . in situ analysis of primary amines is additionally of great interest for investigating planetary chemistry [ 7 , 8 ] as well as the synthesis and origin of prebiotic amino acids . for compositional amine analysis , a separation method many applications require field - deployable amine compositional analyses , such as clinical devices , detection of harmful biological agents , and in situ astrobiology and planetary science experiments . capillary electrophoresis ( ce ) , when used in conjunction with laser - induced fluorescence ( lif ) , provides a fast and easily miniaturizable technique . ce - lif also provides the opportunity to incorporate the separation and detection steps in line with extraction instruments and microfluidics devices . this is an attractive feature for lab - on - a - chip approaches and a necessary step for developing field - deployable detection instruments for the clinic , biowarfare , and in situ astrobiology experiments . in fact , ce is already a targeted implementation of lab - on - a - chip analysis for bioterrorism defense , ce - lif has been fully automated and miniaturized towards future in situ martian and other planetary missions [ 7 , 11 ] , and ce - lif 's direct compatibility with dialysates and biological fluids has been applied to clinical samples and in vivo biomedical research [ 2 , 12 , 13 ] . however , the latter two applications often require detection of amines with varied solubility in aqueous media . micellar electrokinetic chromatography ( mekc ) enables separation and therefore analysis of hydrophobic longer - chain amines , while preserving analytic capability of shorter and more hydrophilic amines . mekc - lif , only differing from ce - lif by addition of a surfactant , presents the opportunity to extend implementation of these established approaches to field - deployable instrument development to detect a broader spectrum of targets in complex samples . fluorescence detection of primary amines provides a quick and potentially highly sensitive , quantitative analysis . particularly , fluorescence detection of amines by ce - lif with excitation at 488 nm has demonstrated limits of detection ( lod ) from m to nm depending on the target amine , fluorescence probe , and optimization conditions . the speed of fluorescence detection ensures that the rate - limiting step for analysis is upstream in sample collection , preparation , or separation . however , to use this method , amines without autofluorescent properties must be chemically derivatized with a fluorescent probe . the commercially available dye , alexafluor 488 ( af488 ) , is optimally excited with a 488 nm laser line ( extinction coefficient of 73,000 cm m at 494 nm ) and has an emission maximum at 525 nm , making it easy to apply with standard light sources , instrumental settings , and filters . low af488 self - quenching enables highly sensitive analysis , and the n - hydroxysuccinimidyl ester ( nhs - ester ) functionality makes it highly amine - specific . in aqueous and biological samples , the spontaneous derivatization reaction of af488 nhs - ester circumvents the need for additional reagents while the high amine specificity provides advantages over other amine - reactive dyes such as isothiocyanates , which also react with sulfhydryl groups , and dyes which react directly with amines and change their chemical structure and fluorescent properties upon derivatization ( e.g. , 4-chloro-7-nitro-1,2,3-benzoxadiazole ) . the derivatization through amine esterification , promoted by the nhs leaving group , allows for modular selection of other fluorophores to optimize fluorescent properties for varied equipment . af488 is a water soluble probe that is ph insensitive over a range that extends from below 4 to above 10 , making it suitable for a range of applications . the dye is negatively charged overall , facilitating separation of tagged analytes by electromigration . here , we describe a novel method for amine analysis using mekc - lif in conjunction with a labeling protocol employing af488 nhs - ester . we prepared and analyzed samples of nonylamine , hexadecylamine , and octadecyl amine to test the applicability of this method to aliphatic amines with reduced solubility in aqueous media and no detectable autofluorescence . samples were separated and detected using a commercial beckman coulter p / ace mdq system with 488 nm lif detection . labeling conditions were optimized , including organic solvent , the concentration of the base diisopropylethylamine ( diea ) , and the incubation time . suitable separation characteristics were found using mekc with sodium dodecyl sulfate as the surfactant , and the resulting analytical technique was characterized . alexafluor 488 succinimidyl ester ( af488 nhs - ester ) was purchased from invitrogen corporation ( carlsbad , ca ) , diluted to 20 mm in n , n - dimethylformamide ( dmf , sigma - aldrich , st . sodium carbonate ( naco3 , sigma - aldrich ) was used to prepare 50 mm aqueous solutions with 18 mcm water . the ph was adjusted using 1 m naoh ( sigma - aldrich ) and measured using a glass electrode and a digital ph meter ( orion 290a , thermo ; waltham , ma ) . sodium dodecyl sulfate ( sds ) was acquired from sigma aldrich and used to prepare a 100 mm stock in 18 mcm water . amines for standard solutions , including nonylamine ( c9-nh2 ) , dodecylamine ( c12-nh2 ) , hexadecylamine ( c16-nh2 ) , and octadecylamine ( c18-nh2 ) , were purchased in pure form from sigma aldrich and used to prepare 10 mm solutions in ethanol ( sigma - aldrich ) . n , n - diisopropylethylamine ( diea , sigma - aldrich ) was diluted to 10 mm in ethanol , dmf , and dimethylsulfoxide ( dmso , sigma - aldrich ) . labeling reactions were conducted by combining the appropriate volumes of 20 mm af488 , 10 mm diea , amine , and solvent . reactions were incubated in the dark overnight ( 1624 hrs ) unless otherwise indicated . after incubation , reactions were diluted into the separation buffer at a 5 : 100 ratio unless otherwise indicated . capillary electrophoresis ( ce ) separations were conducted on a beckman coulter p / ace mdq capillary electrophoresis system equipped with 488 nm laser - induced fluorescence ( lif ) detection . the capillary was rinsed using pressure with the separation buffer for two ( 2 ) minutes , and then the sample was injected ( pressure ) for 5 seconds . after separation , the capillary was rinsed using pressure with pure water for five minutes . capillary conditioning using 1 m naoh was conducted with a 5-minute rinse as needed . separation buffers tested included 10 mm carbonate ( ph 10 ) and 10 mm carbonate with 12 mm sds ( ph 10 ) . resulting electropherograms were generated and exported in comma - separated values format using 32 karat software ( beckman coulter inc . ) . these files were imported into peakfit ( systat ) for smoothing ( 0.1% loess ) and baseline correction prior to peak fitting . the resulting smoothed and baseline corrected electropherograms or data from peak fitting was imported into origin ( originlabs ) to generate figures . figure 1 shows the labeling reaction between af488 nhs - ester and a primary amine . this reaction is base - catalyzed and was found to proceed for c9-nh2 and shorter amines in 10 mm aqueous carbonate , ph 10 . longer - chain amines ( c12-nh2 and longer ) were found to be insoluble in aqueous solutions without surfactant and therefore did not label to any detectable extent . for this reason , we examined organic solvents for labeling reactions with diea included to provide a basic environment . the fluorescence intensities of amines labeled in 10 mm diea in ethanol , dmf , and dmso and then separated in 10 mm carbonate , 12 mm sds , ph 10 , are shown to be normalized to the dmso fluorescence intensity in figure 2 . labeling proceeded to nearly the same extent , within error , in all three organic solvents . dmso is often favored in extraction and sample preparation for its solvating ability and stability , so it is encouraging to see that labeling proceeded optimally in dmso . however , these results also indicate that choice of solvent can be dictated by concerns such as downstream analysis method , safety , and ease of evaporation with marginal reduction in labeling . to explore the impact of diea concentration on labeling efficiency , its concentration in ethanol was varied from 0 to 48.75 m in a solution that contained 1 m amine and 25 m af488 nhs - ester . figure 3 shows the results of ce separation after an overnight incubation of the solutions . while very low levels of amine were labeled without any diea , there is no change , within error , of the amount labeled in solutions containing between 12.5 and 48.75 m diea . however , some contamination in the shorter chain amine region ( c2-nh2 , c3-nh2 ) was observed to increase with increasing diea concentration . these results indicate that diea concentration can and , when possible , should be kept to a minimum . while overnight incubations are logistically simple for an operator and for potential automated implementations , sometimes it is preferable to obtain results from a sample within a shorter window of overall time . therefore , we examined the impact of incubation time on the labeling reaction extent . figure 4 shows the relative fluorescence intensity of the amine peaks diluted into separation buffer and immediately separated via ce at incubation times from 1 hr to 30 hr . over 90% final intensity while the samples were prepared in such a way to potentially yield pseudo - first - order kinetics , the data do not fit a rate law first - order in amine concentration and in fact most closely fit a rate law second - order in amine concentration . this does not seem like a physical likelihood given stoichiometry of the reaction and the standard mechanism proceeding via a tetrahedral intermediate . therefore , we can not make any statements about true kinetic parameters of the reaction based on our data and recommend a true kinetics study as part of future work with the af488 nhs - ester fluorescent probe . based on the above experiments , the optimum conditions for our work using af488 nhs - ester as a fluorescent probe for amine analysis are 12.5 m diea in dmf with at least 6 hr and preferably overnight incubation . electropherograms of amines separated in 10 mm carbonate , 12 mm sds , ph 10 , after labeling with the optimized conditions , are shown in figure 5 . while the separation conditions were not optimized nor studied in great detail in this work , separation characteristics are given in table 1 . n = 3 ) were determined using the optimized labeling conditions and these separation conditions : 17 nm c16-nh2 and 5.7 nm c18-nh2 . a contamination issue with our c9-nh2 stock prevented determination of its lod during the timeframe of this work . this work provides a foundation for further work more fully optimizing an af488-based amine assay for a desired application . amine detection using lif with af488 as the reactive probe enables potentially novel analytical techniques as it provides an alternative to other probes ' fluorescent at 488 nm . the optimized reaction conditions are mild and fast ( 90% efficiency within 6 hrs ) while enabling excellent limits of detection ( nm or ppb ) . while af488 is not fluorogenic and therefore can not be used as a fluorescent probe without some form of postreaction separation , we have demonstrated that this separation can be achieved using a simple , standard mekc solution and a commercial ce instrument . the limits of detection achievable using the commercial system and initial ce separation conditions were in the low nm range , or single parts - per - billion , making this technique more than sufficiently sensitive for multiple applications . additionally , the current separation method is insufficient for the simultaneous analysis of c16-nh2 and c18-nh2 ; thus , further work is required to develop application - specific optimized separation methods .
pterygium is a wing - shaped , fibrovascular growth of the bulbar conjunctiva that crosses the limbus and extends over the peripheral cornea.1 this invasion of the corneal surface can lead to significant visual morbidity caused by irritation of the ocular surface , irregular astigmatism , obstruction of the visual axis , and loss of corneal transparency.2 the incidence and prevalence of this condition vary among different populations and are influenced by a variety of factors including age , sex , and geographic location.3 to date , the majority of research evaluating the prevalence of pterygium has focused on population - based studies , with few studies providing a more global understanding of the burden of disease . in a recent meta - analysis by liu the pooled worldwide prevalence of pterygium was found to be 10.2%,3 with prevalence rates ranging from 2.8% in a study by wu et al4 and 33% in a study by mccarty et al.5 the prevalence of pterygium in men was also higher than that in women , with rates of 14.5% and 13.6% , respectively.3 pterygium was more prevalent with increasing age up to 69 years.3 populations living in geographic latitudes ranging from 20 to 30 also had a higher prevalence of pterygium compared with any other area.3 the nasal limbus is the most common site for pterygium formation . this predilection has been attributed to the focusing of light passing through the anterior chamber at the nasal limbus , causing damage to the limbal stem cells and inducing oxidative stress.6,7 many population - based studies have also revealed an association between pterygium formation and outdoor occupation and activities , most likely a result of exposure to ultraviolet ( uv ) radiation , the pathogenesis of which has been described.8,9 conjunctival uv autofluorescence , a biomarker of ocular exposure to uv light , has been shown to be higher in individuals with pterygium than those without.10 an increase in prevalence has also been noted in rural populations when compared to urban populations , likely reflecting differences in lifestyle and lifetime exposure to uv radiation.3,11,12 many tumor - like features , including the propensity to invade normal tissue , the high rate of recurrence , and the coexistence with premalignant lesions , challenge the idea that pterygia are benign lesions.6 in one study , including 100 cases of pterygia , concurrent ocular surface disease included five cases of ocular surface squamous neoplasia , six cases of primary acquired melanosis , and two compound nevi ( one of which was suspicious for melanoma).6 it is thus recommended that all pterygia be sent for histopathology to rule out concurrent ocular surface disorders , including those with malignant potential.6 the treatment of pterygia is surgical ; however , because of the high rate of recurrence , careful consideration of the risks and benefits for surgery is necessary before primary excision is undertaken . indications for treatment include any one or more of the following : vision loss secondary to astigmatism or progressive encroachment on the visual axis , restriction of ocular movement , or discomfort and irritation.13 current management options for pterygium include excision , conjunctival autografting , and the use of adjuvant therapies including mitomycin c , 5-fluorouracil , anti - vascular endothelial growth factor ( anti - vegf ) agents , and -irradiation . the technique of excising a pterygium without repairing the remaining defect is called bare sclera excision . this technique is no longer recommended because of its high rate of recurrence , which ranges from 38% to 88%.14 this recurrence rate is higher than for any other treatment modality.13 moreover , there are no advantages conferred by this technique except for its simplicity and short surgical time.13 primary closure is a technique that involves excision of the pterygium , followed by suturing of the remaining conjunctiva on either side of the wound over the bare sclera , to close the defect . this procedure has also been reported to have an unacceptably high rate of recurrence compared to newer techniques ( 45%70%).14 given the unacceptable recurrence rates of both bare sclera and primary closure techniques , advances in pterygium excision have focused on the use of grafts and adjuvant therapies , of which conjunctival autografts are the most commonly used . in this method , the pterygium is excised and the remaining defect closed with the patient s own grafted conjunctiva and attached using fibrin glue or sutures ( figure 1 ) . although the ipsilateral superior conjunctiva is typically used , both superior and inferior conjunctival autografts have been found to be reasonable options.15 recurrence rates for this procedure have been cited between 2% and 20%.14 alpay et al13 observed that all recurrences in patients who had undergone conjunctival autografting in their case series ( 3/18=16.65% ) occurred in eyes that had undergone previous pterygium surgery ( no primary pterygia treated with conjunctival autografting recurred ) . interestingly , syam et al16 found that 36.66% of patients developed conjunctival scarring at the site of the donor conjunctiva . technique , which stands for pterygium extended removal followed by extended conjunctival transplantation , was pioneered by lawrence hirst and differs from traditional conjunctival autografting by the extensive removal of tenon s layer after pterygium excision . this technique was shown by hirst , in a prospective trial of 250 consecutive patients with primary pterygia , to have a recurrence rate of 0% and a good cosmetic outcome.17 in another study , he went on to show that the p.e.r.f.e.c.t . technique could also be used for excision of recurrent pterygia with a recurrence rate of 0% and few complications.18 limbal conjunctival autografting involves transplantation of limbal stem cells in addition to autologous conjunctiva in order to cover the defect created from excision of the pterygium . the benefit of this method is that , in addition to decreasing recurrence rates , the limbal stem cells promote healing.14 sutured limbal conjunctival autografts have a recurrence rate ranging from 0% to 14.29%.14 the use of fibrin glue was shown in one study to significantly decrease the rate of recurrence.19 a conjunctival flap requires undermining of the conjunctiva at the donor site without detaching the tissue from its origin . the surgeon then rotates the flap to cover the defect left by excision of the pterygium . few complications have been reported for this procedure , apart from conjunctival cyst formation and flap retraction.13 alpay et al13 found a recurrence rate of 33.33% with this technique and noted poor cosmesis , which improves with time . an amniotic membrane ( am ) graft can also be used to cover bare sclera following pterygium excision ( figure 2 ) . these grafts are thought to promote healing and reduce rates of recurrence because of their anti - inflammatory properties , their promotion of epithelial growth , and their suppression of transforming growth factor ( tgf- ) signaling and fibroblast proliferation.14 recurrence rates of pterygia following amniotic membrane grafting ( amg ) are cited between 14.5% and 27.3%.14 the use of postoperative steroid injections following amg also reduces the rate of recurrence.14 when compared to conjunctival and limbal autografting , recurrence rates are higher for amg.20 amg shows particular promise over the other grafting procedures in certain circumstances , such as when preexisting conjunctival scarring precludes the harvesting of donor conjunctiva for an autograft . amg is also helpful when the superior conjunctiva must be spared for future glaucoma filtering surgery , as well as in cases of large or double - headed pterygia.21 while grafts have greatly improved recurrence rates following pterygium surgery , they are not without their complications . reported complications include wound dehiscence , tenon s granuloma , conjunctival cysts , necrotizing scleritis , and subconjunctival fibrosis from the donor site.9 adjuvant therapies , including the use of mitomycin c , have been used both independently and in conjunction with grafting techniques to reduce the rate of recurrence following pterygium surgery . mitomycin c is an antibiotic from the bacteria streptomyces caespitosus that inhibits dna , rna , and protein synthesis.14 its use has been shown to lower the rate of recurrence when used in conjunction with bare sclera , limbal autograft , conjunctival autograft , and amg techniques . however , many serious complications have been noted with its use , including scleral melt , punctate keratitis , infectious scleritis , corneal perforation , secondary glaucoma , cataract , iritis , and a possible effect on corneal endothelial cells.9,14 a safe , minimal , and effective dose has not been established.13 for this reason , caution must be taken when using mitomycin c to ensure that the risk of treatment does not outweigh the benefit of a reduced recurrence rate . -irradiation has also been found to be a relatively well - tolerated procedure , with recurrence rates similar to chemotherapeutic agents and conjunctival autografting . rare but significant complications of this procedure include scleral thinning , ulceration , infection , and radiation - induced cataract.22 other medical therapies are also currently being investigated , including the chemotherapeutic agent 5-fluorouracil , and anti - vegf agents such as ranibizumab and bevacizumab.23 these treatments are beyond the scope of this review . amniotic membrane transplantation ( amt ) was first described by davis24 for use as a surgical material in skin transplantation.24,25 in the 1940s , its use in the treatment of ocular surface conditions was described.25 since 1995 , it has been increasingly used to treat a variety of ocular surface conditions,26 including persistent corneal epithelial defects , acute chemical burns , and cicatrizing conditions such as stevens johnson syndrome and ocular cicatricial pemphigoid.26 amt has been used in the reconstruction of fornices , as a covering following excision of conjunctival lesions , and in limbal stem cell deficiency with concomitant limbal stem cell grafting.26 several characteristics of am make it useful for the treatment of ocular surface conditions . it has been shown to promote epithelialization , it contains important growth factors including epithelial growth factor and keratocyte growth factor ( both of which promote wound healing ) , it inhibits scarring by interfering with the tgf- signaling cascade in corneal and conjunctival fibroblasts , and it inhibits inflammation by releasing anti - inflammatory cytokines from its epithelium and stroma , such as interleukin-10 and interleukin-1 receptor antagonists.26 furthermore , because am does not express hla - a , b , or dr antigens , tissue rejection seldom occurs.25 am can be prepared fresh or preserved using either freeze - drying of the membrane ( dry am ) or cryopreservation . fresh am is more commonly used in the developing world , where preservation techniques are not easily performed.27 unfortunately , the use of fresh am is less advantageous , not only because it must be used in a limited time and does not exploit the size of the membrane for multiple tissue transplantations , but also poses a greater risk of transmitting infection.27 this is because the donor , who may or may not be screened when the membrane is retrieved , is not rescreened following a period of 6 months for communicable infectious diseases , which may only manifest after this period of time has elapsed.27 cryopreservation of am is achieved by freezing fresh am in either phosphate - buffered saline in dimethylsulfoxide or in eagle s minimum essential medium ( mem ) with glycerol , both at 80c.27 this precludes its use outside of large medical centers capable of maintaining this temperature and results in greater cost expenditure.28 once frozen , many of the beneficial soluble factors are depleted from the tissue , and this may cause a decrease in its efficacy.28 despite this drawback , this type of preservation is most commonly in use.28 dry am is made by freeze - drying fresh am and rehydrating it before use.27 conventionally , this would require the membrane to be frozen before being dried , which is thought to cause damage to beneficial factors , such as epidermal growth factor and tgf-1 , in the membrane.28 because am is usually < 100 m thick , the membrane can be dried in a freeze - dryer vacuum without prefreezing , thus resulting in improved factor retention.28 the membrane can then be maintained at room temperature.28 allen et al28 compared dried and cryopreserved am as an ocular surface dressing and found that dried am was superior to cryopreserved am because of the effect of the preservation process on the tissue . they found that preservation affected the biochemical and structural composition of the am . using electron microscopy , they showed that dried am was more similar to fresh am than to cryopreserved am . this was even more evident when the dried am was pretreated with trehalose , a lyoprotectant , which inhibits destruction of intracellular organelles.28 their study also showed that the biochemical composition , including the amount of factors such as epidermal growth factor and tgf-1 , was much more similar to fresh am than to cryopreserved am . dried am , as a preservation method , was found to decrease devitalization of epithelial cells and decrease cellular damage . in addition , the optimization of dried am with the lyoprotectants trehalose and raffinose was found to maintain the integrity of the tissue even more than drying alone.28 animal models using rabbits have also shown that dried am is at least as efficacious as cryopreserved am when used as a substrate for ocular surface reconstruction.29 in addition to the aforementioned effects on the host tissue , amt has also been observed to decrease postoperative pain . pires et al30 studied patients with symptomatic bullous keratopathy with poor visual potential and observed that 90% of patients with intractable pain secondary to their disease preoperatively were pain free following amt . pirouzian et al31 treated three pediatric patients with amg who had previously undergone deep lamellar excision of grade 1 limbal dermoid lesions . they found that postoperative pain was eliminated with the use of am and attributed this finding to the covering of the corneal epithelial defect ( and by extension , the exposed corneal nerves ) , as well as to the inhibition of inflammation provided by the membrane.31 in another study by hamza et al,32 83.3% of patients treated for various ocular surface conditions reported no pain 1 month following amt , whereas 90% of patients in the same study had reported pain on the same questionnaire before transplantation.32 in a study by uhlig et al,33 72.1% and 78.3% of patients suffering from corneal ulcers treated with amt ( with overlay or sandwich techniques , respectively ) reported either no pain or an improvement in their comfort . the efficacy of dry amg in pterygium surgery has been compared to the other standard techniques in many previous studies . significant parameters for comparison include recurrence rate , time to recurrence , complications , and cosmesis . a meta - analysis by li et al34 showed that the recurrence rate of pterygium after primary excision was significantly lower with conjunctival autografting than with amg . however , the recurrence rates were equal when these techniques were used for treatment of recurrent pterygia.34 in another study , it was concluded that recurrence rates of pterygia were significantly lower after limbal conjunctival autograft transplantation when compared to amt.35 yet another comparative study showed that while limbal conjunctival autograft and conjunctival autograft techniques were not significantly different from one another in terms of recurrence , both were significantly better in reducing recurrence rates than amg.20 the technique of using a limbal conjunctival flap was also shown to be superior to amt in terms of recurrence rate in one study by kurna et al.36 prabhasawat et al37 showed significantly higher recurrence rates for primary , recurrent , and all pterygia treated with amg compared to conjunctival autografting , and the time to recurrence was delayed in conjunctival autograft transplantation when compared to amt . despite a body of evidence suggesting that the recurrence rate after the use of amg is higher than the aforementioned techniques , there is also evidence that suggests that the use of am is at least equivalent . for example , in a study by ma et al,38 the use of am was compared retrospectively to the use of both conjunctival autograft and topical mitomycin c. the study showed the recurrence rate of amt to be 3.8% , compared to 5.4% and 3.7% in the conjunctival autograft and topical mitomycin c groups , respectively . the time to recurrence was found to be 12.3 months in the am group , compared to 3 and 5.5 months in the conjunctival autograft and topical mitomycin c groups , respectively . the study showed no significant difference in either the recurrence rate or the time to recurrence in any of the three groups.38 in another study , amt was retrospectively compared to conjunctival autograft transplantation . this study found a recurrence rate of 25% in the conjunctival autograft group and a 35% recurrence rate in the amt group , though statistically , this was not a significant difference.39 in the same study , the mean time to recurrence was significantly shorter for the conjunctival autograft group than for the am group , at 2.30.9 and 3.21.0 months , respectively.39 adjuvant therapies combined with the use of amg have also been studied . soloman et al21 found that the rates of recurrence following pterygium surgery were improved when an intraoperative depot corticosteroid injection was used to control postoperative inflammation following amt , with recurrence rates of 3.0%9.5% . the use of fibrin glue instead of sutures to attach the graft has also been shown to reduce the recurrence rate in amt , with recurrence rates of 9.4% in the fibrin glue group and 10.5% in the vicryl suture group.40 the use of topical mitomycin c in combination with amt has also been shown to lower the rate of pterygium recurrence.16 however , in another study by ma et al,41 the same conclusion was not confirmed to be true . a significant weakness in the data presented here is that , in many of the references cited , the inclusion criteria included patients who were followed for < 1 year before conclusions were made about recurrence rates.16,20,21,37,3942 in several studies , the differing arms of the study had significantly different follow - up times , likely underestimating recurrence rates in those groups followed for shorter periods . in addition , the definition of pterygium recurrence varies from the standard definition of any new fibrovascular growth across the limbus in some studies . these consider pterygium recurrence to occur only once the fibrovascular growth has extended > 2 mm across the limbus , again underestimating the rate of recurrence . amt following pterygium excision has been found to be a very safe and well - tolerated procedure , without any major complications reported in the literature . because am lacks the expression of hla - a , -b , and -dr antigens , the risk of immunologic graft rejection is minimal.25 the am is obtained from potential donors undergoing elective cesarean section.42 these donors are screened for communicable infectious diseases including hiv , hepatitis b and c , and syphilis.27,42 serological screening of donors is again recommended at 6 months following procurement of the membrane , as some infectious diseases may not be detectable within this transmission window period.42 when both of these serological screens are negative , the am is released for use . the membrane is prepared under sterile conditions and washed with antibiotics including penicillin , streptomycin , neomycin , and amphotericin b.42 the amnion and chorion are separated by blunt dissection.42 microbial infection rates after amt have been reported as low as 1.6% and occur primarily with gram - positive organisms.43 clinical complications of amt are relatively minor . in one study by ma,38 no major complications were noted in 80 eyes of 71 patients who were treated with amg . of these , one case ( 1.25% ) developed a pyogenic granuloma , and one case ( 1.25% ) developed an iatrogenic microhyphema , which was caused by an inadvertently deep limbal suture . in one of the comparative arms of the same study , where patients were treated with conjunctival autografting , two cases developed a pyogenic granuloma ( 3.6% ) and four cases ( 7.3% ) developed a conjunctival inclusion cyst . in the third arm of the study , where patients were treated with topical mitomycin c , one case developed a pyogenic granuloma ( 1.8% ) and one case ( 1.8% ) developed scleral ischemia.38 in another study , amt resulted in pyogenic granuloma ( 4.3% ) , epithelial defects ( 4.3% ) , and dellen ( 4.3% ) and showed no significant difference when compared to complications from conjunctival autograft.39 all complications resolved spontaneously.39 other complications that have been reported include increased foreign body sensation , eyelid edema , conjunctival hyperemia , and symblepharon.16 cosmetic outcomes and patient satisfaction are also important considerations when assessing the effectiveness of amt in the treatment of pterygia , in addition to minimizing recurrence rates and surgical complications . conjunctival autografting following pterygium excision has been shown to be significantly superior to amg in final cosmetic appearance.37,44 in one study , cosmetic appearance was graded from 1 to 4 , where grade 3 was an unacceptable cosmetic outcome and grade 4 was true recurrence . of the patients who underwent conjunctival autografting , 10% had an unacceptable cosmetic outcome , compared to 21.1% of patients who had undergone amt ; this result was statistically significant.44 in a study by hirst,45 cosmetic results of the p.e.r.f.e.c.t . for pterygium technique were compared to the same results from the study by prabhasawat et al,37 which examined conjunctival autografting , amg , and primary closure . a cosmetic result graded 3 or 4 ( which represent poor cosmetic result and frank recurrence , respectively ) occurred in 5.7% of cases undergoing the p.e.r.f.e.c.t . technique , 9% of cases with conjunctival autografting , 32.6% of cases with amg , and 85% of cases with primary closure.45 in another study , kucukerdonmez et al40 compared the cosmetic outcome of using amt following pterygium excision with either fibrin glue or vicryl sutures and found that while there was a decrease in surgical time and postoperative symptoms , there was no difference in the cosmetic outcome between the two groups . of note , patient tolerability was much improved in the fibrin glue group compared with the vicryl suture group in all subjective symptoms studied , including epiphora , foreign body sensation , and irritation.40 am has also been used to cover conjunctival defects following the removal of ocular surface neoplasms . in one study , which followed eight patients having undergone tumor excision of ocular surface neoplasms , all eight patients were reported to have satisfactory cosmetic results following amt.46 the role of amt in the management of postoperative pain has been detailed earlier . amg in the treatment of pterygium has been shown to be less effective than , or at best , equivalent to other grafting procedures including conjunctival and limbal autografting.20,3439 in particular , amt has been shown in many studies to have a higher rate of recurrence compared to both conjunctival and limbal autografting.20,34,35,37 despite these findings , there is still a role for this technique in the treatment of pterygia . amg can be useful in the covering of wide ocular surface defects , such as in the case of large or double - headed pterygia.21 in this case , the amount of autologous conjunctival tissue that can be harvested is limited , whereas am can cover any sized ocular surface defect.42 am also provides an advantage in cases where the conjunctiva is scarred and can not be harvested16 or when the bulbar conjunctiva must be spared to allow for future glaucoma filtering surgery . the preservation process allows for the tissue to be quarantined during a window period of 6 months , allowing for screening of infectious diseases , including hiv , hepatitis b and c , and syphilis , from donors who may not screen positive at the time that the membrane is harvested.27 dry am has also been shown to be a more gentle preservation method than cryopreservation , as it allows for greater maintenance of structural and biochemical integrity , and improves the retention of beneficial factors , which give the membrane its useful properties.28 dry am is also less expensive to maintain than cryopreserved membrane because it can be maintained at room temperature , as opposed to cryopreservation which requires temperatures of 80c.28 this also makes its use more versatile outside of large hospital settings , as it can be used in the developing world and in military environments.28 therefore , while the use of am in the treatment of pterygium may not be the ideal choice in all circumstances , there are certainly situations where its use may be most beneficial to the patient , and surgeons should keep this technique in their armamentarium of treatment options .
the cloudsat 2cice product describes the vertically resolved microphysical properties of ice clouds [ deng et al . , 2010 , 2013 ] . the algorithm is a synergetic ice cloud retrieval derived from the combination of the cloudsat radar reflectivity ( ze ) and the cloudaerosol lidar and infrared pathfinder satellite observation ( calipso ) lidar attenuated backscatter ( ) using a variational method for deriving profiles of extinction coefficient ( ) , ice water content ( iwc ) , and particle effective radius ( re ) . the 2cice product has been evaluated with collocated aircraft in situ measurements and compared with the radar / lidar ( dardar ) [ delano and hogan , 2008 ] and other cloud products [ deng et al . , a recent study [ matrosov , 2015 ] to evaluate retrievals of total ice content in precipitating cloud systems from groundbased operational radar measurements shows that ice water path derived ( iwp ) from 2cice is in relatively good agreement with iwp from a method developed specifically for thick ice clouds that accounts for ice hydrometers nonsphericity [ matrosov and heymsfield , 2008 ; matrosov and battaglia , 2009 ] . similar to the 2cice product , the dardar cloud product is a synergetic ice cloud retrieval algorithm derived from the combination of the cloudsat radar and calipso lidar using a somewhat contrasting variational method for retrieving profiles of , iwc , and re [ delano and hogan , 2008 ] . one of the important differences between 2cice and dardar is that 2cice utilizes the fact that clouds in the lidaronly region should have equivalent radar reflectivity factor less than the cloudsat radar sensitivity of about 29 dbz . to exploit this information , 2cice parameterizes ze in the lidaronly region which provides an additional constraint on the retrieval and ensures that the numerical inversion can proceed seamlessly through the layer . in deng et al . , this additional weak constraint is shown to provide somewhat superior results in the lidaronly regions compared to dardar . however , parameterizing ze in the lidaronly region is not an easy task . in the 20072008 version of 2cice [ deng et al . , 2010 ] ze is parameterized by mapping the attenuated backscattering for a whole granule before the profiling retrieval , assuming that the height distribution of the ze below the minimum detectable signals would be similar to what is measured by the arm millimeter cloud radar ( mmcr ) [ moran et al . , however , cirrus in the polar regions are much lower and warmer on average than cirrus in the tropics and midlatitudes ; therefore , in the 20092010 version , the height dependence of ze in deng et al . was replaced by a temperature dependence for the ze parameterization . a discontinuity in retrieved cloud properties was then noted in the lidaronly regions due to the different techniques in ze parameterization in the lidaronly region in those two algorithm versions ( p. kablick , personal communication , 2013 ) . furthermore , while the simple constraint of a maximum value of ze in the lidaronly portion of the layer seems to be useful , using additional ancillary information to provide a more robust parameterization of ze has the potential to further constrain the algorithm . therefore , an improved approach for the ze parameterization in the lidaronly region is developed in this paper . the and ze relations has been studied in matrosov et al . to derive vertical profiles of the visible extinction coefficients in ice clouds from measurements of the radar reflectivity collected by a vertically pointing 35 ghz cloud radar : ( 1)m10.0036ze0.58mm6m3 the uncertainties of radarbased retrievals of cloud visible optical thickness are estimated in matrosov et al . by comparing retrieval results with optical thicknesses obtained independently from radiometric measurements during the yearlong surface heat budget of the arctic ocean field experiment . a negative bias is found due to an inherently low sensitivity of the radar measurements to smaller cloud particles that still contribute noticeably to the cloud extinction . another inevitable uncertainty associated with the relation in equation ( 1 ) is that the ice particle microphysical properties are derived from an unknowable particle habit that contributes to errors in crosssectional area and bulk density as a function of particle maximum dimension . while these properties are known to be a function of temperature ( t ) [ hong et al . , 2007 , donovan et al . , 2004 ; heymsfield et al . , 2004 , 2014 ] , the arm program 's raman lidar ( carl ) [ goldsmith et al . , 1998 ] was located side by side with mmcr , at the southern great plains ( sgp ) site and provides an opportunity for independent measurements of vertical profiles of and ze for optically thin cirrus . the arm mmcr was a zenithpointing radar that operated at a frequency of 35 ghz with a vertical sample resolution of 90 m. mmcr could report radar reflectivity of 50 dbz up to 20 km with a mean calibration error of less than 1 db [ moran et al . such a highly sensitive cloud radar collocated with raman lidar provides a unique data set that allows for us to develop an improved relationship between ze and for upper troposphere cirrus clouds . in essence , such a relationship brings additional information in the form of ancillary data to the 2cice retrieval algorithm . in the following , we develop and evaluate a new ze relation for upper tropospheric ice clouds with a goal of improving the 2cice retrieval algorithm . the raman lidar at the arm sgp site transmits a laser wavelength of 355 nm and measures the backscattered elastic return at the same wavelength as well as the water vapor and nitrogen raman shifted returns at 408 and 387 nm , respectively [ goldsmith et al . , the vertical distribution of nitrogen raman signals is proportional to the nitrogen number density profile , which is well determined by temperature and pressure profiles . therefore , nitrogen raman signals can be used to derive the cloud extinction coefficient without assuming a relationship between backscatter and extinction that is typically needed for extinction retrievals from elastic lidar signals [ ansmann et al . , a method similar to that used by ansmann et al . was applied to carl measurements to generate a 1000 h cirrus cloud data set including [ wang and sassen , 2002 ] . a main limiting factor for cirrus accuracy derived from the nitrogen raman a 10 min sliding average is applied to the raman lidar data . during the daytime , the signal in the nitrogen raman channel is often too noisy for the analysis of the entire cirrus layer . we retrieved the cloud extinction profile from the elastic channel with an average extinctiontobackscatter ratio derived from the raman channel in the lower cloud portion and/or with the constraint of total optical depth [ young , 1995 ] . then , extinction profiles from elastic and nitrogen raman channels are combined to provide the final results . although , random errors in the retrieved could be as high as 30% in the data set , the mean bias is small due to effective constraints from nitrogen raman and molecular elastic signals [ wang and sassen , 2002 ] . the cloudsat cloud profiling radar ( cpr ) is a 94 ghz nadirlooking radar which measures the power backscattered by clouds as a function of distance from the radar . other than about 1 db difference between w and ka band reflectivity due to the dielectric properties of water [ doviak and zrnic , 1993 ] , which is accounted for in the parameterization , the ze is independent of radar wavelength if the hydrometeor 's maximum diameter d is much smaller than the radar wavelength and the scattering is described by the rayleigh approximation . this should be a reasonable assumption for upper troposphere thin cirrus clouds with ice mass equivalent radius smaller than 300 m [ wang et al . this attenuation at mmcr ka band is smaller compared to that in cloudsat cpr , which is corrected in the cpr data set . given the cirrus observed by both raman lidar and mmcr at sgp site , the sampled cirrus clouds are most likely to be synopticscale cirrus than anvil clouds . the attenuation in those profiles should be less than ~0.5 db mean bias [ matrosov et al . thermodynamic profiles are acquired from the merged sounding data product provided by the arm program [ troyan , 2011 ] . this product uses a combination of observations from radiosonde soundings , microwave radiometerderived water vapor path , surface meteorological instruments , and the european centre for mediumrange weather forecasts ( ecmwf ) model and employs a sophisticated scaling / interpolation / smoothing scheme in order to define profiles of the atmospheric thermodynamic state [ troyan , 2011 ] . because temperature provides a wellknown constraint on ice cloud properties [ e. g. heymsfield et al . , 2013 ] , we use temperature as an additional parameter to effectively constrain the ice particle habit or particle size distribution variations in the mmcr and raman lidar data sets . figures 1a and 1b show the probability density functions ( pdf ) of cirrus clouds in ze/ and e / t domains based on the multiyear collocated mmcr and raman lidar measurements using a 1 min time resolution at the sgp site [ wang and sassen , 2002 ] . the ze ranges from 10 to 50 dbz over a temperature range from 20 to 60c . data sample distribution is listed in 2 dbz ze bins in table 1 . the overplotted black line in figures 1a and 1c is derived from equation ( 1 ) in matrosov et al . where a negative bias is found compared to the raman lidarmmcr observations in this study . ( a and b ) the pdf of zeextinction ( ) and zet relations from mmcr measurement , raman lidar , and merged sounding data at arm sgp site , and the ( c and d ) parameterized ze and zet relations from equation ( 2 ) . list of comparison between parameterized ze from equation ( 2 ) and arm mmcr observed ze in figure 2a while ze is correlated with t and , there are large deviations existing in both relations . . given the relations shown in figures 1a and 1b , we used a set of polynomial functions to approximate the relationships between ze , , and t , and found that the following function provides an optimal fit in terms of maximizing correlation coefficients and minimizing standard deviations : ( 2)zdbz = a+blog10log10t+clog10t+dlog10where t is in kelvin and is in per meter . the pdf between fitted ze / t and fitted ze/ is shown in figures 1c and 1d . we can see that the relations between the fitted ze / t and ze/ are very similar with observations with certain deviations . the fitted ze has a good correlation with observed ze ( r = 0.65 ) in figure 2 , while the mean bias between the fitted ze and the mmcr observed ze is less than 3 db in table 1 . the parameterized ze in equation ( 2 ) provides a reasonable firstorder estimate of ze in the lidaronly region for the 2cice retrieval . this parameterization effectively leverages the unique ancillary information provided by many years of collocated raman lidar and mmcr data . this value is designated as the uncertainty of the parameterized ze in the lidaronly region in the 2cice retrieval framework . the fitted coefficients of ze parameterization as a function of extinction coefficient and temperature : z e(dbz ) = a + b log 10( ) log 10(t ) + c log 10( ) t + d log 10( ) , where t is in kelvin and is in per meter comparison in pdf between ze parameterized using equation ( 2 ) and the mmcr observed ze at the sgp site . in the 2cice optimal estimation framework , the s represents the total measurement error which includes forward model error , random measurement error , and systematic error [ deng et al . , 2010 ] . given the geographical variation of cirrus clouds and uncertainties in equation ( 2 ) , the corresponding ze error in the lidaronly region is assigned to be ~6 db . this large error assignment ensures that the parameterized radar reflectivity value provides a weak constraint on the actual retrieved ice cloud properties in this portion of the layer , while the actual information that the radar reflectivity is lower than the minimum detectable by the cpr is retained . the raman lidar at the arm sgp site transmits a laser wavelength of 355 nm and measures the backscattered elastic return at the same wavelength as well as the water vapor and nitrogen raman shifted returns at 408 and 387 nm , respectively [ goldsmith et al . , the vertical distribution of nitrogen raman signals is proportional to the nitrogen number density profile , which is well determined by temperature and pressure profiles . therefore , nitrogen raman signals can be used to derive the cloud extinction coefficient without assuming a relationship between backscatter and extinction that is typically needed for extinction retrievals from elastic lidar signals [ ansmann et al . , a method similar to that used by ansmann et al . was applied to carl measurements to generate a 1000 h cirrus cloud data set including [ wang and sassen , 2002 ] . a main limiting factor for cirrus accuracy derived from the nitrogen raman a 10 min sliding average is applied to the raman lidar data . during the daytime , the signal in the nitrogen raman channel is often too noisy for the analysis of the entire cirrus layer . we retrieved the cloud extinction profile from the elastic channel with an average extinctiontobackscatter ratio derived from the raman channel in the lower cloud portion and/or with the constraint of total optical depth [ young , 1995 ] . then , extinction profiles from elastic and nitrogen raman channels are combined to provide the final results . although , random errors in the retrieved could be as high as 30% in the data set , the mean bias is small due to effective constraints from nitrogen raman and molecular elastic signals [ wang and sassen , 2002 ] . the cloudsat cloud profiling radar ( cpr ) is a 94 ghz nadirlooking radar which measures the power backscattered by clouds as a function of distance from the radar . other than about 1 db difference between w and ka band reflectivity due to the dielectric properties of water [ doviak and zrnic , 1993 ] , which is accounted for in the parameterization , the ze is independent of radar wavelength if the hydrometeor 's maximum diameter d is much smaller than the radar wavelength and the scattering is described by the rayleigh approximation . this should be a reasonable assumption for upper troposphere thin cirrus clouds with ice mass equivalent radius smaller than 300 m [ wang et al . this attenuation at mmcr ka band is smaller compared to that in cloudsat cpr , which is corrected in the cpr data set . given the cirrus observed by both raman lidar and mmcr at sgp site , the sampled cirrus clouds are most likely to be synopticscale cirrus than anvil clouds . the attenuation in those profiles should be less than ~0.5 db mean bias [ matrosov et al . thermodynamic profiles are acquired from the merged sounding data product provided by the arm program [ troyan , 2011 ] . this product uses a combination of observations from radiosonde soundings , microwave radiometerderived water vapor path , surface meteorological instruments , and the european centre for mediumrange weather forecasts ( ecmwf ) model and employs a sophisticated scaling / interpolation / smoothing scheme in order to define profiles of the atmospheric thermodynamic state [ troyan , 2011 ] . because temperature provides a wellknown constraint on ice cloud properties [ e. g. heymsfield et al . , 2013 ] , we use temperature as an additional parameter to effectively constrain the ice particle habit or particle size distribution variations in the mmcr and raman lidar data sets . figures 1a and 1b show the probability density functions ( pdf ) of cirrus clouds in ze/ and e / t domains based on the multiyear collocated mmcr and raman lidar measurements using a 1 min time resolution at the sgp site [ wang and sassen , 2002 ] . the ze ranges from 10 to 50 dbz over a temperature range from 20 to 60c . data sample distribution is listed in 2 dbz ze bins in table 1 . the overplotted black line in figures 1a and 1c is derived from equation ( 1 ) in matrosov et al . where a negative bias is found compared to the raman lidarmmcr observations in this study . ( a and b ) the pdf of zeextinction ( ) and zet relations from mmcr measurement , raman lidar , and merged sounding data at arm sgp site , and the ( c and d ) parameterized ze and zet relations from equation ( 2 ) . list of comparison between parameterized ze from equation ( 2 ) and arm mmcr observed ze in figure 2a while ze is correlated with t and , there are large deviations existing in both relations . therefore , parameterizing ze as single functions of either t or is not optimal . given the relations shown in figures 1a and 1b , we used a set of polynomial functions to approximate the relationships between ze , , and t , and found that the following function provides an optimal fit in terms of maximizing correlation coefficients and minimizing standard deviations : ( 2)zdbz = a+blog10log10t+clog10t+dlog10where t is in kelvin and is in per meter . the pdf between fitted ze / t and fitted ze/ is shown in figures 1c and 1d . we can see that the relations between the fitted ze / t and ze/ are very similar with observations with certain deviations . the fitted ze has a good correlation with observed ze ( r = 0.65 ) in figure 2 , while the mean bias between the fitted ze and the mmcr observed ze is less than 3 db in table 1 . the parameterized ze in equation ( 2 ) provides a reasonable firstorder estimate of ze in the lidaronly region for the 2cice retrieval . this parameterization effectively leverages the unique ancillary information provided by many years of collocated raman lidar and mmcr data . this value is designated as the uncertainty of the parameterized ze in the lidaronly region in the 2cice retrieval framework . the fitted coefficients of ze parameterization as a function of extinction coefficient and temperature : z e(dbz ) = a + b log 10( ) log 10(t ) + c log 10( ) t + d log 10( ) , where t is in kelvin and is in per meter comparison in pdf between ze parameterized using equation ( 2 ) and the mmcr observed ze at the sgp site . in the 2cice optimal estimation framework , the s represents the total measurement error which includes forward model error , random measurement error , and systematic error [ deng et al . , 2010 ] . given the geographical variation of cirrus clouds and uncertainties in equation ( 2 ) , the corresponding ze error in the lidaronly region is assigned to be ~6 db . this large error assignment ensures that the parameterized radar reflectivity value provides a weak constraint on the actual retrieved ice cloud properties in this portion of the layer , while the actual information that the radar reflectivity is lower than the minimum detectable by the cpr is retained . the purpose of parameterizing ze from the collocated raman lidar and mmcr measurements is to provide an additional constraint in the lidaronly region in 2cice retrievals . however , the collocated arm data used in development of the parameterization includes the socalled radarlidar overlap region in 2cice because of the high sensitivity of mmcr . therefore , in the following we compare the parameterized and observed ze in the lidarradar overlapped regions in 2cice as a check on our implementation of the parameterization . during the small particles in cirrus [ deng et al . , 2013 ] field campaign [ mace et al . , 2009 ] , the spec inc . the 2cice retrieval along with the dardar retrieval and from calipso were compared with in situ measurements in deng et al . . for the radar and lidar overlap region , dardar and 2cice were found to be in reasonably close agreement with each other and with the in situ measurements . however , flight mean ratios of retrieved to derived from in situ measurements using the 2d stereo ( 2ds ) [ lawson et al . , 2006 ] probe show that retrieved using algorithms implemented by the calipso team [ young et al . , this low bias is probably related to the 5 km averaging technique implemented by the calipso team in situations where the cirrus layers have significant horizontal gradients in . extinction coefficients derived from the calipso algorithms are also found to be biased low compared to synthetic extinction from in situ particle size distributions as reported in heymsfield et al . . here , for the 12 granules used in deng et al . , we apply equation ( 2 ) with from the 2cice ( 2c_ice ) , dardar ( dardar ) , and the calipso ( calipso ) that is available from the calipso archive and represents the calipso team standard data product . temperature profiles from the cloudsat ecmwf_aux product are used to derive the parameterized ze in the lidarradar overlap region , and then we compare the parameterized ze with the cloudsat cprobserved ze . comparison of cloudsat cprobserved ze and parameterized ze using equation ( 2 ) with from ( a ) 2cice , ( b ) dardar , and ( c ) calipso in the lidarradar overlap region . the comparisons are separated among ( a1 , b1 , and c1 ) high latitudes , ( a2 , b2 , and c2 ) midlatitudes , and ( a3 , b3 , and c3 ) tropics . the parameterized ze using 2c_ice in figure 3a is very well correlated with cprobserved ze with a correlation coefficient ( rr ) of 0.85 . this good correlation might be expected since the 2cice retrieval has already assumed the ze parameterization in the lidaronly region although not in the overlap region . the dardar is retrieved independently from calipso and cpr data at the university of lille in france , although the parameterized ze using dardar in figure 3b is also well correlated with cprobserved ze ( rr = 0.81 ) . the mean biases between the parameterized ze with 2c_ice and dardar and the cprobserved ze are small . although the ( calipso ) is calculated independently in the calipso level 2 product [ young and vaughan , 2009 ] from cloudaerosol lidar with orthogonal polarization ( caliop ) only , the parameterized ze using calipso is also correlated with cprobserved ze ( rr = 0.68 ) . however , the parameterized ze with calipso has a negative bias compared to cprobserved ze and the parameterized ze with 2c_ice and dardar , since calipso is smaller compared to 2c_ice and dardar . having been derived from the broadest possible range of dynamics , aerosol , and thermodynamic conditions , we expect that the ice clouds to which we apply the 2cice algorithm are much more diverse in terms of their microphysical properties than the cirrus clouds from the arm sgp site . therefore , the comparison of cloudsat cprobserved ze and parameterized ze in the lidar and radar overlapped regions is separated among high latitudes , midlatitudes , and tropics in figure 3 . we find that the parameterized ze from the 2cice extinction is generally close to the cloudsat cpr observations except for the clouds with large extinction in the tropics in figure 3a . given the strong correlation between the distance of anvil clouds to their parent convective core edge and the cloud base temperature in the recent study of tropical anvil clouds [ deng et al . , 2015 ] , we further separate the comparison for tropical ice cloud layers with cloud bases warmer or colder than 250 k in figure 4 . the ice clouds with cloud base colder than 250 k represent the cirrus that is generally far away from a convective core and have less influence from convection , and therefore have different properties from those with cloud base warmer than 250 k , which are likely often associated with outflow from convection , hereafter anvil clouds . figure 4 shows that the parameterized ze of anvil clouds from 2cice extinction is slightly biased low by about 23 db to the cloudsat cpr observations . this bias might be related to particle size differences or other microphysical assumptions , but the comparison is still within the standard deviation range ( 6 dbz ) of the ze given in table 1 . comparison of cloudsat cprobserved ze and parameterized ze using equation ( 2 ) with from ( a ) 2cice , ( b ) dardar , and ( c ) calipso in tropical ice clouds with cloud base ( a1 , b1 , and c1 ) warmer than 250 k and ( a2 , b2 , and c2 ) colder than 250 k in the lidarradar overlap region . the newly developed ze parameterization in the lidaronly region has been applied to 2cice from 2007 to 2010 . radiative properties and profiles of radiative fluxes along the cloudsat track can be derived using a suite of techniques that were initially developed and applied to a similar suite of groundbased remote sensors [ mace et al . , 2006a , 2006b ; mace and benson , 2008 ] and to atrain data [ mace , 2010 ; berry and mace , 2014 ] . in berry and mace , vertically resolved ice water content , ice particle effective radius , and extinction coefficient profiles from 2cice were used in place of the more primitive cirrus retrievals of the earlier algorithms , and the calculated top of the atmosphere ( toa ) fluxes were compared to clouds and the earth 's radiant energy system ( ceres ) fluxes to provide some understanding of the quality of the cloud properties using toa radiative closure [ berry and mace , 2014 ] . one case study for a relatively thin ice cloud layer on 3 august 2007 is selected to illustrate a radiative fluxes comparison between calculations with the updated 2cice retrieval and ceres measurements in figure 5 . this cirrus cloud layer is above 10 km and is mainly observed by the calipso lidar . our derived values generally compare well with ceres radiative fluxes which are shown in red in figures 5c and 5d . given the optically thin nature of this layer over a relatively dark marine background , only approximately 100 w m of solar radiation is reflected . the outgoing longwave radiation is about 250 w m. errors in 2cice algorithm translate into errors in the radiative property profiles and ultimately to the radiative fluxes . however , we find that the solar and ir fluxes derived from 2cice track the ceres data reasonably well . this is especially evident in the region near 3 and 1.5 south latitude , where variations in the microphysics drive variations in the fluxes that provide a nice match between ceres and 2cice . from the case study here and monthly averaged data analysis in berry and mace , 2014 , errors in the monthlyaveraged cloud radiative effects are assumed to be on the order of 510 w m. comparison of radiative fluxes calculated using 2cice results that include the new ze constraints discussed in the text with flux measurements collected by ceres on 3 august 2007 over south of the bay of bengal . measured ( a ) cloudsat radar reflectivity and ( b ) caliop attenuated backscatter and the calculated upward ( c ) shortwave and ( d ) longwave flux comparison using the new ze constraint in the 2cice retrieval ( dots ) with ceres ( red lines ) . the purpose of parameterizing ze from the collocated raman lidar and mmcr measurements is to provide an additional constraint in the lidaronly region in 2cice retrievals . however , the collocated arm data used in development of the parameterization includes the socalled radarlidar overlap region in 2cice because of the high sensitivity of mmcr . therefore , in the following we compare the parameterized and observed ze in the lidarradar overlapped regions in 2cice as a check on our implementation of the parameterization . during the small particles in cirrus [ deng et al . , 2013 ] field campaign [ mace et al . , 2009 ] , the spec inc . the 2cice retrieval along with the dardar retrieval and from calipso were compared with in situ measurements in deng et al . . for the radar and lidar overlap region , dardar and 2cice were found to be in reasonably close agreement with each other and with the in situ measurements . however , flight mean ratios of retrieved to derived from in situ measurements using the 2d stereo ( 2ds ) [ lawson et al . , 2006 ] probe show that retrieved using algorithms implemented by the calipso team [ young et al . , this low bias is probably related to the 5 km averaging technique implemented by the calipso team in situations where the cirrus layers have significant horizontal gradients in . extinction coefficients derived from the calipso algorithms are also found to be biased low compared to synthetic extinction from in situ particle size distributions as reported in heymsfield et al . . here , for the 12 granules used in deng et al . , we apply equation ( 2 ) with from the 2cice ( 2c_ice ) , dardar ( dardar ) , and the calipso ( calipso ) that is available from the calipso archive and represents the calipso team standard data product . temperature profiles from the cloudsat ecmwf_aux product are used to derive the parameterized ze in the lidarradar overlap region , and then we compare the parameterized ze with the cloudsat cprobserved ze . comparison of cloudsat cprobserved ze and parameterized ze using equation ( 2 ) with from ( a ) 2cice , ( b ) dardar , and ( c ) calipso in the lidarradar overlap region . the comparisons are separated among ( a1 , b1 , and c1 ) high latitudes , ( a2 , b2 , and c2 ) midlatitudes , and ( a3 , b3 , and c3 ) tropics . the parameterized ze using 2c_ice in figure 3a is very well correlated with cprobserved ze with a correlation coefficient ( rr ) of 0.85 . this good correlation might be expected since the 2cice retrieval has already assumed the ze parameterization in the lidaronly region although not in the overlap region . the dardar is retrieved independently from calipso and cpr data at the university of lille in france , although the parameterized ze using dardar in figure 3b is also well correlated with cprobserved ze ( rr = 0.81 ) . the mean biases between the parameterized ze with 2c_ice and dardar and the cprobserved ze are small . although the ( calipso ) is calculated independently in the calipso level 2 product [ young and vaughan , 2009 ] from cloudaerosol lidar with orthogonal polarization ( caliop ) only , the parameterized ze using calipso is also correlated with cprobserved ze ( rr = 0.68 ) . however , the parameterized ze with calipso has a negative bias compared to cprobserved ze and the parameterized ze with 2c_ice and dardar , since calipso is smaller compared to 2c_ice and dardar . having been derived from the broadest possible range of dynamics , aerosol , and thermodynamic conditions , we expect that the ice clouds to which we apply the 2cice algorithm are much more diverse in terms of their microphysical properties than the cirrus clouds from the arm sgp site . therefore , the comparison of cloudsat cprobserved ze and parameterized ze in the lidar and radar overlapped regions is separated among high latitudes , midlatitudes , and tropics in figure 3 . we find that the parameterized ze from the 2cice extinction is generally close to the cloudsat cpr observations except for the clouds with large extinction in the tropics in figure 3a . given the strong correlation between the distance of anvil clouds to their parent convective core edge and the cloud base temperature in the recent study of tropical anvil clouds [ deng et al . , 2015 ] , we further separate the comparison for tropical ice cloud layers with cloud bases warmer or colder than 250 k in figure 4 . the ice clouds with cloud base colder than 250 k represent the cirrus that is generally far away from a convective core and have less influence from convection , and therefore have different properties from those with cloud base warmer than 250 k , which are likely often associated with outflow from convection , hereafter anvil clouds . figure 4 shows that the parameterized ze of anvil clouds from 2cice extinction is slightly biased low by about 23 db to the cloudsat cpr observations . this bias might be related to particle size differences or other microphysical assumptions , but the comparison is still within the standard deviation range ( 6 dbz ) of the ze given in table 1 . comparison of cloudsat cprobserved ze and parameterized ze using equation ( 2 ) with from ( a ) 2cice , ( b ) dardar , and ( c ) calipso in tropical ice clouds with cloud base ( a1 , b1 , and c1 ) warmer than 250 k and ( a2 , b2 , and c2 ) colder than 250 k in the lidarradar overlap region . the newly developed ze parameterization in the lidaronly region has been applied to 2cice from 2007 to 2010 . radiative properties and profiles of radiative fluxes along the cloudsat track can be derived using a suite of techniques that were initially developed and applied to a similar suite of groundbased remote sensors [ mace et al . 2006a , 2006b ; mace and benson , 2008 ] and to atrain data [ mace , 2010 ; berry and mace , 2014 ] . in berry and mace , vertically resolved ice water content , ice particle effective radius , and extinction coefficient profiles from 2cice were used in place of the more primitive cirrus retrievals of the earlier algorithms , and the calculated top of the atmosphere ( toa ) fluxes were compared to clouds and the earth 's radiant energy system ( ceres ) fluxes to provide some understanding of the quality of the cloud properties using toa radiative closure [ berry and mace , 2014 ] . one case study for a relatively thin ice cloud layer on 3 august 2007 is selected to illustrate a radiative fluxes comparison between calculations with the updated 2cice retrieval and ceres measurements in figure 5 . this cirrus cloud layer is above 10 km and is mainly observed by the calipso lidar . our derived values generally compare well with ceres radiative fluxes which are shown in red in figures 5c and 5d . given the optically thin nature of this layer over a relatively dark marine background , only approximately 100 w m of solar radiation is reflected . the outgoing longwave radiation is about 250 w m. errors in 2cice algorithm translate into errors in the radiative property profiles and ultimately to the radiative fluxes . however , we find that the solar and ir fluxes derived from 2cice track the ceres data reasonably well . this is especially evident in the region near 3 and 1.5 south latitude , where variations in the microphysics drive variations in the fluxes that provide a nice match between ceres and 2cice . from the case study here and monthly averaged data analysis in berry and mace , 2014 , errors in the monthlyaveraged cloud radiative effects are assumed to be on the order of 510 w m. comparison of radiative fluxes calculated using 2cice results that include the new ze constraints discussed in the text with flux measurements collected by ceres on 3 august 2007 over south of the bay of bengal . measured ( a ) cloudsat radar reflectivity and ( b ) caliop attenuated backscatter and the calculated upward ( c ) shortwave and ( d ) longwave flux comparison using the new ze constraint in the 2cice retrieval ( dots ) with ceres ( red lines ) . to evaluate the impact of the new ze parameterization in the lidaronly region of the 2cice product , we compare the new 2cice results with previous versions of the algorithm in figure 6 for the lidaronly region and the radar region , which includes the radar and lidar overlap region and radaronly regions . the pdfs of ice clouds in the lidaronly and radar regions for high latitudes ( 6090 ) , midlatitudes ( 3060 ) , and tropics ( 30s30n ) in figures 6a6c show that the fraction of lidaronly region exceeds that of radar region above 220 k in the tropics . ( a c ) ice cloud frequency distributions as a function of temperature in the lidaronly ( red ) and the radar regions ( black ) for high latitude ( figure 6a ) , midlatitude ( figure 6b ) , and tropics ( figure 6c ) regions . ( d f ) mean iwc as a function of temperature in lidar ( red ) and radar ( black ) regions for corresponding latitude bands . the solid red lines show the mean and standard deviation of iwc from the lidaronly region from the new ze constraints discussed in the text . the dashed lines show previous versions ( 20072008 version in red ; 20092010 version in blue ) that include an erroneous discontinuity that is now superseded by the new work reported on here . we can see that the retrieved iwc with new ze parameterization is very close to the old 20072008 version except for the lidaronly region warmer than 240 k in high latitudes , while iwc in the radar region is almost the same . for 20092010 data , the iwc in the lidaronly region in the new 2cice data that uses the more accurate ze parameterization ( equation 2 ) tends to be larger than the iwc in the older now obsolete version by about a factor of 2 to 5 , while iwc in the radar region is almost unchanged . the iwc in the lidaronly region is order smaller than that found in the radar region , which indicates that only a small portion of the total ice mass in the vertical column tends to exist in lidaronly region , and from a mass perspective it is relatively unimportant to global atmospheric ice mass budget statistics [ li et al . , however , the iwc that exists in the lidaronly region is very important to understand variations in cirrus cloud radiative effects [ berry and mace , 2014 ; comstock et al . , 2002 , 2013 ; sassen and comstock , 2001 ] . as a brief example of the richness of this data set , figure 7 shows the tropical monthly zonal mean occurrence , mean re , and iwc of ice at 210 k 1.5 k since january 2007 . the locations of maximum cirrus occurrence frequency at a temperature of 210 k transit from 10s in january to 10n in july on a welldefined annual cycle . the latitudinal migration of ice cloud occurrence and microphysical properties is well correlated with solar insolation and tropical convection . however , the corresponding values of re and iwc are better correlated with each other than with the occurrence frequency . overall , the southern hemisphere , southward of 20s , demonstrates a minimum in ice cloud occurrence during boreal summer associated with the descending branch of the hadley cell . it is interesting to note both annual mean asymmetry in the hemispheres and interannual variability in cirrus occurrence and zonal mean properties . we expect that improvements to the 2cice algorithm due to the ze parameterization will allow for more fruitful study of the thin cirrus observed only by the calipso lidar . the new 2cice product has been used for recent studies of cirrus cloud properties and their radiative effect [ huang et al . , the monthly zonal mean occurrence , mean re , and iwc of ice at 210 k 1.5 k since january 2007 . in summary , utilizing the capability of arm raman lidar to observe and the high sensitivity of the arm mmcr to observe thin cirrus , a new parameterization relating ze and for the 2cice lidaronly region has been developed . the parameterized ze agrees well with cloudsat observed ze in the 2cice lidar and radar overlapped region . results from the updated 2cice algorithm with the new ze parameterization have been examined with radiation closure methods . this new ze parameterization utilizes in addition to temperature , rather than just height , and is now more suitable for global ice cloud retrievals as compared to earlier approaches . the updated 2cice product with the new ze parameterization removes a discontinuity in retrieved cloud properties which has been reported in the lidaronly regions due to different techniques used relating ze and in the lidaronly region .
cough variant asthma ( cva ) , a variant form of classic asthma that presents solely with a cough and responds to bronchodilator treatment , is considered to be the most common cause of chronic coughs . several studies have suggested that the fundamental pathophysiological characteristics of cva , airway inflammation and airway hyperresponsiveness ( ahr ) , are identical to those of classic asthma . furthermore , as a precursor of classic asthma , it has been demonstrated that nearly 30% of untreated cva patients eventually develop classic asthma . inhaled corticosteroids ( icss ) are considered to be the first - line therapeutic approach to cva , which is similar to classic asthma . previous studies have shown that an inhaled steroid can lead to improvements in markers of airway inflammation as well as in forced expiratory volume in 1 second ( fev1 ) and pc20 in mild asthmatics . furthermore , long - term ics may prevent the development of classic asthma from cva by reducing bronchial responsiveness . it is well known that asthma is defined as a complex and chronic inflammatory disorder that is characterized by activation of t helper type-2 ( th2 ) t cells , ige production , and eosinophilia . interleukin- ( il- ) 5 is the predominant cytokine associated with eosinophilic inflammation and is of key importance in the pathogenesis of asthma . more recent studies have demonstrated that atopic / asthmatic individuals also have an inherent defect in regulatory t cells , which suppress potentially harmful immune responses by releasing anti - inflammatory cytokines such as il-10 and tgf- . nevertheless , the changes associated with the improvements in ahr and airway inflammation including regulatory markers in cva responding to ics treatment , particularly as compared with those in mild classic asthma , have not yet been elucidated . moreover , the time course of these changes during treatment is not known . to our knowledge , this is the first study investigating the changes in cytokine levels and hyperresponsiveness during ics treatment in cva patients compared with classic asthma patients . in the present study , the levels of th2-associated cytokines , il-5 and cytokine synthesis inhibitory factor ( il-10 ) , were analyzed . eosinophils ( eos ) in sputum and airway hyperresponsiveness were also determined in cva patients during the period of ics treatment . changes in inflammatory markers and pd20-fev1 ( the accumulated provocative dose resulting in a 20% decrease in fev1 ) in response to histamine challenge were analyzed to evaluate the efficacy of ics treatment over time in cva patients compared with mild asthmatics . thirty - five patients with cva and 26 patients with mild classic asthma who were attending the respiratory medicine outpatient clinic of the huashan hospital were recruited into this study from november 2010 to may 2011 . mild asthma was diagnosed according to the global initiative for asthma ( gina ) guidelines and cva was diagnosed according to the recommendations of the japanese cough research society . smokers , pregnant women , and individuals diagnosed with upper respiratory tract infections during the preceding 2 weeks were excluded . a separate group of healthy volunteers ( 14 men and 10 women ) attending physical examination department of huashan hospital served as healthy controls , all of whom had no past history of asthma or other respiratory diseases and had no current respiratory symptoms . the study protocol was approved by the ethics committee of huashan hospital , and written informed consent was obtained from all patients and control subjects . prior to the study , all subjects were required to undergo pulmonary function tests , bronchial challenge to histamine , and examination of induced sputum . after participating in these tests , all patients were treated with a daily dose equivalent of 400 g budesonide . the total follow - up period lasted for twelve months . levels of il-5 and il-10 , the percentage of eos in induced sputum , and the value of pd20 were examined every 3 months after the medication . this study comprised a longitudinal comparison of the changes in airway inflammation and bronchial hyperresponsiveness in cva and classic asthma patients to estimate the effect of ics treatment . fev1 and forced vital capacity ( fvc ) were measured utilizing a computerized spirometer ( master screen body , jaeger , germany ) according to the standardization criteria of the american thoracic society . to be eligible for the study , forced expiratory volume in 1 s ( fev1 ) was required to be 80% or more of the predicted value . bronchial responsiveness was tested using an aerodynamic particle sizer ( aps ) aerosol provocative system ( master screen body , jaeger , germany ) . pd20-fev1 was measured by inhalation histamine challenge with different concentrations of histamine ( 0.037.8 mg / ml ) delivered by the quantitative aerosol dosimeter of the aps system . initially , baseline fev1 was measured using a spirometer 2 minutes after inhalation of a control solution ( 0.9% saline ) . subjects inhaled a histamine aerosol from a nebulizer with tidal breathing whilst wearing a nose clip for 2 min . total inhalations of each concentration were administered , and fev1 was measured three minutes after each period of inhalation . the test was stopped in the event of a fall in baseline fev1 of 20% compared with the control inhalation solution . pd20 was calculated as the provocative dose of histamine causing a 20% fall in fev1 . ahr was considered to be present in subjects with pd20 values less than 2.2 mg . subjects received two puffs ( 200 g ) of salbutamol from a metered dose inhaler following the histamine challenge test . sputum was induced with an aerosol of hypertonic saline solution according to a previously described protocol . the volume of mucus plugs was measured prior to incubation with four volumes of 0.1% dithiothreitol ( dtt ) . samples were then vortexed gently and incubated in a shaking water bath at 37c for 15 min to ensure complete homogenization . the resulting suspensions were filtered through nylon gauze ( 48 m ) , and total leukocyte cell counts and cell viability were determined . filtrates were centrifuged at 750 g for 10 min , and supernatants were aspirated and stored in eppendorf tubes at 80c for il-5 and il-10 assays . cell suspensions were adjusted to 1.0 10/ml and a sample ( 60 l ) was used for cytocentrifuge preparations . slides were stained with giemsa stain , and 400 differential nonsquamous cell counts were performed . concentrations of il-5 and il-10 in sputum supernatants were determined using a solid - phase sandwich enzyme - linked immunosorbent assay ( elisa ) ( peprotech , nj , usa ) . the sensitivities of the assays as quoted by the manufacturer were 15 pg / ml and 10 pg / ml , respectively . all data were expressed as the mean standard deviation ( sd ) or median ( m ) with the range . a general linear model was applied for comparison of the time course of sputum marker levels and pd20 before and after treatment in patients with cva and asthma . a time course of repeated measurements was used to compare groups . statistical significance identified between groups or time points by post hoc analysis in the current study , 28 patients with cva , 23 classic asthmatics , and 22 healthy subjects successfully completed the sputum induction procedure and follow - up period . higher serum total immunoglobulin e ( ige ) levels and blood eosinophil counts were detected in the two asthma groups compared with the control group . there were no significant differences between the cva and asthma groups with respect to age , sex , total ige , fev1% predicted , fev1/fvc , hemocytes , and atopy ( table 1 ) . initially , no significant differences were identified between patients with cva and classic asthma in terms of pd20-fev1 in bpt . pd20-fev1 significantly increased from 0.58 0.73 mol to 1.19 0.69 mol in cva patients after receiving ics for 3 months . ics treatment for 12 months significantly improved airway hyperresponsiveness in patients with cva and classic asthma although the overall level of pd20-fev1 in cva patients was higher than that in classic asthma patients after treatment ( figure 1 ) . compared with the control group , the cva and classic asthma groups were found to have higher baseline levels of il-5 and percentage of eos in addition to lower concentrations of il-10 in induced sputum(figures 2 and 3 ) . levels of inflammatory markers were improved to some extent in the two asthmatic groups following treatment with budesonide . however , the changes in inflammatory mediators in induced sputum differed between the two groups during the period of medication . at each time point , levels of inflammatory markers in the cva group also differed from those in the classic asthma group ( p < 0.05 ) . the overall level of il-5 in the cva group was lower than that in the classic asthma group . besides , the il-5 levels in the cva group decreased after treatment for 3 months while those in classic asthma group began to decrease after treatment for 6 months . higher levels of il-10 were detected in the cva group compared with those observed in the classic asthma group throughout the observation period . il-10 values significantly increased in the cva group after ics treatment for 6 months , whereas levels in the classic asthma group increased after treatment for 12 months . the percentage of eosinophils ( eos% ) in sputum was lower in the cva group compared with that in the classic asthma group after treatment . the eos% significantly decreased after ics treatment for 6 months in the cva group , while a change was observed in the classic asthma group only after treatment for 12 months ( figures 2 , 3 , and 4 ) . cough variant asthma is essentially an occult phase of classic asthma that shares similarity in fundamental pathologic and pathophysiologic characteristics . evidence suggests that an imbalance in th1 and th2 lymphocyte responses plays a critical role in the pathogenesis of chronic asthma . the potential of th2 cells to promote allergic immunopathology is amplified by production of a range of mediators , which together promote the salient features of asthma such as ige production , ahr , inflammation , and tissue remodeling . in addition to the traditionally accepted th2 immune response mechanism of asthma , recent advances in both immunology and clinical phenotyping of asthma have raised the possibility that other mechanisms may drive pathology in some patients or coexist with th2-type inflammation . there is now evidence for a variety of suppressive or regulatory t cell ( treg ) subsets that suppress potentially harmful immune responses through the release of various anti - inflammatory cytokines including il-10 and tgf- [ 7 , 14 , 15 ] . however , the effect of regulatory t cells has not been specifically detected in patients with cva . therefore , on the basis of detailed prior studies of the immunopathogenesis of asthma , we hypothesized that cva exhibits homogeneity with classic asthma . the current study was conducted to further assess inflammation in cva by analysis of induced sputum levels of anti - inflammatory th2-cytokines ( il-5 and il-10 ) in addition to measurement of the numbers of eosinophils as the immune effector cells in this condition . furthermore , changes in the levels of these inflammatory mediators during the period of ics medication were investigated . it is believed that ics forms the basis of maintenance therapy for patients with cva . the effect of ics on cva in terms of airway hyperresponsiveness and sputum inflammatory markers was investigated in contrast to those in classic asthma . in this study , the basic level of il-5 and percentage of eosinophils in cva patients and mild asthmatics were clearly higher than those in the control group although no statistically significant differences were detected between the two asthmatic groups . il-5 , one of the most important th2-associated cytokines , is critical for the survival , differentiation , proliferation , and maturation of eosinophils within the bone marrow . activated eosinophils possess secondary granules containing four primary cationic proteins : major basic protein ( mbp ) , eosinophil cationic protein ( ecp ) , eosinophil - derived neurotoxin ( edn ) , and eosinophil peroxidase ( epo ) , all of which participate in the processes of airway inflammation . increased eosinophil counts in sputum have been reported by niimi et al . . therefore , our results are consistent with previous studies indicating that eosinophilic inflammation is involved both in cva and in classic asthma . it is remarkable that not all asthma can be explained by the predominant th2 and eosinophil - dominant inflammation . accumulating evidence has suggested that th17 cells and th17 cytokines are involved in the regulation of asthmatic inflammation as well [ 20 , 21 ] . th17 is a third subset of cd4 t - helper lymphocytes characterized by the production of the il-17 family cytokines like il-17a and il-17f . animal models of asthma denote that th17 cells may promote th2 immunity and exacerbate airway hyperresponsiveness [ 22 , 23 ] . additionally , sputum neutrophils were the highest in moderate - severe asthmatics compared with mild asthmatics and il-17a necessarily led to neutrophilic inflammation in severe asthma . taken together , these findings showed that th17 plays a key role in asthma pathogenesis , particularly in the severe form of asthma . in the present study , we observed elevated sputum il-5 and eosinophils ( % ) in cva and mild asthmatics , perhaps merely supporting that eosinophilic inflammation is involved dominantly in the mild phase of the disease . we speculate that the role of th2 and th17 cells might correlate with the severity of asthma . moreover , there are also different inflammatory subtypes in asthma according to the assessment of induced sputum . apart from eosinophils independent impact , matsuoka et al . reported that eosinophil could act in synergy with neutrophils as well . they also found that inflammatory subtypes in cva were associated with the maintenance ics doses . eosinophilic subtypes required higher maintainenance dose of ics but represented a short - term preferable response of treatment with ics than noneosinophilic asthma . in the present study , we did not assess inflammatory cells like neutrophils , just aiming to principally elucidate the response of eosinophilic inflammation with respect to long - term ics treatment . a good yet dissimilar improvement was found in eosinophilic inflammation under 12-month ics treatment between cva and classic asthma , providing some insight into the efficacy of long - term ics on eosinophilic inflammation in cva . we also observed a kind of immune modulator , the level of induced sputum il-10 in the three studied groups . il-10 , an anti - inflammatory cytokine secreted predominantly by immune regulatory cells in the lung , inhibits the transcription of many proinflammatory cytokines and chemokines borish et al . reported that il-10 secretion by alveolar macrophages may be impaired in asthmatic patients . in our study , lower concentrations of il-10 were observed in cva patients and mild asthmatics compared with control subjects , suggesting the existence of deficient immune regulation in cough variant asthma as well as classic asthma . these data also indicate that ics treatment for 12 months in cva patients significantly altered the levels of induced sputum il-5 and il-10 and reduced the number of eosinophils . despite the similarity in the pattern of inflammatory sputum markers in patients with asthma and cva , the disparity of the two groups is yet detected in terms of improvement of the same item under glucocorticoid treatment . the present study clearly demonstrated greater amelioration of airway inflammation in cva compared with mild classic asthma , by the apparent differences in levels of the markers investigated over the period of ics medication . these data indicate that airway inflammation in cva is highly responsive to early ics treatment and that may also be true for cases of mild classic asthma . in this study , the improvement of airway hyperresponsiveness in patients with cva and classic asthma was also investigated . challenge tests are an important measurement for diagnosis and evaluation of the severity of cva . histamine was used as the provocative agent in this study . in accordance with previous reports , the pd20-fev1 of cva significantly increased after budesonide treatment for only 3 months , and , furthermore , therapy continued for 12 months resulted in improved airway hyperresponsiveness such that it was detected only to an extremely mild degree . in contrast , the improvement in pd20-fev1 observed in classic asthma was delayed and was only apparent after treatment for 9 months . it is postulated that these differences are due to thickening of the subbasement membrane in cva patients compared with healthy controls , although the degree of thickening was less than that detected in classic asthma . this study further demonstrated that early and sufficient treatment with ics effectively reduces ahr and suppresses airway inflammation in cva patients . moreover , this accounts for the close association between ahr and chronic airway inflammation with airway remodeling . treatment with ics for 3 months may significantly decrease ahr in cva , although , for most patients , long - term ics treatment remains indispensable for complete elimination of airway inflammation . there are certainly other antiasthma mediations such as cysteinyl leukotriene ( cys - lt ) receptor antagonists ( ltras ) . ltras alleviate clinical symptoms and improve pulmonary function as well as airway inflammation in classic asthma , but montelukast for 4 weeks was yet unable to change pulmonary function , ahr , and sputum mediator levels in cva other than sputum eosinophil count and cough sensitivity . but for the long - term outcome according to our study , we were satisfied to notice that ics for 12-month monotherapy predominantly attenuates ahr , sputum mediator levels as well as sputum eosinophil count both in cva and classic asthma . thereof our study supported the concept that ics is superior to montelukast with respect to sputum eosinophil and hyperresponsiveness . nevertheless , some limitations of our study should be noted . in the present study , we lack determining the changing trend of cough sensitivity in cva under long - term ics treatment . actually , there is evidence that cough threshold did not change whether taking or not taking ics . thus we may draw a preliminary conclusion that , compared with ltras , inhaled corticosteroids play a better role in ahr and airway inflammation rather than cough receptor sensitivity in cva . the intriguing comparison of budesonide with montelukast further confirmed that ics possibly better ameliorates the degree of sputum inflammatory mediators in cough variant asthma . to summarize , ics treatment provides greater alleviation of th2-associated allergic responses and repair of regulatory mediators in cva than in classic asthma . the current study has indicated that airway hyperresponsiveness can be rapidly improved as a result of short - term ics treatment , although complete control of airway inflammation requires longer - term ics treatment . further studies , including randomized placebo - controlled trials , are required to confirm the effective maintenance period and dose of ics treatment in cva .
childhood obesity is related to a wide range of psychosocial and physical problems resulting in larger health care costs [ 2 , 3 ] . since obesity , once established , is difficult to treat , prevention is the main priority and is therefore receiving increasingly more attention . traditionally , the prevention and treatment of overweight and obesity have focused on stimulating changes in the behaviour of individuals . evidence showed that behaviours such as increased consumption of high energy density beverages and foods , low consumption of vegetables and fruits , less physical activity , and more sedentary leisure time activities contribute to overweight and obesity [ 57 ] . however , overweight and obesity develop within a sociocultural and physical environment in which these behaviours are made possible and sometimes even stimulated [ 8 , 9 ] . so far obesity prevention efforts targeting individual behaviour ( e.g. , education , treatment , and improving skills ) have unfortunately had limited effects [ 11 , 12 ] . since overweight and obesity are caused by multiple interacting determinants , it is important to address both distal and proximal determinants of obesity [ 1618 ] using an integrated community - wide intervention approach ( icia ) . this approach aims to conduct multiple interventions , in a collaborative effort with multiple stakeholders , that work in conjunction with each other in multiple settings and are directed towards multiple target groups in a community [ 12 , 1823 ] . such approaches are believed to be more effective because they target multiple individual and environmental determinants of obesity . an exemplary icia is the french epode programme ( ensemble prvenons l'obsit de enfants or together let 's prevent childhood obesity ) . epode aims to prevent overweight and obesity in children aged 012 years and their families through a multiactivity , multisetting , and multistakeholder approach [ 24 , 25 ] . the focus is on promoting healthy behaviours related to overweight and obesity , for example , healthy eating and regular physical activity through multiple organised activities to the children , their families , and intermediaries [ 2426 ] . at the community - level , this programme manager not only is trained by the central coordination team but also is provided with tools and instruments facilitative to local implementation [ 24 , 25 ] . in netherlands , the nationally coordinated icia ( in dutch : jongeren op gezond gewicht , with the acronym jogg ) has been implemented in more than 100 municipalities since 2010 . pillars : ( 1 ) political commitment , ( 2 ) public - private partnerships , ( 3 ) use of social marketing principles , ( 4 ) scientific evaluation and dissemination , and ( 5 ) linking prevention and healthcare [ 25 , 27 , 28 ] . to improve an icia such as the jogg - approach , the definition of programme evaluation refers to the systematic and objective assessment , analysis , and reporting of information on an on - going programme . it starts with the design and follows the implementation of the programme in order to understand the process and impacts of interventions , to make it possible to adapt the programme given interim findings and to inform decision - making . when started simultaneously with the implementation of an icia , evaluation can increase stakeholder and community participation and provide information allowing programme managers to improve the programme . furthermore , it can increase accountability , as well as strategic and financial support , and enhance the sustainability of resources . moreover , programme evaluation may assist in the dissemination of knowledge of the programme and of elements that are proving successful to other areas and professionals [ 11 , 30 ] . although important for programme success , the evaluation of icias is challenging . icias are complex interventions , and as a result their evaluation is also complex [ 3134 ] . this complexity demands evaluators to go beyond the traditional notions of evaluation research and experimental design [ 3538 ] . it also demands that the evaluation be tailored to the characteristics of a specific icia , to the needs of evaluation users and stakeholders , and to the expected outcomes [ 3941 ] . professionals who are responsible for the programme evaluation of their icia often struggle to carry out such evaluation satisfactorily [ 4244 ] . within the jogg - approach , these professionals are the programme managers ( usually policymakers from local government ) or epidemiologists ( employed by the regional public health services ( rphs ) ) . in order to assist them with programme evaluation of the jogg - approach , evaluation support tools have been disseminated by the national coordination office of the jogg - approach ( jogg - office ) . the evaluation manual describes a six - step evaluation process for programme evaluation of icias , similar to the cdc evaluation framework and the outline for evaluating an initiative by the kansas university community toolbox . the design of the evaluation manual is based on the outcomes of a comprehensive appraisal of existing evaluation frameworks . it also provides a logic model for the jogg - approach ( the jogg - model ) , capacity building exercises , an evaluation planning matrix , checklists , and examples of evaluation practice . the evaluation training consists of four modules following the evaluation approach as described in the evaluation manual . the reasons for this study were the outcomes of the pretraining assessment forms and discussions with programme managers and epidemiologists involved in the local jogg - approaches in 2012 and 2013 during their evaluation training . moreover , programme managers and epidemiologists participating in the evaluation training reported having to continuously juggle their desire to customise evaluation to their local context , needs , and assets , which necessarily costs time , effort , and money on the one hand , with their need to standardise the evaluation using existing datasets , monitors , and standard evaluation instruments ( since hardly any or indeed no evaluation budget was available ) on the other hand . as a consequence , only six of the 60 local jogg - approaches had developed evaluation plans by the end of 2013 . we concluded that despite support given ( i.e. , evaluation manual and evaluation training ) , there were a lot of barriers to programme evaluation of the local jogg - approach . the aim of our study was therefore twofold : ( 1 ) to explore barriers to and facilitators of programme evaluation of icias and ( 2 ) to understand the experiences of programme managers and epidemiologists regarding the offered evaluation tools . a qualitative study was conducted using a framework approach to collect data on both aims of this study . for the exploration of barriers to and facilitators of the programme evaluation of icias , the behaviour change wheel from michie et al . was used to guide semistructured interviews and their analysis . for the second aim , experiences of the evaluations tools theory by rogers [ 50 , 51 ] to guide focus groups and to analyse our findings . a purposeful sampling technique was used to recruit participants for the semistructured interviews and the focus groups . for the semistructured interviews , participants were recruited from municipalities who had been involved in the jogg - approach for at least one year ( n = 20 ) ( figure 1 ) . this criterion was chosen to ensure that they had sufficient experience with the jogg - approach and in the hope that participants would provide experiential knowledge . due to the already complex intervention approach , we choose to include representatives from jogg - municipalities that had one programme manager and one involved epidemiologist . participants were invited by email and arrangements for conducting the interviews were made by phone . the final sample consisted of eight programme managers and seven epidemiologists from nine different municipalities . one consisted of programme managers and epidemiologists involved in jogg - approaches that had commenced at least one year ago ( fg1 ) . the other focus group consisted of experts in the field of community health promotion and evaluation ( fg2 ) . to recruit participants for fg1 , an invitation letter was sent to the programme managers and epidemiologists involved in jogg - approaches . additionally , the jogg - office sent out an announcement email to jogg - municipalities with a participation request . participants for fg2 were recruited from the network of the research group and were only included if they were not responsible for the evaluation of local jogg - approaches . if participants did not respond , we called them one week after the invitation was sent . if participants failed to respond after three reminder calls , we excluded them from the study . when participants could not attend the focus groups ( e.g. , due to time limitations ) , we suggested an interview ( n = 4 ) . the final sample consisted of ten professionals for fg1 and the additional interviews ( i.e. , three programme managers and five epidemiologists ) and seven experts for fg2 and the additional interviews . the first author ( mvk ) and two assistant researchers ( rh and eb ) together developed the interview guide for the semistructured interviews ( see supplementary data 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/2385698 ) . this interview guide was based on the behaviour change wheel and was extended with questions regarding a central component of the behaviour change wheel is the com - b system : capability , opportunity , motivation , and behaviour . to illustrate , evaluation ( behaviour ) is enabled if the target populations ( e.g. , programme managers ) have sufficient understanding on how to apply the evaluation manual ( capability ) ; have sufficient time and financial means to apply the evaluation manual ( opportunity ) ; and have a positive attitude towards using an evaluation manual ( motivation ) . the first author ( mvk ) and one assistant researcher ( es ) together developed the topic list for both focus groups and the additional interviews ( supplementary data 2 ) . rogers ' theory , the diffusion of innovation , was used as an initial organising framework to generate topics [ 50 , 51 , 53 ] . rogers ( 2002 ) assumes that innovations ( e.g. , the evaluation manual ) are more likely to be used if ( 1 ) they can easily be tried ( in this case , if the evaluation manual can be used without major changes or consequences in the organisation ) ; ( 2 ) the relative advantage of the innovation is high ( if the use of the jogg evaluation manual requires less time than current procedures or evaluation manuals ) ; ( 3 ) the innovation is compatible with the daily practice of users ( i.e. , those responsible for evaluation ) ; ( 4 ) the innovation is not complex ( if evaluation manual is perceived to be easy to use ) ; and ( 5 ) the outcomes of using the innovation can be observed ( if use of the evaluation manual leads to better goal setting or alignment of stakeholder needs ) [ 50 , 53 ] . since the experts had not been working with the evaluation manual , the focus of their group discussion was not based on their own experience but on their perception of use of the evaluation manual for programme managers and epidemiologist in general . the semistructured interviews were conducted by rh ( with programme managers ) and eb ( with epidemiologists ) between march and june 2014 at a convenient time identified by the interviewees . open - ended questions were used to give participants the opportunity to share their experiences on design and implementation of evaluation in their own words . prior to data collection , all respondents were asked to sign an informed consent form . interviews lasted on average 53 minutes ( with a range within 2768 minutes ) and were audiotaped . in order to check our interpretation of the semistructured interviews , the respondents received a summary of their interview and were asked whether they agreed with our interpretation or wanted to change or add anything ( member check ) . the focus groups were conducted at a central location and the additional interviews took place at a convenient time and location identified by the interviewees . two weeks before the focus groups and interviews , all participants received the evaluation manual and prior to the data collection all signed an informed consent form . focus groups were conducted by an experienced independent moderator ( mk ) and an assistant moderator ( es ) . the interviews lasted on average 67 minutes ( with a range within 53120 minutes ) and were audiotaped and transcribed verbatim by the interviewer ( es ) . the transcripts from the semistructured interviews , the focus groups , and the additional interviews were thematically analysed using open and axial coding and divided between the main themes from the respective theoretical frameworks used . after this separate data analysis , data was merged where it provided additional information or overlapped data within the other framework ( i.e. , focus group data regarding feasibility of use of the evaluation manual was merged with perceived opportunities to conduct programme evaluation within the michie theme opportunity ; focus group data regarding complexity of the evaluation manual was merged with perceived barriers and facilitators to evaluate within the michie theme fragmentation and open coding of the first transcript were done by the interviewers ( rh ; and eb ) and discussed with the first author ( mvk ) and the fifth author ( mw ) . identified categories were divided between the main themes from the behaviour change wheel . this process resulted in a code tree that was then used to code the other interviews ( supplementary data 3 ) . the themes were the basis for describing the barriers and facilitators that programme managers and epidemiologists experienced with programme evaluation and the use of the evaluation tools . in the open coding phase , two researchers ( es and mvk ) independently read the transcripts of the focus groups and interviews and coded fragments . this resulted in memos and an inductive code list with in vivo codes which were subsequently discussed . axial coding was then used to code fragments in sub- or main categories ( es ) ( supplementary data 4 ) . during this iterative process finally , both researchers conducted selective coding by summarizing the focus groups and the additional interviews . five programme managers and four epidemiologists were involved from the start of their jogg - approaches . of the interviewed programme managers , only one was experienced with icias and none had been involved in the evaluation of an icia ( table 1 ) . approximately 65% of the programme managers attended less than 2 training sessions in evaluation and none attended all four sessions . reasons given for this limited attendance were the limited hours a week available for the jogg - approach besides other responsibilities . programme managers had approximately 16 employable hours available per week for the jogg - approach , within these hours evaluation was not a priority and training sessions took too much of their available time during a week . more than half of the programme managers had other responsibilities , such as being a policymaker for the sport or health sector at the municipality or being a public health professional at the rphs . the epidemiologists were better represented at the evaluation training , more than 70% attended more than three ( out of four ) training sessions , although most had less hours available per week for the jogg - approach than the programme managers . they either felt responsible for the evaluation of the jogg - approach or were sent by their programme manager . the epidemiologists in focus group 1 were all employed by the rphs , the programme managers within the municipality . the experts in focus group 2 were three senior researchers in the field of public health from three universities , two entrepreneurs at strategic level in public health , a senior researcher from a research institute specialised in overweight , and a programme evaluation expert employed at a university . programme evaluation is the systematic collection of information about the activities , characteristics , and outcomes of programmes to make judgments about the programme , improve programme effectiveness , and/or inform decisions about future programme development . in general , it consists of a process and an effect evaluation for which an evaluation plan has to be written . since the jogg - approach needs to be adapted to local context and the needs of stakeholders , and available resources , the design and implementation of the jogg - approach differed between municipalities and so does the programme evaluation . most interviewees referred to four types of evaluation that they conducted for their jogg - approach : ( 1 ) a process evaluation that focused on organised activities , ( 2 ) a process evaluation that focused on the quality of the collaboration with implementing partners , ( 3 ) an intermediate evaluation of behaviour change in children , and an ( 4 ) effect evaluation of overweight in children . all but one of the programme managers and epidemiologists conducted either one or a combination of these evaluations . in one municipality , respondents from this municipality showed more capability , conducted multiple evaluation types , and had more resources than other municipalities that started the jogg - approach in the same year . ideally , programme managers should be engaged in evaluation to ensure a common understanding of the purpose and scope , to properly budget for the evaluation activities , and to clearly assign roles and responsibilities to programme stakeholders . although most programme managers were involved in the development of an evaluation plan and some were involved in data analysis , they were never involved in data collection . often programme managers did not consider it their task to coordinate evaluation ( let alone evaluate themselves ) and felt no urge to fulfil these roles . for example , one programme manager completely delegated evaluation to the rphs and to university students ; she did not know who were involved with the evaluation of her programme , what their tasks were , what they were planning to measure , and if they aligned their evaluation to the programme:she [ name of epidemiologist ] is responsible for the research [ ] . moreover , i believe that the programme manager should not have knowledge of all these things [ research and evaluation ] [ ri21]i need to align with the researcher to understand how we are going to use it [ the establishment of the impact assessment ] , because she is more involved in it than i am . [ ri27 ] she [ name of epidemiologist ] is responsible for the research [ ] . moreover , i believe that the programme manager should not have knowledge of all these things [ research and evaluation ] [ ri21 ] i need to align with the researcher to understand how we are going to use it [ the establishment of the impact assessment ] , because she is more involved in it than i am . [ ri27 ] this perception of responsibilities in evaluation and lack of role fulfilment may explain why programme managers were often not participating in the evaluation training and why one epidemiologist ( ri9 ) said that the evaluation work group consisted of one person ( herself ) . as a result there was no shared responsibility for evaluation and motivation to evaluate decreased:it remains a bit tricky when no one feels responsible for it [ monitoring and evaluation ] . and so , who is going to complete the activity monitor ? and who then makes sure that all the data will be collected and inserted ? [ ri29 ] it remains a bit tricky when no one feels responsible for it [ monitoring and evaluation ] . and so , who is going to complete the activity monitor ? and who then makes sure that all the data will be collected and inserted ? [ ri29 ] epidemiologists were mostly involved in an intermediate evaluation of behaviour change or an effect evaluation of overweight in children in which they conducted baseline measurements , follow - up data collection , and data analysis . most epidemiologists and programme managers were struggling when they attempted to conduct a programme evaluation within icias because they were only familiar with outcome evaluations : this [ the evaluation of the jogg - approach ] is a different kind of evaluation and research than what i 'm used to , i 'm not experienced in doing this and i feel i really miss this expertise . [ ri26 ] this [ the evaluation of the jogg - approach ] is a different kind of evaluation and research than what i 'm used to , i 'm not experienced in doing this and i feel i really miss this expertise . [ ri26 ] evaluation was further complicated because implementing an icia like the jogg - approach itself was new for most programme managers and epidemiologists ( table 1 ) . some interviewees therefore said they required different or more specific evaluation support ( e.g. , measurement instruments , indicator overview , budget calculations , information on how to conduct qualitative research , and how to set up a community intervention trial):i expected more of the jogg evaluation training [ ] like , this is what the evaluation looks like and this is the data you need to collect , and this is how we are going to do it [ ri29 ] i expected more of the jogg evaluation training [ ] like , this is what the evaluation looks like and this is the data you need to collect , and this is how we are going to do it [ ri29 ] a majority of epidemiologists understood that setting concrete evaluation goals for their icia was important . however , most believed this to be difficult , as they did not want to be accountable for such specific and unattainable goals:we also set milestones , like the percentage of kids that should exercise , or 10% of our primary schools this year will have a healthy policy . and [ ri21 ] [ ] for that we still haven't formulated a specific objective it remains truly difficult to be presented with a bill for goals that appear not to be feasible in the end . [ ri31 ] we also set milestones , like the percentage of kids that should exercise , or 10% of our primary schools this year will have a healthy policy . and [ ri21 ] [ ] for that we still haven't formulated a specific objective it remains truly difficult to be presented with a bill for goals that appear not to be feasible in the end . [ ri31 ] some programme managers clearly made use of the evaluation manual ; they involved external parties ( e.g. , primary schools and a health centre ) to set such goals and made agreements about who would actually implement , develop , and conduct the evaluation of these goals:we started with a meeting for all stakeholders [ for the evaluation planning ] to create support . in this meeting we set goals and objectives for processes , behaviour and outcomes people could highlight the ones they believed were most important . [ ri25 ] . we started with a meeting for all stakeholders [ for the evaluation planning ] to create support . in this meeting we set goals and objectives for processes , behaviour and outcomes people could highlight the ones they believed were most important . , several programme managers emphasised that clear communication about evaluation towards stakeholders ( e.g. , rphs , epidemiologists , employees of a sport - centre , aldermen , and school directors ) was important ( table 2 ) . they said that clearly explaining the relevance and goals of evaluation and aligning evaluation efforts were most important . however , some programme managers admitted they did not always invest in such communication resulting in less attention to all aspects of programme evaluation , for instance , to process evaluation:i have to say that the process evaluation received less and less attention . although we had made really clear arrangements and activities of what we were going to do . and although we had made really clear arrangements and activities of what we were going to do . and programme managers said the opportunity to evaluate improved if it was possible to discuss the evaluation with a group or person who felt responsible for evaluation . this apprehension caused them to consider collaboration with an epidemiologist , researcher , or evaluation work group . programme managers felt this collaboration to be crucial for the performance of programme evaluation of icias ( table 2 ) . additionally , availability of students , volunteers , activity coordinators , and public - private partners was considered important , since they often collected data for the evaluation . in this way , the programme managers could save time and financial resources:the university has said it would like to contribute to the evaluation of jogg with x number of students . [ ri23 ] the university has said it would like to contribute to the evaluation of jogg with x number of students . and of course this includes some support from the university . [ ri23 ] respondents mentioned that financial resources often imposed a barrier since municipalities had just faced drastic budget cuts and decentralization of tasks to municipalities . moreover , time was a barrier when programme managers needed to invest in new activities and explain the integrated approach to stakeholders ( i.e. , when the jogg - approach was not based on an existing icia ) , when limited professionals were available for the jogg - approach at local level , when no time was allocated for evaluation ( i.e. , some epidemiologists were employed for the jogg - approach for 2 hours a week ) , or when time was used inefficiently due to lack of professionals with experience in the evaluation of icias . one programme manager ( ri23 ) ( who was employed for 18 hours a week to the jogg - approach ) explained this was insufficient to manage such a comprehensive programme with high expectations : jogg itself states that a half fte for a programme manager is sufficient . i have my doubts . [ ri23 ] jogg itself states that a half fte for a programme manager is sufficient . [ ri23 ] most epidemiologists thus considered it important to show the amount of time that was needed for a proper evaluation:it is especially prior to conducting an evaluation that you should take the time and talk to each other about what information do we track ? ' , what do we want to register ? ' , who feels responsible ? ' and who is going to do it ? ' and this all takes quite some time . [ ri29 ] it is especially prior to conducting an evaluation that you should take the time and talk to each other about what information do we track ? ' , what do we want to register ? ' , who feels responsible ? ' and who is going to do it ? ' and this all takes quite some time . [ ri29 ] additionally , one epidemiologist said that even though 10 hours were available for evaluation , he felt these were used inefficiently due to lack of experience ; more time was needed to get acquainted with the programme evaluation process . especially in smaller municipalities , sufficient time to conduct all sorts of evaluation epidemiologists considered programme managers to be responsible for obtaining more financial resources to make evaluation feasible and added that when a municipality considers it important to evaluate , budget is made available . contrastingly , several programme managers stated it was extremely hard to influence what level of resources would be made available from the municipal budget and allocated to process evaluation . to make evaluation more feasible , programme managers from smaller municipalities suggested the organisation of programme evaluation at a regional level and the alignment of data collection . in this way evaluation expertise , resources , and tools ( e.g. , questionnaires ) could be shared : i know that [ a baseline in a sample group ] is far preferable to group data [ the whole population ] but we just do not have time for it . so , we will say that now n percent of the target group drinks water ' and next year so much percent will drink water ' . and that is enough for us and for the local government . [ ri33 ] i know that [ a baseline in a sample group ] is far preferable to group data [ the whole population ] but we just do not have time for it . now n percent of the target group drinks water ' and next year so much percent will drink water ' . and [ ri33 ] to stimulate the sharing of evaluation responsibility , it was considered important that external partners had a stake in evaluation . for example , teachers and directors of primary schools were often interested in diet and physical activity and overweight of their children , and therefore often agreed to participate in evaluation . some programme managers indicated finding such stakeholders was difficult when the jogg - approach was poorly understood:sometimes that penny [ to know what the jogg - approach is ] just has not dropped yet i 'm not the jogg - approach , but they are ! the stakeholders , the ultimate target group , the community , they are the jogg - approach ! [ ri22 ] sometimes that penny [ to know what the jogg - approach is ] just has not dropped yet i 'm not the jogg - approach , but they are ! the stakeholders , the ultimate target group , the community , they are the jogg - approach ! [ ri22 ] moreover , collecting data among 1218 year olds was considered difficult since these youngsters were overwhelmed by surveys , and schools were sometimes concerned about their privacy . similarly , private partners were not always willing to contribute financially to evaluation or to participate in conducting a programme evaluation . additionally , student or private partners sometimes gathered evaluation data of insufficient quality:quite often i get handed rattled off pieces , obviously motivation is lacking . [ ri21 ] quite often i get handed rattled off pieces , obviously motivation is lacking . [ ri21 ] the main motivations of programme managers and epidemiologists to conduct an evaluation were as follows : interest in childhood obesity prevention and icia ; seeing evaluation as a natural part of the work process ; increasing accountability for the jogg - approach towards the aldermen and council ; understanding of why the jogg - approach is effective ; motivation of stakeholders to participate in the jogg - approach ; securing of resources for the jogg - approach ; and the opportunity to present the results on a national level and compare their progress with other municipalities ( table 2 ) : it 's just really fun to work on a project which is doing good and has a national reputation . if the results are called at the national jogg - conference , than it is very nice to hear that our jogg - approach is doing so well . it is often very hard work and we slog at it but it still very stimulating . [ ri22 ] they [ strategic and tactical managers ] would occasionally like to have numbers , they want to show that the programme works and they want to know whether it works . [ ri19 ] it 's just really fun to work on a project which is doing good and has a national reputation . if the results are called at the national jogg - conference , than it is very nice to hear that our jogg - approach is doing so well . it is often very hard work and we slog at it but it still very stimulating . [ ri22 ] they [ strategic and tactical managers ] would occasionally like to have numbers , they want to show that the programme works and they want to know whether it works . [ ri19 ] programme managers and epidemiologists were demotivated by perceived lack of the necessary knowledge and skills to conduct an evaluation:the process evaluation , i find it extremely difficult so it actually is a kind of delay although i do see that 's very important . [ ri26 ] the process evaluation , i find it extremely difficult so it actually is a kind of delay although i do see that 's very important . [ ri26 ] another aspect that can cause a lack of motivation to evaluate is the strong interest of tactical management ( i.e. , aldermen ) and strategic management ( i.e. , department or sector manager ) in the implementation rather than the evaluation of the icias . especially in smaller municipalities , interviewees considered it not worth their while to shift scarce resources away from implementation to conduct process , intermediate , or effect evaluations . also programme managers explained that it did not make sense to start evaluation if activities had not yet been implemented , which epitomises their ( lack of ) understanding of the importance of programme evaluation to the optimisation of programme:i reason very much in the interest of my people and my people have no interest in such monitoring , they have an interest in policy measures , activities , incentives , whatever so that is my priority . [ ri28]i can now emphatically focus on evaluation , but if we do n't do anything , then i actually do not need to evaluate , so the focus is on performing and doing . [ ri23 ] i reason very much in the interest of my people and my people have no interest in such monitoring , they have an interest in policy measures , activities , incentives , whatever so that is my priority . [ ri28 ] i can now emphatically focus on evaluation , but if we do n't do anything , then i actually do not need to evaluate , so the focus is on performing and doing . [ ri23 ] although , in numerous cases , no budget was allocated for evaluation in any form , where municipalities were interested , they tended to prioritise effect evaluation . [ we noticed ] that they [ managers and aldermen ] would occasionally like to have numbers , they want to show that the programme works and they want to know whether it works . [ ri29 ] [ we noticed ] that they [ managers and aldermen ] would occasionally like to have numbers , they want to show that the programme works and they want to know whether it works . [ ri29 ] as a result , programme managers were less motivated to carry out other types of evaluation : so that component [ the effects ] i can measure , we report about this as well , which is why we have determined this our priority . [ ri21 ] so that component [ the effects ] i can measure , we report about this as well , which is why we have determined this our priority . [ ri21 ] most programme managers and epidemiologists knew the evaluation manual but not a single one of the respondents had used it as intended . despite this limited use , most respondents were positive about the existence of the evaluation manual ; it was considered a relevant and valuable innovation to support planning and performance of an icia programme evaluation . experts said the evaluation manual was relevant because they see programme managers and epidemiologists struggle with the evaluation of icias . several reasons for limited use of the evaluation manual were mentioned : almost all respondents said the evaluation manual was too comprehensive ; following the steps in detail would definitely exceed the proposed evaluation budget as mentioned in the evaluation manual ( 1015% of total programme budget ) or available time ; most programme managers and epidemiologists said their financial resources were insufficient to even implement the icia , and therefore they could not find the time needed for evaluation or could not use their time for evaluation purposes:i have 3 hours a week for two municipalities . [ rf1]if a municipality does not recognize the importance of it [ conducting the evaluation following the evaluation manual ] then it does not happen . [ ri32 ] i have 3 hours a week for two municipalities . [ rf1 ] if a municipality does not recognize the importance of it [ conducting the evaluation following the evaluation manual ] then it does not happen . [ ri32 ] however , experts also reasoned that evaluation budget could easily be obtained from funders ( i.e. , municipality , grants ) , if programme evaluation had been included in the initial planning of the icia and requesting funding for it . another reason offered for limited use of the evaluation manual was that some parts were considered to be more useful than others , but opinions on this differed . some respondents were positive about a graphic illustrating growth and achievement of programme goals in a time - line saying it helped them to manage the expectations of stakeholders . others said that the guiding notebooks presented in the evaluation manual helped them to process theory into practice . some programme managers and epidemiologists found the jogg logic model especially relevant , while others said it was more facilitative to develop their own logic model . furthermore , experts suggested the logic model could be used as a guideline for the planning of programme evaluation in addition to the six - step evaluation roadmap:i would recommend using evaluation planning following the jogg - model and maybe less strict through the six - stepped roadmap . and to determine evaluation type from goals on different levels , and provide examples to make this concrete . [ rf9 ] i would recommend using evaluation planning following the jogg - model and maybe less strict through the six - stepped roadmap . and to determine evaluation type from goals on different levels , and provide examples to make this concrete . [ rf9 ] all respondents added that the main reason for only using parts of the evaluation manual was that reading the evaluation manual was too time - consuming for professionals at the operational level . another reason mentioned for this limited use was that it was difficult to use the suggested evaluation approach in an icia that had already started and was in an implementation phase . since the evaluation manual started by defining stakeholders and collaboratively setting goals and objectives the respondents felt as if they were going back to the drawing board if they had to follow the evaluation manual . they wanted to progress and did not see the advantage of a new approach to a programme which had already been approved by funders and management . another reason for limited use was that the evaluation approach was difficult to align with the emphasis of the council or aldermen on effect evaluation:the town council wanted to see a decrease in bmi at any price [ ] the alderman 's ' head is on the block for it . [ rf1 ] the town council wanted to see a decrease in bmi at any price [ ] the alderman 's ' head is on the block for it . [ rf1 ] although some aldermen were open to discussion and liked to have expectations managed,he wants to know that stakeholders are involved in the right way , those stakeholders are important to him and to the programme . [ rf2 ] he wants to know that stakeholders are involved in the right way , those stakeholders are important to him and to the programme . [ rf2 ] all respondents said the evaluation manual could be very useful but that the evaluation approach proposed in the evaluation manual was too linear , theoretical , and comprehensive for daily practice . to improve the evaluation manual , experts suggested emphasising the involvement of stakeholders in programme design and evaluation , underscoring the importance of synchronising ambitions and expectations of several stakeholders in the community and expressing bottom - up strategies with examples in order to involve the community . our main findings indicate that those responsible for programme evaluation , programme managers and epidemiologists , often lack specific knowledge and skills to conduct a programme evaluation ( capability ) in its fullest , perceive limited time and financial resources ( opportunities ) to conduct a comprehensive programme evaluation , and are seldom internally or externally motivated ( lack of incentives ) to conduct a programme evaluation . we also found that professionals involved in icias do not often use the evaluation manual provided and that evaluation training was poorly attended , especially by programme managers . first of all , even though programme managers understood the importance of evaluation , they did not invest in its management . programme managers often felt forced to invest time either in implementation or in evaluation and often prioritised implementation . if they were investing time in evaluation , they focused on assessing outcomes rather than processes . programme managers did not see reflection and adaptation , as part of a process evaluation , as ways to continuously improve their icia . insufficient programme evaluation knowledge and skills seem to explain the limited involvement of programme managers [ 41 , 42 , 57 ] . besides insufficient skills , unfortunately , epidemiologists felt unable to fulfil this role since programme evaluation of icias was new to them . even though they were familiar with quantitative outcome and impact evaluations , they required new skills and support ( e.g. , guidance and management ) to conduct a process evaluation ( as part of a programme evaluation ) . this need for more technical evaluation support was also felt by others [ 30 , 42 , 5759 ] . secondly , time , manpower , and money were insufficiently allocated to evaluation and generating funding ( i.e. , budget ) for evaluation proved difficult . one important reason for poor generation and allocation of evaluation resources was related to insufficient knowledge ; professionals , and especially programme managers , often did not know what and how they had to evaluate and seemed unaware that evaluation requires management and guidance . additionally , some professionals did not see the benefits of programme evaluation ; they perceived more cons than pros ( e.g. , taking too much money and work ) . this is in line with torres and preskill ( 2001 ) who found that professionals were concerned that evaluation would lead to undesirable results ( e.g. , dissatisfaction of capability ) and therefore have a reduced motivation to engage in or manage the evaluation . on the contrary , we also found some professionals , especially epidemiologists , motivated to evaluate ; these were mostly epidemiologists who liked the idea that it was new to work within icia , who believed evaluation was important for the icia or had personal interest in ( reduction of ) childhood obesity prevention . unfortunately , these motivated professionals were often demotivated by strategic and tactical managers allocating scarce resources to the integrated community - wide intervention approach and showing more interest in implementation than evaluation . another reason for limited evaluation resources is the lack of public - private partnerships at the local level . when private parties finance activities , the municipal budget can be allocated for program management and evaluation . when private parties are not involved , the icia budget was mainly allocated to the implementation of activities . thirdly , the lack of time , role related expectations , and poor allocation of evaluation budget also contributed to the limited use of the evaluation manual and may explain why programme managers scarcely visit evaluation training . although the evaluation manual was seen as a useful tool for more concrete and practical programme evaluation , it was considered too comprehensive ( i.e. , taking too much time ) and linear for practical use . moreover , stakeholders perceived programme accountability as achieved through assessing effects at health outcome level , rather than a full programme evaluation . they need to learn how to plan , conduct , and advocate programme evaluation and involve stakeholders throughout the evaluation process . learning these new skills can be stimulated through providing technical support for the design and implementation of the evaluation and an evaluation manual with multiple stratified versions as well as a stronger emphasis on stakeholder involvement . an evaluation training should be made mandatory for programme managers and epidemiologists and provided at the municipality and supported by strategic and tactical managers of the municipality . secondly , we recommend making clear agreements as to the roles and responsibilities between stakeholders in the icia evaluation . programme managers should realize that evaluation requires their guidance and management and they should take charge of the planning and performance of the programme evaluation . epidemiologists should lead outcome evaluation , establish the evaluation methodology and measurement instruments , and involve stakeholders ( e.g. , defining needs and resources ) . policy makers could be involved in the evaluation of the municipal structure and organisation and specify the municipal evaluation budget . therefore , increased communication between all stakeholders is of crucial importance . thirdly , we recommend investing in convincing strategic and tactical managers that the results or benefits of evaluation outweigh the costs . programme managers and epidemiologist can , for example , emphasise that evaluation is useful for programme improvement and has positive consequences for programme continuation and that these outweigh the costs related to planning and performing programme evaluation [ 49 , 60 ] . fourthly , we recommend ensuring that resources are allocated to evaluation . this can be achieved by involving private stakeholders and making an explicit decision to generate and allocate evaluation resources before designing the evaluation . therefore , we recommend that programme managers communicate the evaluation plan and the necessity of programme evaluation to all stakeholders and ensure that roles are clearly divided between them . when multiple stakeholders are involved in planning and conducting the programme evaluation , it is more likely that resources and necessary preconditions for programme evaluation will be put in place . this might be achieved through standardising programme evaluation in policy documents and establishing evaluation workgroups . the jogg - office can stimulate this dialogue by requesting a specific evaluation budget attached to the municipalities ' icia programme description . they should also discuss the relevance of programme evaluation for the success of the jogg - approach in the first exploratory talks between a jogg account manager from the jogg - office and tactical manager who will decide whether or not the municipality will participate in the jogg - approach . the strengths of this study are the open character of the interviews , member checks , and heterogeneity of our data . although respondents knew they would be interviewed on the evaluation of jogg , and some received questions prior the interview , they seemed comfortable with providing sensitive data and were not inclined to give socially desirable answers . moreover , since respondents had different roles in icias and represented both relatively small and large municipalities and five different provinces , we obtained a panoramic view on facilitators and barriers . a limitation of the study is that theoretical saturation was not reached due to the small number of respondents and the context in which the programme manager and epidemiologist worked was not explicitly taken into account . additionally , not one of the respondents had used the evaluation manual as intended which limited their full understanding of its use when conducting a programme evaluation . programme evaluation is an important element of icias such as jogg but is often omitted in the current complex policy and political environment . additionally , evaluating icias is new for many professionals and not well managed . in this context usefulness of an evaluation manual might improve by prioritising evaluation , providing evaluation incentives , and encouraging municipalities to adjust organisational preconditions . additionally , bottom - up strategies such as involvement of the community and synchronising ambitions and expectations of stakeholders are necessary . assigning a coach to the local icia organisation may be a valuable way to implement these bottom - up strategies . this evaluation coach should support the programme manager and the epidemiologist in involving the community ( target group and stakeholders ) , to raise awareness of the importance of evaluation at operational , tactical , and especially the strategic level ( for obtaining resources ) and give support by applying the evaluation manual in the local context given available resources .
only 10% to 20% of tumors are resectable at the time of diagnosis , and even in these patients , survival beyond 5 years can be anticipated in no more than 10% . these data underscore the importance of an optimal palliation that will guarantee the shortest possible hospital stay and as long a survival as possible with a good quality of life . obstructive jaundice is the most common symptom requiring palliation , occurring in 70% of patients . duodenal obstruction , occurring in 10% to 25% of patients , is rarely a presenting symptom ( < 5% ) and usually follows biliary obstruction . careful evaluation of the patient and multidisciplinary expertise are required to select the appropriate approach . several risk factors are usually present : more than 50% of patients are over the age of 70 years , and they often have associated medical illnesses . jaundice and poor nutritional status can further compound the problems associated with the operative treatment . although the mortality for palliative operations has recently improved , morbidity rates have remained high . decision making in the palliation of jaundice and duodenal obstruction due to pancreatic cancer has become more complex in recent years . treatment options have broadened with the advent of endoscopic and percutaneous stenting of the bile ducts and laparoscopic biliary and enteric bypass . the availability of these minimally invasive techniques has also increased the demand for accurate staging . resectability of pancreatic lesions is determined accurately by preoperative imaging in only 47% to 55% of cases . diagnostic laparoscopy and laparoscopic ultrasonography are indicated in patients whose tumor is deemed resectable on preoperative imaging . using laparoscopy and laparoscopic sonography , the resectability rate can be determined correctly in 75% to 100% of patients . in other words , false - positive results , ie , resectable lesions judged unresectable by staging laparoscopy , have not occurred in any of the prospectively reported controlled studies . for these reasons , a laparoscopic approach to the palliation of advanced pancreatic cancer based on the findings of staging laparoscopy and laparoscopic ultra - sonography may have the potential to replace open bypass procedures . in this article , we describe the surgical technique for laparoscopic hepatico- and gastrojejunostomy and discuss the indications for these procedures in patients with advanced pancreatic cancer . although laparoscopic cholecystojejunostomy has been reported by many authors , few have described laparoscopic hepatico - jejunostomy for the palliation of advanced pancreatic cancer . palliation of duodenal obstruction by laparoscopic gastrojejunostomy has also been widely described . when dealing with tumors deemed resectable on preoperative imaging , a complete laparoscopic evaluation of resectability , which includes laparoscopic ultrasonography , is performed . the presence or absence of tumor invasion into the major vessels , especially the portal vein , must be ruled out . using the stomach as an acoustic window , the pancreas the gastrocolic ligament is then opened with the harmonic scalpel ( ethicon endo - surgery , cincinnati , oh ) and access to the anterior surface of the pancreas is achieved ( figure 2 ) . the pancreatic lesion is usually identified , either by laparoscopy ( figure 2 ) or by laparoscopic ultrasonography ( figure 3 ) . using the stomach as an acoustic window the gastrocolic ligament is opened and the probe is placed directly on the anterior surface of the pancreas . the tumor is identified . once the lesion is judged unresectable , and in the presence of an indication for palliation , laparoscopic side - to - side hepatico- and gastrojejunostomy , using a simple jejunal loop , are performed . an antecolic gastrojejunostomy is preferred because it lessens the possibility of subsequent infiltration by a tumor . a small opening is made both into the posterior wall of the stomach and the jejunal loop with the harmonic scalpel ( figure 4 ) . the jaws of an endo - gia stapler ( 3.5 mm/30 mm , us surgical , norwalk , ct ) are inserted into the enterotomies and a wide gastrojejunostomy is created by 3 firings of the stapler ( figure 5 ) . the staple line is carefully inspected for bleeding and the enterotomies are closed with a running suture ( figures 6 and 7 ) . a small opening is made into the posterior wall of the stomach and the jejunal loop . . the hepatoduodenal ligament is further dissected , and the common bile duct , cystic duct , and cystic artery are isolated . the gallbladder is removed to facilitate construction of the common hepatic duct anastomosis and to prevent the possible later complication of cystic duct obstruction and cholecystitis . an incision of approximately 1 cm is performed on the common hepatic duct ( figure 8) . if present , a hepatic duct drain is removed under direct vision ( figure 9 ) . using the same loop of jejunum that was selected for the gastrojejunostomy , a hepatico - jejunostomy is performed distal to the gastrojejunostomy . two stay sutures are applied to fix the loop of jejunum to the common hepatic duct . the jaws of an endo - gia stapler ( 3.5 mm/30 mm , us surgical , norwalk , ct ) are inserted into the common hepatic duct and the jejunal loop ( figure 10 ) . using a single firing of the stapler , insertion of the jaws of an endo - gia stapler into the hepatic duct and jejunum . firing the stapler , a side - to - side hepatico - jejunostomy is created . optimal palliation should alleviate symptoms effectively , thereby improving the quality of life while reducing the duration of hospital stay . palliation of patients with unresectable pancreatic cancer is aimed at 3 major symptoms : obstructive jaundice , duodenal / gastric outlet obstruction , and tumor - associated pain . in a review of 8,000 patients in the english literature from 1965 to 1980 , the relief of biliary obstruction with surgical biliary bypass was associated with longer and more comfortable survival ( 5.4 months ) when compared with diagnostic laparotomy alone ( 3.5 months ) . for these reasons , an attempt to palliate biliary obstruction is indicated in all but the most terminal patients . open palliative surgery has been associated with high mortality and morbidity rates ( 8% to 33% and 20% to 60% , respectively ) . the introduction of endoscopic and percutaneous stenting of the bile ducts has changed the management of these patients . morbidity , mortality , and primary hospital stay were found to be lower after stenting . in particular , a randomized comparative trial ( surgical vs. endoscopic palliation of jaundice ) 204 patients were randomized to either surgery ( n = 103 ) or endoscopic stent placement ( n = 101 ) . technical success was achieved in 94% and 95% of surgical and stent patients , respectively . stented patients experienced lower procedure - related mortality ( 3% vs. 14% ; p = 0.01 ) , major complication rate ( 11% vs. 29% ; p = 0.02 ) , and median total hospital stay ( 20 vs. 26 days ; p = 0.001 ) . however , recurrent jaundice occurred in 36 stented patients and only 2 surgical patients . median survival was similar in the 2 groups ( surgical = 26 weeks , stent = 21 weeks ; p = 0.065 ) . the authors concluded that endoscopic stenting is easier and safer , but surgical bypass provides more durable relief of jaundice . the endoscopic approach , also when compared with percutaneous stenting , demonstrated significantly higher success rates and lower mortality . as for unilateral versus bilateral endoscopic hepatic duct drainage in patients with malignant biliary obstruction , the results of a prospective , randomized study demonstrated that the insertion of more than 1 endoprosthesis is not routinely justified . single stent insertion proved to be effective , while avoiding the risk of further procedure - related complications and mortality . these data suggest that fit jaundiced patients with unresectable pancreatic cancer benefit from surgery , whereas poor operative risk patients should be treated with endoscopic or percutaneous stenting . current opinion is divided regarding the preferred surgical procedure for biliary - enteric bypass . in an analysis of 600 patients who underwent open biliary bypass in 8 reported series , 93 of 400 patients ( 23% ) developed recurrent jaundice after cholecystojejunostomy compared with 14 of 200 patients ( 7% ) after hepatico - jejunostomy . in a prospective controlled trial assessing the efficacy of biliary enteric bypass , seven bypasses failed after use of the gallbladder and only 2 after using the bile duct ( p < 0.04 ) . the authors concluded that hepatico - jejunostomy is a significantly more effective palliative procedure . a meta - analysis by watanapa and williamson found that cholecystojejunostomy had an 89% success rate , whereas the success rate for hepaticojejunostomy was 97% . more importantly , recurrent jaundice or cholangitis occurred in 20% of patients undergoing cholecystojejunostomy . on the basis of these data , most authors have preferred the routine use of the common bile duct for palliative biliary drainage . the issue of duodenal obstruction , its management , and treatment in patients with advanced pancreatic cancer has been addressed by 3 meta - analyses . thirteen percent of those who did not undergo a bypass procedure at their initial operation required gastrojejunostomy prior to death , and an additional 20% died with symptoms of duodenal obstruction . another meta - analysis included 1600 patients and reported a 17% incidence of duodenal obstruction at a mean of 8.6 months after the initial operation , requiring gastrojejunostomy before death . conversely , when gastrojejunostomy was performed as a second operation , the mortality rates approached 25% . despite these data , they received no biliary and gastric bypass procedures and were prospectively followed to determine the frequency of subsequent gastric or biliary bypass , or both , required prior to death . the study revealed that 98% ( 152 of 155 ) of these patients did not require a subsequent procedure to treat biliary or gastric obstruction . however , 35% of patients in this study had tumors in the body and tail of the pancreas , which represents a group with differing needs for palliation . in a recent , prospective , randomized trial , the need of prophylactic gastrojejunostomy in advanced pancreatic cancer was evaluated . eighty - seven patients with no evidence of duodenal obstruction were randomized to receive either a gastrojejunostomy ( 44 patients ) or no gastrojejunostomy ( 43 patients ) . no patients experienced late gastric outlet obstruction in the gastrojejunostomy group , whereas 19% of patients in the no - gastrojejunostomy group required bypass for gastric outlet obstruction ( p < 0.01 ) . this study provided class i evidence confirming the need for routine gastrojejunostomy in surgically explored patients with unresectable pancreatic cancer . the use of minimally invasive operative techniques has been applied to the management of pancreatic cancer . the role of diagnostic laparoscopy and laparoscopic ultrasonography for the intraoperative staging of pancreatic carcinoma has been well established and is the routine at advanced centers . to date , many reports have described the use of laparoscopic techniques for palliation of patients with advanced pancreatic cancer . initial results demonstrate technical success , with acceptable morbidity and mortality and encouraging outcomes . in a case - controlled study , laparoscopic gastroenterostomy and end - to - side hepaticojejunostomy ( roux - en - y ) were performed in 14 unresectable patients . the results were compared with those of 14 matched patients who had conventional palliative procedures . postoperative morbidity was 7% and 43% for laparoscopic and open palliation , respectively ( p < 0.05 ) . no deaths occurred in the laparoscopic group , compared to 29% in the open group ( p < 0.05 ) . mean postoperative hospital stay was 9 days in the laparoscopic group and 21 days in the open group ( p < 0.06 ) . morphine derivatives were necessary for a significantly shorter period following laparoscopic palliation ( p < 0.03 ) . these results strongly support the use of a laparoscopic approach to palliation of advanced pancreatic cancer . reduced pain and early reinstitution of normal bowel motility after laparoscopy may contribute to the lower incidence of cardiopulmonary complications leading to postoperative deaths in these high - risk patients . another study compared the short - term outcomes of open gastrojejunostomy ( 22 patients ) to laparoscopic gastrojejunostomy ( 9 diagnosis - matched controls ) for the palliation of gastric outlet obstruction caused by advanced pancreatic cancer . mortality , overall morbidity , operating time , time to oral solid food intake , nonsteroidal antiinflammatory drug consumption , opioid consumption , and survival were not significantly different between the 2 groups . estimated blood loss and hospital stay were significantly reduced in the laparoscopic group ( p < 0.01 and p < 0.05 , respectively ) . delayed gastric emptying had played a major role in prolonged hospital stay after open palliation , being found in 12% to 67% of cases . further clinical trials will be needed to demonstrate an evidence - based advantage of the laparoscopic approach to palliation of patients with advanced pancreatic cancer . to date , in patients deemed resectable on preoperative imaging , a diagnostic laparoscopic approach seems reasonable . the technique described in this article for laparoscopic double bypass is technically challenging but has the potential to allow for a minimally invasive and durable biliary and gastric drainage . currently , the laparoscopic approach for palliation of advanced pancreatic cancer is commonly used in advanced minimally invasive centers . fit patients can be spared an unnecessary laparotomy while receiving long - lasting biliary or gastric drainage , or both , through a surgical stoma . whether this minimally invasive approach will be extended to a larger number of centers and patients is yet to be determined .
the question of why one person develops high blood pressure ( bp ) and another does not has long been controversial . for example , freud in the 1930s hypothesized that keeping one 's anger in will cause bp to rise . theories of stress and disease came a bit later , and a multitude of experimental and field studies show that stress is indeed linked , both epidemiologically and causally , to physiological dysregulation and chronic illness . the model we present here builds on this research but attempts to take it a step further . it is welldocumented that stress ( broadly and variously defined ) is a risk factor for hypertension ( htn ) and other cardiovascular disease ( cvd ) , including events and all - cause mortality [ 18 ] . the mechanisms that underlie this association , however , have not been as clearly specified . this is due , in part , to the ambiguity concerning the nature of stress . the construct has been used and measured in many different ways , with little standardization across studies . moreover , the notions of chronic and acute stress , and how they relate to one another , have not been clearly delineated , with little theoretical work targeted at identifying the respective domains of each . to the extent that we believe that stress however it is defined is indeed a risk factor for htn , it is crucial that we identify the pathways through which it may lead to sustained elevated bp if we are to develop effective interventions to reduce stress , with the aim of reducing high bp and associated long term disease risk . in this paper , we will attempt to provide specifications concerning the respective domains of acute and chronic stress and to present a model and review evidence that points to rumination the cognitions and negative affect that stem from the experience of an acute stressor as a key mechanism through which stress exerts its pejorative effects on autonomic functioning and the resting bp level . that is , a discrete event that stems from some aspect of one 's immediate situation , and , for the most part , has an impact on the person only to the extent that ( 1 ) it is perceived , ( 2 ) the perception leads to negative cognitions and affect , and ( 3 ) that these cause perturbations in the various physiological stress - mediating systems ( psmss ) including autonomic , hypothalamic - pituitary - adrenal ( hpa ) , and immune systems . by this definition , an acute stressor may also arise internally ( i.e. , a mental representation ) and provoke continuing thoughts and emotions , and , concomitantly , their effects on the autonomic and other psmss . we have noted for the most part as some acute stressors will have effects independent of this mechanism . for example , a pin - prick may cause such perturbations , but via different , more direct , channels not involving cognition . in this paper , we focus on those acute stressors that exert their effects on the psmss via the ruminative ( perception - negative cognition / affect ) pathway . in research examining stress effects on bp , acute stressors have mostly been defined as standardized laboratory tasks such as mental arithmetic or the cold pressor . we and others have used an anger recall task , which we have shown is similarly effective in raising the bp and hr , decreasing heart rate variability ( hrv ) , increasing cortisol , and other outcomes including impaired endothelial function [ 913 ] . we do not currently have a fully useful definition and circumscribed domain for chronic stress ; in the literature , it often appears to be a case of we know it when we see it . thus , caregiver status , low socioeconomic status ( ses ) , an unpleasant low - paying job , and a stint in jail all these seem like obvious sources of chronic stress . but what are the defining characteristics ? certainly , there is a sense that part of it is the ongoingness of the situation and part is the pervasiveness of the situation . thus , one may find oneself continuously in the midst of the situation being in prison is an extreme example , finding oneself further and further in debt with creditors calling at all hours is another . it is not necessarily the case , however , that even under such pervasive and ongoing conditions , one is continuously attending to the situation . thus , the individual instances or moments that do invoke the situation and do provoke a reaction the creditor 's phone call , for example , or a worry episode regarding mounting dept are intermittent , with a greater or lesser frequency depending on the nature of the chronic stressor . and we would regard the individual instances as examples of acute stressors , per the previous section , that arise from ( are potentiated by ) the chronically stressful context . for the purposes of this paper , as with acute stress , we define chronic stress as a situational variable . in this instance , however , situational refers to a background that increases the likelihood that acute stressors relating to the chronic stress situation arise . ( acute stressors may also , of course , arise from sources not related to one 's chronic stress ) . thus , we propose that chronic stress matters largely to the extent that it consists of a background situation that gives rise to acute stressors , with these acute stressors having an impact on the psmss . it is important to note that this chronically stressful situation may affect one 's bp via other pathways ; thus , chronic stress has effects beyond merely being the source of acute stressors . for example , the parental caretaker may begin eating poorly and gain weight , and may increase salt intake , each of which will affect the bp via other pathways ( i.e. , health - related behaviors , in this instance , although these may arise in response to both the chronic stress background and the acute stressors that may arise ) . so far , we have stated that acute stressors arise from environmental conditions , and often more frequently when the environmental conditions are characterized as chronically stressful . an individual exposed to such a situation will likely be exposed to reminders of the situation such as the creditor 's phone call and may be apt to periodically recall the terrible conditions even without environmental cues . we suggest that there is functionally no differentiation between these reminders and the effects of the acute stressors , both produce acute stress , insofar as each leads to negative cognitions and affect and perturbations in the psmss . posttraumatic stress disorder ( ptsd ) offers an example of this , albeit an extreme one . in some cases , a single event , even a brief one , may lead to persistent effects on cognition and affect . these effects can persist for decades as individuals with ptsd continue to deal with intrusive thoughts and emotions that recreate representations of the event ( and associated stress and other physiological responses ) . in fact , evidence suggests that the frequency of these reexposures to the original event is strongly related to ptsd severity . a limitation of most acute stress models is that they do not take into account the duration of exposure . thus , a person that exhibits a larger bp response to an acute stressor is presumed to be at greater risk for htn than one who exhibits smaller responses . stressor - related thoughts that may emerge over time as a result of ( but temporally distal from ) the acute stressor can also cause acute elevations in the psmss . moreover , these physiological responses may persist as long as the recurring cognitions / emotions ( e.g. , anger or anxiety ) persist . relevant to this , glynn and colleagues have shown that recall of a laboratory stressor to which the subject had been exposed in a previous session produced bp responses comparable to those which occurred when the participant was exposed to the stressor itself . thus , the cognitive and affective sequelae of the acute stressor may have as great an effect on the psmss as the original initiating event , long after that event has occurred ( with the limits on the ruminative responses likely only constrained by the verisimilitude and intensity of the internal representation ) . we thus broadly define rumination as affect - laden cognitions that ( 1 ) result from exposure to an acute stressor ( external in the environment or an internally generated representation ) ; ( 2 ) in most individuals cause acute activation of relevant physiological stress - mediation systems ; ( 3 ) outlast the original stimulus / acute stressor ( i.e. , the ruminative thoughts and accompanying negative affect and their effect on physiological activation persist even after the stressor itself is no longer present ) ; ( 4 ) often reemerge in some , over quite long periods of time ; ( 5 ) typically do not lead to productive solutions , but rather resemble an endless loop , that does not lead to insight or resolution of the issue . we also note that , in addition to the term rumination , there are many other related constructs . some are often used interchangeably , such as perseverative cognitions , whereas others imply some differences . for example , distinct from rumination , which typically concerns past stressful events , one may experience anticipatory negative cognitions and affect concerning future events , which are often termed worry or anxiety ; these states , too , can produce sustained effects on the psmss . it is as yet unclear whether these are distinctions that make a difference in terms of physiological consequences and/or htn risk . we thus propose that it is largely the cumulative duration of exposure to this persistent representation of the acute stressor , with its attendant effects on , and eventual potential dysregulation of the stress - mediating physiological systems that contribute to sustained elevated bp and to htn . that is , actual stressors are important , but that the ruminative representations ( and their consequences ) contribute more to observed psmss responses and to htn risk . figure 1 shows a schematic of a model that illustrates these predictions and pathways . . chronic stress ( a situational factor ) sets the stage for and gives rise to the occurrence of acute stressor incidents that , depending on their nature , may lead directly to activation of the psmss ( e.g. , a pinprick ) or may lead to ruminative thoughts and emotions that in turn may produce psmss responses . thus , many variables that have been implicated in stress effects are accounted for before they are perceived as stressful ; and rumination is affected by the composite as a whole . for example , the perceived availability of social support operates before appraisal and thus , in some individuals , reduces the likelihood that the stimulus will activate ruminative thoughts and affect . the thoughts and emotions that are evoked by the acute stressor affects the psmss , causing acute perturbations in the various systems . in those who tend to ruminate ( more frequently and/or for longer duration ) , the effects on the psmss will be prolonged , and thus , the individual is exposed to the effects of the stressor for a more sustained period of time , which may lead to a resetting of homeostatic set points , including elevated resting bp . herein , lies the critical role of duration , which many models of acute stress tend to ignore . the dashed arrow shows a feedback mechanism such that the autonomic activation that results as a function of rumination itself becomes a stimulus that potentiates the negative emotions and cognitions ; these in turn maintain the autonomic activation , and so on . distraction as a potential moderator of this loop leading to termination of the rumination and its effects on the psmss when a distractor of sufficient potency to drive out the ruminative thoughts and replace them with others is encountered . we also show a moderating effect of neural inhibition by the prefrontal cortex on the association between acute stress and rumination / worry . finally , a number of other pathways known to exist , but not addressed as central to this paper , are depicted in greyscale ( as rumination is not the only pathway by which stress may affect htn ) . specifically , addressing these additional pathways is , however , beyond the scope of this paper . one issue is whether the proposed model represents a mere restatement of existing theoretical formulations or offers practical and/or theoretical advance . we are aware that many of the definitions and relationships among constructs herein have been proposed and discussed by others , many of whom developed theories that had a major impact on how stress was conceptualized . thus , the models proposed by selye and by lazarus and folkman are progenitors of the current proposed model . there are , as well , many other theories that address aspects of the questions at issue , and we have borrowed from some of these . the model as we present it , however , has been informed by recent advances in the psychophysiological , health , and social psychological literatures , and addresses issues not formerly considered . moreover , this model is not intended to propose a general theory of stress ; rather , the goal is to understand the factors that lead chronic stress to be a substantial risk factor for htn , and the model we present focuses on this problem and the central role of rumination . although we have been discussing rumination as a largely active process , data from several lines of research suggest that individuals may be unaware of some portion potentially a large portion of their stress - related cognitions . yet , even these unconscious thoughts and feelings can have an activating effect on the autonomic and other stress - mediating physiological systems . ( we operationalize the term unconscious as indicating only that it occurs while one 's attention is not or can not be directed toward it , or is directed elsewhere , that is , out of awareness . ) there is a rich body of research converging on the idea that implicit emotion plays a role in mental and psychosomatic disorders . in the last two decades , developments in cognitive and social psychology have strongly indicated that a great deal of cognition occurs at the unconscious ( or automatic ) level [ 19 , 20 ] . to date , several studies have shown that neurophysiological responses occur in response to threatening information that is shown subliminally , an accepted experimental model of unconscious cognition [ 18 , 21 ] . more importantly for this paper , it has also been shown that such subliminal stimuli can increase bp [ 2224 ] . regarding chronic stress , we have found that ( conscious ) rumination was linked to cardiac activity in daily life , and this activity persisted for hours after the rumination itself had ended . we and others [ 2629 ] also found effects of daily stress and rumination on cardiovascular activity during subsequent sleeping at night , during which conscious rumination is presumably not possible . on the basis of these findings , some researchers [ 21 , 30 ] have recently proposed that stress research may have missed an essential phenomenon by focusing solely on the conscious tip of the iceberg of stress - related thoughts and feelings . unconscious rumination may be responsible for a considerable portion of cardiovascular activity in one 's daily life . pending development of appropriate measures which remains a challenge studying unconscious stress in the context of htn and other stress - related somatic conditions may turn out to be a particularly fruitful pathway for future investigation . the perseverative cognitions that characterize rumination are thought to be under tonic inhibitory control by the prefrontal cortex [ 31 , 32 ] . there are several conditions , including chronic stress or having an anxiety disorder , that lower prefrontal inhibition and thus render one more vulnerable to rumination as well as to prolonged and indiscriminate responses to environmental stimuli . low prefrontal inhibition is characterized by low parasympathetic activation , which can be measured by low heart rate variability ( hrv ) . as such , an excitatory positive feedback loop is allowed to emerge , reflected in the psychological level in hypervigilance and rumination . as a consequence , the normally fine - tuned ability to adjust to changing environmental factors becomes a relatively rigid , inflexible response disposition , which is in fact a continuation of the default defense response in the absence of clear threat signals . this is reflected by a failure to recognize safe / neutral environmental signals and by responding to them as if they are threatening . in support of this idea , patients suffering from generalized anxiety disorder have been shown to have lower tonic levels of hrv , when compared to nonanxious controls . furthermore , people with low hrv have been shown to have an attentional bias for threatening information and interpret ambiguous situations more negatively . in sum , we propose that low prefrontal inhibition , indexed by low hrv , predisposes people to respond with enhanced cognitive , affective , and physiological activity to stressors . this , in combination with the psychological vulnerability factors for rumination discussed above , can cause even neutral stimuli to trigger the stress response . as a consequence , the total time that people ruminate and worry about stressful events increases , thereby adding to the total duration of exposure to stress representations and their physiological effects in daily life . the overarching prediction derived from the model is that an important mechanism underlying the observed relationship between chronic stress and htn is rumination , which typically stems from exposure to an acute stressor . thus , compared to individuals who show a lower tendency to regulate their angry and anxious and dysphoric thoughts using ruminative processes , high trait ruminators should be more likely to have more frequent , intense , and longer - lasting , ruminative thoughts and emotions as well as more ( future ) reoccurrences and occurrences that persist longer into the future . in support of this prediction , rumination has been shown to increase engagement in depressed thinking [ 37 , 38 ] , is related to negative emotions , including anxiety [ 39 , 40 ] , anger [ 4143 ] , and depressed mood [ 4345 ] , and can prolong negative affect [ 42 , 46 ] . rumination appears to be a relatively stable characteristic , as test - retest is high for both anger and depressed rumination scales [ 47 , 48 ] ( for periods ranging from one month to one year ) . for example , ruminators were more likely to experience a depressive episode in the ensuing 18 months than nonruminators , with a similar result found by nolen - hoeksema . furthermore , higher tendencies toward angry rumination were related to increases in experienced and expressed anger and to decreased satisfaction with life on future occasions . increases in rumination have consistently been linked to higher blood pressure during recovery periods [ 4954 ] . for example , compared with nonruminators , ruminators after an anger recall incident were more likely to brood about their anger thoughts during a recovery period in which they were not distracted . importantly , this tendency to ruminate translated - to - slower blood pressure recovery for these ruminators , demonstrating the ability for rumination to sustain high blood pressure . other studies support this view as well ; participants induced to recall a stressor they experienced in the lab ( i.e. , ruminate about the stressor ) showed increases in blood pressure regardless of whether they ruminated about the stressor 30 minutes after it happened or one week later . in other words , the mental representation of the stressor increased blood pressure regardless of temporal distance from the original event . importantly , the relationship between rumination and blood pressure is not just in the moment . compared to nonruminators , individuals who ruminate have higher resting [ 55 , 56 ] and ambulatory blood pressures [ 5658 ] . for example , ottaviani and colleagues had participants engaged in an anger recall interview , which successfully raised blood pressure . next , half of the participants were assigned to a distraction condition where rumination was abolished , while the other half was not distracted and were allowed to continue ruminating about the anger inducing incident . pertinently , not only did this nondistracted , ruminating group have elevated blood pressure during the recovery period immediately after the anger recall , but their blood pressure remained significantly elevated compared to their baseline levels when examining their mean ambulatory blood pressure readings taken over the ensuing 24 hours . thus , there is evidence demonstrating the power of rumination to sustain elevated blood pressure not just over minutes , but hours and days . higher blood pressure tends to beget yet higher blood pressure over time . in strictly biophysical terms , one mechanism by which the blood vessels in the body regulate blood flow as blood pressure increases , either more blood ( i.e. , higher cardiac output ) enters the blood vessels or the blood enters the vessels with greater force ( i.e. , mean arterial pressure ) ; either way , the blood vessels must expand and contract more to regulate blood flow . over time , this process increases the thickness of the muscle around the vessels the more often a person has higher blood pressure , the more the vessels work and the faster and thicker this layer grows . these thicker arterial walls are , however , more resistant to the force of blood flow , requiring the heart to work harder to pump enough blood hard enough for the vessels to properly regulate . with greater effort from the heart , this process explains how having sustained , untreated htn in one 's life is an independent predictor of cardiovascular morbidity . this process is conceptually similar to that of allostatic load , which can be described as a resetting of the physiological set points in an attempt to maintain homeostasis and thus function , but at a new set point ( e.g. , higher blood pressure ) . in the short - run , achieving allostasis is adaptive ( e.g. , the higher blood pressure allows the blood vessels to continue to regulate blood flow ) , but in the long term can have quite negative consequences . importantly , and supportive of the model presented here , the stressors that cause allostatic load are not major , impactful ( but infrequent ) key to this theory is that these frequent , more minor stressors have to produce sufficiently long response durations so as to lead to allostasis . as discussed , the presence of an initial stressor does not automatically lead to the prolonged activation that causes the resetting of the body 's set point or allostasis . rather , it is the presence and persistence of the cognitive representations ( i.e. , rumination , worry , and other perseverative cognitions ) that can later reactivate / recreate a stressor in one 's mind ; this process and the resultant physiological changes are the circumstances that characterize and explain the chronic stress - htn relationship . we are attempting to describe a highly complex process and to understand why some are prone to ruminate and others are not , how attention clearly a key determinant of the nature of one 's conscious thoughts as well as signal detection and hypervigilance for perceived threats and insults is implicated in the process . finally , we note that classical conditioning processes undoubtedly play a role , for example , a conditioned response to an eliciting stimulus may evoke the cognitive and affective response to past unconditioned aversive events . consideration of these factors is beyond the scope of this paper , but will need to be addressed as our understanding of the processes increases . there are many avenues of approach to such interventions , in part because there are several pathways by which stress may cause htn and heart disease . thus , obvious targets are stress - related behaviors that may increase one 's risk , such as smoking , weight gain , lack of exercise , increased salt intake , and interpersonal behaviors that may cause subsequent and ongoing stressful incidents , and so on . the model we propose suggests that an additional useful point of intervention may be the ruminative processes that can convert acutely stressful experiences into recurrent or chronic stress . for example , one approach would be the cognitive reframing of stress - related negative thoughts and affect as a means of undermining their power to evoke subsequent negative thoughts and affect , and thus reducing the duration of exposure to sustained activation of the psmss . even here , several strategies suggest themselves . cognitive behavior therapy [ cbt ] specifically targets such cognitions , and is a well - validated clinical technique . enrichd has indeed used cbt as an intervention to reduce cvd risk ( in conjunction with a social support manipulation ) , but the results were largely negative . rumination was not measured in that study , however , we would suspect that if the frequency and severity of the negative thoughts and cognitions were not reduced , such an intervention would be unlikely effective . clinicians have developed interventions to train people to learn to forgive past perceived transgressions , and others have targeted one 's ability to let go of anger . although intriguing , their effects on rumination and on psmss activation have been the subject of only a handful of studies and thus data are sparse for these approaches . we hope this model spurs additional intervention work that tests a range of intervention targets , potential mediators , and a wider range of outcomes . on a final note , we suggest that a combination of procedures , likely targeting behavior as well as cognition and affect , may have better results than either alone . defining chronic stress and understanding the pathways by which it may contribute to sustained elevated bp and eventual htn is a large we have addressed one pathway , but others , including changes in risk - related behaviors and in the direct effects of chronic stress on htn , are not addressed here , nor are physiological bp regulatory systems including the angiotensin system , short term regulatory systems including the baroreflex , or the role of endogenous opioids . to eventually form a comprehensive model of the development and maintenance of pathogenically high - sustained bp resting level , our model attempts to make a more modest and circumscribed contribution , but we hope to integrate these potential mediators and moderators into the model in future studies . we propose that acute bp and other changes in response to experiencing an acute stressor may be less important than the continuing psmss activation that often persists after the incident itself has long ended and may reoccur when the memory of the stressor is activated . robert sapolsky addressed this issue in his book why zebras do not get ulcers , in which he noted in his evocative example that zebras do not have the capacity to recall the stress ( the failed attack from the lion , e.g. , ) that has beset them ; therefore , they neither ruminate about the past nor worry about the future , and thus do not suffer the psmss consequences of engaging in such mental activity . and thus do not suffer ulcers or , presumably , htn , at least not due to the pathway proposed in this paper . ( the supposition here is that these acute events occur only intermittently for the beleaguered zebra ; if they are occurring many times a day over long periods of time , the distinction between acute and chronic stress becomes blurred ) . for some time , evolution has been shaping and perfecting the fight - or - flight response system that is designed to engage under acute threat . awareness , or consciousness , however , emerged only a short time ago in evolutionary terms , and it was not , according to our model , until this point that mammals humans , at least , and perhaps some infrahumans developed the capacity to self - reflect and , thus , to ruminate . therefore , zebras do not develop ulcers , but humans do ( as well as elevated bp and htn risk ) . it is widely accepted that chronic stress must be filtered in some manner by sensation and more importantly perceptual processes , thus leading to individual differences in response to environmental challenges or stressors . similarly , the role of duration of exposure to the stress or , as we suggest , to the contents of one 's thoughts and emotions is , we argue , the representation of the chronic stress in its purest form ; it is , after all , those thoughts and emotions that largely affect the stress mediating physiology , which itself is a more proximal cause of htn and cvd . do the data support the model ? as reviewed herein , parts of the model have been tested in different laboratories , often using different approaches , and the results seem to hang together to suggest that indeed , rumination is one important pathway by which the effects of chronic stress on the development of htn are transmitted . scores of studies , however , conducted across several laboratories , have turned their attention to the role of rumination and perseverative cognition , and we anticipate that many of the blanks will be filled in as research on this topic progresses . one important line of inquiry is to apply more intensive , within - person data capture approaches that can carefully document the temporal dynamics of these processes ( such as the initial occurrence and response to an acute stressor , and then its reoccurrence via ruminative processes and their associated responses ; see ) . coupling ambulatory psychophysiology ( e.g. , bp ) with ecological momentary assessment techniques is one well - validated approach that holds great promise in this regard . we believe that the model we have proposed represents an advance in the manner in which we conceptualize stress and its effects on blood pressure and cardiovascular disease . partly , this is because of the basis it provides to develop useful interventions as well as precise targets of those interventions . researchers have been ruminating about the nature of stress for some time ; we hope that this research will help turn these ruminations into productive ideas for further exploration .
participants aged between 4 and 50 years with type 1 diabetes on insulin pump therapy with impaired awareness of hypoglycemia participated in a 6-month intervention trial comparing standard pump therapy ( insulin pump only ) to the veo ( insulin pump with continuous glucose monitoring and lgs function ) . hypoglycemia awareness was determined with the use of the modified clarke s questionnaire ( 7 ) , with a minimum score of 4 suggestive of impaired awareness of hypoglycemia . participants randomly assigned to the veo received a 3-h standardized face - to - face education session followed by ongoing patient - led assistance from an educator and doctor via phone or e - mail , together with scheduled visits every 3 months . participants uploaded their pumps to the carelink therapy management system website weekly , allowing evaluation of patient use and patterns of insulin suspension . the lgs threshold was set at 60 mg / dl for study participants . as the sensor glucose falls below this level if the patient does not respond to the alarm , insulin delivery will be suspended for up to 2 h , after which standard basal insulin is resumed . the patient may intervene at any stage throughout this 2-h period to resume insulin delivery . if there is no patient response , the pump will cycle in a continuous 6-h cycle of 2 h of insulin suspension followed by 4 h of basal insulin delivery . in addition to the lgs alarm and suspend function , a low glucose alert was set at 70 mg / dl . at this level , the pump emits an audible sound or vibratory alert only but does not automatically suspend insulin . a high glucose alert also was set at 324 mg / dl for all participants . participants were requested to check for blood ketones if the first morning finger stick blood glucose level was 270 mg / dl and record the level of ketones , either with a diary or via e - mail to study personnel . ketosis was defined as a blood ketone level of 0.6 mmol / l . data regarding sensor glucose values were obtained from the carelink pump upload . an lgs event was defined as an episode of insulin suspension activated by the lgs alarm set at 60 mg / dl . for each individual subject , each lgs event was evaluated to determine the time and duration of each event ; sensor glucose before , during , and after the period of insulin suspension ; and corresponding meter blood glucose entered as a calibration before or after the event . for each 2-h lgs event , for instance , if a patient has responded to the audible alarm and elected to continue insulin suspension , this was considered to be an lgs event with patient response . if a patient has not responded to the audible alarm at all during the entire 2-h duration of insulin suspension , this was considered to be an lgs event with no response . we then further evaluated overnight lgs events with no patient response occurring prior to 3:00 a.m. , which allowed us to determine the pattern of sensor glucose values with no patient intervention during and after the period of insulin suspension . an lgs event was defined as an episode of insulin suspension activated by the lgs alarm set at 60 mg / dl . for each individual subject , each lgs event was evaluated to determine the time and duration of each event ; sensor glucose before , during , and after the period of insulin suspension ; and corresponding meter blood glucose entered as a calibration before or after the event . for each 2-h lgs event , for instance , if a patient has responded to the audible alarm and elected to continue insulin suspension , this was considered to be an lgs event with patient response . if a patient has not responded to the audible alarm at all during the entire 2-h duration of insulin suspension , this was considered to be an lgs event with no response . we then further evaluated overnight lgs events with no patient response occurring prior to 3:00 a.m. , which allowed us to determine the pattern of sensor glucose values with no patient intervention during and after the period of insulin suspension . twenty - four participants were commenced on the system ( [ means sd ] aged 17.4 9.3 years , duration of diabetes 8.7 6.4 years , and duration of pump therapy 3.8 3.0 years ) , with a median duration of use of 89 days ( range 9211 ) . the incidence of severe hypoglycemia for this cohort of 24 participants was 45.8 events per 100 patient - years prior to commencement of the veo system . this improved to 7.4 0.6% at 3 months ( p = 0.015 ) while using the system . the a1c , however , increased from 3 to 6 months to 7.7 0.9% ( baseline vs. 6 months , p = 0.517 ) for the remainder of the intervention phase . the mean hypoglycemia unawareness score at baseline was 5.6 1.3 , which decreased to 4.5 1.9 ( p = 0.019 ) at the end of the 6-month intervention phase . in total , the system was worn for 2,493 of 4,218 patient - days . the median percentage of sensor use was 72% . during the 2,493 sensor days evaluated , 3,128 lgs events occurred ( 1.3 events per day ) . the duration of lgs events was as follows : 13% ( 406 ) lasted the full 2-h duration , 12% ( 376 ) lasted between 1 and 2 h , 13% ( 400 ) lasted between 30 min and 1 h , 14% ( 446 ) lasted between 10 and 30 min , and 48% ( 1,488 ) lasted < 10 min . thirty - six percent of all events occurred overnight . following the end of the main 6-month intervention phase , 79% of patients chose to continue wearing the system for an additional 6 months . in this cohort , two participants ( 8% ) were not able to tolerate the system and discontinued using the system after several weeks . of the full 2-h suspend events , 80% ( n = 324 ) occurred overnight . in 168 ( 52% ) of these nocturnal events , the patient responded to the alarm and elected to continue insulin suspension . in this case , the pump continued insulin suspension and automatically resumed insulin delivery at the end of the 2-h period . in the remaining 156 ( 48% ) nocturnal events , there was no response from the patient despite an alarm of 60 db lasting 20 s occurring every 2 min during the 2-h period . a total of 126 ( 81% ) overnight suspend events that were associated with no patient response occurred before 3:00 a.m. analysis of these events allowed the evaluation of sensor glucose patterns 4 h following 2 h of insulin suspension and 2 h of basal insulin resumption without the influence of additional insulin therapy or carbohydrate intake ( fig . insulin was always suspended if there was no patient response and sensor glucose dropped below 60 mg / l . as shown in fig . the mean sensor glucose at the end of the 2-h suspend period was 99 6 mg / dl ( [ means se ] 5.5 0.3 mmol / l ) . the mean sensor glucose 2 h after insulin delivery resumed was 155 10 mg / dl ( 8.6 0.6 mmol / l ) . the mean first - morning meter glucose entered into the pump following these events was 185 9 mg / dl ( 10.3 0.5 mmol / l ) . the corresponding mean first - morning sensor glucose values was 144 9 mg / dl ( 8.0 0.5 mmol / l ) . the mean difference between sensor and meter glucose values was 41 mg / dl ( 2.3 mmol / l ) . overnight lgs events with no patient response occurring before 3:00 a.m. ( n = 126 ) to evaluate sensor glucose patterns following insulin suspension . a : insulin delivery is automatically suspended as sensor glucose falls below the 60 mg / dl threshold . b : insulin delivery is automatically resumed after 2 h. at this point , the mean sensor glucose after 2 h of insulin suspension was 99 6 mg / dl ( 5.5 0.3 c : the mean sensor glucose following 2 h of insulin resumption was 155 10 mg / dl ( 8.6 0.6 the frequency of overnight full 2-h suspend events in individual participants is shown in table 1 . the frequency ranged from one event every 2.6 days ( participant 22 ) to one event every 52.8 days ( participant 11 ) . twenty - five percent of participants had one full 2-h suspend overnight at least once per week . frequency of overnight 2-h lgs events in individual participants insulin was suspended for the full 2-h duration more than once overnight on 14 occasions , as shown in table 2 . during these occasions , the patient responded to the alarm and elected to continue insulin suspension . after this time , the sensor glucose again fell below the lgs threshold of 60 mg / dl , and insulin delivery was again suspended . multiple overnight lgs events on these occasions , insulin was suspended for an average of 4.2 0.2 h overnight . the mean sensor glucose at the end of the suspended period was 112 18 mg / dl ( 6.2 1.0 mmol / l ) . following these events , the mean first - morning meter glucose recorded in the pump was 239 22 mg / dl ( 13.3 1.2 the sensor glucose tended to read below the meter glucose by a mean difference of 115 mg / dl ( 6.4 mmol / l ) . on waking , the difference in sensor and meter glucose values resulted in calibration error and in a subsequent request for insertion of a new sensor . there were no episodes of severe hypoglycemia , defined as seizure or coma associated with hypoglycemia , during the time evaluated . of the full 2-h suspend events , 80% ( n = 324 ) occurred overnight . in 168 ( 52% ) of these nocturnal events , the patient responded to the alarm and elected to continue insulin suspension . in this case , the pump continued insulin suspension and automatically resumed insulin delivery at the end of the 2-h period . in the remaining 156 ( 48% ) nocturnal events , there was no response from the patient despite an alarm of 60 db lasting 20 s occurring every 2 min during the 2-h period . a total of 126 ( 81% ) overnight suspend events that were associated with no patient response occurred before 3:00 a.m. analysis of these events allowed the evaluation of sensor glucose patterns 4 h following 2 h of insulin suspension and 2 h of basal insulin resumption without the influence of additional insulin therapy or carbohydrate intake ( fig . insulin was always suspended if there was no patient response and sensor glucose dropped below 60 mg / l . the mean sensor glucose at the end of the 2-h suspend period was 99 6 mg / dl ( [ means se ] 5.5 0.3 mmol / l ) . the mean sensor glucose 2 h after insulin delivery resumed was 155 10 mg / dl ( 8.6 0.6 mmol / l ) . the mean first - morning meter glucose entered into the pump following these events was 185 9 mg / dl ( 10.3 0.5 mmol / l ) . the corresponding mean first - morning sensor glucose values was 144 9 mg / dl ( 8.0 0.5 the mean difference between sensor and meter glucose values was 41 mg / dl ( 2.3 mmol / l ) . overnight lgs events with no patient response occurring before 3:00 a.m. ( n = 126 ) to evaluate sensor glucose patterns following insulin suspension . a : insulin delivery is automatically suspended as sensor glucose falls below the 60 mg / dl threshold . b : insulin delivery is automatically resumed after 2 h. at this point , the mean sensor glucose after 2 h of insulin suspension was 99 6 mg / dl ( 5.5 0.3 mmol / l ) . c : the mean sensor glucose following 2 h of insulin resumption was 155 10 mg / dl ( 8.6 0.6 the frequency of overnight full 2-h suspend events in individual participants is shown in table 1 . the frequency ranged from one event every 2.6 days ( participant 22 ) to one event every 52.8 days ( participant 11 ) . twenty - five percent of participants had one full 2-h suspend overnight at least once per week . insulin was suspended for the full 2-h duration more than once overnight on 14 occasions , as shown in table 2 . during these occasions , the patient responded to the alarm and elected to continue insulin suspension . after this time , the sensor glucose again fell below the lgs threshold of 60 mg / dl , and insulin delivery was again suspended . multiple overnight lgs events on these occasions , insulin was suspended for an average of 4.2 0.2 h overnight . the mean sensor glucose at the end of the suspended period was 112 18 mg / dl ( 6.2 1.0 mmol / l ) . following these events , the mean first - morning meter glucose recorded in the pump was 239 22 mg / dl ( 13.3 1.2 the sensor glucose tended to read below the meter glucose by a mean difference of 115 mg / dl ( 6.4 mmol / l ) . there was no clear pattern in terms of sensor life . on waking , the difference in sensor and meter glucose values resulted in calibration error and in a subsequent request for insertion of a new sensor . there were no episodes of severe hypoglycemia , defined as seizure or coma associated with hypoglycemia , during the time evaluated . the lgs function was frequently activated in patients with type 1 diabetes with impaired awareness of hypoglycemia using sensor - augmented pump therapy . most of these events were of short duration , whereby the patient has overridden insulin suspension and resumed insulin delivery . almost 40% of all lgs events occurred overnight , and one in three events lasted the full 2-h duration . lgs events , including multiple activations , were not associated with severe hypoglycemia or diabetic ketoacidosis . the overnight suspend events with no patient intervention provides a unique opportunity to evaluate the sensor glucose profile following full 2-h insulin suspension when the sensor glucose falls below 60 mg / dl . the initial fall in glucose levels was arrested promptly following insulin suspension and rose steadily during the 2-h suspended period . the mean glucose level at the end of the 2-h suspend event was 99 mg / dl , which is a near - normal value . the mean first - morning meter glucose value entered into the pump was 185 mg / dl , and there was no associated ketosis . these analyses suggest that 2-h insulin suspension overnight is unlikely to be associated with adverse outcomes . the pattern of overnight 2-h suspend events varied widely among our participants . the frequency of overnight full 2-h suspend events ranged from one episode every 2 months to up to one episode every 23 days in others . patients having frequent overnight suspend events were surprised at the frequency of the events and indicated that they did not hear the alarms . despite an auditory and vibratory alarm system to warn patients about hypoglycemia , patients are not responding to the current alarm systems . in a previous study evaluating the acoustic arousal threshold in adolescents with type 1 diabetes , we found that adolescents with type 1 diabetes could sleep through an alarm of 120 db , which is equivalent to standing within 60 m of a jet plane at takeoff ( 8) . these findings again indicate that alarm systems need to be further developed , perhaps to include a system based on varying frequencies and loudness to avoid acclimatization . the finding of multiple activations of insulin suspension was previously reported by danne et al . these events were uncommon ; however , patients should be advised that this can occur if they respond to the lgs alarm and continue insulin suspension . this is easily done by pressing the esc ( escape ) then act button on the pump . these steps effectively silence the lgs alarm and continue insulin suspension . in this case , the system considers this to be a patient response . following insulin resumption at 2 h , if the sensor glucose again falls below the sensor threshold , lgs will again be activated and the 4-h basal insulin delivery cycle does not continue in this case . comparison of finger stick meter glucose and sensor glucose values suggest that the sensor tended to underread glucose values rather than overread them . there were no cases of sensor error resulting in failure to suspend and prolonged hypoglycemia during the night or in the morning . in the trial so far , there have been no episodes of prolonged nocturnal hypoglycemia resulting in seizure or coma , although we still are awaiting the final results . these results are in keeping with the reported accuracy data available for the medtronic veo system ( 10 ) , in particular the low rate of false - negative alarms in the hypoglycemia range of < 70 mg / dl . following 6 months of sensor - augmented pump therapy with the lgs function these findings are preliminary , and the final results will be reported at trial completion .
transepithelial elimination of granulomas has been reported in the past ; however , transfollicular elimination has not found a mention in the dermatologic literature . herein we report this curious phenomenon and correlate it with the clinical features of this condition . a 43-year - old male presented with an asymptomatic lesion on his left elbow of duration 6 years with a gradual increase in size . the patient could not recollect any history of penetrating injury or insect bite or any other trauma at the site of lesion . on clinical examination , the lesion was a well - circumscribed , erythematous to brown , scaly , indurated plaque measuring 5 6 cm over the left elbow . skin biopsy revealed pseudoepitheliomatous hyperplasia with hyper- and parakeratosis of the stratum corneum and a thick granular layer . the dermis showed dense inflammatory infiltrate comprising lymphocytes , plasma cells , and epithelioid cells . langhan 's giant cells were also present , few of which were seen within a granuloma engulfed by a hyperplastic follicular infundibulum . antituberculous treatment was started ; however , the patient was lost to follow - up . erythematous scaly plaque over the left elbow histopathological examination showing pseudoepitheliomatous hyperplasia of the epidermis with granulomatous inflammation of dermis ( h and e 10 ) granuloma within a hyperplastic dilated follicular infundibulum ( h and e 40 ) initial reaction of tissues to tubercle bacilli is that of nonspecific inflammation wherein the bacilli multiply and are killed by neutrophils . macrophages then phagocytose the organisms , but their ability to kill the organisms depends on their enhanced activation , which is mediated by cytokines that are secreted by t - cell - mediated reaction to tuberculous antigens . macrophage death , mediated by tumor necrosis factor - alfa and macrophage proteases results in caseation necrosis within the granuloma . this necrotic process inactivates or kills mycobacteria in the lesion , but does not completely eliminate them . similarly the tissue also tries to eliminate the bacilli surrounded by the necrotic granuloma transepidermally and probably transfollicularly , although these phenomena have been rarely reported . the epidermis , as an organ of catharsis , serves actively in percutaneous elimination of materials from the skin by phagocytosis as demonstrated by the langerhans cells , selective uptake and elimination of small particles such as melanin granules through intercellular spaces in the epidermis and eventually shed off with keratin , and lastly by epidermal proliferation as in the case of various perforating disorders , calcinosis cutis , granulomatous disorders etc . the hypothesis explaining the reactive process of epidermal proliferation and elimination of larger particles from the dermis was put forward by mehregan in discussing the pathogenesis of elastosis perforans serpiginosa . he postulated that the area where the epidermis comes in contact with foreign material undergoes marked acanthosis resulting in pseudoepitheliomatous hyperplasia . this allows the hyperplastic epidermis to engulf the noxious material and inflammatory cells associated with it . subsequently this material gets extruded to the surface by forming canals through the hyperplastic epidermis . a reactive hyperplasia of the epidermis is seen in varying degrees in chronic dermal granulomata such as those of tuberculosis , deep fungal infections , calcinosis cutis , and so on . this process seems to be triggered by the presence of certain reactions in the dermis that are ordinarily chronic and usually associated with multinucleate giant cells . once this dermal reaction has proceeded to a certain extent to influence the epidermis , the latter proliferates and its hyperplastic strands invade the dermis , eventually enveloping and engulfing the dermal material and infiltrate . the engulfed material varies and may consist of connective tissue constituents as in elastosis perforans serpiginosa , minerals as in calcinosis cutis , or granulomata in tuberculosis , sarcoidosis , or deep fungal infections . the phenomenon of transepidermal elimination of granulomas through a hyperplastic epidermis was first reported by goette and odmon . in our case , the granuloma is entrapped in hyperplastic follicular infundibulum probably in a process of elimination , that could correlate with the follicular plugs seen on the surface of the lesion clinically . transfollicular elimination of granulomas in cutaneous tuberculosis has not been reported in the past ; we report this rare phenomenon as one of the probable modes of granuloma expulsion .
improved treatment strategies and better supportive care have resulted in increased survival rates for most childhood cancers . currently , close to 80% of children and adolescents with cancer will survive 5 years or more after diagnosis . in the usa , they are at risk for developing secondary neoplasm , cancer recurrence , organ dysfunction , impaired growth and development , decreased fertility , impaired intellectual function and dental problems as well as psychosocial , behavioral problems and post traumatic stress disorder [ 2 , 3 ] . naturally , many survivors become uncomfortable and anxious about their health and unsure of how to cope with life for months and years after treatment . listed in the table 1 are the several very informative policy reports and recent publications regarding the childhood cancer survivorship issues that had subsequently ignited the subsequent studies on survivorship in the united states and that had initiated setting up the survivorship clinics in many cancer centers [ 5 , 6 ] . in 2004 , childhood cancer survivorship study funded by national cancer institute ( nci ) grants confirmed the somber facts that long - term late effects are common , that severe disabling or life - threatening conditions may occur and psychosocial and behavioral problems may also be present . the study also pointed out that both survivors and the medical community need to be educated about these dreadful late effects of childhood cancer treatment . this had set the stage for establishing survivorship centers across the united states to better evaluate the survivors for appropriate care and offer education of the patients and primary health care providers . to this end , several u.s . state cancer grants and some private grants , particularly the lance armstrong foundation awards had funded a number of survivorship centers . one such funded program is the cancer survivorship center established at emory university in georgia that i am personally familiar with . while it provides coordinated multidisciplinary cancer - focused care for the patients as well as clinical research on survivorship issues , the program offers education to health care professionals in the state of georgia through lectures , consultations and cme videos . the program recently established the " survivor link " , also named the georgia pediatric alliance cancer survivors . through this network which includes patients , primary health care professionals and the cancer center specialists , the primary health care professionals get much needed education regarding the survivorship health issues and every pediatric cancer survivor in georgia receives a comprehensive survivorship health care plan as per children 's oncology group ( cog ) guidelines coordinated by the emory cancer survivor team staff . recognizing that survivorship is an important phase of cancer care , the cog made a decision to merge the previous late effects committee and the cancer control discipline into one entity . in the past , the late effects committee 's role was mainly to gather all the necessary information on the survivors ; thus its function was largely observational in nature . the cancer control discipline on the other hand had been interventional in nature with the research studies conducted throughout prevention , diagnosis and treatment phases . by merging the two , the cog is more streamlined in conducting the research to prevent cancer , reduce acute toxicities , improve symptom management and reduce morbidity and mortality in survivors of all childhood cancer . in short , the new combined committee ( cancer control and long - term survivorship , ccls ) works on improving health across the trajectory of childhood cancer from before diagnosis through long - term survivorship and end of life care . one of the major accomplishments of cog is that it has established a guideline for the long - term follow - up of childhood cancer survivors which can be viewed at www.survivorshipguidelines.org . it serves as a helpful resource both for cancer institutions and primary health care professionals alike in carrying out comprehensive yet standardized follow - up care for the survivors . the focus of investigation is to identify treatment - related risk factors in relation to genetic susceptibility of the individuals . the protocol mandated obtaining blood or buccal samples to study the frequencies of gene polymorphism among the survivors who developed key adverse events such as cardiac dysfunction , ischemic stroke , avascular necrosis and subsequent malignant neoplasms . another important study is alte05n1 : umbrella long - term follow - up protocol that was activated on 5/19/08 and so far has accrued 262 patients out of a target sample of 5,000 . main theme of this study is that one single center is designated to collect health care information , and later the designated institution is to distribute the information to each patient 's cog institutions . the long - term follow - up center located in the keck school of medicine at the university of southern california maintains up - to - date demographic and self / parents - reported health status information on each patient and provides updated data to the cog statistics and data center ( sdc ) on a regular basis , and the sdc will subsequently forward this information to each patient 's cog institutions in conducting follow - up examinations and collecting protocol specific data . these data can then be linked with patients ' outcomes , facilitating identification of important associations and concerns for outcomes research . given the complex medical , psychological and behavioral problems experiencing by the childhood cancer survivors , it is imperative that they receive long - tem follow - up care from the health care providers knowledgeable with the long - term survivor issues . the eventual goal of cancer treatment is not only to ensure the patient 's long - term survival but also to help them to maintain physical and psychological health status to function as normally as possible at home , in school and in the work place . one major problem in dealing with the survivorship issue is that it 's not easy to find the proper health care professional to care for the survivors . as the childhood cancer survivors age out of the pediatric health care system , family physicians and internists who are generally not familiar with the complexity of the pediatric cancer treatment are often called to provide long term care . therefore , it is imperative that the multidisciplinary team at each cancer survivor center work as closely as possible with the community based health care providers . in the late 1980 's and 90 's , a number of modern pediatric oncology departments in seoul and around the country were established . these departments were regularly staffed with pediatric oncologists trained mainly in the usa by way of educational tours during their sabbatical year . as a result , the children in korea began receiving state - of - the - art cancer care for more than 20 years now . this resulted in remarkable improvements in survival statistics that are nearly comparable to those of western countries . it is to be emphasized that the success of childhood cancer care in korea could have not come without the dedicated support of families , and hosts of other care providers working in the hospital side by side with the doctors . at the time of the governmental budgetary constraints , it is commendable that in recent years , the korean government has increased the budget allocations for the families of children with cancer . this eased the financial burden on the families that , in turn , has enabled families to seek cancer care more freely . in korea , the annual incidence of cancer in children and adolescents is 1,700 with the 5 year survival rate of 70% in the year of 2001 through 2005 , with the result that close to 1,200 children and adolescents with cancer currently enjoy long - term survival . hence , the cumulative number of childhood cancer survivors living in korea is estimated to be around 20,000 to 25,000 [ 7 , 8 ] . these include starting the registry of long - term survivors , need for long - term follow - up studies , establishing a cancer - focused multidisciplinary team in the cancer center , raising the patient 's awareness of the importance of regular follow - up , education for primary care providers regarding the survivorship , and the need for special education program in the school system . in addition , the tight budgetary situation on the part of government and hospitals adds to the challenges . first , korea is relatively small geographically , and the transportation system is in place so that patients should be able to travel in a relatively short time to receive cancer focused follow - up care practically anywhere in korea . second , children with cancer have been treated in only a handful of pediatric cancer centers . third , there 's unity among all the pediatric oncologists in terms of working together for the same cause , namely nationwide multi - center studies . it is encouraging to note that the nationwide cohort study for childhood cancer survivors was already proposed , and the versions of instruments to measure the quality of life of childhood cancer survivors were validated [ 9 , 10 ] . further , the meeting of the childhood cancer survivors in korea also took place for the first time early this year . one of the biggest challenges and the utmost importance is setting up the weekly multidisciplinary cancer - focused survivorship clinic in major cancer centers to be staffed with pediatric oncologists , pediatric endocrinologists , pediatric neuro - oncologists , neuro - psychologists , and other pediatric specialists . the cognitive , endocrine and the imaging studies are ideally to be done in the same setting when the patients come in for their regular check - ups . in spite of all these hurdles and hardships , i salute the leadership of korean society of pediatric hematology / oncology for setting up the survivorship issues as one of top priorities .
a 55-year - old woman was referred to our institution with sudden onset of chest pain . she had a history of percutaneous vertebroplasty at the 12th thoracic vertebra for osteoporotic fracture 3 weeks before she was referred to the institution . an electrocardiogram ( ecg ) did not show st - segment elevation ; sinus rhythm , creatine kinase , and troponin - i were all within their respective normal limits . the patient underwent a coronary computed tomography ( ct ) scan and transthoracic echocardiogram ( tte ) under suspicion of stable angina . the ct scan demonstrated a long , linear , highly - attenuated segment ( 10.5 cm in length ) in the right atrium and ventricle , as well as fragmented pieces in the right pulmonary artery and its branches , causing a pulmonary embolism ( fig . 1 ) . tte also revealed a piece of long , linear , echogenic material ( 10 cm in length ) in the right atrium and ventricle . the patient was transferred to our department for emergency surgery with a diagnosis of pulmonary embolism and foreign body in the right side of the heart due to an injection of polymethylmethacrylate ( pmma , bone cement ) via an injured azygos vein after percutaneous vertebroplasty . after the right anterolateral thoracotomy through the 4th intercostal space , a cardiopulmonary bypass was performed using the femoral artery , femoral vein , and superior vena cava . immediately after incising the right atrium , a long , needle - shaped segment was located and removed under the beating heart . there were no other pmma segments or fragmented pieces in the right atrium or ventricle . after the right atrial incision was closed , the right pulmonary artery was longitudinally incised and we removed as many fragmented pieces as possible ( fig . a post - operative tte did not reveal residual foreign bodies in the right atrium or ventricle , and the patient did not have pulmonary hypertension . the follow - up ct scan demonstrated no linear , highly - attenuated segments in the intracardiac or right pulmonary arteries ; however , residual fragmented pieces that were observed preoperatively on ct were observed in the anterior segmental artery and middle lobar artery . percutaneous vertebroplasty is a widely performed and a minimally invasive surgical technique used to enhance mechanical strength by injecting pmma into the vertebral body . although this procedure is effective , easy , and minimally invasive , complications , such as leakage of pmma , may occur . a 26% to 65% incidence of pmma leakage from the targeted vertebral body has been reported in the literature ; however , the majority of leakage incidents were asymptomatic and did not require surgical treatment [ 1 - 3 ] . perivertebral venous leakage has also been reported at an incidence rate of 5% to 48% in the literature , and is also usually asymptomatic . however , some serious complications have been reported , such as massive pulmonary embolism and cardiac perforation necessitating surgical treatment [ 4 - 7 ] . we report a case of pulmonary embolectomy and removal of a free - floating pmma segment in the right side of the heart via a right anterolateral thoracotomy without cardiac arrest after percutaneous vertebroplasty . many surgical procedures to repair a perforated right ventricle , tricuspid regurgitation , incision of the main pulmonary artery , and pulmonary embolism have been performed via median sternotomy with cardiac arrest [ 4 - 7 ] . however , in this case , pulmonary embolism was limited to the right pulmonary artery and the pmma segment was located in the right heart . we also wanted to avoid performing a median sternotomy , if possible , due to osteoporosis . presumed that a pmma segment in the right ventricle could not move into the pulmonary artery because of its long and stiff nature . we thought that we could remove the foreign body in the right side of the heart in a single attempt because it was long enough . we safely removed the pmma segment from the right atrium and ventricle , and successfully completed a pulmonary embolectomy .
the study protocol was reviewed and approved by the ethical committee of the barcelona health institute . during the 200910 influenza season , the sentinel network of primary care physicians and pediatricians of navarre obtained nasopharyngeal swab specimens from patients with influenza - like illness ( ili ) for virologic confirmation of influenza by using standard real - time reverse transcription pcr or cell culture ( 6 ) . in early 2010 , a trained nurse telephoned households of persons who had had laboratory - confirmed pandemic ( h1n1 ) 2009 in october and november 2009 while pandemic influenza was active in the area ( index cases ) . when no response was received , the calls were repeated 5 times on different days and at different times . for each household , an attempt was made to talk to the adult who was primarily responsible for the health issues of those who lived there , usually the mother or father . when possible , other adults in the household were also interviewed . detailed information was obtained about index case - patients and all other persons living in the same household ( contacts ) with regard to sociodemographic data , medical history , influenza symptoms , and whether he or she had sought medical care . a question was asked about each of the following signs and symptoms : fever , cough , sore throat , headache , muscular or joint pain , nasal congestion , and vomiting . this analysis included all household residents who had ili for < 7 days with respect to the onset of ili in the index case - patient . ili was defined as fever and either cough or sore throat in the absence of other diagnoses . the first person with ili in the household was considered the primary case - patient , and others , if any , were considered secondary case - patients . persons with index cases who led us to contact the household were excluded from analysis because , given the study design , they had all sought medical care . of 252 households that met the inclusion criteria , 233 ( 92% ) were successfully contacted , and all persons agreed to participate in the study . of 668 household contacts of index cases , 188 ( 28% ) persons had > 1 influenza symptoms and 166 ( 25% ) met the criteria for having ili . the proportion of symptomatic cases that met the criteria for ili was higher among children ( 94% , 92/98 ) than among adolescents and adults ( 82% , 74/90 ; p = 0.021 ) . of 166 persons with ili , only 107 ( 64% , 95% confidence interval [ ci ] 57%72% ) had sought medical care . the percentage of those who sought medical care was lower among adults and adolescents ( 53% , 39/74 ) than among children < 15 years of age ( 74% , 68/92 ; p = 0.006 ) . this percentage was also lower among persons with cases secondary to a previous household case ( 58% , 64/111 ) than among primary case - patients ( 78% , 43/65 ; p = 0.010 ) ( table 1 ) . * ili , influenza - like illness ; or , odds ratio ; ci , confidence interval . by fisher exact test . no differences were detected by sex , rural / urban residence , country of origin , vaccination against seasonal influenza , smoking status , or presence of major chronic conditions . the percentage of secondary case - patients who sought medical care was the same regardless of whether the primary case - patient received antiviral treatment . persons who had sought medical care had a mean sd of 4.0 0.8 symptoms , which was similar to those who had not sought medical care ( 4.0 0.7 symptoms ; p = 0.688 ) . none of the contacts had received antiviral prophylaxis or vaccine for pandemic ( h1n1 ) 2009 . the frequency with which persons with ili sought medical care , by age group and symptoms , frequency of symptoms did not differ between persons who had sought medical care and those who had not sought medical care . * ili , influenza - like illness ; nd logistic regression analysis showed that seeking medical care was more frequent among children than among adolescents and adults ( adjusted odds ratio 2.2 , 95% ci 1.14.3 ; p = 0.019 ) and among primary case - patients than among secondary case - patients ( adjusted odds ratio 2.2 , 95% ci 1.04.8 ; p = 0.038 ) . a similar proportion of contacts with any symptom but who did not meet ili criteria sought medical care ( 59% , 13/22 ) as did those who met ili criteria ( 64% , 107/166 ; p = 0.642 ) . we studied persons who had ili and were household contacts of persons with laboratory - confirmed influenza . it is likely that the cause of symptoms among persons with ili was also infection with influenza virus . care seeking was less frequent among adults and when there had already been another case within the household . epidemiologic surveillance systems and studies of influenza are typically based on cases of medically diagnosed ili . therefore , the proportion of persons not seeking medical care is usually unknown and not taken into account . ideally , epidemiologic surveillance should consider these persons to avoid underestimating the actual magnitude of the disease ( 7 ) . these persons may increase spread of the disease ( 8) and may also be more likely to self - medicate , resulting in possible risks to their health ( 9,10 ) . persons who do not seek medical care do not contribute to the cost of influenza from the health system perspective , but they do from the point of view of society because they can result in lost work hours , more medications used , and increased need for care ( 11 ) . when influenza symptoms were present , adults and adolescents sought medical care less often than children . this finding may explain in part why the incidence of medically diagnosed ili is usually much higher among children ( 4,6,12 ) . therefore , its results may not be generalizable to other influenza seasons ( 4 ) . nevertheless , the study was conducted when pandemic ( h1n1 ) 2009 virus had already been circulating in the population for several months , the initial alarm had abated , and the level of medical care had returned to levels similar to that for seasonal influenza . the probability of making telephone contact may have been higher in households with more members . families in whom none of the members had sought medical care were not included in the study , which may overestimate the proportion of persons who sought medical care . persons with influenza who did not seek medical care should be taken into account in estimations of the actual incidence of influenza and its effect on the general population . these persons may have a major effect on transmission and should be considered in planning prevention and control measures and in evaluations of the effects of this disease .
fine needle aspiration cytology ( fnac ) of the thyroid gland is firmly established as a first - line diagnostic test for the evaluation of goiter and as the single most effective test for preoperative diagnosis of lesions of the thyroid gland . recent studies have also confirmed that fnac is more sensitive as well as specific in segregating neoplastic and non - neoplastic thyroid pathology . identification of predominant cell pattern , cell morphology and background details in fnac smears play an important role in diagnosing various thyroid lesions with accuracy . marginal vacuoles ( mvs ) have been described as irregular cytoplasmic vacuoles measuring 1 - 7 m in diameter , deeply eosinophilic in the periphery with a largely unstained central area on may - grnwald - giemsa ( mgg ) stained smears . they tend to gather peripherally in a group of follicular epithelium and hence are called marginal vacuoles . the term fire flare has also been used because of the resemblance of mvs to a rising or spreading fire . mvs stain poorly or not at all with periodic acid - schiff ( pas ) . they are not positive for acid phosphatases . in wet fixed smears stained with hematoxylin and eosin , the true nature of mvs is not known , although ultrastructural studies have indicated that these are dilated cisternae of endoplasmic reticulum generated by hyperactivity and vacuolar content as dilated phagolysosomes . were the first to discuss marginal vacuoles in the cytoplasm of follicular cells in smears from toxic goiters . they reported that mvs along with a nuclear ring pattern in follicular fragments are fairly specific signs of hyperfunction of the thyroid . subsequent studies , however , revealed that mvs although suggestive of thyrotoxicosis were nonspecific as they are also encountered in nontoxic goiter , hashimoto 's thyroiditis and follicular carcinoma . das et al . , in 2006 , were the most recent to comment on mvs ; according to them , mvs represent diffusing out of thyroid hormones from the basal aspect of follicular cells on their way to interfollicular capillaries . this study was conducted with the aim of looking for the presence of mvs and strength of correlation ( if any ) with thyroid hormone levels and cytological diagnoses in diffuse thyroid enlargements . adequacy of the samples was assessed by using the hamburger criteria , that is , six clusters of cells in at least two slides prepared from separate aspirates . mvs were studied and graded as per the following criteria : no / scanty mvs : distinct mvs exceeding 2 m in diameter demonstrated in < 10% of cells.moderate mvs : mvs seen in 10 - 50% of the cells no / scanty mvs : distinct mvs exceeding 2 m in diameter demonstrated in < 10% of cells . abundant mvs : presence of mvs in > 50% of the cells examined . though both abundant and moderate mvs were treated as separate entities in our study , the presence of at least a moderate degree of mvs was considered as significant for the sake of easy comparison with earlier published studies . the hormonal status of all the patients having diffuse enlargements was taken into account and depending on the values of the thyroid function test , the patients were categorized as euthyroid , hyperthyroid and hypothyroid . based on cytomorphological features , we segregated three broad groups , namely , primary hyperplasia , hashimoto 's thyroiditis and colloid goiter . the presence and degree of grading of mvs was then correlated individually both with the cytological diagnosis and thyroid hormonal status of the patient . the chi - square test and test of proportion were applied to study the strength of association amongst mvs with cytological diagnoses and hormonal status . a p 0.05 was considered as significant and a p 0.01 as highly significant . there were 84 ( 86.6% ) females and 13 ( 13.4% ) males in this study . the mean age was 29.5 13.5 years , with the maximum number of cases belonging to the third and fourth decades . there was no statistically significant correlation between age / sex and the occurrence of mvs . it was observed that among 97 diffuse goiters , hashimoto 's thyroiditis accounted for 52 of 97 cases ( 53.6% ) , followed by colloid goiter in 26 cases ( 26.8% ) . on categorizing the cases according to their hormonal status , euthyroid cases accounted for 51 of 97 cases ( 52.6% ) . those having hyperthyroidism accounted for 32 cases ( 33% ) and the rest , that is , 14 cases ( 14.4% ) were hypothyroid [ table 2 ] . correlation of marginal vacuoles with cytological diagnosis correlation of marginal vacuoles with hormonal status on studying the association of cytological diagnosis with the presence of mvs and applying pearson 's chi - square test and test of proportion , it was observed that primary hyperplasia had a significant correlation with the presence of mvs ( p = 0.004 ) and colloid goiter with the absence of mvs ( p = 0.01 ) . however , hashimoto 's thyroiditis did not show a statistically significant correlation [ table 1 ] . on studying the association of hormonal status with the presence of mvs , it was found that there was an overall significant correlation in all three hormonal groups with mvs ( p = 0.001 ) . on intragroup comparison , it was found that euthyroid and hyperthyroid patients had a significant correlation with a p value of 0.009 and 0.001 , respectively . the correlation in the hypothyroid group was statistically insignificant with a p value of 0.14 [ table 2 ] . in cases of primary hyperplasia ( n = 19 ) , where 16 ( 84.2% ) patients were hyperthyroid biochemically , abundant mvs could be detected in the majority ( 52.6% ) of the smears and significant mvs ( abundant + moderate ) in 89.4% of the smears [ figure 1 ] . primary hyperplasia : thyroid follicular epithelium showing abundant mvs ( mgg , 400 ) as far as hashimoto 's thyroiditis was concerned ( n = 52 ) , 46% patients were euthyroid and the rest were either hypo or hyperthyroid in equal proportion . abundant mvs were observed in 17% , moderate mvs in 36.5% and no mvs in 46% of the cases [ figure 2 ] . hashimoto 's thyroiditis : intraepithelial lymphocytes and hurthle cells with abundant mvs ( mgg , 400 ) majority of the patients with colloid goiter ( n = 26 ) were euthyroid ( 92.3% ) . of all the patients , > 90% had no mvs in their aspiration cytology smears . three cases which showed moderate mvs were those in which besides abundant colloid , follicular epithelium was also present . cytomorphological features were studied with special attention to the presence and grading of mvs and their correlation with thyroid function and cytological diagnosis . the presence of prominent mvs along with a nuclear ring pattern in follicular wall fragments are fairly specific signs of thyroid hyperfunction but their frequency can also be high in hashimoto 's thyroiditis , cellular adenomatoid nodules and follicular neoplasms . it has been suggested that mvs represent the diffusing out of thyroid hormones from the basal aspect of follicular cells . in our study , it was observed that abundant mvs were present in 10 of 19 cases ( 52.6% ) of primary hyperplasia . seventeen of 19 cases ( 89.4% ) showed the presence of at least a moderate degree of mvs in their smears . in the earlier published study conducted by soderstrom and nilsson and jayaram et al . , abundant mvs were present in 40 - 60% cases of primary hyperplasia and could be significantly detected in more than 80% of the cases . results of our study are in fair agreement with both of them . regarding the association between the presence of mvs and hormonal status in hashimoto 's thyroiditis , 28 out of 52 cases ( 53.8% ) showed the presence of significant mvs ( abundant + moderate ) in our study . on correlating this feature with thyroid function , it was found that eight of 28 ( 29% ) cases were hyperthyroid . other workers have not correlated the presence of mvs in hashimoto 's thyroiditis with thyroid function . this frequency is much less than that observed by nilsson who noted mvs in 32.6% of the cases of nontoxic goiter . the results of our study are in fair agreement with those of das et al . who observed mvs in 5.2% cases of colloid goiter . considering the correlation between abundant mvs ( n = 19 ) in diffuse goiters and levels of thyroid hormone , some interesting observations emerged : none of these cases were hypothyroid ; 21% of diffuse goiters with abundant mvs were euthyroid . thus , the mere presence of abundant mvs does not necessarily imply hyperfunction of the thyroid . hyperthyroid cases constituted 79% of this group ; thus , all diffuse goiters with prominent mvs require hormone levels to evaluate hyperfunction of the thyroid . though primary hyperplasia was the single largest contributor to this group , autoimmune thyroiditis was a close second . this feature therefore indicates considerable cytologic overlap between the two conditions . on a detailed search of the literature correlation of mvs as a whole has , however , been done by soderstrom and nilsson , das et al . and jayaram et al . on correlating moderate mvs ( n = 29 ) in diffuse goiters and thyroid hormone levels , it was noted that a significant proportion of these ( 5 of 29 cases ; 17.2% ) were hypothyroid . hyperthyroid cases constituted seven of 29 ( 24.1% ) cases of this group and primary hyperplasia accounted for six of these seven cases . thus , no conclusion could be drawn on the basis of moderate mvs regarding functional status of the thyroid gland . regarding the correlation between the presence of no / scanty mvs ( n = 49 ) in diffuse goiters and levels of the thyroid hormone , it was observed that the majority of such cases belonged to the category of colloid goiter and hashimoto 's thyroiditis , accounting for 46.9% and 38.8% cases , respectively . all the hypothyroid cases were of hashimoto 's thyroiditis and this is anticipated , as the gland is destroyed by an autoimmune process during the course of the disease . euthyroid cases represented more than half of those with no / scanty mvs ( 61.2% ) . we also encountered two cases of primary hyperplasia with no / scant mvs with biochemical evidence of hyperthyroidism . the presence of abundant mvs in diffuse goiters was not associated with a hypothyroid state in this study . moderate / absent mvs in diffuse goiters were not found to correlate with thyroid function . thus , all diffuse goiters with prominent mvs require hormonal evaluation to rule out hyperfunction of the thyroid . it is also inferred that in the presence of abundant mvs in thyroid fna smears , primary hyperplasia should be ruled out .

hemoptysis is one of the symptoms of pulmonary diseases such as parenchymal diseases ( like infections ) and endobronchial lesions ( like tumors ) as well as vasculitis ( like wegener s disease and anti - glomerular basement membrane disease ) . based on its severity , hemoptysis is classified as mild , moderate , and severe . moderate bleeding is attributed to 50100 ml / h bleeding in those without any history of pulmonary disease and 2550 ml / h in those with an underlying pulmonary disease . severe hemoptysis is attributed to 100150 ml / h bleeding in those without a history of pulmonary disease and 50 ml / h bleeding in those with underlying pulmonary problems . in other words , the most common life - threatening pulmonary bleedings are due to malignancies . according to the previous studies , 515% of patients with lung tumors have life - threatening hemoptysis . other causes of life - threatening hemoptysis include bronchiectasis , pulmonary tuberculosis , mycetoma , necrotizing pneumonia , and bronchogenic carcinoma . bronchoscopy is a technique for observing airways and has 2 applications : 1 ) diagnostic : evaluating patients with hemoptysis , probable lung cancer , and chronic coughs and observing abnormalities in the airways and bronchoalveolar lavage and 2 ) therapeutic : removing external objects , blood , and secretions ; applying tumor laser therapy ; treating bronchial stenosis ; and implementing stents for the palliative care of malignancies causing stenosis in the trachea . an exact diagnosis of lung tumor requires several endobronchial biopsies . the susceptibility of tumors with a necrotic center and vascular tumors to bleeding after the first sampling is a major problem during bronchoscopy . in the recent decade , according to the spanish national guideline , cold saline lavage and adrenaline instillation on the bleeding site has been done as a treatment for hemoptysis . nonetheless , not all patients fully respond to this method of treatment . the instillation of hemostatic factors such as tranexamic acid on the bleeding site is a new technique that has been investigated recently . tranexamic acid is a synthetic antifibrinolytic acid applied for the short - term control of bleeding . it inhibits fibrin degradation and consequently prevents blood loss through binding to plasminogen and plasmin . in lethal massive hemoptysis , early investigation , including endobronchial adrenaline use , balloon tamponade , and intubation , is necessary for bleeding control . bronchial artery embolization is the treatment of choice for massive hemoptysis not controlled by another procedure . for example , it has been reported that in patients with malignant mesothelioma , intrapleural tranexamic acid and , during oral surgeries , its oral solution can be used for controlling blood loss . this drug can be used for bleeding control following surgery on the heart , liver , and large veins or surgery necessitated by trauma - related bleedings . furthermore , it has been reported that tranexamic acid can decrease mortality . according to previous studies , oral or intravenous tranexamic acid has been used extensively in the treatment or prevention of mucosal or post - surgical bleedings . the endobronchial administration of tranexamic acid is a potential therapeutic agent to treat acute pulmonary bleeding ; accordingly , it is a safe and useful method before intervention ( balloon occlusion or embolization ) in patients with uncontrolled hemoptysis . the present study was designed to compare the efficacy of endobronchial instillation of tranexamic acid with that of adrenaline in controlling acute endobronchial bleeding . the present study was approved by the ethics committee of kerman university of medical sciences ( k/92/228 ) and was conducted in the research center of afzalipour hospital , kerman , iran , from 2012 to 2013 . among 276 patients who met the inclusion criteria for participation in the current study , 226 were excluded because of successful bleeding control with cold saline , cardiovascular disease , bleeding tendency , or anticoagulant and antiplatelet drug consumption . patients who already had hemoptysis or those who started bleeding after biopsy and could not be controlled with cold saline lavage during bronchoscopy were selected . first , cbc , ptt , pt , and inr were determined in all the patients . in the bronchoscopy room , after a peripheral venous access was obtained , cardiovascular monitoring , oxygen therapy , local anesthesia of the throat with 2% lidocaine ( 2 ml in 5 ml of saline ) , and sedation / anesthesia with an intravenous injection of 2.5 mg of midazolam were performed . during the bronchoscopy procedure , a biopsy specimen was taken through a pentax bronchoscope ( japan ) using type 1 biopsy forceps ( size 10018 ) ( germany ) . based on the census method and the random allocation method , the process of patient selection was performed ( figure 1 ) . fifty patients were divided into 2 equal groups of 25 . in cases of persistent bleeding , after 90 seconds of cold saline lavage , adrenaline ( 1 mg diluted in up to 20 ml of saline ) in the first group and tranexamic acid ( 500 mg diluted in up to 20 ml of saline ) in the second group were directly instilled on the bleeding site . if necessary ninety seconds after the last dose , in cases of bleeding control failure , crossover with a second medicine was done in order to save the patient s life . direct observation of clot through the bronchoscope was considered as bleeding control . in this study , sao2 before , during , and after the procedure was measured by using an oxypleth 520a pulse oximeter ( usa ) in order to ensure that clot did not disturb ventilation . all the patients were followed up for 3 months after the procedure for bleeding recurrence or complications of drugs such as vascular thromboembolism . in the present study , the data were analyzed with spss , version 20 , using the chi - square test and the t test . based on the previous studies such as the one by mrquez - martn e. and by considering the maximum sample value in those studies , the percentage of blood loss in the 2 groups was 18.8% and 58.3% with the statistical ability of 80% and reliability of 95% by applying the average comparison of the 2 samples with 28 members ( =0.0500 , power=0.8000 ; n=2(z1-/2+z1-)/d ) . the present study recruited 50 patients . in terms of gender , the study population was comprised of 69.4% males and 30.6% females , showing a significant difference ( p=0.032 ) . the mean age of the patients had no statistically significant difference between the tranexamic acid group ( 61.4712.86 y ) and the adrenaline group ( 58.1417.48 y ) ( p=0.589 ) . among the patients , 55.1% were cigarette smokers and 44.9% did not smoke ; there was no significant relationship between bleeding control and cigarette smoking ( p=0.256 ) . moreover , 38.8% of the study population reported a history of using narcotics , while 61.2% reported no such history ; no significant relationship was found between bleeding control and the consumption of narcotics ( p=0.773 ) ( table 1 ) . characteristics of the study population according to the type of treatment according to the t test ; according to the chi - square test ; the mean time of bleeding control showed no significant difference between the tranexamic acid group ( 133.977.91 sec ) and the adrenaline group ( 136.6683.5 sec ) ( p=0.908 ) . there was also no significant difference in regard to the number of injections between the tranexamic acid group ( 2.080.66 ) and the adrenaline group ( 1.800.74 ) ( p=0.352 ) ( table 1 ) . in the current study , the patients were divided into iatrogenic and noniatrogenic groups based on the cause of bleeding . from the whole study population , , 52% of the patients with iatrogenic bleeding belonged to the tranexamic acid group and 56% belonged to the adrenaline group , while in the noniatrogenic group , 48% of the patients were in the tranexamic acid group and 44% were in the adrenaline group . these findings showed no significant differences between the 2 study groups with regard to bleeding control in the iatrogenic and noniatrogenic cases ( p=0.77 ) ( table 1 ) . furthermore , all the patients were studied in terms of the relationship between bronchoscopic findings and bleeding control with 2 drugs . the most frequent bronchoscopic findings were respectively endobronchial tumors ( 16 cases ) , anthracofibrosis ( 10 cases ) , bronchomalacia ( 4 cases ) , abnormal mucous ( 4 cases ) , and tracheomalacia ( 1 case ) . fifteen patients did not have any abnormal findings on bronchoscopy ; in these patients , bronchiectasis , pneumonia , and exacerbation of chronic obstructive lung disease were the most frequent causes of hemoptysis . the data revealed no significant relationship between bronchoscopic findings and bleeding control by tranexamic acid and adrenaline ( table 2 ) . in the present study , all the patients underwent pulse oximetry before , during , and after bronchoscopy and in all the cases , mean sao2 was > 94% . bronchoscopic findings in the two groups according to the chi - square test or the fisher exact test the number of patients who needed a second drug for bleeding control was 1 ( 4% ) patient in the tranexamic acid group and 8 ( 32% ) patients in the adrenaline group . although this rate of crossover was lower in the tranexamic acid group , the difference was not significantly different ( p=0.609 ) ( figure 2 ) . the patients who received tranexamic acid as the first medicine needed a second medicine for bleeding control less frequently than did the adrenaline group . all the patients were followed 3 months later ; none of them had bleeding recurrence or complications such as thromboembolism either during hospitalization or after hospital discharge . pulmonary bleeding is a life - threatening complication resulting from pulmonary diseases or following bronchoscopic therapies and biopsy taking . previous studies have emphasized on the oral or intravenous administration of tranexamic acid for bleeding control and preventing bleeding recurrence . tranexamic acid is an antifibrinolytic agent and is effective against blood loss in patients with increased fibrinolysis activity after surgery and in patients with a normal hemostatic pathway . the results of the present study suggested that the efficiency of tranexamic acid and adrenaline in controlling iatrogenic and noniatrogenic bleedings was the same . indeed , in most of our patients treated with tranexamic acid , bleeding was adequately controlled without any need for a second drug . according to the previously published statistics , the rate of bleeding after bronchoscopy is 120% . the rate of bleeding following biopsy is the same for the malignant and benign tumors of the airways . vascular , necrotic endobronchial , and other hypervascular tumors , including carcinoid tumors , often tend to have the highest rate of bleeding during bronchoscopy . an endobronchial injection of epinephrine or other vasoactive agents before biopsy may not prove effective . furthermore , due to systemic absorption and side effects such as vasoconstriction , hypertension , and tachyarrhythmia , the dose of epinephrine and other drugs should be limited . it was recently reported that tranexamic acid was able to reduce transfusion post cardiovascular surgery ; however , seizures and thrombotic events remained side effects of this drug . generally , tranexamic acid is tolerated well and has only a few side effects , including digestive side effects ( nausea , vomiting , and abdominal pain ) following oral consumption and hypotension following rapid intravenous injections . in the present study , the patients tolerated the endobronchial instillation of tranexamic acid well and it was efficient in bleeding control without any side effects . according to a case report , the instillation of tranexamic acid and then its oral administration , following relative response to bronchial artery embolization , in a patient with frequent severe pulmonary bleedings due to cystic fibrosis was able to stop hemoptysis successfully . hemoptysis in cystic fibrosis is common , and major bleeding aggravates the decline in lung function or may even result in death . sometimes , embolization fails to stop hemoptysis ; therefore , tranexamic acid can assist in stabilizing the patient until lung transplantation . another case report highlighted the beneficial effect of oral tranexamic acid in preventing hemoptysis recurrence and , indeed , rendering embolization unnecessary in a patient with cystic fibrosis . a study on patients with hemoptysis compared the efficacy of oral tranexamic acid and placebo and reported that although the result of 1 week s treatment was not different between the 2 groups , bleeding was controlled sooner in the tranexamic acid group . in a similar study designed as a randomized , double - blinded , controlled trial on 46 patients with hemoptysis in 2002 , a 1-week treatment with oral tranexamic acid led to no significant difference with the placebo group insofar as the results did not demonstrate that tranexamic acid shortened the duration of hemoptysis after 7 days . a survey reported the use of oral hemostatic agents such as tranexamic acid as an additional palliative therapy for lung cancer hemoptysis . all the aforementioned studies investigated the effects of oral or intravenous tranexamic acid , while we studied the effects of the endobronchial instillation of tranexamic acid on the bleeding site . in a study on 2 patients with bleeding due to a pulmonary tumor , one following bronchoscopic biopsy and the other following endoluminal electrocautery , first cold saline and adrenaline were used for bleeding control and then because of failure in bleeding control , 5001000 mg of endobronchial tranexamic acid was administered . bleeding was controlled within a few seconds , and the patients were discharged some days later without any side effects or bleeding recurrence . these results are concordant with those in the current study . invasive or surgical procedures to control bleeding have more complications . not only can antifibrinolytic agents be used as a temporary treatment before surgery or embolization of the pulmonary artery , but also they can reduce mortality . in one study on patients with hemoptysis treated with endobronchial tranexamic acid for 2 years , malignancies and bronchiectasis were the most common causes of noniatrogenic bleeding and in patients who had responded to tranexamic acid , tumors were the most common cause of bleeding . in that study , almost all the patients with iatrogenic bleeding showed a good response to endobronchial tranexamic acid and the patients who did not respond to the treatment suffered from bronchiectasis . these findings chime in with our results in as much as tranexamic acid was as efficient as adrenaline in both groups of our patients with iatrogenic and noniatrogenic bleedings . according to a case report on a patient with severe pulmonary bleeding due to vascular malformation , intrabronchial epinephrine and cold saline lavage failed to control bleeding , while the endobronchial instillation of tranexamic acid was successful in bleeding control . as was previously mentioned , in the current study , tranexamic acid was as beneficial as adrenaline in major hemoptysis ; however , a larger sample size might have yielded a different result . in 2009 , the effects of the endobronchial administration of tranexamic acid on 6 patients with pulmonary bleeding were investigated . two patients who had bleeding during biopsy were treated with intrabronchial tranexamic acid and the rest of the patients , who had spontaneous hemoptysis , underwent inhalation of tranexamic acid . all the patients showed good responses to the first dose of tranexamic acid without any side effects and the study showed no difference between the inhalation and endobronchial instillation of tranexamic acid in controlling hemoptysis . the results of that study were similar to our findings , although we did not investigate the effect of the inhalation of tranexamic acid . another study used intrabronchial tranexamic acid for bleeding control after taking biopsies from tumors with a high risk of bleeding and reported that the injection of intrabronchial tranexamic acid was safe and useful . more recent studies , albeit with small sample sizes , have also confirmed that local tranexamic acid is effective in hemoptysis . the same results have been observed from the effects of nebulized tranexamic acid in patients with tracheobronchial cancer and massive hemoptysis . in rare cases , it is deserving of note that in the present study , only 1 patient needed a second medicine in the group of tranexamic acid , whereas 8 patients in the adrenaline group required a second medicine ; the difference , however , did not constitute statistical significance . the absence of discrepancy can be justified by the small sample size , which is the major limitation of the current study . the present study , with a sample size larger than that in the previous studies , compared the efficacy of the local instillation of tranexamic acid with that of adrenaline in bleeding control during bronchoscopy . our data showed that tranexamic acid was as efficient as adrenaline in controlling pulmonary bleeding . tranexamic acid had the same efficacy in controlling iatrogenic and noniatrogenic bleedings . moreover , in the patients treated with tranexamic acid as the first drug , bleeding was controlled without any need for the use of a second drug .
according to the 2010 national survey on drug use and health , 20 percent of us adult population ( 45.9 million ) were estimated to have mental illness in 2010 : 3.8 percent had suicidal ideation ( 8.7 million ) ; 20 percent of the general population ( 9.2 million ) with mental illness met criteria for substance abuse or dependence , and 13.7 percent of population utilized mental health services in the past year . one study has found that the prevalence of mental illness in medical students is 21.2 percent , compared to the general rate in other graduate students or young adults being 8 - 15 percent . 69.1 percent of those who had depression admitted to receiving treatment in the past , and 7 percent of those with mental illness were receiving treatment at the time . not only do medical students experience higher rates of mental illness , but the relevant sequelae to mental illness is also higher . medical students experience higher rates of burnout , higher risk of suicide , and lower quality of life than an age - matched population . to reiterate the issue , medical students experience depression and mental illness at higher levels than the general population . in addition , medical students are less likely to receive treatment due to the stigma of mental illness . the obvious reason is that they do not want to appear weak , or unable to handle the rigors of training , either to their friends , family , or superiors . also , despite legal efforts to protect those applying for jobs from discrimination for having a prior or ongoing mental illness , they feel it will make themselves less competitive for residency due to documentation of any mental illness in any record . thirty percent of first and second year students were found to be reluctant to receive treatment . furthermore , a mayo clinic found in a recent study that the majority of medical students experience at least one event of distress , such as depression , decreased mental quality of life , etc . those who experience distress were more likely to contemplate suicide and dropping out of medical school , with more episodes of distress equating to a higher risk of thoughts of suicide and dropping out . physical activity should be a part of every able bodied person s daily lives , but especially so for those in stressful situations . acute exercise leads to release of neurotransmitters that promotes adherence to an exercise regimen , which is important for students to continue to reap the benefits of exercise , and activation of cortical areas that improve mood . regular exercise also promotes mental health through angiogenesis and neurogenesis , showing that there is some actual scientific evidence to back the empirical notions of prescribing exercise as a means for mental illness prevention and health improvement . neurogenesis is enhanced in animal models , particularly in the hippocampus , which not only gives rise to better mental health in humans , it also gives an added benefit of possible improved memory through exploiting the plasticity of the hippocampus . exercise improves self esteem and encourages positive self viewing , which is commonly compromised in medical students with or without depressive symptoms . exercise has been correlated with psychological well being and life satisfaction , whereas physical inactivity is associated with psychological disorder development . there is a general consensus that physical activity is beneficial to preventing or treating mental illness , as exercise promotes overall general well being . allotted time for exercise in students schedules should be implemented into curriculums as a therapeutic preventative measure for mental illness . exercise is a therapeutic lifestyle change that has been shown to have positive effects on the treatment and prevention of mental illness . thirty minutes allotted at least three times per week , built into students schedules , is more cost effective than eventual counseling , and more pragmatic as it attempts to prevent mental health issues . mental health problems are easier to deal with before they exist . thus , prevention is a key component of improving the overall mental health status of this population . we argue that integrating exercise time into the curriculum as a block of time will be more effective than simply encouraging students to stay active . it has been shown that very few adults actually meet minimum exercise time guidelines , and given that medical students are constantly pushed for time , they fall into a vulnerable group to repeat this trend of the general adult population . placing exercise into the curriculum enforces the concept that exercise is vital to the students well being , and it also implies that the students studies , while important , can not be the sole focus and that time for maintenance of health is equally important . academia is resistant to curriculum changes for many reasons , particularly in health professional education . some of these reasons are long held traditions , lack of data to back claims , fear of the loss of accreditation , protection of curricular time , and lack of perceived change . while our recommendation is certainly not traditional , there is plenty of data to support that regular physical activity will benefit medical students . the time suggested , 90 minutes total a week , will not interfere with the rest of the curriculum , and there is no reason to fear loss of accreditation . as for lack of perceived need to change , this has been debunked with the growing number of professionals admitting that medical school stresses are negatively affecting students mentally and emotionally . in particular , the authors foresee resistance to the ideas presented due to a lack of time to place in the curriculum / students schedules . the prescription of 30 minutes a day , three days a week , is a fair compromise since it is hard to argue that time can not be made for such an important activity as exercise for this relatively short amount of time with regards to the whole week . we believe this compromise to be satisfactory , balancing between enough exercise frequency to elicit benefits , and the crammed schedules medical students have . medical schools have been attempting to increase material retention rates via new learning strategies , such as problem based learning . given the obvious interest in improving test scores and material comprehension , exercise can play a role in promoting mental well being beyond illness prevention and into the realm of performance enhancement . not only does exercise promote improved memory , it also decreases the likelihood of burnout and depression . burnout and depression are not consistent with improved memory , or a successful medical school curriculum . as shown from the research cited , along with prodigious scientific research touting the benefits of exercise with regards to mental health , there is plenty of evidence to support the claims of a need for a more prevention based approach to mental illness , especially in this population . the medical student population , while not usually viewed as such , is actually a more vulnerable population with regards to the susceptibility of developing mental illness . there is empirical and neuroscience evidence to support the claims that exercise helps more by physical means and can transcend into the mental and emotional realm of public health . time for exercise implemented into the curriculum addresses key concerns in public health , while also balancing the opposing forces to its implementation . not only will it assist in preventing mental illness , but it will also improve the general health of the medical student population , which is important for them but also for the health and well - being of the general population with regards to healthcare .
metabolic syndrome ( syndrome x , insulin resistance syndrome ) consists of constellation of metabolic abnormalities which include central obesity , hyperglycemia plus insulin resistance , high triglycerides plus low hdl cholesterol , and hypertension ( the deadly quartet ) . third national health and nutrition examination surveys ( nhanes iii ) revealed the age adjusted prevalence of metabolic syndrome to be 34% in men and 35% in women in the unites states . in a multiethnic study in singapore , 28.8% of indians , 24.2% of malaysians , and 14.8% of chinese had metabolic syndrome . with rapid industrialization and urbanization , the prevalence of metabolic syndrome has increased dramatically . by ncep ( national cholesterol education programme ) criterion , 41.1% of asian indians were suffering from metabolic syndrome whereas the prevalence of metabolic syndrome was 11.2% in chennai urban population . obesity , a key component of metabolic syndrome , occurs due to increased energy intake , decreased energy expenditure , or a combination of both , thus leading to positive energy balance . thyroid hormones up - regulate metabolic pathways relevant to resting energy expenditure , hence , obesity and thyroid functions are often correlated . on one hand , obesity per se causes alterations in thyroid hormones , i.e. increased thyroid hormone levels , increased tsh with no effect on t3 and t4,[79 ] or increase in tsh and t3 with no effect on t4[1012 ] ; on the other hand , subclinical hypothyroidism as a result of slow metabolism can lead to the obesity . it is still not clear whether these alterations in thyroid hormones are a cause or an effect of obesity ( metabolic syndrome ) . one fact is clear that clinicians often interpret increased tsh levels with normal thyroid hormone levels in obese persons as an evidence of subclinical hypothyroidism and prescribe thyroxine replacement therapy to reinforce the euthyroid status which already exists . it has also been noted that the unnecessary use of thyroxine replacement can lead to its toxicity . the mechanism of normal levels of t3 , t4 with increased tsh in metabolic syndrome is not defined , but it has been hypothesized that metabolic syndrome is associated with insulin resistance due to the defect in postreceptor signal transduction in target tissue , a similar mechanism of thyroid receptor resistance might be operating in these obese persons . thus , in order to explore the possibility of thyroid receptor resistance to tsh , this study was designed to find alterations in metabolic syndrome ( obese insulin resistant individuals , defined by the homa model ) . the study was carried out in 40 patients with metabolic syndrome fulfilling three or more ncep atp iii criteria ( namely waist circumference > 102 cm in men and > 88 cm in women , blood pressure 130/85 mmhg , triglycerides 150 mg / dl , hdl cholesterol < 40 mg / dl in males and < 50 mg / dl in females , and fasting glucose 110 mg/ dl ) . we excluded the subjects with a history of thyroid diseases or radioiodine treatment , antithyroid treatment , thyroid autoantibody positive subjects , and pregnant women . all patients gave a written consent , and the institutional review board at the university of health sciences approved the study protocol . body weight ( kg ) and height ( m ) were measured using standardized techniques and equipment . the bmi was calculated as weight divided by squared height ( kg / m ) . the waist circumference was measured with a paper tape horizontally at the level of umbilicus in the standing position . a waist - to - hip ratio was calculated as waist circumference divided by hip circumference . blood pressure was measured from the left arm in the sitting position with apparatus at the level of heart . venous blood samples were taken after 12 h of fasting , separated and frozen at -8c until analysis . the serum levels of tsh ( reference range , 0.174.05 iu / ml ) , t3 ( reference range , 70200 ng / dl ) , and t4 ( reference range , 5.513.5 g / dl ) were measured as indicators of thyroid function using i-125 gamma counter ( ic 4702a , electronics corporation of india , india ) with radioimmunoassay kits ( irmak 9 , riak 4/4a , and riak 5/5a , respectively ; board of radiation and isotope technology , navi mumbai , india ) . the serum insulin levels ( reference range , 130 mu / l ) were also measured using a radioimmunoassay kit ( riak 1 , board of radiation and isotope technology , navi mumbai , india ) . blood sugar levels ( reference range , 60100 mg / dl ) were estimated by the glucose oxidase - peroxidase ( god - pod ) method using the glucose analyzer ( techno 168 , logotech india pvt . ltd . , india ) . fasting serum concentrations of triglycerides and hdl were measured using commercially available kits . to define the insulin resistance , homeostasis model assessment for insulin resistance ( homa ir ) was calculated by the following formula : the data were analyzed using spss software ( version 12.0 for windows , spss , inc . , the p value was calculated for statistical significance . the p value < 0.05 was taken as significant . the study was carried out in 40 patients with metabolic syndrome fulfilling three or more ncep atp iii criteria ( namely waist circumference > 102 cm in men and > 88 cm in women , blood pressure 130/85 mmhg , triglycerides 150 mg / dl , hdl cholesterol < 40 mg / dl in males and < 50 mg / dl in females , and fasting glucose 110 mg/ dl ) . we excluded the subjects with a history of thyroid diseases or radioiodine treatment , antithyroid treatment , thyroid autoantibody positive subjects , and pregnant women . all patients gave a written consent , and the institutional review board at the university of health sciences approved the study protocol . body weight ( kg ) and height ( m ) were measured using standardized techniques and equipment . the bmi was calculated as weight divided by squared height ( kg / m ) . the waist circumference was measured with a paper tape horizontally at the level of umbilicus in the standing position . a waist - to - hip ratio was calculated as waist circumference divided by hip circumference . blood pressure was measured from the left arm in the sitting position with apparatus at the level of heart . venous blood samples were taken after 12 h of fasting , separated and frozen at -8c until analysis . the serum levels of tsh ( reference range , 0.174.05 iu / ml ) , t3 ( reference range , 70200 ng / dl ) , and t4 ( reference range , 5.513.5 g / dl ) were measured as indicators of thyroid function using i-125 gamma counter ( ic 4702a , electronics corporation of india , india ) with radioimmunoassay kits ( irmak 9 , riak 4/4a , and riak 5/5a , respectively ; board of radiation and isotope technology , navi mumbai , india ) . the serum insulin levels ( reference range , 130 mu / l ) were also measured using a radioimmunoassay kit ( riak 1 , board of radiation and isotope technology , navi mumbai , india ) . blood sugar levels ( reference range , 60100 mg / dl ) were estimated by the glucose oxidase - peroxidase ( god - pod ) method using the glucose analyzer ( techno 168 , logotech india pvt . ltd . , to define the insulin resistance , homeostasis model assessment for insulin resistance ( homa ir ) was calculated by the following formula : the data were analyzed using spss software ( version 12.0 for windows , spss , inc . , the p value was calculated for statistical significance . the p value < 0.05 was taken as significant . the mean age of the patients ( group i ) and the controls ( group ii ) was 38.249.17 years and 34.1611.61 years , respectively . a female - to - male ratio ( sex ratio ) was 3:2 in both the groups . the mean waist circumference was 99.108.88 cm in patients and 74.859.44 cm in controls ( p < 0.0001 ) . the mean systolic and diastolic blood pressure were 133.313.8 mmhg and 87.59.8 mmhg , respectively , in group i patients whereas the mean systolic and diastolic blood pressure were 111.611.6 mmhg and 74.29.64 mmhg , respectively , in group ii individuals and the difference was statistically significant . the mean blood sugar levels were 114.49.34 mg / dl in group i individuals as compared to 79.6511.28 mg / dl in controls ( p < 0.0001 ) . similarly , there was a significant difference in triglyceride levels ( 162.7952.44 vs. 108.836.57 mg / dl ; p = 0.0002 ) and hdl cholesterol levels ( 39.245.03 vs. 48.484.9 mg/ dl ; p < 0.0001 ) in both groups [ table 1 ] . individuals with metabolic syndrome ( group i ) and controls ( group ii ) homa ir ( homeostasis model assessment for insulin resistance ) was calculated in both groups . the standardized value of homa ir ( mean+2sd = 5.7 ) obtained in group ii was taken as a reference value to define the insulin resistance . the mean homa ir was 7.781.58 in group i and 3.730.98 in group ii individuals , and the difference was significant ( p < 0.0001 ) . the mean t3 levels were comparable in both the groups ( 117.8227.56 vs. 124.75 + 14.55 ng / dl ; p = 0.31 ) . similarly the difference in mean t4 levels was also nonsignificant in both the groups ( 7.771.37 vs. 7.311.20 g / dl ; p = 0.23 ) . mean tsh levels in group i individuals were higher as compared to group ii ( 5.681.90 iu / ml vs. 2.190.94 iu / ml ) and the rise was statistically significant ( p < 0.0001 ) as shown in table 2 . the mean age of the patients ( group i ) and the controls ( group ii ) was 38.249.17 years and 34.1611.61 years , respectively . a female - to - male ratio ( sex ratio ) was 3:2 in both the groups . the mean waist circumference was 99.108.88 cm in patients and 74.859.44 cm in controls ( p < 0.0001 ) . the mean systolic and diastolic blood pressure were 133.313.8 mmhg and 87.59.8 mmhg , respectively , in group i patients whereas the mean systolic and diastolic blood pressure were 111.611.6 mmhg and 74.29.64 mmhg , respectively , in group ii individuals and the difference was statistically significant . the mean blood sugar levels were 114.49.34 mg / dl in group i individuals as compared to 79.6511.28 mg / dl in controls ( p < 0.0001 ) . similarly , there was a significant difference in triglyceride levels ( 162.7952.44 vs. 108.836.57 mg / dl ; p = 0.0002 ) and hdl cholesterol levels ( 39.245.03 vs. 48.484.9 mg/ dl ; p < 0.0001 ) in both groups [ table 1 ] . homa ir ( homeostasis model assessment for insulin resistance ) was calculated in both groups . the standardized value of homa ir ( mean+2sd = 5.7 ) obtained in group ii was taken as a reference value to define the insulin resistance . the mean homa ir was 7.781.58 in group i and 3.730.98 in group ii individuals , and the difference was significant ( p < 0.0001 ) . the mean t3 levels were comparable in both the groups ( 117.8227.56 vs. 124.75 + 14.55 ng / dl ; p = 0.31 ) . similarly the difference in mean t4 levels was also nonsignificant in both the groups ( 7.771.37 vs. 7.311.20 g / dl ; p = 0.23 ) . mean tsh levels in group i individuals were higher as compared to group ii ( 5.681.90 iu / ml vs. 2.190.94 iu / ml ) and the rise was statistically significant ( p < 0.0001 ) as shown in table 2 . alterations in thyroid functions , though well known yet , are not recognized clinically and there is inconsistency in the thyroid functions in metabolic syndrome . the rise in tsh with normal t3 and t4 is mostly reported,[79 ] but the rise in tsh with alteration in t3 without any effect on t4[1012 ] or increased thyroid hormone levels also have been described in obesity . these disturbances in the thyroid function do not have any physiological basis , because it can not be explained by the feedback mechanism . by this study , we tried to explore the possibility of thyroid receptor rsistance akin to insulin resistance in metabolic syndrome and made interesting observations . forty obese individuals fulfilling the ncep atp iii criteria were taken as cases and 20 healthy individuals served as controls . patients with prior history of thyroid disease , history of antithyroid treatment , with positive antithyroid antibody and pregnancy were excluded . the persons in both groups were made comparable with regards to age and sex but had significant difference with regards to components of metabolic syndrome as displayed in table 1 . keeping in mind that metabolic syndrome is associated with insulin resistance with hyperinsulinemia as a metabolic consequence ; it is possible that these patients might have thyroid receptor resistance resulting in persistent elevated tsh levels . the stimulus for increased tsh could be accounted by the elaboration of certain hormones namely leptin , adiponectin from the fatty tissue which cross the blood brain barrier and stimulate the hypothalamic pituitary axis . to explore the thyroid receptor resistance , we took the help of homa ir to define insulin resistance . the cut off limit of homa ir was derived from the individuals of group ii as 5.7 ( mean+2sd ) . the mean levels of t3 ( 117.8227.56ng / dl ) and t4 ( 7.771.37g / dl ) in group i and mean t3 ( 124.7514.55 ng / dl ) and t4 levels ( 7.311.20 g / dl ) in group ii were comparable ; however , the mean levels of tsh in group i ( 5.68 1.90 iu / ml ) were significantly higher than in group ii ( 2.19 0.94 iu / ml ) . the raised tsh levels with normal t3 and t4 could not be explained on the basis of the feedback system . these findings also do not explain the state of subclinical hypothyroidism as all these patients were antithyroid peroxidase antibody negative . our finding suggest that insulin - resistant obese persons had higher tsh levels as compared to normal individuals but t3 and t4 levels were comparable in both groups . raised tsh thus could be a metabolic consequence of metabolic syndrome rather than a state of subclinical hypothyroidism . from our findings , it can be hypothesized that raised tsh levels in obese persons with insulin resistance ( metabolic syndrome ) could be due to association of thyroid receptor resistance to tsh similar to insulin resistance . the stimulus for rise in tsh could also be due to hormones secreted by adipose tissue . since we did not estimate these hormones ( leptin , resistin , adiponectin , etc . ) in our study , thus are not able to comment on the effect of these hormones on thyroid function tests . to conclude , it is suggested that these findings are preliminary and require evaluation on a large scale with inclusion of various hormones elaborated by adipose tissue . to conclude , it is suggested that these findings are preliminary and require evaluation on a large scale with inclusion of various hormones elaborated by adipose tissue .
a left renal vein passing behind the abdominal aorta is termed a retroaortic left renal vein ( rlrv ) , and this anomaly is a relatively uncommon condition . recent advances in computed tomography and magnetic resonance imaging techniques make it possible to visualize the vascular structures in detail . additionally , the congenital anomalies of the inferior vena cava ( ivc ) and its tributaries have become more frequently encountered in asymptomatic patients . rlrv anomalies , although usually overlooked , are not rare . however , only a few cases showing clinical symptoms with this anomaly have been reported . compression of the rlrv between the aorta and the vertebra is known to be the cause of urological problems such as hematuria , varicocele , and ureteropelvic junction obstruction ( upjo ) . we retrospectively evaluated the type , frequency , clinical significance , management , and long - term follow - up of rlrv in 9 patients who were examined with multidetector computed tomography ( mdct ) owing to urological problems . twelve patients with urological problems , such as hematuria , flank and abdominal pain , varicocele , and upjo , were studied with mdct at our institution from january 2003 to december 2009 . we retrospectively reviewed these patients ' medical records and analyzed their clinical characteristics including sex , age , type of rlrv , urologic symptoms , concomitant disease , treatment , and follow - up results ( table 1 ) . type i , ii , iii , and iv of rlrv join the ivc in the orthotopic position , join the ivc at level l4 - 5 , join at the circumaortic or collar left renal vein , or join the left common iliac vein , respectively ( fig . 1 , 2 ) . the mean age and follow - up duration of the 9 patients were 46.020.1 years ( range , 17 - 65 years ) and 32.820.1 months ( range , 15 - 69 months ) , respectively , and the male to female ratio was 5 to 4 . the urologic symptoms of the initial diagnosis were various ( microscopic hematuria : 5 of the 9 patients ; left flank pain : 4 of the 9 patients ; inguinal pain : 1 of the 5 male patients ; and gross hematuria : 1 of the 9 patients ) , and the concomitant diseases were upjo in 2 of the 9 patients and varicocele in 2 of the 5 male patients . the number of patients with type i , ii , iii , and iv of rlrv were 6 , 2 , 1 , and 0 patients , respectively . in type ii , all patients had upjo and 1 of the patients had varicocele . one of these patients underwent laparoscopic pyeloplasty , and the other patient underwent laparoscopic nephrectomy owing to a nonfunctional kidney with varicocelectomy . symptoms in the five patients with microscopic hematuria continued , whereas 1 patient with gross hematuria had resolved gross hematuria after nephrectomy . the two patients with upjo who underwent pyeloplasty and nephrectomy were treated successfully , and in the other 2 patients , the pain spontaneously resolved . the symptoms in the one patient with varicocele also spontaneously improved ( table 1 ) . however , the microscopic hematuria was not resolved with conservative management for long - term follow - up . the development of the renal veins is a part of the complex developmental process of the ivc . the process starts from the fourth week of conception and ends at about the eighth week . the ivc is formed from a vast network of three pairs of parallel veins in communication . the posterior cardinal veins , the subcardinal veins , and the supracardinal veins are in the order of appearance . during the development of the ivc , there are anastomotic communications between the subcardinal and supracardinal channels that form a collar of veins encircling the aorta . if the dorsal portion of this collar persists , then the left renal vein is posterior to the aorta , forming a rlrv . the diagnostic methods for detecting ivc anomalies in previous reports were autopsy study , renal venography , color doppler ultrasonography , computed tomography , and magnetic resonance imaging . with recent advances in computed tomography technology , mdct has replaced conventional angiography and venography in most clinical conditions . mdct is a reliable , easily applicable , and noninvasive tool for demonstration of abdominal organs and vascular structures . in type i , the ventral preaortic limb of the left renal vein is obliterated , but the dorsal retroaortic limb persists and joins the ivc in the orthotopic position . type ii results from the obliteration of the ventral preaortic limb of the left renal vein , and the remaining dorsal limb turns into the rlrv . the left renal vein lies at the level of l4 to l5 and joins the gonadal and ascending lumbar veins before joining the ivc . this type is due to the persistence of subsupracardial and intersupracardial anastomoses and the dorsal limb of the left renal vein . if all small retroaortic veins that empty into the ivc are considered , the incidence of a circumaortic left renal vein could be as high as 16% . in type iv , the ventral preaortic limb of the left renal vein is obliterated , and the remaining dorsal limb becomes the rlrv . then , the rlrv courses obliquely and caudally behind the aorta to join the left common iliac vein . the incidences of left ivc , double ivc , and retrocaval ureter are 0.2 - 0.5% , 0.3 - 2.8% , and 0.1% . the incidences of rlrv type i , ii , iii , and iv are 0.3 - 1.9% , 0.4 - 0.9% , 1.5 - 8.7% , and 0.16% , respectively . rlrv type i , ii , iii , and iv in our cases occurred in 6 , 2 , 1 , and 0 patients , respectively . during the renal surgery , this kind of anatomical variation influences the technical feasibility of the operation . failure to recognize these anomalies the posterior " nutcracker phenomenon " occurs when a decreased space between the aorta and the vertebra causes compression of the rlrv . it is postulated that compression of the left renal vein leads to hematuria because of elevated pressure in the left renal vein , resulting in congestion of the left kidney and the venous communications [ 15 - 18 ] . it is well known that the gonadal , ascending lumbar , adrenal , ureteral , and capsular veins are potential collateral venous pathways in cases of renal vein compression or obstruction . in addition , vascular dilatation of the afferent venous system can result from increased drainage pressure , which men can suffer from left - sided varicocele and women from pelvic congestion syndrome [ 19 - 21 ] . in our series , 3 patients with microscopic hematuria had type i , 1 had type ii , and 1 had type iii . symptoms continued in all patients with microscopic hematuria , maybe because of increased pressure in the renal vein , that is , the posterior nutcracker phenomenon . in two patients with varicocele , symptoms spontaneously improved in one patient ( type i ) , and the other patient ( type ii ) resolved his problem after nephrectomy . in the two cases of type ii with a left - sided upjo , it was postulated that dilatation of the renal pelvis led to the upjo directly posterior contacting the dilated left gonadal vein ( fig . one patient with partial obstruction underwent pyeloplasty because of severe left flank pain , and the other patient underwent nephrectomy for a nonfunctioning kidney . in adult upjo , the incidence of aberrant vessels is 35 - 39% [ 22 - 24 ] . three of 12 patients were related to dilated gonadal vessels and 1 patient had rlrv type iii . cho et al reported the congenital absence of the ivc as a rare cause of pulmonary thromboembolism . gibo and onitsuka reported a case of rlrv that showed left renal vein hypertension by pullback pressure measurement from the rlrv to the ivc . therefore , congenital anomalies of the venous system might easily induce the congestion of the renal bloodstream , and these anomalies might be the predisposing conditions of clinical symptoms such as hematuria , flank pain , and pelvic congestion . unawareness of this situation during retroperitoneal surgery can result in bleeding , nephrectomy , and even death . surgeons prefer the left renal vein in renal transplantation because of its longer length . because of this , it is important to know the course of the left renal vein and whether it is pre - aortic or not . it is also important to be aware of anomalies of the renal vein for distinctive diagnosis of retroperitoneal tumors , retroperitoneal lymph node pathologies , and aortic dissection . before the renal surgery , careful reading of the preoperative rlrv imaging study helps to avoid fatal complications during the operation . this study was limited by the fact that it was performed retrospectively , and the data were analyzed in selected patients who had urologic symptoms . , we could not reveal the korean incidence , follow - up results , concomitant diseases , or a casual relationship between rlrv and urologic symptoms . it may sometimes cause hematuria , flank pain , and vascular dilations ( varicocele ) . the most common type of rlrv was type i. the microscopic hematuria due to rlrv is thought to be continued in the long - term follow - up . for patients with gross hematuria or flank and inguinal pain , individualized treatment such as conservative care , pyeloplasty , varicocelectomy , and nephrectomy widespread use of diagnostic mdct in retroperitoneal diseases , particularly kidney tumors , can identify changes in the renal vascularization more easily , and thus allows urologists to plan a safe and less complicated surgery .
over the past few decades , percutaneous coronary intervention ( pci ) has become one of the most common medical procedures for patients with coronary artery disease . the benefits of pci are mainly attributable to a reduction of myocardial ischemia . however , noninvasive tests of myocardial ischemia are limited by their poor sensitivity in localizing the lesions . fractional flow reserve ( ffr ) is a pressure - wire based index used during the invasive procedure to identify myocardial ischemia . ffr is now considered as the gold standard for evaluating the functional significance of coronary stenosis . despite increasing evidence demonstrating the utility of ffr in different patient subsets , indeed , the operator 's decision whether or not to use ffr in real - world practice is often based on angiographic findings , which often provide inaccurate data for stenosis . meanwhile , revascularization in patients with coronary artery disease is among the most common major medical procedures in china . approximately , 2.5 million chinese suffer from myocardial infarction , generating hospitalization costs of rmb 4.9 billion yuan as assessed in 2012 . unfortunately , studies assessing the benefits of ffr in chinese real - world practice are scarce . in addition , the use of ffr is still limited in china due to the lack of financial support . hence , this study aimed to test the hypothesis that the use of ffr is associated with improved outcome and reduced cost in chinese real - world clinical practice . this was a retrospective study using the database of second affiliated hospital of zhejiang university , a tertiary and high - volume center in china . ffr measurement was first introduced in this center in 2010 , and patients were recorded in a registry starting july 2010 . therefore , consecutive patients referred for coronary revascularization with adjunct ffr between july 2010 and july 2014 were included in this study . a matched cohort design was used , with the control group matched for gender , age ( in 5-year age bands ) , date of admission ( in 3-month bands ) and gensini score , at a ratio of 1:1 . exclusion criteria comprised myocardial infarction within 5 days of pci ; previous coronary bypass surgery ( coronary artery bypass grafting ) ; cardiogenic shock , rheumatic heart disease , and congenital cardiovascular diseases ; serious concomitant disease , reduced life expectancy ( < 2 years ) ; pregnancy in women . this study was approved by the institutional review board and carried out according to the principles of the declaration of helsinki . invasive coronary angiography was performed using 47 f guiding catheters through the radial or femoral approaches . patients receiving a stent were pretreated with clopidogrel ( 300600 mg ) and aspirin ( 100300 mg ) . then , patients were prescribed statins , lifelong aspirin use , and clopidogrel for 36 months ( after implantation of bare metal stents ) to 12 months ( after implantation of drug - eluting stents ) after pci . the intracoronary pressure of intermediate lesions was measured with a coronary pressure wire ( st . pci was performed in patients with ffr < 0.75 , and deferred in those with ffr > 0.80 . for patients with ffr values between 0.75 and 0.80 , patients were clinically followed up at 1 , 6 , and 12 months , and annually thereafter , via office visits or telephone contact . the primary outcome measure during follow - up was major adverse cardiac events ( maces ) , defined as composite of death from any causes , myocardial infarction , any repeat revascularization , and hospitalization for angina . the study population was analyzed according to the different diagnostic strategies ( ffr and angiography ) used . continuous variables were presented as mean standard deviation ( sd ) or median ( interquartile range [ iqr ] ) . the cost of events during follow - up was presented as median ( min , max ) . group comparisons were assessed using student 's two - sample t - test or the mann - whitney u - test for continuous variables , and pearson 's test or the fisher 's exact test for categorical data . survival curves were constructed using a multivariate cox proportional hazards model , after adjusting for age , gender , coronary risk factors ( hypertension , hypercholesterolemia , current smoker , diabetes mellitus , body mass index , family history ) , and gensini score . this was a retrospective study using the database of second affiliated hospital of zhejiang university , a tertiary and high - volume center in china . ffr measurement was first introduced in this center in 2010 , and patients were recorded in a registry starting july 2010 . therefore , consecutive patients referred for coronary revascularization with adjunct ffr between july 2010 and july 2014 were included in this study . a matched cohort design was used , with the control group matched for gender , age ( in 5-year age bands ) , date of admission ( in 3-month bands ) and gensini score , at a ratio of 1:1 . exclusion criteria comprised myocardial infarction within 5 days of pci ; previous coronary bypass surgery ( coronary artery bypass grafting ) ; cardiogenic shock , rheumatic heart disease , and congenital cardiovascular diseases ; serious concomitant disease , reduced life expectancy ( < 2 years ) ; pregnancy in women . this study was approved by the institutional review board and carried out according to the principles of the declaration of helsinki . invasive coronary angiography was performed using 47 f guiding catheters through the radial or femoral approaches . patients receiving a stent were pretreated with clopidogrel ( 300600 mg ) and aspirin ( 100300 mg ) . then , patients were prescribed statins , lifelong aspirin use , and clopidogrel for 36 months ( after implantation of bare metal stents ) to 12 months ( after implantation of drug - eluting stents ) after pci . the intracoronary pressure of intermediate lesions was measured with a coronary pressure wire ( st . pci was performed in patients with ffr < 0.75 , and deferred in those with ffr > 0.80 . for patients with ffr values between 0.75 and 0.80 , patients were clinically followed up at 1 , 6 , and 12 months , and annually thereafter , via office visits or telephone contact . the primary outcome measure during follow - up was major adverse cardiac events ( maces ) , defined as composite of death from any causes , myocardial infarction , any repeat revascularization , and hospitalization for angina . the study population was analyzed according to the different diagnostic strategies ( ffr and angiography ) used . continuous variables were presented as mean standard deviation ( sd ) or median ( interquartile range [ iqr ] ) . the cost of events during follow - up was presented as median ( min , max ) . group comparisons were assessed using student 's two - sample t - test or the mann - whitney u - test for continuous variables , and pearson 's test or the fisher 's exact test for categorical data . survival curves were constructed using a multivariate cox proportional hazards model , after adjusting for age , gender , coronary risk factors ( hypertension , hypercholesterolemia , current smoker , diabetes mellitus , body mass index , family history ) , and gensini score . baseline characteristics in the two groups were similar : age , gender , coronary risk factors , prior pci , prior myocardial infarction , left ventricular ejection fraction , clinical presentation , and discharge medications except clopidogrel . sd : standard deviation ; ffr : fractional flow reserve ; bmi : body mass index ; pci : percutaneous coronary intervention ; ace inhibitor : angiotensin - converting enzyme inhibitor ; arb : angiotensin ii receptor blocker . angiography and procedural characteristics of patients are summarized in table 2 . in total , 366 patients undergoing ffr were matched with 366 individuals to be treated with angiography - guided pci . the number of indicated lesions and severity of coronary artery disease ( as indicated by gensini scores ) were similar between both groups . a total of 24.9% , 36.6% and 38.5% of patients in the angiography group had three- , two- and one - vessel disease , respectively ; meanwhile , values of 24.6% , 35.8% and 39.6% were obtained for the ffr group ( p > 0.05 ) . however , more patients had left anterior descending artery stenosis in the ffr group ( p < 0.05 ) . in addition , the number of patients that underwent pci was significantly reduced in the ffr - guided arm ( 34.4% vs. 63.1% , p < 0.001 ) ; idem for the mean number of implanted stents and treated lesions , although similar numbers of lesions were obtained in both groups . interestingly , the overall stent length needed was shorter in the ffr - guided group . finally , significant differences in multi - vessel stenting were observed in the angiography - guided pci group compared with patients treated with ffr - guided pci ( 6.3% vs. 1.1% , p < 0.001 ) . sd : standard deviation ; ffr : fractional flow reserve ; lm : left main ; lad : left anterior descending ; lcx : left circumflex ; rca : right coronary artery . economic evaluation for each strategy included costs of initial hospitalization as well as events during follow - up . in this study , guiding catheters , regular wires , pressure wires , balloon dilatation catheters , and stents were taken into consideration . the cost of events during follow - up was calculated for the actual consumption of mace by determining death from any causes , myocardial infarction , any repeated revascularization , and hospitalization for angina . the overall costs of initial hospitalization in the ffr group were similar with those obtained for the angiography group ( rmb 33,000 yuan , ranging from rmb 7393 to 44,700 yuan vs. rmb 21,200 yuan , ranging from 19,100 to 47,100 , p = 0.54 ) . however , significantly reduced costs for mace during follow - up were observed in the ffr - guided arm compared with the angiography group ( rmb 0 yuan , ranging from rmb 0 to 95,181 yuan vs. rmb 0 yuan , ranging from rmb 0 to 102,401 yuan , p < 0.001 ) . costs of ffr and angiography strategies ( rmb , yuan ) * guide catheter , guidewire , pressure wire , balloon catheter , stents and other materials used at initial procedure ; death from any causes , myocardial infarction , any repeat revascularization , and hospitalization for angina ; the sum of the overall costs of initial hospitalization and the cost of events during follow - up . median follow - up durations were 13 months and 13.5 months in the angiography and ffr groups , respectively , indicating no significant difference . the number of patients lost to follow - up were 21 ( 5.7% ) in the angiography group and 16 ( 4.4% ) in the ffr group . of the 804 deferred lesions in the ffr group , 10 ( 1.24% ) were revascularized during follow - up . particularly , among 235 deferred patients in the ffr group , 3 ( 1.27% ) had myocardial infarction ( one patient died ) and 5 ( 2.13% ) repeated revascularizations . cox multivariable model estimates of mace ( 4.9% vs. 12.0% , p < 0.001 ) and hospitalization for angina ( 4.9% vs. 11.2% , p = 0.001 ) at 4 years showed lower values in the ffr arm compared with patients treated with angiography - guided procedure [ figure 1 ] ; on the other hand , the rates of mortality , myocardial infarction , and repeated revascularization were comparable between the two groups [ table 4 ] . ( a ) primary outcome of death from any causes , myocardial infarction , any repeated revascularization , and hospitalization for angina ; ( b ) death from any causes , myocardial infarction , and any repeated revascularization ; ( c ) any repeated revascularization ; ( d ) hospitalization for angina . clinical outcomes between july 2010 and october 2014 * adjusted for age , gender , bmi , smoking history , diabetes , hypertension , hypercholesterolemia , family history , and gensini score . sd : standard deviation ; iqr : interquartile range ; ffr : fractional flow reserve ; hr : hazard ratio ; ci : confidential interval ; mace : major adverse cardiac event ; mi : myocardial infarction ; bmi : body mass index . baseline characteristics in the two groups were similar : age , gender , coronary risk factors , prior pci , prior myocardial infarction , left ventricular ejection fraction , clinical presentation , and discharge medications except clopidogrel . sd : standard deviation ; ffr : fractional flow reserve ; bmi : body mass index ; pci : percutaneous coronary intervention ; ace inhibitor : angiotensin - converting enzyme inhibitor ; arb : angiotensin ii receptor blocker . angiography and procedural characteristics of patients are summarized in table 2 . in total , 366 patients undergoing ffr were matched with 366 individuals to be treated with angiography - guided pci . the number of indicated lesions and severity of coronary artery disease ( as indicated by gensini scores ) were similar between both groups . a total of 24.9% , 36.6% and 38.5% of patients in the angiography group had three- , two- and one - vessel disease , respectively ; meanwhile , values of 24.6% , 35.8% and 39.6% were obtained for the ffr group ( p > 0.05 ) . however , more patients had left anterior descending artery stenosis in the ffr group ( p < 0.05 ) . in addition , the number of patients that underwent pci was significantly reduced in the ffr - guided arm ( 34.4% vs. 63.1% , p < 0.001 ) ; idem for the mean number of implanted stents and treated lesions , although similar numbers of lesions were obtained in both groups . interestingly , the overall stent length needed was shorter in the ffr - guided group . finally , significant differences in multi - vessel stenting were observed in the angiography - guided pci group compared with patients treated with ffr - guided pci ( 6.3% vs. 1.1% , p < 0.001 ) . sd : standard deviation ; ffr : fractional flow reserve ; lm : left main ; lad : left anterior descending ; lcx : left circumflex ; rca : right coronary artery . economic evaluation for each strategy included costs of initial hospitalization as well as events during follow - up . in this study , guiding catheters , regular wires , pressure wires , balloon dilatation catheters , and stents were taken into consideration . the cost of events during follow - up was calculated for the actual consumption of mace by determining death from any causes , myocardial infarction , any repeated revascularization , and hospitalization for angina . the overall costs of initial hospitalization in the ffr group were similar with those obtained for the angiography group ( rmb 33,000 yuan , ranging from rmb 7393 to 44,700 yuan vs. rmb 21,200 yuan , ranging from 19,100 to 47,100 , p = 0.54 ) . however , significantly reduced costs for mace during follow - up were observed in the ffr - guided arm compared with the angiography group ( rmb 0 yuan , ranging from rmb 0 to 95,181 yuan vs. rmb 0 yuan , ranging from rmb 0 to 102,401 yuan , p < 0.001 ) . costs of ffr and angiography strategies ( rmb , yuan ) * guide catheter , guidewire , pressure wire , balloon catheter , stents and other materials used at initial procedure ; death from any causes , myocardial infarction , any repeat revascularization , and hospitalization for angina ; the sum of the overall costs of initial hospitalization and the cost of events during follow - up . median follow - up durations were 13 months and 13.5 months in the angiography and ffr groups , respectively , indicating no significant difference . the number of patients lost to follow - up were 21 ( 5.7% ) in the angiography group and 16 ( 4.4% ) in the ffr group . of the 804 deferred lesions in the ffr group , 10 ( 1.24% ) were revascularized during follow - up . particularly , among 235 deferred patients in the ffr group , 3 ( 1.27% ) had myocardial infarction ( one patient died ) and 5 ( 2.13% ) repeated revascularizations . cox multivariable model estimates of mace ( 4.9% vs. 12.0% , p < 0.001 ) and hospitalization for angina ( 4.9% vs. 11.2% , p = 0.001 ) at 4 years showed lower values in the ffr arm compared with patients treated with angiography - guided procedure [ figure 1 ] ; on the other hand , the rates of mortality , myocardial infarction , and repeated revascularization were comparable between the two groups [ table 4 ] . ( a ) primary outcome of death from any causes , myocardial infarction , any repeated revascularization , and hospitalization for angina ; ( b ) death from any causes , myocardial infarction , and any repeated revascularization ; ( c ) any repeated revascularization ; ( d ) hospitalization for angina . clinical outcomes between july 2010 and october 2014 * adjusted for age , gender , bmi , smoking history , diabetes , hypertension , hypercholesterolemia , family history , and gensini score . sd : standard deviation ; iqr : interquartile range ; ffr : fractional flow reserve ; hr : hazard ratio ; ci : confidential interval ; mace : major adverse cardiac event ; mi : myocardial infarction ; bmi : body mass index . fame and fame2 assessing the value of ffr - guided pci demonstrated reduced rates for maces . however , data regarding the effect of ffr - guided pci in real - world practice are sparse in china . the current study , to our knowledge , examined the largest number of patients treated with ffr - guided pci in china . this cohort study , with median follow - up of 13 months revealed the benefits of ffr - guided pci in an actual patient population . ffr - guided pci , compared with the strategy using angiography , significantly reduced the rate of the primary composite outcome ( mace ) during follow - up . the presence and extent of inducible ischemia are the most important prognostic factor among patients with coronary artery disease . ffr - guided pci probably reduces the rate of hospitalization for angina by allowing a more judicious use of stents and equal relief of ischemia . on the other hand , the high rate of stent implantation deferral obtained after ffr measurement can help avoid the risk of thrombosis , restenosis , and unexpected device - related diseases associated with stent placement . while the primary composite end point was improved by ffr , secondary end points , including death , myocardial infarction , and repeated revascularization , showed no significant differences , in agreement with a recent large cohort study . a recent observational study from the mayo clinic also detected no survival benefit for ffr - guided pci using cox multivariable models . suggested that ffr - guided pci may have a stent sparing effect , which does not translate into a survival benefit . although ffr is now considered as the gold standard for evaluating the functional significance of coronary stenosis , its use is still limited . park et al . reported that routine measurement of ffr in daily practice appears to be associated with improved clinical outcomes , where ffr use during pci increased from 1.9% to 50.7% in 4 years , suggesting that irregular measurement of ffr may weaken the survival benefit of ffr - guided pci in real - world practice . pci is performed without documentation of inducible ischemia and is associated with high economic costs . subgroup analysis of the fame study revealed that ffr - guided pci results in significant cost - savings by reducing stent use , rehospitalization rate , and mace . similarly , the fame 2 sub - study showed an incremental cost - effectiveness ratio of $ 36,000 perquality - adjusted life - year for ffr - guided pci . in the fame study , universal stenting of lesions with angiographic stenosis > 50% can not be justified in real - world practice , let alone economic grounds . in the current study , ffr use did not seem to decrease the overall costs of initial hospitalization . among 366 patients in the angiography - guided group , 134 ( 36.6% ) hence , the overall costs of initial hospitalization of pci - deferred patients are higher in the ffr - guided group , weakening the benefit of fewer stents used . in the subsequent years , mean costs for maces during follow - up were higher in the angiography - guided arm ( p < 0.001 ) . the sample size was determined based on ffr volume in a single - site , and a retrospective matched cohort study has limitations inherent to nonrandomized trials . in addition , coronary lesions were assessed visually , rather than by quantitative coronary angiography . finally , we had no detailed information on costs of resources used at initial procedure ; idem for secondary preventive medications during follow - up . in conclusion , ffr - guided pci in daily practice is associated with the implantation of fewer stents , and improved clinical outcome , which is primarily due to decreased hospitalization for angina . in addition , the ffr - guided strategy also reduced costs during follow - up .
lack of effective cancer treatments and the emergence of drug resistance in progressively growing tumors make cancer immunotherapy a viable alternative to treat and manage metastatic diseases . synthetic peptide - based vaccines that are capable of inducing specific t cell - mediated immunity are emerging as attractive therapeutic strategies for cancers . while such vaccines have proven rather effective in diverse animal studies , multiple human clinical trials implementing this approach have thus far yielded limited success [ 25 ] . although many of these trials were able to elicit significant number of tumor antigen - specific t cells in cancer patients , clinical regressions of disease remain rare . although incompletely understood , the lack of clinical efficacy for peptide - based vaccines may be related to several factors such as poor immunogenicity of the chosen peptides , insufficient numbers or appropriate functional polarity of responder t cells , inefficient trafficking of effector cells into tumor microenvironment , and , more importantly , tumor - induced increase in the number of immunosuppressive regulatory cells . thus , there remains considerable room for improvement in cancer vaccine design in order to maximize the chances of clinical benefit to the patients . many studies have identified highly diverse subset of tumor infiltrating leukocytes , both with pro- and antitumor functions , among lymphocyte , nk cells , macrophages , and neutrophils . naturally occurring and inducible cd4cd25foxp3 t regulatory lymphocytes ( treg ) , suppressive cytokine - secreting tr1 cells , however , these cells can also block anti - tumor / antimicrobial immune responses . particularly in the context of cancer , treg / tr1-cell frequencies and function are important because increased numbers might dampen anti - cancer immune responses and favor tumor growth [ 810 ] . in addition to treg and tr1 cells , myeloid derived suppressor cells ( mdscs ) have been shown to be associated with increased tumor growth [ 1113 ] . recent evidence suggests that endogenous regulatory / suppressor cells invoked in the tumor - bearing state may be largely responsible for preventing effective antitumor immune responses . this paradoxical observation of tumor growth in the face of large numbers of activated , circulating tumor - reactive t cells suggests that their antitumor efficacy is actively attenuated in vivo . evidence for this idea has been accumulating rapidly during the past decade , fueled by the discovery of a number of different immune - regulatory cell types , soluble factors , and mechanisms that limit the strength and duration of anti - tumor immune responses . nevertheless , it has been well recognized that the detection of immune cells infiltrating tumors , such as memory / effector cd8/cd4 t cells or treg , correlated with the prognosis in certain types of cancers [ 7 , 9 , 15 , 16 ] . a successful cancer vaccine , therefore , needs to enhance tumor - specific protective immune responses despite the presence of suppressor cells . previously , we have demonstrated that a vaccine platform technology , vaccimax , can be used to therapeutically deliver antigens and effectively eradicate established tumors in a mouse hpv16 and melanoma tumor model [ 1719 ] . more recently , we have developed a new , clinic friendly liposome and oil - based delivery platform called depovax ( dpx ) that is well suited for delivering antigen with limited immunogenicity potential . dpx-0907 is a human dpx - based vaccine , containing 7 hla - a2-restricted peptides indicated for breast , ovarian , and prostate cancer [ 20 , 21 ] . dpx - based vaccines are known to induce strong peptide - specific immune response in preclinical studies in mice and , unlike gm - csf containing conventional emulsion ( ce ) vaccines , do not promote proliferation of treg / tr1 cells following vaccination . however , the status of tumor - induced immune - suppressor cells in tumor - bearing mice during dpx - vaccine - induced tumor suppression has not been studied previously . the present study examines ( i ) the levels of immune - regulatory cells in blood and spleen during hpv16-c3 tumor progression and dpx - vaccine - induced tumor suppression and investigates the extent of tumor infiltration by such suppressor cells ; ( ii ) using c3 tumor bearing hla - a2 transgenic mice with increased frequency of suppressor cells , this study also evaluates the ability of human dpx-0907 vaccine to induce effective cancer peptide - specific immune responses . the results indicate that dpx - based vaccine - induced tumor suppression is also accompanied with reduced suppressor cell accumulation within the tumor tissue and that this vaccine platform can induce potent immune response in tumor - induced immune - suppressive environment . female c57bl/6 mice , 68 weeks of age , were obtained from charles river laboratories ( wilmington , pa ) . hla - a0201/h2d ( aad ) transgenic mice were obtained from jackson laboratories ( bar harbor , me ) and were bred in house . these aad transgenic mice express a chimeric mhc class i molecule containing human 1 and 2 domains from human hla - a0201 and the transmembrane / cytoplasmic 3 domain from mouse h2d . mice were housed under filter - top conditions and supplied with water and food ad libitum . mice were implanted s.c . with full - length hpv16 transfected , 0.5 10 c3 tumor cell line in the left flank as described earlier . this tumor has been used as a model for epithelial malignances and the mice protected by vaccination are resistant to rechallenge . tumor growth was monitored every 4 - 5 days and tumor size was recorded until termination of experiments at 5 to 6 weeks after implantation . all peptides were synthesized by neomps - polypeptide ( san diego , ca ) at > 95% purity . hpv16 e7 ( h-2d ) peptide rahynivtf4957 ( r9f ) containing ctl epitope was fused to padre containing cd4 t helper epitope to generate the fusion peptide ( fp ) . dpx - based vaccine containing fp peptide and adjuvant was designated as dpx - e7 . human cancer peptide vaccine dpx-0907 containing hla - a2-restricted , seven breast / ovarian / prostate cancer - associated peptides ; their source proteins ; peptide locations within each protein ; and methods used to identify these ctl epitopes have been described recently by karkada et al . the modified tetanus toxin peptide a16l ( aqyikanskfigitel830844 ) , with an alanine residue added to its amino terminus to enhance stability , was used as a t helper ( th ) epitope . vaccines were formulated either as a conventional emulsion in montanide isa51 or as a proprietary depovax formulation . briefly , for dpx-0907 , liposomes were formulated by mixing phosphatidylcholine and cholesterol in a 10 : 1 ratio ( w : w ) ( lipoid gmbh , germany ) with the seven bo peptides ( 50 g each per dose ) , a16l ( 25 g / dose ) , and a proprietary polynucleotide adjuvant ( 20 g / dose ) in phosphate buffer ( 50 mm , ph 7.4 ) . similarly , dpx - e7 was prepared using fp ( 5 g / dose ) and the adjuvant ( 20 g / dose ) during liposome preparation . the liposomal solution was lyophilized and then resuspended in montanide isa51 ( seppic , france ) just before vaccination . control emulsion ( ce ) vaccine was prepared by mixing the seven bo peptides , a16l , and 5 g / dose of gm - csf , and by emulsifying the mixture with montanide isa51 using a 3-way stopcock according to manufacturer 's recommendations . similarly , control ce vaccine for dpx - e7 was prepared with corresponding dose of fp and gm - csf . six days following tumor implantations on the left flank and when the tumor is established , mice were immunized s.c . with vaccines in dpx or ce formulations or with pbs as control injections on the right flank near the base of the tail . injection was given as a single dose in most experiments or as two doses with one week interval for ce formulation . subsequently , 3 weeks and 5 weeks after implantation , blood , spleen , draining lymph nodes ( dln ) , and tumor tissues were harvested from mice . for assessing immune response in tumor - bearing aad mice to peptides in dpx-0907 these tumor implanted aad mice showed comparable tumor size as in pbs injected control c57bl/6 mice at the time dpx-0907 was injected . unless stated otherwise , all antibodies for immunofluorescence staining and flow cytometry were obtained from ebioscience ( san diego , ca ) . three - color staining was performed for detecting treg cells ; briefly , cells were surface - stained with anti - cd4 and anti - cd25 antibodies followed by intracellular staining for foxp3 using a staining kit ( ebioscience ) . mdscs in blood and spleen were detected using two - color staining using antibodies against cd11b and gr1 . il-10/tgf- secreting tr1 cells in the spleen were identified by intracellular staining of fixed / permeabilized cd4+/cd8 + t cells using anti - mouse - il-10 or anti - tgf- antibodies ( r&d systems , minneapolis , mn ) , after the cells were stimulated with peptide r9f for 48 h. to identify antigen - specific cd8+/cd4 + t cells producing ifn- following dpx-0907 injection of aad mice , two - color intracellular staining was performed . dln cells were cultured overnight in a 24-well plate with peptide - loaded dendritic cells ( dcs , see below ) at 10 : 1 ratio and 10 g / ml peptides at 37c in 5% co2 . on the following day , golgistop ( bd bioscience ) was added to each well and the plates were incubated for additional 4 hours . surface and intracellular staining of fixed / permeabilized cells was performed as described previously . stained cells were collected on a facscalibur flow cytometer ( bd bioscience ) and the data analysis was done using winlist 6.0 software ( verity software house , topsham , me ) . cells were isolated from the femur bone marrow of nave aad transgenic mice and cultured in the presence of gm - csf to generate mature dcs over eight days as described . mature dcs were loaded with 10 g / ml of peptide by incubation at 37c for 18 h. activated antigen - specific t cells were detected in the draining lymph nodes of mice using standard ifn- elispot ( bd bioscience ) modified as dc - based elispot assay . pma ( 5 ng / ml ; sigma ) and ionomycin ( 1 g / ml ; sigma ) served as positive controls , while stimulation with irrelevant peptide ( svydffvwl , t2p-2180188 , s9l ) and media alone served as negative controls . the plates were developed on the following day for detecting ifn- secreting cells using standard method and spots were enumerated by three independent personnel or through automated elispot plate reader . to study the effect of mdscs on t cell activation in normal and tumor - bearing mice , tumor infiltrating mdscs were enriched to > 95% purity using macs column ( miltenyi biotech gmbh , germany ) . single cell suspension of tumor - derived cells were treated with biotinylated anti - gr1 antibody , washed , and treated with streptavidin microbeads before sorting on macs column . single cell suspensions from ln of normal mice or tumor - dln of dpx - e7 or pbs injected mice with large tumors were prepared on week 5 after implantation . dln cells were stimulated using plate bound anti - cd3 antibodies , in the presence of 0.5 g / ml anti - cd28 antibody , 20 u / ml il2 , and 20 ng / ml il12 . cell stimulation was carried out for three days in the presence and absence of 1 : 10 ratio of tumor derived mdscs to dln cells . effect of mdscs on t cell activation was measured by the ability of these cells to generate ifn- using intracellular cytokine staining of cd8 t cells as described above . to analyze tumor infiltrating cells , matched volumes of tumor tissues from different groups of mice were homogenized and single cell suspensions were adhered to plastic dishes for 2 hours at 37c , and 50 l nonadherent cells were used to prepare cytospin slides . in the initial experiments , single color staining for cd8 or cd25 was performed on cytospin preparations of tumors from dpx - e7 , ce , and pbs control groups . to identify functionally relevant , glycoprotein - a - repetitions - predominant- ( garp- ) expressing activated treg cells , slides were double stained with anti - cd25-fitc and anti - garp - pe antibodies . to visualize regulatory cells within tumors , tumor tissues were snap - frozen in liquid nitrogen , sectioned , acetone - fixed , and stained for treg and mdscs using combinations of anti - cd4/anti - cd25 and anti - cd11b / anti - gr1 antibodies , respectively . statistical analyses were performed using a student 's unpaired t - test , with p values < 0.05 considered significant . tumor take and tumor growth kinetics for c3 tumors in c57bl/6 mice has been described earlier . aad transgenic mice , which have the same background , also exhibited similar tumor growth kinetics ( data not shown ) . as shown in figure 1(a ) , by week 5 after implantation , pbs control mice developed a mean tumor size of nearly 1000 mm and ce - immunized mice had the tumors in the range of 200400 mm size . in contrast , dpx - e7-immunized mice showed good tumor inhibition with a small percentage of mice developing tumor volume of 100 mm . mean tumor volume was not significantly different between dpx - e7 and ce - vaccinated groups , but control mice had significantly larger tumors compared to both groups of vaccinated mice . since tumor volume is measured only in surviving mice , to get a better picture on vaccine efficacy we determined tumor free mice in each group . dpx - e7-vaccinated group had most tumor free mice ( > 80% ) while about half the mice in ce vaccine group did develop tumors , and all the mice in pbs control group showed tumor growth ( figure 1(b ) ) . similar differences between groups of mice in tumor size / tumor - free status were also seen at week 3 after implantation albeit with lesser tumor volume . to investigate tumor - induced changes in treg cells in blood and spleen , percentage of cd4cd25foxp3 treg lymphocytes increased significantly ( p < 0.02 ) in non - vaccinated pbs control mice compared to nave mice ( figure 2(a ) ) . in contrast , level of treg cells remained significantly lower in dpx - e7 immunized mice over non - vaccinated control mice and was comparable with nave mice . there was some increase in treg cells in ce - vaccinated mice but was not significantly different from pbs control or tumor - free nave mice . however , at three weeks after implantation , level of treg cells in ce - vaccinated mice was as high as that seen in pbs group and was significantly higher than dpx - e7-vaccinated and nave mice ( p < 0.01 , data not shown ) . similar to the observations in the spleen , circulating treg cells in the blood also showed increases in pbs control mice and in mice vaccinated using ce formulation ( figure 2(b ) ) . although mean frequency of treg was relatively higher in pbs control mice the differences were not statistically significant as compared to nave mice . however , mice injected with ce vaccine showed significantly higher treg cells in blood compared to dpx - e7 vaccinated mice ( p < 0.05 ) and the level of treg in the latter group was significantly lower than pbs control mice ( p < 0.01 ) . similar to treg cells , cd11bgr1 mdscs showed significant increases in pbs injected , tumor - challenged control mice , both in spleen ( p < 0.01 , figure 3(a ) ) and blood ( p < 0.01 , figure 3(b ) ) . although , conventional vaccine injected mice had some increase in mdscs levels in the spleen , and more noticeably in the blood , the differences were not statistically significant . in contrast , level of mdscs in dpx - e7 vaccinated mice remained very close to what was seen in tumor - free nave mice . mice that received ce - vaccine also had higher gr1 + cells in the circulation , but the difference was not significant . in order to assess the ability of tumor infiltrating mdscs to inhibit t cell effector function , enriched mdscs from large tumors were co - cultured during anti - cd3 activation of tumor - dln cells . in the absence of mdscs , cd8 t cell activation was slightly decreased by about 25% in dpx - e7 immunized mice compared to tumor - free nave mice ( figure 4 , left panel ; 13.5% versus 9.1% ) . in contrast , ability of tumor dln cd8 t cells from non - vaccinated mice to secrete inf- was markedly compromised ( 13.5% versus 3.8% ) . however , in the presence of tumor - derived mdscs , in vitro cd8 t cell activation was almost completely inhibited in all the three groups of mice tested ( figure 4 , right panel ) . we further investigated both cd4 and cd8 t cells from the spleen for their ability to secrete antigen - induced inhibitory cytokines such as il10 and tgf- in c3 tumor challenge model . as shown in figure 5(a ) , at week 5 of tumor growth , frequency of il10 secreting cells , among both cd4 and cd8 t cells , increased significantly in pbs - treated control mice . interestingly , vaccination using ce platform also resulted in significantly higher il10 cd4 and cd8 t cells compared to nave mice . in contrast , antigen - stimulated spleen cells from dpx - e7 vaccinated mice showed only a marginal and insignificant increase in il10cd4 cells and did not show any increase in il10cd8 cells . unlike il10 secreting cd4 t cells , we were not able to detect cd4tgf- cells in all groups of animals studied ( figure 5(b ) ) . however , a small but significantly higher proportion of cd8 cells in both pbs control mice and ce - vaccinated mice showed intracellular tgf- compared to mice immunized with dpx - e7 . no tgf- was detected in cd8 t cells from nave mice . in order to assess the extent of tumor infiltrating cd8 , treg , and mdscs , tumor tissues from different groups of mice this was done either by isolating cells from tumor tissue for cytospin preparation for immunofluorescence staining ( cd8/treg ; figure 6 ) or by direct staining of snap - frozen tumor sections ( treg / mdscs ; figure 7 ) . as shown in figure 6(a ) , higher cd8 t cells were found to infiltrate tumors from dpx - e7 immunized mice compared to tumors from ce - vaccinated or pbs control mice ( upper panel ) . in contrast , staining with anti - cd25 antibodies suggested least cd25 t cell infiltration of tumors in dpx - e7 vaccinated group compared to tumors from both ce - vaccinated and control mice ( figure 6(a ) , lower panel ) . further , to identify tumor infiltrating treg cells as functionally relevant regulatory cells , cells were double stained with anti - garp and anti - cd25 antibodies . tumors from non - vaccinated pbs control mice had much higher tumor infiltrating cd25garp treg cells compared to dpx - e7-treated tumor - bearing mice ( figure 6(b ) ) . tumor tissues from vaccinated and non - vaccinated mice were also snap - frozen , sectioned , and double stained to identify treg and mdscs in situ . for treg cells , since anti - garp antibody was not compatible to use on frozen / processed / fixed tissues , we had to rely on staining for cd4cd25 t cells . nevertheless , tumors from dpx - e7 vaccinated mice showed considerably less cd4cd25 double positive cells compared to non - vaccinated mice ( figure 7(a ) ) . similarly , dpx - e7 treatment also resulted in much reduced cd11bgr1 mdscs infiltration of tumors compared to non - vaccinated pbs control mice ( figure 7(b ) ) . although no attempts were made to positively identify blood vessels in the tissue sections , presenting morphology appears to suggest accumulation of mdscs in the perivascular tissues , probably migrated out of tumor vasculature ( v ) . type 1 cd8 t cell responses are crucial to the ability of the host to prevent the development and progression of cancer [ 2 , 25 ] . however , it is also critical that vaccines used for immunotherapy need to elicit such cd8 t cell response in cancer - bearing host , most of them with concurrently active immune - suppressive mechanisms . hence , we assessed the efficacy of dpx-0907 in eliciting ifn- producing cd8 and cd4 t cells within vaccine dln 8 days after immunization of late - stage tumor bearing aad mice . as controls , tumor - free aad mice were immunized in parallel . at week five after tumor implantation , aad mice showed similar tumor growth kinetics as c57/bl6 mice ( as in figure 1 ) with comparable increase in the percentages of spleen treg ( 26.8 3.1 versus 14.9 2.4 in naive , p < 0.02 ) and mdscs ( 79.1 9.2 versus 43.3 6.4 in nave , p < 0.001 ) . dpx-0907-induced , peptide - specific immune responses were determined by dc - based elispot detection of ifn- cells in dln and by intracellular staining for inf-cd8 and ifn-cd4 t cells . following dpx-0907 vaccination , both tumor - bearing and tumor - free aad mice showed strong and equivalent number of cancer peptide - specific ifn- secreting cells in the dln , as seen in elispot assay ( figure 8(a ) ) . in both groups of mice , the peptide - specific immune response was > 10-fold higher compared to unstimulated background values . when the peptides are injected in ce formulation , although elispot response was lower than dpx-0907 treated groups , the differences were not statistically significant . intracellular cytokine staining showed that percentages of inf-cd8 t cells from both tumor - free and tumor - bearing , vaccinated mice were much higher than nave mice ( figure 8(b ) ) , and there was no significant difference in ifn-cd8 t cells between tumor free and tumor bearing mice . intracellular ifn- response in cd4 t cells was also analyzed , and as shown in figure 8(c ) , cd4 cells from dpx-0907-vaccinated tumor - bearing mice and tumor - free mice showed equivalent percentages of ifn- cells . unlike elispot results , ce - vaccinated group had significantly lower ifn--secreting cd8 and cd4 t cells compared to dpx-0907 vaccinated , tumor bearing mice . tumor microenvironment , influenced largely by the growing tumor , is dominated by tumor - induced interactions with host tissues and cells . although it has been well documented that cancer patients generate antitumor effector t cells , either endogenously as part of anti - cancer immune surveillance or as a result of cancer immunotherapy , tumor inhibition is often hindered by mechanisms that block t cell functions [ 9 , 10 , 1214 , 26 ] . many cancer researchers believe that the efficacy of immune therapy against established tumors depends on the effector functions of recruited lymphocytes within the tumor bed . hence , the ability to block tumor escape from immunological targeting depends largely on a better understanding of the cellular and molecular pathways within the tumor microenvironment . in this regard , tumor - induced immune - regulatory / suppressor cells and their recruitment into tumor tissue appear to have a major role in neutralizing antitumor effector mechanisms [ 10 , 13 , 14 ] . the present study examines association of regulatory / suppressor cells during the growth inhibition of hpv16-e7 expressing c3 tumors by a potent vaccine platform , dpx - e7 . results presented here indicate that , in this model , tumor inhibition is also accompanied by decreased treg / tr1/mdscs frequency in the peripheral tissue and reduced infiltration of the tumor by such cells . in addition to type-1 cd8 t cells playing a crucial role in the efficacy of immunotherapy for cancer patients , th1 cd4 t cells also play a role in regulating antitumor protective efficacy , either by enhancing ifn--mediated increase in mhc class i expression and antigen - presenting capacity of dc and macrophages leading to the formation of effector / memory cd8 t cells or by dampening the immune response via the action of regulatory t cells [ 14 , 27 ] . however , in many instances , the lack of correlation between the frequency of antigen - specific t cells and prevention of tumor growth suggests that efficacy depends on functional quality of vaccine - activated t cells [ 5 , 28 ] . in many human cancers such as cervical , ovarian , melanoma , lymphoma , gastrointestinal , and head and neck cancer , level of tumor infiltrating cd8 versus cd4foxp3 treg cells provides strong prognostic factor ( reviewed in [ 7 , 10 ] ) . however , improved prognosis is lost if concurrent accumulation of treg is also observed in tumor biopsies along with tumor - specific cd8 t cells [ 9 , 15 , 29 ] . in parallel , many studies in mouse models show that depleting treg cells or reducing their suppressive activity improves spontaneous or immunotherapy - mediated tumor clearance . cd4cd25 natural treg cells that represent approximately 5% to 10% of peripheral cd4 cells , constitutively express cd25 ( il-2r ) , glucocorticoid - induced tumor necrosis factor receptor ( gitr ) , ctl antigen 4 ( ctla-4 ) , and the transcription factor forkhead box p3 ( foxp3 ) . recent phase 3 clinical trial with ipilimumab blockade of ctla-4 has shown some clinical benefits in stage iii / iv melanoma patients , suggesting the potential of suppressor cell inhibition in promoting endogenous anti - tumor immune responses . the exact mechanism of action of treg cells is still under debate , as is the nature of their antigen recognition . however , it should be noted that tumor antigen - specific treg cells occur naturally in certain metastatic cancers [ 31 , 32 ] and can also be induced by vaccination . as shown in this study , tumor infiltrating treg cells were at their lowest level in dpx - e7 vaccinated group , which also had much lower tumor burden . two possible explanations can be put forward for enhanced efficacy of dpx - based vaccine platform . firstly , if a proportion of suppressor cells are induced by growing tumor itself , their number is reduced in dpx - e7-treated mice over ce - treated mice because of effective control of tumor volume in the former group . secondly , it is possible that a proportion of regulatory cells are antigen driven , as has been shown in non - vaccinated and hpv16 peptide vaccinated cervical cancer patients [ 34 , 35 ] . dpx - based vaccines use lyophilized liposomes containing the antigen ( s ) and adjuvant , which are used as carriers that allow for the incorporation of hydrophilic antigens and adjuvant directly into an oil medium . moreover , these vaccines form a depot of antigen / adjuvant at the site of injection and gradually release antigens and adjuvant to be taken up by antigen - presenting cells that are shown to accumulate around the vaccine injection site . it is plausible that , unlike ce vaccinated mice , gradual antigen uptake and processing in dpx - e7 group would allow strong type 1 response without enhancing antigen - driven regulatory cell responses . enhanced suppressor / regulatory cell expansion could be associated with high antigen exposure over short periods of time . furthermore , use of gm - csf as adjuvant in ce formulation , to mimic the most common approach in designing human cancer vaccine delivery system in recent years , may also lead to increased treg / mdscs proliferation . it is interesting to note that the immunosuppressive strength among mdscs subsets , during cd8 t cell activation , is determined by gm - csf . recently , we made similar observations using human cancer peptide immunization of aad mice using ce formulation . the regulatory and immune - suppressor cells associated with cancers are diverse , and quite often mere expression of certain phenotypic markers does not necessarily parallel their inhibitory functions . in this regard , the present study attempted to recognize functionally relevant regulatory and suppressor cells by using anti - garp antibodies to recognize activated treg cells . it has been well documented that expression of garp is necessary for their suppressive function , possibly related to its role as cell surface receptor for tgf- and its ability to induce foxp3 expression [ 24 , 37 ] . mdscs recruitment to tumor tissues most likely results in the inhibition of effector cd8 t cells within tumor microenvironment . although we have not tested the inhibitory function of mdscs derived from a noninflammatory tissue , the finding of strong suppressive function of / inflammatory site - derived mdscs is in agreement with a recent report . we are currently investigating the status of mdscs / treg in breast , ovarian , and prostate cancer patients receiving dpx-0907 and survivin - targeted dpx - survivac peptide cancer vaccine in ovarian cancer patients . it may be feasible to correlate clinical benefits of such dpx - based cancer vaccines and their influence on the frequency of suppressor cells in these patients . similar studies with simultaneous determination of immune response and suppressor cell analysis would validate the ability of dpx - based vaccines to specifically induce potent anti - tumor immune response . in preclinical studies dpx-0907 however , its ability to induce comparable immune response in tumor - bearing mice with expanded suppressor cell populations has not been investigated previously . this aspect is important since many cancer patients with minimal residual disease , even after chemotherapy and radiation , are expected to be in immune - suppressed state . an effective therapeutic vaccine is expected to induce effective immune response under such adverse conditions . current study using tumor - bearing hla - a2 transgenic mice shows that dpx-0907 , a potent adjuvented vaccine platform , is able to induce antigen - specific immune response which is not compromised in the presence of tumor - induced immune - suppressor cells . although c3 tumor model is unrelated with respect to dpx-0907 included antigens , the nonspecific nature of immune suppression would have played an inhibitory role , had dpx-0907 been not a potent vaccine platform . nevertheless , inclusion of other drugs aimed at reducing suppressor cell frequency , in combination of dpx - based vaccines , might alleviate the negative effect of such cells within the tumor bed . efforts are currently underway to assess dpx-0907 in a mouse tumor model that expresses both hla - a2 and dpx-0907 included antigen(s ) . in a therapeutic setting , these findings are expected to provide insight into the effectiveness of dpx - based vaccines in immune - suppressed cancer patients . the present study provides additional information on tumor - induced suppressor cells and the advantages of using dpx - based vaccines over ce - based vaccines as a means of immunotherapy . results from on - going clinical studies with survivin - targeted cancer vaccine dpx - survivac and from animal studies in relevant tumor - bearing hla - a2 transgenic mice are expected to contribute to our understanding of the mechanisms involved in enhanced protection provided by this vaccine platform . full comprehension on the role of treg / mdscs and other suppressor cells might facilitate the development of successful intervention strategies for the immunotherapy of cancer with long - term efficacy .
the use of natural and artificial substances has increased during the past few decades , which indicates the incidence of a new problem in physical and social health . the term addicted individual can be defined as one who has a very strong desire toward the use of addictive substances , regardless of their consequences ( 1 ) . therefore , any type of confrontation and intervention in this field needs to be conducted after extensive investigation . in research on substance dependence , in addition to determining the different aspects of this problem , there are attempts to study and discover the variables that predict these disorders . cash and pruzinsky ( 2 ) and some other authors ( 3 - 5 ) have reported several risk factors related to substance abuse such as family conflict ; educational problems ; weakness in social relations ; and psychological disorders , including depression , anxiety , and personality disorders . furthermore , other significant factors are emotional problems , inability in the cognitive process of emotional information , and emotion - regulation problems ( alexithymia ) ( 6 , 7 ) . alexithymia is a multifaceted construct consisting of difficulty in recognizing and describing emotions , difficulty in distinguishing between emotions and bodily tensions related to emotional excitement , and difficulty in expressing feelings for others . individuals with this emotional problem are aware of their emotional experience but can not distinguish the type of emotion . although the prevalence rate of alexithymia in the general population is reported at about 10% , there are higher levels of this emotional problem in disorders such as depression , anxiety , pain disorders , sexual disorders , educational problems , procrastination , substance abuse , and alcoholism ( 7 - 12 ) . consequently , the focus of the current study was the role of alexithymia in substance abuse and its correlation with other variables linked to substance dependence disorder . one of the most important variables allied to substance abuse is metacognition beliefs , which include the beliefs that individuals harbor about their thoughts . metacognition refers to the beliefs and attitudes of an individual about cognitive events like thoughts , emotions , memories , feelings , and other perceptual forces ( 13 ) . according to the metacognition theory , substance abuse is an effective means of swiftly modifying cognitive events such as emotions , thoughts , and memories . according to berthoz et al . ( 9 ) , swart et al . ( 14 ) , and bahramnejad et al . ( 1 ) , the relationship between emotion and substance abuse may be mediated by metacognition . along the same line , saed et al . ( 13 ) , in their research on substance addicts , found that metacognitive factors , thought suppression , and negative emotions were the predictors of substance dependence . likewise , hoffman and spatariu ( 15 ) , stated that in their study population , comprising individuals with severe emotional and cognitive deficits , there was an extreme tendency toward dangerous behaviors . according to two studies by spada et al . ( 16 , 17 ) , emotion and cognition were the key factors in success and mental health and reducing psychological problems and risky behaviors like substance abuse . ( 18 ) found that addicts with alexithymia showed less interest in self - seeking and analytical / cognitive activities . ( 19 ) and stanghellini and ricca ( 20 ) also showed that deficit in cognitive processing and emotional order and the failure to differentiate internal feelings from physical emotions were reasons for the tendency toward substance abuse in individuals with emotional disorders . in addition to metacognition , body image is another psychological factor related to alexithymia ( 21 ) . body description and image encompass individuals perceptions concerning their body and their attitudes toward thoughts , ideas , emotions , and behaviors ( 22 ) . body image influences the individual s mental / social quality of life , and many reactions of the individual depend on the imagination and the image that the individual has in his / her mind . any negative attitude toward self - causes feelings of inadequacy and inability in various aspects of physical and mental health ( 23 , 24 ) . researchers in one previous study ( 25 ) mentioned that body dissatisfaction can lead to a belief of imaginary defects in the body and that it finally renders the individual unable to cope with emotions and feelings and creates severe emotional problems . cash and pruzinsky ( 2 ) and nye and cash ( 6 ) believed that body dissatisfaction was a major concern inasmuch as it caused depression , obsession , and tendency to drug and suicide among their study populations . petrides and furnham ( 23 ) showed that deviated body image was able to influence psychological and physical health . continuous dissatisfaction from body image is likely to lead to mental disorders such as anxiety , depression , mental distress , severe emotional problems , and tendency to drug abuse ( 22 , 23 , 26 , 27 ) . research indicates that individuals who are competent regarding experience , recognition , and emotion are better equipped to adapt and show reasonable and logical reactions , while individuals with alexithymia tend to have difficulty in identifying and regulating their emotions . additionally , alexithymia renders individuals prone to magnify their normal body emotions , misinterpret these emotions , and exhibit helplessness through physical complaints and high - risk behaviors , all of which are deemed risk factors for psychosomatic and mood disorders . in light of the above - mentioned findings , we sought to investigate the role of metacognition and body image with respect to alexithymia in individuals with substance abuse . the aim of this study was to determine the role of metacognition and body image in predicting alexithymia in substance abusers . in this research , metacognition and body image were considered as the predictor variables and alexithymia as the criterion variable . the community of the study included all addicts referring to the addiction treatment and counseling centers of three iranian cities of zahedan , sari , and neyriz between 2013 and 2014 . a sample of 175 subjects , comprised of 19 ( 10.7% ) females and 81 ( 87.6% ) males , were selected by random sampling . the instruments were the metacognitive strategy questionnaire ( mcq-30 ) , toronto alexithymia scale ( tas-20 ) , and physical self - description questionnaire ( psdq ) . the mcq-30 , designed by wells and cartwright - hatton ( 28 ) , consists of 30 items to which each participant can respond with one of the four options of disagree , somewhat agree , agree , and completely agree . metacognition is made up of the following subscales : 1 ) positive anxiety beliefs ; 2 ) beliefs regarding uncontrollability and risk ; 3 ) beliefs about cognitive qualifications ; 4 ) negative anxiety beliefs ; and 5 ) cognitive self - awareness . cronbach s alpha coefficient of this questionnaire is reported from 0.72 to 0.89 , and its test - retest reliability coefficient ranges from 0.76 to 0.80 with the correlation coefficient of the metacognitive questionnaire between 0.26 and 0.73 according to the anxiety scale of spielberger et al . shirinzadeh dastgiri et al . ( 30 ) measured cronbach s alpha coefficient of this questionnaire for an iranian sample at 0.91 for the total scale and at 0.71 to 0.87 for the subscales . furthermore , after 4 weeks , its test - retest reliability coefficient was 0.73 for the total scale and 0.58 to 0.87 for the subscales . ( 18 ) , is a self - assessment questionnaire with 20 items to assess alexithymia . this questionnaire has three dimensions : 1 ) difficulty in the diagnosis and detection of emotions ( 7 items ) ; 2 ) difficulty in expressing emotions ( 5 items ) ; and 3 ) focusing on outside experiences ( 8 items ) . the questions are based on a 5-point likert scale ranging from 1 = completely agree to 5 = completely disagree ( 31 ) . cronbach s coefficient for alexithymia in the farsi version was 0.85 , and it stood at 0.82 , 0.75 , and 0.72 for the three subscales of difficulty in identifying emotions , difficulty in expressing feelings , and extrinsic thinking , respectively , all of which are good internal consistencies ( 31 ) . ( 32 ) calculated cronbach s alpha coefficient for an iranian sample population at 0.74 for difficulty in identifying emotions , at 0.61 for difficulty in expressing feelings , and at 0.50 for extrinsic thinking ( 32 ) . shahgholian et al . ( 33 ) measured the reliability of the total scale for an iranian sample using split - half and test - retest at 0.74 and 0.72 , respectively , and the validity of the scale at 0.85 . the psdq is a multidimensional , physical self - concept instrument designed to measure 11 scales , viz . strength , body fat , activity , endurance / fitness , sports competence , coordination , health , appearance , flexibility , physical self - concept , and global self - esteem . the revised form of this questionnaire includes 47 phrases and has good reliability and reproduction capacity . body self - description includes 9 specific factors for body concept and self - esteem . self - description includes 6 or 8 phrases and is presented such that the subject answers on a 6-point scale from right to wrong . marsh ( 34 ) studied the validity and reliability of the psdq with two samples , including 315 and 395 high - school students , and observed that this test had the required validity for detecting self - concept factors and that its reliability was 0.80 by cronbach s alpha . ( 35 ) , reported test - retest reliability of ( padq ) about 0.78 . this questionnaire was reexamined by abdolmaleki et al . ( 36 ) , who reported its reliability at 0.87 . the mcq-30 , designed by wells and cartwright - hatton ( 28 ) , consists of 30 items to which each participant can respond with one of the four options of disagree , somewhat agree , agree , and completely agree . metacognition is made up of the following subscales : 1 ) positive anxiety beliefs ; 2 ) beliefs regarding uncontrollability and risk ; 3 ) beliefs about cognitive qualifications ; 4 ) negative anxiety beliefs ; and 5 ) cognitive self - awareness . cronbach s alpha coefficient of this questionnaire is reported from 0.72 to 0.89 , and its test - retest reliability coefficient ranges from 0.76 to 0.80 with the correlation coefficient of the metacognitive questionnaire between 0.26 and 0.73 according to the anxiety scale of spielberger et al . shirinzadeh dastgiri et al . ( 30 ) measured cronbach s alpha coefficient of this questionnaire for an iranian sample at 0.91 for the total scale and at 0.71 to 0.87 for the subscales . furthermore , after 4 weeks , its test - retest reliability coefficient was 0.73 for the total scale and 0.58 to 0.87 for the subscales . the tas-20 , first introduced by bagby et al . ( 18 ) , is a self - assessment questionnaire with 20 items to assess alexithymia . this questionnaire has three dimensions : 1 ) difficulty in the diagnosis and detection of emotions ( 7 items ) ; 2 ) difficulty in expressing emotions ( 5 items ) ; and 3 ) focusing on outside experiences ( 8 items ) . the questions are based on a 5-point likert scale ranging from 1 = completely agree to 5 = completely disagree ( 31 ) . cronbach s coefficient for alexithymia in the farsi version was 0.85 , and it stood at 0.82 , 0.75 , and 0.72 for the three subscales of difficulty in identifying emotions , difficulty in expressing feelings , and extrinsic thinking , respectively , all of which are good internal consistencies ( 31 ) . ( 32 ) calculated cronbach s alpha coefficient for an iranian sample population at 0.74 for difficulty in identifying emotions , at 0.61 for difficulty in expressing feelings , and at 0.50 for extrinsic thinking ( 32 ) . shahgholian et al . ( 33 ) measured the reliability of the total scale for an iranian sample using split - half and test - retest at 0.74 and 0.72 , respectively , and the validity of the scale at 0.85 . the psdq is a multidimensional , physical self - concept instrument designed to measure 11 scales , viz . strength , body fat , activity , endurance / fitness , sports competence , coordination , health , appearance , flexibility , physical self - concept , and global self - esteem . the revised form of this questionnaire includes 47 phrases and has good reliability and reproduction capacity . body self - description includes 9 specific factors for body concept and self - esteem . self - description includes 6 or 8 phrases and is presented such that the subject answers on a 6-point scale from right to wrong . marsh ( 34 ) studied the validity and reliability of the psdq with two samples , including 315 and 395 high - school students , and observed that this test had the required validity for detecting self - concept factors and that its reliability was 0.80 by cronbach s alpha . based on previous studies , khanjani et al . ( 35 ) , reported test - retest reliability of ( padq ) about 0.78 . the mean and standard deviation of the variables are depicted in table 1 , and the coefficients of the variables are given in table 2 . according to the results in table 1 , the mean of positive beliefs about worry and cognitive confidence ( 14.13 ) was higher than that of the other metacognition strategies . values are presented as mean standard deviation . the relationship between metacognitive beliefs , body image and alexithymia as the correlation matrix shows , alexithymia was not correlated with body image , but there was a positive correlation between metacognitive beliefs and alexithymia . the highest correlation was between alexithymia and cognitive awareness ( r = 0.305 ; p < 0.01 ) . furthermore , there were no significant correlations between alexithymia and the subscale of risk uncontrollability . multiple regression analysis was used to determine the effects of the common and specific variables ( metacognitive strategies and body image ) on predicting alexithymia . according to table 3 , in the first stage , the factor of cognitive awareness was entered in the model , and it showed the most significant correlation . in the subsequent stages , positive beliefs about worry , need to control thoughts , and cognitive confidence were respectively entered in the model . multiple regression analysis was used to test whether metacognition and body image significantly predicted the participants alexithymia ; it indicated the three predictors explained 11% of the variance ( r = 0 . 11 , f = 3.981 ; p < 0.01 ) ( tables 3 and 4 ) . the results also demonstrated that cognitive awareness significantly predicted 9% of the variance ( = 0.305 ; p < 0.01 ) and that the other subscales predicted about 2% . in the present study , the findings arising from the relationship between metacognition and alexithymia in the substance abusers were concordant with the studies of cash and pruzinsky ( 2 ) , bagby et al . ( ( 3 ) , lane et al . ( 4 ) , nye and cash ( 6 ) , and dubey et al . ( 7 ) , showing that impaired metacognitive beliefs in their study populations prompted non - beneficial coping strategies , leading to cognitive interactions and ineffective emotional and behavioral reactions forming the basis and context for addiction . also , abbasi et al . ( 37 ) , swart et al . ( 14 ) , saed et al . ( 13 ) , besharat ( 31 ) , hoffman and spatariu ( 15 ) , and bagby et al . ( 18 ) reported that substance abuse was an effectual means of swiftly modifying cognitive events such as feeling , expression , and regulation of emotion in alexithymia . furthermore , hoffman and spatariu ( 15 ) identified emotion and cognition as key factors in the success of mental health and reducing risk behaviors such as substance abuse and psychological problems . our multiple regression analysis results showed that out of all the metacognitive beliefs , cognitive awareness was the best predictor for alexithymia and also for the tendency toward addiction . it can , thus , be argued that metacognitive beliefs and especially the metacognitive beliefs concerning cognitive awareness can cause a tendency toward using drugs by affecting the individual s emotion controlling power and decision - making and causing them to use non - adaptive coping strategies . one probable reason for the emotional problems of the addicts in the current study is body image insofar as individuals perception of their body size and fitness is associated with their thinking and feeling about other people s views of their body ( 38 , 39 ) . ( 40 ) , mohamadi and sajadinejad ( 41 ) , and spangler and stice ( 42 ) , there was no relationship between alexithymia and body image in our sample . it is important to note that in contrast to our study , most of the previously conducted investigations such as those by adami et al . ( 36 ) , moss and harris ( 45 ) , von soest et al . ( 27 ) , phillips ( 46 ) , frederick et al . ( 47 ) , and zojaji et al . ( 48 ) took into account body image perception ( body size ) and psychological problems such as obesity , anxiety , and depression . in addition , most previous studies considered psychosomatic and mood disorders as the most important risk factors for alexithymia ( 27 , 36 , 39 , 43 - 48 ) . we sought to determine the role of metacognition and body image in predicting alexithymia in substance abusers ; however , further research is required to shed sufficient light on this issue . given the concerns regarding the harmful effects of substance abuse and addiction and the old adage prevention is the best treatment , it is vitally important that the causes of tendency toward addiction be identified so that more effective preventive strategies can be devised to assist health care practitioners in the field of primary prevention .
clinical reasoning ( cr ) is one of the most crucial competencies that a physician should achieve . most of specialty societies , licensing bodies and medical schools consider it in the list of competencies that their graduates should attain and they try to ensure its mastery in the learners . many factors may influence ( cr ) as a construct and many researchers try to investigate factors that may improve , predict and illustrate it . although , many factors may affect cr , because of scarcity in time and resources in this study only critical thinking ( ct ) , personality , and emotional intelligent ( ei ) will be studied in depth . concerning the role of ct as a general competency in higher education , many educational workers argue that effective and meaningful education requires that all aspects of curricular and pedagogical strategies should be coordinated to promote cognitive skills associated with ct . they believe that educating students as critical thinkers is fundamental for students and their society . many resources necessitate ct as a crucial competency for nursing education and its association with cr has been magnified . on the other hand , association of ct and cr researches conducted about ei in the last decade , e.i . remains a controversial topic . three main models of ei exist . the first model by peter salovey and john mayer perceives e.i . as a form of pure intelligence , and as a cognitive ability . a second model introduced by reuven bar - on , which regards e.i . as a mixed intelligence , consisting of cognitive ability and personality aspects the third model , introduced by daniel goleman , also perceives e.i . as a mixed intelligence involving cognitive ability and personality aspects . it is found to be a predictor of life satisfaction , academic achievement , and healthy psychological adaptation , positive interactions with peers and family , and higher parental warmth . lower ei has also been found to be associated with violent behavior , illegal use of drugs and alcohol , and participation in delinquent behavior meanwhile , several tests have been devised to measure ei . bar - on ( 2000 ) , however , recently revised his scale ; he now views the general mood factor as a facilitator of ei rather than a part of it . given the diverse factors that comprise the ei , it is important to know if the scales represent a one - dimensional or multidimensional construct . according to funder , personality refers to an individual 's attribute patterns of thoughts , emotions , and behavior . although , there are several different approaches to the study of personality , this paper will focus on the trait and type approach according to trait theories , traits define the nature of personality as well as determine the actual operation of personality . although , thousands of traits have been identified , costa and mccrae suggested that five traits are central to personality : neuroticism , extraversion , openness to experience , agreeableness , and conscientiousness . the selection of these five traits was based on studies that demonstrated that the five traits were the most useful and recurrent when rating personality . one strength of the big five is that the model has shown cross - cultural consistency in studies in which measures based on the model , such as the revised neo personality inventory neuroticism , extraversion , openness to experience personal inventory revised ( neo - pi - r ) , have been devised within one culture , and validated in other cultures . the neo - pi - r has been translated in to most western languages , as well as many asian languages including persian . divergent trace of correlation between personality traits and problem solving can be found . in a study , neuroticism trait of personality perceived as a predictor of problem solving . to date , little empirical research on the relation between cr , personality , ei and ct has been conducted . theoretical links between personality , ei , and ct have been proposed as all of them may influence problem solving . cr is a kind of problem solving in medicine , and all above constructs influence problem solving , so each of them may influence cr , too . the combined influence of personality domains , critical thinking , and emotional intelligence on clinical reasoning does not appear to have been studied . this research was conducted to investigate the relationship between cr , emotional intelligence ( ei ) , ct , and personality traits . the study was of a quantitative descriptive design , and surveyed 69 medical students ( 59% female and 41% male ) in two consecutive groups that enroll pediatrics clerkship rotations . age ranged from 18 to 21 ( m = 19.21 , sd = 2.32 ) years . the intent of this study was to assess the individual and combined influences of personality domains , ct , and ei on cr . the study also explored the relationships that exist between students personality traits , the cognitive abilities comprising ct , and the subscales associated with ei and cr . personality domains , ct , and ei considered as the independent variables , and cr constituted the dependent variable . all participants completed a battery of four measures assessing cr , ct , ei , and personality . all measures utilized in this study ( considering ct , ei and personality ) were chosen as they were available in farsi and their test characteristics were examined thoroughly . these measures were as follow . to measure clinical reasoning ( cr ) competency of medical students a battery test first , they were instructed about different test , then a blue - print devised by them and finally the battery test provided . this co - joint test was consisting of three sections : comprehensive integrated puzzle ( cip ) , key feature exam ( kf ) and clinical reasoning problems ( crp ) . these test formats have been studied in literature thoroughly . to devise a cr battery test , a table of specifications was arranged by faculty members in pediatrics department considering the results of needs assessment . columns were holding clinical problems ; age and sex of the patients , involved organ system , the context of presentation ( ambulatory , inpatient or emergency department ) , the field of clinical work ( information gathering , diagnosis and management ) and finally the type of exam ( cip , kf and crp ) . in the rows , different features of 42 clinical scenarios completed according to the different fields in columns based on the goal and objectives of the course . all scenarios were chosen based on the needs assessment that has been conducted to define most usual and life threatening clinical situations that general practitioners may face with . the faculties devised different part of the test using the protocols provided by the researcher . the left - hand column contains diagnoses or brief clinical vignettes . to complete the cells of the grid the student is required to match , stepwise , the various disciplinary investigations to the diagnoses or clinical vignettes . when the puzzle is completed each horizontal row reflects a coherent medical case . a key - feature problem consists of a clinical case scenario followed by questions that focus on only those critical steps . the questions can be presented to require examinees either to write in their responses or to select them from a list of options . for each question , examinees are instructed to supply or select whatever number of responses is appropriate to the clinical task being tested , and answer keys can comprise one or several responses . the last part of cr battery test consists of six challenging clinical reasoning problems ( crp ) . each problem consists of a clinical scenario comprising presentation , history , and physical examination . based on this information , subjects are asked to nominate the two most likely diagnoses and to list the clinical features that they consider in formulating their diagnoses , indicating whether these features supported or opposed the nominated diagnoses . for shortening the data , in this study , all parts of cr battery tests were added up and each student has a single mark in clinical reasoning . california critical thinking skills test ( cctst ) : a 34 multiple choices measure that assess ct in two different manners . each item has one credit and the maximum credit is 34 revised neo personality five factors inventory ( neo - f fi ) : a 60-item , self - reported questionnaire designed to measure five major personality dimensions . respondents indicate level of agreement with each item on a five point likert scale ( one = strongly agree , five = strongly disagree ) . this widely used inventory assesses the big five personality traits , namely neuroticism , extraversion , openness , agreeableness , and conscientiousness . extraversion / introversion dimension point out if a person like social activities or s / he prefers to act by her / himself alone . openness specifies how a person reacts with new experiments , new foods , and new places . the reliability and validity of the neo - pi - r is well established ( = 0.86 - 0.93 ) . two - year test - retest reliabilities ranged from.83 to.91 for domains and from.64 to.86 for facets . a recent article using the neo ffi to study perfectionism had the internal consistencies at n = 0.85 , e = 0.80 , o = 0.68 , a = 0.75 , c = 0.83 bar - on emotional intelligence questionnaire : a 90- item self - report measure . it requires individuals to rate the extent to which each statement represents them on a five point likert scale . the individual 's responses render a total eq score , as well as scores on the following 5 composite scales and 15 subscales : intrapersonal self - regardemotional self - awarenessassertivenessindependenceself - actualization interpersonal empathysocial responsibilityinterpersonal relationship stress management stress toleranceimpulse control adaptability reality testingflexibilityproblem solving general mood optimismhappiness self - regardemotional self - awarenessassertivenessindependenceself - actualization emotional self - awareness empathysocial responsibilityinterpersonal relationship social responsibility interpersonal relationship stress toleranceimpulse control reality testingflexibilityproblem solving to measure clinical reasoning ( cr ) competency of medical students a battery test were designed by faculties in pediatrics department . first , they were instructed about different test , then a blue - print devised by them and finally the battery test provided . this co - joint test was consisting of three sections : comprehensive integrated puzzle ( cip ) , key feature exam ( kf ) and clinical reasoning problems ( crp ) . these test formats have been studied in literature thoroughly . to devise a cr battery test , a table of specifications was arranged by faculty members in pediatrics department considering the results of needs assessment . columns were holding clinical problems ; age and sex of the patients , involved organ system , the context of presentation ( ambulatory , inpatient or emergency department ) , the field of clinical work ( information gathering , diagnosis and management ) and finally the type of exam ( cip , kf and crp ) . in the rows , different features of 42 clinical scenarios completed according to the different fields in columns based on the goal and objectives of the course . all scenarios were chosen based on the needs assessment that has been conducted to define most usual and life threatening clinical situations that general practitioners may face with . the faculties devised different part of the test using the protocols provided by the researcher . the left - hand column contains diagnoses or brief clinical vignettes . to complete the cells of the grid the student is required to match , stepwise , the various disciplinary investigations to the diagnoses or clinical vignettes . when the puzzle is completed each horizontal row reflects a coherent medical case . a key - feature problem consists of a clinical case scenario followed by questions that focus on only those critical steps . the questions can be presented to require examinees either to write in their responses or to select them from a list of options . for each question , examinees are instructed to supply or select whatever number of responses is appropriate to the clinical task being tested , and answer keys can comprise one or several responses . the last part of cr battery test consists of six challenging clinical reasoning problems ( crp ) . each problem consists of a clinical scenario comprising presentation , history , and physical examination . based on this information , subjects are asked to nominate the two most likely diagnoses and to list the clinical features that they consider in formulating their diagnoses , indicating whether these features supported or opposed the nominated diagnoses . for shortening the data , in this study , all parts of cr battery tests california critical thinking skills test ( cctst ) : a 34 multiple choices measure that assess ct in two different manners . each item has one credit and the maximum credit is 34 revised neo personality five factors inventory ( neo - f fi ) : a 60-item , self - reported questionnaire designed to measure five major personality dimensions . respondents indicate level of agreement with each item on a five point likert scale ( one = strongly agree , five = strongly disagree ) . this widely used inventory assesses the big five personality traits , namely neuroticism , extraversion , openness , agreeableness , and conscientiousness . extraversion / introversion dimension point out if a person like social activities or s / he prefers to act by her / himself alone . openness specifies how a person reacts with new experiments , new foods , and new places . the reliability and validity of the neo - pi - r is well established ( = 0.86 - 0.93 ) . two - year test - retest reliabilities ranged from.83 to.91 for domains and from.64 to.86 for facets . a recent article using the neo ffi to study perfectionism had the internal consistencies at n = 0.85 , e = 0.80 , o = 0.68 , a = 0.75 , c = 0.83 bar - on emotional intelligence questionnaire : a 90- item self - report measure . it requires individuals to rate the extent to which each statement represents them on a five point likert scale . the individual 's responses render a total eq score , as well as scores on the following 5 composite scales and 15 subscales : intrapersonal self - regardemotional self - awarenessassertivenessindependenceself - actualization interpersonal empathysocial responsibilityinterpersonal relationship stress management stress toleranceimpulse control adaptability reality testingflexibilityproblem solving general mood optimismhappiness self - regardemotional self - awarenessassertivenessindependenceself - actualization emotional self - awareness empathysocial responsibilityinterpersonal relationship social responsibility interpersonal relationship stress toleranceimpulse control reality testingflexibilityproblem solving descriptive statistics correlations ( available data using pair wise correlations ) between all measures are shown in table 2 . the reliability of this data was confirmed by comparing with list wise correlations , which revealed markedly similar outcomes . correlation matrix of all measures the results indicate that clinical reasoning is the most significant correlate of some ei subscales ( independence , stress tolerance , emotional self - awareness , reality testing , social responsibility , and assertiveness ) ; but there is low correlation between cr , ct and personality subscales . on the other hand , correlation coefficient for clinical reasoning and problem solving ( subscale of ei ) is only.07 . hierarchical regression analysis estimated shared variance attributable to each ei and personality measure . for each regression model , cr was dependent variable , with personality , ei , and critical thinking measure as independent variables ( step1 = critical thinking ; step 2 = personality ; step 3 = ei measure ) . results of all multiple regression s are summarized in table 3 , which shows the significant contribution to clinical reasoning by ei measures ( r change = 0.46 ) hierarchical regression of cr on ct , personality and ei correlations ( available data using pair wise correlations ) between all measures are shown in table 2 . the reliability of this data was confirmed by comparing with list wise correlations , which revealed markedly similar outcomes . correlation matrix of all measures the results indicate that clinical reasoning is the most significant correlate of some ei subscales ( independence , stress tolerance , emotional self - awareness , reality testing , social responsibility , and assertiveness ) ; but there is low correlation between cr , ct and personality subscales . on the other hand , correlation coefficient for clinical reasoning and problem solving ( subscale of ei ) is only.07 . hierarchical regression analysis estimated shared variance attributable to each ei and personality measure . for each regression model , cr was dependent variable , with personality , ei , and critical thinking measure as independent variables ( step1 = critical thinking ; step 2 = personality ; step 3 = ei measure ) . results of all multiple regression s are summarized in table 3 , which shows the significant contribution to clinical reasoning by ei measures ( r change = 0.46 ) hierarchical regression of cr on ct , personality and ei medical schools and licensing bodies need to understand the nature of this construct and predict it and find influential factors to improve it . this study has investigated the clinical reasoning and other pertaining variables including critical thinking , emotional intelligence , and personality . considering previous literature ct is a meta - cognition competency and is pre - requisite for cr . many studies , especially those pertaining clinical reasoning in nursing , considered cr and ct two interrelated variables , and suppose that ct predicts cr . the results in this study indicate that not only ct is not correlated to cr but also considering the regression model , ct does not contribute to cr variance . articles tried to evaluate critical thinking as a cognitive ability did not interpret it as a cognitive construct . it was presumed that some personality traits ( especially neuroticism ) may predict problem solving and consequently cr . in our study , cr is not only correlated to personality and its subscales , their contribution to cr variance was negligible , the results show that emotional intelligence subscales provide most contribution to cr variance . subscales ( such as problem solving , stress tolerance and self - awareness ) may predict cr variance considerably . clinical reasoning can be considered as a unique and specific construct that may not be predicted easily . although , the sample volume participated in this study may be not sufficient to provide significant results . it is suggested to conduct similar studies in learners with different level and larger samples .
cutaneous t - cell lymphoma ( ctcl ) is a rare group of mature t - cell lymphomas primarily presenting in the skin . between 2000 and 3000 new cases of ctcl occur in the us each year , with mycosis fungoides ( mf ) being the predominant subtype.1 skin manifestations of mf include patches , plaques , tumors , and erythroderma . mf is categorized as limited stage by plaques or patches limited to the skin ( ia , ib , and iia ) and advanced stage by cutaneous tumors and involvement of the blood , lymph nodes , bone marrow , or visceral organs ( iib to ivb).2 szary syndrome ( ss ) , the leukemic variant of mf , is characterized by generalized erythroderma and abnormal lymphoid cells in the blood.3 patients with limited stage disease frequently have an indolent clinical course and may be effectively treated with skin - directed therapies , including topical nitrogen mustard or psoralen plus ultraviolet a therapy.4 treatment of advanced stage disease includes systemic therapy with interferon , bexarotene , photopheresis , denileukin diftitox , vorinostat , cytotoxic chemotherapy , or combined modality therapies . this has led to a need to develop new therapies with targeted mechanisms of action and acceptable safety profiles . romidepsin ( istodax [ astellas pharma inc . , tokyo , japan ] , depsipeptide , fr901228 , fk228 ) is a promising new agent that has shown remarkable activity in the treatment of t - cell lymphomas in preclinical studies and early - phase clinical trials . in november 2009 , it was granted approval by the us food and drug administration ( fda ) for the treatment of ctcl in patients who have received at least one prior systemic therapy . it belongs to the new class of antineoplastic agents known as histone deacetylase inhibitors ( hdacis ) that are known to affect gene and protein function by epigenetic modulation . vorinostat , another hdaci , is approved in the us for the treatment of ctcl in patients who have failed at least two prior systemic therapies . hdacis have shown remarkable signal of activity in some t - cell malignancies such as ctcl and peripheral t - cell lymphoma , and several of these agents , including belinostat , panobinostat , and entinostat , are in various stages of clinical trials . romidepsin was isolated from the fermentation broth of a gram - negative , nonsporing coccobacillus chromabacterium violaceum strain wb968 by fujisawa pharmaceutical company in japan in an effort to identify compounds that can selectively reverse the phenotype of the ras transformed cells.5,6 romidepsin was found to have the ability to reverse the morphology of the ha - ras transformed cells to a normal phenotype . hence , it was isolated and purified and was found to be a colorless prism with the structure of ( e)-(1s , 4s , 10s , 21r)-7-[(z)-ethylidene]-4 , 21-diisopropyl-2-oxa-12 , 13-dithia-5 , 8 , 20 , 23-tetraazabicyclo [ 8 , 7 , 6]-tricos-16-ene 3,6,9,19,22-pentanone , as shown in figure 1 . nakajima et al showed that romidepsin caused the arrest of the cell cycle at both g1 and g2/m phases and the induction of internucleosomal breakdown of chromatin characteristic of apoptosis.7 they also demonstrated that it inhibited intracellular histone deacetylase activity , leading to marked accumulation of acetylated histone ( h1 , h2a , h2b , h3 , and h4 ) in the cell , and established its role as a novel hdaci . furumai et al demonstrated that romidepsin circulates in the serum in its inactive form and penetrates the cell membrane effectively given its hydrophobic nature.8 however , on entering the tumor cell , it gets converted to an active , reduced form ( redfk ) by intracellular antioxidants involving glutathione . it has been proposed that one of the sulfhydryl groups of the reduced form interacts with the zinc ions in the active site of the class i hdac enzymes , leading to potent inhibition of enzymatic activity of both class i and ii hdacs in vivo , but its activity against hdac4 and hdac6 class ii enzymes remains weak . the prodrug form of romidepsin has a half - life of > 12 hours , whereas the redfk form has a half - life of 0.54 hours . this clearly demonstrates that romidepsin is quite stable in the prodrug form in blood , which may be responsible for its strong in vivo hdac inhibition . it is now well established that modulation of histone and protein acetylation alters pathways that promote proliferation , angiogenesis , and survival in cancer cells . a common finding in malignant cells is the high level of expression of hdac isoenzymes and a corresponding hypoacetylation of histones . hdacis work through myriad different mechanisms , including i ) upregulation of cell - dependent kinase ( cdk ) inhibitors like p21/p27 and downregulation of cyclind1 ; ii ) acetylation of nonhistone proteins , including stat-3 , rela / p65 , p53 , hif-1 , bcl-6 , and hsp 90 , in a way that modulates their activity and thus their ability to regulate cell growth and survival ; iii ) direct activation of apoptotic pathways by affecting the balance between the antiapoptotic proteins like bcl-2 and the proapapototic proteins like bax and bak ; iv ) enhanced production of reactive oxygen species along with increased thioredosin levels ; v ) enhanced antitumor immunity through enhancement of trail or upregulation of antigen expression , which could facilitate cancer cell recognition ; and vi ) disruption of dna repair through acetylation or downregulation of proteins such as ku70 , ku86 , brca1 , and rad51.9,10 despite these pleiotropic effects , it has been difficult to assign a precise mechanistic basis to their anticancer effects in any particular tumor type , let alone ctcl . it is peculiar that romidepsin appears to have a class effect in ctcl in contrast to other subtypes of lymphoproliferative neoplasms . gene expression profiling on paired tissue samples and studies of select biomarkers , including gene activation with hdacis , has shown that up to 5%10% of the genome can be affected by hdacis . in one study , the genes that were consistently affected included genes affecting cell cycle ( ccndi , igfi ) , apoptosis ( septin10 , tef , sorbbs2 ) , angiogenesis ( gucy1a1 , angpt1 ) , and immune modulation ( lair1).11 romidepsin demonstrated potent cytotoxicity against human tumor cell lines and in vivo efficacy against both human tumor xenografts and murine tumors.5,6 many human tumors appear to be associated with the expression of activated ras genes . romidepsin was found to reverse the morphology of the ha - ras transfected mouse nih/3t3 ( ras1 ) cells to that of the parent , thereby suggesting inhibitory effects on the growth of tumor cells and potential as an anticancer agent.5 this was through a decrease in the mrna expression of the c - myc oncogene in ras1 cells , but it had no effect on h - ras mrna expression , implying various other potential mechanisms such as activation of the raf / mek / erk axis or the pi3k / akt / gsk-3 pathway , which needs validation in preclinical and correlative clinical studies . in vitro , it induced cell cycle arrest in both g1 and g2/m phases and apoptosis in a number of human tumor cell lines such as a549 , pc-9 lung adenocarcinoma , pc-1 and pc-10 lung squamous cell carcinoma , adh , lx-1 small cell lung carcinoma , mkn28 , mkn74 stomach adenocarcinoma , mcf-7 , zr-751 mammary adenocarcinoma , colo201 , and sw480 colon adenocarcinoma at low ng / ml concentrations.6 romidepsin exhibited weak cytoxicity against human normal endothelial cell he-9 , mouse fibroblast balb / c3t3 , and nih3t3 and demonstrated no activity against human and mouse normal fibroblast cells , thereby suggesting selectivity . other in vitro studies have revealed antiproliferative effects of romidepsin against b - cell chronic lymphocytic leukemia ( cll ) cell lines,12 t - cell lymphoma cells,13 and multiple myeloma cells.14 in vivo , it was found to prolong the life of mice bearing murine ascitic tumors , murine leukemias , and melanoma and inhibited the growth of murine solid tumors such as colon cancer , sarcomas , and human solid tumors implanted in normal and nude mice.6 when given intravenously every 4 days , it inhibited the growth of several human tumor xenografts such as mx-1 breast carcinoma , lc-6 lung carcinoma , lu-65 large cell lung carcinoma , and sc-6 stomach adenocarcinoma.6 this antitumor activity was confirmed by the national cancer institute ( nci ) , and the agent was assigned the nsc number nsc-630176 for further clinical development . romidepsin showed a lack of cross - resistance to several commonly used cytotoxic agents such as vincristine , 5-fluorouracil , mitomycin c , and cyclophosphamide and was identified as a p - glycoprotein ( pgp ) and multidrug resistance - associated protein ( mrp-1 ) substrate by xiao et al.15 it is well established that pgp and mrp-1 are associated with resistance to anticancer drugs . with the help of a romidepsin - resistant cell line ( hct15r ) developed from hct15 colon carcinoma , it was confirmed that efflux of romidepsin via pgp induction is the major mechanism for acquired romidepsin resistance . these data need to be considered when romidepsin is used to pretreat patients who are receiving pgp substrate drugs . this property also raises the possibility of combining romidepsin with pgp inhibitors in developing treatment strategies for patients with ctcl or using pgp inhibitors to reverse the resistance to romidepsin . as a substrate for pgp - mediated drug efflux , romidepsin would likely not penetrate into the central nervous system , as opposed to the other hdacis , which are not pgp substrates.16,17 johnstone et al demonstrated that romidepsin induced the accumulation of 4n dna content , a g2 cell cycle checkpoint in the acute t - cell leukemia cell line cem - ccrf , and caused apoptosis via activation of the intrinsic apoptotic pathway , which was accompanied by the cleavage and activation of caspase-2 and bid.18 romidepsin - induced mitochondrial damage and apoptosis were inhibited by overexpression of bcl-2 but not by the polycaspase inhibitor ( zvad - fmk ) . moreover , induction of a g1-s checkpoint through overexpression of p16 or suppression of de novo protein synthesis inhibited romidepsin - induced cell death . while romidepsin induced the activation of the proapoptotic bcl-2 protein , bid cleavage in response to romidepsin this effect has not been demonstrated with other hdacis such as oxamflatin or saha ( vorinostat ) , demonstrating the ability of these drugs to induce apoptosis in the absence of activation of certain caspases and highlighting functional differences between these structurally dissimilar compounds that are thought to have similar intracellular targets . it was also found that romidepsin was a substrate of pgp and hence less effective against pgp - expressing tumor cells as opposed to oxamflatin and saha . subsequently , johnstone et al used a e-myc mouse model of b - cell lymphoma and showed that romidepsin had the ability to kill e-myc lymphomas that overexpress bcl-2 with delayed kinetics of cell death and could mediate therapeutic responses against these lymphomas.19 of interest , they noted that e-myc cells that overexpressed bcl - xl were resistant to romidepsin as opposed to saha , thereby supporting the hypothesis that hdacis may have subtle yet potentially important differences in molecular activities . the current infusion schedule of administering romidepsin over 4 hours was based on the safety and efficacy of the 4-hour intravenous infusion over the 24-hour infusion observed in beagle dogs and rats , following which this infusion time was incorporated for further studies in phase i clinical trials.20,21 two phase i trials were carried out simultaneously to determine the maximum tolerated dose ( mtd ) and toxicity profile of the 4-hour infusion of romidepsin and to characterize its pharmacokinetics . in the first trial performed by investigators at the nci , the agent was given intermittently on a day 1 and day 5 schedule every 21 days.22 thirty - seven patients with advanced or primary refractory neoplasms were administered the drug at that schedule . based on the preclinical toxicology data , the starting dose of the trial was chosen as 1 mg / m , and dose escalations proceeded through a total of eight dose levels to a maximum of 24.9 mg / m . biologically active serum concentrations were achieved with this dose , and one partial response was observed . dose - limiting toxicities ( dlts ) included grade 3 fatigue ( three patients ) , grade 3 nausea and vomiting ( one patient ) , grade 4 thrombocytopenia ( two patients ) , and grade 4 cardiac arrhythmia ( one patient , atrial fibrillation ) . the mean volume of distribution , clearance , distribution half - life ( t1/2 ) , and elimination half - life ( t1/2 ) at a dose of 17.8 mg / m were 8.6 l / m , 11.6 l / h / m , 0.42 hours , and 8.1 hours , respectively . the mean maximum plasma concentration ( cmax ) at the mtd dose was 472.6 ng / ml . furthermore , romidepsin pharmacokinetics was not significantly different when comparing the first cycle of a dose level with a subsequent cycle of the same dose . as expected , an increased dose led to increased cmax and area under the curve ( auc ) . was not observed , and the dose suggested for phase ii was 17.8 mg / m administered on day 1 and day 5 of a 21-day cycle . the other phase i trial conducted at the nci utilized a different schedule in which romidepsin was administered as a 4-hour infusion given weekly for 3 weeks followed by 1 week of rest in 33 patients with advanced cancers , including colorectal , breast , bladder , sarcoma , and others . this trial established 14 mg / m as the mtd dose , as no significant cardiac or skin toxicity was observed with this dose and schedule.23 the dlts continued to be grade 3 thrombocytopenia and grade 3 fatigue , as seen in the earlier study . a romidepsin dose and schedule of 14 mg / m given on days 1 , 8 , and 15 of a 21-day cycle were established as the phase ii dose for further investigation . pharmacokinetic analysis was performed on 98 patients enrolled in the nci - sponsored phase ii multicenter clinical trial of romidepsin in patients with ctcl in whom the drug was administered at 14 mg / m or 18 mg / m on day 1 during their first treatment cycle.24 using a noncompartmental analysis for the first dose , the mean half - life ( 2.95 hours ) and observed clearance ( 9.6 h / m ) were comparable with previously reported parameters in the phase i studies . after a 4-hour infusion , romidepsin was rapidly eliminated from the circulation with a short half - life of 3.5 hours , as observed in other studies . owing to this rapid distribution and clearance , resulting in plasma concentrations below the limit of quantitation , terminal elimination rates ( kel ) could not be calculated . romidepsin is extensively metabolized in vivo , primarily by the cytochromes p450 ( cyp ) 3a4 , and to a lesser extent by the cyp3a5 , and eliminated through bile and excreted in feces.25,26 drug drug interactions to be considered when administering romidepsin include any strong cyp3a4 or cyp3a5 inhibitors , such as ketoconazole , which has been shown to significantly lower romidepsin metabolism in vitro . two - compartment population modeling was carried out to explore the effects of polymorphic variations in cyp3a4 , cyp3a5 , slco1b3 , and abcb1 , all of which encode genes thought to be involved in the metabolism of romidepsin . despite some minor interindividual variability , no statistical differences in romidepsin pharmacokinetic parameters were found between patients with wild - type genes and those with polymorphisms . pharmacodynamic studies have revealed the ability of serum from patients treated with romidepsin to induce cell cycle arrest and confirmed the histone acetylation increase in circulating peripheral blood mononuclear cells ( pbmcs).27 the maximal accumulation of acetyl h3 histones in pbmcs occurred at 4 hours after the end of the romidepsin infusion . other studies have also reported that in patients with acute myelogenous leukemia ( aml ) and cll treated with 13 mg / m of romidepsin intravenously on days 1 , 8 , and 15 , hdac inhibition and histone acetylation of at least 100% were observed , and the levels of h3 and h4 histone deacetylation were higher at the 4-hour time point than 24 hours in the cll and aml cell lysates . additionally , an increase in p21 expression occurred concurrently with h4 histone acetylation of the p21 promoter.28 in an attempt to find meaningful biomarkers of response to romidepsin , the molecular endpoints of romidepsin infusion were evaluated in 61 patients enrolled in the phase ii trial of romidepsin.29 they were identified as laboratory parameters modified by romidepsin and included histone h3 acetylation and abcb1 gene expression in pmbcs , abcb1 gene expression in tumor biopsy samples , and blood fetal hemoglobin ( hbf ) levels , all of which were increased following romidepsin treatment . a somewhat lower proportion of patients demonstrated increased abcb1 expression in pbmcs . when each surrogate was evaluated for association with either pk parameters or disease response , it was noted that the increase in histone acetylation measured in pbmcs at 24 hours correlated with cmax and auc and inversely correlated with clearance . in addition , histone acetylation in pbmcs at the 24-hour time point appeared to correlate with response , despite a lack of a statistically significant correlation between pk parameters and response . there was no correlation between the levels of abcb1 induction and pharmacokinetic parameters , nor was there an association between induction of abcb1 in biopsy specimens and disease response . in total , 60% of patients also had a greater than four - fold increase in circulating hbf detected in blood . although this was correlated with response , it was also associated with increased time on study . these data suggest that for romidepsin , drug exposure dependent on drug clearance and drug activation are important determinants of response , consistent with data from other hdacis . the first phase i clinical trial of romidepsin was conducted in 2001 at the nci by piekarz et al and included four patients with refractory t - cell lymphomas ( two patients with ss , one patient with tumor - stage ctcl , and one patient with unspecified peripheral t - cell lymphoma).30 the dosing schedule consisted of romidepsin administered at a dose of 12.7 mg / m or 17.8 mg / m as a 4-hour intravenous infusion on days 1 and 5 of a 21-day cycle , as described previously . to confirm biological activity , both patients with ss who were refractory to conventional chemotherapy agents had a rapid decrease in the percentage of circulating szary cells along with improvement in the skin erythema and edema following treatment with romidepsin . the first ss patient was removed from the study due to persistent methicillin - resistant staphylococcus aureus infection , and the second ss patient was removed after five cycles because of erythema and pruritus . there was rapid clearing of all tumors after six cycles of treatment with romidepsin in the third patient , whereas the fourth patient with peripheral t - cell lymphoma was declared to be in complete remission after the eighth cycle of romidepsin , with complete disappearance of his skin lesions and diffuse adenopathy . at the end of this trial , it was concluded that romidepsin demonstrated significant clinical activity in the treatment of t - cell lymphomas and needed further investigation in a phase ii trial . based on the favorable responses observed in patients with ctcl during phase i testing , a phase ii trial to evaluate the efficacy of romidepsin in these patients was initiated at the nci and extended to other centers after initial encouraging observations.31 a total of 71 patients with relapsed , refractory , or advanced ctcl with either mf or ss who had no more than two prior cytotoxic chemotherapy regimens were enrolled , although there was no restriction on the number of prior systemic biologic therapies such as steroids , retinoids , monoclonal antibodies , or topical therapies . a majority of the 71 patients had advanced disease ( stage iib to ivb ) as follows : stage iva ( n = 28 ) , stage ivb ( n = 13 ) , stage iib ( n = 15 ) , stage ib ( n = 6 ) , stage iiia and iiib disease ( n = 3 ) , stage iia ( n = 2 ) , and one patient alone had stage ia disease . the median number of prior therapies was four ( 014 ) , including psoralen plus ultraviolet therapy , retinoids , radiation , total skin electron beam , photopheresis , interferon , and single or combination chemotherapy agents . romidepsin was administered as a 4-hour intravenous infusion at the dose 14 mg / m on days 1 , 8 , and 15 of a 28-day schedule . the average number of cycles received by the patients was four ( 172 ) , and the median number of doses was 12 ( 2141 ) . the response criterion used in this study was a composite score comprising the disease compartment that was involved , ie , skin , lymph nodes , or marrow . disease in skin or viscera was assessed by response evaluation criteria in solid tumors ( recist ) criteria,32 lymph node involvement was assessed using international working group guidelines ( iwgg),33 and bone marrow involvement was recorded as present or absent . generalized erythroderma was scored as present or absent . according to the recist criteria , size of skin lesions the overall response rate for the group of 71 patients was 34% ( 23%46% ) with four complete responses ( 7% ) and 20 ( 28% ) partial responses observed . the median duration of response was notable at 13.7 months ( range 166 + months ) . among the patients with a major response ( complete or partial ) , the median time to response was 2 months ( range 16 months ) . the median time to progression was 15.1 months for patients with a major response and 5.9 months for patients with stable disease . one of the striking features of romidepsin was the long duration of response noted that extended beyond 3 years , which was observed in some patients even after discontinuation of the drug . the complete responders included one patient with mf and three patients with ss . among the 62 patients with stage iv disease , 18 patients ( 29% ) overall , the drug was well tolerated , with toxicities similar to those observed in phase i trials , such as fatigue ( 41% ) , nausea ( 52% ) , vomiting ( 20% ) , anorexia ( 21% ) , leukopenia ( 31% ) , granulocytopenia ( 37% ) , lymphopenia ( 21% ) , thrombocytopenia ( 39% ) , and anemia ( 37% ) . asymptomatic t - wave flattening ( 71% ) or st segment depression ( 9% ) was observed , with no evidence of cardiac damage . of note , a median increase of 14 ms in the qt interval was seen with 0.2% of > 2000 electrocardiograms , demonstrating a qtcb ( bazett s correction ) interval of more than 500 ms . one of the major drawbacks of this study was the tools used to assess response . the recist criteria for solid tumors and the iwgg for lymphomas used in this trial give inaccurate estimations of the disease burden in skin or blood . the severity - weighted assessment tool ( swat ) score developed by stevens et al34 uses the principle of grid - point counting and gives a more comprehensive , dynamic , and accurate measurement of the skin disease burden in mf.35 this score represents the product of the percentage total body surface area ( % tbsa ) involvement of each lesion type ( patch , plaque , and tumor or ulceration ) , multiplied by a weighting factor : swat = ( patch % tbsa 1 ) + ( plaque % tbsa 2 ) + ( tumor or ulcer % tbsa it captures overall physical impressions of disease status on a continuous dimensionless numerical scale , thereby providing a defined , objective , and sensitive measure and hence is regarded as the best scoring system for skin disease in ctcl . while the nci study was being undertaken , gloucester pharmaceuticals started a parallel single - arm , open - label , prospective , phase ii trial of romidepsin in patients with pre - treated ctcl . it was a multicenter trial involving 33 centers in eight countries.36 ninety - six patients ( 59 with mf and 37 with ss ) were enrolled in the study and were administered romidepsin at the dose of 14 mg / m as a 4-hour infusion on days 1 , 8 , and 15 of each 28-day cycle for up to six cycles with the option of extension of treatment in those with stable disease or response . enrolled patients had the following characteristics : stage iva ( n = 24 ) , stage iii ( n = 23 ) , stage iib ( n = 21 ) , stage iia ( n = 13 ) , and stage ib ( n = 15 ) . the median number of prior therapies was four ( 111 ) , which included skin - directed and systemic agents . this study employed a rigorous composite endpoint , which included the swat and erythroderma scores along with the recist criteria for the assessment of nodal disease and flow cytometry for changes in peripheral blood tumor burden to assess responses to romidepsin . pruritus was reported monthly by the patients using a 100 mm visual analog scale ( vas).37,38 these various scores and scales were felt to be a better marker of overall meaningful tumor response . the primary endpoint of overall response rate was 34% , including six complete responses and 20 partial responses , with responses seen in all stages and all compartments of the disease ( skin , blood , and lymph nodes ) . of the 37 ss patients , 12 patients ( 32% ) had a response to romidepsin , including two complete responses . there was a 50% reduction in swat and/or erythroderma scores in 38 patients ( 40% ) . the median duration of response was 15 months ( range 019.8 + months ) with a time to response of 2 months ( range 0.94.8 months ) and median time to complete response of 4 months ( range 0.96.9 months ) . ninety - two percent of the patients showed improvement in their pruritus score . of note , clinically meaningful improvement in pruritus ( assessed by vas decrease of 30 mm or a score of 0 for 2 consecutive days ) was observed in 28 ( 43% ) patients . no serious adverse events were noted , with gastrointestinal intolerance and asthenia being the most common toxicities , and most of those were mild ( grade 1 or 2 ) . a clinically insignificant change in qtc interval was seen , which was attributed to antiemetics rather than to the drug per se . these noteworthy results from two parallel , prospective , single - arm , multicenter trials were presented to the fda and led to the approval of this agent in patients with advanced , refractory ctcl . the outcomes of major clinical trials of romidepsin as monotherapy have been summarized in table 3 . cardiac toxicity associated with hdaci therapy has been the focus of attention in all clinical trials involving these agents and appears to be a class effect . when romidepsin was administered at the dose of 1 mg / kg or 2 mg / kg as a 4-hour intravenous infusion on days 1 , 5 , and 9 or 2 mg / kg as a 24-hour infusion on day 1 in beagle dogs at the nci , mortality occurred in all four dogs that received the 24-hour infusion and in one of the dogs that received the dose of 2 mg / kg over 4 hours . lymphoid atrophy was noted in all three treatment groups.20 this suggested efficacy of the 4-hour infusion and demonstrated toxicity of the 24-hour infusion . at very high doses neither electrocardiogram abnormalities nor cardiac hemorrhages were observed when depsipeptide was infused over 4 hours . no cardiac lesions were detected in rats treated with very high doses.21 unpublished observations of qt interval prolongation and st segment abnormalities were observed in the beagle dogs treated with rapid infusions of romidepsin . this and similar asymptomatic electrocardiographic abnormalities noted in phase i clinical trials involving a 4-hour infusion led to the incorporation of intensive cardiac monitoring in the phase ii trial for ctcl.39 cardiac studies such as serial electrocardiograms , cardiac enzymes , multiple gated acquisition scans , echocardiograms , baseline 24-hour holter analysis , and telemetry monitoring were performed during the first dose of the first cycle . cardiac studies from 282 cycles and 736 doses of romidepsin involving 2051 electrocardiograms and 161 left ventricular ejection fraction evaluations were reviewed . although t - wave flattening ( grade 1 ) and st segment depression ( grade 2 ) were observed in more than half of the electrocardiograms post - treatment , no impact on myocardial function was noted . analysis of the qtc interval before and after treatment demonstrated a median qtc prolongation of 14 ms following treatment with romidepsin . some potential explanations that have been proposed regarding the asymptomatic electrocardiogram abnormalities noted with romidepsin are pretreatment with serotonin inhibitors or concurrent use of medications known to cause qtc prolongation , electrolyte abnormalities of potassium and magnesium known to exist in patients with ctcl , and coexistence of comorbidities or pre - existing cardiac arrhythmias in the treatment population . however , on retrospective analysis , all patients had cardiac comorbidities that predisposed them to risk of sudden death . with regard to cardiac dysfunction , clinical trials have excluded patients with pre - existing cardiac disease , long qt syndrome , or other risk factors for sudden death . unless this class effect of hdacis is further investigated and more experience is gained with their use , it would be reasonable to adopt a safe strategy and avoid them in the patient population mentioned . other unexpected toxicities of romidepsin , including three cases of reactivation of latent dna viruses , have been reported in the patients enrolled in the phase ii multi - institutional study of romidepsin in patients with t - cell lymphoma.40 two cases of epstein barr virus ( ebv ) associated illness and one case of hepatitis b virus ( hbv ) reactivation were observed . the first patient with ebv reactivation died of multiorgan failure from disseminated ebv infection , and the second patient responded well to treatment with single - agent rituximab . the patient with hbv reactivation was started on treatment with single - agent entecavir with a favorable response . it was hypothesized that these complications were likely related to hdaci therapy through transcriptional activation of silenced promoters and induction of immunosuppression via depletion of circulating lymphocytes . nausea ( all grades 52%56% ) , asthenia ( all grades 44% ) , anorexia ( all grades 20% ) , vomiting ( all grades 19%26% ) , and aguesia ( all grades 13% ) were commonly seen in patients receiving romidepsin.31,36 most of these adverse events were mild ( grade 1 or 2 ) . only about 2%7% of the patients experienced grade 3 or 4 toxicity , likely secondary to administration of serotonin inhibitors prior to and after romidepsin infusion . constitutional symptoms of fatigue ( 41% ) and fever ( 25% ) have been observed in patients on romidepsin treatment and felt to be cytokine mediated.31 however , once again , 24% of the patients had grade 1 or 2 fatigue , and grade 3 or 4 was rare . leukopenia ( all grades 30% ) , granulocytopenia ( all grades 36% ) , thrombocytopenia ( all grades 40% ) , and anemia ( all grades 37% ) were transient and rapidly reversible.31 furthermore , romidepsin does not affect the colony - forming units from bone marrow cells , and bone marrow biopsies performed in patients with romidepsin do not demonstrate significant myelosuppression . the mechanism behind the efficacy of romidepsin and other hdacis as a class in t - cell lymphomas remains unclear . it is tempting to speculate that the responsive subset of t - cell lymphomas has its origin in an as yet unknown chromosomal arrangement that recruits the class i hdacis to the promoter of a gene that normally limits cell proliferation . however , such chromosomal aberrations have not been described in patients with t - cell lymphomas . hence , it is possible that hdacis through their pleiotropic effects are merely regulating the balance between cell survival and apoptosis in patients with t - cell lymphomas similar to other disease entities rather than exerting efficacy via tumor - specific mechanisms . better understanding of tumor biology in ctcl and cellular pathways affected by hdacis will be required to better define the role of these agents in ctcl , particularly in combination therapies . why responses seen are far more common in hematologic malignancies than in solid tumors is also not understood . . advanced pharmacodynamic studies with more clinical experience and incorporation of correlative studies into the trials across different tumors may answer some of these questions and expand the spectrum of activity of romidepsin . another pertinent question is the role of synergy between romidepsin and other chemotherapeutic agents in diseases such as ctcl . hdacis in combination with hypomethalating agents have shown clinical activity in early - phase clinical trials in myeloid malignancies.4143 romidepsin has also been investigated in combination with the proteasome inhibitor bortezomib in patients with relapsed and refractory multiple myeloma with encouraging results in a small phase ii trial of 22 patients.44 the study showed good tolerability in the patients , with four complete responses , two very good partial responses , six partial responses , five minimal responses , and one stable disease . not only will the choice of chemotherapeutic agent be an important area of research in such combination strategies but the sequence of administration will pose a greater challenge given the lack of clear understanding of the molecular effects of hdacis . given the plethora of hdacis now available for clinical use , molecular analysis of tumors for signatures that can predict sensitivity response to individual agents would be ideal . a retrospective analysis of pretreatment ctcl skin biopsies found that high nuclear stat1 and phopho - stat3 staining in lymphoma cells correlated with a lack of clinical response to vorinostat.45 although the status of stat1 and phosphor - stat3 as predictors of romidepsin response remains unknown , identification of such biomarkers that could predict the outcome of romidepsin in patients with ctcl should be a focus of future clinical trials . finally , given the promising activity of all hdacis in ctcl , it is important to compare them with one another either as single agents or in combination with other therapies in phase iii trials to see whether one drug emerges to have superior efficacy or use molecular analysis to tailor therapies for individual patients . comparative efficacy , toxicity , and cost effectiveness between the two fda - approved and indicated hdaci therapies romidepsin and vorinostat for ctcl have not been studied . identification of ways to exploit the use of romidepsin in solid tumors and combination with radiation will be needed to select cell types that are sensitive to the poorly understood epigenetic modifications induced by romidepsin . despite multiple unresolved questions with respect to the mechanism of action of romidepsin in ctcl and the fact that it is not a targeted therapy , romidepsin has demonstrated significant clinical activity in patients with refractory neoplasms such as mf and ss with good durability and tolerable side effects . we anticipate future trials comparing its role either as a single agent or in combination with other biologic agents in both frontline and relapsed settings . designing and implementing randomized multicenter clinical trials with laboratory correlates to investigate the efficacy of drugs for rare diseases like ctcl will always be a challenge . hence , it is imperative that insight into the biology of ctcl guides the development of novel agents .
proton pump inhibitors ( ppis ) are a class of drugs that are routinely used in children ; however , stability data for extemporaneous liquid formulations are not readily available for many of these compounds . in a prospective study performed by lugo et al . , which included 21 children 's hospitals , an oral suspension of lansoprazole was the number one reported extemporaneous formulation prepared in the inpatient setting . in the study , 19 of 21 hospitals surveyed reported using a 3 mg / ml lansoprazole oral suspension ; however , there are limited data regarding the stability of this formulation . compromised stability , in regard to commercial and extemporaneous formulations , is defined as loss of more than 10% of the active ingredient . while the lack of stability of lansoprazole in acidic media ( such as apple juice ) has been clearly demonstrated , the studies examining the stability in a basic suspension ( prepared in 8.4% sodium bicarbonate ) are conflicting . according to a study by digiacinto et al . , the reported stability of lansoprazole suspension was eight hours at 22c and 14 days at 4c . in contrast , a study performed by phillips et al . revealed a stability of 4 weeks when lansoprazole was stored in amber plastic vials under refrigeration and 2 weeks at room temperature . thus , the commonly accepted stability of lansoprazole suspension prepared in 8.4% sodium bicarbonate is 14 days . a few reports of lansoprazole quantification by lc - ms / ms can be found in the literature . measured lansoprazole and rabeprazole in human serum for applications in pharmacokinetic studies using a triplequadrupole mass spectrometer . oliveira et al . and wu et al . used similar instrumentation for quantification of lansoprazole for bioequivalence studies . other reported quantitative methods for this drug rely on uv - vis spectroscopy [ 1012 ] . the only published method claiming stability - indicating properties for lansoprazole utilizes thin - layer chromatography and uv - vis spectroscopy . the hybrid ion - trap time - of - flight mass spectrometer configuration is relatively new and was released to the market as a competitor for the orbitrap ( thermo scientific , waltham , mass , usa ) for applications in proteomics . to date , this ms configuration has found applications in the proteomics field , as well as in metabolomics and global metabolite profiling and lipidomics . one quantitative method involving the ms configuration has been published measuring five lignan standards applicable to herbal medicines . these investigators noted similar linearity , precision , accuracy compared to the well - established quantitative powers of the triplequadrupole ms configuration ; however , the it - tof performed at higher sensitivity for all compounds measured . the lansoprazole and omeprazole standards were of usp grade and were purchased from spectrum chemical ( gardena , calif , usa ) . the solvents used included methanol , water , and 0.1% v / v formic acid in acetonitrile . all of these solvents were of lc - ms grade ( burdick & jackson , morristown , nj , usa ) . lansoprazole delayed - release capsules , usp , of 30 mg were used in the pilot stability study ( teva pharmaceuticals usa , sellersville , pa , usa ) . the suspension samples were prepared using usp grade 8.4% w / v sodium bicarbonate ( hospira , inc . , the hplc column was a waters acquity uplc beh c18 column , 1.7 micron , 2.1 100 mm ( milford , ma , usa ) . the shimazdu liquid chromatography system consisted of two lc-20ad pumps with uflc - xr upgrade , sil-20acht autosampler , cto-20a column oven , dgu-20a3 degasser , and cbm-20a communications module . this system was coupled to the shimazdu it - tof mass spectrometer with an electrospray ( esi ) source ( columbia , md , usa ) . all chromatographic separations were performed using the waters acquity uplc beh c18 ( 1.7 micron , 2.1 100 mm ) column . the isocratic separation utilized a mobile phase of 60% water/40% acetonitrile with 0.1% v / v formic acid at a flow rate of 0.200 ml / min . all mass spectrometric measurements were performed using the shimazdu it - tof with an esi source operating in positive ion mode . liquid nitrogen was used as the nebulizing gas at a flow rate of 1.5 l / min . for quantification of lansoprazole and omeprazole , a direct ms / ms method was used , where the transitions specific to the analyte and internal standard were monitored ( m / z 370 252 and m / z 346 198 for lansoprazole and omeprazole , resp . ) . calibration and validation standards were prepared in 50/50 v / v water / methanol mixture . the calibration curve consisted of five points 5 , 10 , 15 , 20 , and 25 g / ml lansoprazole . these calibration standards were prepared using a 100 g / ml stock solution of lansoprazole in 50/50 water / methanol . all calibration and validation samples were filtered using a 0.22-micron syringe filter prior to injection . five replicates of each of the calibration points were prepared on each day of validation to assess precision and accuracy . precision was calculated as the relative standard deviation , % rsd = 100 ( sd / mean , sd standard deviation ) . to reflect the accuracy of the assay , the % error was calculated as the percent difference between the theoretical concentrations and the experimentally determined concentrations of the replicate samples . the method limit of detection ( lod ) was determined using a 3 : 1 signal - to - noise criterion . a lansoprazole suspension ( 3 mg / ml ) was prepared by pouring the contents of ten 30 mg capsules of lansoprazole into 100 ml of 8.4% sodium bicarbonate and stirring on a magnetic stir plate for a minimum of 30 minutes . the sample was stored at room temperature ( 22c ) and sampled at the following time points : 0 hr , 8 hr , 24 hr , 48 hr , 72 hr , and 168 hr . upon sampling , 0.600 ml of suspension was removed by micropipette and added to 8.4 ml of a 50/50 mix of methanol and water and vortex mixed . from this mixture , 100 l was removed by micropipette and added to 800 l of the 50/50 solvent mix . a volume of 100 l of the internal standard stock solution ( 100 g / ml ) , omeprazole , was added to the aforementioned mixture for a final concentration of 10 g / ml omeprazole . this dilution of lansoprazole used was intended to bring the final sample concentration within the calibration range ( 525 g / ml ) . the actual concentration of lansoprazole in each sample was calculated using the calibration curve from that day . before being added to the autosampler vials the method was validated in a range of 525 g / ml . using a 3 : 1 signal - to - noise ratio , the limit of detection ( lod ) was determined to be 2 ng / ml ( 0.002 ng on - column ) . the actual limit of quantification ( loq ) is likely lower than the lowest validation point , as defined by a 10 : 1 signal - to - noise ratio ; however , 5 g / ml was the lowest concentration validated . it is highly likely that the assay could have been validated in a more sensitive range , but the objective was to create a method that was easily compatible with the 3 mg / ml starting concentration of the lansoprazole suspension . since the suspension is a nonhomogeneous matrix , the authors felt it best to minimize the number of dilutions needed for sample preparation as well as maximize the volume of suspension used for the samples . working in the 525 g / ml range allowed for this , thus helping reduce some of the error inherent in sampling from a nonhomogeneous preparation . table 1 shows the intraday precision ( % rsd ) and accuracy ( % error ) for the method ( n = 5 for each concentration for each day ) . the intraday % rsd ranged from 1.28 to 10.54% and the intraday % error from 0.30 to 5.22% . table 2 shows the interday precision over four days ( % rsd ) and accuracy ( % error ) for the method ( n = 20 for each concentration ) . these data indicate a high reliability and reproducibility with an interday % rsd ranging from 1.99 to 3.93% and interday % error from 1.06 to 2.81% . the use of the waters acquity column enabled a separation that was fast ( <3 min ) and baseline resolved as shown in figure 1 . the matrix peak shown likely represents the presence of phthalate contamination , which has been shown to be ubiquitous in laboratory environments ; however , as shown in figure 1 , it is chromatographically resolved from the analyte and internal standard peaks . the direct ms / ms transitions monitored allowed for specificity in quantification of the analyte and internal standard ( figure 2 ) . to demonstrate that the method was capable of quantifying lansoprazole in a pharmaceutical suspension , a one - sample pilot study was run using a 3 mg / ml lansoprazole suspension stored at room temperature . the suspension was sampled periodically and concentration of lansoprazole quantified using the developed lc - ms / ms method . suspension samples were spiked with internal standard ( omeprazole ) and diluted to fall within the calibration range as planned for a scaled - up version of the study . the pilot data , shown in figure 3 , indicate that stability of lansoprazole in an oral suspension , if stored at room temperature of 22c , would be compromised after 72 hours . loss of stability was defined as lansoprazole concentration < 90% of the initial concentration at any time point . the method presented here is the first application reported of uplc coupled with the unique it - tof mass spectrometric detector to quantify this drug . the use of uplc is becoming widely accepted as a means to achieve higher - resolution separations compared to conventional hplc , ultimately resulting in higher sensitivity and fast run times . furthermore , the use of the it - tof mass spectrometer provides the potential for accurate mass data collection on degradation products , something that is not readily achieved using more conventional triple quadrupole instruments . while this method offers a comparable option to existing lc - ms assays for quantification of lansoprazole in terms of accuracy and precision , it stands as one of the few quantitative applications of the hybrid it - tof mass analyzer configuration as well as one of two stability - indicating methods for this drug . ultimately , this method can be applied to monitor the stability of lansoprazole in oral suspensions with confidence of accuracy , precision , and specificity .
chronic obstructive pulmonary disease ( copd ) , a disorder of chronic airflow limitation ( chronic airflow obstruction ) not reversed by bronchodilators , is now the 3 commonest cause of death worldwide . while most of the information on copd prevalence , morbidity , and mortality is available from high - income countries , it is known that low- and middle - income countries already shoulder much of the burden of copd with almost 90% of copd deaths taking place in these countries . spirometrically documented copd is common in kashmir , the northern indian state of jammu and kashmir , with a prevalence of about 19% among males and 14% among females above the age of 40 years ; much higher than reported previously on the basis of questionnaire surveys . the natural history of copd is punctuated by exacerbations that lead to considerable morbidity , mortality and an inexorable decline in pulmonary function . exacerbations of copd form one of the most common medical admissions , especially during winter when respiratory viral infections are common . exacerbations are regarded to be infective in most cases , and viruses are believed to be a common cause of exacerbations . we have recently reported that influenza a and b viruses are associated with about 8% of copd exacerbations . data regarding the contribution of other respiratory viruses in the causation of exacerbations of copd are scant , especially from the developing countries . we herewith report our results of testing of patients for other respiratory viruses in patients with copd exacerbation . a total of 233 consecutive consenting patients admitted with an exacerbation of copd were enrolled from may 2011 to september 2012 for detection of respiratory viruses at a tertiary care facility in srinagar , the summer capital of the northern indian state of jammu and kashmir . kashmir has a temperate geography in contrast with the generally more tropical climate in the rest of the country , and we have earlier reported a temperate seasonality of influenza virus circulation which contributes to the exacerbations of copd in 8% cases . twin nasopharyngeal / oropharyngeal swabs were collected from the patients as described earlier . tested for respiratory viruses including respiratory syncytial virus ( rsv ) a and b , parainfluenza ( piv ) 1 , 2 , 3 , and 4 , human metapneumovirus ( hmpv ) a and b , influenza a and b , enterovirus , corona nl65 , oc43 , and 229e viruses , adenovirus 2 and 4 , rhinovirus , and bocavirus , were performed by in - house standardized duplex real time reverse - transcription polymerase chain reaction ( qrt - pcr ) using previously published primers and probes . the details of the enrollment and testing for influenza among these patients have been reported elsewhere . briefly , nasal and throat swabs were collected from participants at enrollment for testing by rt - pcr for influenza a and b viruses , and those positive for influenza a viruses were further subtyped for a / h1n1pdm09 , a / h3n2 , and seasonal h1n1 using us centers for disease control and prevention protocols . the patients were managed with routine antibiotics , supportive oxygen therapy , noninvasive or invasive ventilation , and antiviral agents ( oseltamivir ) in case of influenza positivity . antibiotics were withdrawn in ten patients in whom influenza viruses were detected and there was no other clinical or lab parameter suggestive of bacterial etiology such as purulence of sputum , polymorphonuclear leukocytosis , elevated c - reactive protein ( crp ) or detection of bacteria on gram 's staining or culture ; and the patients showed a consistent improvement . the patients were followed for about 30 days for any readmissions and at home mortality . the study was approved by the institute ethics committee , and informed consent was obtained from all participants before recruitment . the recruited patients included 152 male and 81 female patients with age ranging from 40 to 100 ( median 65 ) years . the various symptoms experienced by the patients included cough ( n = 229 , 98% ) , worsening dyspnea ( n = 226 , 97% ) , wheezing ( n = 214 , 92% ) , increased sputum ( n = 135 , 58% ) , fever ( n = 93 , 40% ) chills ( n = 86 , 37% ) , sore throat ( n = 51 , 22% ) , and nasal discharge ( n = 44 , 19% ) . hypertension ( 60.52% ) and heart disease ( 14.16% ) were the commonest comorbid illnesses . there were thirty current smokers ; 50 ( 21.4% ) reported second - hand tobacco exposure , and 50 ( 21.4% ) reporting the use of biomass fuels ( i.e. , wood , crop build ups , or compost ) for cooking / heating . most ( n = 213 , 92% ) of the participants used kangri ( personal heating devices consisting of earthenware pots carried along with live charcoal ) or bukhari for personal heating . seven patients had received influenza vaccine in the current season , and three were on chronic oral steroids whereas 72 were on home oxygen therapy . respiratory viruses were identified in nasal and throat swabs in 46 ( ( 19.7% ) of the 233 cases [ figure 1 ] . more than one virus was isolated in four cases consisting of hmpv - b + adeno-2 + inf - b ; rhino + h3n2 , piv-1 + rhino ; and piv-1 + hmpv - b in one case each . ancillary supportive therapeutic measures included antibiotics and steroids ( n = 194 , 83% ) . noninvasive ventilation was used in 42 cases whereas invasive mechanical ventilation was needed in four cases . three patients ( 1.3% ) died in the hospital whereas one patient was readmitted within 30 days of discharge and another one died suddenly within 30 days of discharge at home . no autopsies were done , and all the patients who died had influenza a / h3n2 recovered from their nasopharyngeal specimens but antibiotics were not withdrawn in any of these . none of the patients who had other respiratory viruses had a readmission or died within 30 days of the hospital discharge . monthly distribution of positive samples for respiratory viruses in acute exacerbation of chronic obstructive pulmonary disease patients various viruses detected among 233 patients with acute exacerbations of chronic obstructive pulmonary disease monthly detection of the various viruses in patients with exacerbation of chronic obstructive pulmonary disease despite the increasing recognition that viruses are frequent in exacerbations of copd ( acute exacerbations of copd [ aecopd ] ) , it is less clear which viruses are involved and to what extent they contribute to exacerbations . our data show that respiratory viruses are frequently associated with hospitalized patients with exacerbations of copd , influenza , and rhinoviruses being the most common ones detected in the respiratory samples of the hospitalized patients . the detection rate of respiratory viruses in our study was similar to previous reports by walsh et al . however , mcmanus et al . reported a detection rate of 37% among their subjects with aecopd . in a recent meta - analysis of 17 eligible studies , weighted overall prevalence of respiratory viruses in patients with aecopd was 39.3% compared to 13.6% in four studies in stable copd , a pooled risk ratio for respiratory viral infection being 4.1 ( 95% confidence interval [ ci ] : 2.08.5 ) for aecopd as compared with stable copd , rhinovirus being the most common detected one . the results suggested that respiratory viruses probably are important etiological agents in patients with aecopd as compared with the stable copd patients . in this study , in a recent systematic review of 19 studies involving 1728 patients with aecopd , rhino / enteroviruses was found to be the most common ( 16.39% ) pathogens involved followed by rsv ( 9.90% ) , influenza ( 7.83% ) coronaviruses ( 4.08% ) , piv ( 3.35% ) , adenovirus ( 2.07% ) , hmpv ( 2.78% ) , and bocaviruses ( 0.56% ) . in the only study from india , mohan et al . reported detection of viruses in 16 of the 137 patients with aecopd and influenza was the most common virus detected ( n = 11 ; 8% ) , followed by parainfluenza virus-1 ( n = 3 ; 3.6% ) , rsv , and piv3 ( n = 1 each ) . three of the patients in our study who died had a / h3n2 influenza viral infection . the detection rates of pathogens in aecopd have been influenced by the diagnostic methods employed . comprehensive rt - pcr has been regarded as a method of choice for detection of the viruses in the respiratory specimens . combining real - time pcr with conventional methods improves the ability to detect infectious etiologies of copd exacerbations including both bacteria and respiratory viruses and may emerge as the standard of care in facilities with capacity to do the testing . recently microarray - based etiology of aecopd pathogens was carried out in 200 patients with aecopd . human rsv ( subtypes a and b ) ( 40.5% ) , influenza virus ( subtypes a , b , c ) ( 11% ) , rhinovirus ( 8% ) and human parainfluenza virus ( subtypes a and b ) ( 7.5% ) were the commonest isolated strains . most of the patients in this study were not vaccinated against influenza and pneumococcus despite clear recommendations for vaccination against these pathogens in patients with copd . this trend of poor vaccination uptake is consistent with our previous report of poor uptake of influenza and pneumococcal vaccination in patients with copd from low - income countries as compared to those from high - income countries . since empirical utilization of antibiotics has prompted disturbing levels of antibiotic resistance , it would be very useful to distinguish between the viral and the bacterial etiology of aecopd so that antibiotic use could be curtailed and properly rationalized . antibiotic use in severe copd exacerbations has been recommended routinely , even as there is a paucity of placebo - controlled clinical trials and up to today , no single study has been powered sufficiently to prove the efficacy of antibiotic treatment in aecopd . in a recent cochrane review of 16 randomized controlled studies involving 2068 patients , the authors concluded that antibiotics for copd exacerbations showed large and consistent beneficial effects across outcomes of patients admitted to an intensive care unit . however , for outpatients and inpatients , the results were inconsistent . the risk for treatment failure was significantly reduced in both inpatients and outpatients when all trials ( 19572012 ) were included but not when the analysis for outpatients was restricted to currently used antibiotics . furthermore , antibiotics had no statistically significant effect on mortality and length of hospital stay in inpatients and almost no data on patient - reported outcomes exist . these inconsistent effects call for research into clinical signs and biomarkers that help identify patients who benefit from antibiotics and patients who experience no effect , and in whom downsides of antibiotics ( side effects , costs , and multi - resistance ) could be avoided . while some clinical and lab parameters have been suggested to distinguish between the two , in a recent study in which viruses were isolated from 22% of 72 enlisted patients of aecopd , bacterial diseases could not be separated from viral disease by white cell number , crp or procalcitonin levels . however , the utility of these parameters needs to be probed further , and there is a distinct need for a biomarker for distinguishing the bacterial from a viral infection . an extensive placebo - controlled study is underway in germany to assess if anti - microbial treatment is at all needed in copd exacerbations . in ten of our patients in whom influenza viruses returned positive on testing , there was absence of evidence of a bacterial infection , we used oseltamivir as stand - alone medication without any complications . all the three patients who died had h3n2 detected in them , and this could argue in favor of starting antiviral medication in patients with aecopd , pending a result of the respiratory specimen , when a patient of aecopd presents with features of influenza - like illness or a severe acute respiratory infection during a time of heightened activity of influenza circulation . according to the advisory committee on immunization practices , antiviral treatment is recommended as soon as possible for patients with confirmed or suspected influenza who have a severe , complicated , or progressive illness or who require hospitalization . antiviral treatment for influenza is recommended especially for patients who are at a higher risk for influenza - associated complications and chronic pulmonary disease for one such group . de - escalation to antivirals alone might be justified if the testing of the respiratory specimens returns positive for influenza . viral infections seem to contribute to the exacerbations of copd in our settings and should be strongly considered in the management of such patients . further studies reproducing our data from other areas could perhaps help reduce the inappropriate empiric use of antibiotics in the event of an influenza viral exacerbations . our data also strongly advocate strenghtening the routine use of influenza vaccination in patients with copd . the study was funded by the sher - i - kashmir institute of medical sciences , srinagar , jammu and kashmir , india . the study was funded by the sher - i - kashmir institute of medical sciences , srinagar , jammu and kashmir , india .
darier 's disease ( dd ) , described independently by darier and white in 1889 , is an autosomal - dominant disease characterized by persistent eruption of hyperkeratotic papules , histological examination of which shows suprabasal acantholysis with distinct overlying dyskeratosis . dd is caused by mutation in the atp2a2 gene at chromosome 12q24.1 , which encodes the sarco endoplasmic reticulum calcium pumping atpase type 2 ( serca2 ) . clinically , the disease is characterized by keratotic papules or plaques in the seborrheic areas with occasional nail or mucosal involvement . the disease shows aggravation during summers and sun - exposure ; heat and humidity may exacerbate the condition . localized pattern of dd was first reported in 1906 . since then , many localized variants like localized , unilateral , linear , segmental or zosteriform dd have been described . we present a case of zosteriform dd confined to the right infra - mammary region in an adult female . a 35-year - old woman presented with mildly pruritic , unilateral , hyperpigmented , keratotic papules localized to the right infra mammary region [ figure 1 ] . for the past 3 years , these lesions occurred strictly in summers , from march to june , and regressed spontaneously afterwards . histopathological examination of the skin biopsy showed hyperkeratosis , elongation of rete ridges , suprabasal acantholysis and marked dyskeratosis in the form of corps ronds and grains [ figures 2 and 3 ] along with a mild lymphohistiocytic inflammatory infiltrate in the dermis consistent with dd . ( a - c ) hyperpigmented , keratotic papules localized to the right infra - mammary region in a zosteriform distribution photomicrograph shows hyperkeratosis , elongation of rete ridges , suprabasal acantholysis and dyskeratosis [ h & e , 40 ] photomicrograph shows characteristic corps ronds and grains [ h & e , 100 ] dd is an inherited autosomal - dominant condition with prevalence of around 1/50,000100,000 . around 10% of the cases of dd present in a localized pattern . , various localized variants like unilateral , linear , segmental or zosteriform have been described . the skin lesions in the localized variants are usually confined to a limited area and other features that are associated with classical dd are usually absent . the lack of family history , absence of other signs of dd and the limited distribution of the lesions favor a nevoid origin . the severity and the histological findings within the streaks do not differ from those of generalized dd . this distribution is the result of genetic mosaicism due to post zygotic somatic mutation early in embryogenesis . if there is associated gonadal mosacism , a patient with segmental manifestation may have an offspring with generalized dd . in the type ii segmental variant , type ii mosacism occurs in patients with heterozygous germline mutation who also have a somatic loss of heterozygosity of wild type allele in a segmental area leading to homo- or heterozygosity and therefore increased severity in a linear array . segmental ( localized , linear or zosteriform ) forms of dd may be clinically and histologically indistinguishable from epidermal nevi with acantholytic dyskeratosis ( aden ) . it is believed that aden possibly represents the localized , mosaic form of dd , and it has been suggested to replace the term aden by segmental dd , induced by post - zygotic mutation . a case of aden on the sole of a 1-year old infant that showed histological features of dd however , cases of segmental dd not associated with serca mutations , mutations of other probably as yet unknown genes and controlling the differentiation of epidermal keratinocytes has been hypothesized . another interesting observation noted in our patient was history of lesions occurring strictly during summers and their complete spontaneous disappearance during the other seasons . a similar observation has also been reported by plantin et al . and
uterine rupture rarely occurs during pregnancy , but it is a critical obstetric complication associated with maternal and fetal mortality and morbidity.1 it is known that the rate of uterine rupture increases in patients with a history of uterine surgery , such as cesarean section or myomectomy . it is however , not yet known what kinds of complications this procedure can cause in pregnant women . here we report a case of spontaneous uterine rupture in the 35th week of gestation after a laparoscopic adenomyomectomy . a 42-year - old , gravida 2 , para 0 woman was referred to our hospital at the 11th week of gestation . before she got pregnant , she had had sterility treatment , but it had nt been successful . the findings in the operation were as follows : the lesion of adenomyosis existed at the posterior wall of the uterus and the lesion and rectum adhered to each other firmly . a vertical incision was made in the middle of the posterior wall of the uterus , and the adenomyosis lesion was enucleated as much as possible ( 50 g in total ) . the patient s singleton pregnancy was uneventful and she was admitted at the 30th week of gestation for the purpose of bed rest . this was done because past reports suggest that uterine rupture after laparoscopic adenomyomectomy typically occurs around the 30th week of gestation . the patient s hospital stay was incident - free , and a cesarean section was due to be performed at the 34th week of gestation to achieve optimal trade - off between fetal lung maturation and fetal prematurity . however , the patient s cesarean section was moved to the 35th week of gestation due to her strong request . on the scheduled date the patient had undergone combined spinal epidural anesthesia , and was experiencing frequent uterine contractions when a weak pain suddenly occurred . after 13 minutes of uterine contractions , vaginal bleeding was observed while sterilizing the vagina . cesarean section was performed immediately and a female neonate , weighing 2,283 g , was delivered 31 minutes after uterine contractions had begun . the neonate s apgar scores were 7 and 8 at 1 and 5 minutes , respectively . umbilical arterial gas analysis indicated no acidosis ( ph 7.267 , base excess 3.4 ) . the rectum and omentum adhered to the posterior uterine wall , where there was a scar from the previous adenomyomectomy . one area did not have a muscle layer and was covered with only uterine serosa adjacent to the ruptured area . this was repaired with a three - layer suture and total sutured length was 78 cm . the patient s postoperative process proceeded without complications and the patient was discharged at a postoperative date of 12 days . informed consent was obtained from the patient orally , in order to retain the anonymity of the patient . two important clinical issues were identified : 1 ) after a laparoscopic adenomyomectomy , a pregnant uterus might rupture easily by rather weak and short uterine contractions ; 2 ) uterine contractions followed by uterine bleeding might be useful for the diagnosis of uterine rupture . first , after laparoscopic adenomyomectomy , a pregnant uterus might rupture easily by rather weak and short uterine contractions . a recent review by morimatsu et al showed that the rate of uterine rupture after adenomyomectomy during pregnancy is 6.0%2 in contrast to the entire rate of uterine rupture during pregnancy , which is 0.035%.3 this is much higher than the rate of complications after cesarean section ( 0.27%0.7%)4,5 or myomectomy ( 2.45%).6,7 we determined some reasons why uterine rupture frequently occurs in pregnant women with prior laparoscopic adenomyomectomies . a lesion of adenomyosis tends to remain around the edges of excisions and the area to be sutured , which might lead to weak connections between sutured edges . if a lesion of adenomyosis is enucleated widely to eliminate the lesion , the uterus will be small and irregular in shape , which leads to a diminished capacity to expand.8 with a laparoscopic adenomyomectomy , it is particularly difficult to delineate the border of the lesion because of a lacking sense of touch and deep sensation.9 only five publications have presented cases of uterine rupture during pregnancy in patients with prior histories of adenomyomectomy ( laparoscopic 4 , laparotomic 1 ) ( table 1).2,8,10,11 according to 96 reported cases ( table 2),2,816 the rate of uterine rupture during pregnancy was 6.25% ; this is similar to the review reported by morimatsu et al.2 it would appear that the frequency of uterine rupture after laparoscopic adenomyomectomy is higher than that of laparotomic adenomyomectomy ( tables 1 and 2 ) , but there is no cohort study to compare these so far . uterine rupture or dehiscence during pregnancy tends to occur more frequently after laparoscopic myomectomy than after laparotomic myomectomy . specifically , the frequency of uterine dehiscence is 1.854.94% in laparoscopic6,7 and 0% in laparotomic ( this finding is extremely limited because it depends on only 69 cases from two reports).6,7 we guess that uterine rupture or dehiscence is similarly more common in laparoscopic than in laparotomic adenomyomectomy cases . second , uterine contractions followed by uterine bleeding might be useful for the diagnosis of uterine rupture . uterine rupture does not have any typical symptoms and is often asymptomatic.17,18 classical symptoms and signs are abdominal pain , external bleeding , vital signs indicating shock , disappearance of labor pain , and signs of fetal distress etc . in this case , the patient experienced both abdominal pain and external bleeding . a pregnant uterus after laparoscopic adenomyomectomy might rupture easily by rather weak and short uterine contractions . furthermore , uterine contractions followed by uterine bleeding might be useful for the diagnosis of uterine rupture . when uterine contractions are followed by uterine bleeding in pregnant women that have had a prior adenomyomectomy , this must be considered a potential sign of uterine rupture . if physicians are planning to perform surgery for treating sterility and infertility , they should carefully choose the modality of surgery and get more detailed informed consent from patients with adenomyosis . further reports should be accumulated to develop more reliable and safe operative techniques , and to establish guidelines for pregnancy after adenomyomectomy in women of reproductive age .

tracheoesophageal fistula ( tef ) is a congenital or acquired life threatening complication of a communication between the esophagus and the tracheobronchial tree or the other mediastinal structures , due to pathological conditions affecting these structures . the most common etiology of nonmalignant acquired tef is a cuff - related tracheal injury in an intubated patient . esophageal cancer is the most common malignant etiology ( 1 , 2 ) . patients with tef on mechanical ventilation tend to have increased secretions , and are at increased risk of aspiration pneumonia from gastric contents that reflux through the tef into the tracheobronchial tree . barrett s esophagus refers to the replacement of normal squamous epithelium in the lower esophagus by columnar epithelia caused by chronic exposure to gastric acidic secretions . this report describes an unusual case of barrett s esophagus presenting with ulcerating tef which was detected while investigating a persistent air leak on a mechanical ventilator . a 44-year - old female was admitted to the hospital with a sudden onset of respiratory distress . she had a three year history of gastro - esophageal reflux disease ( gerd ) but no history of smoking or alcohol use . the patient was intubated and mechanically ventilated . on physical examination , she had bilateral crepitations and rhonchi on chest auscultation . , she was noted to have a constant leak of 150cc of tidal volume with each respiration . suspecting a cuff leak , the treating team decided to replace the endotracheal tube . esophagogastroduodenoscopy ( egd ) confirmed the diagnosis at 30 cm from the incisor teeth ( figure 1 ) . histopathology was consistent with barrett s mucosa with ulceration and active inflammation ( figure 2 ) . computed tomography was obtained to investigate for an associated mass , but showed only a left sided tracheoesophageal fistula with no other mediastinal or abdominal abnormality . primary surgical repair was undertaken with longitudinal suturing of the individual bronchial and esophageal components of the fistula . a small post - operative leak was identified on the barium esophagogram ( figure 3 ) . it was successfully treated with conservative management consisting of percutaneous endoscopic jejunostomy ( pej ) tube feeding and a proton pump inhibitor therapy for 2 weeks . the patient improved and was extubated on post - surgical day 5 and discharge home in a stable condition after 2 weeks . barrett s esophagus is an intestinal metaplastic response of the esophageal squamous mucosa to chronic gastroesophageal reflux ( 3 ) . it can present as tongue - like extensions from the gastroesophageal junction or scattered islands of columnar epithelium in the early stages . an air leak of more than 100 cc per tidal volume in mechanically ventilated patients is very unusual . such a leak can be caused by inappropriate cuff inflation , misplacement of the endotracheal tube , pneumothorax , bronchopleural fistula , chest tube air leak and fistulas in - between the airways and the mediastinal structures . in this case , the etiology was found to be tef . it is likely that the erosive nature of barrett s esophagus and the proximity of the esophagus and tracheobronchial tree anatomically that led to the tef . proton pump inhibitors were used to decrease the gastric acid secretion to aid in epithelial healing . there are only two prior reported cases of acquired tef associated with perforated barrett s esophagitis ( 4 , 5 ) . our experience suggests that the detection of persistent and unexplained air leak in a mechanically ventilated patient should lead the clinician to consider tef as a causative condition . conditions that predispose patients to tef should be taken into account when considering this etiology . recognizing the presence of acquired tef early on prevents the development of complications such as aspiration pneumonia , which could prove to be life - threatening especially in our current population of inpatients with multiple comorbidities .
inguinal hernia repair is one of the most commonly performed surgical procedures with approximately 20 million inguinal herniorrhaphies repairs performed yearly around the globe . the operative approach to inguinal herniorraphy overtime has evolved from primary tissue - based repairs to open tension - free mesh repairs and finally more recently laparoscopic mesh repairs . the latter has gained favor due to the substantial decrease in pain experienced by patients and an earlier return to activity . moreover , within the realm of laparoscopy , robotic assistance offers the operating surgeon considerable advantages in terms of superior visualization , stabilization of instruments and improved ergonomics , making this application an attractive alternative to conventional laparoscopic inguinal herniorraphy . robotic - assisted inguinal hernia repair utilizes the transabdominal preperitoneal approach ( tapp ) which entails development of a peritoneal flap ( pf ) , dissection and reduction of the hernia sac and placement of a mesh in the preperitoneal space thus created . following hernia repair pf should be closed thereby partitioning the mesh from the viscera consequently reducing the risk of mesh erosion into the viscera and bowel obstruction secondary to herniation through the peritoneal defect . there are many methods available for closure of the pf including sutures , tacks , adhesive sealants and staples ; however , suture closure is preferred particularly in robotic tapp as it allows complete exclusion of the preperitoneal space with an improved short - term quality of life . in recent years , self - anchoring barbed sutures have been developed and gained popularity for a variety of tissue applications due to the simplicity of use and knotless application . herein we report an unusual case of post - operative small bowel obstruction following robotic - assisted inguinal hernia repair emanating from the self - anchoring suture used for pf closure . a 61-year - old male with a weight of 88 kg and a bmi of 27 kg / m presented with symptomatic right - sided reducible inguinal hernia . he was offered and subsequently underwent a robotic - assisted tapp inguinal herniorraphy . during the operation , a right - sided direct inguinal hernia was identified , the sac was completely reduced and hernia repaired with right - sided 15 10 cm monofilament polyethylene terephthalate mesh ( progrip ; covidien dublin ireland ) . following successful placement and fixation of the mesh pf was closed using 02 - 00 self - anchoring - barbed suture ( v - loc 180 absorbable wound closure device ; covidien dublin ireland ) . total operative time was 124 min with 5 ml estimated blood loss and the patient was discharged home from post - anesthesia care unit . the patient presented to the emergency department on post - operative day 3 with complaints of nausea , vomiting , abdominal distention and obstipation . physical examination revealed a tender and distended abdomen , further evaluation with cross - sectional imaging revealed a transition point in the right lower ( fig . he subsequently underwent diagnostic laparoscopy where a free end of the barbed suture was noted to be adherent to the small bowel creating a sharp angulation in the bowel leading to obstructive symptoms ( fig . the suture was lysed close to the peritoneum , which led to the relief of bowel obstruction . post - operatively the patient had return of bowel function the following day and was discharged home . figure 1:ct scan demonstrating transition point between dilated proximal and collapsed distal small bowel . figure 2:laparoscopic view of self - anchoring suture adherent to the small bowel causing the obstruction . laparoscopic view of self - anchoring suture adherent to the small bowel causing the obstruction . complete closure of the pf following a tapp inguinal hernia repair is important to prevent post - operative complications . available evidence suggests that closure of pf with running suture is preferable due to a reduction in post - operative pain and incidence of bowel obstruction secondary to herniation through the peritoneal defect or displaced tacks [ 4 , 7 ] . self - anchoring knotless - barbed sutures offer an easier and faster option for pf closure . while these devices facilitate pf closure given their propensity of adherence to intra - abdominal viscera , there remains a risk of adhesive bowel obstruction . there have been some previous reports of small bowel obstruction following ventral rectopexy , sacral colpopexy and laparoscopic tapp suggesting that if these devices are used maximal efforts should be made to ensure that the suture material is not exposed to any intra - abdominal viscera [ 810 ] .
les indices de pharmacorsistance ( ipr ) quantifient leffet cumulatif de la rsistance antimicrobienne sur la probabilit quun pathogne donn soit susceptible un traitement antimicrobien . driver lipr des infections urinaires dorigine non nosocomiale causes par lescherichia coli en colombie - britannique entre 2007 et 2010 et examiner les tendances au fil du temps et selon les caractristiques des patients . les donnes sur les indications dutilisation , tires du systme pharmanet de la colombie - britannique relativement aux prescriptions dantimicrobiens , taient lies linformation diagnostique prleve dans les dossiers dhonoraires des mdecins . les donnes de rsistance relies aux isolats urinaires de coli provenaient des bc biomedical laboratories ( qui font dsormais partie des lifelabs medical laboratory services ) . les ipr taient drivs en multipliant le taux de rsistance un antimicrobien prcis au taux proportionnel dutilisation de cette classe de mdicament et en les regroupant entre les classes de mdicaments . des indices de valeur plus levs indiquaient une plus forte rsistance . les ipr utilisation adapte demeuraient stables au fil du temps , environ 18 % ( 95 % ic 17 % 18 % ) chez les adultes de 15 ans et plus , et environ 28 % ( 95 % ic 26 % 31 % ) chez les enfants de moins de 15 ans . les chercheurs ont observ des rsultats similaires lorsque lutilisation proportionnelle des mdicaments tait restreinte lanne de rfrence ( modle utilisation statique ) . les tendances en fonction des groupes dge laissent supposer une rpartition en u , les ipr les plus levs se produisant chez les enfants de moins de dix ans et les adultes de 65 ans et plus . dans tous les groupes dge , les hommes prsentaient un ipr plus lev que les femmes . daprs la tendance stable des ipr utilisation adapte au fil du temps , les cliniciens adaptent leurs pratiques de prescription pour le traitement des infections urinaires aux profils de rsistance locaux . les rsultats en fonction des groupes dge rvlent une plus forte probabilit de rsistance la thrapie initiale chez les jeunes enfants et les personnes ges . antimicrobial utilization data were obtained from the bc pharmanet database of outpatient prescriptions for oral antimicrobials for systematic use for the years 2007 to 2010 . the pharmanet database includes records of all outpatient prescriptions dispensed from community pharmacies to bc residents . it excludes over - the - counter medications , medications administered to inpatients in acute care hospitals , medication samples dispensed at a physician s office , and medications administered for veterinary or agricultural use . antimicrobial utilization data were classified according to the who s anatomical therapeutic chemical classification system ( 17 ) . to derive indication - specific antimicrobial utilization rates , antimicrobial prescription records were linked to diagnostic codes from the bc medical services plan ( msp ) files . the msp files contain data for all medically necessary services provided by fee - for - service practitioners to bc residents covered under the provincial insurance program . prescription records were linked to msp records based on matching patient and physician identifiers when the prescription dispensing date occurred within five days of the practitioner service date . in instances in which more than one msp record was extracted , the record with the most recent service date was used . indications were defined according to the international classification of diseases , ninth edition ( icd-9 ) ( 18 ) . pharmanet - msp linked data were restricted to icd-9 diagnostic codes for cystitis ( 595 ) and its subcodes ( 595.0 , 595.1 , etc ) . antimicrobial resistance data for e coli urinary isolates were obtained from bc biomedical laboratories ltd ( part of lifelabs medical service laboratories as of 2013 ) , a community - based laboratory practice serving the vancouver coastal and fraser health authorities in bc , for the years 2007 to 2010 . annual population estimates used in the calculation of antimicrobial utilization rates were obtained from the bc health data warehouse ( 20 ) . analyses were performed separately for adults 15 years of age and children < 15 years of age . in adults , antimicrobial utilization rates were calculated as the defined daily dose ( ddd ) per 1000 population per day , where ddd represents the average maintenance dose per day for a drug used in its main indication in adults ( 17 ) . in children , antimicrobial utilization rates were calculated as the total number of prescriptions per 1000 population per day because antimicrobial dosing in pediatric patients varies according to weight . an adaptive - use dri for e coli was calculated according to methods adapted from laxminarayan and klugman ( 16 ) using the formula : r=kpktqktwhere pkt is the rate of resistance to drug class k at time t and qkt is the proportional rate of use for drug class k used to treat cystitis at time t. higher index values indicate more resistance . static use model was used to measure the hypothetical dri trend had proportional antimicrobial use remained unchanged over time ( 16 ) . differences between static and observed adaptive dris help gauge the contributions of physician - adaptation to preserving the value of initial empirical therapy . the static dri was calculated using the proportional rate of use for drug class k fixed to the baseline year of the analysis . to ensure comparability of results with those of previous studies , a sensitivity analysis was also performed using overall prescription data for any indication rather than prescription data specifically for the treatment of cystitis . anatomical therapeutic chemical drug classes were restricted to drug classes relevant to the treatment of utis caused by e coli , where data were available . these included : penicillins with extended spectrum ( j01ca ) , first - generation cephalosporins ( j01db ) , third - generation cephalosporins ( j01dd ) , fluoroquinolones ( j01ma ) , nitrofuran derivatives ( j01xe ) and sulphonamide / trimethoprim combinations ( j01ee ) . an isolate was considered to be resistant to a drug class if it was found to be nonsusceptible to at least one antimicrobial agent within that class ( 21 ) . it should be noted that cefotaxime resistant cut - off ranges were modified in 2010 , but that standards otherwise remained constant throughout the study period . stratified analyses were performed according to patient demographic characteristics ( ie , age group and sex ) . percentile cis were calculated using nonparametric bootstrap methods with m=1000 simulations performed on an approximately 10% sample ( pharmanet = 47,782 observations ; bc biomedical = 13,166 observations ) drawn at random from the full datasets . ethics approval for the present analysis was obtained from the clinical research ethics board at the university of british columbia ( vancouver , british columbia ) . antimicrobial utilization data were obtained from the bc pharmanet database of outpatient prescriptions for oral antimicrobials for systematic use for the years 2007 to 2010 . the pharmanet database includes records of all outpatient prescriptions dispensed from community pharmacies to bc residents . it excludes over - the - counter medications , medications administered to inpatients in acute care hospitals , medication samples dispensed at a physician s office , and medications administered for veterinary or agricultural use . antimicrobial utilization data were classified according to the who s anatomical therapeutic chemical classification system ( 17 ) . to derive indication - specific antimicrobial utilization rates , antimicrobial prescription records were linked to diagnostic codes from the bc medical services plan ( msp ) files . the msp files contain data for all medically necessary services provided by fee - for - service practitioners to bc residents covered under the provincial insurance program . prescription records were linked to msp records based on matching patient and physician identifiers when the prescription dispensing date occurred within five days of the practitioner service date . in instances in which more than one msp record was extracted , the record with the most recent service date was used . indications were defined according to the international classification of diseases , ninth edition ( icd-9 ) ( 18 ) . pharmanet - msp linked data were restricted to icd-9 diagnostic codes for cystitis ( 595 ) and its subcodes ( 595.0 , 595.1 , etc ) . antimicrobial resistance data for e coli urinary isolates were obtained from bc biomedical laboratories ltd ( part of lifelabs medical service laboratories as of 2013 ) , a community - based laboratory practice serving the vancouver coastal and fraser health authorities in bc , for the years 2007 to 2010 . annual population estimates used in the calculation of antimicrobial utilization rates were obtained from the bc health data warehouse ( 20 ) . analyses were performed separately for adults 15 years of age and children < 15 years of age . in adults , antimicrobial utilization rates were calculated as the defined daily dose ( ddd ) per 1000 population per day , where ddd represents the average maintenance dose per day for a drug used in its main indication in adults ( 17 ) . in children , antimicrobial utilization rates were calculated as the total number of prescriptions per 1000 population per day because antimicrobial dosing in pediatric patients varies according to weight . an adaptive - use dri for e coli was calculated according to methods adapted from laxminarayan and klugman ( 16 ) using the formula : r=kpktqktwhere pkt is the rate of resistance to drug class k at time t and qkt is the proportional rate of use for drug class k used to treat cystitis at time t. higher index values indicate more resistance . for contrast , a static use model was used to measure the hypothetical dri trend had proportional antimicrobial use remained unchanged over time ( 16 ) . differences between static and observed adaptive dris help gauge the contributions of physician - adaptation to preserving the value of initial empirical therapy . the static dri was calculated using the proportional rate of use for drug class k fixed to the baseline year of the analysis . to ensure comparability of results with those of previous studies , a sensitivity analysis was also performed using overall prescription data for any indication rather than prescription data specifically for the treatment of cystitis . anatomical therapeutic chemical drug classes were restricted to drug classes relevant to the treatment of utis caused by e coli , where data were available . these included : penicillins with extended spectrum ( j01ca ) , first - generation cephalosporins ( j01db ) , third - generation cephalosporins ( j01dd ) , fluoroquinolones ( j01ma ) , nitrofuran derivatives ( j01xe ) and sulphonamide / trimethoprim combinations ( j01ee ) . an isolate was considered to be resistant to a drug class if it was found to be nonsusceptible to at least one antimicrobial agent within that class ( 21 ) . it should be noted that cefotaxime resistant cut - off ranges were modified in 2010 , but that standards otherwise remained constant throughout the study period . stratified analyses were performed according to patient demographic characteristics ( ie , age group and sex ) . percentile cis were calculated using nonparametric bootstrap methods with m=1000 simulations performed on an approximately 10% sample ( pharmanet = 47,782 observations ; bc biomedical = 13,166 observations ) drawn at random from the full datasets . ethics approval for the present analysis was obtained from the clinical research ethics board at the university of british columbia ( vancouver , british columbia ) . among both patient populations , the highest resistance rates were observed for ampicillin ( 43.8% for adults and 46.9% for children ) in the baseline year , followed by cephalothin ( 25.8% and 20.1% ) , tmpsmx ( 25.1% and 26.2% ) and ciprofloxacin ( 22.7% and 7.1% ) . resistance rates for cefotaxime ( 11.8% and 6.5% ) and nitrofuratoin ( 3.3% and 1.3% ) remained relatively low for both patient populations across all study years . for adults , resistance rates were generally stable over time despite a trend toward increasing resistance to fluoroquinolones ( p>0.05 ) ( figure 1a ) . for children , resistance rates for ampicillin significantly decreased over time ( rho=1.00 ; p<0.01 ) , while resistance rates for ciprofloxacin significantly increased ( rho=1.00 ; p<0.01 ) ( figure 1b ) . resistance rates for all other included drug classes were stable ( p>0.05 ) . among adults , fluoroquinolones comprised the largest proportional use at 37.9% in the baseline year , followed by nitrofuran derivatives ( 32.1% ) and sulphonamide / trimethoprim combinations ( 21.4% ) ( figure 2a ) . proportional use of fluoroquinolones ( rho=1.00 ; p<0.01 ) and sulphonamide / trimethoprim combinations ( rho=1.00 ; p<0.01 ) significantly decreased during the study period , corresponding to a significant increase in the proportional use of nitrofuran derivatives ( rho=1.00 ; p<0.01 ) . among children , sulphonamide / trimethoprim combinations comprised the largest proportional drug use at > 50% across all study years ( figure 2b ) . a significant decreasing trend in proportional use was observed for sulphonamide / trimethoprim combinations over time ( rho=1.00 ; p<0.001 ) , while significant increasing trends were observed for third - generation cephalosporins ( rho=1.00 ; p<0.001 ) and nitrofuran derivatives ( rho=1.00 ; p<0.001 ) . among adults , the adaptive - use dri , when averaged across all study years , was 0.18 ( 95% ci 0.17 to 0.18 ) and remained stable over time ( rho=0.80 ; p=0.20 ) ( figure 3a ) . the static - use dri increased from 0.18 ( 95% ci 0.17 to 0.19 ) in 2007 to 0.20 ( 95% ci 0.19 to 0.21 ) in 2010 ; however , the overall trend was not significant ( rho=0.80 ; p=0.20 ) . dri values for the static - use model did not significantly differ from the adaptive - use model over the four years of study , as indicated by the overlapping 95% cis . dri values did not dramatically differ when total number of prescriptions ( versus ddds ) was used to calculate proportional antimicrobial use for adults ( data not shown ) . among children , the adaptive - use dri peaked at 0.30 ( 95% ci 0.25 to 0.35 ) in 2008 then declined to 0.27 ( 95% ci 0.22 to 0.32 ) in 2010 ; however , this trend was not significant ( rho=0.40 ; p=0.60 ) ( figure 3b ) . fluoroquinolones and sulphonamide / trimethoprim combinations made the largest contribution to the dri among adults across all years , while among children the trend in dri values was predominantly driven by sulphonamide / trimethoprim combinations and penicillins with extended spectrum ( table 1 ) . other drug classes had relatively less impact on dri trends among both adults and children . analyses according to age group and sex show that dri values were consistently higher among males than females across all age groups ( figure 4 ) . for adults , there was a significant increasing trend from 0.09 ( 95% ci 0.08 to 0.11 ) among those 15 to 19 years of age to 0.32 ( 95% ci 0.29 to 0.34 ) among those > 90 years of age ( rho=0.95 ; p<0.01 ) . for children , there was a significant declining trend from 0.35 ( 95% ci 0.27 to 0.43 ) among those < 1 year of age to 0.17 ( 95% ci 0.13 to 0.22 ) among those 10 to 14 years of age ( rho=1.00 ; p<0.01 ) . a sensitivity analysis was performed using overall , rather than indication - specific , prescription data to calculate proportional drug use . among both patient populations , penicillins with extended spectrum were the most frequently used antimicrobial class for any indication in the baseline year ( 43.4% for adults and 75.0% for children ) , followed by fluoroquinolones ( 22.1% and 0.4% , respectively ) , first - generation cephalosporins ( 14.8% and 12.4% , respectively ) , sulphonamide / trimethprim combinations ( 11.5% and 9.6% , respectively ) , nitrofuran derivatives ( 7.5% and 0.4% , respectively ) and third - generation cephalosporins ( 0.7% and 2.2% , respectively ) . among adults , the adaptive - use dri was significantly higher at 0.31 ( 95% ci 0.31 to 0.32 ) when overall prescription data were used . the adaptive - use dri remained stable over time ( rho=0.40 ; p=0.60 ) and did not significantly differ from the static - use model . among children , the adaptive - use dri was significantly higher at approximately 0.40 ( 95% ci 0.37 to 0.43 ) when overall prescription data were used and remained stable over time ( rho=0.80 ; p=0.20 ) . a sensitivity analysis was performed using overall , rather than indication - specific , prescription data to calculate proportional drug use . among both patient populations , penicillins with extended spectrum were the most frequently used antimicrobial class for any indication in the baseline year ( 43.4% for adults and 75.0% for children ) , followed by fluoroquinolones ( 22.1% and 0.4% , respectively ) , first - generation cephalosporins ( 14.8% and 12.4% , respectively ) , sulphonamide / trimethprim combinations ( 11.5% and 9.6% , respectively ) , nitrofuran derivatives ( 7.5% and 0.4% , respectively ) and third - generation cephalosporins ( 0.7% and 2.2% , respectively ) . among adults , the adaptive - use dri was significantly higher at 0.31 ( 95% ci 0.31 to 0.32 ) when overall prescription data were used . the adaptive - use dri remained stable over time ( rho=0.40 ; p=0.60 ) and did not significantly differ from the static - use model . among children , the adaptive - use dri was significantly higher at approximately 0.40 ( 95% ci 0.37 to 0.43 ) when overall prescription data were used and remained stable over time ( rho=0.80 ; p=0.20 ) . the dri reflects the proportion of prescriptions for e coli cystitis in a population that uses a drug to which the organism is not susceptible . our findings show that the overall dri value for community e coli urinary isolates , when averaged across study years , is 18% ( 95% ci 17% to 18% ) among adults 15 years of age . this value is lower than the e coli dri of 30% ( 95% ci 29% to 32% ) reported by laxminarayan and klugman ( 16 ) for 2006 , likely because our study used indication - specific antimicrobial utilization data , which will more accurately reflect the tailored selection of antimicrobials for the treatment of utis . sensitivity analyses based on overall prescribing for any indication found dri values more consistent with previously reported values at 31% ( 95% ci 31% to 32% ) , largely driven by the greater proportional use of extended - spectrum penicillins . our analyses were stratified according to age group , which may also partially explain the discrepancy with previous findings . while resistance rates were similar between adults 15 years of age and children < 15 years of age , the higher proportional use of extended - spectrum pencillins and sulphonamide / trimethoprim combinations among children increased the relative contribution of these drug classes to the age - specific dri . for children , the overall dri value across all study years was 28% ( 95% ci 26% to 31% ) . geographical variability may also be a factor because resistance rates are known to be significantly higher in the united states in comparison with canada ( 8,9 ) . adaptive - use dri trends remained stable over time , and dri values based on a static - use model that fixes proportional antimicrobial use to the baseline year did not significantly differ from an adaptive - use model . however , even over this short study period , the static index appears to be trending upward . a contrast between the value of the observed adaptive and static dri is consistent with physicians increasing the proportionate use of nitrofurantoin . physicians appear to have been increasing their use of this drug at the expense of tmp - smx and ciprofloxacin . this change could be in response to evolving guidelines , emphasis in continuing education sessions or individual physician attention to antibiograms . whatever the driver , physicians may be adapting prescribing practices to local resistance patterns ( 16 ) . however , the limited availability of data makes it difficult to assess long - term trends . others have noted rising resistance rates over time associated with the clonal spread of virulent , multidrug - resistant strains ( 7,22 ) , including extended - spectrum beta - lactamase - producing strains of community - acquired e coli ( 23,24 ) . a longer duration of surveillance will be required to monitor the impact of extended - spectrum beta - lactamase - producing e coli strains on dri trends in bc . consistent with recent guidelines ( 4,25 ) , our data suggest that nitrofurantoin is an appropriate treatment for acute uncomplicated cystitis given its low observed resistance rates . while this may be good news for cystitis , nitrofurantoin is not indicated for the treatment of pyelonephritis due to inadequate concentrations achieved in renal tissues and should not be used if early pyelonephritis is suspected ( 4,25 ) . despite earlier research suggesting that nitrofurantoin has lower clinical cure rates and more side effects than traditional first - line agents , namely tmp - smx and fluoroquinolones ( 26 ) , more recent studies suggest that empirical treatment with nitrofurantoin is as effective as these drugs ( 25 ) . given the concerning resistance trends observed for ciprofloxacin and tmp - smx , our findings support calls made by others for more judicious use of these drugs ( 8,2527 ) . routine testing data were not available for our study period for other , alternative treatments for utis , such as fosfomycin , which has only recently become available again in canada . dri trends according to age group suggest a u - shaped distribution with the lowest rates occurring among adults < 50 years of age and the highest rates occurring among young children and the elderly . elderly adults are at greater risk for developing a drug - resistant infection , likely as a result of cumulative lifetime exposure to antimicrobials , particularly to fluoroquinolones , higher frequency and longer duration of hospital stays , increased likelihood of developing complicated infections and increased prevalence of comorbidities ( 7,11,12,14 ) . while acute , uncomplicated utis are generally self - limiting and not associated with long - term medical sequelae in adults in the absence of underlying conditions ( 1,28 ) , among pediatric patients , utis can be associated with significant morbidity , including impaired renal function and end - stage renal disease ( 28,29 ) . males had consistently higher dri values than females across all age groups , a finding that is consistent with resistance trends observed nationally ( 79 ) . these trends are largely driven by the greater proportional use of fluoroquinolones for cystitis and the higher rates of ciprofloxacin resistance among men ( data not shown ) . the high prevalence of complicated utis among men , combined with the longer duration of therapies and older age at diagnosis , likely explains the higher observed resistance rates ( 7,30,31 ) . in children , this sex difference is likely the result of congenital abnormalities , which are more prevalent among males than females ( 29,30 ) . the present study extends previous work in this area in a number of important ways . access to prescription drug use through bc pharmanet allows for examination of antimicrobial utilization in community settings for the entire population of bc . we have adopted standardized methods of measuring antimicrobial use in units of ddds ( 17 ) and established linkage methodologies to connect antimicrobial prescriptions to icd-9 diagnostic codes to derive indication - specific antimicrobial prescribing ( 32,33 ) . however , despite these strengths , certain limitations should be acknowledged . prescription data from pharmanet are restricted to oral antimicrobials dispensed in out - patient settings . while these data comprehensively measure prescription use in the community the main focus of our study they do not capture antimicrobial use for agents that are administered parenterally and/or primarily used in inpatient settings . as a result , utilization rates for certain drug classes may be underestimated in our study or , in the case of carbapenems and aminoglycosides , excluded completely . the accuracy of data on indication - specific prescribing will depend on the manner in which physicians code the diagnosis when billing . we focused on the majority of utis for which a cystitis code ( icd-9 595 ) was coded to assure specificity of that diagnosis in describing treatment patterns , utis identified by less specific codes ( eg , icd-9 599 ) were excluded . nonetheless , our linked data analysis reflects patterns of prescription for an entire population . bc biomedical laboratories are located in only two of five of the regional health authorities in bc , and , although one of the largest laboratory service providers in bc , they are not the sole provider of outpatient laboratory services in these areas . future analyses will begin with more complete sampling of laboratory data through collaboration with broader laboratory systems . routine susceptibility testing of penicillins with beta - lactamase inhibitors ( eg , amoxicillin - clavulanate ) was only performed from 2009 onwards ; consequently , this antimicrobial class was excluded from our analysis to ensure comparability of dri values over time . culture samples are generally not indicated for young , otherwise healthy , premenopausal women . as such , isolates may be more representative of certain patient populations with higher resistance rates such as those with complicated utis ( 79 ) . finally , dris for adults and children in the present analysis may not be directly comparable due to differences in their calculations ( refer to the methods section ) . dris improve our ability to quantify and communicate the cumulative impact of antimicrobial resistance on the likelihood that an organism will be susceptible to initial antimicrobial treatment . for clinicians , they provide information on the effectiveness of alternative treatment regimens and the relative adaptability of these treatments to local resistance patterns . for policymakers and other nonexpert groups , dris function as an important communication tool for translating knowledge about antimicrobial resistance into practice . our findings show that the overall dri value for community utis caused by e coli in bc is 18% for adults and 28% for children for the years 2007 to 2010 . the likelihood that a uti caused by e coli will be susceptible to initial antimicrobial therapy is highest among adult females , while concerning dri values were observed among young children and the elderly . further work is required to develop a dri to summarize the impact of resistance on all relevant pathogens causing uti in a population .
stroke is the major cause of chronic disability and the third leading cause of death in humans after cancer and coronary heart disease in developed countries . because stroke causes severe neurological complications , new strategies for patients suffering from stroke are needed . the capacity for neuroprotection depends on multiple factors including the use of an appropriate agent at the appropriate time and during the appropriate interval.1 a variety of animal models have been developed for modeling ischemic stroke . among them , the most common model of focal ischemia in rats is the occlusion of the middle cerebral artery ( mcao ) with an intraluminal suture , which mechanically occludes the major cerebral arteries.2 in contrast with other models of cerebral ischemia that involve mechanical occlusion of blood vessels , the rat photochemical cortical lesion models show endothelial damage , resulting in platelet aggregation , thrombosis , and permanent cerebral vascular occlusion.3 considering that vascular thrombosis is responsible for more than 70% of human stroke cases and that only one food and drug administration ( fda)-approved therapy exists for the treatment of acute ischemic stroke ( the thrombolytic tissue plasminogen activator , or tpa ) , this model may be useful for understanding the pathobiology of ischemic stroke.4 cerebral ischemia / reperfusion ( i / r ) injury triggers multiple and distinct but overlapping cell signaling pathways that may lead to cell survival or cell damage.5 compared with global ischemia , focal ischemia is a reduction in blood flow to a very specific brain region as in embolic occlusion of the specific vessels.6 there are also significant differences in the modes of cell death between global and focal cerebral ischemia . in focal cerebral ischemia , most of the cells in the ischemic core undergo necrosis , but the rim of brain tissue that is hypoperfused surrounding the core is called the ischemic penumbra , which has the capacity to recover if perfusion is improved.7 in the penumbra , multiple mechanisms lead to apoptosis : excitotoxicity and ionic imbalance , oxidative stress , and apoptotic - like cell death may all occur and in both caspase - dependent and caspase - independent manners that may contribute to delayed ischemic cell death.8 a quite simplistic view implies poor prospects regarding cell survival in the core of the cerebral infarction and therapeutic expectations to control cell death and cell survival in the penumbra . in fact , focal cerebral ischemia triggers a cascade of molecular events that produce neuronal death ranging from immediate death to that occurring many days later , and this phenomenon raises the hypothesis that a " therapeutic window " exists during which interventions may improve neurological outcome.9 the aim of neuroprotection is to prevent delayed neuron apoptosis in the zone of the ischemic penumbra . on the basis of our current knowledge , many experimental trials have been reported to restore this process.10 - 12 in the photochemical stroke model , a ring lesion , which creates a cortical region of viable tissue surrounded by a circular rim of damaged tissue , has been described for the study of histological , biochemical , and molecular changes associated with the ischemic penumbra.13 tunel stain is used to demonstrate apoptotic and nonapoptotic features.14 although the expression of pro- and anti - apoptotic factors and apoptotic cell death sequences have been extensively investigated , the time point changes in the mechanisms of cell death in photochemically induced focal cerebral ischemia remain inadequately defined . previous investigations have reported on changes in the bcl-2 family in this model.14 - 16 therefore , we examined changes in the expression of bcl-2 , bax , caspase-3 , phosphorylated akt ( pakt ) , and survivin by western blot 1 , 3 , and 7 days after photochemically induced focal cerebral ischemia in the rat . all surgical procedures and postoperative care were performed in accordance with the guidelines of the chonnam national university animal care and usage committee . twenty male sprague - dawley rats weighing between 200 and 250 g were used in this study . these animals were maintained on a 12-h light / dark cycle and were allowed free access to food and water . each rat was anesthetized with 5% isoflurane and was maintained with 2% isoflurane in an oxygen / air mixture with the use of a gas anesthesia mask in a stereotaxic frame ( stoelting , wood dale , il , usa ) . focal cortical ischemia was induced by photothrombosis of the cortical microvessels using rose bengal ( rb ; sigma chemical co. , st . louis , mo , usa ) with cold light ( zeiss kl1500 lcd , jena , germany ) as previously described with minor modifications.3,17 the expression of each apoptosis associated protein was measured 1 , 3 , and 7 days after the onset of ischemia . body temperature was maintained during surgery at 370.5 with a heating pad controlled with a rectal probe . for illumination , a 4.5-mm fiber optic bundle from a cold light source was positioned on the exposed skull 0.5 mm anterior to the bregma and 3.7 mm lateral to the midline over the left sensorimotor cortex . the brain was illuminated for 10 min after infusion of 50 mg / kg of rb in normal saline into the right femoral vein via a microinjection pump within 1 min . the scalp was sutured and the rats were allowed to awake before being returned to their cages . the sham - operated control group received the same vein injection of rb and the same surgery , but without exposure to light ; thus , no photochemical reaction was induced.3 after decapitation , rat brains from different reperfusion time points ( sham and days 1 , 3 , and 7 ) were quickly removed ( each group , n=5 ) . samples were flash - frozen with liquid nitrogen and were stored at -80 until analyzed . brain tissues were homogenized and proteins were purified by using 50 mm tris - cl ( ph 7.4 ) , 150 mm nacl , 1 mm edta , 60 mm n - octyl--d - glucopyranoside ( ogp ) , and 1 mm phenylmethylsulfonyl fluoride ( pmsf ) supplemented with a protease inhibitor cocktail ( sigma ) . to prepare lysates , tissues were sonicated for 4 s for a total of five times and the homogenate was spun at 20,000g for 20 min at 4 to remove insoluble material . protein concentrations were determined with the bca protein assay reagent kit ( pierce , usa ) using bovine serum albumin ( bsa ) as the standard . equal amounts of nuclear protein ( 20 g ) for bcl-2 , bax , caspase-3 , pakt ( serine-473 ) , and survivin were separated on 6% to 12% sds - polyacrylamide gels ( usb fueling innovation , cleveland , oh , usa ) . a pageruler plus prestained protein ladder ( fermentas life sciences , hanover , md , usa ) was used as a size reference . proteins were transferred to pure nitrocellulose membranes ( bio - rad , richmond , ca , usa ) . the membranes were incubated in blocking buffer , 5% nonfat dry milk in tbst ( 200 mm tris - hcl , ph 7.4 , 500 mm nacl , 0.2% tween 20 ) , at room temperature for 1 h with agitation . the membranes were then incubated with rabbit bcl-2 polyclonal antibody ( santa cruz , 1:2,000 in tbs with 0.2% tween 20 ) , rabbit bax polyclonal antibody ( millipore , 1:3,000 in tbs with 0.2% tween 20 ) , mouse caspase-3 monoclonal antibody ( millipore , 1:1,000 in tbs with 0.2% tween 20 ) , polyclonal rabbit anti - phopho - akt antibody ( serine-472 , santa cruz , 1:5,000 in tbs with 0.2% tween 20 ) , and mouse survivin monoclonal antibody ( santa cruz , 1:1,000 in tbs with 0.2% tween 20 ) overnight at 4. following washes , the membranes were incubated with peroxidase - conjugated goat anti - mouse or anti - rabbit immunoglobulin ( santa cruz biotechnology , 1:8,000 ) in tbs with 0.2% tween 20 for 1 h. after rinses in washing buffer , signal of bound antibodies was developed by an enhanced immoblion western chemiluminescent hrp substrate ( millipore , bedford , ma , usa ) . quantification of each band was performed by densitometry analysis ( scion , nih software , bethesda , md , usa ) of the protein signal using las 3000 . mouse anti - gapdh monoclonal antibody conjugated with hrp ( sigma , 1:25,000 ) was detected on immunoblots as a loading control for protein quantitation . to analyze the amount of the bcl-2 , bax , caspase-3 , pakt , survivin , and gapdh , we used a relative protein ratio to control , and quantitative data were expressed as meanssd . differences between means were determined by one - way anova followed by dunnett 's post hoc test for multiple comparisons . all surgical procedures and postoperative care were performed in accordance with the guidelines of the chonnam national university animal care and usage committee . twenty male sprague - dawley rats weighing between 200 and 250 g were used in this study . these animals were maintained on a 12-h light / dark cycle and were allowed free access to food and water . each rat was anesthetized with 5% isoflurane and was maintained with 2% isoflurane in an oxygen / air mixture with the use of a gas anesthesia mask in a stereotaxic frame ( stoelting , wood dale , il , usa ) . focal cortical ischemia was induced by photothrombosis of the cortical microvessels using rose bengal ( rb ; sigma chemical co. , st . louis , mo , usa ) with cold light ( zeiss kl1500 lcd , jena , germany ) as previously described with minor modifications.3,17 the expression of each apoptosis associated protein was measured 1 , 3 , and 7 days after the onset of ischemia . body temperature was maintained during surgery at 370.5 with a heating pad controlled with a rectal probe . for illumination , a 4.5-mm fiber optic bundle from a cold light source was positioned on the exposed skull 0.5 mm anterior to the bregma and 3.7 mm lateral to the midline over the left sensorimotor cortex . the brain was illuminated for 10 min after infusion of 50 mg / kg of rb in normal saline into the right femoral vein via a microinjection pump within 1 min . the scalp was sutured and the rats were allowed to awake before being returned to their cages . the sham - operated control group received the same vein injection of rb and the same surgery , but without exposure to light ; thus , no photochemical reaction was induced.3 after decapitation , rat brains from different reperfusion time points ( sham and days 1 , 3 , and 7 ) were quickly removed ( each group , n=5 ) . samples were flash - frozen with liquid nitrogen and were stored at -80 until analyzed . brain tissues were homogenized and proteins were purified by using 50 mm tris - cl ( ph 7.4 ) , 150 mm nacl , 1 mm edta , 60 mm n - octyl--d - glucopyranoside ( ogp ) , and 1 mm phenylmethylsulfonyl fluoride ( pmsf ) supplemented with a protease inhibitor cocktail ( sigma ) . to prepare lysates , tissues were sonicated for 4 s for a total of five times and the homogenate was spun at 20,000g for 20 min at 4 to remove insoluble material . protein concentrations were determined with the bca protein assay reagent kit ( pierce , usa ) using bovine serum albumin ( bsa ) as the standard . equal amounts of nuclear protein ( 20 g ) for bcl-2 , bax , caspase-3 , pakt ( serine-473 ) , and survivin were separated on 6% to 12% sds - polyacrylamide gels ( usb fueling innovation , cleveland , oh , usa ) . a pageruler plus prestained protein ladder ( fermentas life sciences , hanover , md , usa ) was used as a size reference . proteins were transferred to pure nitrocellulose membranes ( bio - rad , richmond , ca , usa ) . the membranes were incubated in blocking buffer , 5% nonfat dry milk in tbst ( 200 mm tris - hcl , ph 7.4 , 500 mm nacl , 0.2% tween 20 ) , at room temperature for 1 h with agitation . the membranes were then incubated with rabbit bcl-2 polyclonal antibody ( santa cruz , 1:2,000 in tbs with 0.2% tween 20 ) , rabbit bax polyclonal antibody ( millipore , 1:3,000 in tbs with 0.2% tween 20 ) , mouse caspase-3 monoclonal antibody ( millipore , 1:1,000 in tbs with 0.2% tween 20 ) , polyclonal rabbit anti - phopho - akt antibody ( serine-472 , santa cruz , 1:5,000 in tbs with 0.2% tween 20 ) , and mouse survivin monoclonal antibody ( santa cruz , 1:1,000 in tbs with 0.2% tween 20 ) overnight at 4. following washes , the membranes were incubated with peroxidase - conjugated goat anti - mouse or anti - rabbit immunoglobulin ( santa cruz biotechnology , 1:8,000 ) in tbs with 0.2% tween 20 for 1 h. after rinses in washing buffer , signal of bound antibodies was developed by an enhanced immoblion western chemiluminescent hrp substrate ( millipore , bedford , ma , usa ) . quantification of each band was performed by densitometry analysis ( scion , nih software , bethesda , md , usa ) of the protein signal using las 3000 . mouse anti - gapdh monoclonal antibody conjugated with hrp ( sigma , 1:25,000 ) was detected on immunoblots as a loading control for protein quantitation . to analyze the amount of the bcl-2 , bax , caspase-3 , pakt , survivin , and gapdh , we used a relative protein ratio to control , and quantitative data were expressed as meanssd . differences between means were determined by one - way anova followed by dunnett 's post hoc test for multiple comparisons . ischemia caused changes in the expression of various apoptosis - associated proteins depending on the time of study ( fig . notably , the expression of bax , caspase-3 , and pakt proteins was markedly increased in the ischemic hemisphere 1 day after the operation compared with that in the sham controls . the expression of bcl-2 and survivin was slightly reduced at day 1 and then gradually increased by day 3 . bax expression , the bands at 21 kda , reached peak levels at day 3 and gradually decreased by day 7 . caspase-3 expression , identified at 32 kda , was abruptly increased from day 1 to day 3 compared with the sham control and decreased at day 7 . it was slightly reduced at day 1 , but gradually increased from day 3 , and was rising on day 7 . however , the variation in expression of survivin , which is an inhibitor apoptosis protein ( iap ) , was insignificant compared with the others . changes in the density of each apoptosis - associated protein at each time point are shown in fig . bcl-2 protein was increased 1.9-fold at day 3 ( p<0.05 versus control ) and increased 2.8-fold at day 7 ( p<0.05 versus control ) in the ischemic hemisphere . bax and pakt were increased 1.6-fold at day 3 and 1.8-fold at day 1 , respectively ( p<0.05 versus control ) . caspase-3 was abruptly increased 8.4- to 9.9-fold from day 1 to day 3 ( p<0.01 versus control ) , respectively , after the operation . no significant differences in survivin levels were found between sham controls and the experimental groups . focal and global cerebral ischemia in rats triggers apoptotic cell death within the ischemic lesion . in many previous reports,1,18 comparison of human small cortical infarcts reveals identical histological patterns to photochemically induced rat brain lesions as well as naturally occurring human infarcts.19 comparatively little is known about the apoptotic process in the photochemically induced model , so we evaluated the time point expression patterns of the major apoptosis regulatory proteins . the bcl-2 family of proto - oncogenes encode specific proteins such as bcl-2 , bcl - xl , and bax that regulate apoptosis . the anti - apoptotic effect of bcl-2 occurs by prevention of cytochrome c release into the cytoplasm.5 many studies have shown that over - expression of bcl-2 can reduce ischemic brain injury in animal models of stroke.12,20 activated bax promotes cell death , unless it is bound by either bcl-2 or bcl - xl.21 however , the two molecules can act independently to exert their effects on cell death.5 in this study , we found strong induction of bax protein 1 day after infarction in the ischemic hemisphere and it reached a peak on day 3 . compared with bax expression , bcl-2 protein expression was slightly reduced at day 1 and then abruptly increased at day 3 and was sustained at day 7 . in a previous report using the same stroke model,15 bax protein was in the cytoplasm of degenerating neurons in the center of the infarction between 4 h and 3 days after photothrombosis , and at the same time points , the levels of bcl-2 and bcl - xl proteins these findings suggest that bcl-2 and bax protein expression switches around day 3 in this model . interestingly , in the mcao mouse model with preconditioning , bcl-2 and bcl - xl were increased after sublethal forebrain ischemia but bax remained unchanged.22 these differential expressions of the bcl-2 family were associated with animal modeling , such as preparing of preconditioning , and reproducibility of lethal legion induction . the slight decrease in bcl-2 expression within 1 day might also be attributed to postischemic protease activation.23 caspases are cysteine proteases that are constitutively expressed as zymogens or pro - caspases and that are specifically activated during apoptotic stimuli.24 several previous studies reported that the role of caspase-3 is as a key mediator of apoptosis in ischemic stroke.25 briefly , upregulation of caspase-3 mrna in rat brain occurs 1 h after the onset of focal ischemia , whereas caspase-3 and its cleavage products in mouse brain are activated during early reperfusion 2 h after mcao . we observed a significant increase in the cleaved active form of caspase-3 from day 1 of operation until day 3 , when the maximal intensity was observed ( fig . 2c ) . these findings are in agreement with an extended treatment window for caspase inhibition after stroke.18 akt is a subfamily of serine / threonine protein kinases and is activated via phosphatidylinositol-3 kinase ( pi3k ) . after phosphorylation , akt promotes cell survival and prevents apoptosis by inactivating several targets . in the mcao model,26 phosphorylation of akt ( pakt ) was temporally accelerated at serine-473 in the ischemic cortex after transient focal cerebral ischemia and was then decreased by 24 h. however , in the ischemic core , pakt was decreased only after ischemia . pakt - positive cells were colocalized with neun - positive cells in the cortex , but pakt - positive cells did not co - localize with tunel - positive cells . in this study , pakt expression was increased about 2-fold at day 1 and was decreased by day 3 compared with the sham control . this change in pakt expression the infarct develops in the superficial cortex , whereas the higher - order branches of the larger patent vessels in the deeper cortical layers are occluded by tissue compression caused by the vasogenic edema associated with this procedure.27 further , complete wedge - shaped coagulative necrosis was formed 24 h after ischemia according to histopathological examination.28 thus , as in previously reported studies,26 pakt expression in the photochemical - induced stroke model might also depend on the severity of the ischemia . survivin is a member of the iap family that serves multiple functions related to both cell survival and cell division within many cell types and only recently has come under study in relation to ischemia in heart or brain.29 recently , zhang et al.30 reported that this protein is a target for the pi3-kinase / akt pathway and can antagonize the effects of apoptosis - inducing factor . on the basis of current knowledge , we hypothesized that survivin would be significantly changed in our stroke model , but as noted , the changes in survivin expression evaluated by western blot were insignificant . mice at 24 h and 3 d were not significantly different from those of survivin mice , which suggests that the role of survivin in neuroprotection is minimal.29 in this study , we evaluated the expression of apoptosis regulatory proteins in photochemically induced focal cerebral ischemic rat brain . to interpret these changes , we have to consider the changes in cerebral blood flow ( cbf ) after photothrombosis . in rats subjected to a photothrombotic stroke lesion , cortical cbf at the center of the region at risk however , a significant partial restoration of blood flow compared with ccbf values at 24 h was observed at 72 and 96 h after irradiation . hence , cortical cbf was 56% of preischemic levels at 72 h and 87% at 96 h after ischemic induction . these cbf changes could explain the meaningful changes in bcl-2 , bax , and caspase-3 around day 3 . in conclusion , targeting and preventing apoptosis in the penumbra seems to be a rational therapeutic goal for reducing cerebral infarct volume after clinical stroke , and the present study may provide the basis for the design of therapeutic interventions using the photochemical thrombosis stroke model . however , the expression of each apoptosis regulatory protein as well as the regional distribution and association with dna fragmentation should be assessed further .
rising interest in health and regular medical checkups have promoted detection of early cancer . likewise , in stomach cancer , detection rates for early stages are higher than those for progressive cancer . traditionally , gastrectomy has generally been regarded as the standard therapy for treatment of gastric cancer . however , endoscopic mucosal resection ( emr ) or endoscopic submucosal dissection ( esd ) has shown effectiveness equivalent to that of gastrectomy and has emerged as a popular technique for curative treatment of gastric cancer . by removing only the lesion , stomach conserving is possible and the quality of life is much improved . however , noncurative resection or resection beyond the indication may lead to lymphatic and extended organ metastasis resulting in loss of the opportunity for full recovery . retrospective studies have reported similar results between gastrectomy and endoscopic resection.1 because the results were similar between patients treated by absolute indications and expanded indications,2,3 many hospitals tend to apply endoscopic removal following expanded indications . not only treatment indications but also the patients ' age , underlying disease , and the clinicians ' preference are all carefully considered in the selection of endoscopic removal . on occasion , clinicians contemplate the course of therapy when endoscopic resection is performed outside the indications or curative resection is not achieved despite complete resection . in this section , we discuss further treatment options in the situation of noncurative resection . to understand the definition of noncurative resection , one should be aware of the indications of endoscopic resection . endoscopic resection should be performed only in cases where lymphatic metastasis is unlikely and en bloc resection is possible . according to korean gastric cancer guidelines , absolute indications for endoscopic resection are : 1 ) differentiated cancer confined to the mucosa ; 2 ) longer diameter than 2 cm ; 3 ) no evidence of an ulcer or ulcer scar ; and 4 ) no lymphatic invasion of cancer cells.4 the japanese gastric cancer association recommends emr / esd in such cases.5 expanded indications include : 1 ) differentiated not ulcerous mucosal cancer , regardless of size ; 2 ) differentiated ulcerous mucosal adenocarcinoma smaller than 3 cm ; 3 ) poorly differentiated not ulcerous mucosal adenocarcinoma smaller than 2 cm ; and 4 ) differentiated adenocarcinoma in which invasion depth is less than 500 m ( sm1 ) . according to the japanese gastric cancer association guidelines , sm1 differentiated adenocarcinoma is not included in the expansive indications ( it is still included in the indication for noncurative resection ) . if the expanded indication is satisfied , investigational treatment by esd ( not emr ) is suggested . in korean guidelines , however , attempts to expand the indications on treatment by esd have been reported ( table 1).6 - 8 although no large scale clinical study has been conducted and no hard evidence has been found , most clinics perform esd as a standard therapy for complete cure rather than experimental treatment . because there are difficulties in officially recommending esd by expanded indication , careful interpretation by expanded indications should be required . curative resection is only defined as when the adenocarcinoma is completely resected and lymphatic metastasis is not risky . absolute indications are : 1 ) en bloc resection ; 2 ) less than 2 cm ; 3 ) differentiated type ; 4 ) confined to the mucosa ; 5 ) complete resection along the horizontal margin ( hm0 ) and the vertical margin ( vm0 ) ; and 6 ) no lymphovascular metastasis ( ly ( - ) , v ( - ) ) . curative resection after esd according to the expanded indications are : en bloc resection , hm0 , vm0 , ly ( - ) , v ( - ) should be possible ; 1 ) larger than 2 cm , differentiated type confined to the mucosa without an ulcer ; 2 ) smaller than 3 cm , differentiated type confined to the mucosa with an ulcer ; 3 ) smaller than 2 cm , poorly differentiated mucosal cancer without an ulcer ; and 4 ) differentiated cancer smaller than 3 cm and submucosal depth invasion less than 500 m ( sm1 ) . therefore , noncurative resection is defined as cases that do not satisfy the indications described above . in practice it is particularly confusing when differentiated cancer and poorly differentiated cancer are mixed . despite weak supporting evidence : 1 ) differentiated mucosal cancer larger than 2 cm without ulcers mixed with poorly differentiated cancer larger than 2 cm ; 2 ) differentiated mucosal cancer with ulcers smaller than 3 cm mixed with poorly differentiated cancer ; and 3 ) poorly differentiated cancer with submucosal invasion are classified as noncurative resection . as mentioned above , size , differentiation , and ulcers are important factors in classifying the indications and judging curative resection . the pathologist 's report is important in defining ulcers ; however , endoscopic or radiological judgment is also referred to.5 if complete resection is not possible , it is called ' incomplete resection ' or ' impossible to judge ' ( complete resection according to endoscopy but impossible to judge by pathology).9 therefore , ' complete resection ' is often decided according to the subjective opinion of the physician performing the endoscopy . since the definition of ' ulcer ' differs between scholars , it seems appropriate to judge each case individually and yet comply with the basic principles . according to national comprehensive cancer network guidelines , history taking and physical examination should be performed every 3 to 6 months for the first 1 to 3 years , every 6 months during 3 to 6 years , and yearly afterwards for long - term observation after complete resection . clinical chemistry tests , radiologic and endoscopic tests should be performed when needed.10 korean gastric cancer standard treatment guidelines recommend yearly endoscopic follow - up for the possibility of synchronous or metachronous cancer . the japanese gastric cancer association recommends helicobacter pylori eradication and endoscopic follow - up every 6 months or yearly after complete cure by absolute indication . h. pylori eradication , endoscopy , and computed tomography scan or ultrasonography is recommended after treatment by expanded indication.5 as discussed above , close observation and examination after curative resection is the appropriate treatment option . however , these guidelines are not established surveillance guidelines for noncurative resection . the most favorable treatment after noncurative resection would be surgery . among the expanded indications , mucosal cancer larger than 3 cm with ulcers , deep submucosal invasion , and lymphatic metastasis are typical indications.11 recently , there have been many reports on the outcome of additional surgery after endoscopic resection . in a domestic research study analyzing patients who underwent radical gastrectomy after esd , nine out of 13 patients with positive margin had residual cancer . fifty percent of patients who underwent surgery for lymphatic invasion of esd specimen showed positive lymph node metastasis.12 in another study involving 28 patients who underwent additional surgery after endoscopic resection , eight patients ( 28.6% ) had residual cancer and one had lymphatic metastasis . the study found that piecemeal endoscopic resection or diffuse type of cancer , or positive vertical resection margin are risk factors for residual cancer.13 in a research study on indications for surgery after noncurative resection , 43 patients underwent additional surgery ; 65.1% of them showed submucosal invasion , 39.5% had residual cancer , and 9.3% had lymphatic invasion . curiously , margin negative sm1 differentiated cancer corresponding to the expanded indication had no lymphatic metastasis or residual cancer . however , one case of 4 cm sized mucosal cancer showed lymphatic metastasis.14 fibrosis was found in pathology analyzed after endoscopic resection . this may be the result of biopsy performed before endoscopic resection ; however , it may also be the result of ulcerous lesions . therefore , over interpretation should be carefully considered . a multicenter domestic study regarding additional surgery after endoscopic resection the patients were grouped according to five categories : submucosal invasion group , positive resection margin group , unknown resection margin positivity group , high probability of lymphatic invasion group , and local recurrence group . among 44 resection margin positive patients , 11 had no signs of residual cancer or lymphatic metastasis.15 among the total 86 patients , 56 ( 65.1% ) had residual cancer and five ( 5.8% ) had lymphatic metastasis . , this study contains many suggestions indicating that other treatment strategies rather than surgery were possible for certain groups . surgery is a desirable treatment ; however , other treatment methods may be recommended based on the patient 's general condition , underlying disease , age , and patient 's refusal of surgery . in particular , when the possibility of lymphatic metastasis is low ( after resection , for example ) , when differentiated mucosal cancer has positive margin only , when complete resection is achieved although piecemeal resection has been performed , etc . , additional endoscopic resection , cauterization using laser or argon plasma coagulation ( apc ) and simply looking forward to the burn effect are all considered treatment methods while postponing additional treatment and closely observing the patient.16 according to japanese gastric cancer association guidelines , focally recurred adenocarcinoma after emr / esd should be treated with additional esd . however , since supporting evidence is weak , this is just one experimental treatment modality . favorable treatment outcomes of esd for local recurrence after endoscopic resection have been reported . in a retrospective analysis of 64 patients who had local recurrence of early gastric cancer ( egc ) after emr , patients treated with esd had higher rates of complete resection and curative resection than patients who underwent repeated emr . in the case of curative resection , there was no recurrence during follow - up.17 in another japanese report , 15 patients underwent esd for local recurrent egc and curative resection rate was 93.9% . there was no recurrence during follow - up durations.18 in a study of 1,150 patients treated with esd as primary treatment , eight out of 33 patients with positive margin underwent esd once more and there were no signs of recurrence during follow - up . recurrent tumors were observed in four out of 12 patients who had close follow - up without additional treatment.19 recently , a researcher in korea reported the results of 1,012 egc patients ; 107 of the patients had a positive hm or a positive vm ; 45 patients underwent immediate surgery , and one patient received additional esd . thirty - nine out of 56 patients who did not undergo additional treatment showed no recurrence during observation . among 17 patients who had recurred , three received apc , and five underwent additional esd . in one patient who received apc after recurrence , there was cancer progression . the authors concluded that although the residual cancer volume is larger in positive horizontal resection margin than positive vertical resection margin and the possibility of recurrence is higher ( odds ratio , 2.05 ; 95% confidence interval , 1.22 to 3.44 ) , lymphatic metastasis is rare . therefore , they emphasized that immediate additional esd before fibrosis can lower the risk of recurrence.20 in another domestic study of 427 egc patients , 58 patients had incomplete removal after endoscopic resection . three patients who underwent additional esd showed satisfactory results . however , both patients treated with apc showed recurrence.21 as in the previously mentioned research studies , this study supports that apc appears to be less effective . however , in other studies using apc as initial treatment , only 10% showed recurrence and many showed favorable results . this is more likely when treated by a well trained and experienced endoscopy expert.22 therefore , in high risk patients for endoscopic resection or surgery , rescue treatment with apc is thought to be the optional treatment . some reports have noted that margin positive cancer shows no recurrence with surgery or observation.15,19,20 therefore , close observation for recurrence may be another alternative for treatment . endoscopic resection by emr / esd is an important treatment method for egc . because of the increasing incidence of egc , endoscopic resection rates have increased and there are many ongoing research studies regarding the expanded indications . therefore , it is an important issue to decide on the range of curative resection in the endoscopic resection field . additional noninvasive strategies such as endoscopic resection , surgery , and apc are performed when curative resection is not achieved after resection . salvage therapy for complete cure in surgery or conservative endoscopic therapy is effective in some patients . in the future , prospective research studies and observations are expected to verify the effectiveness of noninvasive treatments .
worldwide , more than 4 million people each year are estimated to have a non - st - segment elevation myocardial infarction ( nstemi ) , and long - term mortality is higher in patients with non - st - elevation acute coronary syndrome ( acs ) than in those with st - elevation acs . in clinical practice , for patients who present with non - st - elevation acs , different therapeutic strategies may significantly affect short and long - term outcomes . the influence of different therapy strategies on mortality after non - st - elevation acs is still a matter of controversy . to our knowledge , few studies have evaluated the long - term outcomes ( > 2 years ) of invasive and conservative strategies in patients with acute nstemi in china . with the development of modern percutaneous coronary intervention ( pci ) technology , we aimed to determine the long - term prognosis and disparities for nstemi patients in the real - world setting of different therapeutic strategies in the current era . this was a retrospective study that was approved by the ethics committee of dalian medical university . from december 2006 to december 2012 , the study included 1194 consecutive patients survived nstemi and discharged from the cardiology department of the first affiliated hospital of dalian medical university . we recorded patient characteristics , the treatment process , and adverse events during the patients hospital stay . the thrombolysis in myocardial infarction ( timi ) risk score ( trs ) was calculated from the initial clinical history , electrocardiogram ( ecg ) , and laboratory values collected on admission . unified follow - up questionnaire was performed by outpatient or telephone contact from november 1 , 2013 to november 30 , 2013 . we excluded patients with coronary artery bypass grafting ( cabg ) on admission ( n = 37 ; 3.1% ) and those lost to follow - up ( n = 181 ; 15.2% ) . nstemi diagnosis was defined as ecg st - segment depression or prominent t - wave inversion and/or positive biomarkers of necrosis ( e.g. , troponin i 1 g / l in our laboratory ) in the absence of st - segment elevation and in an appropriate clinical setting ( chest discomfort or angina equivalent ) . exclusion criteria : pci - related myocardial infarction or cabg related myocardial infarction ; other diseases affecting the long - term prognosis including other serious heart diseases ( severe primary cardiomyopathy , valvular heart diseases , and congenital heart diseases ) , severe liver dysfunction ( liver cirrhosis ) , kidney dysfunction ( serum creatinine [ scr ] 443 the invasive group contained patients who underwent pci during hospitalization ; in contrast , the conservative group contained patients who did not receive pci during hospitalization . the secondary endpoint was a composite of death , myocardial reinfarction , recurrent angina or new york heart association class iv heart failure . hypercholesterolemia was defined as a history of hypercholesterolemia and use of lipid - lowering agents or levels of total cholesterol 6.22 mmol / l or low - density lipoprotein cholesterol 4.14 mmol / l . myocardial reinfarction was based on the recurrence of chest pain , new ecg changes indicative of ischemia , and an increase in creatine kinase ( ck ) , ck - mb , or troponin i that was 50% or higher than the previous value . recurrent angina was defined as clinical features of angina with ischemic change in ecg findings or related symptoms only released by anti - ischemic agents but not satisfying the diagnostic standard of myocardial infarction . for statistical analyses , we used spss 13.0 ( spss inc . , chicago , il , usa ) . continuous data were described with mean standard deviation ( sd ) and categorical data with median and interquartile range ( 25th to 75th ) . for comparisons between two groups of continuous data , we used t - tests and for comparisons of categorical data we used chi - square tests . we controlled for confounding effects by performing multivariate cox regression analyses for the primary and secondary endpoints . the regression model was adjusted for the patient 's demographics ( gender and age ) , medical history ( hypertension diabetes , hypercholesterolemia , smoking , etc ) , hospital - related characteristics and therapies . we calculated risk ratios ( rrs ) and 95% confidence intervals ( 95% cis ) and considered p < 0.05 to represent statistical significance . this was a retrospective study that was approved by the ethics committee of dalian medical university . from december 2006 to december 2012 , the study included 1194 consecutive patients survived nstemi and discharged from the cardiology department of the first affiliated hospital of dalian medical university . we recorded patient characteristics , the treatment process , and adverse events during the patients hospital stay . the thrombolysis in myocardial infarction ( timi ) risk score ( trs ) was calculated from the initial clinical history , electrocardiogram ( ecg ) , and laboratory values collected on admission . unified follow - up questionnaire was performed by outpatient or telephone contact from november 1 , 2013 to november 30 , 2013 . we excluded patients with coronary artery bypass grafting ( cabg ) on admission ( n = 37 ; 3.1% ) and those lost to follow - up ( n = 181 ; 15.2% ) . nstemi diagnosis was defined as ecg st - segment depression or prominent t - wave inversion and/or positive biomarkers of necrosis ( e.g. , troponin i 1 g / l in our laboratory ) in the absence of st - segment elevation and in an appropriate clinical setting ( chest discomfort or angina equivalent ) . exclusion criteria : pci - related myocardial infarction or cabg related myocardial infarction ; other diseases affecting the long - term prognosis including other serious heart diseases ( severe primary cardiomyopathy , valvular heart diseases , and congenital heart diseases ) , severe liver dysfunction ( liver cirrhosis ) , kidney dysfunction ( serum creatinine [ scr ] 443 the invasive group contained patients who underwent pci during hospitalization ; in contrast , the conservative group contained patients who did not receive pci during hospitalization . the secondary endpoint was a composite of death , myocardial reinfarction , recurrent angina or new york heart association class iv heart failure . hypercholesterolemia was defined as a history of hypercholesterolemia and use of lipid - lowering agents or levels of total cholesterol 6.22 mmol / l or low - density lipoprotein cholesterol 4.14 mmol / l . myocardial reinfarction was based on the recurrence of chest pain , new ecg changes indicative of ischemia , and an increase in creatine kinase ( ck ) , ck - mb , or troponin i that was 50% or higher than the previous value . recurrent angina was defined as clinical features of angina with ischemic change in ecg findings or related symptoms only released by anti - ischemic agents but not satisfying the diagnostic standard of myocardial infarction . for statistical analyses , we used spss 13.0 ( spss inc . , chicago , il , usa ) . continuous data were described with mean standard deviation ( sd ) and categorical data with median and interquartile range ( 25th to 75th ) . for comparisons between two groups of continuous data , we used t - tests and for comparisons of categorical data we used chi - square tests . we controlled for confounding effects by performing multivariate cox regression analyses for the primary and secondary endpoints . the regression model was adjusted for the patient 's demographics ( gender and age ) , medical history ( hypertension diabetes , hypercholesterolemia , smoking , etc ) , hospital - related characteristics and therapies . we calculated risk ratios ( rrs ) and 95% confidence intervals ( 95% cis ) and considered p < 0.05 to represent statistical significance . a total of 976 patients were enrolled in the study , which involved 390 ( 40% ) in the invasive group . patients in the invasive group were younger and were more likely to be males , with a history of smoking , prior pci , family history of coronary heart disease , st - segment change , a history of taking aspirin within 1-week prior to admission and higher ejection fraction ; less likely to have hypertension , a killip class 2 . there were higher n - terminal pro - brain natriuretic peptide ( nt - probnp ) , scr and serum uric acid ( ua ) levels in the conservative group . there was no difference in cardiac troponin i ( the highest values during hospitalization ) and timi score between the two groups [ table 1 ] . baseline characteristics of patients with acute non - st - segment elevation myocardial infarction chd : coronary heart disease ; pci : percutaneous coronary intervention ; cabg : coronary artery bypass grafting ; timi score : thrombolysis in myocardial infarction ( timi : low risk 0 - 2 ; intermediate risk 3 - 4 ; high - risk 5 - 7 ) ; scr : serum creatinine ; ua : uric acid ; bnp : brain natriuretic peptide ; crp : c - reactive peptide ; lvdd : left ventricular end - diastolic diameter ; ef : ejection fraction ; ck : creatine kinase ; ck - mb : creatine kinase - mb ; tpn - i : troponin - i ; aspirin consumption : aspirin consumption in the previous 7 days ; sd : standard deviation . there was no difference in -blocker , angiotensin - converting enzyme inhibitor ( acei)/angiotensin receptor blocker and calcium antagonists therapies between the two groups . however , there was more use of aspirin , clopidogrel , statin , and lmwh in the interventional therapy group and more use of diuretics in the conservative group [ table 2 ] . concomitant drug therapy during hospitalization ( n ( % ) ) lwwh : low molecular weight heparin ; acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; ccb : calcium antagonists . the median follow - up time was 29 months ( interquartile range : 1851 months ) . there was no difference between the two groups in follow - up time ( median 31 months vs. 28 months , p = 0.166 ) . mortality was 28.7% ( n = 168 ) in the conservative group and 2.1% ( n = 8) in the invasive one , crude rr for conservative group 14.93 ( 95% ci : 7.3530.63 , p < 0.001 ) . the secondary endpoint was 59.0% ( n = 346 ) in the conservative group and 30.3% ( n = 118 ) in the invasive one , crude rr for conservative group 2.43 ( 95% ci : 1.972.99 , p < 0.001 ) [ figure 1 ] . after multivariable adjustment , patients in the conservative group had higher mortality rates than in the invasive one ( adjusted rr = 7.795 ; 95% ci : 3.79616.006 , p < 0.001 ) . the similar result was also seen in the secondary endpoint ( adjusted rr = 2.102 ; 95% ci : 1.6942.610 , p < 0.001 ) . meier curves for primary and secondary endpoints . in the subgroup analysis according to each trs , log - rank analysis showed lower mortality and secondary endpoint rates in the invasive group with the intermediate and high - risk patients ( trs 37 ) . however , for patients in the low - risk group ( trs 02 ) , there was no significant difference in the long - term outcomes between the two groups [ table 3 and figure 1 ] . the long - term outcomes according to trs ( n ( % ) ) timi : thrombolysis in myocardial infarction ; trs : timi risk score . a total of 976 patients were enrolled in the study , which involved 390 ( 40% ) in the invasive group . patients in the invasive group were younger and were more likely to be males , with a history of smoking , prior pci , family history of coronary heart disease , st - segment change , a history of taking aspirin within 1-week prior to admission and higher ejection fraction ; less likely to have hypertension , a killip class 2 . there were higher n - terminal pro - brain natriuretic peptide ( nt - probnp ) , scr and serum uric acid ( ua ) levels in the conservative group . there was no difference in cardiac troponin i ( the highest values during hospitalization ) and timi score between the two groups [ table 1 ] . baseline characteristics of patients with acute non - st - segment elevation myocardial infarction chd : coronary heart disease ; pci : percutaneous coronary intervention ; cabg : coronary artery bypass grafting ; timi score : thrombolysis in myocardial infarction ( timi : low risk 0 - 2 ; intermediate risk 3 - 4 ; high - risk 5 - 7 ) ; scr : serum creatinine ; ua : uric acid ; bnp : brain natriuretic peptide ; crp : c - reactive peptide ; lvdd : left ventricular end - diastolic diameter ; ef : ejection fraction ; ck : creatine kinase ; ck - mb : creatine kinase - mb ; tpn - i : troponin - i ; aspirin consumption : aspirin consumption in the previous 7 days ; sd : standard deviation . there was no difference in -blocker , angiotensin - converting enzyme inhibitor ( acei)/angiotensin receptor blocker and calcium antagonists therapies between the two groups . however , there was more use of aspirin , clopidogrel , statin , and lmwh in the interventional therapy group and more use of diuretics in the conservative group [ table 2 ] . concomitant drug therapy during hospitalization ( n ( % ) ) lwwh : low molecular weight heparin ; acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; ccb : calcium antagonists . the median follow - up time was 29 months ( interquartile range : 1851 months ) . there was no difference between the two groups in follow - up time ( median 31 months vs. 28 months , p = 0.166 ) . mortality was 28.7% ( n = 168 ) in the conservative group and 2.1% ( n = 8) in the invasive one , crude rr for conservative group 14.93 ( 95% ci : 7.3530.63 , p < 0.001 ) . the secondary endpoint was 59.0% ( n = 346 ) in the conservative group and 30.3% ( n = 118 ) in the invasive one , crude rr for conservative group 2.43 ( 95% ci : 1.972.99 , p < 0.001 ) [ figure 1 ] . after multivariable adjustment , patients in the conservative group had higher mortality rates than in the invasive one ( adjusted rr = 7.795 ; 95% ci : 3.79616.006 , p < 0.001 ) . the similar result was also seen in the secondary endpoint ( adjusted rr = 2.102 ; 95% ci : 1.6942.610 , p < 0.001 ) . meier curves for primary and secondary endpoints . in the subgroup analysis according to each trs , log - rank analysis showed lower mortality and secondary endpoint rates in the invasive group with the intermediate and high - risk patients ( trs 37 ) . however , for patients in the low - risk group ( trs 02 ) , there was no significant difference in the long - term outcomes between the two groups [ table 3 and figure 1 ] . the long - term outcomes according to trs ( n ( % ) ) timi : thrombolysis in myocardial infarction ; trs : timi risk score . acute coronary syndrome has been categorized into unstable angina , nstemi and st - segment elevation myocardial infarction . st - elevation acs represents an increasingly frequent cause of hospital admission , as it is the most frequent presentation of coronary instability in patients with prior cardiac events or coronary revascularizations . so far , an increasing number of studies have demonstrated that an invasive treatment strategy rather than a conservative approach improves the outcomes of non - st - elevation acs patients , and the two guidelines recommended an invasive approach for the higher - risk st - elevation acs patients , however , some studies showed an invasive strategy could not provide comparable benefits of all - cause mortality and long - term survival when compared to conservative therapy . our results showed that invasive strategy is still the most effective treatment to reduce long - term complications in nstemi patients . in our study , patients in the invasive therapy group were younger and were more likely to have higher ejection fraction and lower nt - probnp , scr and serum ua levels , but less likely to have a killip class 2 . these results indicated more comorbidities and higher baseline risk in a conservative group that could lead to worse outcomes . according to heart association task force on practice guidelines , medical therapy remains a cornerstone in managing patients with non - st - elevation acs . in our study , patients in the conservative group received less evidence - based medical therapy than those underwent the invasive management , including the use of aspirin , clopidogrel , statin , and lmwh , which should be considered as another main reason for worse primary and secondary endpoints . our main finding in this study assessing the long - term impact of the invasive strategy demonstrated a sustained advantage for invasive management in the subsequent primary or secondary endpoints . after adjustment for confounding factors including the above comorbidities , baseline risk , and evidence - based medical care , the trend toward decreased all - cause mortality or the secondary endpoint in patients with an invasive strategy was still observed . this is almost consistent with the findings from the recent meta - analysis , particularly in term of reduced long - term mortality rates , but is in disagreement with those of the randomized invasive versus conservative treatment in unstable coronary syndromes ( ictus ) trial . to avoid other diseases affecting the long - term prognosis , the admission criteria excluded patients with other serious diseases . previous studies showed cardiac troponin i ( the highest values during hospitalization ) , c - reactive protein , and timi score might be better discriminator of patients of acs who remain at high - risk , however , these markers have no difference in between the two groups . for most patients who stabilized after an acs , in - hospital coronary intervention was not associated with reduced risk compared with medical therapy . the korea acute myocardial infarction registry indicated early invasive ( within 48 h ) rather than late invasive treatment improved 1-year clinical outcomes in patients with high trs ( 5 points ) . however , in the fir database of patients presenting with st - elevation acs , the timing of angiography was not related to 5-year cardiovascular mortality or myocardial infarction . the present study did not focus on the optimal timing of pci during hospitalization and showed lower mortality and secondary endpoint rates in the intermediate and high groups ( trs 37 ) from the studied population of nstemi patients suggesting that we should focus on a treatment - risk paradox in clinical practice where most interventions are performed in lower risk patients . however , the ictus showed that an early invasive strategy was not better than an early conservative strategy , even for the higher risk patients , on the short - term and long - term clinical follow - up . hence , the heterogeneity of different results clearly calls for clinical investigation . in previous trials , st - elevation acs patients in the conservative group also received invasive therapy during the index hospitalization when medical therapy failed or if substantial residual ischemia was documented . a major strength of this study is that we strictly distinguished or defined between conservative and invasive strategies during hospitalization . we performed the present retrospective study to analyze the benefits between invasive and conservative strategies for nstemi patients and had no crossover from conservative treatment to pci during hospitalization and eliminated the interference from the two therapies with one another , and reflected real - life setting in nstemi prevalence and outcome . different from the majority of trials , patients with unstable angina were not enrolled in our study which could exclude possible residual confounding by different st - elevation acs diseases . the samples were restricted to patients discharged from our hospital with a successful follow - up , which may have resulted in selection biases and conclusions with limited generalizability . there was a tendency for cardiologists to perform conservative treatment for high - risk patients which might have affected the final results . finally , this study can not exclude possible residual confounding by other measured and/or unmeasured factors including the treatment decisions of patients when nstemi occurred , which is an important source of prognosis . in conclusion , our findings suggested that an invasive strategy could improve long - term outcomes for nstemi patients , especially for intermediate and high - risk ones ( trs 37 ) .
surgery for pancreatic cancer is probably the most demanding and risky operative procedure in abdominal surgery . remarkable progress has been achieved during the last three decades and pancreatic surgery can nowadays be performed safely with low morbidity and mortality in various specialized hospitals around the world . this is in part a result of regionalization of pancreatic surgery into high volume centers resulting in an increased resection rate in the presence of pancreatic carcinoma which is nowadays approximately 60% and to an extension of indications for resection . consequently , vascular resection , considered a contraindication for pancreatic resection a couple of years ago , is now implemented in the armamentarium of pancreatic surgeons to realize r0-resections when clearance of the portal or superior mesenteric vein can not be realized without vascular resection . the results following synchronous resection of liver metastasis together with resection of periampullary or pancreatic adenocarcinoma or pancreatic resection for m1 carcinoma are not equivocal . although these procedures can be performed safely , the median survival time ranges between 6 and 13 months [ 3 , 4 ] . therefore , synchronous metastasectomy of periampullary cancer is rarely performed when extended disease has already been found preoperatively . however , within daily clinical practice different scenarios occur which may require a decision for resection in an m1 situation . in a young patient with advanced disease , resection may provide a small but important increase of survival . in addition , metastatic disease may become overt when the point of no return has already been passed as it may be the case in the presence of positive interaortocaval lymphnodes , or metastatic disease will be detected during operation despite negative imaging results preoperatively . finally , a strong patient desire may force the surgeon to go further than consented guidelines may summarize existing evidence . since curative r0 resection is the most important prognostic factor for long - term survival even a small potential to achieve r0 resection may provide the legitimisation for extended resection despite the fact that overt systemic disease is already present . so far , there is little data available on the value of synchronous metastasectomy together with pancreatic resection in patients with pancreatic carcinoma . patients who undergo resection of metastases together with pancreatic resection seem to have a higher complication rate than patients with multivisceral liver resections . an extended lymphadenectomy is associated with an increased postoperative complication rate , but has no impact on long - term survival . the fact that despite r0 resection long - term survival does not exceed 25% even in the most experienced pancreatic centers may prove that carcinoma of the pancreas is a systemic disease . further improvement of survival can only be achieved by adjuvant treatment ( picozzi et al . , 2003 ) . it seems logical , that in the presence of metastatic disease a low tumor load may increase the success of adjuvant chemotherapy although the absolute increase in survival time may be very low . we therefore hypothesized that radical resection of m1-pancreatic carcinoma may be justified in well - selected patients as long as these operations can be performed with a low morbidity and mortality . thus , we aimed at reviewing our experiences with pancreatic resections in patients with m1 periampullary cancer of the pancreas after having launched a new pancreatic program in our hospital . there were 280 patients who underwent either pancreatoduodenectomy or left - sided pancreatic resection for histologically proven pancreatic adenocarcinoma or periampullary cancer . among this patient population we identified 20 ( 7,1% ) patients ( 9 men , 11 women ; mean age 60 years ) in our prospective database who underwent either a pancreaticoduodenectomy ( pd ) , or a left - sided distal pancreatectomy together with resection of metastasis for pancreatic adenocarcinoma . resection of metastatic disease was defined as the resection of any tumor deposit outside the regional lymph nodes or resection of infiltrated surrounding organs in order to achieve a r0 resection . magnetic resonance imaging , magnetic resonance cholangiography , or endoscopic retrograde cholangiopancreatography were performed when indicated . in patients who were found intraoperatively to harbour metastatic disease the decision for resection was based on the impression to reach a r0 situation with synchronous resection of metastasis and a good clinical performance status of asa iii or better . patients with suspected metastatic disease preoperatively were operated with potentially curative intention or with palliative intention . reasons for resection were good performance status and patient 's will to receive maximal treatment . all pathologic specimen were reviewed by a single pathologist ( at ) to confirm the diagnosis of pancreatic adenocarcinoma or cancer of the papilla vateri . a pancreatic fistula was defined as a prolonged drainage with amylase activity of more than 10.000 wound infection was defined as any wound requiring reopening for the drainage of pus together with a positive wound culture . mortality was defined as any death during postoperative hospitalization or within 30 days of surgery . follow - up information was obtained through direct contact with the patient and review of hospital charts and operative notes . the results of the study group were compared with a matched - pair control group of 20 patients with pancreatic adenocarcinoma , who were matched according to age and tumor location and who did undergo only pancreaticoduodenectomy or distal pancreatic resection for stadium iib or iii pancreatic adenocarcinoma . all statistical analyses were performed using spss - software ( version 15 ) . the distribution of age at operation , blood loss , and postoperative hospital stay were presented as median with range . comparisons between subgroups were performed with fisher 's exact test and differences between means were tested by the t - test . cases were matched with control group ( resection without multivisceral resection ) in a 1 : 1 fashion . specifically , cases and controls were matched on primary tumor characteristics and age of the patient . morbidity , mortality , and overall survival were compared between cases and control group . the nonparametric product limit method ( kaplan - meier estimations ) two - sided p values were always computed and an effect was considered statistically significant at p .05 . the primary tumor was located in the pancreatic head in 9 ( 45% ) patients , in the pancreatic tail in 9 ( 45% ) , and at the papilla vateri in 2 ( 10% ) . synchronous liver metastasis was present in 14 ( 70% ) patients , 4 ( 20% ) patients had peritoneal metastases , one patient had a metastasis in the transverse mesocolon , 2 ( 10% ) patients had a metastasis in the greater omentum , and 3 ( 15% ) patients had macroscopically lymph node metastases . six patients ( 30% ) had metastatic disease at more than one location . in 7 ( 35% ) metastatic disease was known before operation based on preoperative imaging in 5 of 20 patients ( 25% ) . of the 20 patients , 8 ( 40% ) underwent a pylorus - preserving duodenopancreatectomy and 2 ( 10% ) a classic whipple - procedure . in 8 ( 40% ) patients a distal pancreatic resection was performed and 2 ( 10% ) patients underwent a total pancreatectomy . details of the localisation of the tumor and the metastases , tnm - staging , r - status postoperatively , and type of surgery performed are listed in table 2 . an r0/r1-resection could be achieved in 11 patients ( 55% ) , whereas in the remaining patients only an r2 resection was performed due to remaining metastatic disease in liver . there was no perioperative mortality , and complications occurred in 9 ( 45% ) patients . the median intraoperative blood loss was 1000 ml ( range 3002500 ml ) and the median postoperative length of hospital stay was 20.7 d ( 1171 d ) . this was statistically not different to a matched - pair group of patients with ductal adenocarcinoma of the pancreas without metastatic disease ( tables 3 and 4 ) . all patients received postoperative adjuvant or palliative chemotherapy , two patients underwent neoadjuvant radiochemotherapy prior to surgery . the median postoperative survival was 10.7 months ( range 2.637.8 mo ) which was not significantly different to the control group who had a median survival time of 15.6 months ( p = .11 ; figure 1 ) . all deaths were caused by recurrent cancer.there was no difference in the median survival between patients with liver metastases ( median survival time 11 months ) compared to patients who had metastases at other locations ( median survival 14.1 months ; p = .62 ) . pancreatic cancer still carries a dismal prognosis and the potential to cure a patient can only be achieved when the primary tumor can be completely resected . however , this scenario can only be accomplished in about 5%25% of patients presenting with locally resectable cancer . patients with metastatic pancreatic cancer who receive palliative chemotherapy with gemcitabine have a median survival time of 5.6 months . various protocols with gemcitabine - based regimen or multidrug regimen tested in prospectively randomised phase iii clinical trials have not altered this situation substantially making metastatic pancreatic cancer one of the most frustrating malignancies to investigate and treat . therefore , when metastatic disease is recognized preoperatively , an operative procedure is usually avoided except for surgical palliation . in addition , according to the s-3 guidelines of the german cancer society pancreatic resections should be avoided in the presence of intraoperative metastatic disease . however , locally advanced disease may be resected as long as an r0 resection can be achieved . both statements have a grade of evidence of 3 which means that only systematic reviews and individual case control studies are available to scientifically support this statement . it seems reasonable , that postoperative chemotherapy may be more effective , when no gross tumor is left in situ . in addition , the high recurrence rate even after r0 resection is an indirect prove for early systemic spread which is not treatable by surgery but by chemotherapy . our study gives support to the hypothesis despite the relatively small number of patients that in the presence of metastasis of a pancreatic adenocarcinoma radical resection is possible and safe and that overall survival is comparable to patients who do not have metastatic disease . this relatively good result is in part consequence of restriction of the procedure to well suited patients who were expected to tolerate even a significantly expanded procedure . the mean age of our patients was 58 years , and all patients were either asa 1 or 2 ( data not given ) . therefore , it was expected that these patients would tolerate pancreatic resection with synchronous metastasectomy without significant increase in morbidity or mortality . nevertheless , the median survival of 10.6 months is low , but we believe that we could offer these patients additional life time , although quality of life was not evaluated . however , these procedures should be restricted to well - suited patients and only be performed at centers with significant expertise in pancreatic surgery . a shortcoming of the study may be the fact that patients had various presentation of the m1 category . most of our patients had liver metastasis , but there were also patients with peritoneal or lymph nodes metastases who were classified as m1 or the tumor involved surrounding organs . since there is no significant difference in survival according to the location of the metastasis of pancreatic carcinoma , we believe that an analysis of this group was appropriate . according to the results of at least 3 randomized trials extended lymphadenectomy has not been effective in improving survival of patients with pancreatic adenocarcinoma with 1 and 3 years survival between 51% and 77% and between 16 and 41% , respectively . therefore , in an nonmetastasized pancreatic adenocarcinoma a standard lymphadenectomy is adequate . when locally advanced cancer with suspected venous infiltration is present extensive surgery can be performed safely with morbidity and mortality rates comparable to conventional resections . in a recent analysis of 136 patients with locally advanced pancreatic cancer yekebas et al . could demonstrate that patients with concomitant vascular resections had the same median ( 15 versus 16 mo ; p = .86 ) and two year survival ( 34% versus 36% : p = .9 ) compared to patients who did not undergo vascular resection . even in multivariate analysis vascular infiltration was not considered a negative prognostic factor . even arterial encasement of the celiac trunk in the presence of a pancreatic corpus carcinoma is not considered a contraindication for surgery . recently published a series of 23 patients with pancreatic body cancer with infiltration of the celiac trunk . negative surgical margins were obtained in 91% of patients , postoperative mortality was 0% , and the median survival was 21 months . however , early hepatic recurrence in 6 patients was a hint that this procedure may be indicated for the treatment of less advanced disease . in a recently published study shrikhande et al . reported a series of 29 patients with m1 ductal adenocarcinoma of the pancreas who underwent pancreatic resection with synchronous resection of metastases . of these 11 patients had metastatic disease to the liver and 9 to the peritoneum . the authors concluded , that concomitant resection of primary pancreatic tumor and metastases can be performed safely . however , a risk benefit ratio should be carefully assessed since the overall increase in survival is moderate . in our study the survival time of patients with liver metastasis was 11 months compared to 14.1 months when metastases were located at other regions ( p = .62 ) . in a study of 22 patients who underwent pancreatic resection and synchronous liver resection for metastasised pancreatic adenocarcinoma , gleisner et al . found that the median survival was only 5.9 months , which was not statistically different from patients who underwent palliative bypass alone ( 5.6 months ; p = .46 ) . they concluded from the results of their study that simultaneous resection for patients who present with synchronous liver metastasis from pancreatic adenocarcinoma is not justified . in our study we found a median survival of 10.6 months for the complete group , while the 14 patients with liver metastases had a median survival of 11 months . despite this small increase we believe that in highly selected patients after careful counselling this aggressive therapy is still an option that should be offered to the patient . the fact that there was no significant difference in survival between the study and the matched - pair control group despite an objective difference in the median survival of 5 months is probably due to the limited number of patients . with an increased number of patients we would have expect this difference to reach statistical significance . compared to published results of patients receiving palliative chemotherapy who have a median survival time of approximately 5.48.4 months [ 13 , 14 ] , the increase in survival in patients with m1 ductal adenocarcinoma of the pancreas is about 2 - 5 months . we believe that patients who may benefit must be carefully selected and therapeutic options and expected survival should be discussed with the patients and their families . in the presence of pancreatic neuroendocrine carcinomas extended resections or even debulking operations seem to be associated with increased survival . reported on a series of 23 patients with pancreatic islet - cell carcinomas with synchronous hepatic metastases who underwent synchronous hepatic resections without perioperative mortality . this demonstrates , that extended resections in this subset of metastasized patients with primary pancreatic tumors are justified . the preoperative or intraoperative differentiation between a pancreatic adenocarcinoma and a neuroendocrine carcinoma may be difficult when typical carcinoma associated syndromes are missing preoperatively . however , intraoperatively the differentiation may be easier from the macroscopic point of view , and when histology has not been determined preoperatively , intraoperative frozen section may identify the tumor so operative strategy can be planned appropriately . currently , a monocentric prospective randomized trial on the value of pancreatic resection in the presence of liver metastasis is under way in germany ( nct00855634 , pamevitum ) . the results of this study may give further evidence whether pancreatic resection in this scenario is justified . the results of our study demonstrate , that resection of pancreatic adenocarcinoma with synchronous metastases can be performed safely . justification for combined resection should be made on an individual basis for each patient only . there is probably a small increase in survival compared to patients who undergo palliative chemotherapy and there was no statistically significant difference in survival compared to patients who underwent resection for stadium iib or iii tumors . the decision to resect a patient in an m1 situation should be carefully assessed and may be an option in highly selected cases .
benzo[a]pyrene ( b[a]p ) , a polycyclic aromatic hydrocarbon compound , is produced by incomplete combustion of organic compounds and high - pressure processes . this compound is commonly present in motor vehicle exhaust , tobacco smoke , grilled , smoked and broiled foods , and emissions from residential and industrial heating sources . biotransformation of b[a]p occurs as it undergoes initial oxidation by cytochrome p450 enzymes and consequently forms reactive metabolites , for example , b[a]p-9 , bap-7,8-dihydrodiol 9,10-epoxide and quinones . the active intermediates are capable of covalently bonding to dna and form dna adducts . also , they can undergo redox cycling and generate excessive reactive oxygen species , which may alter cell signaling and damage cellular membranes resulting in apoptosis . previous studies in animal models have demonstrated that direct exposure to b[a]p could cause toxic effects on male reproduction and has been implicated in the pathobiology of adverse reproductive health . b[a]p has been related to decreased spermatozoa quality [ 3 , 4 ] and germ cell apoptosis after chronic exposure . to date , limited data has appeared available to depict the impact of exposure to b[a]p on spermatozoa quality in relation to testicular germ cells during spermatogenesis . spermatogenesis represents a complex and dynamic process of proliferation and differentiation of the transformation of spermatogonia into mature spermatozoa in three major stages , the mitotic stage , the meiotic stage , and the maturation stage . alterations occurring in any of them could lead to the production of abnormal spermatozoa and reduce proliferation of spermatozoa . thus , the understanding of processes related to spermatogenesis is critical for assessment of male reproductive health . the objective of this study was to investigate the toxicological impact of b[a]p exposure on testicular germ cells during spermatogenesis by assessing concentrations of and apoptosis in testicular germ cells and mature spermatozoa . hsd : icr ( cd1 ) 10-week - old male mice , weighing 3040 g , were used ( harlan laboratories , inc . ) . mice were caged individually with 12 hr light / dark cycles , given rodent chow ( global rodent diet number 2018 ) , water ad libitum , and enrichment items . all procedures were performed in accordance with protocols approved by the old dominion university 's institutional animal care and use committee . each mouse was weighed every other day and was examined daily for behavioral and clinical symptoms . upon arrival mice ( n = 8 per group ) were randomly assigned to the exposure group and control group . the exposed mice were gavaged with 1 , 10 , 50 , and 100 mg / kg / day of b[a]p dissolved in 100% corn oil for 30 days . control animals were only dosed with 20 l of 100% corn oil . on day 30 , mice were anesthetized using intraperitoneal injection of sodium pentobarbital ( 50 mg / ml ) to the surgical plane , confirmed by the toe - pinch method . reproductive tissues , including testes , seminal vesicles , prostate , epididymis , and vase deferens , were removed and placed on a petri dish and weighed . the left testes were used for collection of germ cells and stored in the freezing media - rpmi 1640 and 5% glycerol at 80c until germ cell collection . the testes were fixed with 10% formalin in a 1% phosphate buffer at room temperature for 2448 hours . , serial sections ( 8 m ) were cut from the middle of each testis using a microtome . these sections were placed in a water bath at 47c and transferred to charged microscope slides . sections were dried overnight on glass slides at 35c and deposited in a storage box at room temperature until processed for histology . for staining and morphometric evaluation , sections were deparaffinized and rehydrated using 100% , 95% , and 70% graded ethanol . images were acquired using an olympus cx41 microscope with a dp72 camera and cellsens standard software . testes were digested in a 50 ml conical tube containing 10 ml d - hanks solution with 0.5 mg / ml collagenase and 25 g / ml dnase , then incubated at room temperature for 10 minutes with gentle oscillation . next , the suspension was filtered through a 150 mesh ( 104 m ) filter to collect the seminiferous tubules . the filtrate was centrifuged at 300 g for 10 minutes to concentrate the interstitial cells . cells were pelleted by centrifugation ( 300 g for 10 minutes ) , and the supernatant was removed . the cells were then suspended in 0.2% bovine serum albumin ( bsa ) in rpmi+ by gentle pipetting . sedimentation at unit gravity was used to separate each type of germ cell , including spermatogonia , pachytene spermatocytes , round spermatids , and elongated spermatids . the procedures were performed according to those developed for murine spermatogenic cells [ 7 , 8 ] with some modifications . a small sta - put chamber and sample loading chamber ( pro science glass co. , ontario , canada ) were prepared by sterilization . both chambers were primed by applying 25 ml rpmi+ through the sample loading chamber using the pump to advance the volume into the sta - put chamber . a 10 ml cell suspension was loaded onto a 5 ml cushion of 0.2% bsa in rpmi+ in the sample loading chamber , and then 0.2% bsa in rpmi+ was added at a flow rate of 20 ml / min . the tubing was reversed to allow 10 ml fractions to be drawn from the chamber at a flow rate of 10 ml / min . each fraction was centrifuged at 900 g for 10 min , and then supernatant was removed . each fraction was next assessed for cell types , which were identified based on morphology . elongated spermatids were identified based on shape , placement of cytoplasm , condensed nucleus , and the tail . each type of cell was counted using a hemocytometer and stored at 80c until apoptosis analysis . a consistent length of each vas deferens , 1.5 cm , was flushed with 0.5% bsa in rpmi+ to recover spermatozoa . spermatozoa quality , including concentration , viability , motility , and morphology , was assessed . the percentage of motility was determined by counting both motile and immotile spermatozoa . for viability analysis , at least 100 spermatozoa per sample were assessed from an eosin stained preparation . for morphology assessment , at least 100 spermatozoa were classified as either normal , abnormal head , or curly tail . apoptotic cells were detected by terminal deoxynucleotidyl transferase mediated deoxy - utp nick end labeling ( tunel ) , using an in situ detection kit ( roche diagnostic , manheim , germany ) . to detect apoptotic germ cells in seminiferous tubules , testes were fixed in formalin for 24 hours and then embedded in paraffin , and testicular sections were sliced in six m ribbons on a leica microtome . the tissue sections were washed with 1% hsa in a 1x phosphorous buffer solution ( pbs ) , permeabilized using 0.1% ( v / v ) triton x-100 , 0.1% ( w / v ) sodium citrate in 1x pbs , and incubated at room temperature for 10 minutes . the presence of germ cell apoptosis related to dna strand breaks was evaluated by terminal deoxynucleotidyl transferase mediated d'utp nick - end labeling by means of the in situ cell detection kit with fitc - labeled d'utp . a tunel mixture ( roche ) slides were incubated for 60 minutes at 32c , in the dark , with humidity , and were monitored for wetness by adding 1% hsa in 1x pbs as needed . slides were analyzed immediately using a fluorescent microscope ( nikon eclipse 80i ) with a cool snap ez camera and nis elements br3.2 software . a minimum of 20 fields of view at 1000x magnification were randomly selected for analysis of cells . to detect apoptotic spermatozoa , 200 l of spermatozoa at 30 10/ml collected from the previous step were washed twice with 1x pbs/1% hsa . spermatozoa were fixed to the slides , permeabilized with 0.1% triton x-100 in 0.1% sodium citrate at 4c for two minutes . each test included both positive and negative controls to ensure the performance of the assay . cells in the positive control were treated with 50 l of dnase solution , while cells in the negative control did not get treated with the tunel mixture . after the cells were incubated one hour at 37c , the cells were washed twice with 1% has in pbs . a minimum of 100 spermatozoa were counted as either positive ( green ) or negative ( absence ) at 1000x under oil immersion , using a nikon eclipse 80i fluorescent / brightfield microscope with the x - cite series 120 and fitc filter . all data were tested for normality and variance before statistical analysis took place and presented as mean standard deviation ( sd ) . multivariate analysis of variances ( manova ) and lsd post hoc tests were utilized to assess statistically significant differences in spermatozoa quality , reproductive organ weight , and apoptotic cells between the exposure group and the control group . the weight of testes and epididymis did gradually decrease as b[a]p doses increased ( figures 1(c ) and 1(d ) ) . the cross sections of the seminiferous tubules of mice that showed that exposure to b[a]p at 100 mg / kg / day resulted in the atrophy of seminiferous tubules , reduced width of adluminal compartments of the seminiferous tubules , and altered morphology of spermatogonia and spermatocytes ( figure 2 ) . figure 3 shows spermatogenic cells recovered from the testes of mice exposed to b[a]p and their concentrations are shown in figure 4 the b[a]p - treated mice yielded lower concentrations of germ cells , including spermatogonia , pachytene spermatocytes , spermatids , and elongated spermatids in the tubules , as b[a]p doses increased . mg / kg / day and 100 mg / kg / day induced a significant decrease in the concentrations of germ cells as compared to the control ( p < 0.05 ) . mice exposed to 50 and 100 mg / kg / day of b[a]p yielded significantly lower concentrations of spermatozoa than the control group ( figure 5(a ) ) . for motility and viability , only b[a]p at 100 mg / kg / day induced a significant decrease in percentages of motility and viability as compared with the control ( p = 0.04 and 0.04 , resp . ) percentages of morphology of spermatozoa with normal head decreased but did not reach the significant level . the percentages of spermatozoa with abnormal head and curly tail remained stable with no significant changes ( figure 5(c ) ) . a cross section of a seminiferous tubule of mice exposed to b[a]p at 100 mg / kg / day showed apoptosis in germ cells as detected using the tunel assay as compared with the control ( figure 6 ) . mg / kg / day had less than 0.6% of cells with apoptosis ( figure 7 ) . the apoptosis percentages of those cells started increasing as b[a]p reached 50 mg / kg / day and 100 mg / kg / day . at 100 mg / kg / day , spermatogonia , pachytene spermatocytes , and spermatocytes had significant percentages of apoptosis ( 3.0% , 4.1% , and 3.0% , resp . ) as compared with the control groups ( 0.05% , 0% , and 0% , resp . ) elongated spermatids were the type of germ cells with the highest percentages of cells with apoptosis . the percentages of elongated spermatids from mice exposed to 1 , 10 , and 50 mg / kg / day were not significantly higher than those in spermatogonia , pachytene spermatocytes , and round spermatids . at 100 mg / kg / day of b[a]p , elongated spermatids , however , had a significant increase in the percentage of apoptosis ( 19.2% ) as compared with the control ( 2.1% ) ( p = 0.01 ) . mature spermatozoa with apoptosis had a similar pattern as elongated spermatids , whose apoptotic cells increased at 10 mg / kg / day of b[a]p and reached a significant level of increase at 50 mg / kg / day and 100 mg / kg / day ( 8.7% and 27.6% , resp . ) as compared with the control groups ( 1.4% ) ( p = 0.04 and 0.009 , resp . ) the current study used rodents for the experimental paradigm to provide knowledge about the influence of exposure to b[a]p on germ cells during spermatogenesis . each type of germ cell represents steps of the spermatogenic process involving mitotic cell division , meiosis , and the process of spermiogenesis . exposure to b[a]p , particularly at 50 mg / kg / day and 100 mg / kg / day for 30 days , induced a significant decrease in the levels of germ cells , including spermatogonia , pachytene spermatocytes , round spermatids , and elongated spermatids . b[a]p could have affected the proliferation of spermatogonia and pachytene spermatocytes in the early spermatogenesis . such effect led to the lower concentrations of round spermatids and elongated spermatids in the later spermatogenesis . our results suggest that exposure to b[a]p did not significantly affect remodeling of spermatids during spermiogenesis , which produces the mature streamlined spermatid form . histological assessment revealed that b[a]p exposure at 50 mg / kg / day and 100 mg / kg / day for 30 days had a significant impact on the seminiferous tubules : reduced width of the adluminal of the seminiferous tubules and the size of the sertoli cell basal membrane , lost integrity of cellular membranes and the atrophy of seminiferous tubules . that ran parallel with a reduction in the weight and size of testes of mice exposed to b[a]p at 100 mg / kg / day . the basal compartment of seminiferous tubules is occupied by spermatogonia and early spermatocytes and forms a niche for the developing spermatogonia . the proliferation and differentiation of spermatogonia up to meiosis occurred within the basal compartment of the seminiferous tubules . a reduction in the basal membrane of sertoli cells may impact spermatogonial proliferation and survival . also , bap could cause cellular changes and affect cell signaling pathways of sertoli cells , which involve regulation of growth and differentiation of spermatogenic cells . another possible mechanism related to the reduction of germ cells could be associated with the destruction of testosterone production in leygid cells . in mammalian male reproduction , leygid cells are the major source of testosterone , which is a critical hormone for the initiation and maintenance of spermatogenesis . b[a]p metabolites have been detected upon accumulation in the male reproductive system , especially in the testis and epididymis of exposed animals . accumulation of the metabolites of b[a]p could reduce the number of leydig cells via apoptosis . that may lead to altering intratesticular testosterone production in leydig cells , which causes testicular atrophy and an accompanying decrease in the number of germ cells [ 5 , 14 ] . based on the histological assessment , leydig cells appeared to have increased dna strand breaks ( data not shown ) . testosterone levels from mice exposed to b[a]p at 50 mg / kg / day and 100 mg / kg / day decreased , which was in line with increased percentages of apoptosis in spermatogonia and pachytene spermatocytes and decreased levels of the germ cells . b[a]p promotes apoptotic cell death of four types of germ cells . with doses of b[a]p at 1 mg / kg / day and 10 mg / kg / day , the apoptotic responses of spermatogonia , pachytene spermatocytes , and round spermatids remained stable , while elongated spermatids with apoptotic responses increased . although the percentages of apoptotic elongated spermatids were not significant higher than those in other germ cells , the elongated spermatids from mice exposed to 100 mg / kg / day had a more significant increase in apoptosis than other germ cells . during the normal spermatogenesis cycle , elongated spermatids have been known to rarely undergo apoptosis . our results suggest that at the b[a]p doses for 30-day exposure , spermatogonia and spermatocytes with b[a]p - induced dna damage underwent apoptotic cell death rather than repair . then , during the course of exposure , dna damage was passed on and accumulated in elongated spermatids . similar results were observed in van loon 's study , showing increased dna breaks in the elongated spermatids after radiation exposure . the increased dna damage might be due to a cumulative effect in the absence of repair . the repair mechanisms , for example , nucleotide excision repair [ 16 , 17 ] , of spermatogonia seemingly failed to overcome the insults from b[a]p exposure and led to a reduction in the proliferation of pachytene spermatocytes . in addition , the high dose of 100 mg / kg / day could damage the blood - testis barrier ( btb ) and directly insult the elongated spermatids . germ cells during spermatogenesis were susceptible to being insulted by 100 mg / kg / day of b[a]p exposure for 30 days . in the testis , junctional proteins of the sertoli cells participated in forming btb , which divides the seminiferous epithelium into basal and adluminal compartments . increased levels of b[a]p - dna adducts were detected in germ cells of mice exposed to 50 mg / kg / day and 100 mg / kg / day ( data not shown ) . our results suggest that b[a]p at 50 mg / kg / day and 100 mg / kg / day could compromise the function of btb for entry into germ cells , while directly reaching germ cells in the spermatogonia and early spermatocytes . b[a]p can undergo a futile redox cycle in the presence of nadph to generate ros , which could directly react with germ cells and cause damage to their dna . spermatozoa production in the testis is a regulated balance between germ cell proliferation and germ cell loss . mice exposed to b[a]p experienced an increase in germ cell loss and a decrease in their proliferation . the reduction in germ cell levels appeared to be correlated with depletion of spermatozoa levels in the epididymis and vas deferens . also , the percentage of apoptosis in germ cells was positively correlated with dna damage in spermatozoa . low concentrations of spermatozoa with poor morphology were more likely to show high levels of tunel positivity . our data simply reflect that b[a]p could decrease the proliferation of germ cells that eventually leads to alteration in spermatozoa production and motility in the later stages of spermatogenesis ( figure 5 ) . we observed that the apoptosis of germ cells appeared to be associated with the depletion of spermatozoa concentrations in the epididymis and vas deferens . our present study demonstrated that mice exposed to b[a]p at or greater than 50 mg / kg / day for 30 days result in pathological changes such as decreasing spermatogonial cell proliferation and modulating germ cell apoptosis during spermatocytogenesis , the early stage of spermatogenesis . the results suggest that b[a]p affects the apoptotic process during the early stage of spermatogenesis and leads to decreased spermatozoa quality during sperm maturation . exposure to b[a]p at higher than 50 mg / kg / day for 30 days influenced germ cell quality by decreasing their concentrations and increasing apoptotic response . b[a]p can impact germ cells during spermatogenesis , which could lead to decreased proliferation of spermatids and mature spermatozoa .
ectodermal dysplasia , as first described by thurman,1,2 is a hereditary disorder occurring as a consequence of disturbances in the ectoderm of the developing embryo . the triad of nail dystrophy ( onchodysplasia ) , alopecia or hypotrichosis ( scanty , fine light hair on the scalp and eyebrows ) , and palmoplantar hyperkeratosis is usually accompanied by a lack of sweat glands ( hypohidrosis ) and a partial or complete absence of primary and/or permanent dentition.25 ectodermal dysplasia represents a large and complex group of diseases comprising more than 170 different clinical conditions.3 the incidence of this condition is 1:100,000 , with a mortality rate of 28% in males up to 3 years of age.1 when at least 2 types of abnormal ectodermal features occur , such as malformed teeth and extremely sparse hair , the patient is diagnosed with ectodermal dysplasia syndrome.3,5 there are 2 major types of this condition depending on the number and functionality of the sweat glands : ( 1 ) x - linked anhidrotic or hypohidrotic , where sweat glands are either absent or significantly reduced in number ( christ - siemens - touraine syndrome ) , and ( 2 ) hidrotic , where sweat glands are normal and the condition is inherited as autosomal dominant ( clouston s syndrome).1,3,5 the dentition and hair are affected similarly in both types , but the hereditary patterns and nail and sweat gland manifestations tend to differ.2 christ - siemens - touraine syndrome , with x - linked recessive inheritance , is the most frequently reported manifestation of ectodermal dysplasia.2,7,8 depending on the severity of clinical manifestations , christ - siemens - touraine syndrome can be classified as either hypohidrotic or anhidrotic ectodermal dysplasia.8 oral traits of ectodermal dysplasia ( ed ) may be expressed as anodontia or hypodontia , with or without a cleft lip and palate . anodontia also manifests itself by a lack of alveolar ridge development;7 as a result , the vertical dimension of the lower face is reduced , the vermilion border disappears , existing teeth are malformed , the oral mucosa becomes dry , and the lips become prominent . the face of an affected child usually has the appearance of old age.79 genetic studies regarding the etiology of ed reveal that mutations in the ectodysplasin - a and ectodysplasin - a receptor genes are responsible for x - linked and autosomal hypohidrotic ectodermal dysplasia.2 the clinical diagnosis is based on clinical and radiological manifestations such as the number and distribution of sweat pores , the amount of sweat produced , the structural and biochemical characteristics of hair , skin biopsy , and characteristics of lacrimal secretions.10 alopecia areata , incontinentia pigmenti , werner syndrome , focal dermal hypoplasia , familial simple anhidrosis , and dyskeratosis congenita are some of the conditions through which the differential diagnosis is made.11 the most frequent prosthetic treatment for the dental management of ectodermal dysplasia is removable prosthodontics . since alveolar bone development is dependent on the presence of teeth , children with ectodermal dysplasia have little or no bone ridge upon which to construct dentures ; therefore , restoring function and appearance is more challenging than usual.3 follow - up by a multidisciplinary team involving pediatric dentistry , orthodontics , prosthodontics , and oral - maxillofacial surgery specialists is advocated to be the most appropriate approach in such cases.8 in this case report ; it is aimed to describe the prosthetic rehabilitation of 2 young boys with anhidrotic ectodermal dysplasia associated with severe anodontia . an 8-year old male patient was referred to gazi university faculty of dentistry department of pediatric dentistry due to lack of teeth as well as speech and mastication problems . parental history revealed that the patient was diagnosed with anhidrotic ectodermal dysplasia after skin biopsy at gazi university faculty of medicine department of pediatrics 5 years ago . the patient was the only child and there were no other cases of ectodermal dysplasia in the family . the lack of primary and permanent teeth in the oral cavity resulted in dietary problems ; in addition , the child did not accept any previous dental intervention due to anxiety . during extraoral examination , the child exhibited the typical features of anhidrotic ectodermal dysplasia : saddle nose ; soft , dry and light colored skin ; increased pigmentation ; as well as thin , linear wrinkles in the peri - oral region ( figures 1 and 2 ) . intraoral examination revealed the complete absence of primary and permanent teeth , thin alveolar ridges , reduced vertical bone height , and loss of sulcus depth in the posterior regions of maxillary and mandibular jaws ( figures 3 and 4 ) ; complete anodontia was also confirmed by panoramic radiography ( figure 5 ) . in order to improve appearance , mastication , and speech , removable complete maxillary and mandibular dentures were determined to be the best treatment choice . preliminary impressions were made with irreversible hydrocolloid , and then custom trays were prepared for functional impression . acrylic bases with wax rims were made on the master casts in order to establish maxillomandibular relations . after making the maxillomandibular records , the casts were mounted in an articulator . rather than primary tooth forms , primary tooth forms were not adequate to fulfill the ideal vertical dimensions because the patient is in the transition period from mixed to permanent dentition . after the final insertion , routine hygiene instructions for the dentures were given to both the child and his parents ( figures 6 and 7 ) . the patient was advised to maintain a soft diet for the first few days , and to remove the dentures at night to promote healing of the oral tissue . despite the initial lack of compliance , the child tolerated the dentures quite well . in order to accommodate growth and development , the patient was scheduled for ongoing follow up visits every 3 months . at recall appointments , good retention was observed and the parents reported a significant improvement in terms of speech and mastication ; in addition , they discovered that he enjoyed wearing the dentures . continued follow - up is suggested for modification or replacement of the dentures to fit the patient s developing maxilla and mandible . a 3year old male patient was referred to gazi university faculty of dentistry department of pediatric dentistry due to the lack of primary teeth . the major complaint of his parents was mastication issues that resulted in dietary deficiencies ; the parents further reported that he was unable to speak . in the clinical extraoral examination , prominent forehead , sparse , very fine scalp hair and eyebrows , saddle nose , protuberant lips , large chin , soft , dry and light colored skin could be observed ( figures 8 and 9 ) . intraoral examination revealed complete absence of primary and permanent teeth , underdeveloped maxillary and mandibular ridges and relatively enlarged tongue ( figures 10 and 11 ) . radiographic examination confirmed complete absence of both primary and permanent teeth as well as underdevelopment of alveolar ridges ( figure 12 ) . parental history revealed that there were no other cases of ectodermal dysplasia in his family . since clinical findings suggest the presence of ectodermal dysplasia , the patient was referred to gazi university faculty of medicine department of pediatrics . the patient was diagnosed anhidrotic ectodermal dysplasia by performing skin biopsy . to improve appearance , mastication and speech , it was decided that removable complete maxillary and mandibular dentures would be appropriate for the patient . primary tooth forms were used and the occlusal vertical dimension was increased by 1 mm in order to improve the balance of both the dentures and facial profile . after the initial insertion , oral hygiene instructions for the dentures were given to the parents . initially , the patient had some difficulty in accepting the dentures and was unable to keep them in his mouth due to his young age . after a few months , he was fully adapted to using the dentures , and his parents reported that he was able to eat ; in addition , his speech improved and he was quite happy with the dentures ( figures 13 , 14 and 15 ) . further follow - ups have taken place every 3 months . further adjustments were made to eliminate interferences at recall appointments ; future treatment will include relining , rebasing , or remaking the dentures in order to accommodate growth and development . an 8-year old male patient was referred to gazi university faculty of dentistry department of pediatric dentistry due to lack of teeth as well as speech and mastication problems . parental history revealed that the patient was diagnosed with anhidrotic ectodermal dysplasia after skin biopsy at gazi university faculty of medicine department of pediatrics 5 years ago . the patient was the only child and there were no other cases of ectodermal dysplasia in the family . the lack of primary and permanent teeth in the oral cavity resulted in dietary problems ; in addition , the child did not accept any previous dental intervention due to anxiety . during extraoral examination , the child exhibited the typical features of anhidrotic ectodermal dysplasia : saddle nose ; soft , dry and light colored skin ; increased pigmentation ; as well as thin , linear wrinkles in the peri - oral region ( figures 1 and 2 ) . intraoral examination revealed the complete absence of primary and permanent teeth , thin alveolar ridges , reduced vertical bone height , and loss of sulcus depth in the posterior regions of maxillary and mandibular jaws ( figures 3 and 4 ) ; complete anodontia was also confirmed by panoramic radiography ( figure 5 ) . in order to improve appearance , mastication , and speech , removable complete maxillary and mandibular dentures were determined to be the best treatment choice . preliminary impressions were made with irreversible hydrocolloid , and then custom trays were prepared for functional impression . acrylic bases with wax rims were made on the master casts in order to establish maxillomandibular relations . after making the maxillomandibular records , the casts were mounted in an articulator . rather than primary tooth forms , primary tooth forms were not adequate to fulfill the ideal vertical dimensions because the patient is in the transition period from mixed to permanent dentition . after the final insertion , routine hygiene instructions for the dentures were given to both the child and his parents ( figures 6 and 7 ) . the patient was advised to maintain a soft diet for the first few days , and to remove the dentures at night to promote healing of the oral tissue . despite the initial lack of compliance , the child tolerated the dentures quite well . in order to accommodate growth and development , the patient was scheduled for ongoing follow up visits every 3 months . at recall appointments , good retention was observed and the parents reported a significant improvement in terms of speech and mastication ; in addition , they discovered that he enjoyed wearing the dentures . continued follow - up is suggested for modification or replacement of the dentures to fit the patient s developing maxilla and mandible . a 3year old male patient was referred to gazi university faculty of dentistry department of pediatric dentistry due to the lack of primary teeth . the major complaint of his parents was mastication issues that resulted in dietary deficiencies ; the parents further reported that he was unable to speak . in the clinical extraoral examination , prominent forehead , sparse , very fine scalp hair and eyebrows , saddle nose , protuberant lips , large chin , soft , dry and light colored skin could be observed ( figures 8 and 9 ) . intraoral examination revealed complete absence of primary and permanent teeth , underdeveloped maxillary and mandibular ridges and relatively enlarged tongue ( figures 10 and 11 ) . radiographic examination confirmed complete absence of both primary and permanent teeth as well as underdevelopment of alveolar ridges ( figure 12 ) . parental history revealed that there were no other cases of ectodermal dysplasia in his family . since clinical findings suggest the presence of ectodermal dysplasia , the patient was referred to gazi university faculty of medicine department of pediatrics . the patient was diagnosed anhidrotic ectodermal dysplasia by performing skin biopsy . to improve appearance , mastication and speech , it was decided that removable complete maxillary and mandibular dentures would be appropriate for the patient . primary tooth forms were used and the occlusal vertical dimension was increased by 1 mm in order to improve the balance of both the dentures and facial profile . after the initial insertion , oral hygiene instructions for the dentures were given to the parents . initially , the patient had some difficulty in accepting the dentures and was unable to keep them in his mouth due to his young age . after a few months , he was fully adapted to using the dentures , and his parents reported that he was able to eat ; in addition , his speech improved and he was quite happy with the dentures ( figures 13 , 14 and 15 ) . further adjustments were made to eliminate interferences at recall appointments ; future treatment will include relining , rebasing , or remaking the dentures in order to accommodate growth and development . oral rehabilitation of the ectodermal dysplasia patient is necessary to improve both the sagittal and vertical skeletal relationship during craniofacial growth and development as well as to provide improvements in esthetics , speech , and masticatory efficiency.2 although removable prostheses are the most common treatment method , dental implants are also considered to be a treatment option . dental implants combined with implant - supported dentures for adolescents over 12 years of age are recommended as a treatment choice in literature . in situations where implant therapy is indicated , the main problem is insufficient bone ; if bone atrophy progresses in these already alveolar - deficient patients , implant placement may not be possible without bone grafting.7 conversely , implantation reconstruction surgery is subject to a greater risk of failure compared to more conservative prosthetic treatment , besides its psychological aspects particularly in young children.8,12 early implant placement in a growing child may cause cosmetic problems because the implants act like ankylosed teeth . with the vertical development of the jaws , implant over - structures may not meet with the teeth of the opposite jaw , and may result in prosthetic infraocclusion.7,13 therefore , the use of implants in young children should be considered carefully , taking into account the above - mentioned issues , especially dental and skeleton maturation as compared to the chronologic age of the patient . in both of the above cases , implant therapy was not the treatment choice due to ongoing growth and development and insufficient alveolar bone support . it is well - known that dental findings in ectodermal dysplasia may range from hypodontia to anodontia of the primary or permanent teeth . however , the congenital absence of primary teeth is relatively rare;5,9,10 nevertheless , complete anodontia involving primary and permanent dentitions was observed in both cases . in the second case , the parents were unaware of their child s condition prior to a routine dental visit , after which anodontia and extra - oral findings were thought to be related to ectodermal dysplasia and the patient was then referred to a pediatrician . there is little research regarding the impact of extensive hypodontia or anodontia on young children;14 it is known that from around 9 years of age , children with disabilities realize that they will remain different from other children . therefore , the prosthetic intervention at the age of 8 in case 1 is thought to be beneficial in terms of psychological development . although the definite time to initialize treatment is still controversial , till and marquez15 recommend that an initial prosthesis could be fitted when the child starts school , so that he may enjoy a better appearance and will have time to adapt to the prosthesis . due to the lack of compliance in case 1 , the patient could not receive any dental treatment prior to his referral to our clinic . however , consequences of the delay in regards to speech , mastication , and psychological development were not determined to be significant . after using appropriate behavior management techniques , it was eventually possible to achieve prosthetic treatment . in case 2 , early prosthetic treatment led to significant improvements in appearance , speech , and masticatory function . during the first month following the initiation of prosthetic treatment , it was difficult for the 3-year old patient to adapt to the complete dentures ; however , he was accustomed to using the dentures and was able to eat adequately within a few months . although dentures are poor alternatives to healthy dentition , they create conditions for the maintenance of a normal , satisfactory diet for the child . this is very important , considering that the establishment of lifelong dietary patterns occurs during childhood.2 our observations in case 2 comply with this view . dental prostheses may also improve the tone of the muscles of mastication and may compensate for the reduced vertical dimension.10 difficulty with mastication has been referred to as a major problem arising from loss of teeth.3 as seen in both cases , the facial profile and expression improved significantly with complete dentures ; in addition , mastication and dietary patterns also improved . learning and reinforcement of articulation is known to continue until 8 years of age.3 therefore , complete agenesis of primary teeth may result in speech abnormalities , as is seen in both cases . regarding the dental treatment , it was possible to improve speech and communication skills and , as a consequence . thereby , a higher self - esteem and better social acceptance was promoted with the establishment of complete dentures . these case reports highlight the importance of accurate treatment planning as well as the influence of anodontia on the diagnosis of ectodermal dysplasia . since the oral rehabilitation of these cases is often difficult , particularly in pediatric patients , treatment should be administered by a multidisciplinary team involving pediatric dentistry , orthodontics , prosthodontics , and oral - maxillofacial surgery . the clinical manifestations of ectodermal dysplasia cause considerable social problems in individuals affected by the condition . in this case report , the prosthetic rehabilitation of 2 young boys with anhidrotic ectodermal dysplasia associated with severe anodontia was described . since oligodontia or complete anodontia leads to atrophy of the alveolar bone , prosthetic treatment is of great value to these patients from functional , psychological , and psychosocial standpoints .
chronic renal failure is a progressive , irreversible kidney disorder in which the body s ability to maintain liquid and electrolytes is lost ; it is considered a major problem in the health system , and is one of the leading causes of death and inability worldwide ( 1 ) . the prevalence of chronic renal failure is 242 cases per one million people and this increases 8% annually worldwide ( 2 ) . methods for the treatment of end - stage renal disease include long - term treatment with dialysis and kidney transplantation . the best method is kidney transplantation , but in most countries there is an insufficient number of donated kidneys ; hence , hemodialysis is the most common treatment worldwide ( 3 ) . in iran , there are more than 13,000 dialysis patients , and 150,000 dialysis sessions are conducted each month ( 4 ) . although hemodialysis as the main treatment for chronic renal failure increases dialysis patients lifespans , it causes numerous psychosocial problems for them ( 5 - 7 ) . economic problems , therapy - related stresses , dietary restrictions , and family problems are among factors that makes hemodialysis patients prone to anxiety ( 8) . in addition , because of multiple and complex drug therapies , various difficulties and serious changes in lifestyle affect their psychosocial performance ( 9 ) . the length of the disease course , the period of treatment , and dysfunction in the support system of hemodialysis patients leads to their inability to adapt to stressful situations ( 10 ) . these conditions provide for increasing anxiety in patients undergoing hemodialysis , so that they experience high levels of stress , anxiety , and depression ( 11 ) . in fact , it can be said that depression and anxiety are the most important reactions of patients with chronic renal failure ( 8) . anxiety is a common psychological problem in patients with end - stage renal disease ( 10 - 12 ) ; it consists of unpleasant mental feelings , worry , and ambiguous tension along with physical symptoms such as perspiration , headache , restlessness , and heart palpitations . however , sometimes anxiety becomes escalated and changes into a mental disorder ; such patients suffer from excessive anxiety ( 13 ) . according to the world health organization , from among the 870 million people who live in europe , the prevalence of 12-month anxiety disorder is 17.7% ; lifetime anxiety disorder in women is 30.5% , and lifetime anxiety disorder in men is 19.2% ( 14 ) . studies show that stress has a dramatic effect on hemodialysis patients and can increase illness , mortality , frequency of hospitalization , and cost of treatment ( 1 ) . it also prevents adherence to diet and recommended therapies , and has a negative impact on self - care and treatment outcomes ( 11 - 15 ) . in addition , it increases vulnerability to suicide of patients undergoing hemodialysis ( 16 ) . in addition to anti - anxiety drug treatments for these patients ( 7 ) , there are several ways to treat anxiety , including psychological treatment and complementary medicine treatments . complementary medicine and herbal medicine have developed globally , and these new treatments have gained a special status and value ( 17 ) . complementary medicine treatment is accepted as a part of nursing care in the united kingdom . nurses in over 30 countries are licensed to use complementary medicine therapies , such as aromatherapy , in nursing care ( 18 ) . aromatherapy is a type of complementary medicine in which the volatile oil of plants is used to promote the level of physical , spiritual , and physiological health ( 19 , 20 ) . aromatherapy is used in several forms , including massage , inhalation , compresses , baths , or topical application . inhalation and massage are the most widely used forms , especially in nursing interventions ( 20 , 21 ) . a large number of plants have been found useful for aromatherapy , including tea oil , chamomile , lemon balm , and rose oil ( 22 ) . damask rose is a native iranian plant whose water has been used to improve physical and mental health since ancient times ( 23 , 24 ) . according to the studies conducted concerning this substance , it can relieve thirst , stop bladder bleeding , control pests , and enhance digestion . also , it has antispasmodic , anti - bacterial , anti - virus , and hematopoietic effects . the damask rose can also be used to strengthen the nerves and to treat anxiety and depression ( 24 - 26 ) . nursing care of the hemodialysis patients is very important , taking into account the high prevalence of chronic renal failure , the significant increase of hemodialysis patients , and their significant need for physical and psychological care . rose water inhalation could be used as a safe , simple , and low - cost method in nursing care if it can be proved to be effective for the reduction of anxiety in hemodialysis patients . however , in light of the tranquilizing and anxiolytic effects of rose water noted since ancient times , the pleasant scent of this substance , and its availability , possible production , and processing in good quality within iran , we decided to study the effects of rose water on the anxiety of hemodialysis patients in birjand . this study aimed to investigate the effect of inhaling rose water aromatherapy on anxiety in hemodialysis patients . this study is a randomized clinical trial that was conducted in the hemodialysis center of birjand vali - asr hospital in 2014 . the population included hemodialysis patients from birjand and the sample was selected from the hemodialysis patients referred to the hemodialysis center . the necessary sample size for both the control and treatment group was calculated as 22 patients , and increased to 25 patients for each group given the likelihood of patients not completing the study . convenience sampling was conducted and patients who had the inclusion criteria were divided randomly into two groups based on a previously prepared random list . inclusion criteria included undergoing dialysis 2 times a week and dialysis treatment 6 months . exclusion criteria included having an acute stressful event ( such as the death of a beloved one , etc ) over the previous 6 months ; using tranquilizers , anti - anxiety drugs , or other therapeutic interventions such as herbal essences ; respiratory problems and smelling difficulties ; not being able to cooperate or go on dialysis ( kidney transplant , coma , death , etc ) for any reason ; or not having consent to continue the study . during the study , two participants were unwilling to continue cooperation : one patient underwent kidney transplant , and another patient went into a coma . thus , 46 patients participated in the study . the first section included demographic / clinical characteristics , and the second was the spielberger s state - trait anxiety inventory . the spielberger questionnaire included 40 items and consisted of two equal parts for measuring state and trait anxiety . each item was scored from 1 to 4 , and the achieved score was between 40 and 160 , with higher scores indicating more anxiety . its validity and reliability were confirmed in a study by behdani et al . ; the cronbach s alpha coefficient was 90% ( 27 ) . this clinical trial began after obtaining approval from the ethics committee of the birjand university of medical sciences and registering in the iranian registry of clinical trials ( irct2013052613476n1 ) . the patients in the active - treatment group were instructed to put three drops of rose water ( at a concentration of 25% ) on a clean absorbent cotton handkerchief using a dropper each night before sleeping and also 15 - 20 minutes before beginning dialysis sessions . they were requested to put the handkerchiefs in front of their noses for 15 to 20 minutes and breathe normally . the intervention continued for 4 weeks , during which time the control group received no intervention . the data were collected by questionnaires after the research goal was completely explained to the subjects and their informed consent was obtained . the questionnaires were completed for both groups three times : before the intervention and at the end of the weeks 2 and 4 after the intervention . the data were analyzed with spss software ( version 18 ) using the independent t - test , paired t - test , repeated measures analysis of variance ( anova ) , kruscal wallis , and one - way anova statistical tests at = 0.05 . this study was conducted in the vali - asr hospital of birjand on 46 hemodialysis patients who were assigned to two equal active - treatment and control groups . the average age for patients in the treatment group was 50.3 19.4 years compared with 59.1 10.1 years for the control group . in terms of age , there were no significant differences ( p = 0.06 ) between the groups . in each of the groups , there were 12 men ( 52.2% ) and 11 women ( 47.8% ) ( p = 1.00 ) . among those in the treatment group , 4 patients were single ( 17.4% ) , 17 were married ( 73.9% ) , and 2 were widowed ( 8.7% ) ; in the control group , 21 patients were married ( 91.3% ) , 2 were widowed ( 8.7% ) , and there were no single patients . there was no significant difference between the groups in terms of marital status ( p = 0.21 ) . the two groups were similar with reference to education ( p = 0.7 ) and employment ( p = 0.21 ) . the mean duration of dialysis for the patients in the treatment group was 2.26 2.29 years and in the control group was 3.26 2.52 years . in the treatment group , the cause of hemodialysis in 16 patients ( 69.6% ) was pre - renal failure ( 21.7% ) , in 5 was renal failure , and in the others was post - renal failure ; in the control group , 18 of the patients ( 78.3% ) had pre - renal failure as the cause , 3 had renal failure ( 13% ) , and the others had post - renal failure . thirteen ( 56.5% ) of the patient in the treatment group had undergone dialysis 2 times a week , and the rest had done so 3 times a week . in the control group , 14 patients ( 60.9% ) had undergone hemodialysis 2 times a week , and it had been done three times a week for the rest . regarding clinical characteristics , the groups were not significantly different regarding the duration of dialysis ( p = 0.14 ) , the cause of dialysis ( p = 0.88 ) , and the number of dialysis sessions per week ( p = 0.76 ) . the scent of rose significantly reduced the mean state of anxiety in the treatment group from 47.47 7.68 before the intervention to 37.1 6.5 in week 4 after the intervention ( p < 0.001 ) , whereas the control group did not show any statistically significant difference ( p < 0.43 ) . independent t - test results also indicate that there was a significant difference ( p<0.001 ) between the mean state anxiety scores of the treatment group ( 37.1 6.5 ) and the control group ( 49.5 9.4 ) after week 4 of the intervention ( table 1 ) . the results indicate that the scent of rose reduced the trait anxiety of the treatment group significantly ( p < 0.001 ) , from 49.65 13.88 before the intervention to 42.91 10.1 in week 4 after the intervention , whereas there is no such statistical difference in the control group ( p = 0.38 ) . independent t - test results also indicate that there is a significant difference ( p < 0.001 ) between the mean trait anxiety scores in the treatment group ( 42.91 10.1 ) and the control group ( 52.8 12.4 ) after week 4 of the intervention ( table 2 ) . results showed that the mean change in state anxiety scores ( before and after the intervention ) were independent of sex , age , education , marital status , and disease duration . the only significant factor affecting the mean change in state anxiety score was the cause of dialysis ( p = 0.009 ) ( table 3 ) . abbreviation : anova , analysis of variance ; sd , standard deviation . the mean change in trait anxiety scores ( before and after the intervention ) were independent of sex , age , education , marital status , cause of dialysis , and duration ( table 4 ) . in the present study , the scent of rose water was used to study the effects of aromatherapy on anxiety in hemodialysis patients . the results showed that the levels of trait and state anxiety in hemodialysis patients after the intervention were reduced significantly in the treatment group compared with the control group , whereas before the intervention there was no significant difference between the groups . currently , much clinical research is being done on various applications of aromatherapy and the use of herbal oils around the world ( 28 ) . roses , lavender ( lavandula ) , bergamot , oranges , and lemons , etc , are examples of essential aromatic oils that are commonly used to relieve anxiety , stress , and depression ( 29 ) . several studies that examined the effect of rose scents have obtained results similar to our study . the results showed that aromatherapy , in the form of inhalation of a blend of rose and lavender essences , was effective on students anxiety . their study was conducted on 260 students who were randomly placed into the control and treatment groups . students receiving aromatherapy showed a significant decrease ( p < 0.001 ) in the symptoms of anxiety and depression at the end of weeks 2 and 4 compared with the control group ; anxiety scores before the intervention in the treatment and control groups were 99.6 17.1 and 15.7 99.3 , respectively , and their depression scores in the treatment and control groups were respectively 9 28.4 and 9.9 29.1 , respectively . ( 2012 ) conducted a study to evaluate the effects of aromatherapy on anxiety level ; the study was done on 130 veteran students randomly assigned to treatment and control groups . after the administration of the spielberger s state - trait anxiety inventory as a pre - test to all subjects , an oil rose and lavender blend was inhaled every night for half an hour in the treatment group for 4 weeks . the control group received only sesame oil inhalation . at the end of weeks 2 and 4 , the results indicated a significant reduction in anxiety symptoms of the students receiving treatment compared with the control group , thus showing the effectiveness of aromatherapy ( 30 ) . some studies have also investigated the effects of other herbal oils , such as lavender aromas , on the anxiety of hemodialysis patients ; they achieved results similar to the present study . ( 1 ) studied the effects of aromatherapy on the anxiety of patients undergoing hemodialysis in 2010 . the aromas used in his research included lavender , which , similar to rose , has anxiolytic effects . this clinical trial was conducted on 65 hemodialysis patients who were randomly divided into treatment and control groups . the results showed that the mean state and trait anxiety of patients were reduced after the use of aromatherapy at the end of week 4 , suggesting that aromatherapy , in the form of inhaling lavender aromas , could significantly reduce anxiety levels in hemodialysis patients ( 1 ) . in another study by kanany ( 2012 ) , the effects of aromatherapy on anxiety in patients undergoing hemodialysis was examined using orange essence . the anxiolytic effects of orange essence are similar to those of rose , and it has been confirmed in studies , including the present study , that aromatherapy can significantly reduce trait and state anxiety ( p < 0.001 ) compared with the time before the intervention . the mean state and trait anxiety in patients in the treatment group were reduced from 9.7 46.9 and 9.2 46.5 to 8.7 35.9 and 8.9 26.0 , respectively , at the end of week 4 ( 31 ) . itai et al . exposed hemodialysis patients to a condition in which they inhaled the scent of aromatic oils and to a condition without scents , and measured the impact of each condition on the anxiety and moods of hemodialysis patients ( 7 ) . aromatherapy to reduce anxiety in hemodialysis and other patients was studied by oh et al . in which patients with presurgical anxiety were treated with aromatherapy in the form of a lavender and bergamot blend , the effectiveness of aromatherapy on the reduction of anxiety in those patients was confirmed ( 32 ) . ( 2010 ) investigated the effects of inhalation aromatherapy on patients anxiety for heart and abdominal surgery . they used a lavender essence , which significantly reduced the anxiety of the subjects after the intervention ( p = 0.001 ) ( 18 ) . wilkinson et al . conducted a study ( 2006 ) on 288 patients with cancer to investigate the effects of aromatherapy on the control of depression and anxiety in cancer patients in england . sampling was done randomly and patients with cancer were divided randomly into control and treatment groups . the treatment group was treated with massage aromatherapy , and the controls were treated with common medical therapies . the results indicate a positive but short - term impact of aromatherapy on the anxiety and depression of cancer patients ( 33 ) . ( 2003 ) examined the effects of inhalation aromatherapy on the anxiety of cancer patients during radiotherapy . unlike the present study , this study did not confirm the effectiveness of aromatherapy on the reduction of the subjects symptoms of anxiety and depression , which may be due to the difference in the kind of diseases under study ( hemodialysis and cancers patients ) ( 34 ) . conrad et al . ( 2009 ) , for instance , did one study on 28 postpartum women in which aromatherapy using an essential oil blend of rose otto and lavandula angustifolia was applied both through inhalation and the hand m technique for 4 weeks . according to the results of the present study , rose water has a significant effect on the anxiety of hemodialysis patients ( p < 0.001 ) . consequently , inhalation of rose water can be used to improve the patients psychological condition during hemodialysis . we suggest that there should be further studies to compare the effects of rose water with other herbal oils and essences on hemodialysis patients and to investigate the effects of rose water and its anxiolytic property in other chronic illnesses
curing a medical disease generally implies completely removing the pathogenic agents : e.g. , killing microbes with specific antibiotic therapy or surgically removing cancer . in t1d , cure implies reversing new - onset t1d and restoring glucose homeostasis . initial surgical success using the edmonton protocol in islet transplantation1 was tempered by later results showing recurrence of t1d in these grafts . ( refer to the curing t1d : antigen - specific approaches section ) . in contrast to cure , in most clinical autoimmune diseases ( such as rheumatoid arthritis ) , current approaches do not cure the disease : the disease is suppressed or halted by immunosuppressive therapy ( e.g. , anti - tnf therapies in rheumatoid arthritis ) . usually ( although not always ) , when the treatment is stopped , autoimmune disease recurs in rheumatology . this raises a major issue in t1d therapeutics : there is already a treatment for the disease . there has been progress in controlling blood glucose ( bg ) using rapid - acting or long - acting insulin analogs and sensor - augmented insulin pumps for long - term management of hyperglycemia and hypoglycemia.25 given that many t1d patients are children otherwise in good health , the existence of insulin therapy makes the risks of many treatment approaches ( e.g. , bone marrow transplantation with chemoablation or strongly immunosuppressive drugs ) prohibitive . the risks of ongoing t1d are considerable , but these risks must be matched up against the risk of any proposed therapy . ultimately , one must ask in each case of a proposed new therapy what is the risk / benefit of daily treatment over a lifetime , compared to the same risk / benefit analysis of insulin therapy ? this is one reason why many have emphasized trying to cure t1d : to avoid the risks of chronic treatment . evidence that t1d is an organ - specific autoimmune disease has accumulated for over 45 years.6 cell - mediated immunity to insulin in human t1d was shown in 1975.7 the development of t - cell cloning8 and the discovery of the t - cell receptor genes9 rapidly led to the cloning of human t cells autoreactive to insulin.10,11 this led to the concept that t1d could be cured by eliminating specific t cells without affecting the entire t - cell repertoire ( and thus inducing global immunosuppression).12 an enormous number of studies focused on insulin reactive t cells in t1d , with the hope that eliminating or otherwise changing the function of these autoreactive t cells could cure the disease without affecting the global t - cell response to infections ( i.e. , without inducing global immunosuppression ) . the idea seems simple : why not just remove those t cells that express autoreactive t - cell receptors from the t - cell repertoire ? unfortunately , this approach has failed in human t1d for both insulin and other autoantigens . more than 20 different human trials of various insulin preparations , given with the hope of either eliminating or anergizing insulin - specific t cells and thus stopping t1d , have failed.13 antigen - specific therapy in general has had no effect on the disease process it did not halt t1d let alone cure it . although antigen - specific immunotherapy remains an area of intense interest and investigations , these repeated failures in human trials have dampened initial optimism about this approach . the most likely explanation is that by the time of acute disease in humans , epitope spreading has occurred ; in other words , t cells reacting to many islet - related autoantigens are attacking the islet , so that eliminating one set of t cells is unable to affect the progression of the disease.14 even if insulin is the autoantigenic target , in this scenario , targeting insulin would only work very early in disease ( refer to the preventing t1d : antigen - specific and nonspecific approaches section ) . many different nonantigen - specific , but adaptive immune - directed ( i.e. , directed primarily to t and b cells ) , immunotherapeutic approaches have been tried in the past 30 years . one of the first was cyclosporine a ( csa ) , which inhibits calcineurin and reduces t - cell inflammatory cytokine production . csa was quite effective in retrospect , with 20%65% initial t1d reversal , but severe side effects ( renal toxicity ) were reported and t1d recurred after the reagent administration ceased.15,16 another approach to adaptive immunity , targeting t cells with anti - cd3 antibodies , has been thoroughly studied . anti - cd3 ab induced 60% reversal of newly diagnosed t1d in nonobese diabetic ( nod ) mice17 by modulating both effector t cells and regulatory t cells . anti - cd3 ab depleted up to 50% of peripheral t cells in a dose - dependent manner18,19 by inducing effector t - cell apoptosis.20 this treatment also induced anergy in both cd4 and cd8 t cells.21 beta - cell - specific regulatory t cells were differentiated in the periphery,17 and these regulatory t cells were protected from the anti - cd3-mediated depletion or inactivation.20 however , these promising results in mice were not replicated in humans ; diabetic patients did not obtain long - term preservation of endogenous insulin secretion and showed only a small decrease in exogenous insulin use.22,23 a large number of other agents targeting adaptive immunity have been tested in human t1d trials , including anti - cd20 , ctla4-ig , and many others ( well reviewed recently by kolb and von herrath24 ) . many of these approaches can temporarily halt loss of beta - cell mass / function ( as measured , e.g. , by c peptide preservation ) , but none of them can halt the disease process . new therapeutic approaches are clearly needed , including combination therapies that can synergistically target multiple different immune pathways.25 the failure to find effective immunotherapies to reverse acute t1d has focused attention on other approaches , including prevention . one impetus for considering prevention is the rapid increase in t1d incidence in many countries since the mid-20th century , which is not explicable by the known genetic predisposition to disease.26 in europe , for example , the incidence of t1d cases has been increasing on the average 3.9% per annum , which is a near epidemic pace.27 if environmental factors are driving this increase , they could be targeted to prevent the disease . one explanation is the hygiene hypothesis , which suggests that insufficient exposure to microbial agents in immune system development predisposes the adaptive immune system to hyperactivity , resulting in increased autoimmune and allergic diseases.28 evidence for the hygiene hypothesis in t1d is increasing . recently , it was shown that finnish children , located across the border from russian , have a much higher incidence of t1d and that this could be explained by exposure to an altered form of lipopolysaccharide ( lps ) in altered bacterial flora found in a western european village.29 the authors concluded that early colonization by immunologically silencing microbiota may thus preclude aspects of immune education,29 which can lead to hyperactive adaptive immunity . besides hygiene hypothesis , there are several other reasons to consider preventative approaches in t1d . one reason is found in the nod mouse model : it is very easy to prevent t1d in nod mice . a peculiarity of the nod literature is the exaggerated attention paid to prevention vs. disease reversal studies . it was already shown over 12 years ago that 440 studies had been done showing prevention of nod t1d , while only 23 published protocols attempted to reverse the disease.30 in fact , many agents that can prevent nod t1d can not reverse it ( a good example is lps : it can prevent nod t1d but is ineffective after age 10 weeks and does nothing to reverse acute disease31 ) . apart from the mouse model , it is now possible to predict human t1d on the basis of autoantibodies : two positive islet autoantibodies ( iaas ) predict a rate of development of clinical disease at ~11% progression per year.32 thus , the presence of two iaas in children is now considered preclinical human stage 1 t1d.33 finally , genetic risks of t1d are increasingly well defined . ninety percent of european t1d patients have disease susceptibility dr3 , dr4 , or dq hla loci.34 four single nucleotide polymorphisms ( snps ) , in dr3 , dr4 , ctla4 , and ins , can predict t1d with an auc of 0.72 ; a full panel of 40 snps increases the auc to 0.87.35 these favorable developments , combined with the failure to effectively treat disease , have led to a renewed emphasis on t1d prevention , as well as many t1d prevention clinical trials.3638 unfortunately , however , there are serious problems facing any attempt to prevent human t1d . first , prevention trials in high - risk individuals ( defined by both autoantibody status and genetic risk ) have already failed . the trigr trial , which tested the hypothesis that exposure to cow s milk increased t1d incidence and substituted hydrolyzed casein in infant formula , failed to prevent autoantibody formation.37 the diabetes prevention trial ( dpt-1 ) to prevent t1d using insulin to tolerize t cells failed , and nicotinamide failed to prevent progression to t1d in the endit trial.37,39 these failures could be attributed to the wrong preventative agent , indeed trialnet is conducting many additional prevention trials ( www.diabetestrialnet.org ) . however , there are several other major obstacles facing t1d prevention trials : 1 ) although , as noted previously , individuals with two iaas progress to t1d at ~11% per year , 15%20% of these individuals still do not develop t1d at 20 years.36 this is a major clinical and research problem : how to develop a trial to deal with such variable and prolonged disease development . moreover , is it feasible to treat children for 10 years or longer with immunosuppressive agents ( anti - cd3 antibodies and ctla4-ig are examples of agents currently being tested in prevention trials ) to achieve prevention what would the side effect profile be over this prolonged period ? 2 ) many current studies are performed in populations enriched in t1d , however , the vast majority of patients ( ~85% ) have no family history of t1d.36 thus , prevention studies would require expansion of autoantibody screening to the entire population . while this is certainly possible , the performance of assays and predictive value of iaas in the general population ( as opposed to enriched populations ) is not known . finally , of course , we simply do not know if the ease with which t1d is prevented in the nod mouse is translatable to humans . all of this emphasizes the need to continue to try to develop new approaches for treating the acute disease . the nod mouse , which spontaneously develops autoimmune t1d , has been studied extensively since its introduction in 1982 , and a large nod literature has been published showing that virtually every aspect of the immune system is involved.40,41 studies of adaptive immunity have predominated in the nod literature , due to the spectacular correlation between human and mouse with respect to involvement of mhc class ii , autoreactive t cells against the same autoantigens ( insulin , glutamic acid decarboxylase , etc . ) in mouse and man , and the presence of similar autoantibodies prior to disease onset.41 in addition , genetic studies have found that many non - mhc loci related to immunity were shared between nod and human t1d patients.42 more recently , however , focus has shifted to the role of innate immunity in nod pathogenesis . the role of environmental factors was originally highlighted by the observation that nod mice housed in cleaner facilities had increased disease incidence.43 in fact , nod mice housed in germ - free facilities had 100% incidence of t1d ( compared to 80% in nod female mice housed under conventional conditions).44 wen et al44 pursued these ideas and showed that nod mice lacking myd88 ( an adapter protein that mediates signaling from multiple innate molecules , including toll - like receptor [ tlr ] 4 ) were completely protected from t1d in the presence of normal flora , but developed 100% t1d incidence under germ - free conditions . furthermore , they showed that the myd88 effect was mediated through the microbiome : treating the germ - free mice with the microbiota from protected mice ameliorated disease.44 this revolutionary study initiated an explosion of studies on the role of microbiota in autoimmunity , which has been recently reviewed.4547 in t1d , attention was initially focused on the role of tlr4 , which was investigated by making nod tlr4/ mice . however , the effect of tlr4 knockout on t1d development has been inconsistent , with studies showing increased , decreased , and no effect on t1d incidence.44,48,49 this contradictory evidence is probably explained by the fact that tlr4/md-2 signaling is mediated by both tir - domain - containing adapter inducing ifn- ( trif ) and myd88 adaptor proteins.50 chervonsky et al51 confirmed this by showing that dual myd88/trif nod knockout mice are not protected from t1d in the presence of conventional microbiota ( unlike the nod single myd88 knockout ) . this result supports a balanced signal hypothesis , meaning that both inflammatory and regulatory responses are induced by the microbiota and that tlr4-mediated trif signaling causes a tolerizing immune response , which protects against t1d development.51 these extremely important studies bring studies of innate immunity , and potential therapeutic approaches to tlr4 signaling , to the forefront in t1d research . we decided a few years ago to focus our efforts on reversing t1d in nod mice . while this approach seems obvious , for some reason ( perhaps because prevention experiments are easier to do , are more successful , and generate more articles ) , the field is dominated by prevention studies in nod . many of the agents that have been shown to prevent nod t1d demonstrably can not reverse acute disease in nod . one reason for the ineffectiveness of preventative agents to reverse disease is that prevention approaches may target earlier , more permissive checkpoints in the disease prior to significant inflammation , whereas reversal must address the acute disease.52 as discussed previously , by 2005 , only 23 attempts to reverse nod t1d had been reported , with 16 successes , compared to 440 prevention trials.30 we estimate that by now there have been published ~1000 articles showing prevention of nod t1d and perhaps 4050 articles on reversal . clearly , there is much room to define novel reversal pathways in nod t1d . the hygiene hypothesis and accumulating evidence showing that environmental influences , particularly the microbiota signaling effect on the tlr4 pathway , played a key role in t1d , suggested to us that innate immunity might be an underdeveloped target in t1d therapy antigen - presenting cells ( apcs ) critically affect adaptive immune responses53 and the balance of pathogenic vs. protective t - cell responses54 ; and macrophages / dendritic cells ( dcs ) are among the first cell types found infiltrating the islet.55 nod mice have developmental defects in macrophages.56 altered immune stimulation of apcs by environmental agents , including variant forms of lps , could profoundly affect apcs and either enhance or reduce diabetogenesis , in fact this was subsequently demonstrated as mentioned previously.29 the invading macrophages in nod t1d promote disease pathogenesis57 by producing proinflammatory cytokines such as tnf-.55 proinflammatory apcs that do not regulate effector t cells could explain epitope spreading and thus the inability to treat t1d by targeting a single autoantigen . this critical cross - talk between innate and adaptive immunity is thus an attractive therapeutic target in autoimmunity.58 we have focused on tlr4 given the literature discussed previously ; other tlrs and innate molecules may be suitable targets.59 tlrs are expressed on innate immune cells including dcs and macrophages . ten tlrs have been identified in humans ( tlr110 ) and 12 ( tlr19 and tlr1113 ) in mice.60 there are two tlr subfamilies : cell surface tlrs ( tlr1 , tlr2 , tlr4 , tlr5 , tlr6 , and tlr10 ) and intracellular tlrs , localized in the endosome ( tlr3 , tlr7 , tlr8 , tlr9 , tlr11 , and tlr12 ) . one mechanistic explanation for the hygiene effect , as discussed previously , is that tlr4 interacts with the microbiome to mediate protective / tolerance inducing pathways via the trif signaling pathways.51 another major immunoregulatory mechanism that attracted our attention to agonist signaling via tlr4 is the induction of endotoxin tolerance by submaximal tlr4 signaling . endotoxin tolerance is induced in hepatic macrophages due to persistent low - level exposure to lps ; this reduces subsequent induction of macrophage activation and reduces inflammation.61,62 endotoxin - tolerized immune cells have a tolerogenic immune response that has profound downregulatory effects on adaptive immunity.63 we hypothesized that converting islet infiltrating macrophages to a tolerogenic phenotype ( e.g. , by inducing endotoxin tolerance ) could alter adaptive t - cell responses and reverse the acute inflammatory state in the diabetic islet . to alter apc function via tlr4 in vivo in acute t1d , we used agonistic tlr4/md-2 directed antibodies ( tlr4-ab ) that had been shown to induce endotoxin tolerance.64,65 we tested the effect of tlr4-ab in acute t1d in mice with serum bg between 200 and 400 mg / dl . our approach was extremely effective at reversing acute t1d ( even with bg in the 300400 mg / dl range ) in the vast majority of mice , while control antibody - treated mice or untreated mice rapidly progressed to end - stage t1d ( bg > 500 mg / dl).66 notably , bg decreased even though we did not treat mice with exogenous insulin . investigation of islet pathology gave valuable clues to the mechanism of action : tlr4-ab - treated mice demonstrated increased numbers of islets with reduced inflammation , and the amount of insulin per islet increased compared to mice at the onset of t1d ( untreated ) or mice treated with control antibody.66 investigation of the cellular target of tlr4-ab revealed that it did not directly affect either t cells or treg cells , but increased the numbers of macrophages and dcs . in addition , interleukin ( il)-10 production increased significantly , along with il-2 , il-4 , and il-33.66 two weeks after tlr4-ab treatment , t regulatory cells increased in the periphery . finally , transfer studies suggested but did not prove that b cells were not necessary for the disease protective effect . these dramatic results indicate a profound reorganization of the immune response in tlr4-ab - treated diabetic mice . we are beginning to unravel some of the mechanisms of this treatment effect . we performed rnaseq at 1 week following the second tlr4-ab treatment in vivo to try to understand this issue . rnaseq revealed a highly significant induction of genes associated with cd11b+ly6-g+ apcs and a downregulation of t - cell - related genes ( w ridgway , university of cincinnati , unpublished data , february , 2017 ) . this raised the possibility that rather than simply tolerizing existing apcs , we were inducing an influx of myeloid - derived suppressor cells ( mdscs ) . mdscs are a relatively new class of apcs that act to actively suppress t effector cells.67 mdscs have been predominantly studied in oncology ; infiltration of mdscs in the tumor environment can suppress the effect of t - cell - mediated immunity against cancer cells.6870 increasingly , however , it is appreciated that deficient mdsc induction may play a role in autoimmunity.71,72 induction of mdscs can inhibit autoimmunity , most likely by interacting and suppressing t - cell effector responses.73,74 notably , this is highly consistent with the studies by ghazarian et al75 who showed that while coxsackievirus b4 virus can accelerate t1d , natural killer t cell induced mdscs that infiltrated into the islet , downregulated effector t cells , and prevented t1d . although tlr4-ab is given systemically , therefore , it may act by mobilizing mdsc from the bone marrow whereupon they expand in multiple sites including the pancreatic islet . how they home to the islets , however , is currently unknown . at this point , therefore , our leading hypothesis is that tlr4-ab induces mdscs that can actively suppress diabetogenic t cells and reverse t1d . many agents targeting innate immunity have been tested to prevent nod t1d ; however , as mentioned previously , this has little correlation with successful treatment of active disease . however , one approach that was highly successful in reversing t1d in nod included granulocyte - colony stimulating factor ( g - csf ) along with an anti - t - cell agent.76 we suspect , but do not know , that this approach may mobilize similar suppressive apcs as our tlr4-ab . a few other approaches to innate immunity have reversed acute t1d in nod mice.77 both aralast , a serine protease inhibitor , and imatinib , a tyrosine kinase inhibitor , have reversed t1d , although their exact effect on innate immunity is not well understood.78,79 other therapeutic approaches to t1d may have heretofore unrecognized effects on innate immunity . for example , we have shown that an agonistic antibody to cd137 prevented t1d,80 while the splice variant of cd137 , soluble cd137 ( scd137 ) is an immunosuppressive molecule that can both prevent t1d81,82 and ameliorate active t1d ( w ridgway , university of cincinnati , unpublished data , february , 2017 ) . our emphasis has been on t cells and tregs in these studies , but since both cd137 and cd137 ligand are expressed on apcs , the innate immune system may also play a role that needs to be investigated . in terms of human clinical trials , the vast majority have targeted adaptive immunity . g - csf combination therapy has recently been tested in humans , where it was well tolerated.83 both aralast and imatinib human t1d trials have been initiated , but the full results are not yet known.84 therefore , the field of targeted immunotherapy directed to innate immunity is in its infancy in human t1d . overall , our results and the abovementioned studies suggest that many promising approaches will soon be developed targeting innate immunity as a therapeutic pathway in t1d . the ultimate goal is to replicate in humans the intricate immune system re - education that we see over the course of tlr4-ab treatment in mice : an alteration of the numerical and functional immune components active in disease to downregulate autoimmune t cells ( thus overcoming the limitations of antigen - specific therapies that can not address epitope spreading ) and upregulate t regulatory and apc suppressive functions . our tlr4-directed approach is promising because it appears to target the same pathways that are understimulated in environments showing a dramatic increase in t1d in the last half century ( as explained by the hygiene hypothesis of increased t1d incidence ) . since tlr4 agonists are already being tested in other human diseases,85 this approach could be translated to human application relatively quickly , whether alone or as a combination therapy with agents that also target the adaptive immune component .
of all the functional groups in organic chemical space , the olefin must be regarded as one of the most privileged from the vantage point of utility in synthesis . as a result , methods for olefin synthesis and functionalization have been extensively developed and applied in both academic and industrial sectors . most olefin - forming reactions rely on preoxidized starting materials and fall into four main categories : functionalization of ketones or aldehydes ( aldol condensation , wittig olefination , etc . ) ; modification of other alkenes ( olefin metathesis , metal - catalyzed coupling reactions , etc . ) ; reductive transformations of alkynes ( stereoselective reduction , reductive coupling , etc . ) ; or synthesis by elimination reactions ( from alcohols , halides , etc . ) . a less explored strategy for olefin synthesis is the direct desaturation of the parent alkane . within this category , the dehydrogenation of activated aliphatics ( leading to enones , dienes , styrenes , etc . ) , generally a more facile process , has been broadly utilized and continues to be investigated . in contrast , the efficient oxidation of unactivated alkanes directly to alkenes remains an unmet challenge and approaches to this problem have only rarely surfaced in methodological studies . select examples of reported strategies for alkane dehydrogenation are shown in figure 1 . among these , breslow s groundbreaking work awakened the community to the strategic value of a remote desaturation reaction and provided extensive studies on steroid frameworks . in general , these approaches employ high - energy radicals ( figure 1a , b ) or transition metals ( figure 1c , d ) to overcome the high kinetic stability of the c h bond . while these pioneering studies have clarified the difficulties in achieving such a transformation ( regiocontrol , product isolation , functional group tolerance ) , most of these methods lack the generality and practicality required for wide use in complex systems . important limitations include the use of inconvenient starting materials ( e.g. , peroxides ) , poor substrate scope , overoxidation of the resulting olefin , large substrate excesses or harsh reaction conditions . therefore , the invention of a broadly applicable , mild protocol to achieve regio- and chemoselective desaturation of unactivated aliphatics remains highly desirable . our interest in a direct desaturation reaction was sparked during the synthesis of the eudesmane terpenes using a cyclase / oxidase synthetic strategy ( figure 2a ) . in order to achieve the formal dehydrogenation of substrate 13 to 16 , three individual steps were required , involving ( 1 ) n - bromination with acetyl hypobromite ( 1314 ) ; ( 2 ) homolysis / recombination under photochemical conditions to provide a tertiary alkyl bromide ( 1415 ) ; and ( 3 ) base - mediated elimination ( tetramethylpiperidine , 80 c ) ( 1516 ) to give the desired olefin . in stunning contrast , nature is able to use desaturase enzymes to achieve direct , selective and controlled oxidations of aliphatic carbon chains with remarkable precision . this overall process is believed to occur through a number of discrete steps including ( 1 ) c-9 specific alkane hydrogen abstraction ; ( 2 ) single - electron oxidation of the resulting alkyl radical to a carbocation ; and ( 3 ) stereoselective loss of a proton to provide olefin 20 . given the inherent difficulty of controlling an intermolecular desaturation , as in the case of enzymes , a directing group ( portable desaturase ) that could be appended onto commonly encountered alcohol and amine functional groups to guide dehydrogenation of unactivated alkanes was designed . in this article , the development of a portable desaturase is presented together with a protocol for desaturation that requires no added metal salts , proceeds under mild reaction conditions and most importantly avoids overoxidation of the olefin product . three basic criteria were taken into account when designing the portable desaturase : ( 1 ) reactivity the employment of a highly reactive species capable of controlled h - abstraction ; ( 2 ) practicality short , scalable synthesis and stability ; ( 3 ) ease of installation and versatility of the resulting functionality . when considering the reactivity criterion , aryl radicals were identified as highly reactive , very short - lived intermediates ( rate of h - abstraction by aryl radicals is of the order of 10 m s ) , which have rarely been employed in c h functionalization reactions due to difficulties associated with their efficient generation and promiscuous reactivity . an underutilized tactic for generating aryl radicals is the reductive dissociation of aryl triazenes in the presence of acid and catalytic metal salts . traditionally used as protecting groups for anilines and linkers in solid - phase synthesis , aryl triazenes can be prepared in high yields from the corresponding anilines and are stable to basic , reductive and alkylating conditions , even in complex settings . in order to append an aryl triazene to alcohols and amines , a functionalized aryl sulfonyl chloride was deemed appropriate , thus leading to a triazene sulfonyl chloride ( o - tosyl triazene chloride , tzcl , 21 ) as our portable desaturase of interest . the proposed set of events for the guided desaturation is outlined in figure 2c . after installing the directing group onto the desired substrate to give b , treatment with acid in the presence of a single - electron reductant this inherently high - energy radical could abstract a proximal hydrogen atom to generate a lower energy alkyl radical d that could be oxidized to a carbocation e and finally terminated to the desired alkene f. additionally , if a suitable metal salt could participate in both the reduction and oxidation steps , a redox cycle could be envisioned to allow for catalysis . the final design element involves the directing group transforming into a simple tosylate or tosylamine at the end of the reaction , a functional group commonly employed in modern organic synthesis . the triazene sulfonyl chloride ( tzcl , 21 ) was prepared in two steps from commercial 2-bromo-5-methylaniline ( see supplementary information ) . gram - quantities of this directing group can be obtained and stored indefinitely at ambient temperature without notable decomposition . after attaching the directing group to the pilot substrate 2-cyclopentylethanol under straightforward reaction conditions ( dmap , dcm , rt , 90% yield ) to give 22 , studies toward alkane desaturation began ( table 1 ) . when considering metal catalysts for the desired desaturation reaction , copper salts were the obvious starting point due to their ability to promote the reductive dissociation of triazenes ( sandmeyer chemistry ) and to rapidly oxidize alkyl radicals to carbocations . indeed , preliminary screening demonstrated the ability of catalytic copper(ii ) bromide in the presence of tfa to provide the desired olefin 23 in 20% yield when acetonitrile was used as solvent ( table 1 , entry 1 ) ; however , this product was accompanied by the inseparable reduction ( 24 ) and sandmeyer ( 25 ) byproducts . importantly though , under these reaction conditions byproducts resulting from olefin overoxidation were not observed . switching from acetonitrile to nitromethane was key to suppressing 24 ( entry 2 ) , a byproduct which seemingly arises by h - abstraction from the solvent . during further investigations of the reaction , tempo ( 1 equiv ) was added in an attempt to trap a radical intermediate or to inhibit the reaction altogether , but it surprisingly resulted in a higher yielding transformation ( entry 3 ) . intrigued by this result , a control experiment was performed in the absence of copper salts ( entry 4 ) to reveal that tempo alone could facilitate the desaturation in slightly better yield and without the sandmeyer byproduct 25 . when no additives were employed and the reaction was run only with acid ( entry 5 ) , trace amounts of the desired product were observed , accompanied by mostly nonspecific decomposition . in the absence of acid , no reaction took place and the starting material was recovered intact ( entry 6 ) . these exciting metal - free desaturation conditions prompted further optimization of the reaction with respect to acid , temperature , concentration and nitroxide radical . using three equivalents of tfa as the acid , the temperature can be lowered to 60 c and the reaction time shortened to 1.5 hours to fully consume the starting material and to give the desired olefin 23 and along with minor amounts of 24 in 68% isolated yield ( entry 7 ) . when the stronger triflic acid ( 2 equiv ) was employed , the reaction proceeded efficiently at room temperature over 3 hours to give 23 in 54% yield ( entry 8) . increasing finally , other nitroxide radical species were tested with the hope to increase the reaction efficiency , but both the less hindered azado and the more hindered adamantyl nitroxide were inferior to the cheaper , commercially available tempo ( entries 9 and 10 ) . thus , with two sets of conditions in hand ( entries 7 and 8) , the substrate scope of the newly developed desaturation reaction system was explored . as shown in table 2 , a variety of primary and secondary alcohols are viable substrates for the guided dehydrogenation reaction , giving moderate to good yields of olefinic products . in these systems , the desaturation reaction takes place most efficiently when a tertiary carbon center is in a 1,3 relationship to the functionality carrying the portable desaturase and this selectivity implies a 1,7 h - abstraction by the intermediate aryl radical . such a process has been only briefly described in the literature , with aryl radicals usually preferring the 1,5 or 1,6 h - abstraction mode of reactivity . when simple aliphatic secondary alcohols were employed in the guided desaturation ( 2629 ) , good selectivity for tertiary alkyl positions was detected and desired alkenes were obtained as the only isolable , oxidized products in moderate yields . for these substrates , the reaction proceeded best when tfa was used as acid , whereas tfoh generally led to decomposition . on a substrate designed to test for oxidation at a tertiary alkyl site versus a benzylic site , the desaturation reaction provided the olefin product 27 ( 36% yield ) and no styrene derivatives were observed . this data does not rule out the possibility of h - abstraction at the benzylic position , but such potential products may be unstable under the reaction conditions ( vide infra ) . furthermore , because the portable desaturase favors h - abstraction at proximal tertiary alkyl positions , site - selective oxidation can be performed ( e.g. 28 ) and functional groups such as olefins are also tolerated in the reaction ( e.g. 29 ) . an ideal substrate for the guided desaturation is menthol , which cleanly gives isopulegol tosylate 33 in 92% yield in an efficient desaturation process that is accommodated by the more rigid molecular organization of the substrate . alkenol tosylates 30 and 31 were obtained in good yield as mixtures of olefin isomers due to uncontrolled elimination from the corresponding tertiary alkyl cations . cyclic substrates behave well under the desaturation conditions , giving products such as 23 and 32 in good yields ( 68% and 51% respectively ) . these substrates reiterate a limitation of the method , the formation of small amounts of reduction product ( e.g. , 24 ) that are difficult to separate from the desired alkene . notably , when applied to alcohol - derived substrates , the desaturation reaction provided only homoallylic tosylates and no allylic tosylates were isolated . however , given the modest yields obtained in certain cases ( e.g. 2629 ) , it is likely that allylic tosylates are being generated ( by elimination of the alkyl cation towards the tosylate group ) , but simply decompose during the reaction . in support of this possibility , tosic acid is always observed as a byproduct in the crude reaction mixture of the alcohol derived substrates . additionally , this mode of elimination is operative in the case of the allylic tosylamine 35b , the nitrogen analog of 26 . therefore , substrates leading to allylic tosylates ( e.g. alcohols in a 1,2 relationship to a tertiary center ) are not well suited for this method . in addition to primary and secondary alcohols , aliphatic amines are also competent substrates for the guided desaturation reaction , leading to either homoallylic ( 3437 ) or allylic tosylamines ( 38 and 39 ) . it was found that for amine - derived substrates , tfoh performs slightly better in the desaturation reaction compared to tfa . indeed , menthylamine behaves well to give 34 in 59% yield , while 35 is obtained in 60% yield as a 10:1 mixture of homoallylic ( major ) and allylic ( minor ) tosylamines . furthermore , allylic tosylamines such as 39 can be prepared selectively in good yield ( 50% ) and amino esters are selectively desaturated to provide valuable dehydroaminoesters ( 3638 ) in useful yields . with the goal of exploring this methodology in more complex settings , a series of natural product derived substrates although similar in substructure with the simpler menthol , 40 contains two equidistant , but sterically different tertiary sites available for oxidation . using procedure a , the desaturation reaction generated a 2:1 mixture of inseparable regioisomers in 46% yield ( figure 3a ) . this result suggests that h - abstraction with an aryl radical is indifferent to the steric environment around the c h bond as long as a favorable geometry for abstraction is possible . dehydroabietyl amine derivative 42 poses an interesting challenge for selective c h functionalization , given two reactive benzylic sites , a hindered tertiary ring junction and an otherwise unfunctionalized decalin system . treatment of 42 with tempo ( 1 equiv ) in the presence of tfoh ( 2 equiv ) at a temperature as low as 4 c gave two oxidation products ( figure 3b ) . the major product was the rearranged and cyclized sulfonamide 43 ( 30% yield ) , which arises by an interrupted desaturation process . meerwein rearrangement occurs to provide the more stabilized benzylic cation , which is then trapped intramolecularly by the tosylamine moiety . the minor product 44 ( 16% yield ) can be accounted for by initial abstraction from an unactivated methylene c h bond and subsequent termination to the disubstituted olefin . thus , the guided desaturation affords oxidation products at sites other than the more reactive benzylic positions that would be targeted in intermolecular oxidation processes . to demonstrate the ability to generate dienes in complex settings , triterpene derivative 45 containing a trisubstituted olefin and a free secondary alcohol 46 was obtained in 47% yield as the major product of the reaction after allylic 1,8 h - abstraction , oxidation and elimination to the most stable diene system . it is of note that the secondary alcohol remains untouched under these conditions and the sensitive diene functionality is safely contained in the final product . following the successful desaturation of aminoesters ( 36 - 38 , table 2 ) , this protocol was further tested on a more complex tetrapeptide . when 47 was subjected to the reaction conditions , the desaturated product 48 was isolated in 35% yield without racemization of any of the four chiral centers ( figure 3d ) . additionally , the portable desaturase allows for site - selective oxidation on substrates such as 47 that are distinguished by strong chelating ability , multiple sites available for oxidation and reactive functional groups . preliminary experiments to clarify both the mechanism of the reaction and the role of tempo are shown in figure 4 . the first goal was to confirm the h - abstraction event that productively leads to the olefin product through deuterium labeling ( figure 4a ) . thus , when deuterium was incorporated at the tertiary alkyl site in 49 , deuterium transfer was indeed observed in the desaturated product 50 , but the reaction is less efficient ( 34% yield for 50a , vs. 68% for 23 , see table 1 ) and a new minor product was observed that is tentatively assigned to the olefin isomer 50b . presumably the stronger deuterium carbon bond diverts the aryl radical towards the normally less reactive methylene c h bond . the second goal was to provide evidence for the formation of the aryl radical intermediate from the triazene precursor ( figure 4b ) . thus , when 51 was subjected to the reaction conditions , both cyclization and desaturation took place to give 54 in 47% yield . this result is consistent with the intermediate aryl radical adding across the alkyne to produce a reactive vinyl radical 52 , which performs a favorable 1,5 h - abstraction to generate tertiary alkyl radical 53 that is further oxidized and terminated to an olefin . thirdly , in order to clarify the role of tempo in the reaction , aniline substrate 55 was prepared and converted into the corresponding diazonium salt , a presumed intermediate in the reaction ( figure 4c ) . when tempo ( 1 equiv ) was added at room temperature , the dehydrogenation event took place and olefin 56 was isolated in 44% yield ( entry 1 ) . when the tempo loading was lowered to 0.1 equiv ( entry 2 ) , the reaction provided the product in only a slightly lower yield ( 31% , ton = 3 ) ; meanwhile , the absence of tempo led to nonspecific decomposition of the starting material ( entry 3 ) . this data indicates that tempo can support a catalytic cycle , but is inefficient under the current reaction conditions . importantly , these transformations proceed under acid - free conditions , implying that tfa is not required in the oxidation events , but merely to liberate the diazonium salt from the starting triazene . based on our experimental results and literature precedent , the proposed reaction mechanism for the tempo - mediated desaturation reaction is presented in figure 4e . tempo then promotes its reductive decomposition to aryl radical 59 , an event concomitant with the generation of n2 and the oxidation of tempo to tempo . once formed , 59 undergoes h - abstraction to generate an alkyl radical intermediate 60 which is oxidized to the corresponding carbocation 61 by tempo previously formed in the reaction . spontaneous elimination from 61 leads to the olefin product 23 , with tempo acting as a single - electron shuttle in the overall process . finally , based on the proposed mechanism , the reaction should be catalytic in tempo and preliminary results show that this is true ( figure 4c , ton = 3 ; figure 4d , ton = 2 ) , but suffers from low efficiency . a new chemical moiety , tzcl ( 21 ) , has been designed to mimic processes observed in nature , leading to desaturated aliphatics . the chemistry performed by this directing group is centered on the high reactivity of an aryl radical masked as an aryl triazene . this application expands the chemistry of the aryl radical , as it exploits this reactive intermediate as a useful tool for c h functionalization . the desaturation reaction described herein is applicable on simple substrates derived from saturated alcohols and amines , giving olefin products in a predictable fashion without any overoxidation . some of the drawbacks of this method are the modest product yields , the formation of minor amounts of inseparable reduction products and the occasional difficulties in purification . nonetheless , the guided desaturation reaction can be successfully applied in complex settings , as it shows very good functional group tolerance while leading to useful oxidized products . additionally , the intriguing reaction mechanism suggests that tempo can act as a single - electron shuttle , providing impetus to further investigate a truly catalytic version of this transformation . studies to expand the scope of this transformation and apply it in two - phase terpene total synthesis will be forthcoming .
juxtaglomerular cell tumor ( jgct ) , also known as reninoma , is an extremely rare kidney tumor of the juxtaglomerular cells that typically secrete renin . it was first described , in 1967 , by robertson et al . , and was first named , in 1968 , by kihara et al . since then , approximately 100 case reports have been published . we report a case of renin secreting tumor diagnosed after nephrectomy through histological and immunohistochemical investigations . a 35-year - old woman who suffered from hypertension for 3 years that was poorly controlled by medical treatment with angiotensin converting enzyme ( ace ) inhibitors laboratory tests showed hypokalemia with a potassium level of 2.63 mmol / l ( normal range 3.6 - 5 abdominal computed tomography revealed a low - density tissue lesion indicating a tumor in the right kidney [ figure 1b ] . a ) renal ultrasonograpy shows a round cortical lesion of the right kidney ( arrow ) . b ) abdominal computed tomography reveals a low - density tissue lesion ( arrow ) indicating tumor in the right kidney . in the pathological examination , the surgical specimen showed a yellow tan color and mid - renal lesion ( 4 cm in diameter ) . microscopically , the tumor consisted of sheets of polygonal and spindle shaped cells with a central regular nucleus . a complex vascular hemangiopericytic pattern and rare tubular elements entrapped were also present [ figure 2 ] . 35-year - old woman with uncontrolled hypertension diagnosed with juxtaglomerular cell tumor . hematoxylin and eosin stained tumor tissue - a ) at 10 magnification shows sheets of polygonal and spindle - shaped cells with a complex vascular hemangiopericytic pattern and b ) at 40 magnification , shows the tumor cells with eosinophilic cytoplasm and a central regular nucleus . in the immunohistochemical investigation , the tumor cells were found to be immunoreactive for renin , vimentin , and cluster of differentiation molecule 34 ( cd34 ) [ figure 3 ] . immunostaining of the tissue demonstrates tumor cells positive for - a ) vimentin and b ) renin . the first example of a jgct was reported in 1967 by roberston et al . , who described a young male patient with hypertension and a small renal neoplasm . , reported a similar neoplasm in a young female patient and proposed the term juxtaglomerular cell tumor . the majority of these patients are women between the ages of 20 and 30 years . the diagnosis of jgct is usually suspected in patients with uncontrolled hypertension , marked hypokalemia , and hyperaldosteronism , although one patient had normal blood pressure . in our case , tests to determine aldosteronism were not conducted and the final diagnosis was made after surgery based on histological features . in such a context , radiological studies are often helpful in ruling out other causes of hypertension . renal arteriography shows the majority of jgct to be hypervascular and helps to rule out renal artery stenosis . computed tomography shows a hypovascular and solid mass in most cases . renal vein catheterization and selective measurement of renin values may help to detect small tumors . grossly , jgct is a solid , well - circumscribed lesion that has a yellow tan color . the tumor is usually smaller than 3 cm in diameter , but cases ranging from 2 mm to 9 cm have been reported . histologically , it is made of sheets of polygonal or spindle shaped tumor cells with central round regular nuclei , distinct cell borders , and abundant granular eosinophilic cytoplasm . typically , tumors have a complex vascular hemangiopericytic pattern . papillary architecture , microcystic pattern , and prominent tubular elements , either neoplastic or entrapped , are also present . marked nuclear atypia and mitotic activity are uncommon . it is important to distinguish these morphological features from those seen in glomus tumor , hemangiopericytoma , metanephric adenoma , papillary renal cell carcinoma , collecting duct carcinoma , urothelial carcinoma , renal epithelioid angiomyolipoma , and wilms tumor . in these cases , immunohistochemical investigation and ultrastructural studies using electronic microscopy ultrastructural features include abundant rough endoplasmic reticulum , a well - developed golgi apparatus , and numerous peripherally located sharply angulated rhomboid renin protogranules . majority of the cases with jgct are benign , and neither local recurrence nor metastasis has occurred after either radical or partial nephrectomy . the complete tumor resection by radical or partial nephrectomy is the best modality for jgcts . antihypertensive agents should be the treatment for hypertension until accurate diagnosis is made . blood pressure and plasma renin level usually normalize after nephrectomy . in summary , primary jgct is a distinct mesenchymal tumor entity distinct from other renal tumors . there are only a few genetic studies of jgct because of the rarity of this disease . it is a benign tumor , but may affect the vital prognosis due to complications of associated hypertension .
preterm birth ( ptb ) is a leading cause of neonatal morbidity and mortality , and it is well recognized that the risk decreases with increasing gestational age of delivery . the benefits of antenatal corticosteroid ( acs ) administration to patients at risk of delivery < 34 weeks to mitigate the risks of prematurity have been well researched [ 1 , 2 ] . as a result , the american congress of obstetricians and gynecologists ( acog ) recommends acs to patients between 24 and 33 6/7 weeks at risk of ptb [ 2 , 3 ] . they specifically , recommend either two 12 mg doses of betamethasone given 24 hours apart or four 6 mg doses of dexamethasone given 12 hours apart to reduce the incidence of respiratory distress syndrome , intraventricular hemorrhage , necrotizing enterocolitis , and/or neonatal death [ 2 , 3 ] . late - preterm infants , delivered 3436 6/7 weeks , are at less risk of adverse outcomes compared to infants delivered at earlier gestational ages . despite earlier beliefs that outcomes for these infants were not much different from term infants and that fetal lungs reach surfactant maturity by 34 weeks , recent evidence has revealed that late - preterm infants face significantly greater risk of adverse respiratory outcomes compared to term infants [ 69 ] . nevertheless , there are minimal data to suggest whether acs might benefit patients who need delivery at 3436 6/7 weeks . as a result , given the prevalence of acute respiratory morbidity between 34 and 36 6/7 weeks and the biologic plausibility for steroids to be beneficial at these gestational ages , our objective was to use decision - analytic and cost - effectiveness analysis to determine whether acs is cost - effective in late - preterm infants at risk of delivery . due to the decreasing prevalence of acute respiratory morbidity with increasing gestational age , we hypothesized that the preferred strategy for infants < 36 weeks would include acs while the preferred strategy for infants 36 weeks would not . decision - analytic and cost - effectiveness analysis is designed to assess the costs and utilities of alternative strategies compared to a model of usual practice . although absent of patient - level data , this form of analysis integrates existing data from the literature in an effort to make population - level healthcare decisions . prior to performing our analyses we constructed a decision tree a visual tool to illustrate the relationship between possible treatment strategies and outcomes ( figure 1 ) . three separate decision trees were created , one to represent each gestational age week during the late - preterm period ( 34 , 35 , and 36 weeks ) . models were constructed for women with singleton pregnancies who present and subsequently deliver within the same gestational age week . the choice node of each tree involved the decision of whether or not to administer acs to patients at risk of ptb , as the latter represents the currently accepted standard of care at these gestational ages . in either strategy , we assumed that the infant might or might not experience acute and/or chronic respiratory disease . furthermore , we assumed that the infant might also experience nonrespiratory outcomes including adverse neurodevelopment , death , or health . we chose these outcomes because we believed that these were the most medically relevant outcomes to late - preterm infants and because there are data in the literature to support that acs could mitigate their occurrence . adverse maternal outcomes that might occur secondary to acs including fever requiring antibiotics , admission to the intensive care unit , hypertension , and/or side - effects of treatment were not included in the models because they were thought to be very rare events and not likely due to the administration of acs alone . furthermore , other potential adverse neonatal outcomes that may occur with acs including decreased weight , height , head circumference , hypothalamic - pituitary - adrenal axis function , educational attainment , and/or intellectual parameters , increased blood pressure and/or cholesterol , or visual and/or hearing impairments were not included based upon their insignificant association with acs . we conducted a literature review to estimate the probabilities of relevant neonatal outcomes , namely , acute and/or chronic respiratory disease , adverse neurodevelopment , death , or health , both with and without acs ( tables 1 and 2 ) . we defined acute respiratory disease as a diagnosis of respiratory distress syndrome ( rds ) and estimated the baseline prevalence of rds among infants delivered at 34 weeks [ 2 , 6 , 12 ] . probabilities and relative risks ( rr ) of acute respiratory disease among infants not exposed to corticosteroids were estimated using data from a multicenter observational study of more than 20,000 neonates who were not routinely administered corticosteroids . to find gestational age - specific relative risks of rds among infants exposed to corticosteroids we used data from the 2010 cochrane review on antenatal corticosteroids which provided information on the risk of rds in infants exposed to corticosteroids at various gestational ages compared to infants not exposed to corticosteroids . we specifically , used data for babies born 34 weeks to represent 34-week deliveries , babies born < 36 weeks to represent 35-week deliveries , and babies born 36 weeks to represent 36-week deliveries . the baseline prevalence of chronic respiratory disease was estimated by using the prevalence of current asthma . since gestational age - specific data for chronic respiratory disease was not available , we used the relative risk of chronic respiratory disease associated with acs exposure in all babies to estimate the risk at all gestational ages . we then determined the baseline prevalence estimates as well as probabilities and relative risks of long - term , nonrespiratory childhood outcomes [ 15 , 16 ] . infant mortality rates were estimated using 2009 united states center for disease control data ( 6.42 infant deaths/1000 live births ) . the rate of significant neurodevelopmental delay was estimated by using the prevalence of cerebral palsy from 19912000 new england data of singleton infants ( 2.46 cases of cerebral palsy/1000 neonatal survivors ) . because gestational age - specific relative risks were not available for death or neurodevelopmental delay in childhood , we used the relative risk of each outcome associated with acs exposure in all babies to estimate the risk at all gestational ages . we used information derived from the literature to determine the utility in quality - adjusted life years ( qalys ) and duration of effect of the various neonatal outcomes ( table 3 ) . a health utility value , ranging from 0 ( death ) to 1 ( perfect health ) , was assigned based upon published data quantifying perceived preferences for various health states [ 15 , 17 , 20 , 21 , 24 ] . qalys were computed by multiplying the duration of effect ( anticipated number of life years in each health state ) by the utility associated with that health state . utilities for neonatal outcomes were estimated based upon a study of over 4,000 parent and guardian preferences of various pediatric morbidities . because the utility of a diagnosis of rds has not specifically been previously studied , we decided that 10-day intensive care unit admission was an acceptable alternative since infants with rds are managed in the neonatal intensive care unit . applied utilities for both chronic respiratory disease and adverse neurodevelopment were those for moderate persistent asthma and moderate cerebral palsy , respectively . the duration of effect of acute childhood illness was estimated based upon latency to 40 weeks gestation ( 6 , 5 , and 4 weeks duration for 34 , 35 , and 36 week infants , resp . ) . the duration of effect of child health , chronic lung disease , and death in childhood were estimated to be 78.2 years based upon 2009 life expectancy data . duration of effect of neurodevelopmental delay in childhood was estimated to be 17 years based upon data describing the life expectancy for patients with cerebral palsy . finally , we estimated the costs in us dollars of various neonatal outcomes as well as the cost of potential medical interventions [ 1820 , 22 , 23 ] . of note , we estimated that the cost of acs was more than simply the cost of the medication . instead , we assumed that patients who would receive acs like most patients < 34 weeks who currently receive acs would require inpatient management due to either their risk of relatively imminent spontaneous ptb or their need for medical attention prior to iatrogenic ptb . thus acs potentially doubles the total length of hospital stay compared to a patient without acs who has an uncomplicated spontaneous vaginal delivery and is discharged on postpartum day two [ 22 , 23 ] . all cost estimates were made from a single payer perspective and adjusted to 2011 us dollars . when more than one data source was available to describe a health state , the point estimate was calculated as a weighted mean by sample size and the range was represented by the lowest and highest values from published sources . when estimates were derived from a single source , the range was defined by the 95% confidence interval that was calculated from the binomial distribution . we performed base - case cost - effectiveness analysis to calculate incremental cost - effectiveness ratios ( icers ) to compare strategies within each gestational age week . since many infants will deliver prior to the completion of 48 hours of steroid prophylaxis at these gestational ages , we next evaluated the cost effectiveness of steroid administration if a full course was not administered . we adjusted our estimates and repeated our base - case analyses to determine the preferred strategy associated with receiving only a partial course of steroids at each gestational age week of delivery . we assumed that one dose of steroids resulted in only a 50% reduction in the rate of complications and was associated with a 50% reduction in hospital costs . we then studied a hypothetical cohort of late - preterm , singleton infants to determine both the number of adverse events that could be prevented as well as the cost that could be saved with the administration of acs to all patients at risk of delivery at a particular gestational age . univariable sensitivity analyses were performed to determine whether varying each estimate of probability , utility , and/or cost impacted which strategy was considered optimal . finally , we replaced point estimates for each probability variable with beta distributions ( due to the dichotomous nature of all probabilities in the model ) and point estimates for each cost and utility variable with normal distributions . then , 10,000-iteration second - order monte carlo simulation ( probabilistic sensitivity analysis ) was performed to simultaneously and randomly vary all variables across their plausible ranges to determine over what range of values we could be 95% confident that one strategy was of good value compared to the alternative . these calculations were also used to determine how frequently the optimal strategy was consistent with the results of the base - case analysis . for all analyses , we chose the standard and accepted threshold of $ 100,000/qaly as the willingness - to - pay threshold for all models . therefore , it was considered exempted by the institutional review board at the hospital of the university of pennsylvania . at 34 , 35 , and 36 weeks of gestational age , the administration of acs to patients at risk of imminent ptb was the more cost - effective strategy and was dominant compared to not administering acs to those at risk of ptb ( table 4 ) . given that many infants will be delivered prior to the completion of 48 hours of acs prophylaxis , we then adjusted our estimates and repeated our base - case analyses to determine the preferred strategy associated with receiving only a partial course of acs at each gestational age week of delivery . for late - preterm infants , the administration of a partial course of acs with labor was not cost - effective at any gestational age week ( icer $ 131,233.39/qaly , $ 133,117.42/qaly , and $ 133,654.76/qaly at 34 , 35 , or 36 weeks , resp . ) . next , we studied a hypothetical cohort of late - preterm , singleton infants to determine both the number of acute ( table 5 ) and chronic ( table 6 ) adverse events that could be prevented as well as the cost that could be saved with administering acs to all late - preterm patients at risk of delivery . based on 2008 united states census data , the live birth rate is approximately 4.25 million infants per year of which 7.8% are late - preterm singletons . therefore , there are approximately 331,500 late - preterm singleton infants born annually in the united states . under our base - case assumption , if each infant in this hypothetical cohort received acs , the rate of rds would decrease by approximately 50% for both 34 and 35 week infants ( from 14,498 to 7,072 cases at 34 weeks and from 7,072 to 3,536 cases at 35 weeks ) and by 40% ( from 3,536 to 2,122 cases ) for 36 week infants . while this would generate savings of approximately $ 32 million for 34 and 35 week infants , it would be associated with a loss of $ 3.4 million for 36-week infants due to the relatively greater cost of hospitalization associated with universal steroid therapy compared with the cost saved by reducing acute respiratory disease at this gestational age given its low prevalence ( table 5 ) . finally , acs could also potentially save $ 166.2 million annually due to a reduction in chronic medical comorbidities ( table 6 ) . one - way sensitivity analyses were performed for all probabilities , costs , and utilities in each gestational age week tree . despite variations in all variables associated with acute respiratory disease across their plausible ranges , however , when sensitivity analyses were performed with variables associated with long - term adverse outcomes across their plausible ranges , all models were sensitive to changes in variables associated with chronic respiratory disease such that small changes in probabilities and utilities resulted in an efficacy of treatment that no longer justified the cost . at 34 , 35 , and 36 weeks , specifically if the probability of chronic respiratory disease with acs was 6.45% , or the probability of chronic respiratory disease without acs was 6.60% , or the utility associated with chronic respiratory disease was 94.0% , then the efficacy of treatment with acs was no longer sufficient to justify the cost and the icer exceeded the willingness - to - pay threshold . ten - thousand iteration second - order monte carlo simulation ( probabilistic sensitivity analysis ) was performed to simultaneously and randomly vary all acute and chronic disease distributions across their plausible ranges to determine for which values we could be 95% confident that one strategy was of good value compared to the alternative . at 34 , 35 , and 36 weeks , acs was the dominant strategy resulting in greater effectiveness for < $ 100,000/qaly only 47.35% , 47.33% , and 46.98% of the time , respectively . furthermore , at each gestational age week there was no willingness - to - pay threshold for which we could be 95% confident that the two therapies acs administration and nonadministration differed significantly in value . when analyses were restricted to only those distributions associated with acute respiratory disease , we found that in 100% of the 10,000 iterations , acs was the dominant strategy resulting in greater effectiveness for < $ 100,000/qaly at 34 , 35 , and 36 weeks gestational age . furthermore , we could be 95% confident that acs represented good value compared to the alternative strategy of not administering acs for all willingness - to - pay thresholds > $ 64,677 , > $ 65,700 , and > $ 65,819 at 34 , 35 , and 36 weeks , respectively . to date , we are the first group to approach the question of whether to administer acs to late - preterm infants at risk of ptb through a decision - analytic and cost - effectiveness analysis . our findings suggest that , contrary to our hypothesis regarding the cost - effectiveness of steroids at 36 weeks , under our base - case assumptions , a full course of acs is cost - effective for patients 3436 weeks at risk of ptb . furthermore , our sensitivity analyses and monte carlo simulations confirm that , when the goal is to reduce acute respiratory disease , acs is the dominant strategy . a review of the literature reveals a single brazilian randomized controlled trial from 2011 in which 320 women with late - preterm pregnancies at risk of imminent ptb were randomized to receive acs or placebo . although powered to see a 50% difference between exposure groups , acs did not reduce the incidence of respiratory disorders in late - preterm infants . however , the authors acknowledge that the results of their small , single - centered trial should not be interpreted in isolation . instead , they recommend the incorporation of their findings into future meta - analyses and cochrane reviews to draw more robust conclusions this manner of data modeling allows for the generation of pretrial data without incurring the cost or time associated with a clinical study . furthermore , through sensitivity analysis and monte carlo simulation , one can determine whether modifications in point estimates that might occur in various clinical scenarios and/or patient populations could alter the preferred strategy . our study was not without limitations . by nature of the decision - analytic model , assumptions regarding probability , utility , and cost of various disease states must be made . the strength of the decision - analytic model and the validity of the results are intrinsically associated with the accuracy of these assumptions . while the quality of the literature we reviewed reassured us regarding the accuracy of our assumptions as a result , we had to assume that the point estimate referred to infants of all gestational ages . furthermore , the data used to estimate the probabilities of acute respiratory disease among late - preterm infants exposed to acs included data from steroid administration that did not occur immediately prior to delivery . however , if the mechanism of action of acs in late - preterm infants is similar to that of preterm infants < 34 weeks , this will bias our results towards the null . in our model , all patients were assumed to deliver within the gestational age week in which they presented and all health states and disease pathways were seen as absolute , which we recognize may not reflect real - life disease processes . finally , although decision analysis yields results in a cost to benefit ratio , this value may not be what determines whether an intervention is deemed medically appropriate on an individual or population level . while awaiting the results of the ongoing , large , multicenter , maternal - fetal medicine units network trial ( alps trial - antenatal late preterm : randomized placebo - controlled trial ) , our findings suggest that acs to patients at risk of delivery between 34 and 36 weeks could significantly reduce both the cost and acute morbidity associated with late - preterm births .
porcelain - fused - to - metal ( pfm ) restorations are the most commonly used crowns due to high strength and accuracy of the cast metal and esthetic properties of the porcelain . factors affecting the bracket bond strength to enamel have been extensively studied and include enamel surface preparation techniques , adhesive systems , and bracket - related factors such as the size and design of bracket base . some studies confirm the effect of bracket base design on bond strength to enamel while some others have rejected this theory . type of porcelain surface treatment , the adhesive system used and size and design of bracket base are believed to affect the bracket bond to porcelain . there is a gap of information about the effect of bracket base design on the bond strength of bracket to porcelain . thus , this in vitro study sought to assess the effect of bracket base design on the shear bond strength ( sbs ) of the bracket to feldspathic porcelain . bond strength of brackets with four different base designs to feldspathic porcelain was evaluated . considering the type i error of 0.05 and type ii error of 0.2 ( power of 80% ) , the sample size was calculated to be 10 specimens in each group ( a total of 40 ) . mandibular premolars of both quadrants on a mandibular dentate model ( nissin , tokyo , japan ) received the standard preparation for pfm crowns . each type of bracket was randomly allocated to one group , and brackets were bonded to pfm crowns under similar conditions . the four types of brackets used were as follows : discovery stainless steel brackets with laser - structured base ( dentaurum , ispringen , germany)mini master stainless steel brackets with photochemically micro - etched base ( american orthodontics , wi , usa)mini twin stainless steel bracket with maximum injection molding base ( henry schein orthodontics , ca , usa)4radiance ceramic bracket with patented radiance mechanical lock base ( american orthodontics , wi , usa ) . discovery stainless steel brackets with laser - structured base ( dentaurum , ispringen , germany ) mini master stainless steel brackets with photochemically micro - etched base ( american orthodontics , wi , usa ) mini twin stainless steel bracket with maximum injection molding base ( henry schein orthodontics , ca , usa ) 4radiance ceramic bracket with patented radiance mechanical lock base ( american orthodontics , wi , usa ) . the porcelain surface was first cleaned with a brush and then completely dried . next , 9% hydrofluoric acid ( porcelain etch , ultradent , ut , usa ) was applied to the buccal surface of crowns using an applicator . after 2 min , the etchant was removed by rinsing with water , and the surface was air dried for 15 s using air spray to yield a porous , chalky porcelain surface . using the sweeping motion of an applicator , silane ( ultradent porcelain repair kit , usa ) was applied to the porcelain surface . self - cure composite resin ( 3 m unitek , usa ) was applied to the posterior surface of the bracket in 1 mm thickness according to the manufacturer 's instructions , and the bracket was placed on the porcelain surface using 150 n load measured by an orthodontic gauge ( dentaurum , ispringen , germany ) . before composite polymerization , the excess composite resin was removed using the sharp tip of an explorer . crowns on their respective dies were embedded in auto polymerizing acrylic resin ( meliodent , germany ) [ figure 1 ] . crowns on their respective dies embedded in acrylic blocks all specimens were thermocycler ( dorsa , tehran , iran ) for 5000 cycles at 555c with 20 s of dwell time in each water bath and 20 s of transfer time . next , the specimens were stored in an incubator ( pars azma , tehran , iran ) at 37c and 100% humidity for 24 h. the bracket - porcelain sbs was measured using utm instron machine ( zwick roell z020 , germany ) . the load was applied at a crosshead speed of 1 mm / min to the bracket - porcelain interface . to apply a load , each specimen was fixed by clamps to maintain the bonding interface parallel to the blade . load was applied until fracture , and the load at fracture was recorded in newton ( n ) . to convert the sbs values to megapascals ( mpa ) , the load in n was divided by the cross - sectional area of the bracket base ( m ) provided by the manufacturers ( 12.93 mm for discovery , 11.04 mm for mini master , 9.42 mm for mini twin , and 13.94 mm for radiance ) . after separation of brackets from the porcelain , the bracket base and porcelain surface were examined under a stereomicroscope ( olympus szx9 research stereomicroscope system , japan ) at 10 magnification . to assess the adhesive remnant index ( ari ) describing the amount of adhesive remained on the surface , the classification of bordeaux et al . wilk test was used to assess the normality of the sbs data and revealed that the data had a normal distribution . levene 's test was used to assess the equality of variances for sbs values and confirmed it . one - way anova was applied for overall comparison of groups while the tukey 's honest significant difference ( hsd ) test was used for pairwise comparisons . type i error was considered as 0.05 and p < 0.05 was considered statistically significant [ figure 2 ] . mandibular premolars of both quadrants on a mandibular dentate model ( nissin , tokyo , japan ) received the standard preparation for pfm crowns . each type of bracket was randomly allocated to one group , and brackets were bonded to pfm crowns under similar conditions . the four types of brackets used were as follows : discovery stainless steel brackets with laser - structured base ( dentaurum , ispringen , germany)mini master stainless steel brackets with photochemically micro - etched base ( american orthodontics , wi , usa)mini twin stainless steel bracket with maximum injection molding base ( henry schein orthodontics , ca , usa)4radiance ceramic bracket with patented radiance mechanical lock base ( american orthodontics , wi , usa ) . discovery stainless steel brackets with laser - structured base ( dentaurum , ispringen , germany ) mini master stainless steel brackets with photochemically micro - etched base ( american orthodontics , wi , usa ) mini twin stainless steel bracket with maximum injection molding base ( henry schein orthodontics , ca , usa ) 4radiance ceramic bracket with patented radiance mechanical lock base ( american orthodontics , wi , usa ) . next , 9% hydrofluoric acid ( porcelain etch , ultradent , ut , usa ) was applied to the buccal surface of crowns using an applicator . after 2 min , the etchant was removed by rinsing with water , and the surface was air dried for 15 s using air spray to yield a porous , chalky porcelain surface . using the sweeping motion of an applicator , silane ( ultradent porcelain repair kit , usa ) self - cure composite resin ( 3 m unitek , usa ) was applied to the posterior surface of the bracket in 1 mm thickness according to the manufacturer 's instructions , and the bracket was placed on the porcelain surface using 150 n load measured by an orthodontic gauge ( dentaurum , ispringen , germany ) . before composite polymerization , the excess composite resin was removed using the sharp tip of an explorer . crowns on their respective dies were embedded in auto polymerizing acrylic resin ( meliodent , germany ) [ figure 1 ] . all specimens were thermocycler ( dorsa , tehran , iran ) for 5000 cycles at 555c with 20 s of dwell time in each water bath and 20 s of transfer time . next , the specimens were stored in an incubator ( pars azma , tehran , iran ) at 37c and 100% humidity for 24 h. the bracket - porcelain sbs was measured using utm instron machine ( zwick roell z020 , germany ) . the load was applied at a crosshead speed of 1 mm / min to the bracket - porcelain interface . to apply a load , each specimen was fixed by clamps to maintain the bonding interface parallel to the blade . load was applied until fracture , and the load at fracture was recorded in newton ( n ) . to convert the sbs values to megapascals ( mpa ) , the load in n was divided by the cross - sectional area of the bracket base ( m ) provided by the manufacturers ( 12.93 mm for discovery , 11.04 mm for mini master , 9.42 mm for mini twin , and 13.94 mm for radiance ) . after separation of brackets from the porcelain , the bracket base and porcelain surface were examined under a stereomicroscope ( olympus szx9 research stereomicroscope system , japan ) at 10 magnification . to assess the adhesive remnant index ( ari ) describing the amount of adhesive remained on the surface , the classification of bordeaux et al . was used . the shapiro wilk test was used to assess the normality of the sbs data and revealed that the data had a normal distribution . levene 's test was used to assess the equality of variances for sbs values and confirmed it . one - way anova was applied for overall comparison of groups while the tukey 's honest significant difference ( hsd ) test was used for pairwise comparisons . type i error was considered as 0.05 and p < 0.05 was considered statistically significant [ figure 2 ] . comparison of the mean sbs values with one - way anova revealed statistically significant differences among the four groups ( p < 0.001 ) . pairwise comparison of groups with tukey 's hsd test revealed that groups one , two , and four were not significantly different regarding the mean sbs while the mentioned three groups had significantly higher mean sbs values than group 3 ( p < 0.001 for all three ) . the highest mean sbs values belonged to groups 1 ( 11.61 mpa ) , 4 ( 11.38 mpa ) , and 2 ( 10.68 mpa ) , respectively , which were not significantly different . group three had the lowest mean sbs ( 6.16 mpa ) , which was significantly lower than the corresponding values in the remaining three groups ( p < 0.001 ) [ table 2 ] . the shear bond strength values in the four groups comparison of the shear bond strength values among the four groups using tukey 's honest significant difference test the results of the ari scores in the four groups based on the fisher 's exact test are shown in table 3 . the ari scores were significantly different among the four groups ( p < 0.001 ) . as seen in table 3 , type iii ari was the most frequent in groups one and two . however , in groups three and four , types i and ii indicative of bracket - adhesive and mixed failures , respectively were more common [ figure 3 ] . adhesive remnant index scores in the four groups according to the fisher 's exact test electron micrograph of discovery bracket base following laser irradiation comparison of the mean sbs values with one - way anova revealed statistically significant differences among the four groups ( p < 0.001 ) . pairwise comparison of groups with tukey 's hsd test revealed that groups one , two , and four were not significantly different regarding the mean sbs while the mentioned three groups had significantly higher mean sbs values than group 3 ( p < 0.001 for all three ) . the highest mean sbs values belonged to groups 1 ( 11.61 mpa ) , 4 ( 11.38 mpa ) , and 2 ( 10.68 mpa ) , respectively , which were not significantly different . group three had the lowest mean sbs ( 6.16 mpa ) , which was significantly lower than the corresponding values in the remaining three groups ( p < 0.001 ) [ table 2 ] . the shear bond strength values in the four groups comparison of the shear bond strength values among the four groups using tukey 's honest significant difference test the results of the ari scores in the four groups based on the fisher 's exact test are shown in table 3 . the ari scores were significantly different among the four groups ( p < 0.001 ) . as seen in table 3 , type iii ari was the most frequent in groups one and two . however , in groups three and four , types i and ii indicative of bracket - adhesive and mixed failures , respectively were more common [ figure 3 ] . adhesive remnant index scores in the four groups according to the fisher 's exact test electron micrograph of discovery bracket base following laser irradiation the purpose of this study was to compare the sbs of metal and ceramic brackets with different base designs to porcelain crowns and showed that the discovery bracket had the highest mean sbs followed by the radiance ceramic bracket and mini master bracket . mini twin bracket had the lowest mean sbs , showing significant differences with the values in other groups . this confirms the effect of bracket base design on sbs to porcelain . in the fabrication process of discovery brackets , the base surface is selectively melted in a controlled fashion by laser irradiation in the final step of fabrication causing droplets of variable sizes . these structures are probably responsible for high sbs of this bracket to porcelain demonstrated in this study . the manufacturing process of radiance ceramic bracket is similar to that of other monocrystalline ceramic brackets . for the fabrication of mini master brackets , 80-gauge mesh is placed over an etched foil base and is subjected to photochemical etching . mini twin brackets , with the lowest sbs in this study , are fabricated as a single mass using maximum injection molding technique and then the base is subjected to micro etching to provide retention . however , in all four groups , the mean sbs values were higher than the minimum required bond strength value of 68 mpa and thus , they may be safely used in the clinical setting . the ari scores in this study confirmed our sbs results . in groups one , two , and four ( almost ) with the stronger bond between bracket and composite , the ari score was mostly type iii . in contrast , in group three with the lowest sbs , fractures were mostly type i and at the bracket - adhesive interface . as observed in this study , ceramic brackets had high - bond strength comparable to that of metal brackets , which is in line with the results of reddy et al . , and stumpf ade et al . samruajbenjakul andkukiattrakoon , in 2009 , evaluated the effect of the base design of ceramic and metal brackets on the sbs to feldspathic porcelain . they showed that bracket base design significantly affected the sbs , which confirms present findings despite some differences between their study and the present study . bishara et al . , in 2004 , assessed the sbs of two brackets with single and double mesh bases to enamel in vitro and revealed no significant difference in bond strength . they showed that the difference between the two base designs did not affect the bond strength . evaluated the effect of base size and mesh size on sbs in an in vitro study and reported no significant difference between brackets with different base sizes and similar mesh size . since this was an in vitro study , further clinical studies are required to better elucidate this subject . the bracket base design significantly affects the sbs of brackets to feldspathic porcelain , and debonding characteristics are related to bracket base design .
the study was approved by the medical ethics committee of jinling hospital and adhered to the tenets of the declaration of helsinki . all donor tissues were obtained within 12 h after enucleated eyeballs from healthy young adults with accidental death without any disease . twenty eyes of 17 patients , who are with moderate to advanced keratoconus without acute keratoconus , the corneas were clear and there was no edema in dm , were enrolled in this study . the patients were divided into femtosecond laser - assisted dalk with 75% of stromal dissection ( predescemetic group ) and using big - bubble technique with total stromal resection ( descemetic group ) . there were 10 eyes of 9 patients ( 8 males and 1 female ) with mean age of 29.4 8.8 years ( range , 2042 years ) in predescemetic group and 10 eyes of 8 patients ( all males ) with mean age of 20.6 6.4 years ( range , 1532 years ) in descemetic group . all treated eyes were examined preoperatively and postoperatively in terms of the uncorrected visual acuity ( ucva ) and best - corrected visual acuity ( bcva ) with the standard logarithm of the minimum angle of resolution chart . anterior segment optical coherence tomography ( oct ) ( carl zeiss meditec , jena , germany ) combined with the 50-mhz ultrasound corneal pachymetry was used to measure the thinnest corneal thickness in the recipient cornea . slit - lamp examination , endothelial cell density ( ecd ) , corneal topography , and dilated fundus examination in dilated pupil were also evaluated . the preoperative data in two groups all procedures of the two groups were performed by a sole fully qualified and highly experienced surgeon ( zhen - ping huang ) . the whole eyeball was placed in the eyeball fixation ( which we previously described ) and received the treatment with the 500-khz visumax femtosecond laser ( carl zeiss meditec ag , jena , germany ) . in the predescemetic group , the mean donor lenticule thickness and diameter were 399 55.27 m ( range , 300510 m ) and 7.73 0.21 mm ( range , 7.38.0 mm ) , respectively ; the mean recipient lenticule thickness , lenticule diameter , and residual stromal bed thickness were 309.5 53.87 m ( range , 230380 m ) , 7.54 0.16 mm ( range , 7.37.8 mm ) , and 84.4 8.67 m ( range , 74105 m ) , respectively . the recipient corneal button was lifted , and the donor lenticule was placed on the recipient residual corneal stromal bed and sutured by continuous or 16 interrupted stitches with 10 - 0 nylon sutures under retrobulbar anesthesia . in descemetic group , we use the laser settings in pkp mode for the donor and recipient to achieve the vertical side cut according to the thinnest corneal thickness . the mean diameter of lamellar was 7.52 0.14 mm ( range , 7.47.8 mm ) in the donor and 7.38 0.10 mm ( range , 7.27.5 mm ) in the recipient . after femtosecond laser procedure , the patient was transferred to the operating room with retrobulbar anesthesia . a 27-gauge needle bent into 60 with a 5 ml syringe filled with air was inserted into the deep stroma . air was then injected into the stroma to achieve a big bubble between the dm and posterior stroma . the anterior chamber paracentesis was performed to lower the intraocular pressure , and a small air bubble was injected into the anterior chamber to check the baring of dm . then , a crescent disposable knife was used to cut the corneal stroma into half , and an ophthalmic viscosurgical device was used to refill the space and protect the dm . the residual stroma was excised by corneal scissors to expose the smoothly transparent dm . after peeling off dm and endothelium using forceps from the donor lamella , prepared by femtosecond laser , the donor lamella was placed on the host stromal bed and sutured by 16 interrupted stitches with 10 - 0 nylon sutures . the antibiotics and corticosteroids of intravenous injection were used once a day for 3 days . tobramycin dexamethasone and ofloxacin eye drops were applied six times and four times / day for 2 weeks , respectively . fluorometholone and cyclosporine a eye drops were instilled three times / day for 3 months to prevent graft rejection . statistical analysis was performed with spss software ( version 17 , spss , inc . , data were calculated as mean standard deviation differences of bcva , myopia , corneal astigmatism , keratometry , central corneal thickness ( cct ) , and ecd between two groups were analyzed using two independent samples t - test . all procedures of twenty eyes were uneventful while one eye has encountered intraoperative dm microperforation in the predescemetic group without converted to pkp operation intraoperatively . 1a ] in the descemetic group , and the regular surface of predescemetic plane was detected in the predescemetic group [ fig . alignment of dm with the donor tissue was much better in the descemetic group at day 2 postoperatively [ fig . the clear and smooth descemet 's membrane was observed after stromal removal in descemetic group . ( b ) the regular surface of predescemetic plane was detected in predescemetic group the excellent alignment of descemet 's membrane with the donor tissue was observed through anterior segment optical coherence tomography at 2 days after surgery in descemetic group the alignment of descemet 's membrane with the donor tissue was detected through anterior segment optical coherence tomography at day 2 postoperatively in predescemetic group the postoperative parameter between two groups was shown in table 2 . the differences of bcva , corneal astigmatism , corneal curvature , cct , and loss rate of ecd between two groups were not statistically significant ( all p > 0 . 05 ) while the loss rate of ecd was slightly higher in the descemetic group ( 6.4% ) than the predescemetic group ( 4.6% ) . however , the mean postoperative myopia was 9.43 7.44 diopter ( d ) in the predescemetic group and 1.03 1.13d in the descemetic group , which was statistically significant ( p = 0.002 ) . the postoperative data in two groups at the last examination in term of postoperative complications , there was one patient with wound dehiscence in the predescemetic group , which has been resutured and one eye with stromal rejection , which has been treated with the application of topical steroids ; there was one eye with high intraocular pressure postoperative induced by the application of steroid in descemetic group , which has been treated by medical therapy . it has gained widespread acceptance that lamellar keratoplasty ( lk ) is necessary for the treatment of moderate to advanced keratoconus , and the acute advanced keratoconus with ruptured dm is required to be treated with pkp . the important benefit from lk is that it can decrease the risk of endothelial rejection . in the previous study , the ucva and bcva have been improved by the femtosecond laser - assisted predescemetic lk for the patients with moderate to advanced keratoconus . as the stroma is unhealthy for the patients with keratoconus , we performed the femtosecond laser - assisted descemetic lk with big - bubble technique for the treatment of keratoconus . dalk with big - bubble technique was first reported by anwar and teichmann in 2002 , which the 60%80% thickness of the cornea was removed first and injected air between deep stroma and dm to separate the cornea with the advantages of shortening the surgical time , reducing the risk of perforation , and exposing the smooth surface . in contrast to anwar 's technique , we use the 500-khz visumax femtosecond laser to cut the donor and host peripheral cornea with regular and smooth incision . our results indicate that the differences of bcva , corneal astigmatism , keratometry , cct , and the loss rate of ecd between two groups are not statistically significant ( all p > 0 . the results of postoperative bcva are similar to results by abdelkader and kaufman , sarnicola et al . , and schiano - lomoriello et al . , which have indicated that no difference in bcva between the predescemetic and descemetic groups is observed , while the visual recovery time is faster in the descemetic group . however , authors have not compared the postoperative myopia between the predescemetic and descemetic groups in their reports . the preoperative mean thinnest corneal thickness , evaluated with ultrasound corneal pachymetry and with anterior segment oct , was 393.90 49.85 m ( range , 319.0454.0 m ) in the predescemetic group and 361.90 51.54 m ( range , 280.0430.0 m ) in the descemetic group . the postoperative corneal mean thickness was 483.40 53.70 m ( range , 389.0598.0 m ) and 471.5 41.68 m ( range , 430.0550.0 m ) in the predescemetic group and descemetic group , respectively . although there was no statistically significant difference in the cct of the two groups , both groups have an improvement of thickness and restoring the corneal optical integrity and structure . the mean postoperative myopia is 9.43 7.44d ( 8 of 10 eyes 6.0d ) in the predescemetic group and 1.03 1.13d ( all of 10 eyes < 3d ) in the descemetic group in our results , which is statistically significant ( p < 0.05 ) . the results of descemetic group are consistent with buzzonetti et al . 's paper , who have reported the mean postoperative myopia was 1.50 1.70d at 12 months after surgery following the 60-khz intralase femtosecond laser - assisted dalk with big - bubble technique for keratoconus . girard et al . reported that the postoperative myopia has been decreased when the diameter of donor graft is 0.25 mm smaller than the recipient graft in pkp for keratoconus . . stated , to some extent , the postoperative refraction could be manipulated toward acceptable ametropia with altering the size of the donor graft button , and they have demonstrated that the average of hyperopic 2.2d would resulted from decreased 0.25 mm of diameter for the graft . some papers have indicated that the diameter of donor graft is depending on the axial length of eyeball . if the axial length is more than 24.5 mm , the diameter of donor graft should be equal to the recipient . if not , the diameter of donor graft should be 0.25 mm larger than the recipient . in addition , when the diameter of donor grafts was smaller than the recipient or the tight sutures were used intraoperatively , there will be hyperopic diopters for the patients and vice versa . have verified that the postoperative visual acuity was better in the patients with same size of the donor and recipient graft than the patients with donor graft 0.25 mm larger of than recipient for the keratoconus patients with longer axial length of eyeball . in this study , we set the mean diameter of donor graft 0.19 mm larger than the recipient in the predescemetic and 0.14 mm larger in the descemetic group referring to mosca et al . 's paper , which they set the average diameter of donor graft 0.2 mm larger than the recipient . wang et al . who performed predescemetic lk and big - bubble descemetic lk for keratoconus manually , which there was no significant difference of postoperative myopic diopter between two groups ( p > 0.05 ) . it was different from our results while it was possible to be significant difference of postoperative myopic diopter between predescemetic and descemetic groups using femtosecond laser as the postoperative corneal pattern is better in the patients with total stromal resection . moreover , ardjomand et al . reported that the postoperative visual acuity following dalk was similar to pkp when the residual stromal thickness was < 20 m , while it would be affected in the case of > 80 m of the residual stromal thickness . in our study , the residual stromal thickness was 84.4 8.67 m in the predescemetic group and 15 m in the descemetic group ( the thickness of dm was about 10 m and the endothelium was 5 m ) ; however , there was no significant difference in terms of bcva and ucva between two groups ( p = 0.758 and 0.445 , respectively ) . the excellent alignment of dm with the donor tissue was noticed in the descemetic group through anterior segment oct at early postoperative examination while there was interface observed between the recipient dm and the donor tissue in the predescemetic group . hence , we believe that there will be different results in different diameters of donor and recipient graft with or without femtosecond laser . in this paper , we have not evaluated the preoperative manifest refraction and axial length of eyeball in the study population , further study should include these data and increase the studied patients . our preliminary results suggest that femtosecond laser - assisted descemetic lk with big - bubble technique could be the alternative procedure to treat the patients with keratoconus rather than femtosecond laser - assisted predescemetic lk . this work was supported in part by young medical talent project grant qnrc2016905 from jiangsu province , china . this work was supported in part by young medical talent project grant qnrc2016905 from jiangsu province , china .
central retinal artery occlusion ( crao ) leads to severe visual disturbances and is generally resistant to treatment . the fundus changes are characterized by retinal whitening , a cherry red spot , and a pale disc.1 the visual prognosis in eyes with crao is generally very poor , and the degree of visual damage has been suggested to be related to the duration of retinal ischemia before treatment and occlusion of the retinal ciliary artery.26 a crao results from one or more pathological processes , ie , retinal emboli , vessel narrowing and thrombosis , arterial spasm , vascular narrowing caused by extravascular disease , and reduction of blood flow caused by carotid or ophthalmic artery obstruction . however , atypical cases with good visual recovery have been reported.7,8 fundus findings were common in appearance of multiple cotton wool patches and scattered retinal whitening . hayreh et al investigated fundus findings that appeared frequently in three types of crao , ie , the permanent type with or without cilioretinal artery sparing and the transient type defined by the findings of fundus examination and fluorescein angiography.1 the more frequent findings were retinal opacity , a cherry red spot , and a pale disc in both transient and permanent crao . only nine ( 3% ) of 248 eyes showed cotton wool patches , indicating that cotton wool patches are a rare manifestation of crao . purtscher retinopathy was first described in patients suffering from sudden visual loss after severe head injury , and is characterized by cotton wool patches , retinal whitening , and superficial retinal hemorrhages . since then , cases with sudden visual loss associated with acute pancreatitis , collagen vascular diseases , and renal failure had been reported as having fundus alterations similar to those of purtscher retinopathy . these cases are considered to be the same entity as purtscher retinopathy , and are grouped together as purtscher - like retinopathy.9 the pathological mechanism for purtscher retinopathy remains unknown , but several mechanisms have been hypothesized based on the pathogenesis of more general diseases , eg , occlusion and infarction of the microvascular bed due to small emboli,10,11 disseminated intravascular coagulopathy,12 and leukocyte aggregation due to complement c5a activation.13,14 raised intrathoracic pressure and venous dilatation have also been suggested to be the cause of purtscher - like retinopathy.15,16 previously , gass proposed that purtscher - like retinopathy is a subtype of crao cause by an endogenous emboli.17 however , the distinction between atypical crao and purtscher - like retinopathy has not been made . we present three cases of crao with retinal changes resembling purtscher - like retinopathy , although the exact pathological mechanisms remain unknown . case 1 was a 55-year - old man who noticed blurred vision in his right eye when he woke and was referred to our hospital on the same day . at initial examination , his best - corrected visual acuity ( bcva ) was 0.06 od and 1.5 os . fundus examination revealed multiple cotton wool patches surrounding the optic disc and retinal whitening in the macular area of the right eye ( figure 1a ) . goldmann perimetry showed a paracentral scotoma and a large absolute scotoma in the lower nasal field ( figure 1e ) . fluorescein angiography showed that the arm - to - retina circulation time was 33 seconds ( normally 15 seconds in our clinic ) , and choroid circulation to the nasal retina was delayed . the areas of occlusion of the retinal arterioles corresponded to the areas where the cotton wool patches were found ( figure 1b ) . there were no plaques in the arteries or apparent spasm of the retinal vessels ( figure 1b ) . in the late phase of fluorescein angiography no significant stenosis of the carotid artery was found by ultrasound examination ( internal carotid artery plaque score 1.7 ) . systemic examination showed no diseases except for hypertension , that was well controlled by medication , and no traumatic history . based on the clinical findings , the case was diagnosed as crao combined with ischemic optic neuropathy . an intravenous drip of methylprednisolone ( 1 g each day for 3 days ) , antiplatelets , and vasodilator therapy was started after our initial examination . one month after initiation of therapy , bcva in the right eye improved to 0.8 , and the retinal lesions were noticeably improved . case 2 was a 47-year - old man who complained of blurred vision in his left eye and came to our hospital 3 days later . at initial examination , the bcva of both eyes was 1.2 , and the intraocular pressure was within the normal range . fundus examination showed multiple cotton wool patches surrounding the optic disc and slight retinal whitening around the fovea of the left eye ( figure 2a ) . fluorescein angiography showed that the arm - to - retina circulation time was 23 seconds , and the areas of occluded retinal capillaries corresponded to the areas of the cotton wool patches ( figure 2b ) . staining of the retinal arteries was not detected in the late phase of fluorescein angiography ( data not shown ) . humphrey static perimetry showed several paracentral scotomas ( figure 2c ) , and optical coherence tomography ( stratus oct 3000 , humphrey zeiss ) showed a slight thickening of the inner retina but not the retinal ganglion cell layer due to edematous changes ( figure 2d ) . from the clinical findings three weeks later , the cotton wool patches and retinal whitening disappeared without any medication ( figure 2e ) , and the thickness of the inner retinal layers was within the normal range in the optical coherence tomography images ( figure 2 g ) . the humphrey field analyzer findings improved and most of the scotomas were resolved ( figure 2f ) . on systemic enquiry , there were no systemic diseases except for hypertension that was well controlled with medication . case 3 was a 66-year - old man who noticed decreased vision in his left eye and visited our hospital . at initial examination , his bcva was 1.2 od and 0.7 os , and the intraocular pressures were 18 mmhg od and 14 mmhg os . no abnormalities were detected in either fundus , and we followed him as an outpatient without any medication . he had hypertension and diabetes mellitus that were well controlled with medication and had no history of traumatic injury . four days later , his left bcva suddenly decreased to 0.4 and the critical flicker frequency was 23 hz . the direct pupillary light reflex in the left eye was weak and a relative afferent pupillary defect was detected in the left eye . fundus examination showed cotton wool patches surrounding the optic disc and slight retinal whitening in the macular area ( figure 3a ) . fluorescein angiography showed that the arm - to - retina circulation time was 28 seconds , and the choroidal circulation to the nasal retina was delayed ( figure 3b ) . the areas of the occluded retinal capillaries corresponded to the cotton wool patches but there was no staining of the retinal arteries . goldmann kinetic perimetry showed a central scotoma and a defect in the lower nasal field ( figure 3c ) . based on the clinical findings , it was believed that the patient had disturbances of both the retinal and choroidal circulations . the rapd , decreased critical flicker frequency , and central scotoma were related to ischemic optic neuropathy rather than retinal artery occlusion . after diagnosing our patient as having crao combined with ischemic optic neuropathy , an intravenous drip of methylprednisolone ( 1 g each day for 3 days ) and antiplatelet therapy two weeks later , bcva in his left eye improved to 0.9 and critical flicker frequency to 31 hz . the central scotoma and lower nasal field defect were almost gone ( figure 3d ) . repeat fluorescein angiography showed a marked improvement in both retinal and choroid circulations ( figure 3e and f ) . purtscher retinopathy is a vaso - occlusive retinopathy associated with traumatic injury , acute pancreatitis , renal failure , and vasculitic diseases , such as dermatomyositis or lupus erythematosus.1824 the lesions are characterized by purtscher flecken that consist of many discrete areas of retinal whitening and cotton wool patches that are retinal microinfarctions at the level of the nerve fiber layer with or without retinal hemorrhages . all cases had good visual recovery to at least 0.9 , even though disturbances of the proximal retinal and choroid circulation were initially present . the underlying diseases in our cases were only mild hypertension and diabetes mellitus . in cases 1 and 2 , relatively large cotton wool patches were found surrounding the optic disc and a whitening of the retina around the fovea accompanied by a cherry red spot . in case 3 , multiple cotton wool patches were detected surrounding the optic disc but the retinal whitening was mild . the fundus changes in our three cases resembled that of purtscher - like retinopathy but without any traumatic injury or other systemic diseases that have been associated with purtscher retinopathy ( table 1 ) . fluorescein angiography showed that the arm - to - retina circulation time of all cases was delayed , and two of the cases had a filling defect of the choroid circulation which has not been reported in eyes with purtscher retinopathy . in the early phase , retinal capillaries were blocked in the areas corresponding to retinal whitening and the cotton wool patches that were seen surrounding the optic disc . these findings suggest the presence of capillary obstructions at more proximal sites than the arterioles ; the arterioles had also been obstructed , and the radial peripapillary capillary network might also be disturbed . regarding the pathological mechanisms , an experimental study has shown that intermediate 150 m emboli can cause retinal infarctions resembling purtscher retinopathy.10 another experimental study found that when the central retinal artery is partially obstructed , cotton wool patches appear around the optic disc.25 the ophthalmoscopic and fluorescein angiographic findings in all of our cases were characterized by both retinal and choroidal circulation disturbances and multiple cotton wool patches surrounding the optic disc . our findings suggest that the pathogenesis of rare subtypes of crao , such as our cases , involves partial occlusion of the central retinal artery , and the findings are similar to those of purtscher - like retinopathy , even though there are no more general diseases which can be linked to purtscher - like retinopathy . iwamasa et al described two cases of crao with good recovery of vision and visual fields.7 their fundus changes were very similar to that of our cases , ie , multiple cotton wool patches surrounding the optic disc and retinal whitening in the macular area . fluorescein angiography showed a delay of arm - to - retina circulation time and the retinal capillary nonperfused areas corresponding to the cotton wool patches . these findings are also similar to those of purtscher - like retinopathy except for the delay in retinal circulation time . interestingly , the visual fields of two of their cases recovered by 70 days after onset , as did our cases . thus , it appears that the course of the signs and symptoms is quite different from those in eyes with typical complete crao . the resemblance of the clinical course and findings probably indicates similar pathogenesis of these cases . several studies have reported that steroid pulse therapy and intravenous administration of steroids has beneficial effects in purtscher retinopathy.26,27 on the other hand , a retrospective study showed that purtscher retinopathy can recover spontaneously , with an improvement of at least two lines on the snellen visual acuity chart at 1 year after onset.28 inflammatory reactions can develop secondarily in the nonperfused areas . an optical coherence tomographic study showed subretinal fluid in the nonperfused areas corresponding to the cotton wool patches in a case of purtscher retinopathy associated with acute pancreatitis.29 likewise , in our case 2 , optical coherence tomography showed a hyper - reflective area in the nerve fiber and retinal ganglion cell layer which probably represented secondary edema . although we did not examine how effective steroid therapy alone is against retinal edema in crao , steroid therapy probably suppressed the inflammation and improved not only the retinal condition but also the optic nerve ischemic condition . in conclusion , we report three cases of crao with fundus appearances resembling purtscher - like retinopathy . our findings support the proposal by gass17 that there are subtypes of crao , with some cases having a good visual prognosis . thus , when cases of crao with cotton wool patches are encountered , special attention should be paid to the regimen of steroid therapy .
neurodegenerative disorders represent a global health problem , being a major cause of high mortality among elderly populations . one of the most common degenerative diseases of the nervous system is parkinson 's disease ( pd ) . it is related to the death of dopaminergic neurons in the basal ganglia of the brain . regardless of the etiology of this disease , including genetic or environmental factors , inflammatory processes are associated with the progression of neurodegeneration [ 2 , 3 ] . in fact , several lines of evidence confirm the interdependence between neuroinflammation and neurodegenerative disorders , including an increased cytokine release and an imbalance of molecules involved in oxidative stress [ 25 ] . these substances , produced mainly by microglial and dendritic cells , act on astrocytes and neurons , inducing secondary responses that can lead to uncontrolled inflammation in nervous tissues , resulting in cell apoptosis . moreover , the ability of neuronal cells to release inflammatory mediators and reactive oxygen species ( ros ) in response to stimulus has also been reported . therefore , an indirect self - destruction of neurons can not be excluded in neurodegenerative disorders . current studies have shown that kinins , potent proinflammatory peptides , can be produced by cells of the nervous system , even by neuronal cells [ 68 ] , and their presence is correlated with neuroinflammation [ 911 ] . differentiated and inf--stimulated neuroblastoma cell line imr-32 was able to produce quickly significant amount of bradykinin and its metabolites , the peptides without arginine residue at the c - terminus that act through a specific kinin receptor type 1 ( b1r ) , enhancing inflammatory responses . in addition , an increasing number of studies indicated the involvement of kinin receptors in neurological disorders including those associated with cell degeneration [ 1115 ] . it was demonstrated that kinins can activate not only nonneuronal cells , such as astrocytes and microglia , but also neurons , causing proinflammatory reactions [ 1618 ] . recently , interesting investigations on anti - inflammatory therapies in parkinson 's disease were reported . despite the fact that no direct evidence of the participation of kinin peptides in pd has been presented so far , the examination of the impact of these peptides on degenerative processes seems to be interesting . in the present study , we investigated the influence of bradykinin ( bk ) and des - arg - kallidin ( dakd ) on apoptosis markers ( such as caspase activity ) or cell viability in a pd cellular model . neuron - like cells , obtained from retinoid acid - differentiated human neuroblastoma cell line sk - n - sh , treated with a neurotoxin , 1-methyl-4-phenylpyridinium ( mpp+ ) , showed enhanced degenerative response . the treatment with kinin peptides enhanced this effect and was accompanied by increased cytokine release and generation of oxidative molecules . rabbit polyclonal antibodies against kinin receptor 1 , mouse monoclonal antibodies against kinin receptor 2 ( b2r ) , fitc - conjugated goat polyclonal antibodies against rabbit , and tritc - conjugated rabbit polyclonal antibodies against mouse were supplied by abcam ( england ) . elisa kits for detection of cytokines , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , and tumor necrosis factor- ( tnf- ) , were purchased from bd biosciences ( usa ) . 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid ( mgta ) was supplied by calbiochem ( usa ) and fluorescence mounting medium from dako ( denmark ) . sybr green kit for real - time pcr was purchased from kapa biosystems ( usa ) . a kit for the determination of caspase 3/7 activity ( caspoglo3 ) and m - mlv reverse transcriptase kit were from promega ( usa ) . rabbit polyclonal antibodies against receptor d2 ( d2r ) were purchased from santa cruz biotechnology ( usa ) . 2,3-diaminonaphthalene ( dan ) , 2,7-dichlorodihydrofluorescein diacetate ( dcfh2-da ) , icatibant ( hoe ) , des - arg - icatibant ( deshoe ) , captopril , bacitracin , protease inhibitors , retinoid acid ( ra ) , 1-methyl-4-phenylpyridinium ( mpp+ ) , resazurin sodium salt , 4,6-diamidino-2-phenylindole ( dapi ) , tri - reagent , and standard chemicals were supplied by sigma ( usa ) . antibiotics , antimycotics , cell culture media , fetal bovine serum ( fbs ) , nonessential amino acids , and sodium pyruvate were purchased from thermo scientific ( usa ) . the human neuroblastoma cell line sk - n - sh supplied by atcc ( usa ) was cultured in mem medium supplemented with 1% nonessential amino acids , 1 mm sodium pyruvate , 1 u / ml penicillin , 1 g / ml streptomycin and 2.5 g / ml amphotericin b , and 10% fbs in a humidified atmosphere containing 5% co2 at 37c . sk - n - sh cells were differentiated with 5 m ra into neuronal - like cells according to previously published method . the pd cellular model was obtained by stimulation of ra - differentiated cells with 2.5 mm mpp+ in 1% fbs mem medium for 24 hours . changes in neurite length were observed with an inverted phase contrast microscope ( nikon eclipse ts100 , japan ) equipped with toupcam industrial digital camera ( tp605100a , china ) before and after stimulation with neurotoxin . the effect of kinin peptides on cell viability in the cellular pd model was evaluated by using alamarblue assay . after differentiation , cells were incubated with bk and dakd at different concentrations ( 1 nm or 1 m ) in 1% fbs cell medium supplemented with kininase inhibitors ( 10 m mgta , 20 m captopril , and 500 m bacitracin ) for 24 hours . on the next day , cells were treated with 2.5 mm mpp+ in fresh medium containing kinin peptides . briefly , the stimulating medium was discarded and 100 l of 10% resazurin in phenol red - free medium was added to each well . then , cells were incubated for 4 hours at 37c and after that time the fluorescence of supernatants was measured using synergy h1 microplate reader ( biotek instruments , usa ) at 560 nm excitation and 590 nm emission wavelengths . ra - differentiated cells ( 6 10 ) were incubated with bk or dakd at 1 nm or 1 m concentration for 24 hours . next , the samples were stimulated as during the viability test ( see above ) and after further 24 hours caspase 3/7 activity was measured according to the manufacturer 's instructions . some samples were first preincubated with antagonists for kinin receptors , icatibant ( hoe ) , and des - arg - icatibant ( deshoe ) at 1 m concentration for 2 hours before treatment with kinins and the antagonists were also added during the further incubation with kinins and mpp+ . total rna from untreated cells or cells incubated with 5 mm mpp+ , 1 m bk , or 1 m dakd for several hours was isolated with tri - reagent . expression of mrna for b1r , b2r , d2r , il-1 , il-6 , inducible no synthase ( inos ) , and tnf- was analyzed with real - time pcr procedure . expression of elongation factor 2 ( ef-2 ) mrna was also determined in each sample for subsequent quantitative analysis . cdna was synthesized with m - mlv reverse transcriptase kit according to the manufacturer 's instructions . then , the amplification of cdna was performed with sybr green kit and specific primers , using the 7500 fast real - time pcr system thermocycler ( life technologies , usa ) . the annealing temperature of real - time pcr was 58c for b1r , b2r , and ef-2 , 60c for d2r and il-6 , and 62c for il-1 , inos , and tnf-. the pcr conditions were 95c for 10 min , followed by 95c for 15 s , 15 s at the corresponding annealing temperature , and 72c for 30 s ( 40 cycles ) . the measurement was based on a comparison of analyzed gene expression relative to the housekeeping gene expression ( ef-2 ) . the relative value of gene expression ( q ) was calculated with 7500 fast real - time software by using the equation : q = 2 , where ct is the threshold cycle . intracellular level of ros was determined with a fluorometric assay using 2,7-dichlorodihydrofluorescein diacetate ( dcfh2-da ) dye sensible to oxidation . sk - n - sh cells ( 1.3 10 ) were seeded in a clear bottom opaque microplate , precoated with 1% gelatin . after differentiation , cells were pretreated with kinins as described above and next incubated for 30 minutes with 100 m then , cells were washed three times and stimulated with 1 m dakd or 1 m bk in the presence of 2.5 mm mpp+ or without neurotoxin . the changes in fluorescence signal were measured every 30 seconds during 20 minutes at 37c , using microplate synergy h1 reader at excitation and emission wavelengths of 485 nm and 528 nm , respectively . a fluorometric assay was used to analyze the influence of kinin peptides on the production of nitrites ( one of the final no metabolites ) . confluent ra - differentiated cells were treated with kinins as described above and next incubated with 1 m bk and 1 m dakd in the presence of or without 2.5 mm mpp+ for 15 and 30 minutes . after this incubation time briefly , 100 l of supernatant was mixed with 10 l 0.05 mg / ml dan in opaque microplate and incubated for 30 minutes in the dark . the reaction was stopped by the addition of 5 l 2.8 m naoh and fluorescence was measured in synergy h1 microplate reader at excitation and emission wavelengths of 365 and 450 nm , respectively . the nitrite concentrations were calculated on the basis of two different standard curves ( with and without mpp+ ) . sk - n - sh cells ( 4 10 ) were seeded in a 12-well plate , differentiated for 3 days with ra , and then stimulated for 6 or 24 hours with 1 m bk or 1 m dakd in the presence of 2.5 mm mpp+ or without neurotoxin . the assessment of cytokine release ( il-1 , il-6 , and tnf- ) into the medium was evaluated with elisa kits according to the manufacturer 's instructions . absorbance was measured with the use of powerwave microplate reader ( biotek instruments , usa ) at 450 nm . to determine cell surface receptors , b1r , b2r , and d2r , cells ( 5 10 ) were seeded on glass cover slides in a 12-well plate and after ra - differentiation were stimulated with 5 mm mpp+ for 1 , 2 , and 5 hours . additional experiments aimed to analyze the effect of kinin peptides on d2r expression were also performed . differentiated cells were incubated with 1 m bk or 1 m dakd for 30 minutes , 1 hour , and 3 hours . next , following fixation with cold methanol and 1-hour blocking with 2% fbs , cells were incubated overnight at 4c with 50-fold diluted primary antibodies : rabbit polyclonal anti - b1r , mouse monoclonal anti - b2r , and rabbit polyclonal anti - d2r . after washing , samples were incubated for one hour with diluted ( 1 : 250 ) secondary antibodies : goat fitc - labeled anti - rabbit or rabbit tritc - labeled anti - mouse . then , 2 m dapi was added for 10 minutes and , after extensive washing , cover slides were mounted on microscope slides using fluorescent mounting medium . the samples were analyzed with epifluorescence dmire2 microscope ( leica , germany ) and images were taken at 40x magnification under oil immersion . the intensity of fluorescence was measured in at least three randomly chosen fields of every picture using imagej software . the measured area was selected with brightness slicing procedure , with a constant lower threshold value . the values for negative control samples all values are presented as means standard deviation from at least three experiments ; the statistical significance was analyzed by student 's t - test . a suitable cellular model of parkinson 's disease was obtained after stimulation of the neuron - like cells with neurotoxin mpp+ . the ra - differentiated sk - n - sh cells showed increased neurite degeneration , accompanied by decrease of cell viability and an enhanced activity of caspases after 24-hour incubation with 2.5 mm mpp+ ( figure 1 ) . the well - developed neurite net was broken ( see arrows in figure 1(a ) ) ; cells presented changed morphology being more round and some cells were even detached from the plastic support . the cytotoxic effect of neurotoxin on these cells was assayed with alamarblue test , showing a significant decrease of cell viability ( by 38% ) , as compared with nonstimulated cells ( figure 1(b ) ) . kinin peptides , bk as well as dakd , at the higher concentration ( 1 m ) caused a slight , insignificant decrease of cell viability ( by 10% ) while at the lower concentration ( 1 nm ) no toxicity on cells was observed ( results not shown ) . cells treated with bk and dakd at 1 m final concentration were more vulnerable to the mpp+ action . in this case , likewise , meaningful changes in caspase activity were observed after mpp+ stimulation , without and with bk or dakd treatment ( figures 1(c ) and 1(d ) , resp . ) . the effect of kinin peptides at 1 nm concentration was smaller ( results not shown ) ; thus , in the following experiments only the higher concentration was used . neuron - like cells , stimulated with neurotoxin , showed an increase in enzyme activity by 100% , as compared to control cells ( cells without stimulation were assumed to present 100% of basic enzyme activity ) . bk and dakd caused only insignificant increase in caspase activity ( by 20% and 32% , resp . ) . bk - treated cells showed a 132% augmentation whereas dakd - treated cells presented an even higher caspase activity after mpp+ coincubation ( 156% ) . in addition , the use of antagonists of kinin receptors , hoe and deshoe , produced an inverse effect , suggesting the direct involvement of kinin receptors . incubation of cells with these substances caused an inhibition of bk- and dakd - induced caspase activity but it did not affect the mpp+ action significantly . the intracellular production of ros depended on activation time with kinin peptides and on the neurotoxin stimulation . all experiments were performed simultaneously showing time - dependent influence both in neuronal cell model and in pd cellular model . results are presented as percentage changes induced by peptides compared to values from cells without or with mpp+ stimulation , untreated with kinin peptides ( figures 2(a ) and 2(b ) , resp . ) . surprisingly , the response of the cells treated with kinins and mpp+ is much greater ( up to eightfold ) than that of the cells treated only with kinin peptides . a slight increase of ros level by bk ( by 40% ) after 30 seconds was observed while dakd effect ( enhanced ros production by 50% ) appeared later ( after 2 minutes ) . further incubation showed a tendency of ros level to decrease even below the basis level detected in control cells . however , after 10-minute incubation the cells achieved a level similar to that observed in untreated cells . in the case of the cellular pd model ( cells were additionally incubated with mpp+ ) the effect of kinin peptides on ros production was stronger and delayed . the maximal ros release by both peptides was observed after 5 minutes and increased ca . the increased ros level was largely retained especially after dakd activation , achieving values similar to control cells after 15 minutes from the stimulation start . the effect of kinin peptides on oxidative stress in neuron - like cells was also estimated by measuring inos mrna expression and no release by cells . the inos mrna expression was significantly elevated by dakd and mpp+ ( figure 3(a ) ) . the dakd effect was the highest , showing a 2-fold increase of mrna expression , whereas mpp+ and bk caused an increase in mrna expression by 75% and 40% , respectively . it should be pointed out that this effect was time - dependent ; kinin peptides enhanced mrna expression of inos quickly ( after 30 minutes ) while the maximum response to neurotoxin appeared after 4-hour incubation . the no release by neuron - like cells after stimulation with bk , dakd , and mpp+ is presented in figures 3(b ) and 3(c ) . no significant effect of bk was detected whereas dakd caused a significant increase ( by 60% ) after 30 min incubation . mpp+ induced a higher no release , by 80% and 100% , after 15-minute and 30-minute incubation , and this effect was significantly enhanced in the presence of dakd but not of bk . the simultaneous activation with mpp+ and dakd led to an increase in no metabolites by 50% , as compared to treatment with mpp+ alone . the ability of bk and dakd to induce cytokine expression in neuron - like cells was analyzed by real - time pcr and elisa techniques ( figure 4 ) . increased mrna expression for tnf- , il-1 , and il-6 was registered after just 30 minutes of incubation with peptides . the highest values were obtained for tnf- mrna expression after 30-minute stimulation with bk and dakd , showing over 25-fold increase as compared to nonstimulated cells ( figure 4(a ) ) . the effect of mpp+ on the induction of tnf- mrna in cells was more moderate but significant , showing a 14-fold increase . the effect on tnf- protein release by cells after incubation with peptides and mpp+ was quick and after 6 hours protein production was enhanced by 25% , 38% , and 23% , respectively , for bk , dakd , and mpp+ , as compared with untreated cells ( figures 4(b ) and 4(c ) ) . moreover , a slight augmentation of tnf- protein was observed when cells treated with kinin peptides were stimulated with neurotoxin ; the amount of this protein was then 20% greater than in cells stimulated with mpp+ alone . in the case of il-1 , kinin peptides ( bk , dakd ) and mpp+ caused considerable increase in mrna expression , 4- , 7- , and 2-fold , respectively ( figure 4(d ) ) . the maximal mrna expression was observed after 30-minute stimulation with bk and dakd and after 4-hour neurotoxin incubation . nevertheless , il-1 protein release after 24 hours was only slightly induced by kinin peptides ( 1.5-fold and 2-fold for bk and dakd , resp . ) while neurotoxin effect was significantly higher , about 3-fold ( figures 4(e ) and 4(f ) ) . nonetheless , a synergic effect of bk or dakd with mpp+ on il-1 release was observed . the amount of protein in supernatants was higher than that from cells incubated with neurotoxin alone ( by 54% and 72% for bk / mpp+ and dakd / mpp+ , resp . ) . besides , the expression of il-6 mrna , induced by bk and dakd , was relatively lower ( 3-fold and 2-fold , resp . ) , albeit these changes were significant in comparison to nonstimulated cells ( figure 4(g ) ) . mpp+ caused a slight but significant enhancement increment of il-6 mrna ( by 1.7-fold ) . the protein release was also significantly induced by kinin peptides ( by 160% and 190% for bk and dakd treatment , resp . ) and by mpp+ ( over 3.5-fold ) ( figures 4(h ) and 4(i ) ) . similar as in the case of other cytokines , simultaneous cell treatment with kinin peptides and mpp+ also led to an improved protein production ( about 420% as compared with untreated cells ) . the samples treated with antagonists inhibited the production of cytokine proteins , demonstrating that the increment achieved by bk and dakd in the presence of mpp+ was caused by the activation of the kinin receptors . the effect of mpp+ on receptors ' expression in the neuron - like cells was analyzed at gene and protein level . significant changes were observed after 4-hour incubation when the amount of d2r mrna decreased by 40% ( figure 5(a ) ) . these observations were in accord with protein expression of this receptor that was diminished to 37% after 5 hours ( figures 5(b ) and 5(c ) ) . on the other hand , the expression of kinin receptors was also affected by neurotoxin . the induction of mrna expression of b1r by mpp+ is undisputable ( figure 6(a ) ) . a significant increase in mrna expression as compared with control cells ( by 200% ) was observed after one hour . these changes were preserved after further 3 hours , at a lower but still significant level . likewise , the amount of b1r protein in the cell membrane was enhanced , leading to above 2-fold augmentation after five hours ( figures 6(b ) and 6(c ) ) . conversely , the expression of b2 receptor in neuronal cells showed a decreasing tendency after neurotoxin treatment ( figure 7 ) . a slight diminution of b2r mrna was observed after 4 hours ( by 20% ) but it was not significant as compared to control cells ( figure 7(a ) ) . nevertheless , the decrease of b2r protein in neuron - like cells observed after mpp+ stimulation ( achieving a maximal decrease after 5 hours by 25% ) was significant in comparison to untreated cells ( figures 7(b ) and 7(c ) ) . d2r expression at mrna and protein level was analyzed after stimulation with kinin peptides at different time intervals . mainly , cell incubation with bk and dakd caused a decrease of d2r mrna ( figure 8) . however , this effect was decidedly stronger and significant in the case of dakd stimulation ( figure 8(b ) ) . bk induced a decrease of mrna ( over 60% ) but only after 3-hour incubation ( figure 8(a ) ) , whereas dakd achieved a similar effect faster , just after 30 minutes of incubation . at the protein level , the quantitative analysis of the pictures obtained with immunofluorescent technique showed a significant diminution of d2r expression in cells . changes of the amount of d2r protein were observed after 3-hour bk stimulation ( by 20% ) and after 1- and 3-hour dakd stimulation ( by 20% and 32% , resp . ) . a possibility that tissues of the central nervous system , through the production of inflammation mediators , may contribute to the regulation of neurodegenerative processes incites the reconsideration of these processes in terms of well - known cellular processes associated with inflammation . kinins and the components necessary for their production are abundantly present in nervous tissues [ 11 , 12 , 24 ] . thus , it is likely that these peptides may contribute to neuronal degeneration by enhancing inflammatory response . hence , in this study we analyzed the influence of the main kinin peptides , bk and dakd , in the promulgation of neurodegenerative processes in a cellular model of pd . for this purpose , a popular cellular model related to the human neuroblastoma cell line ( sk - n - sh ) was differentiated into neuron - like cells with ra . the use of this substance as a proapoptotic factor in cellular and animal pd models has been frequently reported [ 26 , 27 ] . the pd model obtained in this study showed significantly diminished cell viability , with the loss of neurites and enhanced caspase activity ( figure 1 ) . with the help of this model the effect of kinin peptides on apoptosis could be appreciated . neither bk alone nor dakd alone did influence significantly the viability of neuron - like cells . however , cell death was significantly enhanced when cells were treated with these peptides before mpp+ incubation , suggesting a better predisposition for neurodegeneration in tissues rich in kinin peptides . in addition , kinin peptides were also able to trigger apoptotic processes through activation of caspases in the pd cellular model . despite a slight insignificant increment in caspase 3/7 activation by bk and dakd caspase 3 and caspase 7 , as executioner enzymes , once activated , can accelerate a feedback loop of caspase activation leading to cell death . the results obtained in this study argue in favor of a proapoptotic effect of both peptides , bk and the active metabolite dakd , in the pd cellular model . however , a distinct role of b2r and b1r has been proposed in neuronal apoptosis induced by organophosphates . the authors demonstrated that b1r can promote cell damage while b2r may exert a protective role during neurotoxin - induced toxicity . a neuroprotective role of bk against nmda - mediated excitotoxicity was also suggested . nevertheless , in our study , the bk - induced effect on the increase of caspase activity in mpp+-treated cells was downregulated by the b2r specific antagonist ( hoe ) , showing an inversion of the bk - induced effect ( figure 1(c ) ) . it should be emphasized that the used concentration of bradykinin was very high , usually considered as pathologic . hence , the observed effects could take place in vivo only if kinin peptides were in abundance in the nervous tissues . it should be noted that in these experiments cells were treated with bk for extended time before neurotoxin stimulation . pathologic amount of kinin peptides can stimulate ros production and cytokine synthesis , even in neuronal tissues [ 24 , 31 ] . in fact , we were able to observe different effects on ros production induced by bk and dakd in the pd cellular model in comparison with the effect induced in neuronal cells ( figure 2 ) . in the latter case , ros production was immediately induced by kinins and this effect was quickly reversed while in the pd model these peptides caused a much stronger and longer effect . in this context , the difference between our findings and those describing the protective role of bk on cell apoptosis could also be attributed to the different cell response following neurotoxin stimulation . a long exposure of nervous tissues on pathological quantities of bk could make these cells more vulnerable to neurotoxins . the level of no in the central nervous system is regulated by a relevant enzyme , neuronal no synthase , whereas the inducible form ( inos ) is not expressed . however , in pathological situations the expression of this enzyme becomes elevated . indeed , the mediation of oxidative stress , through ros and reactive nitrogen species such as no , in apoptosis is related to a mitochondrial impairment . in addition , acute and chronic oxidative stress may be involved in diverse neurodegenerative diseases , including pd . accordingly , our pd model using mpp+ also displayed increased expression of inos mrna with significant no production ( figure 3 ) . however , our results demonstrate only a slight increase in inos mrna induced by bk in neuron - like cells , without no production ( figure 3 ) . in addition , bk was unable to induce no release also in the pd cellular model . hence , the observed bk - induced decrease in cell viability and increased caspase activity can not be unequivocally assigned to the deregulation of oxidative stress . probably , bk can activate different cellular pathways involved in apoptosis , including those associated with inflammatory response . the study of inflammatory response in our pd cellular model could provide a hypothesis on bk action . there is emerging evidence that inflammatory processes involving microglia and astrocytes can mediate neurodegeneration , including that in parkinson 's disease [ 2 , 3 , 23 ] . nevertheless , in this study we demonstrated that neuronal cells could also respond to exogenous stimuli with significant production of proinflammatory molecules . actually , kinin peptides as well as neurotoxin mpp+ induced a high release of tnf- , il-1 , and il-6 ( figure 4 ) . in addition , simultaneous treatment of cells with mpp+ and peptides led to enhanced cytokine production . bk evokes a high production of tnf- , il-1 , and il-6 , the cytokines of crucial importance for development of inflammatory response in nervous tissues [ 34 , 35 ] . therefore , we can suggest that bk - induced cytokines , especially tnf- , produced by neurons may promote inflammatory processes , causing cell death in our pd cellular model . this assumption is in agreement with the observation obtained in a study using transgenic mice that lacked tnf- receptors ; the animals were completely protected against neurotoxicity of mpp+ . the other peptide , dakd , was able to induce enhanced inos mrna expression in neuron - like cells and also caused overproduction of ros and no , which could contribute to starting the extrinsic oxidative stress pathway , especially in the presence of exogenous mpp+ ( figure 3 ) . like bk , this peptide also caused the release of large amounts of cytokines in neuron - like cells , as well as in the pd cellular model ( figure 4 ) . hence , our observations argue for the mediation of kinin peptides , especially the b1r agonist , in degenerative processes occurring in the pd cellular model through increased oxidative reactivity and prolonged inflammatory processes . the dopamine receptor 2 ( d2r ) plays an important role in parkinson 's disease . the existence of two types of d2 receptor has been reported [ 37 , 38 ] . the long isoform differs from the short type by the presence of 29 additional amino acids in the third intracellular loop . these receptors possess different functions : the longer receptor is associated with postsynaptic effects , where in cooperation with the dopamine receptor type 1 it modulates the dopaminergic responses producing locomotor activation . on the other hand , the shorter isoform is present , mainly in presynapses , where it acts as an autoreceptor , regulating dopamine secretion by dopaminergic neurons . here , we report the decrease of d2r expression by mpp+ and suggest that the loss of these receptors implies neurodegeneration . in fact , a protective role of d2 autoreceptors against mpp+ has been reported in drosophila primary neuronal cells . it seems that at the early phase of degenerative processes both receptor d2 isoforms are important for neuroprotection . the lack of postsynaptic d2r may result in degeneration of nervous tissues due to disturbances of the dopaminergic pathways . in turn , the loss of d2 autoreceptors can also lead to cell degeneration because the regulation of dopamine production and release is perturbed , leading to their oxidation by monoamine oxidase that may trigger enhanced redox processes in cells . therefore , the observed lower expression of d2r mrna and protein induced by mpp+ corroborate these reports , demonstrating that our cellular model can reflect neurodegenerative features described in pd . to explain the kinin action in the pd cellular model , the expression of their specific receptors was analyzed after mpp+ stimulation . kinins and their metabolites without the c - terminal arginine residue ( des - arg kinins ) are recognized by two types of receptors , b1r and b2r . the first type of receptor preferably binds des - arg kinin peptides , whereas b2r primarily recognizes bk and kallidin . the b2r is ubiquitous in numerous types of cells and plays a pivotal role in several physiological processes while b1r is mainly induced by certain stimuli during pathological disorders . the b2 receptor plays an important role in acute inflammation while b1r is rather associated with chronic inflammation . this study shows a large increase in b1r mrna expression that is translated into protein ( figure 6 ) . therefore , enhanced inflammatory processes in neuronal cells caused by mpp+ are also associated with functioning of this receptor . after activation , it can propagate processes leading to cell degeneration . interestingly , in the case of the effect of mpp+ on b2r expression , the results were less clear . the mrna expression of this receptor was not changed by mpp+ ( figure 7 ) . these results can indicate , as in the case of d2r , that b2 receptors play an essential role in the protection against mpp+ . indeed , the mediation of bk through b2r in the neuroprotection has recently been proposed . nevertheless , the expression of b2r is less vulnerable to changes ; this receptor after agonist binding and internalization may recycle to the cell surface . in addition , bk can be converted to des - arg metabolites by carboxypeptidases , present in different tissues , including the brain [ 6 , 41 ] . hence , the observed effect of bk on cell viability and caspase activity can partially be associated with their transformation to des - arg metabolite , which directly triggers inflammatory and redox reactions leading to neurodegeneration . finally , the observations concerning the diminishing of d2r expression by kinin peptides ( figure 8) may confirm a hypothesis of the mediation of these peptides in neurodegenerative processes related to pd . these observations , combined with those obtained from the effect of mpp+ on b1r expression , allowed us to attribute a main role in the development of pd to the des - arg peptides . the effect of bk could be also attributed to des - arg bradykinin formation , since in these experiments kininase inhibitors were not used . however , the role of bk in neurodegeneration is not absolutely clear at present and this issue requires additional , more profound studies . in this study we examined the effect of kinin peptides on inflammatory and oxidative reactions in a pd cellular model . kinins as well as their metabolites without arginine residue at the c - terminus were able to induce significant changes in the release of ros and proinflammatory cytokines . these changes are accompanied by intensified apoptotic processes . induced expression of b1r by mpp+ may also contribute to a potentiation of inflammatory response . greater abundance of kinin peptides in nervous tissues , produced by neurons as well as by astrocytes and microglia , promotes degeneration of nervous tissue during neuroinflammation . the mediation of kinin receptors may be critical for offering new possibilities for the development of effective therapies against neurodegeneration through their agonists / antagonists .
this evaluation illustrated that without the implementation of an appropriate scaling system , the work - up to rule out pe is of low yield . consequences of excessive testing include unnecessary exposure to radiation and financial wastefulness . the implementation of clinical prediction rules will improve the diagnostic approach , management , and utilization of resources . the use of a scaling system will also account for the possibility of normal physiologic or pathologic conditions , other than pe , that may attribute to the elevation of d - dimer values . pulmonary embolism ( pe ) is a very dangerous disease state , as it presents with nonspecific symptoms that can lead to death within a few hours of onset . as many as 200,000 deaths per year in the united states are due to pe , making this the third leading cardiovascular cause of death of inpatient groups.1 any patient who is at risk for venous thrombosis must be watched carefully , since 50% to 60% of these patients can develop pe.1 there has been a corresponding rise in diagnostic studies being conducted based on suspicion of pe . the rates of unexpected death due to pe are second only to cardiac arrest . of those patients who develop pe , approximately 10% will die within the first hour , while another 30% will die from a recurrent case of emboli.2 with improved and increased accuracy of diagnosing pe , 25% of those who currently die from pe could be saved.2 however , the concern of missing the diagnosis of pe should not give way to unnecessary orders for tests without adequate basis for those orders . it has been noted in the past that the d - dimer value can be systematically elevated in cases of deep venous thrombosis or pe . however , the d - dimer value can be elevated in a number of other normal physiologic , as well as pathologic states . hence finding the d - dimer level to be elevated rather , clinical suspicion should direct the investigation for the presence of pe . with the aid of appropriate labs and imaging studies , the diagnosis of pe can be made quickly and adequate treatment can be initiated in a short period of time . the purpose of this study is to demonstrate that an elevated d - dimer value alone often results in extensive pe workup , which has a low diagnostic and economical yield . a retrospective analysis of the diagnosis of pe at jackson park hospital ( jph ) was conducted . jph is a short - term , comprehensive care facility serving the south side of chicago , il . the analysis focused on the use of the d - dimer and ct scan of the thorax with contrast . in an attempt to minimize bias the charts of 220 patients having a d - dimer test ordered at the hospital were randomly selected for review . factors evaluated for these patients included levels of the hemosil assay of the d - dimer and subsequent tests ordered , including ventilation / perfusion scan ( v / q scan ) , doppler studies and ct scan of the thorax with contrast . to assess the consistency in the approach to diagnosing a pe , the sequence of events leading to the suspicion and ultimate diagnosis was considered in the patients diagnosed with a pe . the factors for each of the cases that were considered included clinical suspicion , d - dimer , the use of arterial blood gases , v / q scan , doppler studies , and ct of the thorax with contrast . while it was possible to investigate these tests , the current lack of a widely accepted and used pe protocol at the hospital resulted in limitations to objectively identifying the ordering physician s clinical and laboratory rationale behind the tests . the study consisted of reviewing 220 patients who had one or more d - dimer tests ordered . of the 220 patients , 217 patients had d - dimer values . 118 out of the 217 patients ( 54.4% ) had elevated d - dimer values greater than 254 ng / ml . of these 118 patients , five were diagnosed with pulmonary emboli , resulting in a positive predictive value of 4.2% . ninety - two follow up tests were ordered for the 118 patients with elevated d - dimer values . some of these patients may have had more than one follow - up test ordered while some patients had no further tests ordered . thirty - four of the 92 tests ordered were venous doppler studies ( 36.9% ) , 36 were ct of the thorax ( 39.1% ) and 22 were v / q scans ( 23.9% ) . out of the 118 patients with elevated d - dimer , five were diagnosed with pe . when considering the group of patients who had the d - dimer test ordered , those having elevated values often had subsequent tests ordered , including v / q scans , ct scans of the thorax with contrast and lower venous doppler studies . summarized in the table 3 is average cost and reimbursement ( as per medicaid ) for the tests ordered in the 118 cases of elevated d - dimer values . the prevalence of pe and the associated mortality undoubtedly warrant sufficient work - up when there is clinical suspicion . the current approach to diagnosing pe has been inefficient , as there is an overly liberal use of the d - dimer lab test and chest ct scan . this exposes the patient to high doses of radiation , increases costs , and delays the disposition of patients.3 it is evident that the diagnostic approach must be altered to efficiently and effectively diagnose pulmonary emboli . the d - dimer lab test is considered to be highly sensitive , but non - specific.4 as a result , the role of this test has typically been limited to ruling out a pe in cases of low suspicion.4 however , there are various formulations of the d - dimer test itself , each with different sensitivities . the hemosil assay , used at jph , has sensitivities comparable to the gold standard vidas elisa assay.5 due to such high sensitivity , when a d - dimer assay result is negative , pe can be confidently ruled out . sensitivity , negative predictive value , turnaround time for the results and cost all play important roles when selecting a particular d - dimer assay . turnaround time is important since a pe needs to be ruled out as quickly as possible . although the elisa may be the gold standard assay , since the sensitivity , negative predictive value , turnaround time , and cost of the new generation latex agglutination d - dimer assays are comparable and in some instances better , they are more advantageous than the elisa . there has been great confusion resulting from multiple commercial assays having various techniques , cut - offs , systems of units , operational characteristics , and clinical validity . since d - dimer is a complex mixture of degradation products of different sizes , standardization has not been possible.6 while many assays typically use cut - off values of 500 ng / ml , studies found that the lower cut - off values with the hemosil assay demonstrated not only safe exclusion of venous thromboembolism , but also proved specificity and exclusion rates similar to other formulations.5 as a result , at jph , the threshold for the d - dimer value is 254 ng / ml . however , according to the literature , d - dimer values greater than 500 ng / ml are considered positive.7 if the threshold for an elevated d - dimer had been increased to 500 ng / ml in this study of the 217 patients from the d - dimer group , 66 would have had an elevated d - dimer , of whom 5 were diagnosed with a pe . although a normal d - dimer value is used to rule out pe , an elevated d - dimer value alone can not indicate a pe . there are many factors that can contribute to the elevation of a d - dimer lab value . d - dimer can be elevated in advanced age , pregnancy , trauma , post - operative periods , inflammatory states , and cancer4 ( table 1 ) . also , it has been found that the d - dimer values are markedly higher in african - americans , who are four times more likely to have an extreme value ( > 600 ng / ml ) than caucasians.8 this is significant because the vast majority of the patient population at jph is of african - american origin . in a study by harper et al,9 it has been found that the median d - dimer concentration increased with age from 294 ng / ml in people aged 1640 years ; to 387 ng / ml in those aged 4060 years ; to 854 ng / ml in those aged 6080 years ; to 1397 ng / ml in those aged 80 + years . the assay specificity decreased with age from 70% in patients < 40 years to less than 5% in patients > 80 years.9 this trend is of particular interest because age is a risk factor for pe as well as contributing to an elevated d - dimer . the d - dimer values of the 220 patients in different age groups , both in the literature8 and at jph , is presented in table 2 . while the average value is increased in the older groups , it is possible that in a larger follow - up analysis , the trend may correlate more with that seen in the study conducted by harper et al.9 douma et al10 found that an age - adjusted d - dimer cut - off point , combined with clinical probability , generally increased the proportion of older patients in whom pe could be safely excluded . raising the cut - off value of the d - dimer test for older patients to values between 600 ng / ml and 1000 ng / ml increases the test s specificity , but at the cost of safety . they suggested a new d - dimer cut - off value to be ( patient s age 10 ) g / l in patients older than fifty years.10 of the five patients diagnosed with a pe , four were older than 60 years . more than 95% of the 220 patients investigated were african american . of the patients studied , 17.1% were between the ages of 1639 years , 46.1% were between the ages of 4059 , 28.6% were between the ages of 6079 , and 7.8% were over 80 years of age . the literature indicates that the positive predictive value of the d - dimer test in diagnosing a pe is approximately 20%.11 at jph the ppv of 4.2% was markedly less . this may be due to the elevated d - dimer values in the predominantly african american and elderly population at the hospital . the combination of the present clinical judgment and rationale by different physicians in the evaluation of pe has resulted in very low yields from the tests . the common practice has been either over - defensive , neglected to account for other possible causes of elevated d - dimer values , or a combination of both . this disparity further supports the need to consider normal physiologic and pathologic conditions , other than pe , that can cause an elevated d - dimer . moderate sensitivity and specificity of clinical signs and symptoms of pe prevent the diagnosis from being made solely on a clinical basis . submitting all patients suspected of having a pe to imaging would not be cost effective . to efficiently and cost - effectively work - up a suspected pe , institution - wide clinical prediction rules must be implemented.12 due to the high false positive rate of the d - dimer , there has been an increase in the use of radiological imaging , length of patient stay , and false positive diagnoses.13 the diagnosis of any illness always begins with a thorough patient history and physical exam . the first step for the diagnosis of a pe should also be the same . these initial guidelines require diagnostic tests , such as chest x - ray or arterial blood gas analysis on room air , that are not always available . more recent rules , like the revised - geneva scoring scale , are based only on clinical elements and it has been demonstrated that patients can be safely managed based on clinical assessment utilizing these scores . implementing a scoring system ( figure 2 ) would establish an appropriate index of suspicion and improve the diagnostic approach towards pe.14 it would also reduce the financial burden of over - testing for suspected pe by increasing the yield of the work - up . the most significant change in the diagnostic approach to acute and chronic pe during the past years has been the utilization of the spiral ct scan . many clinicians have used the spiral ct scan in situations when other diagnostic techniques , for example the ventilation / perfusion scan , have failed to yield a definite diagnosis . according to a study by ost et al,15 the spiral ct has led to an improvement in the diagnosis rate of pe from 1.8 per 1000 hospital admissions to 2.8 per 1000 admissions . in addition to this diagnostic improvement , the perception of the spiral ct being a non - invasive technique has made it favourable . even with the diagnostic improvement of the spiral ct , only one third of patients suspected of pe actually were diagnosed with a pe in the study by ost et al . the remaining two thirds often had such conditions as aortic dissection , pneumonia , lung cancer , metastasis and pneumothorax , all of which are conditions that could be diagnosed with the aid of the ct scan . despite this advancement , there are numerous inconclusive ct scan results.15 these results may be attributed to the fact that current techniques for ct arteriography have the greatest sensitivity and specificity for detecting emboli in the main and lobar arteries.16 the spiral ct scan has a sensitivity of 57%100% and specificity of 78%100% , both of which vary with the location of the emboli . both values are approximately 90% in the main and lobar pulmonary arteries however there is a marked decrease in the segmental and sub - segmental pulmonary vessels.4 even though ct scans are of great advantage , they expose the patient to substantial amounts of radiation which can lead to health risks associated with radiation exposure . although ct scans can be useful in diagnosis of pe , an increased d - dimer should not be the sole reason for a ct scan to be ordered . the average annual natural background radiation exposure is 2.5 msv compared to 7.0 msv of radiation exposure with one ct scan . this is equivalent to about 140 chest radiographs.17 patients can experience a lifelong impact with the increased risk of developing cancer due to the cumulative effects of radiation exposure , whether from a chest radiograph , ct scan or environmental . the current approach has had low yields , has been financially wasteful and has caused unnecessary radiation exposure to patients . it is imperative that patients should not be worked - up for a pe based primarily on an elevated d - dimer value , as there are numerous conditions and pre - disposing factors , other than a pe , that can cause an elevated d - dimer value . two very prominent factors that pertained to the population examined included age and african - american origin . it is proposed that implementing a scoring system , like the revised - geneva scale , will establish a better index of suspicion that will ultimately guide the medical team to improve their diagnostic approach , management , and utilization of resources . such a scale will enable patients with a low risk for pe to have a d - dimer ordered with the objective of ruling out a pe . using similar scales , patients who are suspected to be highly likely to have a pe may have a ct scan of the thorax done . such a system would be systematic and reflect sound scientific practice that would be beneficial to the patient and hospital , alike .
a study conducted in a saudi hospital assessed the opinion of nurses on self report of pain as the indicator of pain intensity revealed that nurses underestimated pain intensity in smiling patient and overestimated it in the grimacing patient ( kaki et al . ( 2000 ) conducted a survey of nurses ' decisions about assessment and treatment of pain in two patient situations and confirmed that nurses continue to under treat severe pain . as shown in previous studies , nurses may be more influenced by the patient 's behavior than the patient 's self - report of pain . surveys have shown that patient self - report of pain is often not considered as a reliable indicator of pain ( pederson 1997 , sloman et al . in a recent report , desai , chaturvedi ( 2009 ) reported that majority of the nurses considered pain behaviors and ignored the subjective component of pain when they were asked to rate the pain on two case vignettes . this study analyses nurses rating of intensity of pain after assessing two mock clinical situations using role plays . the sample was formed by the participants of series of workshop on chronic pain that are being conducted for the health professionals . a workshop was conducted for the postgraduate nursing students at national institute of mental health and neuroscience , bangalore india in november 2009 . two role plays were done by the participants on assessment of pain when the other participants were asked to assess the severity of pain . the participants were asked to observe the interview and rate the severity of pain and suffering on a visual analogue scale ( vas ) of 0 - 10 in both the cases . a 35-year - old woman has been admitted with history of pain in lower back since 1 year . you are asked to assess the severity of pain and rate the pain on vas . you are a 35-year - old woman who has been suffering from pain in her lower back since 1 year . you have been having very severe pain at times . on the day of your assessment your pain is severe and you are unable to sit comfortably , moaning in pain , holding your back with your hands . a 39-year - old man has come with history of pain the upper back since 1 year . you are asked to assess the severity of pain and rate the pain on vas . on examination vitals you are a 39-year - old man who has been suffering from pain in his upper back since 1 year . you have been having very severe pain at times . on the day of your assessment put 1 next to the rating you would record for case 1 and 2 next to the rating you would record for case 2 pain . 0 - --------1 - --------2 - -------3 - -----4 - -----5 - -----6 - -----7 - ------8 - -----9 - ------10 give reasons for your rating : how would you rate the suffering for case 1 and 2 ? put 1 next to the rating you would record for case 1 and 2 next to the rating you would record for case 2 . 0 - --------1 - --------2 - -------3 - -----4 - -----5 - -----6 - -----7 - ------8 - -----9 - ------10 give reasons for your rating : the sample consisted of 14 ( 61% ) women and 9 ( 39% ) men . in case scenario 1 , only 4 ( 17% ) participants rated the correct reported intensity of pain as 7 . in case scenario 2 also , four ( 17% ) rated the intensity of pain as 7 . the pain was underrated , more so in the case scenario 2 . in case scenario 1 : only 2 ( 8.6% ) participants received a score 2 - 3 , 15 ( 65.2% ) received a rating of 4 - 6 . in case scenario 2 : 10 ( 43.5% ) participants received a score 2 - 3 , 8 ( 34.7% ) received a rating of 4 - 6 . the mean , median , and mode for the above cases are shown in table 1 . in both the cases , mean , median , and mode of the observer rating scores of pain as compared to the patient 's rating of pain the mean for suffering for the above cases were 5.48 1.53 for case 1 and 4.30 2.12 for case 2 . in both cases the mean rating for suffering is below 7 which is the expected rating . the reasons for rating pain intensity in case scenario 1 : patient showed grimacingappeared uncomfortablecause was clear patient showed grimacing appeared uncomfortable the reasons for rating of pain in clinical scenario 2 : functioning was not much affectedthe patient was not using analgesics on daily basisthe patient did not show discomfortthe patient did not express pain and feelings functioning was not much affected the patient was not using analgesics on daily basis the patient did not show discomfort the patient did not express pain and feelings the findings illustrate that self report of pain is often not relied for rating the pain intensity by professionals . nurses relied on their observation of the patient 's behavior rather than on the self - report . the observed factors which they took into consideration for rating were facial expression , discomfort in maintaining posture during interview , interference with daily activities , use of analgesics and the cause of the pain . this study corroborates the findings of studies done in other clinical settings ( mccaffrey et al . the mean , mode , and median scores were lower for case 2 who was not showing signs of distress and was trying to distract from pain . despite pain being highly subjective experience , the self - report is not taken into account as indication of pain intensity . a recent report used the written case scenarios methods and concluded that overt behavior and emotions determined pain ratings ( desai , chaturvedi 2009 ) , and this report using clinical interview by role play method confirms that pain ratings are based on many factors and not actually by self report . this exercise was done as a part of microteaching , hence role play was used and the observers were asked to do the assessment of severity of pain along with assessment of other aspects of pain . this method for assessing observer bias in pain assessment the above findings highlight the need for increasing awareness among nurses regarding assessment of pain . health professionals and nurses should not get misled by overt behaviors and emotional distress expressed or suppressed , while assessing pain intensity , and should give credence to the subjective rating mentioned by the person in pain . gender bias in assessment of pain is a possibility that has been raised . in order to maintain anonymity of the responses the participants were asked not to mention their personal details including gender . this is to have an authentic and reliable response by the participants . it is also important to study other factors which may influence the nurses ' assessment of pain . a manual of chronic pain assessment and management describes various methods for pain measurements ( chaturvedi , desai 2009).[for a copy of the manual , please contact the author ] the study highlights that the overt behaviors of pain are given more precedence than the subjective report of the patient in assessment of pain by the nurses , which is misleading . a comprehensive pain assessment is more desirable and is likely to be accurate measure of pain . since nurses play an important role in pain assessment and management it is important to increase the awareness among them by various educational methods .
birth weight is dependent on several parameters , the chief being the gestational age at delivery . of the non - pathological variables , maternal ethnicity , height , and weight , as well as parity definition of small for gestational age baby depends on accurate criteria for expected mean weight and standard deviation . country of birth is often used as a proxy for ethnicity where the population is ethnically relatively homogeneous . infants who are small for gestational age , generally defined as having birth weight below the 10 percentile at a particular gestational week , have a higher risk of various adverse outcomes in perinatal period . creation of a generic reference for fetal weight and birth weight that could be readily adapted to local populations was recently described . the present paper is validation of the reference for birth weights adapted to the local population . information on maternal demographics [ table 1 ] was retrieved from pregnancies from women registration for antenatal care at a charitable maternity hospital in mumbai , india . all pregnancies were dated on crown - rump length obtained before 14 weeks in keeping with accepted nice guidelines . demographic details of women participating in the study we used mean birth weight at 40 weeks and the coefficient of variation for india from the 2004 - 2008 who global survey on maternal and perinatal health , in order to construct the weight percentiles for the calculation of 5 , 10 , 50 , 90 , and 95 centiles . the mean birth weight at 40 weeks of gestation was 2984 g. the results of fitting the observed data to hadlock et al . reported that the use of hadlock reference ranges would lead to 60% of newborns in india being classified as small for gestational age . in our study , the use of hadlock reference ranges led to 47.6% of newborns below the 10 centile and only 1.1% above the 90 centile . we concluded that the hadlock reference ranges did not fit our data and significantly overpredicted birth weights . the data were fitted to who global reference range adapted for india , using the weight percentile calculator , and the results are shown in figure 2 . distribution of birth weights using generic reference range adapted for india the observed distribution of birth weight does not fit the reference range derived from the global reference range adapted to indian population based on the who survey . it identifies approximately one - quarter of all babies to weight above the 90 centile , and therefore was significantly underpredicting birth weights . the reason for this could be the following : mild differences in maternal characteristics as compared to the who study which , however , have been shown to play only a minor role in variation of birth weightsin the who survey , women were included even if the pregnancies were not dated by an early ultrasound scan which may have resulted in inaccuracy of birth weight at 40 weeks . it is well known that women overestimate the gestational age in the absence of early ultrasound dating . mild differences in maternal characteristics as compared to the who study which , however , have been shown to play only a minor role in variation of birth weights in the who survey , women were included even if the pregnancies were not dated by an early ultrasound scan which may have resulted in inaccuracy of birth weight at 40 weeks . it is well known that women overestimate the gestational age in the absence of early ultrasound dating . consequently , we searched for reference ranges of other countries in the who generic reference ranges which might fit our data . sri lankan population is closer to south indian in terms of ethnicity and the median maternal weight and the height for sri lankan women is 154 cm and 60 kg , respectively . table 2 and figure 3 show the use of generic reference adapted to sri lanka for our data . we found that 48.1% were below the 50 centile and 15.9% were above the 90 centile and 12.2% were below the 10 centile . use of generic reference adapted to sri lanka distribution of birth weights using generic reference range adapted for sri lanka we have shown that global reference curves adapted on the basis of who data for india do not fit all the population in india , and the charts need validation . we have also shown that the hadlock reference curves do not fit all the population in india . reference charts modified on the basis of data for sri lankan population fit our data much better , and therefore are more appropriate to use in clinical practice . the strength of our study is that all the participants had secure dating in pregnancy by means of an ultrasound scan in the first half of the pregnancy . weakness of our study includes a relatively modest numbers of participants , and these preliminary results on a small sample size need to be further validated with larger numbers of patients .
a 30-year - old man was admitted to our clinic with complaints of abdominal pain and fever . he had a history of ewing 's sarcoma diagnosed 18 months earlier on his chest wall and he had undergone pneumectomy with negative excision margins , followed by chemotherapy . on physical examination , his saturation was 91% despite oxygen treatment given at a 10/min rate through an oxygen mask . after oral administration of methylene blue , leakage of the substance from the thoracic tube was detected . a contrast - enhanced computed tomography and barium study showed a gastropleural fistula between the stomach and the thorax . gastroscopy confirmed the diagnosis of a fistula , with a massive amount of pus coming from the thorax . treatment with ampicillin / sulbactam and fasting relieved his abdominal pain and fever . after oral feeding was started , his symptoms worsened ; therefore , the patient was administered parenteral nutrition . the gastropleural fistula was in the greater curvature of the stomach in the fundic area . wedge resection of the fistula was performed by three trocars and a 60 gia stapler ( ethicon endo - surgery , cincinnati , oh , usa ) , and the diaphragm was repaired with a prolene graft . the thoracic tube was extracted on the second postoperative day , and the patient was discharged on the fifth postoperative day . pathologic evaluation of the specimen demonstrated the recurrence of ewing 's sarcoma , which may have played a role in the formation of the gastropleural fistula . gastropleural fistula ( gpf ) is a rare condition that can occur as a consequence of prior pulmonary surgery , trauma , or malignancy . they described the causes of gastropleural fistula as intrathoracic perforation of the stomach in hiatal hernia , traumatic diaphragmatic hernia with perforation of the stomach , and intraperitoneal gastric perforation with erosion of a subphrenic abscess via the diaphragm . fistulation between the stomach and the pleural cavity through the diaphragm is an uncommon diagnosis . it has been previously reported as a complication of pulmonary resection , trauma ( especially due to traumatic diaphragmatic hernia ) , peptic ulcer disease , and malignancy . some case reports have indicated a predisposition to gpf after the oral intake of steroids or anti - inflammatory drugs , when the cause is gastric perforation . ewing 's sarcoma is a rare cause of chest wall tumors that comprises 5% of all thoracic tumors and 0.04% of all primary tumors . because of its high grade malignancy , a multidisciplinary approach including wide resection of the tumor , chemotherapy , and also radiotherapy after the resection should be considered . according to a recent review , subphrenic pathologies less frequently lead to formation of a gpf , generally due to diaphragmatic erosion . supradiaphragmatic conditions , such as infections , intrathoracic operations , and fistulas due to procedures ( forceful intercostal tube insertion or nasogastric tube placement after gastric adenocarcinoma resection ) can also result in gpf . to our knowledge , this is the first reported case of a gpf caused by ewing 's sarcoma invasion , which presented 1.5 years after the operation . the diagnosis of gpf is usually determined by contrast radiology , upper gastrointestinal endoscopy , and pleural fluid testing . the use of methylene blue as a marker , and the testing of the pleural fluid for ph or bile salts have been suggested as easy bedside tests for gpf . conservative management usually fails in gpf , and gastrectomy and even thoracotomy are often required , especially in debilitated patients . in conclusion , although gpfs are relatively uncommon , they require prompt surgical intervention . laparoscopic treatment of a gpf may be associated with less morbidity , especially in debilitated patients , and should probably be considered as the initial procedure of choice .
in mid - april 2010 , influenza - like illness was reported in pigs in a north yorkshire gilt ( female pig intended for breeding that has not farrowed ) grower unit of 1,200 animals . gilts were brought into the unit in batches of 100 animals at 5 months of age . the first batch of gilts arrived in mid - january 2010 ; previously , the unit did not contain animals for > 4 months . gilts were housed in stable groups of 20 in a naturally vented building with a straw yard and remained in the unit for 70 days . the nearest pig farm was 3 miles away . a persistent moist cough and signs typical of epizootic swine influenza seven days after the onset of clinical signs , nasal swabs and serum samples were obtained from 6 pigs , and serum samples were obtained from 4 acutely affected pigs . convalescent - phase serum samples were obtained from 9 pigs in the same batch 21 days later . total rna was extracted from swab eluant and amplified by using an m gene real - time reverse transcription pcr ( rt - pcr ) capable of detecting pandemic ( h1n1 ) 2009 virus ( 9 ) ; 4 of 6 swabs were positive . none of the samples were positive for pandemic ( h1n1 ) 2009 virus with a modified real - time rt - pcr specific for the ha gene ( 9 ) . only the sample positive by real - time rt - pcr with the lowest cycle threshold value yielded virus when inoculated into embryonated fowl eggs ( 10 ) . egg - grown virus was identified as subtype h1n2 by using hemagglutinin inhibition ( hi ) and na inhibition with standard methods ( 10 ) and designated a / swine / england/1382/10 ( h1n2 ) . a / swine / england/1382/10 was characterized by using whole genome sequencing and phylogenetic analysis . gene fragments were amplified by using a 1-step real - time rt - pcr ( qiagen , hilden , germany ) , and ha and na genes were sequenced by using subtype h1n2 virus specific primers ( 5 ) . partial internal gene segment sequencing was initially performed by using primer pairs ( 5 ) . full sequencing of internal gene segments used universal ( np , m , and nonstructural genes ) and pandemic ( h1n1 ) 2009 virus specific primers ( pb2 , pb1 , acidic polymerase , and np genes ) . analysis of sequence data by blast analysis ( http://blast.ncbi.nlm.nih.gov ) determined the closest similarity to influenza virus isolates in the genbank database . a / swine / england/1382/10 had ha and na genes closely related to uk swine subtype h1n2 viruses ( table 1 ) . all genes encoding internal proteins showed the highest similarity to pandemic ( h1n1 ) 2009 viruses ( table 1 ) . * pb , basic polymerase ; pa , acidic polymerase ; ha , hemagglutinin ; np , nucleoprotein , na , neuraminidase ; m , matrix ; ns , nonstructural ; a , genes with closest homology to european avian - like swine influenza viruses ( h1n1 ) ; h , genes with closest homology to european swine influenza viruses ( h1n2 ) ; nd , gene not sequenced but typed as n2 by using standard na inhibition assay ; p , genes with closest homology to pandemic ( h1n1 ) 2009 virus . closest matching homologous genes were determined by analyzing nucleotide sequences ( minimum of 180 bases ) against the national center for biotechnology information genbank database ( http://blast.ncbi.nlm.nih.gov ) . ha and na genes of a / swine / england/1382/10 grouped within the european swine subtype h1n2 cluster , specifically , with contemporary subtype h1n2 isolates from england . the closest matching isolate for ha and na was a / swine / england/1428/09 , which is reported in this article ( figure ) . phylogenetic analysis of influenza a virus hemagglutinin ( a ) and neuraminidase ( b ) genes . solid diamonds indicate a / swine / england/1382/10 genes from virus isolated in this study , and open diamonds indicate genes from other viruses reported in this study . percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1,000 replicates ) is shown next to the branches for values > 70% ( 11 ) . the m gene of a / swine / england/1382/10 had the s31n amantadine - resistance mutation , typical of pandemic ( h1n1 ) 2009 viruses . it also had 627e and 701d mutations in the pb2 gene and mutation 591r , a basic amino acid that reportedly compensates for lack of the 627k mammalian - adaptive mutation ( 14 ) . the pb1-f2 open - reading frame encoded a truncated pb1-f2 protein of 11 aa , consistent with other pandemic ( h1n1 ) 2009 viruses . the na gene has mutations 119e and 292r , which are associated with susceptibility to oseltamivir in n2 subtypes . since the emergence of pandemic ( h1n1 ) 2009 virus , 5 other subtype h1n2 viruses have been detected in pigs in the united kingdom . partial sequencing of internal genes of these viruses showed they were not reassortants ( table 1 ) . reassortment was not detected in a european avian - like swine subtype h1n1 virus isolated from another pig unit in the same region in april 2010 , or in a pandemic ( h1n1 ) 2009 virus isolated from another pig unit in the same region in june 2010 ( table 1 ) . the 10 acute - phase and 9 convalescent - phase serum samples were subjected to standard hi tests ( 10 ) with antigens from a / swine / england/195852/92 ( avian - like subtype h1n1 ) , a / swine / england/1353/09 pandemic ( h1n1 ) 2009 virus , a / swine / england/438207/94 ( subtype h1n2 ) , and homologous a / swine / england/1382/10 ( table 2 ) . acute - phase serum samples were positive for antibodies against pandemic ( h1n1 ) 2009 virus . antibody titers to endemic and reassortant subtype h1n2 viruses were negligible in acute - phase serum samples but increased 14-fold and 16-fold , respectively , in convalescent - phase serum samples . * acute - phase serum samples were obtained from 10 pigs in the same batch during the time when clinical signs were apparent ( 2 wk after arrival at the unit ) . convalescent - phase serum samples were obtained from 9 pigs in the same batch 21 d later . standard hemagglutinin inhibition assays were conducted with subtype h1n1 , pandemic ( h1n1 ) 2009 , and subtype h1n2 virus antigens derived from uk swine isolates . we report detection of a novel reassortant virus between pandemic ( h1n1 ) 2009 virus and a swine subtype h1n2 virus . in contrast to an earlier report of a reassortant virus that contained the na gene of pandemic ( h1n1 ) 2009 virus ( 8) , in this study all genes encoding internal proteins of a / swine / england/1382/10 virus are derived from pandemic ( h1n1 ) 2009 virus . appearance of clinical signs 2 weeks after arrival in the unit suggests that pigs were not previously infected with either a precursor or reassortant virus . however , detection of antibodies against pandemic ( h1n1 ) 2009 virus in pigs coinciding with appearance of clinical signs suggests earlier subclinical infection with pandemic ( h1n1 ) 2009 virus preceding co - circulation of subtype h1n2 or reassortant h1n2 viruses once pigs arrived at the unit . therefore , it is unclear whether a / swine / england/1382/10 can be transmitted between pigs or has any selective advantage . our serologic results and those of others ( 15 ) indicate that antibodies against pandemic ( h1n1 ) 2009 virus or subtype h1n2 virus produced during natural infection of pigs do not show cross - reactivity in hi tests . therefore , pandemic ( h1n1 ) virus and subtype h1n2 virus may continuously circulate in pigs in europe , providing additional opportunities for reassortment .
recurrence of bladder cancer is common , and is reported to occur in 50%-70% of cases , and approximately 10%-15% of patients will experience disease progression . detection of bladder cancer when it is still at an early stage is important for effective treatment , and currently the standard approach in detection of bladder cancer is urethrocystoscopy and urine cytology . urine cytology has a reasonable sensitivity for detection of high - grade bladder cancer ; however , it is restricted in its capacity for detection of low - grade urothelial carcinoma , with sensitivity and specificity of 8.5% and 50% , respectively . unfortunately , biomarkers for bladder cancer that can provide reliable information about diagnosis , progression , and surveillance have not been clearly established . development of a bladder tumor biomarker with high sensitivity and specificity would be a useful adjunct tool for detection or follow - up of cases of bladder cancer . a bladder cancer usually originates from cells lining the bladder transitional cells , which could be exposed to several cytokines during chronic inflammation and dna damage . the urinary bladder is a sac which acts as a reservoir containing metabolites , waste products , and several releasing proteins from the cells . interestingly , it was reported that redox regulating and dna repair protein such as apurinic / apyrimidinic endonuclease 1/redox factor-1 ( ape1/ref-1 ) was increased with occurrence of cancer . ape1/ref-1 is a multifunctional protein involved in both base excision dna repair and transcriptional regulation . there is growing evidence linking heterogeneous ape1/ref-1 expression to a wide range of pathological conditions , including metabolic and differentiation disorders such as cancers . ape1/ref-1 is mainly localized in the nucleus , but cytoplasmic and mixed nuclear / cytoplasmic localization of ape1/ref-1 has been reported in several tumor types . active secretion of ape1/ref-1 into the circulation also occurs in response to lipopolysaccharides or intracellular acetylation , so that it could act as a serologic biomarker . ape1/ref-1 secretion from cells is also supported by the presence of auto - antibody against ape1/ref-1 in blood of lung cancer patients . because tumor biomarkers may originate from tumors or adjacent tissue in response to tumor growth therefore , the aim of the current study was to determine whether serum ape1/ref-1 levels are elevated in bladder cancer patients . the study groups were classified as either non - cancer controls ( n=55 ) , consisting of individuals with no evidence of malignancy , or patients with operable bladder cancer ( n=51 ) . all samples were obtained from the archives of the department of urology , chungnam national university from 2010 - 2011 . this study was approved by the chungnam national university hospital institutional review board , and all participants signed informed consent forms . in the control group , we included patients with benign prostate hyperplasia ( n=22 ) , a urinary tract stone ( n=18 ) , trauma ( n=11 ) , and urethral stricture ( n=4 ) . we excluded patients with chronic or recurrent urinary tract infections , unevaluated gross hematuria , and malignancy . in the bladder cancer group , postoperative histological confirmation of urothelial cell carcinoma , including grade and stage , was recorded . thirty - eight patients underwent transurethral resection of bladder cancer and 13 patients underwent radical or partial cystectomy . blood ( 8 - 10 ml ) was drawn from an antecubital vein in patients with bladder cancer or healthy control subjects using a 22-g needle and placed in a vacuum tube . serum was separated by centrifugation at 3,000 rpm for 10 minutes at room temperature , and then re - centrifuged at 5,000 rpm for 5 minutes to obtain cell - free serum , which was stored in liquid nitrogen until use . the clinical and pathological characteristics of the bladder cancer subjects , which were divided according to nonmuscle invasive bladder cancer ( nmibc ) and muscle invasive bladder cancer ( mibc ) , are shown in table 1 . clinical staging of bladder cancer was performed using cystoscopy , computed tomography , bone scanning , and simple chest radiography . all tumor tissues were analyzed by a single genitourinary pathologist according to a standardized protocol . pathological staging was recorded in accordance with the 2003 tnm classification and the tumors were assigned a grade according to the world health organization classification . tumors were thus classified as ta ( noninvasive papillary carcinoma ) , t1 ( invasive up to the subepithelial connective tissue ) , t2 ( invading the muscle ) , t3 ( invading the perivesical fat ) , and t4 ( invading an adjacent organ ) . tumors were also graded based on the likelihood of recurrence and progression into grade i ( tumors may recur but have only a low risk of progression ) , grade ii ( tumors are more likely to recur and progress compared with grade i ) , and grade iii ( tumors are very likely to recur and progress ) . bladder cancer patients were followed - up , and cystoscopy and urine cytology were performed every 3 months for the first year , every 6 months for the second year , and annually thereafter . recurrence was defined as transitional cell carcinomas with lower or equivalent pathologic stage after treatment . for mibc patients who had undergone partial cystectomy or radiation treatment , reappearance of bladder cancer including ta and carcinoma in situ 96-microwell plates ( nunc , penfield , ny ) were precoated overnight with 100 l of a 1:1,000 dilution of a rabbit anti - ape1/ref-1 antibody ( abcam , cambridge , uk ) in coating buffer ( 0.5 m carbonate buffer , ph 9.6 ) in each well . after blocking with blocking buffer ( 5% bovine serum albumin in phosphate buffered saline [ pbs ] containing 0.05% tween 20 [ pbs - t ] ) at room temperature for 60 minutes , 100 l of sample was added to the wells and the plates were incubated at 4c for 90 minutes , and then washed five times with pbs - t . this was followed by addition of 100 l of a 1:1,000 dilution of mouse anti - ape1/ref-1 antibody ( abcam ) , and further incubation at room temperature for 2 hours . the plate was then washed seven times with pbs - t , and 100 l of horseradish peroxidase conjugated secondary antibody ( 1:5,000 ) was added , followed by incubation at room temperature for 30 minutes . after further washing , 100 l of freshly prepared tetramethyl benzidine substrate was added to the wells . the color development reaction was stopped by addition of 100 l of 2.5 m h2so4 , and the absorbance was measured at 450 nm using an automatic microtiter plate reader ( sunrise xfluor 4 , tecan systems inc . , san jose , ca ) . each sample was assayed in duplicate , and mean values were determined . to establish a standard curve , purified recombinant human ape1/ref-1 ( 1 mg / ml ) was serially diluted ( 5-fold ) and used in a concentration series from 0.16 - 20 ng/100 l . human full length ape1/ref-1 dna was inserted into the pet28b expression vector ( novagen , gibbstown , nj ) , containing a 6-histidine tag for easy purification . pet28b - ape1/ref-1 plasmids were then transformed into the bl21(de3 ) strain of escherichia coli . following induction with isopropyl -d-1-thiogalactopyranoside ( iptg ) , the cells were sonicated in lysis buffer ( 100 mm nacl , 20 mm hepes ) , and the recombinant protein was purified on a nickel - nitrilotriacetic acid agarose column ( qiagen , valencia , ca ) . after washing , the isolated ape1/ref-1 protein was eluted with 250 mm imidazole buffer followed by desalting on a pd-10 column ( amersham pharmacia biotech , liverpool , uk ) in pbs , and frozen in 10% glycerol at 80c . imunohistochemical staining of ape1/ref-1 was determined in paraffin - embedded sections of bladder tissues , which were stored in the tissue bank of chungnam national hospital . tissue specimens were cut into 4-m sections from randomly selected tumor and non - tumor blocks . distant , apparently normal tissue samples from the same patient were used as the non - tumor blocks . a monoclonal antibody against human ape1/ref-1 was used at a 1:600 dilution ( novus biological , littleton , co ) . the sections were lightly counterstained with hematoxylin and mounted using immu - mount ( thermo shandon , midland , canada ) . negative control slides were incubated with the mouse igg1 negative control reagent provided with the kit for evaluation of nonspecific staining . tumor tissues from bladder cancer patients were obtained from the tissue bank of chungnam national hospital . distant , apparently normal tissue samples from the same patient were used as the non - tumor tissues . tissues were suspended in pbs containing protease and phosphatase inhibitors , cut into small pieces , and homogenized using a polytron . the homogenates were centrifuged at 14,000 rpm for 40 minutes and the resultant supernatant fractions were used for immunoblotting . the proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane . after blocking with 5% skimmed milk powder , the blots were incubated with anti ape1/ref-1 antibody ( 1:1,000 , abcam ) . each membrane was stripped and re - probed with an anti glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) antibody to ensure equivalent protein loading . changes in protein levels were quantified by densitometric scanning of the immunoreactive band and normalized against gapdh loading control . a statistical evaluation was performed using a one - way analysis of variance and post - hoc analysis . differences were considered statistically significant if the null hypothesis could be rejected with > 95% confidence interval ( p < 0.05 ) . nonparametric receiver operating characteristic ( roc ) curves were generated by plotting the sensitivity value against the false - positive rate ( 1specificity).we assessed the potential predictive value of ape1/ref-1 for bladder cancer as well specific cancer stages and grades , and recurrence , by calculating the area under the curve ( auc ) . we estimated the sensitivity and specificity of ape1/ref-1 at the optimal cutoff value ( youden index ) to maximize the sum of sensitivity and specificity . the study groups were classified as either non - cancer controls ( n=55 ) , consisting of individuals with no evidence of malignancy , or patients with operable bladder cancer ( n=51 ) . all samples were obtained from the archives of the department of urology , chungnam national university from 2010 - 2011 . this study was approved by the chungnam national university hospital institutional review board , and all participants signed informed consent forms . in the control group , we included patients with benign prostate hyperplasia ( n=22 ) , a urinary tract stone ( n=18 ) , trauma ( n=11 ) , and urethral stricture ( n=4 ) . we excluded patients with chronic or recurrent urinary tract infections , unevaluated gross hematuria , and malignancy . in the bladder cancer group , postoperative histological confirmation of urothelial cell carcinoma , including grade and stage , was recorded . thirty - eight patients underwent transurethral resection of bladder cancer and 13 patients underwent radical or partial cystectomy . blood ( 8 - 10 ml ) was drawn from an antecubital vein in patients with bladder cancer or healthy control subjects using a 22-g needle and placed in a vacuum tube . serum was separated by centrifugation at 3,000 rpm for 10 minutes at room temperature , and then re - centrifuged at 5,000 rpm for 5 minutes to obtain cell - free serum , which was stored in liquid nitrogen until use . the clinical and pathological characteristics of the bladder cancer subjects , which were divided according to nonmuscle invasive bladder cancer ( nmibc ) and muscle invasive bladder cancer ( mibc ) , are shown in table 1 . clinical staging of bladder cancer was performed using cystoscopy , computed tomography , bone scanning , and simple chest radiography . all tumor tissues were analyzed by a single genitourinary pathologist according to a standardized protocol . pathological staging was recorded in accordance with the 2003 tnm classification and the tumors were assigned a grade according to the world health organization classification . tumors were thus classified as ta ( noninvasive papillary carcinoma ) , t1 ( invasive up to the subepithelial connective tissue ) , t2 ( invading the muscle ) , t3 ( invading the perivesical fat ) , and t4 ( invading an adjacent organ ) . tumors were also graded based on the likelihood of recurrence and progression into grade i ( tumors may recur but have only a low risk of progression ) , grade ii ( tumors are more likely to recur and progress compared with grade i ) , and grade iii ( tumors are very likely to recur and progress ) . bladder cancer patients were followed - up , and cystoscopy and urine cytology were performed every 3 months for the first year , every 6 months for the second year , and annually thereafter . recurrence was defined as transitional cell carcinomas with lower or equivalent pathologic stage after treatment . for mibc patients who had undergone partial cystectomy or radiation treatment , reappearance of bladder cancer including ta and carcinoma in situ a sandwich enzyme - linked immunosorbent assay ( elisa ) was used to quantify serum ape1/ref-1 levels . briefly , 96-microwell plates ( nunc , penfield , ny ) were precoated overnight with 100 l of a 1:1,000 dilution of a rabbit anti - ape1/ref-1 antibody ( abcam , cambridge , uk ) in coating buffer ( 0.5 m carbonate buffer , ph 9.6 ) in each well . after blocking with blocking buffer ( 5% bovine serum albumin in phosphate buffered saline [ pbs ] containing 0.05% tween 20 [ pbs - t ] ) at room temperature for 60 minutes , 100 l of sample was added to the wells and the plates were incubated at 4c for 90 minutes , and then washed five times with pbs - t . this was followed by addition of 100 l of a 1:1,000 dilution of mouse anti - ape1/ref-1 antibody ( abcam ) , and further incubation at room temperature for 2 hours . the plate was then washed seven times with pbs - t , and 100 l of horseradish peroxidase conjugated secondary antibody ( 1:5,000 ) was added , followed by incubation at room temperature for 30 minutes . after further washing , 100 l of freshly prepared tetramethyl benzidine substrate was added to the wells . the color development reaction was stopped by addition of 100 l of 2.5 m h2so4 , and the absorbance was measured at 450 nm using an automatic microtiter plate reader ( sunrise xfluor 4 , tecan systems inc . , san jose , ca ) . each sample was assayed in duplicate , and mean values were determined . to establish a standard curve , purified recombinant human ape1/ref-1 ( 1 mg / ml ) was serially diluted ( 5-fold ) and used in a concentration series from 0.16 - 20 ng/100 l . human full length ape1/ref-1 dna was inserted into the pet28b expression vector ( novagen , gibbstown , nj ) , containing a 6-histidine tag for easy purification . pet28b - ape1/ref-1 plasmids were then transformed into the bl21(de3 ) strain of escherichia coli . following induction with isopropyl -d-1-thiogalactopyranoside ( iptg ) , the cells were sonicated in lysis buffer ( 100 mm nacl , 20 mm hepes ) , and the recombinant protein was purified on a nickel - nitrilotriacetic acid agarose column ( qiagen , valencia , ca ) . after washing , the isolated ape1/ref-1 protein was eluted with 250 mm imidazole buffer followed by desalting on a pd-10 column ( amersham pharmacia biotech , liverpool , uk ) in pbs , and frozen in 10% glycerol at 80c . imunohistochemical staining of ape1/ref-1 was determined in paraffin - embedded sections of bladder tissues , which were stored in the tissue bank of chungnam national hospital . tissue specimens were cut into 4-m sections from randomly selected tumor and non - tumor blocks . distant , apparently normal tissue samples from the same patient were used as the non - tumor blocks . a monoclonal antibody against human ape1/ref-1 was used at a 1:600 dilution ( novus biological , littleton , co ) . staining the sections were lightly counterstained with hematoxylin and mounted using immu - mount ( thermo shandon , midland , canada ) . negative control slides were incubated with the mouse igg1 negative control reagent provided with the kit for evaluation of nonspecific staining . tumor tissues from bladder cancer patients were obtained from the tissue bank of chungnam national hospital . distant , apparently normal tissue samples from the same patient were used as the non - tumor tissues . tissues were suspended in pbs containing protease and phosphatase inhibitors , cut into small pieces , and homogenized using a polytron . the homogenates were centrifuged at 14,000 rpm for 40 minutes and the resultant supernatant fractions were used for immunoblotting . the proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane . after blocking with 5% skimmed milk powder , the blots were incubated with anti ape1/ref-1 antibody ( 1:1,000 , abcam ) . each membrane was stripped and re - probed with an anti glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) antibody to ensure equivalent protein loading . changes in protein levels were quantified by densitometric scanning of the immunoreactive band and normalized against gapdh loading control . a statistical evaluation was performed using a one - way analysis of variance and post - hoc analysis . differences were considered statistically significant if the null hypothesis could be rejected with > 95% confidence interval ( p < 0.05 ) . nonparametric receiver operating characteristic ( roc ) curves were generated by plotting the sensitivity value against the false - positive rate ( 1specificity).we assessed the potential predictive value of ape1/ref-1 for bladder cancer as well specific cancer stages and grades , and recurrence , by calculating the area under the curve ( auc ) . we estimated the sensitivity and specificity of ape1/ref-1 at the optimal cutoff value ( youden index ) to maximize the sum of sensitivity and specificity . serum levels of ape1/ref-1 in bladder cancer patients were significantly elevated compared to those of the control group ( 3.5480.333 ng/100 l [ n=51 ] for bladder cancer vs. 1.5470.319 ng/100 l [ n=55 ] for the control group ) ( fig . the us national cancer institute recommends use of roc curve for evaluation of the performance of potential cancer detection markers . serum samples from 55 non - cancer controls and 51 patients with bladder cancer were used for the roc curve analysis to determine the sensitivity and specificity of ape1/ref-1 in detection of bladder cancer . the roc curve for all tumors resulted in an auc of 0.824 ( 95% confidence interval , 0.74 to 0.90 ) ( fig . the range of sensitivity and specificity at these different values was 0.51 - 0.95 and 0.51 - 0.86 , respectively ( table 2 ) . the sensitivity and specificity of ape1/ref-1 were estimated at the optimal cutoff value in order to maximize the sum of sensitivity and specificity . on this basis , the optimal combination of sensitivity and specificity were determined to be 0.93 and 0.59 , using a cut - off value of 2.83 ng/100 l ( table 2 ) . the grade of bladder cancer provides important prognostic information and can help guide treatment , and is based on the degree of cellular abnormality . we found that serum ape1/ref-1 levels were associated with the grade of bladder cancer ( fig . 2a ) . in particular , the mean values of serum ape1/ref-1 levels of patients with grade ii and iii bladder cancer ( 2.7190.465 ng/100 l for grade ii , n=12 ; 4.8910.389 ng/100 l for grade iii , n=23 ) were significantly greater than those of the control group ( 1.5470.319 ng/100 l , n=55 ) . the roc - auc for grade i , grade ii , and grade iii cancers were 0.70 , 0.71 , and 0.96 , respectively ( fig . 2b ) , suggesting that serum ape1/ref-1 may serve as a predictor of tumor grade . in addition to grade , bladder cancer is also staged by depth of invasion into the bladder wall . thus , we attempted to determine whether serum ape1/ref-1 is also associated with the tumor stage ( fig . 2c ) . serum ape1/ref-1 levels were significantly elevated among patients with ta ( 2.4500.365 ng/100 l ) , t1 ( 3.2510.305 ng/100 l ) , and t2-t3 ( 5.7110.437 ng/100 l ) bladder tumors compared to the control groups . the roc auc for ta , t1 , and t2-t4 were 0.67 , 0.87 , and 0.99 , respectively ( fig . 2d ) . serum ape1/ref-1 levels in mibc ( 5.7110.437 ng/100 l ) were significantly elevated , compared with nmibc ( 2.8010.249 ng/100 l ) ( fig . the roc - auc for nmibc and mibc were 0.76 and 0.99 , respectively ( fig . next , we attempted to determine whether serum ape1/ref-1 levels were also elevated in bladder cancer recurrence . among the 51 patients with bladder cancer , 20 patients ( 39% ) had recurrent bladder cancer the mean serum ape1/ref-1 level titer in these patients was ( 4.4800.430 ng/100 l ) , significantly greater than in patients with non - recurrent tumors ( 2.9400.324 ng/100 l ) ( fig . the auc for non - recurrent tumors was 0.75 , and the auc for recurrent tumors was 0.93 ( fig . 2 g ) , suggesting a high predictive value of serum ape1/ref-1 for recurrent bladder tumors . serum ape1/ref-1 levels in patients with multifocal tumors did not differ significantly from those who had single tumors ( data not shown ) . to determine whether ape1/ref-1 protein was differentially expressed in bladder cancer , immunoblotting for ape1/ref-1 was performed in non - tumor tissues and tumor tissues of bladder cancer ( fig . differences in protein level were quantified by densitometric scanning of the immunoreactive bands and normalized against the gapdh loading control . this revealed that ape1/ref-1 protein was expressed at a far higher level in tumor tissue ( t ) compared to non - tumor tissue as normal ( n ) ( fig . ape1/ref-1 protein expression in tumor and non - tumor tissues of bladder cancer was visualized using immunohistochemistry ( fig . 3b ) . ape1/ref-1 in non - tumor tissues and tumor tissues were mainly localized in the nucleus . in addition , mononuclear cells including monocytes and lymphocytes were infiltrated close to tumor regions . serum levels of ape1/ref-1 in bladder cancer patients were significantly elevated compared to those of the control group ( 3.5480.333 ng/100 l [ n=51 ] for bladder cancer vs. 1.5470.319 ng/100 l [ n=55 ] for the control group ) ( fig . the us national cancer institute recommends use of roc curve for evaluation of the performance of potential cancer detection markers . serum samples from 55 non - cancer controls and 51 patients with bladder cancer were used for the roc curve analysis to determine the sensitivity and specificity of ape1/ref-1 in detection of bladder cancer . the roc curve for all tumors resulted in an auc of 0.824 ( 95% confidence interval , 0.74 to 0.90 ) ( fig . the range of sensitivity and specificity at these different values was 0.51 - 0.95 and 0.51 - 0.86 , respectively ( table 2 ) . the sensitivity and specificity of ape1/ref-1 were estimated at the optimal cutoff value in order to maximize the sum of sensitivity and specificity . on this basis , the optimal combination of sensitivity and specificity were determined to be 0.93 and 0.59 , using a cut - off value of 2.83 ng/100 l ( table 2 ) . the grade of bladder cancer provides important prognostic information and can help guide treatment , and is based on the degree of cellular abnormality . we found that serum ape1/ref-1 levels were associated with the grade of bladder cancer ( fig . 2a ) . in particular , the mean values of serum ape1/ref-1 levels of patients with grade ii and iii bladder cancer ( 2.7190.465 ng/100 l for grade ii , n=12 ; 4.8910.389 ng/100 l for grade iii , n=23 ) were significantly greater than those of the control group ( 1.5470.319 ng/100 l , n=55 ) . the roc - auc for grade i , grade ii , and grade iii cancers were 0.70 , 0.71 , and 0.96 , respectively ( fig . 2b ) , suggesting that serum ape1/ref-1 may serve as a predictor of tumor grade . in addition to grade , bladder cancer thus , we attempted to determine whether serum ape1/ref-1 is also associated with the tumor stage ( fig . 2c ) . serum ape1/ref-1 levels were significantly elevated among patients with ta ( 2.4500.365 ng/100 l ) , t1 ( 3.2510.305 ng/100 l ) , and t2-t3 ( 5.7110.437 ng/100 l ) bladder tumors compared to the control groups . the roc auc for ta , t1 , and t2-t4 were 0.67 , 0.87 , and 0.99 , respectively ( fig . 2d ) . serum ape1/ref-1 levels in mibc ( 5.7110.437 ng/100 l ) were significantly elevated , compared with nmibc ( 2.8010.249 ng/100 l ) ( fig . the roc - auc for nmibc and mibc were 0.76 and 0.99 , respectively ( fig . next , we attempted to determine whether serum ape1/ref-1 levels were also elevated in bladder cancer recurrence . among the 51 patients with bladder cancer , 20 patients ( 39% ) had recurrent bladder cancer the mean serum ape1/ref-1 level titer in these patients was ( 4.4800.430 ng/100 l ) , significantly greater than in patients with non - recurrent tumors ( 2.9400.324 ng/100 l ) ( fig . the auc for non - recurrent tumors was 0.75 , and the auc for recurrent tumors was 0.93 ( fig . 2 g ) , suggesting a high predictive value of serum ape1/ref-1 for recurrent bladder tumors . serum ape1/ref-1 levels in patients with multifocal tumors did not differ significantly from those who had single tumors ( data not shown ) . to determine whether ape1/ref-1 protein was differentially expressed in bladder cancer , immunoblotting for ape1/ref-1 was performed in non - tumor tissues and tumor tissues of bladder cancer ( fig . differences in protein level were quantified by densitometric scanning of the immunoreactive bands and normalized against the gapdh loading control . this revealed that ape1/ref-1 protein was expressed at a far higher level in tumor tissue ( t ) compared to non - tumor tissue as normal ( n ) ( fig . ape1/ref-1 protein expression in tumor and non - tumor tissues of bladder cancer was visualized using immunohistochemistry ( fig . 3b ) . ape1/ref-1 in non - tumor tissues and tumor tissues were mainly localized in the nucleus . in addition , mononuclear cells including monocytes and lymphocytes were infiltrated close to tumor regions . in this report , we describe a new potential diagnostic marker for bladder cancer . in clinical practice , the differential diagnosis of bladder cancer grade using non - invasive techniques is difficult . our study demonstrates for the first time that ape1/ref-1 protein is detectable in the serum of patients with bladder cancer , and is associated with tumor stage , grade , and recurrence . the underlying mechanism for serum ape1/ref-1 existence was completely uncovered . in the current study , our data showed increased expression of ape1/ref-1 in tumor tissues and mononuclear cell infiltration close to tumor tissues . ape1/ref-1 can be secreted from tumor cells itself and its secretion can also be evoked by cellular signaling between tumor and inflammatory cells . a previous study showed that intense inflammation occurs in patients with invasive bladder carcinoma , and it is likely that metalloproteinase-9 is derived from inflammatory cells infiltrating tumors . in addition , nitric oxide ( no ) is elevated in the urine of patients with bladder cancer . no influences the subcellular localization of ape1/ref-1 by nitrosation , and may similarly lead to extracellular translocation of ape1/ref-1 . in previous reports we showed that ape1/ref-1 was secreted into the circulation in response to lipopolysaccharide , which can induce no and tumor necrosis factor production . thus , the release of ape1/ref-1 by tumor cells into the circulation might be a result of tumor - mediated inflammation . for measurement of secretory ape1/ref-1 into blood , we developed a new elisa assay and obtained roc curve by applying to human serum samples . the roc curve is used to evaluate the usefulness of a biomarker for classifying disease status . we used roc curve based on the youden index , the sum of sensitivity and specificity minus one , for setting an optimal cut - point for use in diagnostic testing . a cut - off ape1/ref-1 value of 2.83 ng/100 l resulted in a sensitivity of 93% and a specificity of 59% . in the current study , we showed that increased serum ape1/ref-1 levels are associated with bladder cancer severity , suggesting that it could act as a potential serologic biomarker for bladder cancer . in studies of nuclear maxtrix protein 22 ( nmp-22 ) as a urinary marker of bladder cancer , the results vary widely depending on the reporting group : values of 47%-100% for sensitivity and 58%-91% for specificity with this test have been previously reported . in a recent prospective test for nmp-22 test they concluded that nmp-22 assessment alone can not be recommended for bladder cancer screening in a high - risk group nor as a reliable alternative to cystoscopy during follow - up . urine cytology is a more widely accepted noninvasive tumor - specific marker for bladder cancer . however , despite being highly specific for carcinoma in situ and high - grade urothelial carcinoma , it has a relatively low sensitivity for low grade carcinoma . the majority of bladder cancers were diagnosed low grade and stage tumors , the relative value of cytology is limited . collectively , we have demonstrated that ape1/ref-1 protein is detectable in the serum of patients with bladder cancer , and its level is associated with tumor stage and recurrence . our findings provide a good foundation for development of a new serological biomarker for bladder cancer .