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breast density is defined as the proportion of fibroglandular tissue in the total breast volume. it can be influenced by age, menopausal status, and ethnicity (123). the proportion of dense breasts tends to decrease as patients age and go through menopause (123). dense breasts are well known as a risk factor of breast cancer, and it can also negatively influence the accuracy of breast cancer screening with mammography (24). mammography is the primary imaging modality for breast cancer screening worldwide. despite being acknowledged as a first - line tool, multiple studies have reported that its sensitivity may be as low as 30% to 48% for dense breasts (456). the reason is that the dense breast tissue can obscure breast cancers on mammography, and this can give false - negative results. this is an important issue for women who have dense breast tissue because a negative mammography does not confirm that the patient does not have breast cancer. this means that women at high risk for breast cancer may need further work - up for breast cancer screening, such as breast ultrasonography or breast magnetic resonance imaging. recently, several states in the united states have enacted a law requiring radiologists to notify a patient about her breast density, and medical insurance covers more screening examinations for high - risk women ; moreover, the number of states with this policy is increasing (7). therefore, it is important to recognize the exact current prevalence of dense breasts and breast density alterations by age group in our country. mammographic density can be measured by qualitative visual assessment or quantitative computer - aided methods (18). mammographic breast glandular tissue is routinely visually assessed and categorized by four different patterns : type a, entirely fatty ; type b, scattered fibroglandular ; type c, heterogeneous dense ; and type d, extremely dense, according to the guidelines of the breast imaging reporting and data system (bi - rads) (9). however, this visual assessment is subjective so it shows varying degrees of inter - observer agreement (10). in comparison, the quantitative computer - aided measurement is objective and can be roughly categorized into area - based measurement and volumetric measurement methods (11). the fully automated volumetric assessment (fava) system has recently been developed, and several reports have shown a relatively good correlation between fava and the bi - rads visual density category (81112). applying the results of fava to breast density would be more helpful for personalizing breast cancer screening and developing appropriate national breast cancer screening strategies because breast density is an important risk factor (24). there was a previous study which compared breast density on mammograms between korean and western women according to age (13). however, it was an only report and there has been no study regarding the objective breast density by age using fava, which could be used for standard reference. therefore, the purpose of this study was to present the mean breast density of korean women according to age groups using fava and to compare it with that of women in other countries. between august 20 and october 7, 2013, a total of 6,751 women who underwent screening mammography with full - field digital mammography (ffdm) at our institution for private and national breast cancer screening programs were enrolled. this included some women younger than 40 years because they want to perform screening ffdm, although the korean national breast cancer screening program targeted at women older than 40 years. the exclusion criteria in this study were as follows : 1) women were younger than 30 years (n = 70) ; 2) they had a history of breast surgery (n = 68) ; and 3) they showed the bi - rads category results of 0 (incomplete), 3 (probably benign), 4 (suspicious abnormality), or 5 (highly suggestive of malignancy) (n = 646). finally, a total of 5,967 women who were over 30 years of age and showed bi - rads category 1 (negative) or bi - rads category 2 (benign) results were included. all ffdms were performed with standard craniocaudal and mediolateral oblique views of both breasts on ffdm units (senographe ds, ge healthcare, milwaukee, wi, usa ; brestige, medi - future, seongnam, korea). the volume of mammographic density was calculated using volpara imaging software (version 1.5.5, mtakina technology, wellington, new zealand), which is a fda - approved program for ffdm images. this software calculates the breast density by computing the x - ray attenuation at each pixel on ffdm by comparing the pixel value at the reference point to the pixel value at each pixel. by adding up all the values in the density map over the image, it can calculate the fibroglandular tissue volume (ftv) in cubic centimeters, breast tissue volume (btv) in cubic centimeters, and therefore the percentage of the breast that consists of fibroglandular tissue the volumetric breast density (vbd). we divided the vbd into four grades as follows : 0%4.7%, volpara density grade (vdg) 1 ; 4.8%7.9%, vdg 2 ; 8.0%14.9%, vdg 3 ; and 15.0% or higher, vdg 4. patients with vdg 1 and 2 were considered to have fatty breasts, while those with vdg 3 and 4 were considered to have dense breasts (8). all statistical analyses were performed using pasw statistics 18.0 (ibm, armonk, ny, usa). the kolmogorov - smirnov normality test was used to assess the distribution of data. we calculated the average values of ftv, btv, and vbd of both breasts for each patient. the pearson correlation was used to evaluate the relationships among the mean values of ftv, btv, and vbd. moreover, we calculated the mean, median, and interquartile ranges of age, ftv, btv, and vbd for all subjects. we divided the enrolled subjects into six groups according to age as follows : 3039 years of age, 4049 years, 5059 years, 6069 years, 7079 years, and 8089 years. we evaluated the proportion and the mean ftv, btv, and vbd values of fatty and dense breasts in each age group. recently, the korea centers for disease control and prevention reported that the mean age of menopause in korean women is 49.4 5.1 years (3). taking this into consideration, we also subdivided all subjects into groups younger or older than 50 years (14), and evaluated the mean, median, interquartile ranges of vbd, and distribution of the four vdgs in each group, using the chi - square test to assess statistical significance. 2014 - 01 - 080) and required neither patient approval nor informed consent for the review of patient images and records. between august 20 and october 7, 2013, a total of 6,751 women who underwent screening mammography with full - field digital mammography (ffdm) at our institution for private and national breast cancer screening programs were enrolled. this included some women younger than 40 years because they want to perform screening ffdm, although the korean national breast cancer screening program targeted at women older than 40 years. the exclusion criteria in this study were as follows : 1) women were younger than 30 years (n = 70) ; 2) they had a history of breast surgery (n = 68) ; and 3) they showed the bi - rads category results of 0 (incomplete), 3 (probably benign), 4 (suspicious abnormality), or 5 (highly suggestive of malignancy) (n = 646). finally, a total of 5,967 women who were over 30 years of age and showed bi - rads category 1 (negative) or bi - rads category 2 (benign) results were included all ffdms were performed with standard craniocaudal and mediolateral oblique views of both breasts on ffdm units (senographe ds, ge healthcare, milwaukee, wi, usa ; brestige, medi - future, seongnam, korea). the volume of mammographic density was calculated using volpara imaging software (version 1.5.5, mtakina technology, wellington, new zealand), which is a fda - approved program for ffdm images. this software calculates the breast density by computing the x - ray attenuation at each pixel on ffdm by comparing the pixel value at the reference point to the pixel value at each pixel. by adding up all the values in the density map over the image, it can calculate the fibroglandular tissue volume (ftv) in cubic centimeters, breast tissue volume (btv) in cubic centimeters, and therefore the percentage of the breast that consists of fibroglandular tissue the volumetric breast density (vbd). we divided the vbd into four grades as follows : 0%4.7%, volpara density grade (vdg) 1 ; 4.8%7.9%, vdg 2 ; 8.0%14.9%, vdg 3 ; and 15.0% or higher, vdg 4. patients with vdg 1 and 2 were considered to have fatty breasts, while those with vdg 3 and 4 were considered to have dense breasts (8). all statistical analyses were performed using pasw statistics 18.0 (ibm, armonk, ny, usa). the kolmogorov - smirnov normality test was used to assess the distribution of data. we calculated the average values of ftv, btv, and vbd of both breasts for each patient. the pearson correlation was used to evaluate the relationships among the mean values of ftv, btv, and vbd. moreover, we calculated the mean, median, and interquartile ranges of age, ftv, btv, and vbd for all subjects. we divided the enrolled subjects into six groups according to age as follows : 3039 years of age, 4049 years, 5059 years, 6069 years, 7079 years, and 8089 years. we evaluated the proportion and the mean ftv, btv, and vbd values of fatty and dense breasts in each age group. recently, the korea centers for disease control and prevention reported that the mean age of menopause in korean women is 49.4 5.1 years (3). taking this into consideration, we also subdivided all subjects into groups younger or older than 50 years (14), and evaluated the mean, median, interquartile ranges of vbd, and distribution of the four vdgs in each group, using the chi - square test to assess statistical significance. the institutional review board in our institution approved this retrospective study (irb no. 2014 - 01 - 080) and required neither patient approval nor informed consent for the review of patient images and records. there were 4,611 patients from private screening programs and 1,356 patients from the national breast cancer screening program at our institution who were included in our study. the distribution of each age group was as follows : 3039 years (n = 1,484, 24.9%), 4049 years, (n = 2,706, 45.3%), 5059 years (n = 1,138, 19.1%), 6069 years (n = 489, 8.2%), 7079 years, (n = 138, 2.3%), and 8089 years (n = 12, 0.2%). the data of ftv, btv, and vbd met the hypothesis of normality. the mean and median values of ftv, btv, and vbd are presented in table 1. the mean age and vbd were 46.2 9.7 years (range, 3089) and 15.8% 7.3%, respectively. there was a significant correlation between the mean values of vbd and ftv (p < 0.001, r = 0.459), between vbd and btv (p < 0.001, r = -0.454), and between ftv and btv (p < 0.001, r = 0.459) (fig. the mean values of ftv, btv, and vbd, and the proportion of dense and fatty breasts in each age group are summarized in fig. 2 and table 2, respectively. ftv, fibroglandular tissue volume ; btv, breast tissue volume ; vbd, volumetric breast density ; sd, standard deviation. correlations of mean fibroglandular tissue volume (ftv), breast tissue volume (btv), and volumetric breast density (vbd). mftv, mean fibroglandular tissue volume ; mbtv, mean breast tissue volume ; mvbd, mean volumetric breast density. when patients were divided into the two categories by 50 years of age, there were 4,313 patients (72.3%) in the younger group and 1,654 patients (27.7%) in the older group. the proportions of vdg 1, 2, 3, and 4 were 0.8% (mean vbd, 4.3% 0.8%), 6.9% (mean vbd, 6.7% 0.9%), 29.2% (mean vbd, 11.6% 2.0%), and 63.1% (mean vbd, 22.1% 4.4%), respectively, in the younger group, and 7.7% (mean vbd, 4.2% 0.6%), 32.6% (mean vbd, 6.3% 0.9%), 40.3% (mean vbd, 10.8% 1.9%), and 19.5% (mean vbd, 20.5% 4.7%), respectively, in the older group ; and these differences were statistically significant (p < 0.001, fig. the mean values of vbd were 17.8% 6.8% (median, 17.9% ; interquartile range [iqr ], 12.2%23.2%) and 10.7% 5.8% (median, 9.1% ; iqr, 6.3%13.3%) in the younger and older groups, respectively. composition of the volpara density grade (vdg) in korean women younger than 50 years and women older than 50 years. fat appears dark, whereas fibroglandular tissue appears bright due to different attenuation of x - rays. extensive bright breast density may lower the sensitivity of screening mammography by obscuring masses (1115). in this study, more than 90% of korean women under 50 years of age, 72% of women in their 50s, and about 40% of women in their 60s and 70s showed had dense breasts. the proportion frequency of dense breasts was about 83.2% (4,967/5,967) in all of the enrolled subjects in our study ; and our values are higher than those kim s study (13). kim. (13) showed the distribution of mammographic density according to age group in asymptomatic korean women with qualitative visual assessment ; and their results were as follows : 78.3% (4044 years), 61.1% (4549 years), 30.1% (5054 years), 21.1% (5559 years), and 7.0% (6064 years) of women were found to have dense breasts in 2000 (fig. there result of the overall tendency with aging was similar to our results, but their number of dense breasts per each age groups was small. the overall frequency of breast density might have been increasing for the past 15 years ; and it may be due to changes in diet and environment or hormone replacement therapy. when considering that the method used in our study, fava, is more objective. we can conclude that most korean women have dense breasts, as the frequency in our study was 83.2%, and this can be a cause of hindrance to lowered sensitivity of breast cancer screening with screening mammography. in korea, a national breast cancer screening program was launched in 2002, and interest in screening mammography has greatly increased. when we consider that mammographic density has an influence on the sensitivity of mammography, the higher breast density in women in their 40s and 50s can be a major factor in reducing the overall breast cancer screening sensitivity in korea, compared to other western countries (1316). moreover in korea, the reported incidence rates of breast cancer have continued to increase and are highest in patients in their late 40s and early 50s, whereas the incidence rates in western women continue to increase with age (17). the incidence rates in korea differ from those in the united states or united kingdom (44.7, 92.9, and 95.0 per 100,000 women, respectively) (1819). (a) mammographic parenchymal density difference between current korean women of our study and western women. (b) chronological changes of mammographic parenchymal density in korean women : comparison of previous (1998) and current (2014, our study) women. data from stomper. (20) in 1996. (13) in 2000. in our study, the frequency of women with dense breasts in korea was higher than 91% until patients reached their late 40s, and then there was a sharp decrease in frequency between the ages of 40 and 59 years. this characteristic change was unique compared to western women, and similar to results observed in a study by kim. 4). in the study of kim. (13), there was a much greater proportion of dense breast, with an abrupt decrease in patients aged between 40 and 54 years. in western women, the proportion of women with dense breasts tends to decrease steadily with age but without abrupt changes ; this is true even for patients who are in the 4054 year age group (1520). after menopause, as a normal aging process, mammographic breast density decreases due to regressed glandular tissue (22122). the mean age of menopause is different among countries, which is especially earlier in asian than in western women. reports have shown that the values are 5051 years in italy, iran, slovenia, and the united states, while 4750 years in korea, lebanon, singapore, greece, morocco, mexico, taiwan, and turkey (23). we evaluated breast density in the younger and older than 50 year age groups because the recently reported mean age of menopause was 49.4 years in korea (14). in our study, the younger group had a much greater prevalence of dense breasts (92.3%) with higher vbd (mean, 17.8%) than the older group (59.8% with 10.7% vbd). our study only considered age and not menopause ; however, we suggest that menopausal status itself also plays a role in changing mammographic density. we demonstrated that there is a much higher proportion of dense breast in korean women than in women in other countries, including other asian countries. (24) reported a higher density in asian women, and a lower density in african american women, and this difference was statistically significant. however, the vbd was much higher in our study than in the saharan african, asian, white, and african american groups in the study by carmen. (26) reported that among japanese women, the frequency of dense breasts was 43% at a mean age of 53.3 years. extensive bright breast density may also increase cancer risk by about 46-fold (1115). several breast cancer risk assessment models have been developed using bi - rads density (22227) ; however, this is a subjective method that does not consider exact numerical values. the recently developed fava method is a highly reproducible volumetric method that takes breast thickness into consideration, and several reports have shown relatively good agreement between bi - rads density and fava (81112). moreover, several studies have reported a high correlation of fava with magnetic resonance imaging, a 3-dimensional objective method (2829). (1) reported the distribution of breast density in asymptomatic new zealand women according to ethnicity and age using fava. this was the first study written in english, which demonstrated an accurate vbd value in screening women using volpara imaging software. it reported median vbds according to age, with results of 5.6% for 55-year - old europeans, 6.3% for 52.5-year - old pacific islanders, and 10.2% for 52-year - old asians. in our study, the median vbd according to age was much higher at 15.3% for 44-year - old women, even though asian women have the greatest risk of dense breasts. a younger median age could be the cause of the higher vbd ; however, the mean vbd of our 5059-year - old women was 12.4%, which was higher than that of 52-year - old asian women in new zealand. this fact also supported our hypothesis that korean women have much higher breast densities and greater prevalence of dense breasts. the ftv, btv, and vbd values are obtained by calculating the difference between the amount of x - ray absorbed by the fibroglandular tissue and fat tissue (30). we evaluated the mean ftv, btv, and vbd of both breasts. by summing up quantitative values across the breast, fava can calculate the volume of fibroglandular tissue and the volume of the breast in cubic centimeters. the vbd is then calculated by multiplying ftv by 100 and dividing the product by btv. additionally, a positive correlation was found between vbd and ftv, whereas a negative correlation was found between vbd and btv. first, it was a retrospective study and included only patients with normal or benign results on screening mammography during a 1.5-month period of our cancer - screening program ; thus, a selection bias might exist. however, this is overcome by the relatively large sample size, as well as inclusion of patients from both the national and private screening programs at our institution. second, we applied the average values of both breasts into one representative level with ftv, btv, and vbd. moreover, we used two different ffdm units ; thus, a generalized error might also exist. however, gweon. third, we evaluated only fava data and age without considering other multiple factors that can influence breast density, such as parity, menopausal status, history of hormone replacement therapy, body mass index, etc. despite these limitations, our study is meaningful in that it is the first english - written study regarding mammographic density in a large sample of korean women using quantitative fava. in the future, the mean vbd decreased with aging or menopause, and was about 16% for 46-year - old korean women, much higher than those in women from other countries. the proportion of dense breasts sharply decreases in korean women between 40 and 69 years of age.	the purpose was to present mean breast density of korean women according to age using fully automated volumetric assessment. this study included 5,967 screening normal or benign mammograms (mean age, 46.2 9.7 ; range, 3089 years), from cancer - screening program. we evaluated mean fibroglandular tissue volume, breast tissue volume, volumetric breast density (vbd), and the results were 53.7 30.8 cm3, 383.8 205.2 cm3, and 15.8% 7.3%. the frequency of dense breasts and mean vbd by age group were 94.3% and 19.1% 6.7% for the 30s (n = 1,484), 91.4% and 17.2% 6.8% for the 40s (n = 2,706), 72.2% and 12.4% 6.2% for the 50s (n = 1,138), 44.0% and 8.6% 4.3% for the 60s (n = 89), 39.1% and 8.0% 3.8% for the 70s (n = 138), and 39.1% and 8.0% 3.5% for the 80s (n = 12). the frequency of dense breasts was higher in younger women (n = 4,313, 92.3%) than older women (n = 1,654, 59.8%). mean vbd decreased with aging or menopause, and was about 16% for 46-year - old - korean women, much higher than in other countries. the proportion of dense breasts sharply decreases in korean women between 40 and 69 years of age.
nasal intermittent positive pressure ventilation (nippv) is a form of non - invasive ventilatory assistance using a nasal interface to provide respiratory support. nippv has been shown to be superior to nasal continuous positive airway pressure (ncpap) or conventional mechanical ventilation, as a method of reducing the incidence of extubation failure and pulmonary morbidities including bronchopulmonary dysplasia (bpd). nippv may be synchronized (snippv) or non - synchronized to the infants breathing efforts. many randomized controlled trials (rct) have been conducted proving the efficacy of snippv / nippv in keeping the infant extubated and/or decreasing bpd compared with other modes of respiratory support. the effectiveness of snippv could be due to the decrease in the thoraco - abdominal motion asynchrony and flow resistance through nasal prongs, with improved stability of chest wall and pulmonary mechanics. addition of peak inspiratory pressure (pip) above positive end expiratory pressure (peep) by using snippv leads to increased intermittent distending pressure above peep, with increased flow delivery in the upper airway. moretti reported that application of snippv was associated with increased tidal and minute volumes when compared with ncpap in the same infant. the goal of the present study was to compare the clinical outcomes of infants who were managed with snippv versus those infants who were on nippv anytime during their stay in the newborn special care unit (nbscu) at yale. at yale, prior to 2007, nippv was delivered through a ventilator which synchronized breaths with infant s respiratory efforts and was termed snippv. snippv was delivered using the infant star ventilator with starsync (infrasonics inc.), and synchronization with infant s respiratory efforts was achieved utilizing the graseby capsule. nippv replaced snippv, as the infant star ventilator was phased out of production in the united states. from 2007, nippv has been utilized using the bear cub 750 psv (carefusion inc., san diego, ca) ventilator. clinical retrospective data were collected on infants (n=410) admitted to the nbscu at yale - new haven children s hospital, new haven, ct, usa from jan 1, 2004 to dec 31, 2009. the criteria for inclusion were all infants who were admitted to nbscu and received snippv / nippv anytime during their stay. data collection was approved by the yale institutional review board (human investigation committee). antenatal steroid treatment was defined as at least a 12h interval between maternal dosing and subsequent delivery of the infant. resuscitation after birth was defined as being given bag and mask ventilation and/or intubation and/or chest compression and/or drugs. intraventricular hemorrhage (ivh) was determined according to papile s classification of blood in the germinal matrix or ventricular system with or without ventricular dilatation and parenchymal extension. periventricualr leukomalacia (pvl) was defined as cerebral ultrasound findings of increased echogenicity and cystic lesions in the periventricular white matter. necrotizing enterocoloitis (nec) was defined as stage 2 as per modified bell s criteria. infants were classified into two different groups, based on the type of respiratory support they received, snippv (2004 to 2006) and nippv (2007 to 2009). the main outcome of the study was incidence of bpd / death in the groups. maternal, perinatal and neonatal characteristics of infants in the two groups of respiratory support (snippv and nippv), as well as unadjusted associations between potential risk factors and bpd / death were compared using chi - square or wilcoxon rank sum test. adjusted analyses for the probability of having bpd / death were performed using generalized linear mixed models, taking into account correlation among infants of multiple gestation. antenatal steroid treatment was defined as at least a 12h interval between maternal dosing and subsequent delivery of the infant. resuscitation after birth was defined as being given bag and mask ventilation and/or intubation and/or chest compression and/or drugs. intraventricular hemorrhage (ivh) was determined according to papile s classification of blood in the germinal matrix or ventricular system with or without ventricular dilatation and parenchymal extension. periventricualr leukomalacia (pvl) was defined as cerebral ultrasound findings of increased echogenicity and cystic lesions in the periventricular white matter. necrotizing enterocoloitis (nec) was defined as stage 2 as per modified bell s criteria. infants were classified into two different groups, based on the type of respiratory support they received, snippv (2004 to 2006) and nippv (2007 to 2009). the main outcome of the study was incidence of bpd / death in the groups. maternal, perinatal and neonatal characteristics of infants in the two groups of respiratory support (snippv and nippv), as well as unadjusted associations between potential risk factors and bpd / death were compared using chi - square or wilcoxon rank sum test. adjusted analyses for the probability of having bpd / death were performed using generalized linear mixed models, taking into account correlation among infants of multiple gestation. out of the total 410 infants included in the study, 172 were classified into snippv group and 238 into nippv group. as already mentioned, the classification was based on the type of nippv, synchronized (20042006) or non - synchronized (20072009). there was no significant difference in the mean gestational age (ga) and birth weight (bw) between the two groups. there was also no difference in gender, maternal age, antenatal steroids, small for gestational age (sga), and apgar scores in the two groups. more infants in the nippv group were given resuscitation (63 vs. 44.2%, p < 0.001) while more infants in snippv group needed surfactant administration (84.4% vs. 70.2%, p < 0.001). there was no significant difference in the duration of ett ventilation or snippv / nippv in infants in the two groups, but infants in nippv were exposed to ncpap for slightly longer duration of time (p<0.02) (table 1). no differences were noted in the rate of sepsis, pda, ivh, pvl, rop, and nec in the two groups (table 1). there were eight deaths in the snippv group and nine in the nippv group. there were no instances of intestinal perforation in the snippv group ; however, there were twelve instances of intestinal perforation in the nippv group out of which ten were spontaneous intestinal perforation (sip). details have been provided in table 2. based on unadjusted analyses, bpd / death was significantly increased in the snippv group (63.4% vs. 51.6%, p<0.02). after adjusting for variables such as bw, gender, race, given resuscitation, sepsis, surfactant administration, and days on tpn (table 3), use of nippv, as compared with snippv, was not associated with increased probability of bpd / death (or 0.74 ; 95% ci 0.42, 1.30). with the increased interest in nasal ventilation as the primary mode of respiratory support in premature infants to reduce extubation failure and/or bpd, it is important to know if synchronization has any added benefit. studies have been conducted and are ongoing comparing snippv / nippv to other modes of ventilation, but no study has reported on detailed clinical outcomes, comparing snippv to nippv. moretti conducted a study in which infants weighing < 1251 g with respiratory distress syndrome (rds) requiring mechanical ventilation at 48h of age were extubated randomly to snippv or ncpap, once the criteria were met. infants in snippv group had a higher incidence of successful extubation compared to those in ncpap group (90 vs. 61%, p=0.005). in our previous studies, it has been shown that snippv decreased the duration of intubation and the need for supplemental oxygen as compared to mechanical ventilation in premature infants with rds. in a subsequent rct, comparing snippv to ett ventilation, it was shown that infants in snippv group had fewer outcomes of bpd / death compared to those in ett group (20 vs. 52%, p=0.03). the long term outcomes of premature infants managed on snippv were comparable to those of infants managed on conventional ventilation. in a rct done using snippv, no differences were reported in the mental or psychomotor developmental index scores on follow up between the infants managed with snippv or continued on conventional mechanical ventilation (cmv). in another large retrospective study done to evaluate use of snippv in infants 1250 g, it was observed that, infants who received snippv (compared with those who received ncpap) in the bw category 500750 g were significantly less likely to have the long term outcomes of bpd, bpd / death, neurodevelopmental impairment and neurodevelopmental impairment / death. it is again worth mentioning that no study has been done reporting on long term outcomes on infants managed with snippv vs. nippv. kugelman showed that infants treated initially with nippv needed less ett ventilation (25 vs. 49%, p < 0.05), had decreased incidence of bpd (2 vs. 17%, p < 0.05), compared to infants treated initially with ncpap. similarly, in another rct comparing infants with rds receiving nippv within six hours, to those receiving ncpap, it was shown that infants on nippv had lower rates of extubation failure, but had similar rates of bpd. conducted a rct comparing nippv and ncpap, in which premature infants were randomized to either of the two groups after extubation. the primary outcome of reintubation rates and other measured outcomes such as apnea, abdominal distension, sepsis, and nec were not significantly different in both groups. however, the two groups of nippv and ncpap were not well matched : infants in nippv group had lower mean bw, higher rates of rds and antenatal steroid use. also, after extubation, in the nippv group, the pip was not increased by 4 cm h2o above that required during manual ventilation, as has been recommended. the timing of initiation of snippv / nippv is also a key factor in the outcome of bpd / death. in a recent study conducted by our group, it was observed that infants who were on nippv for most of the time in the first week of life compared to being on ett, had a decreased incidence of bpd / death. there have been 2 reports, comparing snippv to nippv, evaluating short - term effects. recently, chang. studied the effects of synchronization during nasal ventilation comparing nasal intermittent mandatory ventilation (nimv), synchronized nimv (snimv) and ncpap in a randomized manner, each for 1h. owen studied effects of nippv on spontaneous breathing and proposed that synchronization of nippv pressure peaks with spontaneous inspirations may increase the benefits of nippv. our present study compares snippv vs. nippv by examining the clinical characteristics of infants and the clinical outcome of bpd / death. neither mode of nasal ventilation was found to be superior with respect to these outcomes. the most serious complication associated with use of snippv has been associated with a reassuring absence of gastrointestinal side effects. in the present study,. however, these perforations occurred with the infants being on different modes of ventilation. hence, we were unable to specifically associate gastrointestinal perforations with snippv or nippv use, as had been reported in the pre - surfactant era publication. the study by sai sunil kishore. noted a slight increase in abdominal distension in the nippv group vs. cmv, but this difference was not statistically significant. furthermore, there was no difference in the tolerance of feeds. in our study, thus, it would appear that the suggested guidelines on the technique of providing (s)nippv are safe. however, the strengths of the study include the large sample size, detailed evaluation of clinical outcomes based on mode of respiratory support, and the use of nih consensus definition for bpd. use of snippv relative to nippv did not show a significantly different impact on clinical outcomes in premature infants.	backgrounduse of nasal intermittent positive pressure ventilation (nippv) in the neonatal intensive care unit (nicu) has shown promise with better clinical outcomes in premature neonates. it is not known if synchronization makes a significant clinical impact when using this technique.objectiveto compare clinical outcomes of premature infants on synchronized nippv (snippv) vs. nippv in the nicu.design/methodsretrospective data were obtained (1/04 to 12/09) of infants who received nippv anytime in the nicu. snippv (infant star with starsync) was utilized from 200406, while nippv (bear cub) was used from 200709. bpd was defined using the nih consensus definition. unadjusted associations between potential risk factors and bpd / death were assessed using the chi - square or wilcoxon rank sum test. adjusted analyses were performed using generalized linear mixed models, taking into account correlation among infants of multiple gestation.resultsthere was no significant difference in the mean gestational age and birth weight in the 2 groups : snippv (n=172 ; 27.0w ; 1016 g), nippv (n=238 ; 27.7w ; 1117 g). there were no significant differences in maternal demographics, use of antenatal steroids, gender, multiple births, sga, or apgar scores in the 2 groups. more infants in the nippv group were given resuscitation in the delivery room (snippv vs. nippv : 44.2% vs. 63%, p<0.001). surfactant use (84.4% vs. 70.2% ; p<0.001) was significantly higher in the snippv group. there were no differences in the rate of pda, ivh, pvl, rop, and nec in the 2 groups. after adjusting for the significant variables, use of nippv vs. snippv (or 0.74 ; 95%ci : 0.42, 1.30) was not associated with bpd / death.conclusionsthese data suggest that use of snippv vs. nippv is not significantly associated with a differential impact on clinical outcomes.
dentigerous cyst is defined as an epithelial - lined developmental cyst formed by accumulation of fluid between the reduced enamel epithelium and crown of an unerupted tooth. it is formed due to an alteration in the reduced enamel epithelium, and is observed enclosing the crown of an unerupted tooth at the cementoenamel junction. the dentigerous cyst is the second - most common odontogenic cyst and literature shows an occurrence of 24% among all true cysts of jaws. it is most commonly associated with an impacted mandibular third molar, followed by the maxillary canine and maxillary third molar. dentigerous cysts are usually an incidental discovery when radiographs are taken to investigate a failure of tooth eruption, a missing tooth or mal - alignment. most dentigerous cysts are solitary and unicystic involving one side mandibular third molar area and found rarely bilaterally. occurrence of dentigerous cyst in both the jaws and bilaterally is never been mentioned in the literature. there is usually no pain or discomfort associated with the cyst unless it becomes secondarily infected. ct scan show well - defined, unilocular, well - corticated, hypodense lesions that are often associated with the crowns of impacted teeth. the hypodensity is attached at an acute angle to the cervical area of an unerupted tooth. the radiographic differentiation between a dentigerous cyst and a normal dental follicle a normal dental follicular space is 3 - 4 mm whereas a dentigerous cyst can be suspected when the space is more than 5 mm. the presented case is rare, unique and probably the first reported case of its kind to be mentioned in literature as occurrence of dentigerous cyst simultaneously in both the jaws and bilaterally in each jaw is not found in literature. a 24-year - old male reported to the department of oral and maxillofacial surgery in mayya multispecialty hospital, bangalore, india, complaining of asymptomatic facial swelling of 1 year. the swelling extended from the first premolar to third molar region on the right, and from the first premolar region to the retromolar trigone on the left [figures 1 and 2 ]. the swelling obliterated the buccal vestibule bilaterally and there was associated mobility of the first and second molars on both sides of mandible. the overlying mucosa appeared normal in color, non - tender, hard in consistency, and smooth on palpation. all the sensations were found to be normal in lower lip area on clinical evaluation. vitality was found to be present in first and second molars, premolars, and anterior teeth after thermal stimulation test using hot gutta percha application on teeth. preoperative clinical photographs showing intraoral swellings in the retromolar area on right side preoperative clinical photographs showing intraoral swellings in the retromolar area on left side a radiographic investigation by ct scan revealed a unilocular well - defined corticated hypodense lesion involving impacted mandibular third molars bilaterally [figure 3 ]. the dimensions of the hypodensities were 31 and 42 mm anteroposteriorly on the right and left, respectively. the supernumerary teeth were small in size than third molars with single conical root and were placed coronally in close relation to third molars on both sides. incidentally another bilateral hypodense lesion associated with the crowns of maxillary third molars was observed. ct - scan revealed a well - defined and corticated margin was observed surrounding the unilocular hypodensity. in maxilla, ct - scan revealed the dimensions of hypodensities around the crowns of third molars to be 15 and 18 mm in diameter on the right and left side, respectively [figures 4 and 5 ]. in all four quadrants the hypodense lesions were attached to the cementoenamel junction of the third molars at an acute angle which is one of the classical radiographic finding in cases of dentigerous cyst. coronal sections and three - dimensional ct revealed bilateral lingual and buccal cortical perforations in mandible [figure 6 ]. on aspiration a straw - colored fluid was obtained from each of these cystic lesions which is diagnostic of a dentigerous cyst [figure 7 ]. ct image showing bilateral expansile radiolucent lesion surrounding both the mandibular and maxillary third molar regions along with supernumerary teeth ct image shows maxillary radiolucent lesion surrounding the right impacted third molar ct image shows maxillary radiolucent lesion surrounding the left impacted third molar the coronal view of mandible in ct image shows bilateral lingual bone perforation cystic fluid aspirate shows straw - colored fluid after clinical and radiological examination a provisional diagnosis of dentigerous cyst was made. multiple incisional biopsies were taken and histopathological examination revealed a cystic lumen lined by two to three - layer thick non - keratinized stratified squamous epithelium that resembled reduced enamel epithelium. the connective tissue wall was composed of a fibrous capsule which confirmed the diagnosis of dentigerous cyst [figures 8 and 9 ]. histopathological section showing the cystic epithelial lining typically of dentigerous cyst another histopathological section showing the cystic epithelial lining typically of dentigerous cyst under general anesthesia the cystic lesions in all four quadrants were enucleated along with impacted third molars and supernumerary teeth. also in both quadrants of mandible extraction of the first and second molars were carried out as they were mobile. the resultant cavities [figure 10 ] in the mandible after enucleation were packed with bismuth, iodoform, paraffin paste, and healing was by secondary intention [figure 11 ]. follow - up dressing was done after 24 hours postoperatively for the first 2 weeks and then follow - up of intraoral wound irrigation and dressing was carried out once a week for next 2 months for healing of the bony defects in mandible intraorally. six months postoperatively the dimensions of the cystic cavity were reduced both clinically and radiographically [figures 12 and 13 ]. the patient is still under routine follow - up and will be closely monitored for long - term outcome. intraoperative clinical photograph showing resultant bony cavities after enucleation of dentigerous cysts in mandible postoperative clinical photograph showing the gauze packed in to the bony cavities after cyst enucleation intraoral clinical view after 6 months showing the healing of bony cavities postoperative panoramic view after 5 months of enucleation showing significant amount of bone formation dentigerous cysts are the second - most common true cysts of the jaws following radicular cysts. majority of the dentigerous cysts are associated with the permanent teeth during the second decade of life. bilateral and multiple cysts are usually found in association with a number of syndromes including cleidocranial dysplasia and maroteaux - lamy syndrome. occurence in the mandibular third molar region is more common compared to maxillary third molar region. the presented case is unique as it shows dentigerous cysts involving all the four quadrants and not associated with any syndrome symptoms like underdeveloped or absent clavicle and frontal bossing or open fontanales as seen in cliedocranial dysplasia. a review of literature shows no report of any case with dentigerous cyst involving all four quadrants., dentigerous cyst expands the outer cortical plate more than the lingual plate. in this case many times the enlarging cyst may displace the mandibular canal and may case paresthesia due to compression of nerve. in the present case there was no history of paresthesia in the lower lip region or loss of vitality of adjacent teeth indicating no compression or nerve damage. it is important to perform radiological examination for unerupted teeth in which panoramic radiograph may be used primarily. however, in extensive lesions, ct scan is considered as the gold standard. thus in this case a ct scan was chosen as a radiological investigation of choice. radiographic evaluations of dentigerous cysts are classically seen as radiolucent shadows associated with unerupted teeth and seemingly attached to the cementoenamel junction. ct scan imaging gives information about the origin, size, content, cortical plate thickness, and relationship of the lesion to adjacent anatomical structures. ct could be beneficial to determine whether roots of the adjacent teeth were located within the radiolucent space or not associated with the lesion. according to the location of radiolucency around the crown of an unerupted tooth, there are three main types of dentigerous cyst ; they are central, lateral, and circumferential type. it becomes important to differentiate between the dentigerous cyst and other unilocular radiolucent lesions presenting in the third molar region. keratocystic odontogenic tumor appears as a less expansile radiolucent lesion and also the root resorption of adjacent teeth is rare as compared to dentigerous cyst. in this presented case the root resorption of adjacent teeth adenomatoid odontogenic tumor also shows similar features as dentigerous cyst ; however, it is differentiated by the presence of intracystic radio - opaque structures. in younger patients, the periapical radiolucencies associated with deciduous teeth may mimic pericoronal radiolucencies of succeeding permanent teeth and may give a false impression of dentigerous cyst. dentigerous cyst is lined by non - keratinized stratified squamous epithelium resembling reduced enamel epithelium, odontogenic remnants, and rarely sebaceous cells. it has the potential for bone destruction due to the multipotent nature, as the lining is derived from the dental lamina. wall of the dentigerous cyst can give rise or may be associated with various entities like ameloblastoma, squamous cell carcinoma, mucoepidermoid carcinoma, and rarely other tumors. in this presented case the histopathological view showed a similar picture in all the four samples of cystic wall lining that is non - keratinized stratified squamous epithelium and a fibrous wall surrounding the lining from outer side with a inflammatory infiltrate in this fibrous area. the treatment of choice for dentigerous cyst is enucleation along with extraction of the impacted teeth. although in pediatric patients marsupialization has been considered to save the impacted tooth and developing tooth bud. it has been seen that tooth eruption potential is more in children who have open apices in the involved teeth. in this case the complete enucleation was done with the primary closure of the maxillary defects by using resorbable synthetic suture material (vicryl 4 - 0), and the mandibular bone defects after enucleation were packed with povidone iodine - soaked gauze and was left open for secondary healing. in marsupialization, a window is created in the cystic wall to evacuate its contents and the lining of cyst is sutured in continuity with the oral mucosa. the disadvantage of this technique is that the pathological lining is left behind. in this case enucleation is the treatment of choice, as there is no potential for eruption of the impacted third molars either due to age, inverted position, or root resorption of adjacent teeth.	the dentigerous cyst is a developmental odontogenic cyst which usually occurs in the second and third decade of life. dentigerous cyst is one of the most prevalent types of odontogenic cysts associated with partially erupted, developing, or impacted teeth. the mandibular third molars have a high predictability followed by maxillary canines. occurrence of dentigerous cyst bilaterally is generally observed in syndromic cases. non - syndromic dentigerous cyst occurring bilaterally or involving both arches at the same time is very rare. this presented case is rare and unique in which all four quadrants shows the presence of dentigerous cyst. in this case there are well - defined cysts associated with impacted molars as well as with supernumerary teeth in all the four quadrants of jaws. the clinical documentation of such a case in available literature is found to be first time.
leishmaniasis is a global zoonosis from the tropics and subtropics, with humans serving as accidental hosts. due to disease prevalence, one - tenth of the world 's populations (700 million people) globally, there are 12 million cases, and the incidences of visceral (vl) and cl infections are approximately 0.5 and 11.5 million new cases each year, respectively [1, 2 ]. in a sample population (n = 162), parasites were obtained from 85 patients (52.5%), and were isolated from the blood by cultures of 50 patients (30.9%). isoenzyme analysis confirmed that the organisms in blood and skin were the same, which underlines the invasive potential of the parasite that escaped the skin. extracellular procyclic promastigotes in the vector mature to metacyclic promastigotes (motile), that evolve to amastigotes (nonmotile) once they enter cells in the vertebrate host after the insect bite. the amastigote eventually evolves back to the promastigote form in the vector after a blood meal in infected hosts, closing the cycle. the mature infective metacyclic promastigotes have surface glycosyl inositol phospholipid (gipl) and lipophosphoglycan (lpg), virulence factors which inhibit the action of the complement system. once inside the host, metacyclic promastigotes are taken up by macrophages through binding to complement receptors 1 and 3 or c - reactive protein receptor and, after 2472 hours, transform into intracellular amastigotes with no surface gipl or lpg. the amastigotes begin to multiply in the parasitophorous vacuole in the macrophage, suppressing interferon gamma (ifn) and the production of nitric oxide (no) and superoxide. the immunological response in humans and experimental animals is induced by the amastigote form and not by the promastigote, which enters the host target cell immediately after infection and is not seen by the host immune system. amastigotes inhibit antigen presentation by repressing the expression of class i and class ii mhc, both basally and following stimulation with ifn [57 ]. the insoluble antigenic fraction from parasites primarily stimulated cd4 + t cells, while the soluble fraction showed a mixed profile, with cd4 + t cells being the main responsible for th2 cytokines and cd8 + t cells for th1 cytokines. residual parasites remain in the host forever and can be reactivated by aids [9, 10 ]. the challenge is to identify antigens and understand how humoral and cellular immune mechanisms cooperate for immunoprophylaxis, immunotherapy, and clinical remission of lesions [11, 12 ]. the control measures are early case detection and chemotherapy which has been hampered by the toxicity of drugs, severe side effects and by drug resistance in parasites. candidate antigens, including killed promastigotes, live attenuated parasites, crude parasites, pure or recombinant leishmania proteins or dna encoding leishmanial proteins, and immunomodulators from sand fly saliva, have been used ; however, very few candidate vaccines have progressed beyond the experimental stage [1, 13 ]. increased synthesis of heat shock protein (hsp) occurs in prokaryotic and eukaryotic cells when they are exposed to stress, to protect themselves from lethality, and represent target antigens of the immune response. interestingly vt vaccine also induced clinical remission of psoriasis [15, 16 ], psoriatic arthritis [17, 18 ], and rheumatoid arthritis, a serendipity finding. in this paper we present evidence of the immunoprophylactic and immunotherapeutic effects of insoluble proteins from amastigotes, grown in a liquid culture medium without mammalian cells, and the analysis of humoral and cellular immune responses by elisa and immunoblottings in vt vaccinated and glucantime treated volunteers before and after clinical remission of lesions in primary and secondary infections in human beings. the following leishmania strains were used : l(l)amazonensis (la : ifla / br/67/ph8), l(l)venezuelensis (lv : mhom / ve/80/h16), l(v)brasiliensis (lb : mhom / ve/75/h27), and l(l)chagasi (lch : mhom / br/74/pp75). first generation polyvalent antigens and second generation monovalent antigens (la, lv, lb, and lch) vaccines were prepared as published [1520 ]. the final first generation polyvalent immunogen contained 1 mg / ml or 250 g of each leishmania specie antigens in pbs supplemented with rehydragel and 4 g / ml of gentamicin. the concentration of alumina was 0.25 ml per mg (v / w) of parasitic protein. cases with ulcers or nodules in patients from el ingenio and la planta from january, 1994 to august, 2000 were considered as potential cl patients detected in house per house visits every 15 days. diagnosis was established by idr > 5 mm with polyvalent leishmanine and elisa with amastigotes antigens. concomitant bacterial infections in ulcers were treated with flucoxaciline (floxapen) at 100 mg / kg / day orally every 8 hours for 10 days. patients with idr > 5 mm were selected, a biopsy taken with dermal punch of 6 mm in diameter, from the edge of the ulcer, and aliquots processed for microscopic analysis of amastigotes in macrophages after may - grunwald - giemsa staining, injection of 0.1 ml in mice footpads, and parasite culture in o'daly 's medium. peripheral blood from patients was also taken by venous puncture in the left arm, with vacutainer for elisa and immunoblottings. an effective clinical remission was considered the total involution of ulcers or nodules and the appearance of a retractile scar without any inflammatory signs and no relapse for a follow - up period of 6 months after remission. the study group population (n = 87) was in average 25.1 17.1 years old range 5 to 62 years old 45.5% were females and was distributed in several group samples that received different treatments as explained in tables 1, 2, and 4. immunoprophylaxis (vt) was applied with amastigotes proteins to susceptible volunteers by personnel from the ivic immunobiology laboratory, in el ingenio and la planta in guatire miranda state, venezuela, from october to december, 1993. polyvalent first generation vaccines were injected intramuscularly in the deltoid area in 0.20 ml of pbs containing 200 g of amastigotes proteins monthly, for three times, one month apart, with previous signature of informed consent. for immunotherapy, volunteers with cl from january, 1994 to august, 2000 with lesions > 3 months evolution, received 500 g / dose of vt, weekly, up to 12 doses intramuscularly in the deltoid region. house per house visit was done every 15 days for finding efficacy of treatment (tables 1, 2, and 4). glu treatment was done at the tropical medicine institute, central university of caracas (ucv), venezuela, doses calculated at 10 mg / kg / day and injected during 10 days, with a follow up every 15 days. eligible subjects included males and females 5 to 62 years of age. for immunoprophylaxis subjects were idr and with no previous cl. additional inclusion criteria were use of a medically accepted form of contraception throughout the study and a negative pregnancy test (females of child - bearing potential). for immunotherapy, idr+ volunteers with lesions present for at least 3 months were diagnosed and treated as explained above. any female subject pregnant or lactating all trial participants were evaluated for a history of documented immunodeficiency, hiv status, opportunistic infections, or ongoing uncontrolled infections and were a basis for exclusion. subjects, whose medication involved any vaccines, allergy desensitization products, or use of topical therapy (except emollients) for cl within 2 weeks preceding the first administration of the study medication, were excluded from trial participation. subjects with a known hypersensitivity to gentamicin or any other ingredient in the study medication, local anesthesia, or diagnostic agents used for tests pertaining to the trial protocol and a documented history of alcohol abuse were excluded from trial participation. the idr skin test with leishmania antigens provides a dth reaction and is a good marker of the cellular immune response in leishmaniasis. leishmania skin test solutions (polyvalent leishmanine) were prepared with equal concentrations of four leishmania species (la, lv, lb, and lch). also, monovalent leishmanine was prepared for each leishmania species. the final concentration of the solution in each case was 40 g / ml in pbs. four g in 0.1 ml was injected intradermally in the volar surface of the right arm. the reaction was measured 48 hours later by the ball - point - pen technique. 50 g of each leishmania specie for coomassie blue staining and 60 g for silver staining were electrophoresed in invitrogen 420% nupage tris - acetate sds - page precast gels under reducing conditions with tris - glycine sds running buffer in a xcell surelock mini - cell (invitrogen life technologies) following the manufacturer 's instructions as published [16, 19, 23 ]. proteins were stained with silverxpress silver or coomassie brilliant blue (r-250) staining kits (invitrogen) and analyzed in a biorad image system with quantity one 1d analysis software. antileishmania antibodies to amastigotes in sera from volunteers were performed before and after treatments, as published [17, 19, 20, 23 ]. the antigens were derived from amastigotes of the four leishmania species that constitute vt drug substance, tested as polyvalent extracts, and separately not pooled, as found in the vt drug product (la, lv, lb, and lch). the wells of each elisa plate were coated with a solution containing 200 g of antigens per ml. after drying, plates were washed with pbs containing 0.05% tween 20 and postcoated with pbs containing 0.05% tween 20 and 1% bovine serum albumin. the plates were washed, and serum samples (diluted 1 : 1,000) in pbs were added to each well and incubated. another wash was followed by the addition of antihuman igg horseradish peroxidase linked (fab)2 fragment from sheep and incubation overnight. a final wash was followed by the addition of the substrate abts (2 - 2-azino - di - benzothiazoline sulfonate) and 10 l h2o2. the assay was read in an automatic elisa reader (titertek multiskan plus) at 450 nm. a bovine serum albumin calibrator diluted from 6.25 to 400 ng / ml was added to assigned wells. any replicate wells with an average of 3 standard deviations above the average concentration recorded for the negative controls (100 ng / ml), was considered a positive reaction. all values represent the average of duplicate assays of three different experiments performed with sera of patients and healthy controls idr. protein concentration was determined by lowry or bca [24, 25 ]. 420% nupage tris - acetate sds - page precast gels under reducing conditions with tris - glycine sds running buffer in a xcell surelock mini - cell (invitrogen life technologies) as published [17, 19, 20, 23 ]. afterwards transfer to nitrocellulose paper (schleicher & schull, keene, nh, usa) was performed in a mini - trans - blot (biorad) following manufacturer 's instructions. after reaction with the primary antibody from various patients ' sera and controls, staining was done with a secondary anti - human immunoglobulin horseradish peroxidase linked (fab)2 fragment from sheep, (amersham, uk) at 1 : 1000 dilution for 2 hours at room temperature. finally after a wash with sst (tris - hcl 0.05 m, 0.15 m nacl, ph 9.5)-tween 20, a solution with 0.05% diaminobenzidine (w / v), 0.03% h2o2 (v / v), and 0.03% cocl was added, waiting until color was developed, washed with distilled water, and photographs were taken. immunoblottings after staining were aligned side by side from each patient 's sera, digitalized with a scanner scan jet 4c and deskcan ii, 2.3 program (hewlett packard 19911995) and then analyzed with gel - pro 98, 3.0 media cybernetic 1995 - 1996. the integrated optical density (iod) values corresponding to the area under the curve from different bands was obtained and coefficient of variation calculated before and after clinical remission as follows : od posttreatment / od pretreatment. values > 1.0 revealed increase in od antigens and values 5 mm. idr was higher in active secondary infections and significantly different to patients with active primary infections (p 0.05) and were higher than values post - clinical remission of lesions. similar values were obtained in patients with remission in post - sr, post - vt and post - glu treatment (p > 0.05), which confirmed that treatments with vt or glu were effective and similar to natural sr (golden standard) in cl patients. interestingly, od values post - glu in patients that had no remission of lesions were higher than post - sr, post - vt or post - glu with remission of lesions (p 0.05). on the contrary, sera from chagas disease and/or active cl had higher significant (p 10 units by densitometry analysis. antigenic bands revealed by sera from patients with active cl and post - clinical remission in the same patients were compared within the same group with idr() controls (i.e., la in active or remission versus la in idr controls, lb in active or remission versus lb in idr controls, and so on with lv and lch). common bands present in active, remission, and control idr groups are in red bold numbers. mw bands revealed by sera from active and clinical remission patients, not found by sera from controls, at the respective homologous leishmania specie are in bold blue numbers (table 5). this is evidence that amastigote antigens sensitized b cells receptors and expanded specific parasites ' clones in active and after clinical remission of lesions not revealed by healthy control sera. total number of antigens decreased postclinical remission in all species as compared to patients with active lesions (table 5). the shared common antigens by controls and patients explain the location of intracellular amastigotes in the vertebrate host, since promastigotes are immediately recognized by normal immunoglobulin after injection of parasites by the vector in the skin and afterwards phagocytose by apc, neutrophils, and macrophages. specific amastigotes antigens not found by control sera and recognized by sera from active lesions were as follows : 8 in la ; 11 in lb ; 4 in lv ; 6 in lch. in bold green numbers specific antigens found in one leishmania specie only, had the following mw in active lesions : la : 132 ; lb : 115 kda ; lv : none ; and lch : 128 kda ; after clinical remission in the group mw were : la : 100 ; lb : 108 ; lv : 80 ; and lch : 86 kda, respectively. in bold black numbers antigens mw 45 kda were found in controls in la, lb, lv, and lch by 100% sera, also mw 96 kda in lb by 100% control sera. both antigens were not recognized by any patient 's sera (table 7), only by healthy controls. in normal back numbers, mw 86 kda in la and lv, 90 kda in la and 128, and 132 kda in lb are in 80100% controls sera but not in sera from cl patients in the respective leishmania group. bands frequency in the sr natural process revealed by sera from four volunteers showed fourteen antigens (56%) mw 142, 132, 128, 120, 115, 104, 100, 98, 90, 86, 80 53, 45, and 27 kda decreasing after remission of lesions. only one antigen (4%) mw 33 kda increased in band frequency ; the rest ten antigens (40%) mw 136, 108, 74, 65, 60, 57, 50, 40, 35, and 22 kda frequency remained unchanged. band frequency before and after glu revealed by sera from three volunteers, showed twelve antigens (41.4%) 145, 132, 128, 104, 100, 70, 53, 33, 31, 24, 17, and 15 kda decreasing while four antigens (13.8%) 83, 80, 57, and 29 kda increased after remission of lesions, the rest thirteen antigens (44.8%) 98, 86, 78, 74, 65, 60, 50, 43, 40, 35, 27, 22, and 20 kda remained unchanged. band frequency before and after vt revealed by sera from 3 volunteers showed twelve antigens (37.5%) 142, 120, 96, 80, 57, 35, 29, 22, 20, 17, 15, and 13 kda decreasing while two antigens (6.25%) 90 and 60 kda increased after remission of lesions ; the rest of eighteen antigens (56.25%) 145, 136, 132, 128, 122, 115, 104, 100, 98, 86, 74, 65, 50, 45, 40, 33, 27, and 24 remained unchanged. decrease in bands ' frequency predominated in low mw antigens from 57 to 13 kda in post - glu and post - vt treatment in comparison to natural sr. molecular weights bands 80 to 145 mw also decreased, eleven antigens post - sr, followed by six post - glu and four post - vt (table 8). coefficient of variation was calculated as (1)area curve od post - treatmentarea curve od pre - treatment. integrated optical density (iod) area values > 1.0 correspond to increase in antigen density, while values 1.0 for cl surveillance useful to know the prevalence and incidence in endemic and hyperendemic regions. positive idr in residents living in endemic areas without previous cl in skin are defined as subclinic infections. in corguinho, do sul, brazil, an endemic region for lb, 15.7% had positive idr to leishmanine with no previous infections ; also in endemic regions for l(l)peruviana in lima, ancash, and piura in peru, 17.0% idr+ cases were recorded and in northeast ecuador with high prevalence of l(l)guyanensis and l(l)panamensis 17.2% positivity was observed ; all values are in agreement with data in venezuela : 22.0% idr+ volunteers to polyvalent amastigote leishmanine in healthy subjects, with no previous cl infection. the association of protective immunity with idr response and cellular immunity explains the higher incidence of cl in idr (59%) as compared with idr+ (27%) volunteers in the study group. proteases play essential roles in the pathogenesis of leishmaniasis, helping parasites to invade and multiply in mammalian host cells. however, few studies have addressed serine proteases in leishmania infection and their role in host pathogenesis. the level of internalization of parasites into host macrophages after treatment amastigotes with the anti-115 kda antibody was significantly reduced, suggesting that this serine protease probably plays a role in the infection process. the basis of vt vaccine is inhibition of serine proteases with tlck, which induced immune prophylaxis and immunotherapy in cl patients. prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, through which host - parasite interactions occur. vt vaccine does not have prohibitin since amastigotes were extracted with np-40 and supernatant discarded ; thus no antibodies or cellular immunity to surface proteins could be found [1720 ]. specific antibodies decreased post - clinical remission of lesions, confirmed by elisa, bands ' frequency, and iod bands ' area in immunoblottings, pointing out to the fundamental role of cellular immunity in controlling infection. three months was the time waited in cl patients to start vt or glu treatments to avoid the self - healing leishmaniasis effect. size of skin ulcers in relapses was lower than in primary lesions ; thus, a vaccine was possible to control cl. vt vaccine had remission time evidence vt amastigotes vaccine was useful for cl treatment. furthermore vt vaccinated subjects had lower cl cases in idr and idr+ volunteers than idr nonvaccinated subjects. the persistence of these specific antibodies and an acute increase in their levels might be a sentinel of a vl relapse. elisa has been used for diagnosis of cl using soluble (sf) and membrane enriched (mf) antigen fractions obtained from lb. sensitivity was 89.5% for each fraction, and specificity was 89.5% for sf and 93.4% for mf. similar results were obtained with vt vaccine insoluble amastigote antigens from four species lb, ld, lv, and lch. elisa with monovalent amastigotes evidenced that antibodies were induced on b cells in primary active infection and decreased significantly post - clinical remission, once parasites were under control. this underlines that cellular immunity played a key role in control of parasites in secondary infection as evidenced by a high positive dth in active and postclinical remission of lesions. elisa in active lesions, pre - sr and pre - vt was similar in primary infection evidencing that b cell clones were activated and reacting to all monovalent amastigotes. antibodies decreased significantly post - sr, post - vt, and post - glu treatment, since vt was stimulating mainly cellular immunity. interestingly antibodies did not decrease in one group of patients without clinical remission post - glu treatment. the od values in this group, compared with values that had clinical remission, were significant (p < 0.05), a very good internal control. the reason why these patients could not switch on cellular immunity and decrease humoral immunity is under investigation. microorganisms replicate in macrophages, facilitated by surface igg antibody increasing intracellular infection through fc-receptor by phagocytosis. the ligation of monocyte or macrophage fc receptors by igg immune complexes, suppresses innate immunity, increases production of il-10, and bias t - helper-1 (th1) responses to th2 responses, leading to increased infectious output by infected cells [35, 36 ]. normal igg antibodies recognized many parasite antigens, isolating them from the immune system, directing promastigotes to the intracellular site. cellular immunity in chronic infection was probably mediated by a cd8 + t cells and th1 response once intracellular amastigotes were under control and lesions disappeared. parasites remain in the host forever, in proliferative equilibrium inside cells, without cells ' lysis, controlled by a permanent cellular immunological response, which once it is stopped, parasites can be reactivated and disseminated as in aids [9, 10 ]. bands frequency analysis revealed decreased numbers of bands in 56% antigens recognized by sera after sr, 41% post - glu, 37% post - vt, respectively, explaining od decreasing in elisa, in sr and after glu or vt treatments. the highest decrease was in low molecular weight antigens post - glu and post - vt treatment. decrease in bands ' frequency and serum antibodies may be explained by inhibition of antigen presentation by repression of class i and class ii mhc on infected host cells [5, 6 ], by interfering with the loading of antigens onto the mhc class ii molecule or sequestering the mhc ii molecules and antigens within the phagolysosome. the iod area in amastigote antigens revealed by sera from sr volunteers and post - glu treatment decreased significantly, and only one antigen 70 kda increased after sr in secondary infections. sera post - glu had increased iod area in low mw antigens 78, 65, 57, 50, 40, and 29 kda (26%) after clinical remission of lesions. this was probably due to parasites ' death and liberation of low mw antigens to the host, inducing b cell sensitization, antibody production, and immune protection, similar to findings in immunized and infected balb / c mice. after vt vaccination, iod area in majority of proteins decreased in idr patients, while low and high mw antigens increased in idr+ subclinical infections, and in idr+cl+, vt vaccinated volunteers with previous healed cl suggesting vt vaccination also killed parasites inducing nk cells liberating antigens in subclinical and clinical infections, similar to glu treatment. to recognize amastigote antigens, no previous lymphocyte sensitization is required. leishmania promastigotes sonicates induced in vitro proliferation and ifn production in peripheral blood mononuclear cells (pbmc) from individuals that never had contact with leishmania parasites. the proliferating t cell population was cd2 + in a frequency < 1 : 10,000 a response that could be abolished after depletion of cd45ro+ memory cells from the pbmc. thus, normal cd2 + t cells and normal immunoglobulin from healthy volunteers that never experienced leishmania infection reacted with leishmania antigens in the immunoblotting assay. interestingly psoriatic patients, dth negatives to amastigote antigens were also recognized in the blastogenic assay leishmania antigens and were distributed in two groups, low and high responders to amastigote antigens before treatment. furthermore, a high dth response with lb and lch protein chromatography fractions in vivo after treatment with vt polyvalent vaccine was found in human volunteers, confirming the absence of immunosuppression. the lymphocyte stimulation indexes obtained by protein fractions were 5070% lower than the values induced by intact living leishmania amastigotes. this suggests in vitro stimulation appears to be selectively focused on a particular subset of lymphocytes which may be regulatory cd8 + t cells [1720, 41 ]. surprisingly, vt amastigotes antigens induced clinical remission of all forms of psoriasis, psoriatic arthritis (psa), and rheumatoid arthritis in human volunteers and collagen induced arthritis in mice a serendipity finding. the effect of vt was analyzed in patients with psa after treatment, finding that c - reactive protein (crp), complement 5a, tnf, and 1l-1 decreased in human volunteers. interestingly, proliferation of cutaneous t cell lymphoma in vitro was also inhibited by vt monovalent lch amastigotes antigens in a dose / response relationship, which open new roads for the application of this vt treatment to wide spectra of diseases. due to genetic diversity in target populations, including both dogs and humans, a multiple - antigen vaccine will likely be essential. however, the cost of a vaccine to be used in developing countries must be considered. the polyprotein (ksac) was found to be immunogenic and capable of inducing protection against l(l)infantum, responsible for human and canine vl, and against l(l)major, responsible for cl. it is a cost - effective vaccine capable of protecting both humans and dogs against multiple leishmania species. the vt polyvalent vaccine for cl, ml, and vl has a very low cost of production, is feasible to be applied to developing countries worldwide. hsp long - term confrontation of the immune system similar in the host and microbes invaders may convert the immune response against these host antigens and promote and/or decrease autoimmune diseases including psoriasis [4345 ]. there is evidence that recognition of self - hsp60 can have beneficial effects in arthritis and may offer new strategies for improved control measures in the inflammatory processes by administration of peptides cross - reactive to self - determinants. hsp60, hsp70, and gp96 function as host derived ligands for toll - like receptors (tlr2) and play a role in the pathogenesis of ra and psoriasis [44, 45 ]. leishmania antigens are produced after a heat shock in promastigotes that become amastigotes in a liquid culture medium. the mw of chromatography fractions from lb that induced remission of psoriasis was similar to the range of most hsp host ligands (5070 kda) and could be inhibiting the symptoms of psoriasis, psa, and cia [19, 20 ] by competing with peptides in the respective receptors. similar mechanisms could be acting in immunoprophylaxis and immunotherapy of cl as shown in this paper. amastigotes from four leishmania species, cultured in a free cells culture medium without cells treated with tlck (vt) induced clinical remission of cl after six 500 g / dose, weekly, up to 12 doses intramuscularly in the deltoid region in 7 weeks and protected volunteers from cl in six years of followup. humoral immunity decreased after clinical remission of lesions, measured by antibodies in elisa, frequency of antigenic bands, and density area densitometry in immunoblottings. vt vaccine induced mainly cellular immunity, measured by dth intradermic reaction, postremission of lesions as compared to active infection. vt vaccine induced clinical remission of all forms of psoriasis, psa, and rheumatoid arthritis in human volunteers and collagen induced arthritis in mice, a serendipity finding, and inhibited proliferation of cutaneous t cell lymphoma in vitro which open new roads for the application of this vaccine to wide spectra of diseases.	amastigotes from l. (l.)amazonensis (la), l. (l.)venezuelensis (lv), l. (v.)brasiliensis (lb), and l. (l.)chagasi (lch) were cultured in a free cells liquid culture medium. patients (n = 87) from a cutaneous leishmaniasis (cl) hyperendemic region receiving different treatments were followed up from january 1994 to august 2000. time for remission of lesions were spontaneous remission (sr) 7 weeks ; glucantime (glu) chemotherapy 9 weeks ; immunotherapy with la, lv, lb, and lch amastigotes tosyl - lysil chloromethyl - ketone (tlck) treated and nonidet p-40(np-40) extracted (vt) 7 weeks. delayed type hypersensitivity (dth) response with leishmanine intradermic reaction (idr) was higher in cl patients than healthy controls (p < 0.05) and increased in active secondary versus primary infection (p < 0.001) with diagnostic value 1.74 for active infection and 1.81 postclinical remission. antibodies to amastigotes characterized by enzyme linked immunosorbent assay (elisa) decreased in sera postclinical remission versus active infections (p < 0.001), with a diagnostic value from 1.50 to 1.84. immunoblottings antigenic bands frequency as well as integral optical density (iod) area densitometry decreased with sera from sr, after glu or vt treatments in cl volunteers. intracellular parasitism is due to normal antibodies recognizing parasite antigens after inoculation by vector. vt vaccine induced mainly cellular immunity, for remission of lesions and protection from cl infection.
	modification of cytosine - guanine dinucleotides (cpgs) is a key part of mammalian epigenetic regulation and helps shape cellular identity. tet enzymes catalyze stepwise oxidation of 5-methylcytosine (mc) in cpgs to 5-hydroxymethylcytosine (hmc), or onward to 5-formylcytosine (fc) or 5-carboxylcytosine (cac). the multiple mc oxidation products, while intricately linked, are postulated to play independent epigenetic roles, making it critical to understand how the products of stepwise oxidation are established and maintained. using highly sensitive isotope - based studies, we newly show that tet2 can yield fc and cac by iteratively acting in a single encounter with mc - containing dna, without release of the hmc intermediate, and that the modification state of the complementary cpg has little impact on tet2 activity. by revealing tet2 as an iterative, de novo mc oxygenase, our study provides insight into how features intrinsic to tet2 shape the epigenetic landscape.
hirschsprung 's disease (hscr) was first described by harald hirschsprung a century ago. the basic pathological feature of hscr is the absence of ganglion cells in the distal bowel, which results in the functional obstruction manifesting severe chronic constipation and abdominal distention. the most common and most severe complication of hscr is hirschsprung's - associated enterocolitis (haec). in recent years, one - stage pull - through surgery has been commonly performed to deal with hscr, and the methods have developed from laparotomy to minimally invasive surgery such as transanal pull - through operations and laparoscopy - assisted operations. however for children in a poor condition such as total colonic aganglionosis, low birth weight, and severe haec, a multistage surgery is more conservative. patients with haec usually manifest abdominal distention, diarrhea, fever, and even shock. without timely and effective treatment, haec may cause the death of children with hscr. many reports and studies have been published up to now. due to the different diagnostic criteria, the morbidity of haec is from 17% to 50%, with an average of 25%. researches should not be limited to recognize clinical manifestations and take the treatment which seems effective. some differences were found among haec children, hscr children without haec and normal controls, which may interpret the development of haec. however, results are not so satisfactory because of the fact that no conclusion can completely and clearly explain the pathogenesis of haec. mucus barrier, intestinal microbiota, and immune function are major factors that are related to the intestinal inflammation. in the present article, we gave a comprehensive description mainly from these three parts, in spite of the inconsistent results, to inspire readers to achieve more new theories about haec. on the surface of intestinal epithelia, mucins composed of highly glycosylated proteins constitute a protective mucus. the former is the habitat of intestinal microbiota and is essential to form the intestine microenvironment. mattar. collected stool samples from hscr patients and found that muc-2 was obviously lower in hscr group, and interestingly, the expression of muc-2 in haec group could not be detected. have studied mucins and goblet cells in hscr patients and ednrb/ mice (hscr mice models). what was different to the study of mattar. was that they detected no change of muc-2, but a reduction of muc-4 in the distal colon of hscr patients, as well as in ednrb/ mice. besides, they found that in the distal colon of hscr patients, although goblet cells got more, mucins inside goblet cells reduced furthermore, goblet cells of ednrb/ mice showed differences in both aganglionic and ganglionic segments. goblet cells of aganglionic segments were larger and more, while in the ganglionic segments they were found smaller and less. sam - pointed domain - containing ets - like factor (spdef) and math1 factors which are relative to the differentiation of goblet cells increased., no difference was found between hscr patients and the control. besides, the expressions of muc 1, 2, 3, 4, 5ac, 5b, 6, 7, and 8 in hscr group were similar to those of control group. besides, a study showed that radioactive mucin precursors [35s]-sulfate and [3h]-glucosamine, which are turnover of mucin precursors, were reduced in hscr patients in both aganglionic and ganglionic segments. in earlier studies, researchers focused on neutral mucin type and sulfomucin type. fujimoto found that neutral mucins and sulfomucins were both significantly reduced in the piebald lethal mouse model of hscr. but teitelbaum. the abnormal composition of mucins will lead to the dysfunction of mucus barrier. as a consequence these researches showed that mucins might be associated with haec, but there was no direct evidence between aganglionosis and mucins production. the result showed that for passive particle transport, the distal colon, as well as the proximal colon, showed a significantly reduced transport rate. furthermore, compared with the controls, the particle showed a more regular track and a shorter distance, which meant they were hindered. moreover for active microbe transport, by real - time tracking, escherichia coli transport showed a reduced rate in the proximal colon rather than in the distal colon. the abnormality of the proximal colon matched the fact that haec happened even after the definitive pull - through surgery. numerous symbionts and commensals, including bacteria, fungi, and virus, exist in human bowels. most of these microbiota are bacteria, with 1010 inhabiting in the large bowel and a smaller group, 1010 in the small intestine. they play a vital role in maintaining the intestinal homeostasis and keeping normal immune function. resident microbiota is essential in the development of immune response. as described in many previous studies, plenty of diseases, such as ulcerative colitis, crohn 's disease (cd), functional gastrointestinal disorders, and colorectal cancer, are all relative to the unbalance of intestinal microbiota. a concept microbiota - gut - brain axis emerges nowadays, which means that intestinal microbiota interact with the nervous system and have an impact on it. the central and enteric nervous system (ens) also communicate with intestinal microbiota by means of neural, endocrine, immune, and humoral links to influence their composition and behavior. according to this, we may speculate that aganglionosis will lead to an abnormal distribution of microbiota. in recent years, with the development of technique, dna sequencing is used more frequently in the analysis of intestinal microbiota. in a multicenter study of frykman., not only bacteria but also fungus were different in haec children compared with hscr children without haec. they found the modest reductions in firmicutes and verrucomicrobia, but the increases in bacteroidetes and proteobacteria. specifically, candida sp. was increased, but malassezia and saccharomyces sp. were reduced. shen. indicated that bifidobacteria were reduced in haec group compared with hscr and control groups, and they also found that lactobacilli in both haec and hscr groups were markedly decreased compared to control group. similarly, pierre. found that for p21 - 24 ednrb/ mice, a reduction of lactobacillus was observed. in order to study bacteria from different colonic segments, yan. furthermore, haec group had a greater variety in proximal segments than that in distal segments. in contrast, in haec group, proteobacteria occupied the largest portion with a proportion of 55%. besides, the colon from postnatal day 7 (p7) ednrb/ mice was dominated by coagulase - negative staphylococcus species, which were rare in the control. wang. used oral probiotics, composed of bifidobacteria, lactobacilli, and enterococcus as a precaution of haec. results showed that the probiotics would not only reduce the morbidity of haec but also make it less severe. but a consensus has not been reached. in a prospective study of el - sawaf., they declared that the morbidity of haec between probiotics and placebo treating group was of no difference. in some earlier studies, however, conclusions also seem to be contradictory. some reported a significantly higher isolation rate of c. difficile of haec children. but contradictorily, others declared that no difference existed between hscr children with diarrhea and those without diarrhea. a research declared that pseudomembranous colitis complicating hscr caused by c. difficile had a mortality as high as around 50%. found that fecal short - chain fatty acid (scfa) changed in haec children. compared to patients without haec, they showed a 4-fold decline in total scfa concentration. moreover related to this, most of the butyrate - producing genera demonstrated a multifold expansion, while acetate - producing genera also showed this phenomenon. decreased acetate production, or increased acetate absorption, or bacteria utilizing acetate for a large compartment of immune cells resides in the intestine. innate and adaptive immunity work together to eliminate pathogens including bacteria, fungi, viruses, and helminth and to maintain intestinal homeostasis. it mainly includes gut - associated lymphoid tissue, which is consisted of peyer 's patches, solitary follicles, aggregate follicles, and diffusely distributed lymphoid cells in the lamina propria of the gut where priming adaptive immune cells reside. besides, macrophages, dendritic cells, innate lymphoid cells, paneth cells found only in the small intestine, goblet cells and intestinal epithelial cells all contribute to intestinal innate immunity and then adaptive immunity. abnormality of parasympathetic nervous system has been found in cd patients and dextran sodium sulfate(dss)/2, 4, 6-trinitrobenzene sulfonic acid(tnbs)-treated mice. besides abnormality of ens and parasympathetic nervous system in hscr may play a role in the dysfunction of intestinal immunity. found that small spleen phenotype and splenic lymphopenia existed in ednrb/ mice, which was characterized by 5- to 20-fold reductions of total spleen cells, cd19 mature b - cells, cd4 t - cells, and cd8 t - cells. moreover, there was a strong inverse correlation between histopathological enterocolitis (ec) scores and total spleen cells, as well as b - cells. their further research showed that severe haec led to thymic involution, splenic lymphopenia, and suppression of b lymphopoiesis. this supports the fact that hscr patients who have not been timely treated are always in a severe situation. they also illuminated that lymphopenia was related to colonic aganglionosis, while it had no relationship with the ednrb gene. abnormal splenic microarchitecture with lymphopenia was observed in agh - ednrb/ rats, but not detected in le and f344 genetic background ednrb/ rats. they speculated that the development of t - cells and b - cells might be relative to endothelin signaling pathway and be influenced by the genetic background. igm igd (mature) b lymphocytes related to the production of secretory iga decreased in peyer 's patches. they also studied the spleen of ednrb/ mice and had the same conclusion that b - cells decreased. however, they found mature b - cells increased, which was opposite to the study of cheng. in contrast to these studies, turnock. found that t - cells, b - cells, and immunoglobulin - containing cells in intestine mucosa showed no difference in haec children. detected an obvious increase of igm and igg, without an increase of iga in aganglionic and transitional segments. imamura. demonstrated that iga, igm, and j chain containing plasma cells, cd68 positive monocyte / macrophages, and cd45ro positive leukocytes increased in haec children. secretory component in the intestine lumen reduced, which reflected a reduction of secretory iga. in addition, nk cells in ganglionic segments increased but showed no difference in aganglionic segments. in the piebald lethal mouse model of hscr, immune cells and immunoglobulin - producing cells, especially iga - containing cells, were significantly increased in acute illness group compared with the relatively chronic illness group. furthermore in the latter group, iga - containing cells are distributed mainly in the deep layer of lamina propria. wilson - storey. discovered a decrease of secretory iga and these patients were with an obvious deficiency of the transfer of secretory iga. pierre. demonstrated that at the late time point (p22) of ednrb/ mice, the expression and activity of secretory phospholipase a2, an antimicrobial protein secreted by paneth cells in small intestine, were reduced. the dysfunction of innate immunity in the small intestine may explain the reason why haec happens beyond the aganglionic segments and even after the definitive surgery. these findings are not restricted to the aganglionic segments, which mean that even after the resection of the diseased colon, abnormal intestinal immunity is still there to cause haec. the basic manifestation of hscr is the functional obstruction in the distal colon, which leads to fecal stasis and then the reproduction of pathogens. as a consequence, found that in the piebald lethal mouse model of hscr, obstruction of the distal colon resulted in a loss of colonic mucosal barrier integrity, allowing intraluminal pathogens to move through the mucosal surface and enter the circulation. lui. detected that expressions of caudal type homeobox gene-1 and -2 (cdx-1 and cdx-2), which control the proliferation and differentiation of mucosal cells, were reduced in children with haec. showed that the itgb2 (cd18) immunomodulation - related gene coding for the -subunit of leukocyte adhesion molecule lymphocyte function - associated antigen 1 was found with genetic variations in those patients presented with haec symptoms. in addition, others researched the most important susceptibility gene for crohn 's ec, nod2. hackam. referred that postoperation haec were related to many risk factors, such as anastomotic stricture or leak, and postoperative intestinal obstructions. but claimed that these risk factors were not common after the surgery, which meant that they should not be responsible for postoperation haec. what led to the high morbidity of postoperation haec was the existence of intestinal neuronal malformations in reserved intestines postoperations. a research of dembowski. found that the reduction of albumin existed both before and after haec in ednrb/ rats. in the piebald lethal mouse model of hscr, researchers also found that albumin declined gradually at the time around p50. lloyd - still and demers used cholestyramine to treat an infant with haec and found it effective. cholestyramine caused a significant reduction of prostaglandin e (pge) in the colostomy drainage - fluid. according to this, they speculated pge activity, enterotoxin, and bile acid malabsorption might contribute to the development of haec. a study showed that neuroendocrine cells (ne cells) cells of aganglionic segments were significantly higher than that of ganglionic segments. but in the latter, ne cells in patients with haec showed a lower level than patients without haec. haec, as a common and severe complication of hscr, must be understood thoroughly. mucus barrier, intestinal microbiota, and immune function are major factors that are related to intestinal inflammation. in haec children, abnormal mucus barrier can not prevent the permeation of bacteria and help to build a normal microenvironment. it is worthy of note that not only an insufficient innate immune system but also a dysfunctional adaptive immune system exist in the intestine of hscr. then, increased pathogenic microorganism whose production is allowed as a result of alterations of intestinal microbiota will enter the deeper layer of intestine and even the circulation system. and maybe interaction of mucus barrier, microbiota, immune function, and abnormal ens is the main trigger of haec [figure 1 ]. the maintenance of intestinal homeostasis is attributed to a well - organized cooperation of microbiota, mucus barrier, immune system, and nervous system. unbalance of microbiota, alteration of mucus composition and mucin production by goblet cells, and abnormity of immune system which includes a deficient distribution of immune cells, failing iga transfer leading to a decrease of secretory iga, a decrease of secretory phospholipase a2 secreted by paneth cells, and a reduction of b - cells in peyer 's patches, together with abnormal enteric nervous system and parasympathetic nervous system, trigger the occurrence and development of hirschsprung's - associated enterocolitis. however, the inconsistency of the results and the uncertainty of treatment due to these theories still confuse doctors and researchers. the present viewpoints are still far from impeccable. due to the lack of clear explanations for the whole process of haec the direct evidence of the effect of ens malformation on mucus barrier, microbiota, and immune system should be checked out in haec. the role of probiotics on microbiota need to be studied in model animals to find whether it is effective in haec. researches have demonstrated abnormities in intestinal immune system. furthermore, crosstalk between these immune cells should be studied. people also need to find the immune state in the environment of haec and its recover after haec. in the future, scientists should dig deeper in the areas of mucus barrier, microbiota, and immune function, or even a completely new field. this study was supported by a grant from national natural science foundation of china (no. this study was supported by a grant from national natural science foundation of china (no.	objective : to systematically summary the updated results about the pathogenesis of hirschsprung's - associated enterocolitis (haec). besides, we discussed the research key and direction based on these results.data sources : our data cited in this review were obtained mainly from pubmed from 1975 to 2015, with keywords hirschsprung enterocolitis, hirschsprung 's enterocolitis, hirschsprung's - associated enterocolitis, hirschsprung - associated enterocolitis, haec, and ec.study selection : articles regarding the pathogenesis of haec were selected, and the articles mainly regarding the diagnosis, surgical approach, treatment, and follow - up were excluded.results:several factors, mainly including mucus barrier, intestinal microbiota, and immune function, as well as some other factors such as genetic variations and surgical reasons, have been found to be related to the pathogenesis of haec. changed quantity and barrier property of mucus, different composition of microbiota, and an abnormal immune state work together or separately trigger haec.conclusions:the maintenance of intestinal homeostasis is due to a well cooperation of microbiota, mucus barrier, and immune system. if any part presents abnormal, intestinal homeostasis will be broken. meanwhile, for patients with hirschsprung 's disease or haec, dysfunction of these parts has been found. thus, the happening of haec may be mainly attributed to the disorders of intestinal microbiota, mucus barrier, and immune system.
brown - vialetto - van laere syndrome (bvvls) or progressive pontobulbar palsy with deafness is a rare degenerative disorder characterized by slow or rapid onset progressive bilateral deafness and cranial nerve involvement, usually motor components of the 7, 9, 10, 11 and 12 this syndrome was first described in 1894 by brown and subsequently by vialetto in 1936 and van laere in 1966. sathasivam reviewed all reported cases until 2007 and was able to retrieve 58 cases. by now, no case has ever been reported from iran. herein, we report 4 iranian cases of bvvls seen from 1999 to 2008 with significant clinical improvement in some of them and we tried to find similarities and differences of our patients who all had caucasian ethnicity with previously reported patients with bvvls. a 17-year - old female, unmarried and student presented with speech problem and progressive hearing loss since the age of 12. she was admitted in our hospital in 1999 with liquid dysphagia and progressive weight loss in the last 6 months. the tongue was atrophic with fasciculation and gag reflex was diminished and the limbs were atrophic with mild proximal weakness. biochemical lab tests revealed no abnormality ; but on audiometry, there was severe bilateral sensorineural hearing loss. electromyography and nerve conduction studies (emg - ncv) were compatible with motor neuron disease with prominent involvement of cranial muscles ; in addition, repetitive nerve stimulation was negative. other investigations including chest x - ray, brain magnetic resonance imaging (mri) and abdomino - pelvic sonography were normal. this 17-year - old girl was admitted for the evaluation of slowly progressive dysphagia and choking from 6 months before admission in 2000. she had an episode of febrile seizure at the age of 3 and slowly progressive hearing loss from the age of 12. physical examination was normal. on neurologic exam, there was bifacial weakness, diminished gag reflex, atrophic tongue with fasciculation and proximal weakness. routine laboratory tests, peripheral blood smear, creatine phosphokinase (ck), and lactate dehydrogenase (ldh) were all normal, as well as cerebrospinal fluid (csf) analysis. there was bilateral low frequency sensorineural hearing loss on pure tone audiometry (pta) and motor neuron involvement mainly in cranial innervated muscles on emg - ncv. other investigations including chest x - ray, brain mri, tensilon test, repetitive nerve stimulation (rns) and abdomino - pelvic sonography were negative and genetic testing did not show deletion in smn1. one year after the beginning of gabapentin, 900 mg / day, she reported some improvement in dysphonia and dysphagia, which continued for the next 4 years, when she was lost to follow up. this 21-year - old female was admitted in december 2008 because of hoarseness, difficulty in swallowing, choking and fatigue in the recent 3 months. she had weight loss, difficulty in walking and bilateral hearing loss since 2 years ago which aggravated gradually. she also reported chronic bifrontal throbbing headache that was more prominent in mornings and history of several generalized tonic clonic seizures in the past few years. in the last months, she occasionally had mild dyspnea during sleep. there was bifacial weakness, wasted tongue with fasciculation, diminished palatal and left vocal cord movements and nasal speech. routine lab tests, creatine phosphokinase (cpk), lactate dehydrogenase (ldh) and acetylcholine receptor antibody disclosed no abnormality. findings of brain mri, chest ct - scan and abdomino - pelvic sonography were unremarkable. there was bilateral low frequency sensorineural hearing loss and electrodiagnostic study was compatible with anterior horn cell disease. a 22-year - old female, presented with hoarseness, sore throat, dysphagia, and hearing loss from 3 years before our first visit in december 2008. in the last 2 months, weakness developed over lower and then upper limbs associated with muscle wasting, fasciculation and exertional dyspnea. she reported 2 episodes of seizures at the age of 11 and her parents were first cousins and all 4 sisters and her brother were healthy. 1), palatal weakness, reduced motor force more in lower limbs, bilateral foot drop and generalized brisk dtrs. lab data revealed hypochromic microcytic anemia with normal serum iron and total iron binding capacity. nerve conduction studies were normal and emg of limbs and tongue muscles was compatible with motor neuron disease. one year after the treatment with 900 mg gabapentin, her swallowing subjectively improved and motor strengths became completely normal. a 17-year - old female, unmarried and student presented with speech problem and progressive hearing loss since the age of 12. she was admitted in our hospital in 1999 with liquid dysphagia and progressive weight loss in the last 6 months. the tongue was atrophic with fasciculation and gag reflex was diminished and the limbs were atrophic with mild proximal weakness. biochemical lab tests revealed no abnormality ; but on audiometry, there was severe bilateral sensorineural hearing loss. electromyography and nerve conduction studies (emg - ncv) were compatible with motor neuron disease with prominent involvement of cranial muscles ; in addition, repetitive nerve stimulation was negative. other investigations including chest x - ray, brain magnetic resonance imaging (mri) and abdomino - pelvic sonography were normal. this 17-year - old girl was admitted for the evaluation of slowly progressive dysphagia and choking from 6 months before admission in 2000. she had an episode of febrile seizure at the age of 3 and slowly progressive hearing loss from the age of 12. physical examination was normal. on neurologic exam, there was bifacial weakness, diminished gag reflex, atrophic tongue with fasciculation and proximal weakness. routine laboratory tests, peripheral blood smear, creatine phosphokinase (ck), and lactate dehydrogenase (ldh) were all normal, as well as cerebrospinal fluid (csf) analysis. there was bilateral low frequency sensorineural hearing loss on pure tone audiometry (pta) and motor neuron involvement mainly in cranial innervated muscles on emg - ncv. other investigations including chest x - ray, brain mri, tensilon test, repetitive nerve stimulation (rns) and abdomino - pelvic sonography were negative and genetic testing did not show deletion in smn1. one year after the beginning of gabapentin, 900 mg / day, she reported some improvement in dysphonia and dysphagia, which continued for the next 4 years, when she was lost to follow up. this 21-year - old female was admitted in december 2008 because of hoarseness, difficulty in swallowing, choking and fatigue in the recent 3 months. she had weight loss, difficulty in walking and bilateral hearing loss since 2 years ago which aggravated gradually. she also reported chronic bifrontal throbbing headache that was more prominent in mornings and history of several generalized tonic clonic seizures in the past few years. in the last months, she occasionally had mild dyspnea during sleep. there was bifacial weakness, wasted tongue with fasciculation, diminished palatal and left vocal cord movements and nasal speech. routine lab tests, creatine phosphokinase (cpk), lactate dehydrogenase (ldh) and acetylcholine receptor antibody disclosed no abnormality. findings of brain mri, chest ct - scan and abdomino - pelvic sonography were unremarkable. there was bilateral low frequency sensorineural hearing loss and electrodiagnostic study was compatible with anterior horn cell disease. a 22-year - old female, presented with hoarseness, sore throat, dysphagia, and hearing loss from 3 years before our first visit in december 2008. in the last 2 months, weakness developed over lower and then upper limbs associated with muscle wasting, fasciculation and exertional dyspnea. she reported 2 episodes of seizures at the age of 11 and her parents were first cousins and all 4 sisters and her brother were healthy. 1), palatal weakness, reduced motor force more in lower limbs, bilateral foot drop and generalized brisk dtrs. lab data revealed hypochromic microcytic anemia with normal serum iron and total iron binding capacity. nerve conduction studies were normal and emg of limbs and tongue muscles was compatible with motor neuron disease. one year after the treatment with 900 mg gabapentin, her swallowing subjectively improved and motor strengths became completely normal. bvvls is a rare neurological disorder considered as a type of motor neuron disorders which is characterized by bilateral hearing loss (the most common presenting symptom) accompanied by a variety of other mainly motor cranial nerve dysfunction. in spite of rarity, the disease has widespread distribution and cases have been reported in the literature from europe followed by asia and south america. the age of onset of the syndrome varies from infancy to the third decade ; but is more common in the first and second decades. the diagnosis is mainly based on clinical features and electrophysiological studies and no diagnostic biochemical or genetic defect has been reported., as they identified c20orf54 gene located in 20p13 in a study of seven families with bvvls (9 cases which 4 had no consanguinity), although the function of the gene in the nervous system is still not known. previously search for the mutations associated with spinal muscular atrophy (sma) has been done in two patients including the survival motor neuron (smn) gene and neuronal apoptosis inhibitory protein (naip) gene which was negative. half of the cases reported in the last century were sporadic, in the familial cases the vast major inheritance was autosomal recessive ; in some families autosomal dominant inheritance or probable x - linked inheritance has been postulated. all 9 cases reported by green had recessive mutations (homozygous or compound heterozygote, nonsense or missense mutations). herein we presented 4 cases of bvvls which consanguinity was present in 2 of them (cases 1 and 4). clinical feature of our patients with brown - vialetto - van laere syndrome umn : upper motor neuron the first presentation of all cases was hearing loss either isolated or accompanied by other neurological deficits (like speech problem in case 1 and difficulty in walking and weight loss in case 3). other first presentations rather than hearing loss have rarely been reported in the literature ; however, hearing loss has eventually developed in all. noted a girl with weakness of right foot dorsiflexion and then progressive hearing loss 7 years later. summers. depicted difficulty climbing stairs in a young girl one year before onset of deafness, but he also suggested madras type of anterior horn cell disorder as an alternative diagnosis. madras type of motor neuron disorder closely resembles bvvls and is almost confined to southern india. in contrast to bvvls, there is male preponderance or equal distribution, rare familial inheritance, more reported lower and upper motor neuron (umn) signs (more than 75% of patients) and a benign course. umn signs are infrequent in bvvls, although this happened in one of our cases (case 4). another rare feature of the disease is epilepsy which was seen in two of our cases (cases 3 and 4). the usual course reported in most cases is gradual deterioration followed by periods of stability or abrupt worsening. four of the previously reported cases showed some improvement : two in spontaneous activities and swallowing following steroid therapy and two others from respiratory failure. clinical improvement developed in two of our cases was mainly in swallowing with great recovery of motor strength to normal function in case 4. whether this is related to effect of gabapentin or the course of disease itself is unknown. grandis. showed evidence of improvement in motor and even sensory action potentials which are unusual in typical motor neuron disease. brain mri in cases did not show any abnormal changes ; however, atrophy of brain stem and cerebellum, hyperintensity in the brain stem, cerebellar peduncles and subcortical white matter has been previously reported ; all were non - specific changes. the genetic study of our three patients revealed four novel c20orf54 mutations which was published elsewhere. in conclusion, this is the first report of bvvls from iran. patients presented with hearing loss as initial symptom and developed gradual deterioration mainly in bulbar and motor function in their clinical course, although some improvement was noted in two of them. hopefully, in the recent future, molecular studies will clarify a better concept of the disease pathogenesis.	brown - vialetto - van laere syndrome (bvvls) is a rare neurological disorder. we report our finding about four patients clinically and electrophysiologically diagnosed as bvvls and denoted their clinical features with comparison to previous reports. the first symptom was bilateral hearing loss and the onset of other cranial nerves involvement varied between 0 - 15 years. our patients represented some rare features like upper motor neuron signs in one patient and two cases interestingly developed some clinical improvement. this is the first report of bvvls from iran. our patients which represent caucasian population had generally similar features like previously reported cases.
diffuse mesangial sclerosis (dms) is one of the rarer causes of nephrotic syndrome in infantile and childhood period. it is characterized by progressive sclerosis of the mesangial matrix with minimal or absent mesangial cell proliferation, hypertrophy of podocytes in early disease, thickened basement membranes, diminished patency of capillary lumen and, crown - like appearance of vacuolated podocytes in advanced disease. genetic forms of nephropathy including dms is usually resistant to immunosuppressive therapy and rapidly progresses to end - stage kidney disease (eskd). genetic mutation in genes wt1 and plce1 are found in of dms.[24 ] on pubmed search, we found only four cases of dms reported from india.[57 ] we report two cases of dms, one presenting in the first year of life and another in second decade of life. both of them had fatal outcome. an 8-month - old male child presented to a local practitioner with facial puffiness of 2 weeks duration. child weighed 7 kg and was a full - term product of an uncomplicated pregnancy, born to a 33-year - old g3p3l3 mother. however, after 4 months child was referred to a nephrologist with symptoms of anasarca, vomiting, and oliguria. urine examination at this time showed 4 + albuminuria, inactive sediments, and granular casts. hemogram revealed hb14.3 gm / dl, total leukocyte count 14,500 cells / mm and platelets 800 10/l. serum albumin and total cholesterol were 0.7 gm / dl and 3.9.75 mg / dl, respectively. kidney biopsy revealed 19 normal - sized glomeruli showing global and segmental mesangial sclerosis with obliteration of capillary lumina. tubulointerstitial compartment showed marked chronicity diffusely containing granular, leukocytic casts in the dilated lumen [figure 1 ]. rest of the panel (igg, iga, c3, c1q, and fibrin) were negative. child was put on supportive hemodialysis (twice a week) and treated with low - dose prednisolone 40 mg / m, nutritional supplements. four glomeruli displaying global mesangial sclerosis with variably increased mesangial cells, obliteration of capillary lumen, and patent bowmans space (black arrows, a). few capillaries had patent lumen with slightly thickened basement membranes (yellow star, a and d). interstitial space reveals lymphomononuclear infiltrates (a - h and e 20, b - pas, c - masson trichrome, d- pasm stain) a 16-year - old female presented with pedal edema and facial puffiness of 2 months duration. hemogram revealed hb 15.6 gm / dl, total leukocyte count 27,000 cells / mm and platelets 600 10/l. serum albumin and cholesterol was 0.5 gm / dl and 346.87 mg / dl, respectively. ultrasound examination showed normal - sized kidneys with increased echotexture and maintained cortico - medullary differentiation. kidney biopsy revealed eight normal sized glomeruli, all of which had global mesangial sclerosis and crowning of podocytes over the sclerosed tufts. immunofluorescence was negative for the panel (igg, iga, c3, c1q, and fibrin). she was treated with oral nitazoxanide 500 mg bd and intravenous piperacillin - tazobactam 2.25 gm tid. however, six days later she succumbed to severe sepsis despite active treatment. glomerulus showing mesangial sclerosis globally with obliteration of capillary lumen and patent bowmans space (40, pas stain) an 8-month - old male child presented to a local practitioner with facial puffiness of 2 weeks duration. child weighed 7 kg and was a full - term product of an uncomplicated pregnancy, born to a 33-year - old g3p3l3 mother. however, after 4 months child was referred to a nephrologist with symptoms of anasarca, vomiting, and oliguria. urine examination at this time showed 4 + albuminuria, inactive sediments, and granular casts. hemogram revealed hb14.3 gm / dl, total leukocyte count 14,500 cells / mm and platelets 800 10/l. serum albumin and total cholesterol were 0.7 gm / dl and 3.9.75 mg / dl, respectively. kidney biopsy revealed 19 normal - sized glomeruli showing global and segmental mesangial sclerosis with obliteration of capillary lumina. tubulointerstitial compartment showed marked chronicity diffusely containing granular, leukocytic casts in the dilated lumen [figure 1 ]. rest of the panel (igg, iga, c3, c1q, and fibrin) were negative. child was put on supportive hemodialysis (twice a week) and treated with low - dose prednisolone 40 mg / m, nutritional supplements. four glomeruli displaying global mesangial sclerosis with variably increased mesangial cells, obliteration of capillary lumen, and patent bowmans space (black arrows, a). few capillaries had patent lumen with slightly thickened basement membranes (yellow star, a and d). interstitial space reveals lymphomononuclear infiltrates (a - h and e 20, b - pas, c - masson trichrome, d- pasm stain) a 16-year - old female presented with pedal edema and facial puffiness of 2 months duration. hemogram revealed hb 15.6 gm / dl, total leukocyte count 27,000 cells / mm and platelets 600 10/l. serum albumin and cholesterol was 0.5 gm / dl and 346.87 mg / dl, respectively. ultrasound examination showed normal - sized kidneys with increased echotexture and maintained cortico - medullary differentiation. kidney biopsy revealed eight normal sized glomeruli, all of which had global mesangial sclerosis and crowning of podocytes over the sclerosed tufts. immunofluorescence was negative for the panel (igg, iga, c3, c1q, and fibrin). she was treated with oral nitazoxanide 500 mg bd and intravenous piperacillin - tazobactam 2.25 gm tid. however, six days later she succumbed to severe sepsis despite active treatment. glomerulus showing mesangial sclerosis globally with obliteration of capillary lumen and patent bowmans space (40, pas stain) diffuse mesangial sclerosis (dms) was first described by habib and bios in 1973. dms is one of the causes for nephrotic syndrome (ns) that occurs more commonly in first two decades of life. although rare cases of spontaneous remission in other causes of congenital nephrotic syndrome have been described, it is unheard of in patients with dms. this includes antiproteinuric measures and medical or surgical nephrectomy in nonresponders to mitigate proteinuria, nephrotic syndrome, and its complications. this is followed by renal allograft transplant as soon as the child attains 10 kg of weight. the phenotypic finding of a dms is not a single entity but is associated with many genotypic disorders including the wagr syndrome (wilms tumor, aniridia, genitourinary anomalies and mental retardation), denys drash syndrome (ns, male gonadal dysgenesis and wilms tumor), frasier syndrome (ns, normal female external genitalia, streak gonads, xy karyotype), pierson syndrome (ns with microconia), and galloway mowat syndrome (ns, microcephaly, gyral abnormalities, and hiatal hernia) or an isolated diffuse mesangial sclerosis.[24 ] acquired cytomegalovirus infection is also linked to causation of dms. plce1 gene is located on chromosome 10q23, encodes for a protein plc1 that is a member of phospholipase family of enzymes. study in infantile or childhood patients developing ns, and dms as the histologic lesion. chernin. concluded that patients with kts mutations presented at a significantly older age and with a slower progression toward eskd compared with missense mutations. analyzed a worldwide cohort of 1368 children with ns, and detected plce1 mutation as the most frequent (28.6%) and wt1 mutation as the second most frequent (8.5%) cause of isolated dms. three of these cases are reported from a single center with an incidence of 1% over a period of 10 years, studying 290 biopsied cases of the idiopathic nephrotic syndrome under the age of 16 years. we found similar incidence (1%) over a period of 28 months evaluating 196 cases of adequate biopsies under the age of 16 years with similar presentation. demography details on previous cases are available in only three of them (age range 3 to 9 months ; male to female this could probably be due to a mutation that presents at an older age than usual. clinical details and histologic findings are available in only one case wherein the 9-month - old child had nephrotic syndrome, hypertension, and normal renal function at presentation. kidney biopsy had revealed 44 glomeruli showing variable degree of mesangial sclerosis in majority of them. follow - up information on this case though incomplete in duration, mentions complete remission. family history and screening of family members is not available in any of the cases reported. both of our cases presented with nephrotic syndrome and renal insufficiency. the presentation with nephrotic syndrome in case 1 and case 2 was at the age of 8 months and 15 years, respectively. the siblings and family members of case 1 were screened for renal abnormalities and found to be normal. differential diagnosis of dms includes collapsing variant of focal segmental glomerulosclerosis (fsgs - col). the latter is usually a focal process possessing diagnostic features such as de - differentiation of podocytes in the form of hyperplasia (> 2 cell layer), proliferative marker ki-67 nuclear positive podocytes, and loss of wt1 nuclear expression ; and accompanied with collapse of underlying tufts. tubular microcyst formation with or without simplified epithelial lining is more commonly seen with fsgs - col than dms. electron microscopy of kidney tissue from paraffin block was tried ; however, the attempt was unsuccessful. as there were no extra - renal features associated with various syndromes, it is wise enough to diagnose isolated dms ; however, genetic analysis for at least wt1 and plce1 genes would have given more in depth knowledge of the disease. dms must be suspected in a child that presents with ns and/or rapidly progressive renal insufficiency, especially if there is family history of renal disease. there is an immense need for development of genetic analysis in this part of the world, especially for pediatric patients presenting with nephrotic syndrome and renal insufficiency, and their asymptomatic family members.	diffuse mesangial sclerosis (dms) is a rare cause of nephrotic syndrome in the infantile and childhood period. dms is a phenotypic expression of syndromic entities such as wagr syndrome (wilms tumor, aniridia, genitourinary anomalies and mental retardation), denys drash syndrome, pierson syndrome, frasier syndrome, or galloway mowat syndrome. we report two cases of dms, one presenting in first year of life and another in second decade of life. both of them had fatal outcome. recognition of the disease is very important in modifying the management of patient and active surveillance of family members.
cost - utility analysis (cua) is a form of economic evaluation that focuses specifically on the quality of health outcomes produced by health programs or services.1 cua has increasingly been used in the economic evaluation of health care for research or decision - making purposes, particularly when decisions on the allocation of health resources must be made.2 in cua, the incremental cost between two programs is compared with the incremental health improvement attributed to these programs. health improvement is normally measured in the quality - adjusted life - years (qalys) gained or using other generic outcome measures, such as disability - adjusted life - years (dalys). the results of cua are typically expressed as cost per qaly gained.1 the estimated incremental cost - effectiveness ratio (icer) derived is usually compared with a reference value to determine a program s cost effectiveness ; a health system will only fund activities that cost equal to or less than this value. this reference value is also named the cost - effectiveness threshold value, which is usually estimated in terms of societal willingness - to - pay (wtp) for a qaly. for instance, the threshold value set by the national institute for health and clinical excellence in the united kingdom has a threshold value of gbp 20,00030,000 per qaly gained, whereas ireland s threshold is euro 20,000 per qaly gained.35 additionally, the slovak republic uses a threshold ranging from euro 18,00026,500 per qaly for drug reimbursements.5 in current practice in malaysia, however, decisions regarding the coverage of new health care technologies are made without transparent decision criteria. in a review of 14 malaysian economic evaluation papers from 1999 to 2011,619 it was found that most reported cost - effectiveness outcomes were based solely on the average cost - effectiveness ratio without considering the icer. although some papers discussed cost - effectiveness threshold values, decisions were made without using a transparent and solid threshold value. the funding and reimbursement process in malaysia uses an arbitrary threshold value of one to three times the regional gross domestic product (gdp) per capita, as recommended by the world health organization (who).20,21 it was also noted that of the 14 reviewed papers, only two papers17,19 discussed the use of cua in decision making. this finding indicates that the use of cua in decision making for health care programs is not popular in malaysia, despite the global trend of rapid growth in the publication of cua papers, in which many experts have recommended the application of cua in health economics and outcomes research.22,23 hence, through this paper, we hope that the use of cua in decision making for health care interventions will be encouraged in malaysia, via a recommendation in the pharmacoeconomic guideline for malaysia.24 in addition, by exploring a cost - effectiveness threshold value in malaysia, estimated in terms of societal wtp per qaly (wtp / qaly), health care decision making will be improved. the contingent valuation method (cvm) is a widely used technique for estimating the value of nonmarketed commodities, where it creates a hypothetical marketplace in which no actual transactions are made.25,26 this method is applied as a reasonable approach for determining the monetary value that society places on a qaly.3,27,28 as a threshold should represent the value a society attaches to a qaly, individual valuations of personal health gains can be directly elicited using contingent valuation (cv). in the recently performed european value of a quality - adjusted life - year (eurovaq) study, cvm with a chained approach combining the answers of both utility and wtp values would allow respondents wtp / qaly gained to be estimated.29 in cv surveys, the use of a dichotomous - choice format is widely accepted due to its way of inducing respondents to reveal their true preferences and its simplification of the cognitive task faced by respondents (where there are only two possible responses : yes or no).26 nevertheless, it is vital to identify some potential influences on the results, due to the starting - point bias and outliers. this study was the first attempt in malaysia to explore the use of cvm in estimating the value of wtp / qaly and to assess the general factors associated with the wtp amount for a qaly gained. it is anticipated that the findings from this study could serve as a basis for determining a valid cost - effectiveness threshold value for malaysia, which could, in turn, assist health authorities in making informed funding and reimbursement decisions and in formulating health policies. a cross sectional cv study was conducted among adults from the general population in penang, malaysia, in august 2010. penang is one of 13 states in malaysia and consists of two separate areas, penang island and seberang perai, on the peninsular malaysia mainland. it has a high level of urbanization (91.4%) and had a total population of 1.56 million residents in 2010.30 this study was carried out through a face - to - face survey, with one family member in each household, using a predesigned questionnaire that measured wtp for 1 additional year of survival with perfect health. the family member could be the head of household, the spouse of the head of household, son or daughter of the head of household, or someone else. in the questionnaire, a double - bound dichotomous - choice approach was applied in which respondents were asked whether they would be willing to pay a specified amount (which was randomly chosen from three starting bid values) for a particular treatment that would be capable of extending their life by 1 year, with perfect health. a minimum sample size of 286 was required for a multiple regression test, using the rule of thumb for a small effect size (30 participants per variable plus 50) expected with seven predictors (monthly household income, ethnicity, visual analog score, presence of disease, satisfaction with provided medical care, satisfaction with medical care available in the country, and total expenditure on health for the last 3 months) and a dropout rate of 10%.31 in the absence of a population - level sampling framework, this exploratory study adopted random walking to reduce biases and introduce randomness into the sampling. gridlines (1 km) were overlaid on a google earth map of penang, resulting in 192 grids. sixty - nine grids were excluded because they were designated as natural forests and were geographically uninhabitable areas. ten grids were randomly selected from the remaining 123 grids, using microsoft excel 2007 software. within each grid, the initial direction for data collection was determined using a random pen - throw method. we developed questionnaires, in both english and malay, to collect respondents sociodemographic information ; to assess quality of life, using the euroqol visual analog scale (eq - vas)32 (a thermometer - like visual analog scale [vas ] ranging from 0 to 100, with 0 signifying the worst health state imaginable and 100 signifying the best health state imaginable) ; and to determine the cost - effectiveness threshold value, by collecting the information on subject s wtp for one additional qaly. the scenario for the cv was modified from shiroiwa.3 in the scenario (figure 1), respondents were asked whether they were willing to pay for medication a, which would treat a serious illness and give them 1 year of perfect health. if a positive response was obtained, respondents were asked to give a wtp amount for medication a, using a double - bound dichotomous choice bidding game approach with three different starting bids (myr [malaysian ringgit ] 20,000, myr 40,000, and myr 100,000). the first three from six starting bid values used by shiroiwa were chosen because they were close to the range of the cost - effectiveness threshold in malaysia set by the who.21 the bidding values were converted to malaysian currency, and subsequent bidding values were modified, contingent on the response to the initial bid. in the elicitation of wtp values, it is important to distinguish between true zero values and the protest zero values provided by respondents. some respondents who have a positive wtp amount for the commodity may provide protest zero for instance, some respondents may think that the treatment cost should be covered by the government or insurance companies and thus give a wtp value of zero. in this case, the value is identified as a protestor and should be excluded from the analysis.33 conversely, those with true zero values should be included in the analysis. excluding all true zero and protest zero values from the analysis may reduce the number of samples being analyzed and introduce some degree of selection bias. all data were analyzed using stata 9.0 (statacorp, college station, tx, usa). wallis test was applied to assess the association of independent variables with wtp, with the p - value set as 0.05. the heckman selection model was employed in the estimation of the mean wtp, to account for a selection bias due to data clumping at zero. in heckman s two - step approach, the probability of observing a positive wtp was predicted by a binary probit model in the first step, while in the second step, a quantile regression on the observations above zero wtp was estimated.34 a cross sectional cv study was conducted among adults from the general population in penang, malaysia, in august 2010. penang is one of 13 states in malaysia and consists of two separate areas, penang island and seberang perai, on the peninsular malaysia mainland. it has a high level of urbanization (91.4%) and had a total population of 1.56 million residents in 2010.30 this study was carried out through a face - to - face survey, with one family member in each household, using a predesigned questionnaire that measured wtp for 1 additional year of survival with perfect health. the family member could be the head of household, the spouse of the head of household, son or daughter of the head of household, or someone else. in the questionnaire, a double - bound dichotomous - choice approach was applied in which respondents were asked whether they would be willing to pay a specified amount (which was randomly chosen from three starting bid values) for a particular treatment that would be capable of extending their life by 1 year, with perfect health. a minimum sample size of 286 was required for a multiple regression test, using the rule of thumb for a small effect size (30 participants per variable plus 50) expected with seven predictors (monthly household income, ethnicity, visual analog score, presence of disease, satisfaction with provided medical care, satisfaction with medical care available in the country, and total expenditure on health for the last 3 months) and a dropout rate of 10%.31 in the absence of a population - level sampling framework, this exploratory study adopted random walking to reduce biases and introduce randomness into the sampling. gridlines (1 km) were overlaid on a google earth map of penang, resulting in 192 grids. sixty - nine grids were excluded because they were designated as natural forests and were geographically uninhabitable areas. ten grids were randomly selected from the remaining 123 grids, using microsoft excel 2007 software. within each grid, the initial direction for data collection was determined using a random pen - throw method. we developed questionnaires, in both english and malay, to collect respondents sociodemographic information ; to assess quality of life, using the euroqol visual analog scale (eq - vas)32 (a thermometer - like visual analog scale [vas ] ranging from 0 to 100, with 0 signifying the worst health state imaginable and 100 signifying the best health state imaginable) ; and to determine the cost - effectiveness threshold value, by collecting the information on subject s wtp for one additional qaly. the scenario for the cv was modified from shiroiwa.3 in the scenario (figure 1), respondents were asked whether they were willing to pay for medication a, which would treat a serious illness and give them 1 year of perfect health. if a positive response was obtained, respondents were asked to give a wtp amount for medication a, using a double - bound dichotomous choice bidding game approach with three different starting bids (myr [malaysian ringgit ] 20,000, myr 40,000, and myr 100,000). the first three from six starting bid values used by shiroiwa were chosen because they were close to the range of the cost - effectiveness threshold in malaysia set by the who.21 the bidding values were converted to malaysian currency, and subsequent bidding values were modified, contingent on the response to the initial bid. in the elicitation of wtp values, it is important to distinguish between true zero values and the protest zero values provided by respondents. some respondents who have a positive wtp amount for the commodity may provide protest zero for instance, some respondents may think that the treatment cost should be covered by the government or insurance companies and thus give a wtp value of zero. in this case, the value is identified as a protestor and should be excluded from the analysis.33 conversely, those with true zero values should be included in the analysis. excluding all true zero and protest zero values from the analysis may reduce the number of samples being analyzed and introduce some degree of selection bias. all data were analyzed using stata 9.0 (statacorp, college station, tx, usa). to summarize the data, the kruskal wallis test was applied to assess the association of independent variables with wtp, with the p - value set as 0.05. the heckman selection model was employed in the estimation of the mean wtp, to account for a selection bias due to data clumping at zero. in heckman s two - step approach, the probability of observing a positive wtp was predicted by a binary probit model in the first step, while in the second step, a quantile regression on the observations above zero wtp was estimated.34 a total 510 households were approached, and 347 households (68.04%) participated in this survey. the respondents sociodemographic data are summarized in tables 1 and 2. through a cross - comparison of malaysia s population with penang s population, it was noted that the results for age, sex, and employment status were very similar but ethnicity and marital status were not. in this survey, it was noted that n=124 (35.7%) of the respondents were over 51 years of age. a total n=177 (51%) of the respondents were chinese, and the majority of the respondents (n=253 [72.9% ]) were married. of the 347 respondents in this study, 106 (30.5%) earned an estimated monthly household income of myr 2,001 to myr 4,000. in the valuation of the respondents current health statuses using the vas, the mean current health status of the respondents was found to be 76.06, with a standard deviation (sd) of 14.64. the descriptive statistical analysis showed that 152 respondents were not willing to pay any amount for the treatment in the given scenario. the reasons given for not being willing to pay are presented in table 3. from the reasons provided, affordability (n=45 [29.6% ]) was the main concern affecting the respondents wtp. some of the respondents claimed that they could not afford any additional treatment costs, due to financial constraints. there were also some respondents (n=5 [3.3% ]) who felt that the treatment cost should be covered by the government or insurance companies. excluding all zero wtp values, we performed a preliminary analysis and noted that the mean value of wtp / qaly was estimated to be myr 53,629, or approximately usd 17,000 (myr 3.2usd 1), with an sd of myr 106,805. the minimum and maximum results for wtp for an additional qaly were myr 20 (usd 6) and myr 1,000,000 (usd 310,000), respectively, with a median value of myr 20,000 (25th percentile = myr 5,000 ; 75th percentile = myr 40,000) (figures 2 and 3). however, we also found that the mean wtp / qaly value was myr 30,137 (sd = myr 84,297) when we included the positive wtp values together with all of the zero values. by fitting all of the observations using the heckman selection model, the estimated mean wtp for a qaly value was found to be myr 29,080 (usd 9,000). the probit estimation results from the heckman selection model (table 4) showed that the respondents visual analog scores and current disease statuses significantly affected their wtp. from the second - stage regression estimate of the model, it was found that respondents who had incomes more than myr 6,000 or who were of chinese ethnicity were more likely to elicit a higher wtp amount. for a long time, many studies in malaysia employed the threshold value recommended by the who in the choosing interventions that are cost - effective (choice) project, which uses gdp per capita as an indicator.20,21 following this recommendation, any new intervention would be considered cost - effective if it were below the recommended value of three times the gdp per capita threshold of myr 66,704 (usd 20,845).21 many policymakers and local researchers are still using this arbitrary threshold as a reference when making decisions, though the value is over 10 years old. based on this problem, this study was performed with the intention of finding an empirical threshold value for malaysia. moreover, the acceptance of the who - recommended threshold value remains controversial because it depends on the robustness of the assumptions behind the estimation of the regional gdp per capita. in addition, the use of such a generalized threshold value may not be entirely relevant in every country as different countries may have distinct sociodemographic and disease burdens, despite having similar gdps per capita. in using gdp per capita as an indicator, a nation s average wealth for example, the incomes of some individuals in rural areas may remain low, although they live in high - income countries, and may not fairly represent a nation s wealth.35 in this case, these individuals may have a different wtp from the average. thus, the validation method suggested by choice may only be relevant for countries that have a fairly even distribution of wealth. rather than applying arbitrary decision rules, such as league tables or proposed icer thresholds, without actual societal representation, it may be more reasonable to allocate health care resources based on actual societal wtp for health care benefits.27 we found that the cost - effectiveness threshold value estimated in this study was lower than the value recommended by the who. this finding is consistent with that of king,27 in whose study the wtp / qaly ratios, calculated using preference - based data collected from a population, were lower than the proposed cost - effectiveness threshold. the person trade - off technique for evaluating health benefits has been proposed as one way to address the trade - off that may exist between efficiency and equity. economists have typically argued that individuals are the best judges of their own well - being and that social welfare depends only on the welfare of individuals in society.36,37 it is also defined as the utilitarian concept of the sum of individual happiness, where individual utilities are aggregated in a direct and transparent manner.36 in our study, both the numerator (wtp) and denominator (calculated qaly) were obtained from respondents in the general population and should indicate actual societal preferences. in the preliminary analysis of the estimation of the wtp for an additional qaly, the mean wtp / qaly values would be considerably higher due to the exclusion of the zero wtp values. on the contrary, the mean wtp / qaly value was noted to be low when all of the values were included. here, the application of the heckman selection model is important in the analysis of mean wtp / qaly values, where only protestors were excluded. the capabilities of the heckman selection model, which range from specifying a selection function to obtaining an estimate for the bias term, make it an intuitive, appealing tool that can be used to correct for the selection bias problem. the value estimated by the heckman selection eliminates the sample selection bias that arises from the specification error of the zero data.34 in the reported heckman model, a rho value of 0.3257 and a statistically significant likelihood ratio test indicates correlation between the two - part model and the presence of selection. this procedure has the advantage of minimizing skewed outcome distributions, which is robust against potential outliers. additionally, it tends to model two independent decisions that may display distinct effects on the different dependent variables being considered. the quantile regression s ability to give a more complete description of the impact of covariates on the outcome distribution, by analyzing different quantiles, adds to the appeal of using this procedure in exploring the analysis.33 as a result, the heckman selection model is preferred because it gives a better estimated value of wtp / qaly. malaysia is a unique multiracial nation whose predominant ethnic groups are bumiputera (67.4%), chinese (24.6%), and indian (7.3%). different ethnicities have biological, demographic, and social environment differences as well as differences in psychological and behavioral characteristics, all of which contribute to one s health. differences in sociocultural beliefs among different races may affect various forms of self - care and eventually affect health outcomes.38 chinese respondents, with different beliefs and cultural backgrounds, may contribute to the significant difference in their beliefs about paying for health care, as demonstrated by their wtp results. after adjustments, it was found that chinese respondents, on average, offered an additional myr 21,339 per qaly gained relative to other ethnic groups. such diversity in the sociodemographic characteristics in malaysia suggests that the generalized regional wtp / qaly value based on three times the gdp per capita, as recommended by the who, may not be entirely appropriate. in addition, estimated monthly household income was found to be significantly associated with the wtp results. according to the malaysia household income and basic amenities individuals with incomes below myr 2,000 can be considered a low - income group, while those with incomes of myr 6,000 or higher are categorized as a high - income group. logically, within the context of wtp, those with greater wealth have a greater ability to pay.27,40 some research has assessed the reciprocal relationship between income and health status and found that individuals with better health have more household income than individuals with poor health.38,41 in this study, respondents income statuses were found to be positively related to their magnitude of wtp / qaly ; thus, the internal validity of this cv survey is ensured. as we move through the income scale, respondents with income first, the use of just a single scenario might not be practical and illustrative in eliciting wtp amounts for the determination of wtp per qaly values. the design was based on the notion of a qaly is a qaly is a qaly, implying that all qalys have the same value. qaly serves as a measure of health and is a unit on a value function scale.42 for instance, 2 years at a quality of 0.5 is judged as equivalent to 1 year at a quality of 1.0. using weight as another example, the weight of 1 kg of apples is equivalent to the weight measuring 1 kg of watermelon, regardless of the size of the fruits. however, some studies have found that the valuation of wtp might be different in the cases of chronic diseases or when respondents are faced with death.3 for instance, in a study by thavorncharoensap, the value of a qaly varies depending on treatment and prevention scenarios and the health condition under consideration.43 in the eurovaq project, it was found different people do not attach equal value to qalys for example, a qaly gained in a person who is severely ill may be valued differently than a qaly gained in a person who is only mildly ill.29 therefore, we intend to explore these issues in future studies. second, the results in this study only accounted for the valuation by penang s population, where penang is just one of the 13 states in malaysia. although the sociodemographic characteristics of respondents in this survey were marginally comparable with those of the penang population,30 it has an overrepresentation of chinese respondents, despite the fact that the predominant ethnic group in malaysia is bumiputera. however, again, this study serves as our first attempt to determine a validated cost - effectiveness threshold for malaysia. this may be due to the nature of elderly respondents in answering questionnaires, as some of them have no formal education. this issue will be addressed in the inclusion criteria for our future studies, where we plan to have an age limit of 60 years. last but not least, convenience sampling was performed in this study in the absence of a population - level sampling frame. more rigorous population sampling, such as multistage stratified cluster sampling using data derived from computerized household lists from malaysia s department of statistics census, should be applied in future studies. the wtp for an additional qaly gained among respondents in penang, malaysia was estimated to be myr 29,080 (usd 9,000). among the independent variables, ethnicity and income level are key elements that affect wtp responses. it is crucial to obtain the social value of a qaly from the perspectives of the public as different beneficiaries of health care have differing values of a qaly, due to variations in sociodemographic factors. the findings from this study will provide empirical evidence for the determination of the cost - effectiveness threshold in malaysia and, in turn, facilitate the development of health technology assessment for the country.	introductionthe incremental cost - effectiveness ratio (icer) is typically compared with a reference value to support the cost - effectiveness of a decision. one method for estimating this value is to estimate the willingness - to - pay (wtp) for a quality - adjusted life - year (qaly). this study was conducted to explore the wtp for a qaly in the malaysian population.methodsa cross - sectional, contingent valuation study was conducted in penang, malaysia. respondents were selected from randomly chosen geographical grids of penang. respondents sociodemographic information, qualities of life, and wtp for one additional qaly were collected using predesigned questionnaires in face - to - face interviews. wtp values were elicited using a double - bound dichotomous choice via a bidding game approach. the heckman selection model was applied to the analysis of the mean wtp / qaly values, where the protest zero values, which may contribute to selection bias, were excluded.resultsthe mean value of wtp for an additional qaly gained was estimated to be myr (malaysian ringgit) 29,080 (~usd 9,000). key factors that affected the wtp include ethnicity and estimated monthly household income.conclusionthe study findings suggested that the cost - effectiveness threshold value as studied in penang, malaysia was estimated to be myr 29,080.
aberrant hypermethylation in promoter regions of genes is now recognized as an important and early event in carcinogenesis 1. to date, cpg island hypermethylation has been shown to inactivate more than 70 genes in breast tumor tissues 2. among these genes, breast cancer - related tumor suppressor genes, brca1, cdh1 and rar are frequently hypermethylated in breast cancer 3 - 5. numerous studies have confirmed the hypothesis that aberrant methylation of specific genes contributes to the malignant phenotype of breast cancer 6 - 9. it is increasingly recognized that tumor dna can be found in the bloodstream of cancer patients and that this dna frequently contains the same genetic and epigenetic alterations as dna isolated from an individual 's tumor 10 - 12. this suggests that detection of tumor dna in blood may serve as an early and more accessible marker for diagnosis of breast cancer. however, the frequency of aberrant methylation in white blood cells (wbc) as a potential biomarker of risk has not been extensively investigated. we hypothesized that aberrant promoter methylation of brca1, cdh1 and rar would be detectable in wbc dna of breast cancer patients and there would be a correlation between methylation in tumor tissue and blood dna but with more frequent methylation in tissue dna. in the present study, we determined whether methylation in brca1, cdh1 and rar in wbc dna differed between cases and controls in the long island breast cancer study project (libcsp). since tumor methylation for these genes was available for a large subset of the cases, we also determined the correlation between methylation status in tumor and wbc dnas from cases. we utilized the resources from the case - control component of the libcsp, a population - based investigation. details of the study participants and design have been described previously 13 - 15. in brief, eligible case participants included english speaking adult female residents of nassau and suffolk counties on long island, ny. eligible case women were of all ages and races and newly diagnosed with in situ or invasive breast cancer between august 1, 1996, and july 31, 1997. potentially eligible controls were frequency - matched to the expected age distribution of the cases by 5-year age groups and identified through random digit dialing for women age 10% 23. only family history of breast cancer, final models were therefore adjusted for each of these variables as well as 5-year age group. the kappa coefficient (and corresponding 95% confidence intervals) was used to compare the proportion of subjects defined as hypermethylated in wbc vs. tumor tissue for each of the three breast cancer - related tumor suppressor genes, brca1, cdh1 and rar 24. we utilized the resources from the case - control component of the libcsp, a population - based investigation. details of the study participants and design have been described previously 13 - 15. in brief, eligible case participants included english speaking adult female residents of nassau and suffolk counties on long island, ny. eligible case women were of all ages and races and newly diagnosed with in situ or invasive breast cancer between august 1, 1996, and july 31, 1997. potentially eligible controls were frequency - matched to the expected age distribution of the cases by 5-year age groups and identified through random digit dialing for women age 10% 23. only family history of breast cancer, final models were therefore adjusted for each of these variables as well as 5-year age group. the kappa coefficient (and corresponding 95% confidence intervals) was used to compare the proportion of subjects defined as hypermethylated in wbc vs. tumor tissue for each of the three breast cancer - related tumor suppressor genes, brca1, cdh1 and rar 24. brca1, cdh1 and rar were rarely methylated in wbc dna from either cases or controls with only 6, 2 and 1 subjects for brca1, cdh1 and rar, respectively, when values of > 10% were considered as positive. using 1% as the cutoff for positive methylation, methylation frequencies of brca1, cdh1 and rar were 1.8%, 2.0% and 1.5%, in cases and 1.3%, 1.1% and 1.5% in controls, respectively. in contrast, when we used the 0.1% cutoff to define positive methylation status we observed more frequent hypermethylation in wbc dna for both cases and controls (table 1). we found little evidence of association between any of the genes and breast cancer risk modeling methylation as a continuous variable (table 1). given the low proportion of women classified as positive using 1% or 0.5% methylated definitions, our binary exposure models revealed non - significant associations between hypermethylation of brca1, cdh1 and rar and breast cancer risk. we did, however, find evidence of association upon classifying women with 0.1% methylation as positive. hypermethylation of brca1 (or : 1.31 ; 95% ci : 0.98 - 1.75) was associated with increased risk of breast cancer, while hypermethylation of cdh1 (or : 0.65 ; 95% ci : 0.54 - 0.79) and rar (or : 0.67 ; 95% ci : 0.55 - 0.81) was associated with reduced risk (table 1). our three - level categorical model revealed similar modest inverse associations with breast cancer risk for rar (table 1). we previously analyzed promoter hypermethylation in tumor tissues for brca1, cdh1 and rar 16, 17. tumor tissues were more frequently hypermethylated (defined as > 4% methylation) than blood dna for all genes tested (59.3% for brca1 16, 23.3% for cdh1 17 and 44.5% for rar 17). methylation of all three genes was discordant between tumor tissue and paired wbc dna using 0.1% methylated as the cutoff for positive methylation status in wbc (table 2). in a large population - based, case - control study of women with breast cancer, we found that hypermethylation of brca1 may be associated with breast cancer risk when using very low levels of methylation (i.e. 0.1%) as the cutoff for comparison. although two prior studies reported a significantly higher frequency of wbc brca1 methylation in breast cancer cases 25, 26, two others found no difference 27, 28. in contrast, wbc brca1 methylation at levels as high as 17% has been consistently observed in 5 - 43% of women with brca1 mutation - associated pathology but without germline brca1 mutations, suggesting rare constitutional methylation 25, 29. two studies have also suggested that methylation of specific regions in atm may be associated with increased breast cancer risk 30, 31. an analysis of 25 genes reported that methylation of five genes (znf217, neurod1, sfrp1, titf1 and nup155) in wbc was associated with breast cancer risk 32. the original 25 genes were selected because they are estrogen receptor- targets, known to be differently methylated depending on hormone receptor status, known to contain stem cell polycomb group targets or known to be methylated in breast cancer. here we used the methylight assay to determine wbc methylation for three candidate genes frequently methylated in breast cancer. this method allows rapid analysis of large numbers of samples and provides continuous data on % methylation calculated versus a fully methylated previously, we have analyzed methylation for this type of material and found methylation levels ranging from 85 - 95%. this suggests that levels of methylation may not be accurate although relative methylation levels should be valid. in addition, there is no generally accepted cutoff for use in determining hypermethylation in wbc dna. in our prior study of tumor tissue, we used 4% methylation as the cutoff since this value had been used previously 33. however, one recent study used 0.1% methylation as the cutoff with cancer samples 34. thus, for wbc methylation we tested different cutoffs for the definition of hypermethylation including 1%, 0.5% or 0.1%. while the number of subjects with hypermethylation increased as the cutoff was lowered, only for brca1 was there a suggestion of a positive association between hypermethylation and breast cancer risk. the inverse relationships between methylation of cdh1 and rar and breast cancer risk are difficult to explain. many more controls are considered positive for methylation when using the criteria 0.1% in cdh1 (n=684) and rar (n=404) than for brca1 (n=104) and how this impacts results is unclear. the or for cdh1 became inverted as the cutoff was lowered suggesting the data are unstable. in addition, the biological relevance of these very low levels of methylation is also unclear suggesting the results should be interpreted with caution. our results showed a discordance of hypermethylation of the genes tested between wbc and tumor tissue, similar to results observed previously for brca1 35. this finding suggests that a direct link between wbc brca1 methylation and development of breast cancer is still questionable. one of the strengths of our study is the large sample size from a population - based case - control study (1,021 cases and 1,036 controls). in addition, the methylight assay we used is known to be sufficiently sensitive to detect methylated dna present at low levels. however, despite these strengths, analyses were based on small numbers of women with hypermethylation because wbc dna was rarely hypermethylated. furthermore, the relatively modest number of tumor suppressor genes investigated precluded further analyses of the possible relationship between methylation levels and breast cancer risk. thus, further studies with additional genes are needed to establish the usefulness of wbc dna methylation as a marker of risk. in conclusion, we examined methylation levels in tumor and wbc dna from in situ or invasive ductal breast cancer patients and in wbc dna from population - based controls to identify epigenetic markers of breast cancer risk. obtained results suggest that hypermethylation of these three genes in wbc dna may be associated with breast cancer risk, but additional studies on the biological significance of low - level methylation is needed to fully understand the observed results.	the role of gene - specific methylation in white blood cells (wbc) as a marker of breast cancer risk is currently unclear. we determined whether promoter hypermethylation in blood dna of candidate tumor suppressor genes frequently methylated in breast tumors can be used as a surrogate biomarker for breast cancer risk. promoter methylation of brca1, cdh1 and rar was analyzed in wbc dna from a population - based sample of 1,021 breast cancer patients and 1,036 controls by the methylight assay. gene - specific promoter methylation in the dna of 569 tumor tissue samples was also analyzed to determine the correlation of methylation levels with blood from the same individual. hypermethylation of brca1 (or : 1.31 ; 95% ci : 0.98 - 1.75) in wbc was associated with an increased risk of breast cancer when positive methylation was defined as 0.1% methylated. there was lack of concordance between tumor tissue and paired wbc dna methylation. these results provide limited support that hypermethylation of brca1 in wbc dna may be useful for determination of breast cancer risk. additional studies with larger numbers of genes are needed to fully understand the relationship between wbc methylation and breast cancer risk.
the design and implementation of modern control strategies in bioindustry require useful models of the biotechnological processes. in many real - life applications, the bioprocess modeling is a quite difficult task ; still, by using the mass balance of the components inside the process and obeying some modeling rules, a dynamical state - space model can be obtained [13 ]. a practical alternative to the classical modeling is the bond graph method, introduced by paynter in 1961, and further developed in. in the last period, there have been a lot of works on the subject of the theory and application of bond graphs for different kind of systems, such as electrical, mechanical, hydraulic, thermal, and chemical [68 ]. this method provides a uniform manner to describe the dynamical behavior for all types of physical systems. the advantages of bond graph modeling are the following : offers a unified approach for all types of systems ; allows to display the exchange of power in a system by its graphical representation ; due to causality assignment, it gives the possibility of localization of the state variables and achieving the mathematical model in terms of state space equations in an easier way than using classical methods ; provides information regarding the structural properties of the system, in terms of controllability observability, and so forth. the bond graph method uses the exchange power in a system, which is normally the product between the effort and flow variables in the true bond graph. besides this representation there is another one, in which the product effort - flow does not have the physical dimension of power, called pseudo bond graph [7, 8 ]. pseudo bond graphs are more suitable for chemical systems due to the physical meaning of the effort and flow variables. the bond graph modeling of a few biological systems was reported in some works, such as. though, the bond graph modeling of biotechnological processes is not fully exploited yet ; in recent years, only some applications in wastewater treatment bioprocesses were reported [3, 10, 11 ]. to obtain the model of a bioprocess it is necessary to know the fundaments of the bioprocess and to have good knowledge concerning the bond graph methodology. even if for a bioreactor specialist seems to be easily to obtain the model via classical method, the bond graph approach can be applied without difficulty after a short training. the major advantage of the proposed approach is that the obtained bond graph models of the bioprocesses can be easily used and extended to other bioprocesses. for example, the bond graph models can be used in order to obtain complex models of interconnected bioprocesses, and for the direct design of some observers and controllers. the use of modern control techniques for bioprocesses is hampered by the nonlinearity of this kind of processes and the unavailability of several on - line measurements. serious problems appear in the measurement of biological variables, that is, the substrates, biomass and product concentrations, and so forth. in many cases, the state variables (concentrations) are analyzed manually and as a result there is not on - line (and real - time) control. these software sensors can be used not only for the estimation of the concentrations (state variables), but also for the estimation of the kinetic parameters. very important is the estimation of kinetic rates inside a bioreactor, that is, the so - called kinetics of the bioprocess. the growing interest for development of software sensors for bioprocess and biological systems is proven by the numerous publications and applications in this field [13, 1316 ]. one of the first approaches from historically point of view is based on kalman filter which leads to complex nonlinear algorithms. this strategy consists in the estimation of unmeasured state with asymptotic observers, and after that, the measurements and the estimates of state variables are used for on - line estimation of kinetic rates. this method is useful, but in some cases, when many reactions are involved, the implementation requires the calibration of too many parameters. for instance, if we have n components ' concentrations used for the estimation of m kinetic rates, it is necessary to calibrate 2n tuning parameters. in order to overcome this problem, a possibility is to design an estimator using a high gain approach (see [13, 14 ]). the gain expression of the high gain observers involves a single tuning parameter whatever the number of components and reactions. high gain observers have evolved over the past two decades as an important tool for the design of output feedback control of nonlinear systems. the early work on high gain observers appeared in the late 1980s, and afterwards the technique was developed independently by two schools of researchers : a french school lead by gauthier, hammouri, farza, and others, [13, 14 ], and a us school lead by khalil. the modeling techniques and the estimation strategies were used for the design of several control strategies, such as optimal control, sliding mode control, adaptive control [1, 18 ], vibrational control, model predictive control, fuzzy and neural strategies, and so on. generally speaking, due to specificity and nonlinearity of bioprocesses, there is no universal solution to the control problem, and good solutions are given only by studying each particular bioprocess. in this paper, which is an extended work of, three main correlated issues concerning the enzymatic bioprocess of ampicillin are studied : modeling, kinetics estimation, and control. first, a pseudo bond graph approach is proposed for the modeling of an enzymatic synthesis of ampicillin, widely used in bioindustry. ampicillin (6-[2-amino-2-phenylacetamide ] penicillanic acid) is a semisynthetic -lactamic antibiotic which is very stable at acidic conditions, well absorbed and effective against a wide variety of microorganisms, with low minimal inhibitory concentration [2124 ]. these reactions typically involve costly steps, such as very low temperatures and the use of toxic organic solvents like methylene chloride and silylation reagents. enzymatic synthesis is an alternative process ; it has high selectivity, specificity, and activity in mild reaction conditions (aqueous medium, neutral ph, and moderate temperatures) [2527 ]. the main aim of enzyme immobilization is the industrial reuse of enzymes for many reaction cycles [25, 28, 29 ]. thus, simplicity and improvement of enzyme properties have to be strongly associated with the design of protocols for enzyme immobilization. a critical review of enzyme immobilization was presented in. concerning the use of enzymatic methods in production of semisynthetic -lactamic antibiotics, several drawbacks and perspectives they are usually produced by the hydrolysis of natural antibiotics (penicillin g or cephalosporin c). due to the contaminant reagents used in conventional chemical route, as well as the high energetic consumption, biocatalytic approaches have been studied for both steps in the production of these very interesting medicaments during the last decades. the hydrolysis of penicillin g to produce 6-apa catalyzed by penicillin g acylase is one of the most successful examples of the enzymatic biocatalysis. the dynamical model of this complex bioprocess is obtained via the bond graph methodology by using the reaction scheme and the analysis of biochemical phenomena inside the bioreactor. second, because the kinetic rates of the enzymatic bioprocess of ampicillin production are nonlinear and highly uncertain, an on - line estimation strategy is designed. some estimation strategies were proposed in the last years, such as observer - based estimators and the second - order observers (see, e.g.,). in this paper, the design and implementation of high gain observers is proposed, with certain advantages concerning the robustness against disturbances and the simple tuning. the tuning of the proposed observers is reduced to the calibration of a single parameter., farza., focused on deriving global results under global lipschitz conditions. third, an adaptive control law for the enzymatic fed - batch bioprocess of ampicillin production is developed. for bioprocesses taking place into fed - batch reactors, the adaptive control approach is a viable alternative of the optimal control [1, 2 ]. in order to design a control law, the nonlinear controller thus obtained is combined with the high gain estimator for the unknown kinetics, and consequently an adaptive controller is obtained. due to the fact that the implementation of highgain observer and of the adaptive controller requires on - line state estimates, the performances and the behavior of the estimation and control algorithms are studied by using extensive numerical simulations. all these simulations are achieved by using the development, programming and simulation environment matlab (registered trademark of the mathworks, inc. the results obtained in this study show a good behavior of the adaptive controlled ampicillin synthesis bioprocess. the proposed adaptive control scheme is quite simple, because only two tuning parameters were used, one for estimator and one for the linearizing controller. the bond graph modeling, estimation, and control strategies can be also applied to other processes belonging to the nonlinear class of bioprocesses considered in the study. the bioprocesses are highly complex processes that take place inside biochemical reactors (bioreactors). the bioreactors can operate in three modes : the continuous mode, the fed - batch mode, and the batch mode [13 ]. a fed - batch bioreactor (fbb) initially contains a small amount of substrates (the nutrients) and microorganisms and is progressively filled with the influent substrates. a batch bioreactor is filled with the reactant mixture : substrates and microorganisms and allows for a particular time period for the reactions inside the reactor ; after some time the products are removed from the tank. in a continuous stirred tank bioreactor (cstb), the substrates are fed to the bioreactor continuously and an effluent stream is continuously withdrawn such that the culture volume is constant. next, the bond graph method is used in order to obtain the model of the enzymatic synthesis of ampicillin process, which takes place inside an fbb. firstly, after a short presentation of the bond graph method, a simple prototype fed - batch bioprocess is modeled. after that, the enzymatic synthesis of ampicillin is widely studied, and the bond graph model is obtained by using the reaction scheme, the analysis of the phenomena inside the bioprocess and the bond graph modeling rules. each process is described by a pair of variables, effort e and flow f, and their product is the power. the direction of power is depicted by a half - arrow. in a dynamic system one of the advantages of bond graph method is that models of various systems belonging to different engineering domains can be expressed using a set of only nine elements. a classification of bond graph elements can be made up by the number of ports ; ports are places where interactions with other processes take place. there are one port elements represented by inertial elements (i), capacitive elements (c), resistive elements (r), effort sources (se), and flow sources (sf), two ports elements represented by transformer elements (tf) and gyrator elements (gy), and multiport elements effort junctions (j0), and flow junctions (j1). i, c, and r elements are passive elements because they convert the supplied energy into stored or dissipated energy. se and sf elements are active elements because they supply power to the system and tf, gy, 0-and 1-junctions are junction elements that serve to connect i, c, r, se, and sf, and constitute the junction structure of the bond graph model. besides the power variables, two other types of variables are very important in describing dynamic systems and these variables, sometimes called energy variables, are the generalized momentum p as time integral of effort and the generalized displacement q as time integral of flow. in biotechnology, pseudo bond graph models are accomplished starting with processes reactions schemes and using both base bond graph elements and pseudo bonds with effort and flow variables as concentrations and mass flows. one of the simplest biological reactions is the microorganisms growth process, with the reaction scheme given by : where s is the substrate, x is the biomass and is the reaction rate. this simple growth reaction represents in fact a prototype reaction, which can be found in almost every bioprocess. the dynamics of the concentrations of the components from reaction scheme (1) can be modeled considering the mass balance of the components. the dynamical model of process (1) is simple, but if the reaction scheme is more complicated, the achievement of the dynamical model is difficult. in order to model bioprocesses, pseudo bond graph method is more appropriate because of the meaning of variables involved effort (concentration) and flow (mass flow). from the reaction scheme (1) and taking into account the mass transfer through the fbb, using the bond graph modeling characteristics, the pseudo bond graph model of the fed - batch bioprocess is achieved see figure 1. the bond graph model is depicted in the 20 sim environment (registered trademark of controllab products b.v. the directions of half arrows correspond to the run of the reaction, going out from the substrate s towards biomass x. in the bond graph model, the mass balances of the species involved in the bioreactor are represented by two 0-junctions : 01,2,3,4 (mass balance for the substrate s) and 07,8,9 (mass balance for the biomass x). due to the fact that the form of kinetics is complex, nonlinear, and in many cases unknown a general assumption is that a reaction can take place only if all reactants are presented in the bioreactor. therefore, the reaction rates are necessarily zero whenever the concentration of one of the reactants is zero. in order to model the reaction rate, because of the dependency of substrate and biomass concentrations, we have used a modulated two port r element, denoted mr5,6. mass flow of the component entering the reaction is modeled using a modulated source flow element sf1 and quantities exiting from the reaction are modeled using modulated flow sources elements sf represented by bonds 3 and 9. this approach was imposed by the dependency of these elements on fin : the input feed rate, and on v : volume of the bioreactor. from sf constitutive equations we have the accumulations of substrate and biomass in fbb are represented by bonds 2 and 8 and are modeled using capacitive elements c, with the constitutive equations : (2)e2=q2c2=(t(f1f3f4)dt)c2, (3)e8=q8c8=(t(f7f9)dt)c8. by using the constitutive relations of transformer elements tf4,5 and tf6,7, the relations for flows f4 and f7 are obtained : f4 = k4,5f5, f7 = f5/k6,7, with k4,5 and k6,7 the transformers modulus, which are in fact yield coefficients of the bioprocess (their values equal one for this fed - batch bioprocess). in fact, e2 is the substrate concentration, which will be denoted with s, e8 is the biomass concentration x, f5 is proportional to and v, c2 = c8 = v with sf3 = sf9 = fin. therefore, from (2) and (3) we will obtain the dynamical model of the fed - batch bioprocess : (4)vdsdt = vs(t)=finsinf0sv, vdxdt = vx(t)=f0x+v, dvdt = v(t)=fin. the model (4) expresses the equations of mass balance for the reaction scheme (1). taking into account that the dilution rate d = fin / v, the dynamical behavior of the concentrations can be easily obtained from (4) : (5)s(t)=dsinds,x(t)=dx+,v(t)=fin. next, this bond graph procedure is extended to the modeling of the enzymatic synthesis of ampicillin taking place inside a fed - batch bioprocess. ampicillin (6-[2-amino-2-phenylacetamide ] penicillanic acid) is a semisynthetic -lactamic antibiotic which is very stable at acidic conditions, well absorbed and effective against a wide variety of microorganisms, with low minimal inhibitory concentration [2124 ]. for instance, an amino -lactam, such as 6-aminopenicilanic acid (6-apa), having its carboxyl group protected, reacts with an activated side - chain derivative (d - phenylglycine acid chloride, to produce ampicillin). these reactions typically involve costly steps, such as very low temperatures and the use of toxic organic solvents like methylene chloride and silylation reagents. enzymatic synthesis is an alternative process ; it has high selectivity, specificity, and activity in mild reaction conditions (aqueous medium, neutral ph and moderate temperatures). the influence of the substrate structure on the catalytic properties of penicillin g acylase (pga) from escherichia coli in kinetically controlled acylations has been studied in. in particular, the affinity of different -lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis and the rate of hydrolysis of the acylating ester. it has been shown that this approach presents several advantages compared with the classical chemical processes. still, none of the known enzymatic methods have yet been upscaled to industrial applicability, due to the high costs caused by a low yield. penicillin g acylase (pga), for example, can act as a hydrolase as well as a transferase, which means that the same enzyme catalyzes the synthesis of ampicillin as well as the hydrolysis of the activated acyl donor and the hydrolysis of the newly formed antibiotic. the main reactions involved in the enzymatic synthesis of ampicillin are presented in figure 2 [33, 34 ]. the reaction scheme of this complex bioprocess contains three reactions : (a) the ampicillin synthesis : (6)k1s1+s21k5p2+k6p3, (b) the phenylglycine methyl ester hydrolysis : (c) the ampicillin hydrolysis : in the above reactions, s1, s2, p1, p2, and p3 represent 6-aminopenicillanic acid (6-apa), phenylglycine methyl ester (pgme), phenylglycine (pg), ampicillin (amp), and methanol, respectively. 1, 2, and 3 represent the reaction rates of these three reactions, and ki are yield coefficients. considering that the bioprocess takes place inside a fed - batch bioreactor, from the reaction scheme (6)(8), and by using the bond graph elements and modeling rules, a bond graph model of the process is obtained see figure 3. as it is mentioned previously, the meanings of the effort and flow variables of the bond graph model are concentration and mass flow, respectively. the directions of the half - arrows in the bond graph correspond to the progress of the reactions, going out from the components s1 and s2 towards p2 and p3 for the first reaction, from s2 to p1 and p3 for the second reaction, and from p2 towards p1 and s1 for the third reaction, respectively. in bond graph terms, the mass balances of the components involved in the bioprocess are represented by five 0-junctions : 01,2,3,4,33 (mass balance for s1), 06,7,8,9,11 (mass balance for s2), 015,16,17,18,31 (mass balance for p1), 020,21,22,27 (mass balance for p2), and 024,25,26,36 (mass balance for p3). the constitutive relations of these junctions are characterized by the equality to zero of the sum of flow variables corresponding to the junction bonds ; therefore, the next relations are obtained : (9)f1f2f3f4+f33=0, f6f7f8f9f11=0,f15f16f17+f18+f31=0, f20f21f22f27=0,f24f25f26+f36=0. the accumulations of components s1, s2, p1, p2, and p3 in the bioreactor are represented by bonds 2, 7, 16, 22, and 25, and they are modeled using capacitive elements c. the constitutive equations of c - elements are as follows : (10)e2=1c2q2=1c2t(f1f3f4+f33)dt, (11)e7=1c7q7=1c7t(f6f8f9f11)dt, (12)e16=1c16q16=1c16t(f15f17+f18+f31)dt, (13)e22=1c22q22=1c22t(f20f21f27)dt, (14)e25=1c25q25=1c25t(f24f26+f36)dt, where e2, e7, e16, e22, e25 are the concentrations of components s1, s2, p1, p2, p3 (all in mm = 10mol / l), and c2, c7, c16, c22, c25 are the parameters of c - elements : c2 = c7 = c16 = c22 = c25 = v, with v the bioreactor volume (l). the output flows of the components exiting from the reactions are modeled by using flow source elements sf represented by bonds 3, 8, 17, 21, and 26. the constitutive equations of these elements are as follows : f3 = sf3, f8 = sf8, f17 = sf17, f21 = sf21, f26 = sf26, where sf3, sf8, sf17, sf21, sf26 are the parameters of sf - elements. therefore we have sf3 = sf8 = sf17 = sf21 = sf26 = f0, where f0 is the output flow (l / min). mass flows of the components entering the reactions are modeled using source flow elements sf1, sf6, and sf18. consequently, we have f1 = vf1, f6 = vf2, f18 = vf3, where f1, f2 and f3 (all in mm / min) are the feed rates of 6-apa, pgme, and pg, respectively. the transformer elements tf4,5, tf9,10, tf11,12, tf14,15, tf19,20, tf23,24, tf27,28, tf30,31,tf32,33, tf35,36 were introduced to model the yield coefficients. for the modeling of the reaction rates we used three two - port modulated r elements : mr1, mr2, and mr3. from the constitutive relations of the three 1-junction elements, 15,10,19,34, 112,13,23, and 128,29,32 we obtain : f5 = f10 = f19 = f34, f12 = f13 = f23, f28 = f29 = f32, where the constitutive relations of mr elements imply that f34 = 1v, f13 = 2v, and f29 = 3v. also, from the constitutive relations of transformers we obtain the following relations : (15)f4=k4,5f5, f9=k9,10f10, f11=k11,12f12,f15=1k14,15f14, f20=1k19,20f19,f24=1k23,24f23, f27=k27,28f28, f31=1k30,31f30,f33=1k32,33f33, f36=1k35,36f36, with k4,5, k9,10, k11,12, k14,15, k19,20, k23,24, k27,28, k30,31, k32,33, and k35,36 the transformers modulus, which are in fact yield coefficients of the bioprocess. using these notations, from (10)(14) and the above relationships, the following dynamical model of the bioprocess is obtained : (16)vs1=vf1f0s1k4,51v+(1k32,33)3v, vs2=vf2f0s2k9,101vk11,122v, vp1=(1k14,15)2vf0p1+vf3+(1k30,31)3v, vp2=(1k19,20)1vf0p2k27,283v, vp3=(1k23,24)2vf0p3+(1k35,36)1v. taking into account that k4,5 = k1, k32,33 = 1/k2, k9,10 = k11,12 = 1, k14,15 = 1/k3, k30,31 = 1/k4, k19,20 = 1/k5, k27,28 = 1, k35,36 = 1/k6, k23,24 = 1/k7, and using the so - called dilution rate d = f0/v = 1/tr, with tr : medium residence time, the equations (16) can be written in the next form : (17)s1=k11+k23ds1+f1,s2=12ds2+f2,p1=k32+k43dp1+f3,p2=k513dp2,p3=k62+k71dp3. the model of this bioprocess, obtained via bond graph approach, is equivalent with the model obtained via classical method (see e.g.,). the model (17) can be expressed in the form : (18)ddt[s1s2p1p2p3]=[k10k21100k3k4k501k6k70]k[123]d[s1s2p1p2p3]+[f1f2f300 ]. the state vector of the system (18) will be denoted as : =[s1s2p1p2p3]t. the vector of reaction rates is =[123]t, k is the matrix of dimensionless yield coefficients, and the vector of feed rates is denoted with f=[f1f2f300]t. by using these notations, the dynamical nonlinear model (18) can be compactly written as : the dynamical model of the enzymatic synthesis of ampicillin (19) belongs to a large class of nonlinear bioprocesses carried out in bioreactors and is referred as general dynamical state - space model of this class of bioprocesses. the most difficult task for the construction of the dynamical model (19) is the modeling of the reaction rates. the form of kinetics is complex, nonlinear, and in many cases partial or completely unknown. a realistic assumption is that a reaction can take place only if all reactants are presented in the reactor. thus, the reaction rates are necessarily zero whenever the concentration of one of reactants is zero. taking into consideration these aspects, the reaction rates can be written as follows : (20)()=[123]=[s1000s2000p2]h()[123]=h()(), where () is the vector of specific reaction rates. a possible structure of the nonlinear specific growth rates is of monod - type model, and it is given in [22, 23, 34 ]. consequently, the reaction rates can be modeled as follows : for the ampicillin synthesis : (21)1()=s11()=s1ken+s1kcat1cezs2km1(1+p2/kae)+s2 for the phenylglycine methyl ester hydrolysis : (22)2()=s22()=kcat1cezs2km1(1 + p2/kae) + s2 for the ampicillin hydrolysis : (23)3()=p23()=kcat2cezp2km2(1 + s2/kea) + p2, where cez represents the enzyme activity (ui / mlgel), ken, kae, and kea are inhibition constants (mm), kcat1, and kcat2 are kinetic constants (mm / ui min), km1, and km2 are michaelis - menten constants (mm). however, in practice the reaction rates and/or the specific reaction rates given by the relations (21)(23) are imprecisely known. for on - line estimation of these kinetic rates, next, the nonlinear model (19) is expressed as : (24)=kh()(t)d+f, where (t) represents the unknown kinetics of the process. if we suppose that the reaction rates are totally unknown, then (t) = (t) and h() = 1. if the structure of the reaction rates is known () = h()(), but the specific reaction rates are unknown, then (t) = (t) and h() is the matrix given in (20). the high gain observers design necessitates a factorization of the model (24) [13, 14, 20 ]. we will consider that the yield matrix k is of full rank, which is true for our particular model, and for general class of bioprocesses ' models is a generic property. we shall suppose that all state variables are measured (contrarily, a state estimator can be used). since the yield matrix k is of full rank, then the partition (ka, kb) can be considered, such that the submatrix ka has full rank. therefore, a partition (a, b) of the state vector is obtained, and consequently a partition for f is achieved : (fa, fb). then, the system (24) can be written as follows : (25)a = kah(a,b)(t)da+fa,b = kbh(a,b)(t)db+fb, by using this factorization, a highgain observer can be implemented. the design of highgain observers is done in [13, 14 ], with supplementary assumptions regarding global lipschitz conditions, the boundedness of h() diagonal elements ' away from zero, and so forth. the equations of the nonlinear high gain observer for (24) are obtained as : (26)^a = kah(a,b)^d^a+fa2(^aa),^=2[kah(^a,b)]1(^aa). the high gain observer (26) provides on - line estimates ^ for the unknown kinetics. this on - line estimation algorithm is in fact a copy of the process model, with a corrective term. the observer is simple and the tuning of the gain can be done by modifying only one design parameter :. it should be noticed that ^a is an estimate of a, provided by the algorithm in order to be compared with the real state (a is measured or provided by a state observer), and the resulting error to be used in (26). in order to obtain the equations of the observers for our bioprocess, the next factorization of yield matrix and corresponding partition are considered : (27)ka=[k10k21100k3k4 ], kb=[k501k6k70 ], a=[s1s2p1 ], b=[p2p3],fa=[f1f2f3 ], fb=. then, for the enzymatic synthesis of ampicillin, two high - gain estimators can be derived from (26). (a) an estimator for the specific reaction rates. in this case (t) = (t), and h() is the matrix given in (20). the equations of the high - gain observer are : (28)[s^1s^1p^1]=ka[s^1000s^2000p2][^1^2^3]d[s^1s^2p^1 ] + [f1f2f3]2[s^1s1s^2s2p^1p1][^1^2^3]=2[ka[s^1000s^2000p2]]1[s^1s1s^2s2p^1p1 ], (b) a second estimator can be obtained if the entire reaction rate vector is considered unknown. in this case (t) = (t) and h() = 1. then, the equations of the highgain observer are as follows : (29)[s^1s^1p^1]=ka[^1^2^3]d[s^1s^2p^1]+[f1f2f3]2[s^1s1s^2s2p^1p1],[^1^2^3]=2[ka]1[s^1s1s^2s2p^1p1 ]. the high gain observer (28) needs the on - line measurements of s1, s2, p1, and p2. therefore, a state observer is required in order to reconstitute the measurements of p2. in such a case, the estimates of p2 provided by the asymptotic observer will be used in (28). in the case of the high gain observer (29), only the measurements of s1, s2, and p1 are needed. concerning the fed - batch bioprocess control, a typical problem is that of generating the substrate feed rate profile to optimize a performance criterion. for our process this goal can be achieved through an optimal control, that is, the calculation of a feeding rate optimal profile. then, adaptive versions are implemented, considering that the kinetics are unknown, and by using the kinetics estimators described before. the exact linearizing control law for the model (18) (or the model written in the compact form (19)) is obtained in a classical three steps strategy (see for the general point of view and for bioprocesses). the control goal is that the ampicillin concentration y(t) = 4(t) = p2(t) to track the desired substrate trajectory y(t) = p2(t), with the dilution rate as control action : u(t) = d(t). the first step consists in the achievement of an input - output model of the bioprocess, which is obtained directly from (18) : (30)y=p2=4=k51()3()uy. second, we consider a stable and linear reference model for the tracking error y y : (31) (yy)+(yy)=0, >0. finally, the exact linearizing control law is obtained by calculus of usuch that (30) has the same behavior as (31) : (32)u(t)=(1y)(k51()3()(yy))=(1y)(k51()13()y(yy))=(1y)(k51ken+1kcat1cez2km1(1+y / kae) + 2 kcat2cezykm2(1+2/kea) + y(yy)), where we consider y = const. the exact linearizing control (32) can ensure the achievement of control goal only if the involved concentrations are on - line measurable and if the reaction rates (or the specific growth rates, resp.) contrarily, the estimations provided by the estimators are needed, and if they are used in the exact linearizing control law, some adaptive versions of the nonlinear law are obtained. for example, if the estimations ^(t)=^(t) provided by (28) are used in the control law (32), an adaptive version of this law is obtained as follows : (33)u(t)=(1y)(k5^1(t)1^3(t)y(yy)). the entire adaptive control algorithm consists of (28) and (33). regarding the stability and convergence properties of the controlled system, another version of the adaptive control law (33) is obtained if the full vector of reaction rates is considered as unknown. in this case, the high gain estimator (29) is used, and the adaptive control law takes the following form : (34)u(t)=(1y)(k5^1(t)^3(t)(yy)). therefore, the complete adaptive control algorithm consists now of (29) and (34). moreover, when the ampicillin concentration can not be on - line measured, then an asymptotic observer can be used in order to provide the estimates yest, and consequently a version of the adaptive controller (33) is obtained as follows : (35)u(t)=(1yest)(k5^1(t)1^3(t)yest(yyest)). the adaptive control algorithm consists of (28), (35), plus the asymptotic observer. the design of asymptotic observers is based on mass and energy balances without the knowledge of the process kinetics being necessary. more precisely, the design is based on some useful changes of coordinates, which lead to a submodel of the process which is independent of the kinetics. next, the fundaments of the design of an asymptotic observer for the process (24) will be presented. the maximum state information which can be reconstituted is obtained by using the states 1 = [s1 p1 ] considered as measurable. therefore, the vector of unavailable states is 2=[s2p2p3]t ; these states will be estimated. in order to achieve the change of coordinates, the partition (1, 2) induces partitions of the yield matrix k : (k1, k2), also of the rate vector (f1,f2) accordingly. the state partition was chosen such that the submatrix k1 is full rank and dim(1) = rank(k1) = rank(k). then a linear change of coordinates can be defined as follows : with z an auxiliary state vector and g the solution of the matrix equation g k1 + k2 = 0. in the new coordinates, model (19) can be rewritten as (37)1=k1(1,zg1)d1+f1,z=dz+gf1+f2. the main achievement of the change of coordinates is that the dynamics of the auxiliary state variables is independent of the reaction kinetics. the asymptotic observer equations are derived as follows (from (36) and (37)) : (38)[z1estz2estz3est]=d[z1estz2estz3est]+[1k11k3k5k10k6k1k7k3][f1f3]+[f200 ], (39)[s2estp2estp3est]=[z1estz2estz3est][1k11k3k5k10k6k1k7k3][s1p1 ]. the asymptotic observer can be used for the implementation of the adaptive law (35). the behavior and the performance of the proposed estimation and control algorithms were analyzed by using intensive simulations. the fed - batch bioprocess has been simulated by numerical integration of the basic model equations (18), (21)(23). the values of kinetic parameters and of yield coefficients used in simulations are presented in table 1 [23, 31 ]. (i) the exact linearizing control law (32) was implemented for the bioprocess (18), with the design parameter = 3. the closed loop system was tested for a step profile of the ampicillin concentration reference. this simulation case is a kind of benchmark, because all the parameters and the reaction rates are considered to be known (and given by the relations (21)(23)). a parametric disturbance was considered in the feed rate f2, which has a 20% decrease of its nominal value (between t = 50 min and t = 100 min). panel (a) shows the time profiles of 1 = s1, 2 = s2, 3 = p1, and 5 = p3 (i.e., 6-apa, pgme, pg, and methanol concentrations, resp.). in panel (b) the evolution of the specific reaction rates is depicted, while in panel (c) the output (ampicillin concentration) versus the reference profile is shown. panel (d) presents the control action, that is, the evolution of the dilution rate. from these simulation results (ii) in the second simulation scenario, an adaptive version of the control law was implemented in the same conditions as in previous case. this adaptive controller consists of the high gain observer (28) and the control law (33). therefore, the specific growth rates 1, 2, and 3 were considered to be unknown. the kinetic expressions (21)(23) were introduced only for simulation ; so these models were not used in the process of observer design. the main goal of the estimator (28) is to reconstitute the time evolution of 1, 2, and 3 from the measurements of s1, s2, p1, and p2 obtained from the simulation. the on - line estimations provided by the high gain observer (28) were used in the control law (33)in fact only ^1 and ^3 are used here. panel (a) presents the output versus the reference profile, and panels (b), (c), and (d) show the time evolution of the estimated specific reaction rates versus their true profiles. in this scenario, (iii) in order to test the robustness of the proposed estimation and control algorithms to noisy measurements, the behavior of the controlled bioprocess was also analyzed for noisy data of s1 and s2. the measurements of these concentrations are considered to be vitiated by an additive gaussian noise, with zero mean and amplitude equal to 5% of the free - noise values. the same version of the adaptive control law (28), (33) was used in this case. (a) shows the output versus the reference profile, and panels (b), (c), and (d) depict the time evolution of the estimated specific reaction rates versus their true profiles. it can be seen that the behavior of the controlled process is quite good, in spite of the combined action of noisy measurements, parametric disturbance, and kinetics uncertainty. the specific reaction rates 1 and 3 seem to be a little bit more sensible to noise. a lot of supplementary simulations were performed for the other versions of adaptive control laws, such as (29), (34) and (28), (35). in all situations, a good behavior of the closed loop system the results illustrate that the high - gain observer provides accurate estimates of the kinetic rates. it can be seen that the noisy measurement induces some noisy estimates of the kinetics, but the noise effect is limited (however, this effect can be reduced for lower values of the tuning parameter). several comparisons and comments regarding the behavior and the performance of the on - line estimation strategy can be achieved. some remarks can be done by visualization of estimation errors 1=1-^1, 2=2-^2 and 3=3-^3. however, accurate comparisons can be realized by considering a criterion, for example, one based on averaged square estimation errors : (40)i1=1ts0ts12(t)dt, i2=1ts0ts22(t)dt, i3=1ts0ts32(t)dt, where ts is the total simulation time. the values of i1, i2, and i3 computed for different values of tuning parameter and free noise data are given in table 2, and for vitiated measurements in table 3. the problem for a large value of is that the observer becomes noise sensitive. notice that the estimation error can be made as small as wished if we choose greater values of. the problem for a large value of is that the observer becomes noise sensitive. therefore, the value of the tuning parameter is a compromise between a good estimation and the noise rejection. the obtained results concerning the noise sensitivity of the high gain observers are similar with those discussed in several works, such as [3538 ]. the bond graph modeling approach constitutes a noteworthy option to the classical modeling in the case of complex bioprocesses. the ampicillin synthesis process was modeled in a natural way via pseudo bond graphs, and the obtained model was used for estimators and controllers design. one of the key advantages of the bond graph modeling of bioprocesses is the possibility to reuse the models, for example, in the interconnected bioreactors. the application of this feature is beyond of the scope of the present paper, but it is of crucial importance in other bioprocesses. however, it should be mentioned that the obtained model is only validated for the specific enzyme preparation used (but the proposed method can be utilized with adequate modifications when the enzyme is changed). to overcome problems such as the kinetics uncertainties and the lack of on - line measurements, a high gain observer was designed and implemented for the on - line reconstruction of the specific reaction rates. the advantages of this kind of estimator are the simplicity of design, the good convergence and stability properties, and the accuracy of estimates (especially for free noise data). another important advantage is the fact that the tuning of one single design parameter is necessary. the estimation results can be improved if the tuning parameter is chosen higher in value, but only if the measurements are free noise. contrarily, the observer becomes noise sensitive and it is possible that the estimates of kinetics can not be utilized in the control law design. the proposed adaptive control law was obtained by combining an exact linearizing control law with the kinetics estimator. the control goal, that is, the preservation of a high ampicillin concentration, is achieved despite the action of disturbances and noisy measurements. the bond graph modeling estimation and control strategies can be also applied to other processes belonging to the nonlinear class of bioprocesses considered in the present study.	nowadays, the use of advanced control strategies in biotechnology is quite low. a main reason is the lack of quality of the data, and the fact that more sophisticated control strategies must be based on a model of the dynamics of bioprocesses. the nonlinearity of the bioprocesses and the absence of cheap and reliable instrumentation require an enhanced modeling effort and identification strategies for the kinetics. the present work approaches modeling and control strategies for the enzymatic synthesis of ampicillin that is carried out inside a fed - batch bioreactor. first, a nonlinear dynamical model of this bioprocess is obtained by using a novel modeling procedure for biotechnology : the bond graph methodology. second, a high gain observer is designed for the estimation of the imprecisely known kinetics of the synthesis process. third, by combining an exact linearizing control law with the on - line estimation kinetics algorithm, a nonlinear adaptive control law is designed. the case study discussed shows that a nonlinear feedback control strategy applied to the ampicillin synthesis bioprocess can cope with disturbances, noisy measurements, and parametric uncertainties. numerical simulations performed with matlab environment are included in order to test the behavior and the performances of the proposed estimation and control strategies.
patients have been more interested in tooth - colored restorations even in the posterior areas of the mouth as a result of esthetic awareness. several materials and techniques have been introduced for tooth - colored intra - coronal restorations, which include direct composite, indirect composite and ceramic inlays. posterior direct composites provide esthetics and may increase the strength of the remaining tooth structure because of bonding ; however, obtaining adequate proximal contact and occlusal morphology is difficult. the polymerization shrinkage produces contraction forces, which may compromise the bond between the tooth and composite. this leads to marginal opening, pulpal irritation, postoperative sensitivity, marginal staining and secondary caries. to overcome the problems associated with direct composites, composite inlays were introduced in the early 1980s. in this method, the operator has better control over anatomic form and proximal contacts and polymerization shrinkage is limited to the thin layer of resin cement. ceramic inlays have higher esthetics, color stability and stain resistance, but are more expensive, time - consuming and technique sensitive. marginal adaptation of direct composites and esthetic inlays is an important characteristic with a significant role in the long - term clinical performance of the restoration. several studies have shown that if occlusal marginal gap is greater than 100 m, excessive wear of resin cement will occur and if this gap is in the proximal surface of the tooth near the gingiva, the risk of gingival inflammation and periodontal disease will increase. in addition, because of bacterial growth and adhesion on resin cement, secondary caries and pulp damage or marginal deterioration might occur [1012 ]. it is critical to establish a low marginal gap in ceramic and composite inlays because of the inherent limitations of resin cements such as relatively high polymerization shrinkage and high co - efficient of thermal expansion. this study evaluated marginal adaptation of an indirect composite inlay, a glass - ceramic inlay, and a direct composite. seventy - five caries - free, recently extracted human molars were selected for the purpose of this in vitro study and were stored in a 0.5% chloramines t solution for seven days. any calculus and soft tissue deposits were removed from the teeth using a hand scaler. the roots of the teeth were embedded in auto - polymerizing acrylic resin (acropars 200, marlic, tehran, iran) up to 2 mm apical to the cemento - enamel junction. the shape and dimensions of all cubic acrylic blocks were the same (251515 mm). the specimens were randomly divided into three groups (n=25) according to the restorative materials used : group a for indirect composite inlays (gc - gradia, gc corp, tokyo, japan) ; group b for lithium disilicate glass - ceramic inlays (ips - empress 2, ivoclar vivadent, shaan, liechtenstein) ; and group c for direct composites (tetric ceram, ivoclar vivadent, shaan, liechtenstein). class ii mod cavities without bevel were prepared observing the following dimensions : 4.0 mm (sd=0.5) bucco - lingually, 4.0 mm (sd=0.5) occluso - gingivally in the proximal walls and 2.0 mm (sd=0.5) for pulpal wall depth. cavities with divergent walls were prepared in groups a and b with an eight - degree tapered diamond bur (s 856 - 016 - 8 ml, swiss tec, coltene ag, alstatten, switzerland) and parallel - walled cavities were prepared in group c with a cylindrical diamond bur held parallel to the long axis of the tooth (s 835r-014 - 4ml, swiss tec, coltene ag, alstatten, switzerland). the impressions of prepared teeth in groups a and b were taken with condensation silicone (speedex, coltene ag, alstatten, switzerland) using double mixing technique [1618 ]. the impressions were poured after one hour with a type iv dental stone (elite rock, zhermack, rovigo, italy). for group a, indirect composite inlays were fabricated using incremental technique following manufacturer s recommendations. for group b, inlay patterns were waxed up directly onto the stone dies, and then invested and pressed according to the manufacturer s instructions. the cavities were etched with 37% phosphoric acid gel (total etch, ivoclar vivadent, shaan, liechtenstein) for 30 seconds for enamel and 15 seconds for dentin. following rinsing and gentle drying to leave a moist dentin surface, bonding agent (excite, ivoclar vivadent, shaan, liechtenstein) was applied and after 10 seconds, gently air - dried for 13 seconds and light - cured for 20 seconds at 500 mw / cm (coltlux ii, coltene ag, alstatten, switzerland). the cavity was filled with oblique increments of composite (tetric ceram, ivoclar vivadent, shaan, liechtenstein) ; each increment was light - cured for 20 seconds. the finishing procedure was completed with fine (s 8565 - 014 - 8f, coltene ag, alstatten, switzerland) and extra fine (826 - 012 - 8f, coltene ag, alstatten, switzerland) finishing burs. the restorations were then polished using polishing rubbers (2103.1- 050, 2203.1 - 050, coltene ag, alstatten, switzerland). after adjusting the inlays (groups a and b) on the teeth using a control paste (fit - checker black, gc, tokyo, japan), they were placed on the teeth and each specimen was positioned under a stereomicroscope (szx12, olympus, tokyo, japan) in a manner in which the images of occlusal, mesial and distal surfaces with magnification 10 were captured by a digital camera (dpr, olympus, tokyo, japan) and transferred to a computer (fig 1). another magnification 4 was digitally applied to each image, so that a total of magnification 40 was obtained (fig 2). then the marginal gap (the distance between the dental wall and the restoration) was measured by a screen ruler (jr screen ruler pro 3.0) in pixels in four locations at occlusal margins and three locations at each proximal surface (two locations at the proximal margins and one location at the gingival margin). in groups a and b, 37% phosphoric acid gel (total etch, ivoclar vivadent, shaan, liechtenstein) and bonding agent (excite, ivoclar vivadent, shaan, liechtenstein) were applied on the cavities, as previously described. the internal surfaces of inlays were sandblasted with 50-m aluminum oxide particles for 5 seconds. in group a, the internal surfaces of inlays were treated with composite primer (primer, gc, tokyo, japan) and light - cured for 20 seconds, and bonding agent (excite, ivoclar vivadent, shaan, liechtenstein) was then applied. in group b, the internal surfaces of the inlays were etched with 9% buffered hydrofluoric acid (hf, ultradent, utah, usa) for two minutes, rinsed and dried, and then treated with a silane coupling agent (silane, dentsply, pa, usa) for one minute. finally, the bonding agent (excite, ivoclar vivadent, shaan, liechtenstein) was applied. dual - cure resin cement (variolink ii, ivoclar vivadent, shaan, liechtenstein) was mixed and applied to the surfaces of inlays and the teeth. the inlay was seated in place and kept under a pressure of 500 g for 10 minutes. then the cement was light - cured from facial, lingual, and occlusal directions for 60 seconds in each direction. the cement layer was finished and polished as previously described. all the specimens including groups a, b, and c were stored in distilled water at 37c for a week. the teeth were then thermocyled for 2000 cycles at 555c according to iso - tr 11405 standard. each specimen was positioned under the stereomicroscope ; then the images of occlusal, mesial and distal surfaces were captured and transferred to the computer and marginal gaps were measured by the screen ruler in the manner described previously (fig 3). a statistical comparison of occlusal, proximal, and gingival marginal gaps in each group was performed using repeated measure analysis of variance (anova) and post hoc tukey test. to compare the marginal gap of groups a and b together before cementation, independent t - test after cementation, marginal gaps in groups a and b and marginal gaps in group c were compared with the use of one - way analysis of variance and post - hoc tukey test. for comparison of marginal gap before and after cementation in groups the mean marginal gap values of inlays before cementation at occlusal, proximal, and gingival margins are shown in table 1. the mean marginal gap values of indirect composite inlays (46.29 m) were significantly lower than those of glass - ceramic inlays (60.12 m, p<0.001). in both groups, marginal gaps of the gingival margin were significantly greater than both occlusal and proximal margins, according to repeated measure anova and tukey test. the mean marginal gap values of inlays after cementation and direct composites, after thermocycling of all the specimens, at occlusal, proximal, and gingival margins are shown in table 2. mean marginal gap value of direct composites (19.96 m) was significantly lower than that of indirect composite inlays (48.47 m, p<0.001), which in itself was significantly lower than that of glass - ceramic inlays (60.96 m, p<0.001). in all groups, the marginal gap of the gingival margin was significantly greater than that of both occlusal and proximal margins, according to repeated measure anova and tukey test. comparison of marginal gap of inlays before and after cementation and thermocycling revealed that there were no significant differences between marginal gap values before and after cementation (p=0.075 for group a and p=0.766 for group b). close marginal adaptation is crucial to the quality of composite restorations and esthetic inlays luted to teeth with composite resin cement. several studies have reported that the marginal gap of such restorations should be less than 100 m [1012,21 ]. in this study, marginal gaps of indirect composite, glass - ceramic inlays and direct composites in all the margins were less than 100 m. in the present study, the marginal gap in the gingival margin was higher than that in the occlusal and proximal margins in both indirect composite and glass - ceramic inlays. in addition, the marginal gap of indirect composite inlays was significantly lower than that of glass - ceramic inlays. these findings are consistent with the results of a study on one ceramic and three composite inlays, carried out by soares. the marginal gap of indirect composite and glass - ceramic inlays did not increase significantly after cementation and thermocycling. this finding coincides with the results of studies by gemalmaz, who evaluated sintered ceramic inlays and by stappert, who assessed heat pressing glass - ceramic inlays. the marginal gap of direct composite was significantly lower than that of indirect composite and glass - ceramic inlays. in the present study, the marginal gap was defined as the distance between the dental wall and the restoration in the marginal area, which was filled with resin cement in inlays. in direct composite restorations, because of a direct technique on the cavity, the distance between the restoration and the dental wall would be minimum, which is filled with bonding agent. thus, the crucial factor in direct composites is marginal opening as a result of polymerization shrinkage. in this study no marginal opening was detected, which might be attributed to evaluation method (stereomicroscope with magnification 40 without sectioning the specimens). therefore, microleakage evaluation is recommended for precise comparative evaluation of margin quality in direct composites and inlays. under limitations of this in vitro study the following conclusions may be drawn : marginal gap of indirect composite, glass - ceramic inlays and direct composites was less than 100 m, which is clinically acceptable.marginal adaptation of direct composite was better than indirect composite inlay, which in itself was better than glass - ceramic inlay.in comparison of occlusal, proximal, and gingival margins in all the restorations, the marginal adaptation of gingival margin was the worst.marginal adaptation of inlays did not deteriorate after cementation and thermocycling. marginal gap of indirect composite, glass - ceramic inlays and direct composites was less than 100 m, which is clinically acceptable. marginal adaptation of direct composite was better than indirect composite inlay, which in itself was better than glass - ceramic inlay. in comparison of occlusal, proximal, and gingival	objective : this experimental in vitro study compared marginal adaptation of indirect composite, glass - ceramic inlays and direct composite.materials and methods : seventy - five recently extracted human molars were randomly divided into three groups (n=25) and mesio - occluso - distal cavities with the same dimensions were prepared in the teeth. indirect composite and glass - ceramic inlays were fabricated following manufacturer s instructions and the marginal gap was measured by a stereomicroscope at magnification 40 before cementation. after cementation of inlays and restoring the third group by direct composite, all the specimens were thermocycled and the marginal gaps were measured exactly as previously described. repeated measure anova and post - hoc tukey test were used for pairwise comparison of occlusal, proximal, and gingival marginal gaps in each group. one - way anova and post - hoc tukey test were used for comparison of mean marginal gap in the three groups and for comparison of marginal gap before and after cementation in inlays, paired t - test was used.results:the marginal gap of direct composite (19.96 m) was significantly lower than that of indirect composite inlay (48.47 m), which in itself was significantly lower than that of glass - ceramic inlay (60.96 m). in all the restorations, marginal gap in the gingival margin was significantly higher than occlusal and proximal margins. the marginal gap of inlays did not change after cementation and thermocycling.conclusion:this study indicated that the marginal gaps of the evaluated restorations are less than 100 m, which is clinically acceptable.
mast cells form a component of the immune system playing an important role in host defence. they reside in all the vascularised tissues, especially in all the connective tissues and mucosal surfaces of the human body, but mature cells can not be identified in the circulation. they are mobile and contain numerous granules, derived from haemopoietic progenitor cells, which emanate to the tissues to stanch as progenitor cells. they are also known as mastocyte or as labrocyte, derived from the latin word mastocytus, mast meaning well fed or fattening in german. it was first described by paul ehrlich in 1878 as cells belonging to the connective tissue staining purple to blue with aniline blue dye due to the presence of an abundant number of granules. the morphology and cytogenicity of the mast cells differs according to the tissue and hence can be differentiated by staining for their contents. the strategic role of these cells was in mediating type 1 hypersensitivity reaction, acting as effector cells in ige - mediated host immune responses. they are activated by diverse mechanisms and hence secrete their products, thus playing an important role in sustaining health or backing to disease. they have been found to be significantly associated with increased mitotic activity, extracellular matrix degradation, angiogenesis, intensification of microvascular hyper permeability, and recruitment of inflammatory cells including macrophages. a literature search for mast cell - related oral squamous cell carcinoma was conducted in the pubmed database using related medical subject heading (mesh) terms : mast cell and oral cancer and mast cell and oral squamous cell carcinoma. from the late 1980s until the present the research papers were included based on the following : (1) full - text availability ; (2) research papers that were available in english language, and (3) papers having information on mast cell in oral cancer and oral squamous cell carcinoma. research papers were excluded based on : (1) duplication of the titles, (2) mast cell research on non - oral tissue site of squamous cell carcinoma, and (3) research papers that had an incorrect web - link for full text accessibility. twenty - three articles were excluded based on the inclusion and exclusion criteria of the study. finally, twenty - two research papers were included in the present study to summarise the report on the role of mast cells in oral squamous cell carcinoma. mast cells, in both morphology and function, resemble basophils. initially they were thought to be basophils that reside in the tissue, but later they were identified as separate entities due to their development from different haematopoietic lineages. the similarities between them are as follows : both secrete granules containing heparin and histamine, express high affinity receptor for ige fcri under latent conditions, the method of degranulation, and the common precursor positivity for cd34. the features that distinguish them are as follows : basophils that are bi - lobed leave the bone marrow after maturation, and in case of mast cells having single rounded nuclei they circulate in an immature form and then mature at the proper tissue site. basophils are not seen in normal tissue but can be seen at a local site due to stimulation by cytokines released from mast cells or t cells. in contrast, mast cells can not be identified in circulation but can be seen in tissues. the major component of the granules of mast cells and basophils are heparin and chondroitin sulphate, respectively. they are tissue resident sentinel cells and are divided into two types based on the substances present in their granules. the granules secreted by the mast cells have been cloned and sequenced to categorise the clear dissimilarities between the two cell types. an abundant amount of chondroitin sulphate, little histamine, the presence of only tryptase (mct), and absence of chymase are important characteristics of mucosal mast cells. they are found abundantly in intestinal mucosa and alveolar spaces in the lung, ably their incidence is t - cell dependent. connective tissue mast cells contain several neutral proteases, including tryptase, chymase (mctc), cathepsin g - like protease, and carboxypeptidase. they are found abundantly in skin, breast, gastrointestinal tract, myocardium, synovium, and conjunctiva and are t - cell independent. ultra - structurally they are quite large round or ovoid cells that contain abundant membrane - bound granules, about 300 in number per cell. the nucleus is round to oval in shape, which is generally obscured due to their extensive content of electron - dense secretory granules. irregular outline, numerous mitochondria, prominent golgi, few smooth endoplasmic reticulum (ser), and some rough endoplasmic reticulum (rer) are a few important findings. each granule is enclosed by a membrane measuring about 0.2 to 0.8 microns in diameter, which may have electron - dense lamellae, crystalline lattices, or densely packed fine amorphous material. they have a wide array of receptors that sense environmental changes and are able to secrete various effector molecules. the effector molecules are stored in the form of granules such as serotonin, histamine, heparin, tryptase, and chymase. the other molecules synthesised de novo upon stimulation are the lipid mediators, certain prostaglandins, and leukotrienes, which are the chemical mediators of inflammation. biosynthesis of a variety of cytokines that are associated with t - helper 1 (th1) cells includes interleukin 1 (il1-), which is important for biosynthesis of angiogenic factors, interleukin (il-10), and transforming growth factor (tgf-), which have a role in immunosuppression. the other substances produced by mast cells, which deserve attention are the products of cyclooxygenase and lipoxygenase activities that have recently been shown to be a potent intestinal polyposis promoting factor in mice. the action of mast cells and release of all these mediators can occur within minutes of activation or can occur in a delayed fashion (over hours). mast cells will be activated classically via ige receptor whenever a foreign antigen enters into the body by means of different signal transduction pathways through the receptors on its surface [9, 10 ]. neuropeptides, cytokines, growth factors, toxins, complement, lectins, immune complexes, and physical stimuli act as mediators for stimulating the mast cells. few pathogens like bacteria, viral, and fungal organisms possess pathogen - associated molecular patterns recognised by c - kit, toll like receptors (tlr) 1, 7 and 9, nod - like receptors, and retinoic acid inducible gene 1-like receptors on the surface of mast cells. they are activated by crosslinking of fcri (fcri) molecules with antigens bound to respective antibodies. firstly by release of granules exocytosis which helps to increase vascular permeability, stimulates smooth muscle contraction, and degrades microbial substances, tissue damage, and remodelling (fig. the second mechanism is secretion of lipid mediators performing functions like vasodilatation, bronchoconstriction, neutrophil chemotaxis, mucous secretion, and increasing vascular permeability. the last mechanism is biosynthesis and secretion of cytokines, which promotes mast cell proliferation, inflammation, late phase reaction, th2 cell differentiation, eosinophil production, and activation. mast cells correspondingly express receptors for numerous components of complement system like cr3, cr4, and cr5. mast cells vesicles, granules and receptors nonetheless, it has been observed that mast cells can also be activated via other pathways and extrinsic signals. alternate pathways are thought to play an important role in infiltration in tumours (fig. another important aspect of mast cells recently discovered is their ability to change their phenotype according the site and the duration of stimuli to which they are exposed. mast cells have a long life and form a heterogeneous population of cells that seem to have both a positive and negative regulatory effect on the immune system. the physiological function of mast cells in epithelium, endothelium, and the nervous system is immune modulatory. they are ubiquitous in taking part in maintaining homeostasis of the body [17, 18 ]. the mediators induce microbial clearance, debris removal, wound contraction, keratinisation, and proliferation of epithelial cells and fibroblasts. communication among mast cells and the nervous system ensues through the synaptic - like assemblies via adhesion molecules such as n cadherin or syncam. over all activity of mast cells the role of mast cells in cancer in still debatable as to whether it helps in tumour progression or has an anti - tumour effect. the principle concept behind its role against tumours is that it is a cell that participates in innate immunity, and it obviously seems to have an anti - tumorigenic effect. however, some authors have proposed that it has a role in progression of tumours. the presence of mast cells in tumours has been found to be an independent prognostic factor and predictor of poor outcome of prostate cancer, melanoma, pancreatic cancer, and leukaemia [1922 ]. increased expression of c - kit and stem cell factor, which in turn is necessary for the migration, maturation, and survival of mast cells, has been observed in tumours of the breast. other than this increased mast cells count and infiltration of mast cells in the stroma have been associated with merkel cell carcinoma, lung cancer, hepatocellular carcinoma, colorectal cancer, and hodgkin s and follicular lymphoma. as far as oral squamous cell carcinoma (oscc) is concerned, it has been observed that there are increased microvessel density and increased mast cell density in oscc, which can be a reason for poor prognosis [25, 26 ]. the tumour promotion by mast cells is principally considered to be due to its angiogenic ability. evidence of degranulation and angiogenesis due to mast cells in pre - malignancy has also been observed in various mouse model studies. reported an increase in mast cell density, and infiltration into the tumour site was evident, in mice colonic carcinoma (azoxymethane and dextran sodium sulphate induced). there was a significant increase in its presence in the dysplastic lesional areas to carcinomas. tumour necrosis factor (tnf-) is one of the important tumour progression molecules secreted by mast cells at the tumour site. mast cells can also modulate haemostasis and blood perfusion in tumours by production of heparin, which acts as an anticoagulant. the other possible methods in which mast cells promote tumours is by their mitogenic ability, deletion of tumour suppressor genes, and activation of certain oncogenes via the c - kit locus. the secretion of proteases, which contributes to extracellular matrix degradation, formation of vascular tube, release of trapped angiogenic factors, and promotion of metastasis is possibly another mechanism by which mast cells promote the tumours. johansson. suggested that orthotopic mouse models showed activity of peri - tumoural mast cells, which increase angiogenic activity with significant expression of fibroblast growth factor-2, simulating the growth of prostate tumours. chang. utilised the k - ras transgenic spontaneous mouse model to demonstrate early infiltration mast cells in the pancreatic ductal adenocarcinoma, which mimic the tumorigenesis as in humans, and correlated the poor prognosis in pancreatic tumours. the interaction between cancer cells and their microenvironment is very important to both tumour progression and the arrest of its growth. the mast cells have been found to have both direct mitogenic activities on tumour cells as well as an indirect effect on the microenvironment by promoting its invasive potential. much less has been said about the ability of mast cells to promote the immune response against tumour growth, although it is well known that mast cells are antigen presenting cells and they promote migration, maturation, and function of dendritic cells, which interact with t and b cells. many studies were conducted on mast cell association with cancer to state its accumulation in and around the tissue. mast cells play an important role by liberating heady pro - angiogenic and angiogenic factors, which aids in tumour interaction with the host, thus supporting tumour progression. not only was its involvement in tumour progression identified, but also progression of leukoplakia with and without dysplasia to oral squamous cell carcinoma was established. the main factors released are heparin, histamine, chymase, tryptase, basic fibroblast growth factor (bfgf), vascular endothelial growth factor (vegf), and tgf-. epidermal keratinocytes and endothelial cells secrete mast cell growth factor, which aids the migration of the mast cells and moreover the homing of mast cell pioneers to the epithelial tissues. calcitonin gene - related peptide and substance p helps in degranulation of mast cells to release its products favouring tumour progression. copious mast cells are seen at the tumour periphery and are involved in the breakdown of collagenous matrix by provoking fibroblasts to yield excess amounts of collagenase. tryptase helps in the process of neovascularisation by increasing the number of fibroblasts and matrix reorganisations with type 1 procollagen mrna. chymase both directly and indirectly stimulates progelatinase b, which causes proteolysis of extracellular matrix emancipating angiogenic factor vegf, tnf-, heparanase, angiopontin 1, il-18, and fgf. cytokines especially il-8 produced by mast cells induce matrix metalloproteinase 2 (mmp-2) facilitating endothelial cell migration assisting angiogenesis. a greater number of mast cells are found with increased number of micro vessels and direct relation of mast cells was found with vascular tube formation. it has the capacity to respond to certain signal transduction pathways thus providing signals by release of mediators like fgf 2, which increases the mitotic activity of the tumour cells. they also secrete tnf-, which has been implicated in increased expression of mmp-2, mmp-9, and chops type iv collagen helping in invasion (fig. mast cell activity in tumour microenvironment michailidou. mentioned that the presence of mast cells has been correlated with a better prognosis of cancers. the antitumor effects of mast cells include inhibition of cell growth, amplified inflammatory antitumor response, and they also help in secreting substances that decrease tumour cell motility. prostacyclin - synthase, which is secreted by the endothelial cells in response to histamine produced by mast cells, eosinophil recruitment, and survival promoted by mast cell tryptase and il-5, acts as a factor of tumour regression. hence, in the process of cancer, mast cells play an important role in tumour progression, cell proliferation, angiogenesis, extracellular matrix degradation, and hence in invasion and metastasis. until recently, studies, being conducted have revealed that they play a significant role in the progression of cancer. thus it can be said that targeting the survival / function of mast cells can easily influence the behaviour of cancer cells and ultimately its clinical response. mast cells originate from haematopoietic precursor cells in bone marrow tissue. stem cell factor (scf) is responsible for migration and circulation into the blood stream, and further entry into distant peripheral tissues. mast cells that have migrated undergo cellular differentiation and eventually mature in the peripheral tissues. in addition, scf plays an important role in the survival of mast cells because wells stimulate directional motility of mucosal and connective tissue type of mast cells. forty - five research studies have been published from the late 1980s until today on mast cell - related oral squamous cell carcinoma research. however, only twenty - two research papers were available as full text in the pubmed database. the results and inference of those full text research studies are summarised in table 1. reported mast cell - related oral squamous cell carcinoma research the location of mast cells in the tumour - host tissue interface reveals that it plays an important role in host defence. mast cells have the ability to retain preformed mediators that can facilitate immune - regulation, matrix degradation, elastic change, and angiogenesis. on the other hand, some studies demonstrated that the presence, density, and activation of mast cells in oral tissue could be influenced by the chemicals in tobacco smoking. the elevated mast cell population in the tissue plays an imperative role in the modification of the microenvironment during oncogenesis. the presence of mast cells near blood vessels supports in neo - angiogenesis, which is mediated by bfgf, tgf, tnf-, tryptase, and heparin. the mast cells play a role in the release and biosynthesis of mmp, which can lead to collagen tissue degradation. the mast cell population is significantly elevated in oral epithelial dysplasia and oral squamous cell carcinomatous conditions [4751 ]. investigations suggest that the mast cell population is significantly more increased in oral squamous cell carcinomas than in oral epithelial dysplasia. the increase in mast cell population is significantly positive in tumour front as well as intralesional areas of well differentiated oral squamous cell carcinomas compared to moderately or poorly differentiated oral squamous cell carcinoma [53, 54 ]. on the other hand, a study has shown that there is no significant increase among well, moderate, or poorly differentiated squamous cell carcinoma. the mast cell population is significantly increased in advanced stages of epithelial dysplasia and oral submucous fibrosis. a molecular epidemiological study on mast cell population revealed a significantly higher number of mast cells in squamous cell carcinoma of skin than oral mucosa. furthermore, the mast cell population was higher in squamous cell carcinoma of the lip than oral mucosal epithelium. another study showed that the increase in mast cell population was unrelated to the degree of differentiation of the tumour. one study showed serum elevation of mast cell tryptase in patients with oral squamous cell carcinoma, but it was not correlated with the staging of cancer (i.e. nodal involvement and metastasis). mast cell and microvessel densities are increased in squamous cell carcinoma and indicate that mast cells may play a role in up - regulation of angiogenesis in oral squamous cell carcinoma [41, 43, 58 ]. however, three studies revealed that there is no positive correlation between mast cell density and microvessel density in oral squamous cell carcinoma tissues [42, 58, 64 ]. on other hand, one study revealed a positive correlation between mast cell and microvessel densities in well differentiated but not in moderately or poorly differentiated types. contrarily, one study observed statistically significant correlation between mast cell and microvessel densities in poorly differentiated oscc. however, sharma., in 2010, investigated correlation of micro - vascular density with mast cell proliferation and revealed that positive correlation was observed in moderately differentiated lesions but not in well or poorly differentiated types. on the other hand, another study stated that there was significant correlation in mast cell and microvessel density in normal oral mucosa but not in oral squamous cell carcinoma, regardless of the histological grade. however, a recent investigation showed that mast cell in the perilesional and intra tumoural area of oral squamous cell carcinoma expresses cd105, vegf, vegfr1, and vegfr2 and showed positive correlation with angiogenic activity of the tumour. a recent study investigated cancer stem cell population that expresses cd44, cd133, and cd117 at the invasion front and intra - tumoural areas. their results suggest that cd 133- and cd117-positive cells were of mast cell origin and could influence angiogenic activity in addition, cd44 has limited utility in identifying cancer stem cell populations, whereas cd133 and 117 appear to be of more limited utility in identifying cancer stem cell populations. only one study showed that mast cell and mmp-9 densities were positively correlated in oral squamous cell carcinoma and actinic cheilitis. mast cell and lymph vessel density were strongly associated in oral squamous cell carcinomatous tissue, but that association did not show a statistical relationship with histological risk assessment model. mast cells provoke much debate today regarding their role in a variety of physiological and pathological processes, including cancer. they act as gate keepers of the immune system and in turn respond to many signalling pathways, thus contributing to the process of carcinogenesis and metastasis. many studies have revealed that mast cell counts were definitely increased along with tumour progression. new therapies targeting the mediators and receptors of mast cells play an important role in controlling the process of tumour progression and metastasis, thus favouring a good prognosis to the patient.	the mast cells are initial effective lineage in both humoral and adaptive immunity. they are ubiquitous in skin, mucosa, and in function. they contain biologically essential and dynamic mediators in healthy and harmful conditions of tissue. mast cell malfunctioning could be attributed to various chronic allergic diseases. considerately, emerging evidence of mast cell involvement in various cancers shows them to have both positive and negative roles in tumour growth. it mostly indulges in tumour progression and metastasis via angiogenesis, extracellular matrix degradation, and mitogenic activity in the tumour microenvironment. the current paper reviewed research papers on mast cells in oral squamous cell carcinoma through the pubmed database from 1980 to the present date. the present paper is an attempt to summarise the research reports on the role of mast cells in oral squamous cell carcinoma. further to this note, this paper also outlines the role of mast cells in normal physiological processes and tumour biology.
autism is a severe neurodevelopmental disorder of childhood characterized by impairments in social interaction, deficits in verbal and nonverbal communication, and restricted, repetitive, and stereotypical patterns of behavior and interests (dsmiv criteria, american psychiatric association, 1994). many areas of the brain in autism show abnormalities that include decreased purkinje cell counts in cerebellar hemispheres and vermis, loss of granular cells, and purkinje cell atrophy [3, 4 ]. susceptibility to autism is clearly attributable to genetic factors [5, 6 ], but the etiology of this disorder is unknown, and no biomarkers have yet been proven to be characteristic of autism. the btbr t+tfj (btbr) mice have been suggested to be a useful animal model for autism studies since they demonstrate low levels of sociability compared to the c57bl/6j (b6) mice [710 ]. the btbr mice also exhibit an unusual pattern of ultrasonic vocalizations and high levels of self - grooming [9, 1113 ] that may be homologous to the communication deficits and repetitive behaviors observed in autism. thus, the btbr strain of mice is currently a promising model for understanding the mechanisms that could be responsible for the pathogenesis of autism. recently, emerging evidence points to central nervous system (cns) inflammatory and apoptotic mechanisms being responsible for certain neuropsychiatric disorders including autism. several inflammatory cytokines including tumor - necrosis - factor- (tnf-), interferon- (ifn-), il-1, il-6, and il-8 are found to be elevated in the brain, serum, plasma, and cerebrospinal fluid (csf) of autistic subjects [1422 ]. in addition, previous studies, including ours, have found that the antiapoptotic bcl2 protein is decreased, while the proapoptotic p53 protein is increased in the autistic brain [22, 23 ]. we also found that the bdnf - akt - bcl2 anti - apoptosis pathway is compromised in the frontal cortex of autistic subjects. the bdnf - pi3k / akt - bcl2 signaling pathway plays an important role in inhibiting apoptosis and promoting neuronal survival. nuclear factor-b (nf-b) is an important gene transcriptional factor that mediates cellular responses in inflammation, immunity, development, cell proliferation and apoptosis [2430 ]. the inactive form of nf-b is localized to the cytoplasm and consists of dna - binding p50 and p65 subunits and an inhibitory subunit, designated ib. activation occurs via phosphorylation of ib at ser32 and ser36, resulting in the ubiquitin - mediated proteasome - dependent degradation of ib and the release and nuclear translocation of active nf-b dimmers. the key regulatory step in this pathway involves activation of a high - molecular - weight ikappab kinase (ikk) complex, consisting of three tightly associated ikk subunits.. a large number of genes involved in cellular proliferation, apoptosis, and inflammation, are regulated upon activation of nf-b. these genes include antiapoptotic genes (bcl2 and bcl - xl), cell cycle - regulatory genes (cyclin d1), genes encoding adhesion molecules, chemokines, inflammatory cytokines, and genes involved in tumor metastases [2628, 30, 32 ]. tnf induces nf-b p65 polyubiquitination and degradation, as well as termination of tnf-mediated nf-b activation. although previous studies have shown that the bdnf - akt - bcl2 antiapoptotic pathway is downregulated and inflammatory cytokines are increased in autistic brain [22, 35 ], whether the nf-b signaling pathway is altered in the autistic brain has not yet been investigated. in the current study, we examined the entire nf-b signaling pathway in the cerebellum of 7 autistic subjects and their age - matched normal controls. we also determined the activity of nf-b in the frontal cortex of 6 autistic subjects and 6 btbr mice in comparison with the controls. our results show that the expression of ikk, the kinase phosphorylating the inhibitory subunit ib, is significantly increased in the cerebellum of autistic subjects as compared to age - matched controls. we failed to detect the ikk expression in our samples by western blot assay and reckon it is because the expression level of ikk is very low. however, the expression and phosphorylation level of ib, which is the downstream target of ikk and ikk, are not significantly changed in the autistic cerebellum. in addition, our results showed that the phosphorylation / activation of nf-b (p65) at ser536 is not significantly altered in the cerebellum and frontal cortex of both autistic subjects and btbr mice, as compared with the controls. these results suggest that nf-b signaling pathway is not deregulated in the brain of autistic subjects and btbr mice. frozen human brain tissue of seven autistic subjects (mean age 8.1 2.6 years) and seven age - matched normal subjects (mean age 8.4 2.3 years) was obtained from the nichd brain and tissue bank for developmental disorders. donors with autism fit the diagnostic criteria of the diagnostic and statistical manual - iv, as confirmed by the autism diagnostic interview - revised. participants were excluded from the study if they had a diagnosis of fragile x syndrome, epileptic seizures, obsessive - compulsive disorder, affective disorders, or any additional psychiatric or neurological diagnoses. six female btbr t+tfj (btbr) and six b6 mice were obtained from jackson laboratories (bar harbor, me). mice were housed for 24 hours with ad lib food and water to ease the stress before sacrifice. all procedures were conducted in compliance with the nih guidelines for the care and use of laboratory animals. frozen cerebellum and frontal cortex tissues from human and mice were homogenized (10% w / v) in cold buffer containing 50 mm tris - hcl (ph 7.4), 8.5% sucrose, 2 mm edta, 10 mm -mercaptoethanol, and protease inhibitors cocktail (sigma - aldrich). antibodies ikk, ikk, ikb, nf-b (p50/52), nf-b (p65), and phospho - nf - kb (p65) were obtained from commercial sources (cell signaling technology, usa). for western blot analysis, brain homogenate samples in sds sample buffer (20% glycerol, 100 mm tris, ph 6.8, 0.05% w / v bromophenol blue, 2.5% sds (w / v), and 250 mm dtt) were denatured by heating at 100c for three minutes. forty to eighty micrograms of protein per lane per subject was loaded onto a 12% acryl - bisacrylamide gel and electrophoresed for 2 h at 120 v at room temperature (rt). the proteins were electroblotted onto a nitrocellulose membrane for 1 h at 100 v at 4c. protein blots were then blocked with 5% milk in phosphate buffered saline (pbs) with 1% tween (pbst). after blocking, the blots were incubated with primary antibody overnight at 4c (anti - ikk 1 : 1000 ; anti - ikk 1 : 500 ; anti - ib 1 : 1000 ; anti - nf-b (p50/52) 1 : 500 ; anti - nf-b p65 1 : 500 ; anti - phospho - nf-b p65 1 : 500) followed by a secondary antibody incubation for 1 h at rt (goat anti - mouse igg or goat anti - rabbit igg, hrp conjugated, 1 : 5000, sigma). after three washes in pbst (each time for 10 minutes), the blots were visualized using the ecl detection system (amersham pharmacia biotech) and exposed to hyper film ecl (amersham pharmacia biotech). the densities of ikk, ib, and nf-b p65 expression bands, as well as the -actin expression bands, were quantified with background subtraction. statistical analyses were conducted using unpaired t - tests with significance established at p 0.05, figure 3(b)). the positive immunoreaction was similar in both the autistic cerebellum and the controls (figure 3(c)). to further examine the activity levels of nf-b(p65) in the brain of autistic subjects, we examined nf-b(p65) phosphorylation (on ser 536) in the cerebellum and frontal cortex of autistic subjects and the controls with an elisa assay. our results showed the mean values of nf-b(p65) phosphorylation were not significantly changed in the cerebellum (p = 0.966) and frontal cortex (p = 0.535) of autistic subjects, as compared to controls (figures 4(a) and 4(b)). since btbr mice are currently a promising model for understanding the mechanisms that could be responsible for the pathogenesis of autism, we also examined the activity levels of nf-b(p65) in the cerebellum and frontal cortex of btbr mice and b6 mice (control) by determining the nf-b(p65) phosphorylation on ser 536 with an elisa assay. our results showed the mean values of nf-b (p65) phosphorylation were not significantly changed in the cerebellum (p = 0.136) and frontal cortex (p = 0.968) of btbr mice, as compared to the control b6 mice (figures 5(a) and 5(b)). emerging evidence suggests that apoptotic and inflammatory mechanisms may be related to the pathogenesis of autism. a number of studies have shown that apoptosis - related proteins (p53, bcl2) and several inflammatory cytokines are altered in autistic brain [3, 4, 1821 ]. a large number of genes including the apoptosis - related bcl2 and bcl - xl, and inflammation - related cytokines, can be regulated upon activation of nf-b (see illustration of the nf-b signaling pathway in figure 6). to further investigate the mechanisms that underlie the apoptotic and inflammatory changes in the autistic brain, we examined the activity of the transcriptional factor nf-b in the cerebellum and frontal cortex of autistic subjects and their age - matched controls. we also further determined nf-b activities in the cerebellum and frontal cortex of btbr mice that model autism. this is the first study to investigate how the nf-b signaling pathway is regulated in the autistic brain. our results demonstrated that the expression of ikk was significantly increased in the autistic cerebellum as compared to the age - matched normal controls. ikk is a kinase upstream of the nf-b signaling pathway and phosphorylates two serine residues located in the inhibitory subunit ib. this phosphorylation event leads to ib release from the nf-b complex and frees the nf-b complex to enter the nucleus and activate nf-b - dependent gene expression [33, 38 ]. the increased ikk expression in the autistic brain implies a possible increased ikk kinase activity and a possible increased phosphorylation of the inhibitory ib subunit. however, by examining the expression of the ib subunit, as well as the phosphorylation of ib in the cerebellum of 7 autistic subjects and their age - matched controls, we did not detect significant differences in ib expression and phosphorylation between the two groups. to further determine the activity levels of nf-b, we utilized western blot, immunohistochemistry, and elisa approaches to examine the protein expression levels of both the nf-b p65 subunit and the nf-b p50/52 subunit, as well as the phosphorylation / activation levels of the two subunits in the brain of autistic subjects and their age - matched controls. our results showed no significant difference in nf-b p65 expression between the autistic and the control subjects. the phosphorylation levels of nf-b p65 protein at ser536 in the cerebellum and cortex of autistic subjects are also not significantly different from that of control subjects. the btbr strain of mice is currently a promising model for understanding the mechanisms that could be responsible for the pathogenesis of autism since they demonstrate behaviors similar to autism. thus, we also examined the phosphorylation levels of nf-b p65 protein at ser536 in the cerebellum and cortex of btbr mice. we found no significant differences in the phosphorylation levels of nf-b p65 protein at ser536 in the cerebellum and cortex of btbr mice as compared with control b6 mice. all these results point out that the activity of nf-b is not disregulated in the autistic brain. we could not detect protein expression of the nf-b p50/52 subunit in the cerebellum of autistic subjects or in control subjects. the very low expression level of this protein is the likely reason for its not being detectable with the western blot method. nf-b p65 phosphorylation at ser 536 regulates activation, nuclear localization, protein - protein interactions, and transcriptional activities. p65 is a subunit of the nf-b transcriptional factor that actively regulates expression of nf-b - dependent genes involved in apoptosis and inflammation. antiapoptotic bcl2 and bcl - xl genes as well as genes for cytokines have been shown to be regulated through the activation of nf-b p65 [24, 4042 ]. on one hand, constitutively expressed nf-b has been demonstrated to lead to resistance to cell death by different inducers of apoptosis and increased nf-b activity has recently been correlated with progression of different cancers, especially breast cancer, melanoma, and juvenile myelomonocytic leukemia. on the other hand, studies have demonstrated that nf-b is negatively regulated by antiapoptotic bcl2 protein and specifically activated in the course of apoptosis induced by serum withdrawal. this activation of nf-b was demonstrated to be necessary for the execution of the apoptotic program. these findings suggest a complicated role of nf-b signaling in the regulation of apoptosis and inflammation. recently, a number of studies have suggested that apoptosis and inflammation in the central nerve system may be associated with autism [3, 4, 1822 ]. we and others have found that the antiapoptotic factor bcl2 is decreased in the autistic brain [3, 22, 23 ] and the bdnf - akt - bcl2 anti - apoptosis pathway is downregulated in the frontal cortex of autistic subjects. in addition, a number of studies including ours found that the cytokines tnf, il-6, gm - csf, ifn-, and il-8 are significantly elevated in the brain and cerebrospinal fluid (csf) of autistic subjects [18, 21, 22 ]. nf-b was shown to be the transcriptional factor that regulates the gene transcription of bcl2 and inflammatory cytokines such as tnf [24, 29, 42, 44 ]. have also reported that valproic acid inhibits neural progenitor cell death by activation of nf-b signaling pathway, which subsequently enhanced expression of antiapoptotic protein bcl - xl. however, our studies demonstrated that nf-b signaling is not significantly changed in autistic brain as compared with normal controls. these results could be explained as (1) the alteration of inflammatory cytokines and apoptosis - related proteins in the autistic brain may not be enough to stimulate or suppress the nf-b activities and (2) the consequence of dual role of nf-b in the response to inflammatory cytokines and apoptosis - related protein such as bcl2. a number of studies have reported that nf-b can be activated in both transcriptionally activating and repressing forms [38, 46 ]. this dual role of nf-b has presented considerable complexities in understanding the mechanisms of nf-b action. our studies have shown that although the expression of ikk kinase, which is upstream of the nf-b signaling pathway, is significantly increased in the cerebellum of autistic subjects as compared with the age - matched controls, the phosphorylation / activation of the downstream ib inhibitory subunit is not significantly enhanced in the cerebellum of autistic subjects. our results further showed that both the expression and phosphorylation of nf-b p65 are not significantly altered in the cerebellum and cortex of autistic subjects as compared with the control subjects. the phosphorylation levels of nf-b p65 are also not significantly altered in the cerebellum and cortex of btbr mice that model autism.	autism is a neurodevelopmental disorder characterized by problems in communication, social skills, and repetitive behavior. recent studies suggest that apoptotic and inflammatory mechanisms may contribute to the pathogenesis of this disorder. nuclear factor-b (nf-b) is an important gene transcriptional factor involved in the mediation of inflammation and apoptosis. this study examined the activities of the nf-b signaling pathway in the brain of autistic subjects and their age - matched controls. the nf-b activation is also determined in the brain of btbr mice, which is a promising animal model for study of pathogenic mechanisms responsible for autism. our results showed that the level of ikk kinase, which phosphorylates the inhibitory subunit ib, is significantly increased in the cerebellum of autistic subjects. however, the expression and phosphorylation of ib are not altered. in addition, our results demonstrated that the expression of nf-b (p65), and the phosphorylation / activation of nf-b (p65) at ser536 are not significantly changed in the cerebellum and cortex of both autistic subjects and btbr mice. our findings suggest that the nf-b signaling pathway is not disregulated in the brain of autistic subjects and thus may not be significantly involved in the processes of abnormal inflammatory responses suggested in autistic brain.
although the etiology of primary biliary cirrhosis (pbc) is unclear, it is probably related to altered immunoregulation. autoimmune hemolytic anemia (aiha) is characterized by a positive direct coombs ' test. it is idiopathic (primary) in approximately 50% cases or secondary to medication, hematological malignancies and autoimmune disorders. the association between pbc and other autoimmune diseases has been reported, mostly including rheumatoid arthritis, sjgren syndrome, scleroderma, and so on. a 74-year - old woman was admitted to our hospital in december 2006, with a history of fatigue, anorexia and jaundice for about one month. her past medical history included hypertension and hyperlipidemia diagnosed at ages 44 and 54. physical examination revealed mild jaundice, conjunctival pallor and peripheral edema, hepatosplenomegaly and ascites. laboratory data revealed severe anemia : hemoglobin (hb) 6.1 g / dl (range 13.7 - 17.9 g / dl) ; white blood cell count (wbc) and blood platelet count (plt) were normal. liver function test : alt and ast were both in normal range ; total serum bilirubin (t - bil) 47.5 mg / l (range 1 - 10 mg / l) ; direct serum bilirubin (d - bil) 7.7 mg / l (range 1 - 4 mg / l) ; albumin 29 g / l) ; alkaline phosphatase (alp) 186 iu / l (range 40 - 160 iu / l) ; -glutamyltranspeptidase (-gt) 127 iu / l (range 0 - 50 the bone marrow aspirate showed erythroid hyperplasia obviously with ripe erythrocytes polychromasia and inequality of size. g / l (range 7.23 - 16.85 g / l) ; immunoglobulin a (iga) 3.41 g / l (range 0.69 - 3.82 g / l) ; antinuclear antibody (ana) 1:1000, antimitochondrial antibody (ama) 1:1000, m2 subtype positive. on this basis pbc prednisolone (psl) 60 mg and ursodeoxycholic acid (udca) 750 mg (12.5 mg / kg) daily were given. at the same time, the patient was commenced on high dosages of intravenous immunoglobulin (20 g daily) for 5 days, spironolactone 60 mg, furosemide 20 mg and a daily 1 litre fluid restriction. this resulted in 3 kg weight loss with noticeable improvement in her ascites and peripheral edema. three weeks after initiation of therapy, her hb level had recovered to 9.1 g / dl, ret to 1.84%, and liver function had improved : albumin 35.1 tow months later, her hb had risen to 11.6 g / dl. during one - year follow - up her hb was stable and psl 10 mg and udca 750 mg daily were continued. a 60-year - old man was admitted to our hospital in march 2007 with a 2-year history of progressive fatigue and anorexia. he had a small quantity of alcohol consumption (ethanol < 25 g per week). on admission, laboratory data revealed the following : hb 5.4 g / dl ; ret 1.18% ; wbc and plt were in normal range. iu / l. hepatitis b surface antigen and antibody to hepatitis c virus were both negative. ana and ama were both positive at a titer of 1:1000, and ama m2, m4 subtype were both positive. the bone marrow aspirate showed erythroid hyperplasia obviously with ripe erythrocytes polychromasia and inequality of size. blood transfusion with packed red blood cell was instituted for cardiorespiratory distress relief. however, after transfusion his hb descended to 5.1 g / dl and ret to 6.39% ; coombs ' test was performed, and the outcome was positive for anti - igg. the patient was treated with psl 60 mg and udca 750 mg (12 mg / kg) daily. three weeks after initiation of therapy, his hb level had recovered to 8.0 g / dl, ret to 2.36%, and his liver function had improved : alp 45 iu / l ; -gt 63 iu / l. after 3 months of this therapy his hb had risen to 12.2 g / dl with a normal liver function. during a one - year period, psl was gradually tapered to 10 mg per day and udca was continued. a year later, in april 2008, liver needle biopsy was performed ; the outcome showed mild aggregates of lymphoid cells and fibrosis in the portal area, and the bile ducts were scanty. a 55-year - old man was admitted to our hospital in may 2008, with a 4-month history of fatigue. his past medical history included : pbc, immune thrombocytopenia purpura (itp), sjgren syndrome and klinefelter syndrome. in 2002 health examination had revealed that he had thrombocytopenia (plt 10 10/l) and splenomegaly. liver biopsy was performed and the specimen showed moderate to generous aggregates of plasmacytes and lymphocytes with mild piecemeal necrosis and lobular disorganization. schirmer 's test, saliva flow rate and biopsy of salivary gland were performed, and the outcomes were compatible with sjgren syndrome. udca (8.5 mg / kg) was given combined with prednisone 40 mg daily. prednisone was intermittently used and thrombocytopenia was partially controlled. in february 2004, he had splenectomy. after the operation cyclophosphamide (ctx), azathioprine (aza) or cyclosporine a (csa) were successively used. in january 2008, because of extremely fatigue he came to our hospital. on admission, physical examination revealed pallid conjunctiva, rampant caries and testicular maldevelopment. laboratory data revealed : hb 5.8 g / dl, plt 5 10/l, ret 3.6%. udca 600 mg daily did not adjust, but pls 60 mg daily was added, and daily csa 200 mg intravenous injection was given for continuous three days. hb had risen to 8.6 g / dl, plt to 20 10/l, and ret to 2.06%. the diagnosis of pbc is based on three criteria : elevation of liver enzymes (most commonly alp and -gt), presence of detectable ama in the serum, and liver histologic findings that are compatible with the presence of the disease. while aiha is also an immune disease which is caused by antibodies directed against autologous red cells, the diagnosis is based on the presence of anemia, signs of hemolysis with reticulocytosis, increased lactate dehydrogenase, elevated indirect bilirubin, and a positive coombs ' test. all these fourteen cases reported previously with an association of pbc and aiha are summarized in table 1 and table 2. because of language barrier, we could not obtain data from two of the reports, so 12 reports (15 patients) are summarized. for those patients combining pbc with aiha, it is puzzling that besides the liver, the immune attack focuses on red blood cells, where mitochondrial autoantigens are not found. searching pubmed and using autoimmune hemolytic anemia and liver cirrhosis, only 25 articles were found in last 30 years. although pbc is not a common cause of liver cirrhosis, most of the articles are about pbc in combination with aiha. another interesting thing is that none of the 18 patients suffered from aiha before pbc was diagnosed. although the two conditions were detected at the same time in some of the patients, not all patients with pbc received the diagnosis when it was at an early stage because of their asymptomatic status, and it is supposed that aiha occurred after pbc development in these patients. pbc patients are likely to have some conditions which can induce generation of anti - erythrocyte antibodies. with sufficient corticosteroids or immunosuppressant therapy, hemolysis control in these pbc patients achieves a very good treatment effect (table 2). in addition, liver function was improved in all of the 16 patients who had been successfully treated. corticosteroid treatment is not only a preferred therapy for aiha, but an adjuvant therapy that benefits pbc patients. according to the criteria of secondary aiha : (1) aiha would occur on the basis of pbc ; (2) reversal of aiha and liver function improvement of pbc happened simultaneously with the intake of immunosuppressive agents ; (3) both pbc and aiha are known to be immune - mediated diseases. serum bilirubin levels have long been known to be important in assessing the prognosis of pbc, but confounding hemolysis may lead to erroneous conclusions about the severity of pbc. at the same time, compared with primary aiha, this pbc - related aiha is life - threatening but can be controlled. if no attention is paid to this condition, primary pbc will be ignored, as 50 - 60% of pbc patients are asymptomatic at diagnosis. therefore patients with pbc whose serum bilirubin levels rise suddenly should undergo screening for associated hemolysis. the recommended treatment for pbc - related aiha includes sufficient doses of corticosteroids to control the hemolysis at the acute phase, and immunosuppressant (for example ctx and csa) or adequate dose of udca to maintain therapy. to deal with the severe, life - threatening hemolysis, intravenous immunoglobulin and large dosage of corticosteroids impact therapy may be considered. only udca therapy for pbc patients with mild aiha had a successful report, so in mild cases this method could be considered with regular blood cell counts to monitor response. although kaibori. provided a successful case that could confirm liver transplantation and splenectomy is a curative therapy to pbc - related aiha, as retana. reported, hemolysis also occurred after liver transplantation despite well - functioning graft. so immunosuppressant prescribed after liver transplantation and splenectomy would not avoid further production of anti - erythrocyte antibodies in all of the cases. other immunosuppressants, for example rituximab (anti - cd20), appear to be highly effective in patient with warm antibody aiha that is refractory to standard therapy. so it will become a potential treatment for pbc - related aiha, which is refractory to the above treatment. as illustrated in table 1, case reports have been growing in number in recent years. if our three case reports are added, in the last 8 years (since 2000) there have been 13 cases (13/18) reported. thus, further research is required for illustrating the incidence and natural courses of these two organ - specific autoimmune diseases.	the association between primary biliary cirrhosis (pbc) and autoimmune hemolytic anemia (aiha) is uncommon ; only fourteen such case reports have been described. in this report, three patients who developed aiha on the basis of pbc underwent successful therapy with corticosteroids and ursodeoxycholic acid (udca). patient 3 was more complicated, suffering from pbc, evans syndrome, sjgren syndrome and klinefelter syndrome simultaneously. this has not previously been reported in the world literature. review of all fifteen cases showed that there is a prominent occurrence sequence that aiha might take place on the basis of pbc. with sufficient doses of corticosteroids or immunosuppressant therapy, besides hemolysis under effective control, liver function also improved. according to the criteria of secondary aiha, we may call them pbc - related aiha. thus, patients with pbc with serum bilirubin levels rising suddenly should undergo screening for associated hemolysis. recommended treatment for pbc - related aiha includes sufficient doses of corticosteroids to control the hemolysis in the acute phase, and immunosuppressant or adequate dose of udca to maintain therapy. these case reports have been increasing in recent years, so further reserch is needed to illustrate the incidence and natural courses of these two organ - specific autoimmune diseases.
prevalence of subclinical hypothyroidism (sht), defined as elevated thyroid stimulating hormone (tsh) with free thyroxine (ft4) in the normal range, increases with age affecting about 6% of individuals aged 70 to 79 years and 10% of those aged 80 or older [1, 2 ]. patients with sht are associated with increased prevalence of atherosclerotic lesions and cardiovascular events [3, 4 ]. besides, thyroid hormone deficiency may also interfere substantially with various aspects of physical, mental, and social well being and many studies showed changes in functional status (i.e., mobility limitation, disability, and poor fitness level) in patients with sht [6, 7 ]. on the other hand, the evidence for improvement of psychiatric symptoms with hormonal treatment (levothyroxine) of oh and the use of triiodothyronine (t3) to potentiate the response to treatment of depressive disorders suggests a direct relationship between thyroid hormones and psychiatric symptoms [8, 9 ]. neurobiological evidence seems to corroborate the hypothesis of an organic basis of the effects of thyroid hormone on the brain and on psychiatric symptoms. interventions for sht have included pharmacologic agents (i.e., hormonal treatment), psychotherapy, alternative therapies, and physical activity which can improve cardiovascular health, psychiatric symptoms, and health - related quality of life (hrqol) [10, 11 ]. to date, few studies on hrqol in subjects with sht in response to exercise program have been reported [12, 13 ]. it has been proved that regular exercise positively affects the mechanisms of action associated with the physiologic deterioration and transition from subclinical thyroid disease. several authors recommended exercise to be performed within an intensity range of 4085% of maximum oxygen consumption by vo2max. nevertheless, more recent studies emphasize the necessity of exploring the effects of intensity [15, 16 ]. clinical thyroid disease is associated with changes in the cardiovascular system, including changes in heart rate during exercise. considering this, we hypothesized that medium - impact exercise program would provide a more adequate exercise stimulus for improving a number of metabolic factors in females at risk for thyroid disease. therefore, we hypothesized that a medium - impact exercise program can also improve hrqol and cardiorespiratory fitness in females with sht. from january 2012 to september 2012, 17 sedentary colombian women with sht (physical exercise less than once a week) were referred to our hospital for health examination (servicio medico universidad del valle). subjects studied were between 40 and 65 years of age, mean age 43.1 9.7 years. all patients with sht were newly diagnosed and were positive for both antithyroid peroxidase (tpo - ab) and antithyroglobulin (tg - ab) antibodies. the diagnosis of sht was established on the basis of the elevated tsh levels and normal ft3 and ft4 values. in patients with sht, laboratory tests were performed 13 days before and 12 weeks after the initiation of the training program. obese subjects (body mass index (bmi) > 30 kgm), smokers, and individuals with hypertension, clinical detectable coronary artery disease, and other diseases were excluded from the study. none of the patients were taking any medicine, such as estrogen supplements, t4, diuretics, antihypertensive, or hypolipidemic drugs.. preparatory training phase (weeks 16) : the present study began with a 6-week preparatory phase of training to bring all participants up their 12 kcal / kg / wk goal. to accomplish this, all participants began their exercise program at a selected intensity set at a heart corresponding to 4055% of vo2max and a frequency of 3 times per week. implementation of medium - impact exercise program (weeks 712) : exercise prescription was standardized to body weight, and it was estimated that 180 minutes per week of moderate intensity exercise was equivalent to 10 to 12 kcal / kg of body weight per week. exercise intensity was defined between 55% and 80% of vo2max. an aerobic dose of 12 kcal / kg per week was selected for the aerobic group. american college of sports medicine equations (acsm) were used to estimate caloric expenditure rate and time required per session. the exercise prescription used was established from the participants baseline exercise test and corresponded to a speed and grade associated with an upper intensity working level of 50% to 80% vo2max followed by recovery level of 30% vo2max. each session included a 30 min aerobic circuit training guided by an audio recording (tropical and latin music). the entire workout lasted about 30 minutes that, depending on the number of exercise (10 stations), were usually repeated three times. during 15 and 30 minutes throughout the routine, participants were instructed to check their pulse to ensure that they were working within their target heart rate range. each session was preceded and followed by a gradual warm - up and cool down period, both of 10 minutes duration and consisting of walking and light, static stretching (avoiding muscle pain) of most muscle groups (upper and lower limbs, neck, and trunk muscles). resistance exercises were performed through the full range of motion normally associated with correct technique for each exercise and engaged the major muscle groups (abdominal, dorsal, shoulder, and upper and lower limb muscles). they included 5 exercise group circuit training (50 repetitions of each) using barbells (13 kg / exercise) or low - to - medium resistance bands (therabands and balls). adherence to the exercise program was encouraged by the exercise trainer and the physician who supervised each of the group sessions. trainers were physical educators with experience in developing and monitoring exercise programs among clinical populations. in order to maximise adherence to the training program, exercise classes consisted of relevant activities for the group, 35 participants were accompanied by music dancing and performed in a spacious, air - conditioned room (08:00 am). each participant met with the study dietician for nutrition assessment and counselling, and an individualized nutrition intervention plan was developed from the baseline food intake assessment, participant preferences, and the meal plan. the colombian standard version of the medical outcome study short - form health survey (sf-12 version 2) is a questionnaire comprising 12 questions grouped into eight different domains of health : physical functioning, role limitation due to physical problems, bodily pain, general health perception, vitality, social function, role limitation due to emotional problems, and mental health. these eight scales are further clustered into the physical component summary (comprising physical function, role - physical, bodily pain and general health) and mental component summary (comprising vitality, social function, role - emotional, and mental health). the quantification of the mentioned dimensions is values varying from 0 to 100, where 0 corresponds to worse health and 100 to better health. submaximal oxygen consumption (vo2max) was assessed with the rockport walking test. during the test, heart rate was monitored electronically using a polar a-5 pulse meter (polar electro oy, kempele, finland). the cardiorespiratory fitness was calculated by vo2max via the acsm equation : (1)vo2max(mlkgmin1) = 132.8530.0769weight0.3877 height+6.31503.2649 time1565heart rate.statistical methods. it was sought to detect a between - group difference in the change of the sf-12v2 score of 4 points (mental component summary) as it was considered clinically important. assuming that the standard deviation in this score would be 6, similar to what was observed in a similar sample of patients, a total sample size of 15 would provide 80% power to detect a difference of 4 points as statistically significant. the normality of the distribution of scores was confirmed with the kolmogorov - smirnov test. the paired t - test was later used to estimate the difference in each outcome. all analyses were carried out by using the statistical package spss 16 (chicago, il, usa). all subjects were nonsmokers and had a sedentary lifestyle. 90% of patients were females and in the reproductive age group. the anthropometric, cardiorespiratory fitness, and metabolic profile data are listed in table 1. after the 12-week intervention, the participants that performed of a medium - impact exercise program showed improvements in hrqol in most domains, particularly the vitality domain by 7 points (95% ci 2 to 11), the social functioning domain by 10 points (95% ci 4 to 15), the mental health domain by 7 points (95% ci 1 to 12), and mental component summary by 7 points (95% ci 2 to 11). one of the four domains within the physical component summary (general health domain) showed significant effect of the exercise intervention : 6 points (95% ci 1 to 11), table 2. the paired t - test analysis revealed that the participants had a greater cardiorespiratory fitness at the end of the intervention, measured by rockport walk test (p = 0.01) and by the submaximal vo2max (28% ; p < 0.001), figure 1. finally, the subjects participated 28.9 out of 36 (sd 3.2) sessions over the 12 weeks. the purpose of this study was to examine the influence of a medium - impact exercise program on hrqol and cardiorespiratory fitness in females with sht. to the authors ' knowledge, this is the first systematic study evaluating the potential effectiveness of exercise program in sht on hrqol and vo2max. in our clinical experience, we consider that an improvement of 4 points on the sf-12v2 resulting from this intervention is clinically important. however, no threshold has been established empirically for the amount of improvement in the sf-12v2 score that women typically feel makes aerobic training worthwhile. our estimation of the average effect of the training had some uncertainty, with a 95% ci ranging from 1 to 10 points. therefore, even if 4 points is a valid estimate for the smallest worthwhile effect, it must be acknowledged that it is uncertain whether the statistically significant effect of exercise is clinically worthwhile. the median mental component summary and general health scores observed in the present study of women were similar to other studies of patient populations with conditions such as overweight or sedentary. there are very few studies that evaluated hrqol in sht [9, 10 ], and none included cardiorespiratory fitness and health status in the same study. it is believed that some psychological aspects of thyroid hypofunction, when present in sht, may be influenced by physical findings, as suggested by the association between physical aspects of quality of life, in the sf-12 evaluation and cardiorespiratory fitness, specially vo2max, in the present study. one of the primary consequences of thyroid dysfunction is lower tolerance to physical exertion, because of its implications involving the muscle and cardiovascular systems. this interferes directly with the patient 's ability to perform daily activities, thereby reducing his quality of life. the study performed by kahaly. showed that subjects with thyroid dysfunction have reduced workload tolerance at the anaerobic threshold, compared to euthyroid subjects. according to these authors, in hyperthyroidism this exercise intolerance is caused by mitochondria oxidative dysfunction and in hypothyroidism, by inadequate cardiovascular support. following the 12-week exercise program, trends to improvement were seen in most domains of the hrqol questionnaire, with statistically significant changes in the mental component summary and several of its domains. the confidence intervals were not narrow enough to confirm that the benefits would be worth the effort of exercising for these women. nevertheless, given the other benefits of exercise in females with sht, physicians can prescribe exercise expecting that it will improve quality of life. the recommended levels of physical activity were positively associated with one or more domains of health - related quality of life [19, 24 ]. in particular, physical functioning, general health, vitality, social functioning, and mental health are critically affected by the recommended level of physical activity. in the current study, the physical aspects of hrqol, such as mental component summary and general health, seemed to be more closely associated with the amount of physical activity than the physical aspects are. the intervention showed better cardiorespiratory fitness results similar to those previously reported in healthy women with high levels of physical activity [3, 6, 7 ]. so one might speculate that the increase of vo2max observed in the training group could have a beneficial effect in patients with thyroid disease occuring as a result of metabolic status. this study, however, was conceived as preliminary research aiming to evaluate the potential usefulness of exercise in patients with sht. nevertheless, the finding that sht mental symptoms were those showing greater improvement is a fact that argues against a possible placebo effect despite the lack of a control group. in addition, future randomized controlled trials should study the effects of exercise in patients with disorders secondary to thyroid function variations and their implication, as well as therapeutic options for this highly prevalent disease. in summary, a supervised 12-week program of primarily medium - impact exercise in females with sht improves health - related quality of life. moreover, this exercise program proved to have a positive influence on the functional capacity of the subjects, being effective in improving cardiorespiratory fitness.	objective. to examine the influence of a medium - impact exercise program (miep) on health - related quality of life (hrqol) and cardiorespiratory fitness (vo2max) in females with subclinical hypothyroidism (sht). materials and methods. we selected 17 sedentary women with sht (mean age : 43.1 (standard deviation : 9.7) years). participants carried out an miep consisting of 3 weekly sessions of 60 minutes during 12 weeks. before and after the exercise program hrqol was assessed by the sf-12v2 questionnaire, and vo2max was evaluated by rockport walk test. results. after the 12-week intervention, the participants that performed an miep showed improvements in hrqol in most domains, particularly the vitality domain by 7 points, the social functioning domain by 10 points, the mental health domain by 7 points, and the mental component summary by 7 points. one of the four domains within the physical component summary (general health domain) showed significant effect of the exercise intervention : 6 points. moreover, the participants that performed exercise showed a higher vo2max (28% ; p < 0.01). conclusion. after 12 weeks of medium - impact exercise program, there were remarkable improvements in hrqol in most domains. moreover, this exercise program proved to have a positive influence on cardiorespiratory fitness.
les canaux potassiques mitochondriaux calcium - dpendants (mkca) participent la cardioprotection induite par le prconditionnement ischmique. dans la prsente tude, nous avons tent de dcouvrir si le prconditionnement provoqu par la morphine implique galement lactivation des canaux mkca. des curs isols de rats (six groupes ; chacun de n = 8) ont t soumis une ischmie globale pendant 30 min, suivie dune perfusion de 60 min. le prconditionnement provoqu par la morphine (mpc) a t initi par deux cycles de cinq minutes dune perfusion de morphine 1 m, avec une priode de rinage de cinq minutes et une priode de rinage finale de dix minutes avant lischmie. linhibiteur des canaux mkca, la paxilline 1 m, a t administr, avec ou sans administration de morphine (mpc + pax et pax). titre de valeur tmoin positive, nous avons ajout un groupe soumis un prconditionnement ischmique (ipc) seul et combin la paxilline (ipc + pax). la fin de la reperfusion, la taille de chaque infarctus a t dtermine par la coloration du chlorure de tryphnylttrazolium. la taille de linfarctus couvrait 45 9 % (moyenne cart type) de la zone risque dans le groupe tmoin. la taille de linfarctus tait moins importante dans les groupes ayant reu de la morphine ou ayant subi un prconditionnement ischmique (mpc : 23 8 %, ipc : 20 5 % ; chacun de p < 0,05 contre le groupe tmoin). leffet de rduction de la taille de linfarctus a t limin par la paxilline (mpc + pax : 37 7 %, p < 0.05 contre mpc et ipc + pax : mi, que (mpc 6 %, p < 0,05 contre ipc), tandis quadministre seule, la paxilline na eu aucun effet (pax : 46 7 %, aucune diffrence notable comparativement au groupe tmoin). la cardioprotection par prconditionnement provoqu par la morphine est assiste par lactivation des canaux mkca. the investigation conforms to the guide for the care and use of laboratory animals published by the us national institutes of health (nih publication no. 85 - 23, revised 1996) and was performed in accordance with the requirements of the animal ethics committee of the university of duesseldorf, duesseldorf, germany. the rats were maintained on a 12:12 light / dark schedule (lights on at 0600 hr) with food and water provided ad libitum. after thoracotomy, the hearts were excised, mounted on a langendorff system, and perfused at constant pressure (80 mmhg) with krebs henseleit solution containing (in mm) 116 nacl, 4.7 kcl, 1.1 mgso4, 1.17 kh2po4, 24.9 nahco3, 2.52 cacl2, 8.3 glucose, and 2.2 pyruvate at 37c. a fluid - filled balloon was inserted into the left ventricle, and end - diastolic pressure was set at 1 - 4 mmhg. heart rate (hr), myocardial function (isovolumetric left ventricular pressure), coronary flow, left ventricular end - diastolic pressure, and rate of left ventricular pressure development (dp / dtmax) were measured continuously. the data were digitized using an analogue to digital converter (powerlab/8sp, adinstruments pty ltd, castle hill, australia) at a sampling rate of 500 hz, and they were recorded continuously on a personal computer using chart for windows v5.0 (adinstruments). the hearts were assigned randomly to one of six experimental groups (figure 1). the hearts of all groups underwent 30 min of ischemia followed by 60 min of reperfusion. in the control group (con), the hearts were kept under baseline conditions prior to ischemia. to investigate whether morphine induces preconditioning (mpc), morphine 1 m was given in two five - minute cycles, separated by one five - minute washout period, and ending with one final ten - minute washout period prior to ischemia. the morphine concentration was chosen because liang. demonstrated that the preconditioning effect of morphine was maximal at 1 m.12 morphine was dissolved in 0.9% nacl and separately infused into a mixing chamber placed in the perfusion system. as a positive control, the ischemic preconditioning group (ipc) underwent two similar five - minute cycles of ischemia ten minutes prior to ischemia. to test whether mkca channels are involved in the phenomenon of preconditioning, the mkca channel inhibitor, paxilline 1 m,8,9 was given over 25 min together with morphine- and ischemic - induced preconditioning (mpc + pax and ipc + pax). to rule out an effect of paxilline itself, we investigated the effect of paxilline alone (pax).fig. ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline experimental protocol. ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline after 60 min of reperfusion, the heart was cut into transverse slices, which were then stained with 0.75% triphenyltetrazoliumchloride solution. the infarcted area was determined by planimetry using sigmascan pro 5 computer software (spss science software, chicago, il, usa). the sample size was calculated using graphpad statmate version 1.01 (graphpad software, san diego, ca, usa). sample size analysis revealed that a group size of n = 8 was necessary to detect a difference in infarct size of 25% with a power of 80% and an < 0.05. the estimations of the mean difference of 25% and the standard deviation (sd) of 15% were based on our own data.13 data are expressed as mean sd. heart rate (in min) and mean aortic pressure (in mmhg) were measured during baseline, coronary artery occlusion, and reperfusion period. comparisons of hemodynamics between groups or between time points in a group were performed (spss science software, version 12.0.1) using two - way analysis of variance followed by dunnett s post hoc test. a researcher blinded to the infarcts were analyzed by student s t test followed by bonferroni s correction for multiple comparisons. changes within and among groups were considered statistically significant if p < 0.05. the rats were maintained on a 12:12 light / dark schedule (lights on at 0600 hr) with food and water provided ad libitum. after thoracotomy, the hearts were excised, mounted on a langendorff system, and perfused at constant pressure (80 mmhg) with krebs henseleit solution containing (in mm) 116 nacl, 4.7 kcl, 1.1 mgso4, 1.17 kh2po4, 24.9 nahco3, 2.52 cacl2, 8.3 glucose, and 2.2 pyruvate at 37c. a fluid - filled balloon was inserted into the left ventricle, and end - diastolic pressure was set at 1 - 4 mmhg. heart rate (hr), myocardial function (isovolumetric left ventricular pressure), coronary flow, left ventricular end - diastolic pressure, and rate of left ventricular pressure development (dp / dtmax) were measured continuously. the data were digitized using an analogue to digital converter (powerlab/8sp, adinstruments pty ltd, castle hill, australia) at a sampling rate of 500 hz, and they were recorded continuously on a personal computer using chart for windows v5.0 (adinstruments). the hearts were assigned randomly to one of six experimental groups (figure 1). the hearts of all groups underwent 30 min of ischemia followed by 60 min of reperfusion. in the control group (con), the hearts were kept under baseline conditions prior to ischemia. to investigate whether morphine induces preconditioning (mpc), morphine 1 m was given in two five - minute cycles, separated by one five - minute washout period, and ending with one final ten - minute washout period prior to ischemia. demonstrated that the preconditioning effect of morphine was maximal at 1 m.12 morphine was dissolved in 0.9% nacl and separately infused into a mixing chamber placed in the perfusion system. as a positive control, the ischemic preconditioning group (ipc) underwent two similar five - minute cycles of ischemia ten minutes prior to ischemia. to test whether mkca channels are involved in the phenomenon of preconditioning, the mkca channel inhibitor, paxilline 1 m,8,9 was given over 25 min together with morphine- and ischemic - induced preconditioning (mpc + pax and ipc + pax). to rule out an effect of paxilline itself, we investigated the effect of paxilline alone (pax).fig. ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline experimental protocol. ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline after 60 min of reperfusion, the heart was cut into transverse slices, which were then stained with 0.75% triphenyltetrazoliumchloride solution. the infarcted area was determined by planimetry using sigmascan pro 5 computer software (spss science software, chicago, il, usa). the sample size was calculated using graphpad statmate version 1.01 (graphpad software, san diego, ca, usa). sample size analysis revealed that a group size of n = 8 was necessary to detect a difference in infarct size of 25% with a power of 80% and an < 0.05. the estimations of the mean difference of 25% and the standard deviation (sd) of 15% were based on our own data.13 data are expressed as mean sd. heart rate (in min) and mean aortic pressure (in mmhg) were measured during baseline, coronary artery occlusion, and reperfusion period. comparisons of hemodynamics between groups or between time points in a group were performed (spss science software, version 12.0.1) using two - way analysis of variance followed by dunnett s post hoc test. a researcher blinded to the experimental groups determined the infarct sizes. the infarcts were analyzed by student s t test followed by bonferroni s correction for multiple comparisons. changes within and among groups were considered statistically significant if p < 0.05. no differences in body or heart weight were observed between the groups (table 1). in the preconditioning groups, the level of maximal ischemic contracture was significantly lower, and the time of maximal ischemic contracture was significantly higher compared with the control group (p < 0.05 vs con) (table 1).table 1weights and ischemic contracturebody weight (g)heart weight wet (g)heart weight dry (mg)maximal ischemic contracture (mmhg)time of maximal. ischemic contracture (min)control310 251.5 0.2171 1736 314 1ipc315 231.5 0.2175 1820 2 27 1ipc + pax315 241.5 0.2181 1739 315 3mpc309 261.5 0.1180 2020 2 27 1mpc + pax313 271.5 0.1185 1939 313 2pax312 251.6 0.2184 1936 315 1data are mean sdipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline ; p < 0.05 vs control weights and ischemic contracture ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline ; p < 0.05 vs control in the control group, infarct size was (mean sd) 45 9% of the area at risk (figure 2). in the ischemic- and morphine - induced preconditioning groups, infarct size was similar and significantly less than in the control group (ipc : 20 5%, mpc : 23 8% ; each p < 0.05 vs con) (figure 2). the preconditioning effect of ischemia and morphine was attenuated significantly by the mkca - channel inhibitor, paxilline. infarct size was 36 6% and 37 7% in the ipc + pax and mpc + pax groups, respectively ; each p < 0.05 vs ipc and mpc, respectively (figure 2). paxilline alone had no effect on infarct size (pax : 46 7% ; not significantly different from con). there was no significant difference in infarct size between the preconditioning groups with paxilline compared with the control group.fig. histogram showing the infarct size (is) as percent of area at risk (aar) in controls (con, n = 8), morphine preconditioning (mpc, n = 8), ischemic preconditioning (ipc, n = 8), morphine preconditioning with paxilline (mpc + pax, n = 8), ischemic preconditioning with paxilline (ipc + pax, n = 8), and paxilline alone (pax, n = 8). data are mean sd ; p < 0.05 vs con ; # p < 0.05 vs ipc ; p < 0.05 vs mpc infarct size measurement. histogram showing the infarct size (is) as percent of area at risk (aar) in controls (con, n = 8), morphine preconditioning (mpc, n = 8), ischemic preconditioning (ipc, n = 8), morphine preconditioning with paxilline (mpc + pax, n = 8), ischemic preconditioning with paxilline (ipc + pax, n = 8), and paxilline alone (pax, n = 8). data are mean sd ; p < 0.05 vs con ; # p < 0.05 vs ipc ; p < 0.05 vs mpc hemodynamic variables are summarized in table 2. no significant differences in left ventricular end - diastolic pressure and dp / dtmax were observed between the experimental groups during baseline conditions and at the beginning of ischemia (table 2). at the end of the experiment, there was no difference in hr compared with controls at baseline and during reperfusion, with the exception of the paxilline group at time point final ten - minute washout shortly before index ischemia (table 2).table 2hemodynamic variables baselinewashout 2time after reperfusion (min)3153060heart rate (min)control366 37359 35365 29378 5372 15373 14ipc367 11360 23365 22377 2365 20361 24ipc + pax357 31355 32276 73323 47324 47357 28383 28383 28382 30mpc + pax346 28333 34248 215283 142375 24372 17pax336 25280 29 236 126342 71343 69344 68lvedp (mmhg)control12 3212 553 2235 1526 1019 7ipc11 14 452 2224 1319 818 7ipc + pax14 211 1162 2256 3245 3328 18mpc12 315 551 636 729 624 7mpc + pax13 212 457 2343 2438 3134 34pax11 58 1021 820 9dp / dtmax(mmhg sec 1,000)control3.3 0.63.3 0.60.7 0.90.6 0.80.6 0.70.4 0.5ipc3.4 1.13.5 1.10.7 1.01.4 0.71.6 0.5,1.5 0.6,ipc + pax3.3 0.42.7 0.20.1 0.10.3 0.20.2 0.10.1 0.1mpc3.0 0.42.7 0.60.8 0.41.0 0.41.0 0.31.0 0.1,mpc + pax2.6 0.52.5 0.50.1 0.10.3 0.20.4 0.30.3 0.2pax3.5 1.13.0 1.40.2 0.10.4 0.30.5 0.40.5 0.3cf (ml min)control16 315 29 67 67 57 6ipc14 211 57 411 68 69 6ipc + pax13 112 211 25 24 24 4mpc16 415 49 56 35 24 2mpc + pax15 214 213 48 37 46 4pax14 110 2,4 24 23 12 1data are mean sdipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxillinelvedp = left ventricular end - diastolic pressure ; dp / dtmax = rate of left ventricular pressure development ; cf = coronary flow ; p < 0.05 vs control ; p < 0.05 vs baseline hemodynamic variables ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline lvedp = left ventricular end - diastolic pressure ; dp / dtmax = rate of left ventricular pressure development ; cf = coronary flow ; p < 0.05 vs control ; p < 0.05 vs baseline in the control group, infarct size was (mean sd) 45 9% of the area at risk (figure 2). in the ischemic- and morphine - induced preconditioning groups, infarct size was similar and significantly less than in the control group (ipc : 20 5%, mpc : 23 8% ; each p < 0.05 vs con) (figure 2). the preconditioning effect of ischemia and morphine was attenuated significantly by the mkca - channel inhibitor, paxilline. infarct size was 36 6% and 37 7% in the ipc + pax and mpc + pax groups, respectively ; each p < 0.05 vs ipc and mpc, respectively (figure 2). paxilline alone had no effect on infarct size (pax : 46 7% ; not significantly different from con). there was no significant difference in infarct size between the preconditioning groups with paxilline compared with the control group.fig. histogram showing the infarct size (is) as percent of area at risk (aar) in controls (con, n = 8), morphine preconditioning (mpc, n = 8), ischemic preconditioning (ipc, n = 8), morphine preconditioning with paxilline (mpc + pax, n = 8), ischemic preconditioning with paxilline (ipc + pax, n = 8), and paxilline alone (pax, n = 8). data are mean sd ; p < 0.05 vs con ; # p < 0.05 vs ipc ; p < 0.05 vs mpc infarct size measurement. histogram showing the infarct size (is) as percent of area at risk (aar) in controls (con, n = 8), morphine preconditioning (mpc, n = 8), ischemic preconditioning (ipc, n = 8), morphine preconditioning with paxilline (mpc + pax, n = 8), ischemic preconditioning with paxilline (ipc + pax, n = 8), and paxilline alone (pax, n = 8). data are mean sd ; p < 0.05 vs con ; # p < 0.05 vs ipc ; p < 0.05 vs mpc hemodynamic variables are summarized in table 2. no significant differences in left ventricular end - diastolic pressure and dp / dtmax were observed between the experimental groups during baseline conditions and at the beginning of ischemia (table 2). at the end of the experiment, there was no difference in hr compared with controls at baseline and during reperfusion, with the exception of the paxilline group at time point final ten - minute washout shortly before index ischemia (table 2).table 2hemodynamic variables baselinewashout 2time after reperfusion (min)3153060heart rate (min)control366 37359 35365 29378 5372 23365 22377 2365 20361 24ipc + pax357 31355 32276 73323 47324 47357 20mpc367 30349 28383 28383 28383 28382 30mpc + pax346 28333 34248 215283 142375 24372 17pax336 25280 29 236 126342 71343 69344 68lvedp (mmhg)control12 3212 553 2235 1526 1019 7ipc11 14 452 2224 1319 818 7ipc + pax14 211 1162 2256 3245 3328 18mpc12 315 551 636 729 624 7mpc + pax13 212 457 2343 2438 3134 34pax11 58 443 1723 1021 820 9dp / dtmax(mmhg sec 1,000)control3.3 0.63.3 0.60.7 0.90.6 0.80.6 0.70.4 0.5ipc3.4 1.13.5 1.10.7 1.01.4 0.71.6 0.5,1.5 0.6,ipc + pax3.3 0.42.7 0.20.1 0.10.3 0.20.2 0.10.1 0.1mpc3.0 0.42.7 0.60.8 0.41.0 0.41.0 0.31.0 0.1,mpc + pax2.6 0.52.5 0.50.1 0.10.3 0.20.4 0.30.3 0.2pax3.5 1.13.0 1.40.2 0.10.4 0.30.5 0.40.5 0.3cf (ml min)control16 315 29 67 67 57 6ipc14 211 57 411 68 69 6ipc + pax13 112 211 25 24 24 4mpc16 415 49 56 35 24 2mpc + pax15 214 213 48 37 46 4pax14 110 2,4 24 23 12 1data are mean sdipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxillinelvedp = left ventricular end - diastolic pressure ; dp / dtmax = rate of left ventricular pressure development ; cf = coronary flow ; p < 0.05 vs control ; p < 0.05 vs baseline hemodynamic variables ipc = ischemic preconditioning ; mpc = morphine preconditioning ; pax = paxilline lvedp = left ventricular end - diastolic pressure ; dp / dtmax = rate of left ventricular pressure development ; cf = coronary flow ; p < 0.05 vs control ; p < 0.05 vs baseline the main finding of our study is that the opioid, morphine, initiates preconditioning in a similar manner as ischemia, i.e., by activation of mkca channels. ischemic preconditioning (ipc) describes a cardioprotective phenomenon where short periods of myocardial ischemia protect the heart against a subsequent longer period of ischemia and reduce the deleterious consequences of ischemia / reperfusion injury.14 besides ischemic stimuli, volatile anesthetics15,16 and morphine can mimic the cardioprotective effect of preconditioning.17 in contrast to volatile anesthetics, morphine can be administered to patients who are subjected to organ ischemia (vascular surgery, organ transplantation, cardiac surgery) or who recently underwent regional ischemia (stroke, angina pectoris, myocardial infarction, organ transplantation) without the side effect of being opening of mitochondrial atp - sensitive potassium (mkatp) channels that are involved in regulating mitochondrial functions is a key step that mediates cardioprotection induced by both morphine and ischemic preconditioning, possibly through inhibition of mitochondrial permeability transition pore (mptp) opening.3,5 mitochondrial calcium sensitive potassium (mkca) channels seem to be another class of k channels, apart from mkatp channels, that mediate cardioprotection by preconditioning.6,7 in 2002, xu.7 reported that kca - activated potassium channels are located on the inner mitochondrial membrane and mediate cardioprotection against ischemia and reperfusion injury in the isolated perfused guinea pig heart. since this initial observation, mkca - activated potassium channels have been found to be implicated in ischemic preconditioning against ischemia and reperfusion injury in isolated rat and mouse hearts, anesthetized dogs, and isolated cardiac myocytes.6,8,9,18,19 the mkca channel contains a pore forming -subunit and a regulatory -subunit.20 - 22 the -subunit consists of four accessory -subunits (1 - 4). both mkatp and mkca channel activation triggers preconditioning that is independent one from the other and involves the mptp. 8 cao. showed that ischemic preconditioning is triggered by activation of mkca channels and is abolished by the mkca channel inhibitor, paxilline.8 paxilline is a mycotoxin produced by the fungus, penicillium paxilli. it has the ability to block all subunits23 of mkca channels and is a selective inhibitor.24 besides paxilline, there are not many mkca channel blockers available. iberiotoxin, for example, is a mkca channel blocker that is also suitable for in vivo use,13 whereas paxilline is predominantly used for in vitro experiments. besides ischemic preconditioning, our results demonstrate that pharmacological preconditioning with morphine also induces cardioprotection by activation of mkca channels. cao.8 administered the mkca channel blocker, paxilline, at the onset of reperfusion after a prolonged period of ischemia. we applied paxilline during the preconditioning period, suggesting that mkca channel inhibition blocks the infarct size reducing effect of preconditioning during the trigger phase. in a recent study, we could demonstrate that activation of mkca channels not only reduced infarct size by preconditioning but also caused a significant reduction in the mitochondrial respiratory control index.13 co - administration of the mkca channel blocker, iberiotoxin, completely abolished the reduction in the respiratory control index, and we concluded that cardioprotection is mediated by activation of mkca channels leading to mild mitochondrial uncoupling. mild mitochondrial uncoupling during the trigger phase of preconditioning may represent a common characteristic of mitochondria in a conditioned state.7,25 - 27 the involvement of mkca channels in morphine - induced preconditioning was addressed in our study. we demonstrated here that ischemic- and morphine - induced preconditioning reduced the infarct size to a similar extent. the mkca channel blocker, paxilline, abolished both effects, confirming the findings of others that ischemic preconditioning involves activation of mkca channels. furthermore, it supports our hypothesis that mkca channels are involved in the trigger phase of morphine - induced preconditioning.. showed that opening of mkca channels can cause a slight increase in mitochondrial reactive oxygen species generation.28 the mkca channel agonist, ns1619, requires superoxide radical generation during the preconditioning stimulus to induce a cardioprotective effect.29 furthermore, these authors demonstrated that cardioprotection by ns1619 reduces mitochondrial calcium overload and mitochondrial reactive oxygen species production during the subsequent period of ischemia and early reperfusion.29 such a reduction in mitochondrial calcium overload and reactive oxygen species generation has been suggested to prevent mptp opening.30,31 evidence suggests that the mkca channel is located upstream of the mptp, because cardioprotection induced by activation of mkca channels was abolished by opening of the mptp. vice versa, inhibition of the mkca channel with paxilline did not block protection induced by inhibition of the mptp with cyclosporine a (csa).8 whether cardiac preconditioning by morphine is also mediated by regulation of the mptp due to mkca channel activation is yet unknown. the results of the present study have to be interpreted within the scope of some limitations. we can not rule out that morphine confers preconditioning via intracellular pathways leading to activation of mkca channels. however, the means by which morphine regulates mkca channel activation to induce preconditioning is unknown. it has been shown that mkca channel activation is involved in desflurane preconditioning and that protein kinase a (pka) is located upstream of the mkca channel.32 the activity of pka as a possible upstream activator of kca channels depends on the cellular level of cyclic adenosine monophosphate (camp)pka is known as camp - dependent protein kinase. gross. demonstrated that morphine - induced cardioprotection involves glycogen synthase kinase-3beta (gsk3beta) and akt (also called pkb).33 however, we did not investigate in the present study whether these enzymes are related to mkca channels. another limitation of our study is that we did not determine the effect of ischemic and morphine preconditioning on the mitochondria. xi. could show that morphine prevents mptp opening by inactivation of gsk3beta.34 our results showing that morphine confers preconditioning through activation of mkca channels and the fact that enzymes like pka regulate mkca channel activation suggest future directions for investigating the underlying mechanism of morphine - induced cardioprotection. clinically, morphine might be administered to patients who are subjected to organ ischemia (vascular surgery, organ transplantation, cardiac surgery) or who recently underwent regional ischemia (stroke, angina pectoris, myocardial infarction, organ transplantation) without the side - effect of being therefore, unravelling the exact mechanisms of morphine - induced cardioprotection might have clinical consequences. in summary, our results demonstrate that, besides ischemic preconditioning, morphine also initiates cardiac preconditioning via activation of mkca channels.	purposemitochondrial calcium sensitive potassium (mkca) channels are involved in cardioprotection induced by ischemic preconditioning. in the present study we investigated whether morphine - induced preconditioning also involves activation of mkca channels.methodsisolated rat hearts (six groups ; each n = 8) underwent global ischemia for 30 min followed by a 60-min reperfusion. control animals were not further treated. morphine preconditioning (mpc) was initiated by two five - minute cycles of morphine 1 m infusion with one five - minute washout and one final ten - minute washout period before ischemia. the mkca blocker, paxilline 1 m, was administered, with and without morphine administration (mpc + pax and pax). as a positive control, we added an ischemic preconditioning group (ipc) alone and combined with paxilline (ipc + pax). at the end of reperfusion, infarct sizes were determined by triphenyltetrazoliumchloride staining.resultsinfarct size was (mean sd) 45 9% of the area at risk in the control group. the infarct size was less in the morphine or ischemic preconditioning groups (mpc : 23 8%, ipc : 20 5% ; each p < 0.05 vs control). infarct size reduction was abolished by paxilline (mpc + pax : 37 7%, p < 0.05 vs mpc and ipc + pax : 36 6%, p < 0.05 vs ipc), whereas paxilline alone had no effect (pax : 46 7%, not significantly different from control).conclusioncardioprotection by morphine - induced preconditioning is mediated by activation of mkca channels.
glutamate reuptake represents the principal mechanism for inactivation of synaptically released glutamate [1, 2 ]. in the rodent hippocampus, it is mainly accomplished by astrocytic glutamate transporters (eaats : excitatory amino acid transporters), namely, glast (glutamate / aspartate transporter) and glt-1 (glutamate - transporter-1 ; rodent analogues of eaat1 and eaat2, resp. ; [37 ]). glutamate uptake is energized by the concomitant inward transport of three sodium ions and a proton, while one potassium ion is transported outward. consequently, its activation is accompanied by an increase in the intracellular sodium concentration of astrocytes [8, 9 ]. under pathological conditions, astrocytes undergo a complex reaction referred to as reactive astrogliosis, which is seen in diverse preparations and conditions ranging from primary cell culture to the intact brain [10, 11 ]. the hallmarks of reactive gliosis are a massive upregulation of the expression of the intermediate filament glial fibrillary acidic protein (gfap) and a cellular hypertrophy [12, 13 ]. reactive astrocytes display several features of immature astrocytes (e.g., [14, 15 ]) and can partially reenter the cell cycle [16, 17 ]. following traumatic brain injury, a dense glial scar forms at the lesion site due to strong astrocytic reorganization and proliferation [10, 11, 18 ]. at some distance to the lesion and scar, astrocytes usually do not divide, and their morphological reorganization and hypertrophy is less severe. the establishment of a glial scar around injured tissue is regarded as hindrance for the growth and regeneration of axons [19, 20 ]. notwithstanding, reactive astrocytes might also exert a protective role and support regeneration [11, 13, 21 ]. there is evidence that reactive gliosis is also accompanied by an alteration in the expression level of glial glutamate transporters. most studies performed in the rodent and human brain reported an overall downregulation of protein levels of both glast and glt-1 and/or a reduction in functional glutamate uptake in response to brain injury and astrogliosis, respectively (e.g., [2227 ]). the reduction in the expression of glial glutamate transporters has been suggested to contribute to the elevation of extracellular glutamate concentrations and to glutamate - mediated excitotoxicity which is observed under many pathological conditions. injury - induced changes in the expression of the glutamate transporter subtypes might, however, also depend on the degree of astrogliosis. this might be especially relevant and visible upon a mechanical lesion, where reactivity of astrocytes, as judged for example based on their gfap expression and morphology, ranges from severe astrogliosis in the scar region to only moderate - mild astrogliosis distant from the lesion [10, 11 ]. to study this question, we analyzed expression of glast and glt-1 following a mechanical lesion in organotypic tissue slice cultures of the mouse hippocampus. furthermore, we employed ratiometric sodium imaging as functional assay for glutamate uptake in astrocytes. our results show that mechanical tissue injury generates subsets of reactive astrocytes depending on the distance from the lesion site, which differ in both morphological features and their ability to take up sr101. furthermore, these subsets show discrete changes in glutamate transporter expression and glutamate uptake capacity, indicating that glutamate clearance might be largely functional in the periphery of the lesion, but strongly hampered in the scar region. this study was carried out in strict accordance with the institutional guidelines of the heinrich heine university duesseldorf, germany, as well as the european community council directive (86/609/eec). all experiments were communicated to and approved by the animal welfare office at the animal care and use facility of the heinrich heine university duesseldorf, germany (institutional act number : o52/05). in accordance with the german animal welfare act (tierschutzgesetz, articles 4 and 7), no formal additional approval for the postmortem removal of brain tissue was necessary. for generation of acute slices, mice were quickly decapitated (following the recommendation of the european commission published in : euthanasia of experimental animals, luxembourg : office for official publications of the european communities, 1997 ; isbn 92827 - 9694 - 9). acute tissue slices of the hippocampus were prepared from balb / c mice (mus musculus) of both genders at postnatal days 7 to 8 (p7 - 8) using standard procedures. for some experiments, transgenic animals (fvb / n - tg[gfapgfp]14mes / j) expressing green fluorescence protein (gfp) under the gfap promoter were used (obtained from jackson laboratory ; harbor, usa). after decapitation of the animals, brains were quickly excised and hemisected in ice - cold artificial cerebrospinal fluid (acsf) composed of (in mm) 125 nacl, 2.5 kcl, 2 cacl2, 1 mgcl2, 1.25 nah2po4, 26 nahco3, and 20 glucose, bubbled with 95% o2 and 5% co2, and adjusted to a ph of 7.4. hemisections were trimmed, and transverse slices (200 m) comprising the entorhinal cortex, hippocampus, fimbria, and thalamus were prepared using a vibratome (microm hm650v, thermo fischer scientific, walldorf, germany). organotypic hippocampal slice cultures were prepared and cultured according to the protocol introduced by stoppini. with minor modifications. to this end, acute slices were transferred to a millicell culture insert (picm org 50, hydrophilized ptfe, pore size 0.4 m ; merck millipore, darmstadt, germany) and maintained at the interface of a serum - based culture medium free of antibiotics in a humidified incubator atmosphere of 5% co2 at 37c. the culture medium was composed of 30% sterile filtered normal horse serum (nhs ; gibco / life technologies, darmstadt, germany), 30% dulbecco 's modified eagle medium (dmem ; gibco / life technologies, darmstadt, germany), and 40% hank 's balanced salt solution (hbss ; gibco / life technologies, darmstadt, germany) supplemented with 38 mm glucose (ph adjusted to 7.3 - 7.4). the medium was changed three times a week, and the insert surface was washed with medium once a week. after at least 12 days in culture, a mechanical lesion was performed using a sterile scalpel blade. the lesion was positioned in the ca1 area perpendicular to the stratum pyramidale, included the strata oriens, pyramidale, radiatum, and lacunosum moleculare, and spanned the entire depth of the slice (cf. figure 2). after lesioning, slice cultures were maintained for another 6 - 7 days (designated as 6 - 7 days postlesion). unlesioned control slices were cultured in parallel for a corresponding number of days. for visualization of cell death, acsf containing 0.5 g / ml propidium iodide (pi) was applied to the slice surface and incubated for 3 hours at 37c and 5% co2, followed by a wash with acsf. documentation was either accomplished with an epifluorescence microscope (nikon eclipse 90i ; nikon instruments, dsseldorf, germany) or at an olympus fluoview300 laser scanning microscope (olympus, hamburg, germany). astrocyte soma size in organotypic control and lesioned slices was calculated from images of gfp - fluorescence derived from gfp / gfap mice which were obtained at a confocal microscope (olympus fluoview300 ; olympus, hamburg, germany ; see also below). to this end, a semiquantitative approach was used, in which somata of single cells were manually encircled (cf. figure 4(b)), and the resulting area was calculated using imagej software (nih, bethesda, usa). all chemicals were purchased from sigma - aldrich chemical (munich, germany) unless stated otherwise. for imaging experiments, organotypic slices were excised from the millicell inserts and incubated in acsf containing 2.5 m sr101 for 30 min at 35c to stain astrocytes (see above). sr101 is a highly specific and widely used tool for the identification of mature astrocytes in many brain regions including the hippocampus [29, 30 ]. it has recently been shown to be taken up into mature hippocampal astrocytes via an active transport mechanism involving organic anion transport polypeptides. wide - field fluorescence imaging was performed using a variable scan digital imaging system (till photonics, martinsried, germany) attached to an upright microscope (bx51wi, olympus europe, hamburg, germany) and a ccd camera (till imago vga, till photonics, martinsried, germany). sr101 was excited at 575 nm, and emission was collected above 590 nm. excitation wavelength for detection of gfp was 488 nm, and emission was collected above 510 nm. for intracellular sodium imaging, slices were additionally incubated with the membrane permeable form of the sodium - sensitive fluorescent dye sbfi (sbfi - am ; sodium - binding benzofuran isophthalate - acetoxymethyl ester ; molecular probes / life technologies, darmstadt, germany) as described earlier [3234 ]. ratiometric sodium imaging was performed by alternate excitation of sbfi at 340 nm (weakly sodium - sensitive wavelength) and at 380 nm (sodium - sensitive wavelength) at 4 hz. emission (> 440 nm) was collected in defined regions of interest (roi) representing cell bodies. after background correction, the fluorescence ratio (f340/f380) was calculated for the individual rois and analyzed offline using originpro 8 g software (originlab corporation, northampton, ma, usa). changes in sbfi fluorescence ratio were expressed as changes in sodium concentration based on in situ calibrations as reported before [32, 33, 35 ]. to equilibrate extra- and intracellular na - concentrations, sbfi - loaded slices were perfused with saline containing ionophores (3 m gramicidin d, 100 m monensin) and the na / k - atpase blocker ouabain (100 m), as well as different concentrations of na. the glutamate transporter agonist d - aspartate was applied by a pressure application device (pdes-02d, npi electronic gmbh, tamm, germany) coupled to standard micropipettes (hilgenberg, waldkappel, germany) placed 20100 m from cell bodies of selected cells. the following antisera were utilized, which represent well - established, commercially available standard markers : guinea - pig glt-1 antiserum directed against the c - terminus of rat glt-1 (chemicon international / millipore corp., carrigtwohill, ireland ; e.g.,) and guinea - pig glast antiserum directed against the c - terminus of rat glast (chemicon international / millipore corp., carrigtwohill, ireland ; e.g.,). validation and a detailed characterization of both antibodies were provided before [3840 ]. for identification of astrocytes, polyclonal rabbit antibodies against gfap (gfap - pab ; dako cytomation, denmark ; [40, 41 ]) and s100 (abcam, united kingdom ;) were employed. when double staining with the latter antibody was performed, a monoclonal mouse antibody detecting gfap was used (gfap - mab ; sigma aldrich, germany ; [40, 43 ]). standard fluorochrome - conjugated antibodies (alexafluor, invitrogen / life technologies, darmstadt, germany) were employed as secondary antibodies for immunohistochemistry. prior to immunohistochemical processing, organotypic slices were immersion fixed for 30 min at rt in 4% paraformaldehyde (pfa) in phosphate - buffered saline (pbs) following three washes every 30 min with pbs. cell membranes were permeabilized, and unspecific binding sites were blocked in pbs containing 0.25% triton - x100 (tx) and 2% normal goat serum (ngs ; gibco / life technologies, darmstadt, germany) for 90 min at 4c followed by incubation with the primary antibody gfap - pab (1 : 1000 ; dako cytomation, glostrup, denmark), diluted in the same solution over night at 4c. in case of s100/gfap double stainings, slices were incubated with a mixture of the primary antibodies gfap - mab (1 : 1000, dako cytomation, glostrup, denmark) and rabbit - s100 (1 : 100, abcam, united kingdom). after five washes in pbs containing 0.25% tx and 2% ngs, slices were incubated either with guinea pig - anti - glast or with guinea pig - anti - glt-1 (both 1 : 1000, diluted in 0.25% tx/2% ngs / pbs) for 4 hrs at rt. antirabbit - alexafluor594 and antiguinea pig - alexafluor488 or antimouse - alexafluor488 (1 : 100 in blocking solution) were used for visualization of antibody binding and incubated for 2 hrs at rt. the slices were subjected to dapi staining (4,6-diamidino-2-phenylindole ; 0.5 m ; invitrogen), washed three times, and mounted on glass slides with mowiol / dabco (calbiochem, fluka, distributed by sigma - aldrich chemical, munich, germany). negative controls were run in parallel to each staining by either omitting all or just one of the primary antibodies. control stainings in which one of the primary antibodies was omitted showed the identical labeling pattern for the remaining antibody as in the double stainings. omitting documentation of immunofluorescence was either performed with an epifluorescence microscope (nikon eclipse 90i ; nikon instruments, dsseldorf, germany) or a confocal laser scanning microscope (olympus fluoview300 ; olympus, hamburg, germany). the epifluorescence microscope was equipped with a standard dapi (ex 340380 ; dm 400 ; ba 435485), fitc (ex 465495 ; dm 505 ; ba 515555), and tritc (ex 540/25 ; dm 365 ; ba 605/55) filter set. illumination was provided by an intensilight fiber lamp (c - ghfi ; nikon instruments, dsseldorf, germany), and emission was detected with a monochrome digital camera (ds - qi1mc ; nikon instruments, dsseldorf, germany). images were collected with either a 20x/0.75 (planapovc, nikon instruments, dsseldorf, germany) air objective or a 60x/1.40 oil immersion objective (planapovc, nikon instruments, dsseldorf, germany). nis - elements software (nikon instruments, dsseldorf, germany) was used for image acquisition. for confocal microscopy, an olympus bx51wi microscope coupled to a confocal laser scanning system (fv300) equipped with a multiline argon (488 nm) and a helium - neon laser (543 nm, both melles griot, bensheim, germany) was used. images were collected with either a 20x/0.50 (umplanfl, olympus, hamburg, germany), a 40x/0.80 water immersion (lumplan, olympus, hamburg, germany), or a 60x/1.40 oil immersion objective (planapovc, nikon instruments, dsseldorf, germany). a kalman filter 4 was employed at every scan. simultaneous or sequential scanning of both fluorophores revealed no difference in their staining patterns, indicating absence of cross - excitation or spectral bleed through. the thickness of z - plane sections was 1 m, and the number of optical sections varied depending on the preparation. figures show extended focus images as specified in the figure legends, which were calculated from z - stacks of optical sections using imagej software (nih, bethesda, usa). images were overlaid employing adobe photoshop cs2 (adobe systems, cologne, germany). data were statistically analyzed by student 's t - test employing the procedures implemented in originpro 8 g software (originlab corporation, northampton, ma, usa). each set of experiments was performed on at least three tissue slices obtained from different animals. the aim of the present study was to analyze changes in glutamate transporter expression and function in astrocytes in response to a mechanical injury. to this end, we prepared organotypic tissue slice cultures of the mouse hippocampus, a well - established model system in which the layering and basic cellular organization of the neural network are maintained over time. after 1925 days in culture, organotypic tissue slices had flattened to a thickness of 4050 m and were composed of 3 to 4 cell layers. at this stage, propidium iodide assays detected only few dead cells dispersed throughout the slices (3 0.6 cells per ca1 subfield ; 18 3.4 in entire preparation ; n = 6 ; data not shown), indicating that the preparation was viable and in a stable condition. to visualize astrocytes in cultured slices, we used hippocampi of transgenic mice in which green fluorescent protein (gfp) is expressed under the control of the promoter of the astrocyte - specific intermediate filament glial fibrillary acidic protein (gfap ; fvb / n - tg[gfapgfp ] ; gfp / gfap mice). a visualization of the gfp fluorescence demonstrated that the general distribution and organization of astrocytes were preserved in the strata pyramidale and radiatum of the ca1 region of organotypic cultures, confirming earlier reports (n = 3 ; figure 1(a) ; [44, 45 ]). the vast majority of gfp - expressing cells (~95%) also labeled with the astrocyte - specific vital marker sr101 in organotypic slices (n = 3 ; figures 1(a) and 1(b)). furthermore, immunohistochemical stainings for gfap in slice cultures derived from wildtype animals showed that the majority of gfap - positive cells were also positive for s100 (n = 12 ; figure 1(c)), a marker for mature astrocytes. taken together, the nearly complete overlap in the cellular staining pattern for the vital dye sr101 with the expression of gfp in gfp / gfap mice, as well as the overlap in the immunofluorescence for gfap and s100 in organotypic slices indicates that these markers are well suited to identify astrocytes in this preparation. to induce astrogliosis in organotypic slice cultures, we used a scalpel blade and performed a scratch under semisterile conditions through the entire ca1 region oriented perpendicular to the stratum pyramidale (figure 2(a)). 6 - 7 days after the lesion, a general enhancement of gfap immunoreactivity was observed, indicating astrogliosis (figure 2(b)). the greatest enhancement of gfap immunoreactivity occurred within a distance of less than 100 m along the lesion (n = 26 ; figures 2(b) and 2(c)). here, a dense meshwork of thick, gfap - positive labels was present, indicative of the formation of a glial scar. furthermore, the region close to the lesion site exhibited a discernible increase in s100 immunoreactivity (n = 12 ; figure 2(c)). both the prominent expressions of gfap and s100 are characteristics of strongly reactive astrocytes, which represent the major cellular component of the glial scar forming after mechanical tissue injury. more distal to the lesion (> 100350 m), a less dramatic rearrangement of gfap immunoreactivity was observed. a clear difference in astrocyte properties between the scar region and its periphery was also seen following sr101 labeling. in the periphery of the scar (100350 m distance from the lesion), sr101 resulted in a reliable and bright cellular staining, similar to what had been observed in control, unlesioned slices (n = 12 ; figure 3(a), cf. figure 1). in contrast to this, cellular sr101 staining was detectable but only faint within a distance of less than 100 m on both sides of the lesion (figure 3(a) ; mean width of sr101-free area 111 3 m ; n = 5). in slices from gfp / gfap transgenic animals (n = 5 ; figure 3(b)), gfap - positive, weakly sr101-positive scar cells also stained with the sodium - sensitive fluorescence indicator sbfi - am (acetoxymethyl ester of sodium binding benzofuran isophthalate ; see below ; n = 3), indicating that they were able to take up and de - esterify this dye (figure 3(b)). in addition to gfap - positive cells, the scar region hosted microglial cells, identified by their vital staining with texas red - coupled lectin from lycopersicon esculentum (tomato lectin ; n = 4 ; not shown). tomato lectin - positive microglia, however, did not take up sbfi - am (n = 4 ; not shown), confirming earlier reports that microglial cells do not incorporate am - ester dyes in situ. to further characterize reactive gliosis in response to the scratch wound, we determined the area of astrocyte somata in confocal images of the gfp fluorescence in control and lesioned slices obtained from gfp / gfap transgenic animals. these measurements revealed that, compared to astrocytes in control slices, the soma area of astrocytes in the periphery of a lesion was increased by 18% and that of astrocytes in the scar region was increased by 24% (control : 163 2 m ; lesion periphery : 193 3 m ; scar tissue : 201 4 m ; p < 0.001 ; n = 945, 496, and 439 in 7 control and 9 lesioned slices ; figures 4(a) and 4(b)). thus, astrocytes show a second hallmark of reactive gliosis after lesion, namely, a hypertrophy of their cell bodies, which is observed both in the scar tissue as well as in the periphery of the lesion. taken together, these data show that 6 - 7 days after the mechanical lesion, a glial scar has formed along the lesion site. this scar encompasses strongly activated astrocytes, characterized by a robust increase in gfap and s100 expression. these proximal reactive astrocytes, located within a distance of less than 100 m from the lesion, exhibit long gfap - positive processes, have significantly swollen somata, and only weakly stain with sr101. astrocytes in the periphery of the scar (100350 m from the lesion) display swollen cell bodies as well, but show only moderate upregulation of gfap and maintain their ability to efficiently take up sr101. to determine the spatial distribution of the glutamate transporters glast and glt-1 in organotypic slices and changes therein in response to the lesion, immunohistochemistry was employed. in control slices, glast (n = 13 ; figure 5(a)) as well as glt-1 immunoreactivity (n = 11 ; figure 5(b)) appeared as punctate labeling throughout the entire ca1 area. the staining patterns for both transporters in the tissue slice cultures were thus similar to those reported from acute tissue slice preparations of the hippocampus [36, 48, 49 ]. the relatively homogeneous distribution of immunoreactivity throughout the preparation found in control slice cultures was abandoned following a lesion. here, distinct accumulations of glast (n = 17 ; figure 5(a)) and glt-1 immunoreactivity (n = 12 ; figure 5(b)) were found in the scar region. in both cases, these preferentially colocalized with thick gfap - positive bundles running in parallel to the lesion (figure 5). apart from the marked accumulation of glast reactivity in parallel to the scar, glast immunoreactivity appeared generally increased in some lesioned slice preparations (figure 5(a)) but unaltered in others both within the scar as well as in its periphery. for glt-1, a decline in the intensity of immunoreactivity was observed with increasing distance from the lesion (figure 5(b)). in summary, these results reveal distinct alterations in the immunoreactivity of glast and glt-1 following a mechanical lesion. in the scar region, clusters of glt-1 and glast immunoreactivity are present, primarily colocalized with strongly gfap - positive fibers. apart from these distinct clusters, glt-1 immunoreactivity appears to decline with distance from the scar, whereas glast immunoreactivity seems largely uniform throughout the preparation. glutamate uptake is accompanied by the inward flux of sodium, resulting in increase in the intracellular sodium concentration of astrocytes. to monitor its function, we assayed sodium increases induced by d - aspartate, a nonmetabolized substrate of sodium - dependent glutamate uptake which is also transported in astrocytes in the stratum radiatum of organotypic slices. to this end, sr101-stained slices were additionally loaded with the sodium - sensitive fluorophore sbfi - am (figure 6(a), cf. the sbfi fluorescence revealed a linear increase in its fluorescence ratio at intracellular sodium concentrations between 10 and 40 mm (n = 107 ; figure 6(b)) as reported before. repetitive pressure application of d - aspartate for 500 ms induced reliable increases in the intracellular sodium concentration in astrocytes, indicating activation of sodium - dependent glutamate uptake (n = 488 ; figure 6(c)). the experiments were performed in the presence of the sodium - channel blocker ttx (tetrodotoxin, 0.5 m) to suppress action potential generation. the amplitude of d - aspartate - induced sodium increases was dependent on the concentration applied (figure 6(c)) and saturated at about 2 mm (n = 19 ; figure 6(d)), as reported before for mouse cortical astrocytes in culture. at a concentration of 1 mm, the amplitude of d - aspartate - induced sodium transients was near maximum and amounted to 3.8 0.2 mm (n = 161 ; figures 6(c) and 6(d)), a value comparable to that in astrocytes in acute slices obtained in our lab. to probe for changes in glutamate uptake, we compared sodium signals induced by 1 mm d - aspartate between control and lesioned slices (figure 7). in lesioned slices, astrocytes located in the periphery of the lesion (100350 m distance) responded with a sodium transient to application of 1 mm d - aspartate (see cell 2 in figures 7(a) and 7(b)). the amplitude of d - aspartate - induced sodium signals in cells in the lesion periphery was similar to that observed in control slices (3.8 0.3 mm ; n = 111 ; see cell 1 in figures 7(a), 7(b), and 7(c)). application of d - aspartate also evoked sodium transients in weakly sr101-positive, sbfi - filled astrocytes located in the glial scar area (< 100 m from the lesion ; see cells 35 in figures 7(a) and 7(b)). these cells, however, displayed significantly smaller peak amplitudes (1.6 0.21 mm, n = 26 ; p < 0.001 ; figures 7(b) and 7(c)) than astrocytes in the periphery and in control slices. the kinetics of d - aspartate - induced sodium transients differed between all three groups. this was true for the slope of the sodium increase (10%90% change), which was significantly reduced in astrocytes from lesioned slices versus astrocytes from control slices (0.9 0.05 mm / s in control versus 0.6 0.06 mm / s in the periphery and 0.2 0.04 mm / s in the scar region ; n = 161, 111, and 26, resp. ; in addition, the decay back to baseline was significantly slower following lesion (figures 7(b) and 7(c)). the time needed for the signal to decay by 67% was approximately doubled (decay time was 44 2 s in control, = 81 4 s in periphery of the lesion, and = 95 10 s in scar cells ; figures 7(b) and 7(c)). these results show that mechanical injury results in distinct alterations in the kinetics and/or amplitudes of d - aspartate - induced intracellular sodium transients in reactive astrocytes, depending on their distance to the lesion. our results show that within 6 - 7 days after setting a scratch wound in organotypic hippocampal slices, a dense glial scar had formed along the lesion site. up to approximately 100 m distance from the lesion, the scar tissue was characterized by a substantial elevation of gfap expression, a pronounced cellular hypertrophy, and reduced uptake of sr101 by astrocytes. while prominent clusters of glast and glt-1 immunoreactivity were observed along gfap - positive structures in scar cells, d - aspartate - induced intracellular sodium signals were strongly dampened, indicating a significant reduction in glutamate uptake capacity close to the lesion. in the periphery of the lesion (100350 m), astrocytes showed less pronounced reactivity, maintained their ability to efficiently take up sr101, and displayed only minor changes in glutamate transporter immunoreactivity and function. thus, depending on their distance from the lesion, astrocytes showed different grades of reactivity and displayed discrete changes in glutamate transporter immunoreactivity and uptake capacity. organotypic tissue slice cultures of the hippocampus represent a well - established model system, in which the basic tissue architecture is maintained, but which still hosts the major advantages of cell cultures such as good accessibility and control of experimental conditions. they have been used extensively for the analysis of neuronal properties and developing neuronal networks [28, 51, 52 ] or the study of excitotoxic neuronal damage (e.g., [53, 54 ]). recent studies also demonstrated that basic morphological characteristics of astrocytes and the typical glia - synapse organization are well preserved in organotypic slice cultures (e.g., [44, 45, 55, 56 ]). in addition, we found a nearly complete overlap of sr101 staining with the expression of gfp in slice cultures obtained from gfp / gfap mice, demonstrating that sr101 is well suited to identify astrocytes in this preparation as described before for acute tissue slices and in vivo [29, 30 ]. performing a scratch through the ca1 region of organotypic slices resulted in the formation of a glial scar along the lesion site, as judged by the prominent increase in expression of gfap and s100. in addition, astrocyte cell bodies showed a significant hypertrophy. furthermore, astrocytes in the periphery of the scar (100350 m from the lesion) showed mild to moderate reactivity, displaying swollen cell bodies, and only moderate upregulation of gfap. these changes are hallmarks of reactive gliosis observed after tissue injury [10, 13, 57 ]. generally, the degree of injury and the distance of the astrocytes from the site of injury define the degree of their activation [11, 13 ]. our study also revealed that strongly reactive astrocytes in the scar region only weakly stain with sr101. the reduced ability of strongly activated astrocytes to accumulate sr101 indicates that strong glial activation is accompanied by a downregulation of the organic anion transporter responsible for uptake of sr101. this is reminiscent of immature astrocytes in the early postnatal hippocampus, which are sr101-negative and in line with a wealth of experimental evidence suggesting that reactive gliosis represents a process comprising a dedifferentiation of astrocytes. glutamate uptake in the hippocampus is mainly achieved by the glial glutamate transporters glt-1 and glast [1, 2 ]. a central factor in many brain pathologies, including traumatic brain injury, is an increase in extracellular glutamate and excitotoxicity ; it has been suggested that a change in the expression levels of glial glutamate transporters might play a critical role in the failure of glutamate clearance [6, 5860 ]. earlier work has found an overall downregulation of protein levels of both glast and glt-1 following astrocyte activation [2227 ], indicating that this is causal to the elevation of extracellular glutamate. in contrast to this notion, other studies reported increased glutamate transport capacity of reactive astrocytes and suggested a protective influence [61, 62 ]. to visualize possible differences in the spatial expression profile of glt-1 and glast in strongly reactive astrocytes along the scar as compared to moderately reactive astrocytes in its periphery, we performed immunohistochemical stainings. these revealed an accumulation of glt-1 and glast immunoreactivity along thick gfap - positive fibers, which was particularly pronounced for glast. besides these clusters, overall glast immunoreactivity seemed unaltered as compared to unlesioned slices, whereas glt-1 immunoreactivity seemed weaker and declined with increasing distance from the scar. clustering of glutamate transporters has been described by several reports [48, 63, 64 ]. in developing hippocampal astrocytes, cluster formation was preferentially found in branches opposed to synapses and was increased with increased neuronal activity, indicating that it is necessary to cope with synaptic release of glutamate. along the same lines, it was observed that sustained astroglial activation by ciliary neurotrophic factor (cntf) in the rat striatum induced a concentration of glast and glt-1 into raft microdomains and improved glutamate clearance, indicating that cluster formation increased the cellular capacity for glutamate uptake. however, another study found glutamate - induced clustering of glt-1 that induced its endocytosis and intracellular trafficking without changing the total expression levels as detected by western blots, arguing for a decrease in functional glial glutamate uptake capacity. in the present study, antibodies against glt-1 and glast were employed for immunohistochemistry after permeabilization of the plasma membrane. the clusters of glast and glt-1 along gfap - positive fibers in the scar region (as glast and glt-1 immunoreactivity in general) could thus represent glutamate transporters in the plasma membrane or in intracellular compartments or both. consequently, the observed clustering does not allow a prediction about possible functional changes in glutamate uptake. we probed for the functional activation of glutamate transport by application of the transportable agonist d - aspartate, which results in an increase in the intracellular sodium concentration of astrocytes [8, 9 ]. the amplitudes of d - aspartate - induced sodium transients in astrocytes in control organotypic slices were similar to those reported from astrocytes in acute slices obtained in our lab, indicating similar cellular glutamate transport capacity in both preparations. following a lesion, d - aspartate - induced intracellular sodium signals were clearly dampened in scar cells. moreover, the slope of the increase in sodium was significantly reduced, and the decay back to baseline was slowed. because d - aspartate was applied at nearly saturating concentration, the reduction in peak amplitudes suggests a reduction in functional glutamate uptake capacity in cells close to the lesion, which might be mediated by a decrease in the overall number of glutamate transporters available at the plasma membrane. in contrast, the alteration in the kinetics of the d - aspartate - induced sodium signals can be explained by the cellular hypertrophy that was observed in reactive astrocytes. at an equal transport - mediated sodium influx across the membrane, the slope of resulting changes in the sodium concentration will be decreased in cells with a larger volume. furthermore, the decay of intracellular sodium transients back to baseline is mainly governed by the activity of the na / k - atpase as well as by diffusion. the slower recovery is in line with earlier studies reporting a downregulation of the sodium pump following reactive gliosis [65, 66 ]. moreover, sodium is not buffered in the cell and travels solely by means of diffusion in the cytoplasm. hindered diffusion, resulting from increased cytoplasmic protein content or impaired gap junction coupling which both occur upon astrocytic activation, might thus also partially contribute to delayed sodium recovery [12, 34, 67 ]. in contrast to scar cells, the amplitude of d - aspartate - induced intracellular sodium signals was not significantly altered in moderately activated astrocytes in the periphery of the lesion, indicating that the overall number of functional glutamate transporters was not significantly altered. because these cells showed hypertrophy as well, the slowed kinetics of the sodium transients are in line with an increased cellular volume as well as a possible downregulation of na / k - atpase and/or a slowed diffusion as argued above. taken together, our results indicate a significant reduction in glutamate uptake capacity in strongly activated astrocytes in the scar region. thus, the prominent clustering of glast and glt-1 immunoreactivity along gfap - positive structures close to the lesion is likely to reflect a loss of glutamate transporters from the plasma membrane upon transporter internalization as reported by nakagawa.. the authors of the latter study speculated that large and prolonged increases in glutamate concentrations are necessary to induce such cluster formation and endocytosis in the tissue, as they would possibly only occur under pathological conditions. indeed, ambient glutamate concentrations were shown to be significantly increased after traumatic brain injury [6870 ]. along these lines, it can be assumed that in our model system, extracellular glutamate concentrations rose less severely in the periphery of the lesion, preventing a comparable clustering and loss of functional glutamate transport activity in moderately activated astrocytes. our results show complex changes in glutamate transporter expression and function during astrocyte activation following mechanical injury. they confirm that the degree of astrocyte activation depends on the distance from the insult and comprises different endpoints regarding cellular morphology and physiology. furthermore, our results highlight that astrocytes which show different grades of reactivity also display discrete changes in glutamate transporter expression and function. while immunohistochemistry revealed a prominent clustering of glt-1 and glast immunoreactivity in the scar region, our functional assay clearly showed that glutamate uptake capacity is strongly reduced in scar cells, while it is largely maintained in moderately activated astrocytes in the periphery. thus, mild - to - moderate astrogliosis in the periphery of a mechanical lesion does not necessarily seem to be accompanied by a significant change in glial glutamate uptake capacity. at the glial scar itself, in strongly reactive astrocytes, a clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in astroglial glutamate uptake, which may contribute to glutamate - mediated excitotoxicity in this region.	astrocytes express the sodium - dependent glutamate transporters glast and glt-1, which are critical to maintain low extracellular glutamate concentrations. here, we analyzed changes in their expression and function following a mechanical lesion in the ca1 area of organotypic hippocampal slices. 6 - 7 days after lesion, a glial scar had formed along the injury site, containing strongly activated astrocytes with increased gfap and s100 immunoreactivity, enlarged somata, and reduced capability for uptake of sr101. astrocytes in the scar 's periphery were swollen as well, but showed only moderate upregulation of gfap and s100 and efficiently took up sr101. in the scar, clusters of glt-1 and glast immunoreactivity colocalized with gfap - positive fibers. apart from these, glt-1 immunoreactivity declined with increasing distance from the scar, whereas glast expression appeared largely uniform. sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery. our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function. strongly reactive astrocytes in the scar, however, display clusters of glast and glt-1 immunoreactivity together with reduced glutamate transport activity. this reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site.
the recent international consensus conference on the reduction in mortality in cardiac anesthesia and intensive care considered pexelizumab among the drugs that could reduce mortality in cardiac surgery. pexelizumab is a recombinant humanized single - chain monoclonal antibody to the component 5 of the complement system (c5). c5a is a powerful anaphylatoxin and proinflammatory mediator, and c5b is the precursor of the latter is a transmembrane channel involved in thrombosis and inflammation, which also causes direct tissue injury through osmotic lysis. pexelizumab administration on top of conventional treatment has been tested in st elevation myocardial infarction (stemi) and on - pump coronary artery bypass grafting (cabg). in the stemi setting, utility of pexelizumab has been ruled out by the results of our systematic review and meta - analysis. conversely, within the same manuscript we suggested that pexelizumab administration in patients undergoing cabg surgery could significantly reduced the relative risk of all - cause death by 26% : (or 0.74 [0.5 - 0.94 ], p=0.01) with a number needed to treat of 100 (95% ci 33 - 167). the lack of benefit of pexelizumab in the setting of patients with stemi clearly contrast with the apparent benefit observed in patients undergoing cabg. in other words, the diverse effect of pexelizumab might reflect crucially important differences of the two settings in which it has been tested. indeed, in animal models of tissue reperfusion injury (common in stemi setting) an increased accumulation of mac (c5b-9) was demonstrated as well as quite stable levels of c5a during cardiopulmonary bypass. conceivably, in the setting of stemi once microvascular damage and myocardial death due to necrosis, local inflammation, and apoptosis have become irreversible, complement activation could have already led to mac formation, thus nullifying any benefit from pexelizumab. furthermore, penetration of pexelizumab into myocardial tissue may be limited as a consequence of microvascular obstruction and local metabolic derangement. on the other hand, upstream administration of pexelizumab could reduce the generalized inflammatory process accompanying cardiopulmonary bypass. when considering the unconvincing evidences and knowledge of its beneficial effect, it does nt surprise that pexelizumab is no longer on the market and only 32% to 35% of the participants to the consensus conference included it among the life - saving drugs in cardiac surgery. the discouraging results of pexelizumab led to the end of the drug, not to the end of the concept, perhaps the way to an effective antinflammatory / anti - complement drug should probably start from a better knowledge of the underlying mechanisms of such an innate immune response in patients undergoing cabg, and then on possible preventive strategies which might positively affect the outcome.	a recent international consensus conference on the reduction in mortality in cardiac anesthesia and intensive care included pexelizumab, a recombinant monoclonal antibody to the component 5 of the complement system, among the ancillary (i.e. non - surgical) drugs / techniques / strategies that might influence survival rates in patients undergoing cardiac surgery. the consensus conferences state that a subgroup analysis of a meta - analysis of randomized controlled trials suggested that pexelizumab might reduce mortality (longest follow up available, up to 6 months) in patients undergoing coronary artery bypass grafting. pexelizumab was not included among the most important topics of the consensus conference as it was the only topic that did not receive a sufficient percentage of votes from the audience (32% at the first round and 35% at the second round). pexelizumab is no longer on the market, however, the concept of reducing the generalized inflammatory process accompanying cardiopulmonary bypass deserves further investigation.
1. post - kala - azar dermal leishmaniasis is an unusual dermatosis occurring following an attack of visceral leishmaniasis. post - kala - azar dermal leishmaniasis (pkdl) is an unusual dermatosis occurring between 6 months and 5 years following an attack of visceral leishmaniasis (vl). earlier, pkdl was considered to occur in vl cases receiving no treatment or irregular or incomplete therapy with sodium stibogluconate or pentamidine. miltefosine is the current drug of choice for vl and is being used in its elimination program. after thorough search of literature, we could find only two such reports and both of them were published in 2009. a 23-year - old lady was brought to our dermatology outdoor patient department by a health worker with hypopigmented macules on face, upper arms, and abdomen [figures 1 and 2 ]. the peripheral nerves were not tender or thickened. on systemic examination, all findings were normal except mild anemia (9 mg / dl) and high erythrocyte sedimentation rate (esr ; 96 mm in the first hour). biopsy sample from hypopigmented macules did not reveal any acid fast bacilli but showed epidermal atrophy, follicular plugging with keratin, and presence of dense lymphocytic infiltrate in the dermis suggestive of pkdl. there was no demonstrable leishman - donovan (ld) body [figure 3 ]. she was treated for vl with miltefosine, 100 mg / day for 28 days about 6 years back under a phase iv who / tdr / icmr - sponsored clinical trial. after treatment, she became clinically and parasitologically cured and had no complains in follow - up of 6 months. after 4 years of treatment with miltefosine, she developed skin lesions on the face, which spread to upper part of the body. as her daily activities were not hampered, she was reluctant to visit doctor for more than 2 years. a clinical diagnosis of pkdl was made and the patient was put on miltefosine, 50 mg twice daily with complete subsidence of the lesions in 3 weeks. case 1 : hypopigmented macules on face case 1 : hypopigmented macules on face epidermal atrophy, follicular plugging with keratin and presence of dense lymphocytic infiltrate in the dermis (h and e, 10) a 27-year - old patient, farmer by occupation, presented with depigmented macules widely spread all over the body since last 4 years, which were more marked on the front and back of trunk. the lesions on the face were coalescent and they appeared more hypopigmented than depigmented [figures 4 and 5 ]. on touch, sensation was normal and no peripheral nerve was thickened or tender. on systemic examination, all findings were normal except mild raised eosinophils, 85 (normal range, 1 - 6%) and high esr (96 mm in the first hour). biopsy tissue sample from hypopigmented lesion from upper back showed no acid fast bacilli, but epidermal atrophy, a minimal mixed chronic infiltrate consisting of plasma cells, lymphocytes, and histiocytes in periappendageal and perivascular areas of upper dermis, suggestive of pkdl. the history of the patient revealed that he was treated for vl with miltefosine, 100 mg / day for 28 days about 7 years back from a tertiary hospital. then, he had fever and enlargement of liver and spleen (symptoms for vl) and after treatment he was cured. he had appearance of hypopigmented lesions since last 3 years which gradually deepened with time. he was started with miltefosine, 100 mg / day with complete disappearance of the lesions in 5 weeks. both vl and pkdl are caused by same organism, leishmania donovani. the exact mechanism and etiopathogenesis of pkdl more than 90% of the world 's vl cases are found in india, bangladesh, brazil, nepal, and sudan. vl is endemic in the eastern states such as bihar, eastern uttar pradesh, jharkhand, and west bengal. the differential diagnoses of this symmetric hypopigmentation of body were pityriasis alba (in case 1), pityriasis versicolor, pityriasis lichenoides chronica, progressive macular hypomelanosis of trunk, and hypopigmented mycosis fungoides. this is in compliance with other studies where no ld bodies could be demonstrated in macular lesions. rk39 is a rapid dipstick test, based on the rk39 protein, available for rapid diagnosis of vl and pkdl. previous cohort studies of past vl showed that rk39 test stayed positive upto 24 months in sudan and 4 years in india. in a recent study from india, 68.2% were positive after 1 - 5 years, 57.7% after 5 - 10 years, and 42.5% after 10 - 15 years. in our cases, characteristic hypopigmented macules, history of post - kala - azar, positivity for rk39 strip test, suggestive histopathology and disappearance of lesions after starting of miltefosine helped us to diagnose the cases as pkdl. polymerase chain reaction is the best test as it can identify the infection earliest and in any form. when the initial infection was rarely subclinical, pkdl occurred without even a preceding history of kala - azar. parenteral amphotericin b emerged as very effective treatment for vl and incidences of pkdl decreased. it has been found to be equally effective in pkdl but should be used for a longer duration, perhaps for 3 months. as pkdl cases are reservoirs of vl, they are of utmost epidemiological significance. demonstration of ld bodies in slit skin smear or biopsy skin tissue is considered to be the gold standard for diagnosis of pkdl. but these methods are invasive, less sensitive, and difficult to perform at the periphery level. in hypopigmented lesions of pkdl, ld bodies so, it is very important to diagnose all cases of pkdl clinically and start treatment. the initial case reports of pkdl, after treatment of vl with miltefosine, were made from india in 2009. occurrences of more and more pkdl after treatment of vl with miltefosine may require reassessment of overall feasibility of the elimination program. two cases of pkdl are reported here that developed after successful treatment of visceral leishmaniasis with miltefosine.	post - kala - azar dermal leishmaniasis (pkdl) is an unusual dermatosis occurring following an attack of visceral leishmaniasis (vl). there are only few reports of pkdl after successful treatment with miltefosine. we report two cases of pkdl that developed after successful treatment of vl with miltefosine.
	electrostatically tunable negative differential resistance (ndr) is demonstrated in monolithic metal semiconductor metal (al ge al) nanowire (nw) heterostructures integrated in back - gated field - effect transistors (fets). unambiguous signatures of ndr even at room temperature are attributed to intervalley electron transfer. at yet higher electric fields, impact ionization leads to an exponential increase of the current in the 111 oriented ge nw segments. modulation of the transfer rates, manifested as a large tunability of the peak - to - valley ratio (pvr) and the onset of impact ionization is achieved by the combined influences of electrostatic gating, geometric confinement, and heterojunction shape on hot electron transfer and by electron electron scattering rates that can be altered by varying the charge carrier concentration in the nw fets.
celiac disease (cd) is an immune - mediated systemic disorder elicited by gluten and related prolamins in genetically susceptible individuals and characterised by the presence of a variable combination of gluten - dependent clinical manifestations, cd - specific antibodies, hla - dq2 or hla - dq8 haplotypes, and enteropathy. recently, the prevalence of celiac disease across the european countries was shown to be 1.5% based on people who had positive biopsy and ttg results. in the united states, the overall prevalence of celiac disease in children up to 5 years of age is 1 in 104. this disease is quite prevalent in india also with rates of 1 in 96 in north india. lifelong adherence to a gluten - free diet (gfd) is the cornerstone treatment of celiac disease. a gluten - free diet entails strict avoidance of all products containing the proteins from wheat, barley, and rye. it is strongly recommended that gluten elimination from diet must be strict and lifelong not only to control symptoms but also to improve quality of life and decrease the risk of complications. although a well - planned gluten - free diet may provide adequate nutrition, it may be restrictive. strict adherence to gluten - free diet may be more challenging in children and adolescents than in adults. compliance to gfd varies from 45% to 81% in children as reported by the north american society of pediatric gastroenterology, hepatology, and nutrition. noncompliance is a major problem and the greatest challenge which the physicians face is in predicting the compliance to the gluten - free diet in children. noncompliance may occur due to factors like temptation and not liking the taste of gluten - free food and alternative food grains. in adolescents, peer pressure, unclear labelling on ready - to - eat food, and nonavailability of gluten - free food at party, marriages, and so forth an increasingly hectic lifestyle of teenagers has contributed to a greater reliance on packaged foods which often contain gluten, and thus making it inconvenient for them to adhere to restrictive diet. since parents are usually responsible for food preparation for children, low level of knowledge about the diet in the parents, nonavailability of gluten - free foods, and unclear labelling lead to noncompliance in children. many children experience psychological reactions to being placed on a restrictive diet (e.g., feeling deprived, depressed, angry, and anxious) which have been found to further decrease compliance. this study evaluates the impact of celiac disease and the gluten - free diet on the lifestyle and well - being of children with celiac disease and their families, with the aim to identify factors affecting compliance to gfd and predictors of compliance to gfd in children with celiac disease. this study is significant and will contribute to the current body of research by providing health care practitioners with information as to what predicts the compliance to gfd, which may be used to better understand education techniques for dietary instruction so that the children living with celiac disease have less of morbidity and achieve their normal growth potential. participants will contribute to the understanding of celiac disease and the challenges individuals face with the gluten - free diet. this study was conducted in the tertiary care hospital of the department of pediatrics, sms medical college, jaipur, rajasthan, india. during a period of 1 year starting from october 1, 2011, till september 30, 2012, 150 consecutive celiac disease children visiting the gastroenterology super - specialty clinic were studied. 134 consecutive children out of these meeting the following inclusion criteria were enrolled in the study : patients aged between 2 years and 15 years, children diagnosed with celiac disease as per revised espghan criteria for diagnosis of celiac disease 1990, those on gluten - free diet for more than 6 months. patients aged between 2 years and 15 years, children diagnosed with celiac disease as per revised espghan criteria for diagnosis of celiac disease 1990, those on gluten - free diet for more than 6 months. exclusion criteria applied were (1) any child less than 2 years and more than 15 years of age, (2) those who did not have a documented positive serology and/or biopsy suggestive of celiac disease as per revised espgan criteria 1990, (3) those on gluten - free diet for less than 6 months, and (4) those children whose parents did not consent to be included in the study. all children enrolled in the study after signing of the written informed consent form were evaluated for dietary compliance based on a 5-day dietary recall form. a child who had taken even one food article containing gluten in last 5 days was considered noncompliant and those who had strictly taken no gluten in their diet in that period were considered compliant. diet recall was done by parents for children in preschool age up to 5 years since parents were the only one giving the eatables to these children. children, above 5 years of age, going to school and interacting with peers, were actively involved in the dietary recall along with the parents. after the dietary assessment, children and their parents were subjected to an interview by the investigator who was blinded for the compliance status of these children. interview consisted of a self - administered questionnaire which had questions related to demographic profile, history of illness, parents ' knowledge and understanding of disease, barriers to compliance and effect of celiac disease on feelings of children, eating out, and travel. the questionnaire was developed from 4 previously published studies [11, 1416 ] and it was pretested and validated in 20 children in the past in the paediatric gastroenterology unit of kalawati saran children 's hospital, delhi, india. to assess psychosocial problem, standard paediatric symptom checklist containing 35 items the pediatric symptom checklist (psc) is used to screen psychosocial problems in a child, by using a parent completed screening questionnaire as a part of routine primary care visit. it is a screening tool that reflects the parent 's impression of his or her child 's psychosocial functioning [17, 18 ]. each psc item is rated asnever0,sometimes1,often2.item scores are summed and the total score is recorded into a dichotomous variable. psc score of 28 or higher for children aged 6 years and above, and 24 or higher in children between 2 and 6 yr is taken as emotional and psychosocial impairment. if 4 or more items are unanswered, questionnaire was treated as invalid. during the final data analysis, based on the assessment of compliance to gluten free diet this collected data was analysed in microsoft excel 07 and primer 5.00 and classified as per aims and objectives. inference was drawn using chi - square test and mann - whitney u test to compare the compliant and noncompliant groups. p value of 9 years of age. a binary multivariable logistic regression analysis with backward elimination was conducted to narrow down predictors of dietary compliance using all the factors assessed previously (whether found to be significantly associated or not), namely, age, sex, age at presentation, presenting symptom, that is, gi or non - gi, father 's education, mother 's education, family type, per capita income, number of siblings, parental knowledge, parental attitude, child 's attitude, child 's behaviour and psc score as predictors, and using compliance as dependent variable. the wald criterion demonstrated that only age at presentation (with p value = 0.004), family type (with p value = 0.0097), child 's attitude (with p value = 0.0003), and child 's behaviour (with p value = 0.0195) made a significant contribution to prediction. other factors associated with compliance to gfd were not significant predictors (table 6). seeing the odds ratio of the significant predictors, it was demonstrated that the best predictor of compliance is child 's attitude which, if favourable, makes the child 7 times more likely to be compliant. children with a positive outlook and feelings about their condition are 4 times more likely to be compliant ; similarly children of nuclear families are 4 times more likely to be compliant. odds ratio for age is 0.75, that is, with each unit increase in age (one year) chance of compliance becomes 25% less likely. this was in harmony with the rates of compliance as seen in various other studies done in india and outside. strict dietary compliance was shown to vary from 45% to 81% in children by hill., 95% in a canadian study on children 25 years to 41.37% in children above 9 years of age. these results are in accordance with ljungman and myrdal, who also reported compliance rates of 93% at 12 years of age decreasing to 76% in 1517 yr age group. various reasons which may be responsible for increasing noncompliance with increasing age include increased social interaction, increasing peer group pressure, increased outdoor activities, and need for experimentation. compliance was not significantly associated with the sex of the child in our study, just as in a study by errichiello.. this low global level of adherence to a gfd in children with celiac disease is troubling given the known morbidity and mortality associated with long - term untreated celiac disease and the lack of any other effective treatment. effective counselling about the diet is the single most important factor to ensure the required restriction in diet among these patients. our study enumerates factors which are significantly associated with compliance to gfd and understanding the predictors of compliance will help the clinician to target the problem areas and ensure maximum compliance among children. mother 's education is found as a significant factor related with the compliance in our study. it may be because mother is responsible for buying and preparation of food items. with her knowledge, anson. (1990) also found that maternal education is important factor associated with compliance. a statistically significant association of compliance was seen with nuclear family which is coherent with results of chauhan jc. in 2010. joint family may lead to noncompliance as with many people around the child having all varieties of food, tempts the child and leads him to consume gluten containing food. our study also highlights that higher degree of compliance is noted when parents have better knowledge about celiac disease and the gluten containing items, understand importance of gluten - free diet for their child 's overall growth and development, and are able to distinguish gluten containing from gluten - free food so that they handle the menu better. anson. also showed similar correlation of parental knowledge and dietary compliance. there is more evidence that compliance with the gluten - free diet is improved in those who are more knowledgeable about celiac disease and the diet. this study also shows that a parents ' positive attitude towards the child 's condition is associated with higher compliance. dietary noncompliance is more common when preparing gluten - free food items is considered both a burden on self and financial burden by parents. so counselling aiming to increase the knowledge about the disease and awareness of parents regarding cheap and acceptable alternatives to wheat and easy to cook gluten - free food recipes will help ensure compliance to gfd in children. child 's positive attitude towards his condition has also come out as a significant factor associated with higher degree of compliance. difficulty in maintaining dietary compliance at school and at family party and marriages was higher in dietary noncompliant group compared to the dietary compliant. gluten containing food as main dietary item served at above places was a problem for children in both dietary compliant and noncompliant groups. these results highlight that the need of widespread availability of gluten - free food and more clear and apparent labelling of gluten - free items are of as much importance as proper counselling and reinforcement during subsequent visits. the study results show that dietary restriction has effect on child 's feelings and social activities like eating out and travelling. rashid. in their study in canada also reported that more than 50% of children felt left out of activities at school and had problems related to compliance. anson. have reported similar barriers to compliance in relation to child 's feelings. these results indicate that negative feelings in some children owing to their dietary restriction are associated with noncompliance in these children. this is the area where the counselling of the treating doctor is utmost important so that a child can be made to better accept his situation and the efforts of the parents of the child can be better channelized to achieve this. out of all these various factors studied for association to adherence to gfd, our study narrowed down four factors which can serve most closely as the predictors of compliance. child 's attitude (best predictor with or = 7, p value = 0.0003), child 's behaviour (with or = 4, p value = 0.0195), family type (with or = 4, p value = 0.0097), and age at presentation (with or = 0.75, p value = 0.0004) made a significant contribution to prediction of compliance. one child in both compliant and noncompliant group in our study has screened positive for psychosocial problems and so such problems can be encountered in both compliant and noncompliant children. have shown that anxiety is present in celiac disease subjects as a reactive form which decreases with gluten - free diet ; however depressive symptoms still persist in patients on gfd. psc scores are seen to increase as the age increases, more so in noncompliant children. maximum score was seen in children > 9 years of age which is fairly understandable as this is the age when children interact with people other than their parents and develop a defiant attitude, give in to peer pressure, and develop a need for experimentation. the need for psychological support to all patients when they are put on gluten - free diet and continued psychological support through a professional especially to those above 9 years of age is thus emphasised here. our study adds to the available data regarding the significant factors that play a role in compliance to gfd. however, a few limitations of this study are to be mentioned. first, compliance was not confirmed with concurrent histological and/or serological evaluation. however it is not clear if biopsy provides a better assessment of long - term compliance than nutritional evaluation. in fact, prior studies showed only a modest correlation of histology with clinical presentation or assessed dietary adherence [22, 23 ]. while histological and serological relapse has understandably been used to assess compliance, in some studies a more practical and noninvasive approach has been used where compliance was assessed subjectively on the basis of dietary recall by parents and children in last few days. in a study by mustalahti in 2002, the patients ' self - concept of long - term adherence to the gluten - free diet was evaluated by using a visual analogue scale. patients indicated their level of adherence by marking a scale anchored at 0% and 100% compliant. at the end of the study, 2008 in their study defined compliance with gluten - free diet as strict, semistrict, and not on gluten - free diet based on 7-day recall of patient 's intake of amount of gluten assessed subjectively by patient himself or guardian. spatola in his study in 2014 has also used a 4-day dietary record to assess compliance wherein those consuming no gluten - containing item in these 4 days were considered compliant. therefore we have used a noninvasive method of dietary recall of 5 days to assess compliance. due to lack of reliable tools it is challenging to uncover minor dietary lapses or inadvertent gluten intake. to ensure compliance status of our study group, all subjects had been interviewed repeatedly in their follow - up visits in our gastroenterology super - speciality clinic by trained dietician and senior consultants, to uncover hidden transgressions and establish compliance status based on clinical interview besides the 5-day food record. however, the results of this study point to a number of areas, both obvious and obscure, that may be productive targets for interventions aimed at improving dietary adherence in individuals and our future studies will aim to establish the validity of these associations. this is because most doctors currently suspect and diagnose the disease in children presenting with nongastrointestinal symptoms as well as atypical presenters and in at risk asymptomatic children by active case - finding strategy (serologic testing for celiac disease in patients with symptoms or conditions closely associated with celiac disease). for many years, physicians have focused on the diagnosis and molecular and cellular markers of celiac disease, with scarce attention being given to the care and well - being of the patient. patients with celiac disease should be monitored regularly for residual or new symptoms, adherence to gluten - free diet, and assessment for complications. normal growth and development are achievable on a gluten - free diet and should be goals for monitoring children with celiac disease. as per acg, monitoring of adherence to gluten - free diet should be based on a combination of history and serology. hence for monitoring of compliance to the diet by history, it may be stated that interventions specially aiming at improving the child 's understanding and acceptability of celiac disease to promote a favourable attitude and positive feelings may increase the compliance to gluten - free diet. it may be stated that easy availability of palatable, cheap, and socially acceptable gluten - free food and better labelling of food may increase compliance to gluten - free diet. it is evident from our results that if the child lives in a joint family, he needs more of follow - up visits and more efforts from physician 's side to ensure compliance as he is more likely to defy the diet. also since age at presentation predicts compliance, the responsibility lies on the medical fraternity to identify the typically and atypically presenting children and put the child on gluten - free diet at an age which is less affected by the peers and such that the child inculcates the dietary habits required of him, for life. there is a lot of scope for establishment of celiac support groups in india wherein the doctors, ngos, and volunteering individuals can work together to provide the necessary psychological support to the children, where parents are informed of the varied recipe options and upcoming treatments, and a routine assessment can be done from time to time for any psychosocial impairment to all the children, especially those above 9 years of age.	aim. to identify the predictors of compliance to gluten free diet in children with celiac disease. methods. 134 children in the study group were assessed for dietary compliance followed by a questionnaire based interview. psychosocial parameters were assessed by standard pediatric symptom checklist (psc). dietary compliant and noncompliant groups were compared and assessed for factors affecting the dietary compliance. predictability of all of these factors was assessed using binary logistic regression analysis with backward elimination to find out the best predictors of compliance. results. in the study group, 88 (65.67%) were found to be strictly compliant. factors that were found to be significantly associated with compliance were age at presentation, nuclear families, mother 's education, and parents having better knowledge of celiac disease. parents ' and child 's attitude towards his having to follow a restrictive diet and child 's feelings were also shown to be significantly associated with compliance. binary logistic regression analysis with backward elimination demonstrated that age at presentation, family type, child 's attitude, and child 's behaviour made a significant contribution to prediction. conclusions. these results will contribute to the current body of research by providing health care practitioners with a framework for better dietary instruction to ensure maximum adherence to gfd.
the mucosal immune system has been recognized as the first defense of the host against pathogens entering the gastrointestinal or the upper respiratory tracts. mucosal immunization has been well documented and is a highly effective way to stimulate local and systemic immune responses. local - specific iga response, systemic specific igg response, and cell - mediated immunity have been induced by mucosal immunization. however, a close relationship between the adjuvants used and the immune response elicited regarding different antigens has been demonstrated. thus, the development of new vaccines has increased the need for new and more powerful adjuvants. finlay institute has developed a group of adjuvants, of which the afco1 cochleate has resulted particularly effective by both, mucosal and parenteral routes. the intranasal administration of adjuvants has been poorly explored from the toxicological point of view. the fact that only one layer of cells separates the lumen of the nasal cavity from a rich vascular net in the lamina propria raises safety concerns. that is why the objective of the present paper was to carry out single and repeated doses safety studies of afco1 by intranasal administration in sprague dawley rats. the active ingredient of afco1 is a cochleate structure derived from the proteoliposome of neisseria meningitidis serogroup b with a protein a content of 1 mg / ml. every 100 l also contains sodium chloride (292 g), tris (360 g), calcium chloride (73.5 g), thiomersal (10 g), and water for injection as solvent. male and female, 5- to 6 week old, 150 - 200 g body weight, sprague dawley rats supplied by the national center for the production of laboratory animals (cenpalab, cuba) were used. five rats were housed per each t4 (floor area : 1800 cm) polycarbonate cage (tecniplast, italy). sugar cane bagasse sterilized in autoclave (15 minutes, 121c) and changed twice a week was used as bedding. pelleted food produced by cenpalab and fresh drinking water was administered for ad libitum consumption. room temperature (22 - 25c), relative humidity (60 - 65%) and light cycle (10 h light-14 h dark) were controlled and recorded twice a day. animals were killed by intraperitoneal overdose of pentobarbital (100 mg / kg).the protocol of the study was approved by the ethics committee for the care and use of laboratory animals at finlay institute and is in accordance with international standards on the topic. ten rats per sex were inoculated by nasal route with either afco1, the placebo formulation or were not treated at all and functioned as controls. a volume of 50 l (same dose as that proposed for human use) was instilled in each nostril. animals were clinically examined on a daily basis paying special attention to the appearance of the following signs : nose irritation, nasal secretion, sneezing, dyspnoea, head shaking, salivation, tearing, and cutaneous reactions. animals were weighed at the end of quarantine as well as 3, 7, and 14 days after the inoculation. water and food intakes were measured every other day in order to estimate the average consumption. the rats were killed 2 weeks after the inoculation and anatomopathological studies were conducted with special emphasis on the nasal region and the encephalon. based on the degree of edema and congestion, the state of the epithelium and the inflammatory cells infiltrating the nasal mucosa, an irritability index was calculated as described elsewhere. the schedule proposed for human use conceives only there doses, but a further dose was administered to the rats in order to maximize their exposition to the test substance. animal housing and maintenance as well as the experimental groups were as described for the single - dose toxicity study. similarly, clinical symptoms, food and water intakes, and body weight were studied. additionally, hematology, blood biochemistry, and anatomopathological studies were conducted on groups of rats killed 3, 14, and 28 days after the last inoculation. hematology included quantification of hemoglobin, hematocrit, leukocytes, differential count of leukocytes, count of erythrocytes, and platelets. blood serum chemistry measured the levels of glucose, urea, creatinine, alkaline phosphatase, total proteins, triglycerides, cholesterol, direct, and total bilirubin (bil - d bil - t), creatine phosphokinase, transaminases, and urates. all the hemochemical determinations were carried out using commercial kits (centis, havana, cuba) following the procedures recommended by the producer. the anatomopathological studies included the necropsy of all the rats and tissue sampling for histological studies. the relative weight of the heart, thymus, lungs, kidneys, liver, and spleen was calculated. the irritation index caused by the product on the nasal mucosa (2003) ; statistica data analysis software system - version 6, www.statsoft.com) was used. body weights were compared by repeated measures analysis of covariance, using values at the beginning of the assay as covariate. the normality and homogeneity of variance assumptions were tested by means of shapiro - wilk 's w test and levene 's test, respectively, in order to decide whether to conduct parametric (analysis of variance and least significant difference tests) or nonparametric procedures (kruskal - wallis and distribution - free multiple comparisons tests). the active ingredient of afco1 is a cochleate structure derived from the proteoliposome of neisseria meningitidis serogroup b with a protein a content of 1 mg / ml. every 100 l also contains sodium chloride (292 g), tris (360 g), calcium chloride (73.5 g), thiomersal (10 g), and water for injection as solvent. male and female, 5- to 6 week old, 150 - 200 g body weight, sprague dawley rats supplied by the national center for the production of laboratory animals (cenpalab, cuba) were used. five rats were housed per each t4 (floor area : 1800 cm) polycarbonate cage (tecniplast, italy). sugar cane bagasse sterilized in autoclave (15 minutes, 121c) and changed twice a week was used as bedding. pelleted food produced by cenpalab and fresh drinking water was administered for ad libitum consumption. room temperature (22 - 25c), relative humidity (60 - 65%) and light cycle (10 h light-14 h dark) were controlled and recorded twice a day. animals were killed by intraperitoneal overdose of pentobarbital (100 mg / kg).the protocol of the study was approved by the ethics committee for the care and use of laboratory animals at finlay institute and is in accordance with international standards on the topic. ten rats per sex were inoculated by nasal route with either afco1, the placebo formulation or were not treated at all and functioned as controls. a volume of 50 l (same dose as that proposed for human use) was instilled in each nostril. animals were clinically examined on a daily basis paying special attention to the appearance of the following signs : nose irritation, nasal secretion, sneezing, dyspnoea, head shaking, salivation, tearing, and cutaneous reactions. animals were weighed at the end of quarantine as well as 3, 7, and 14 days after the inoculation. water and food intakes were measured every other day in order to estimate the average consumption. the rats were killed 2 weeks after the inoculation and anatomopathological studies were conducted with special emphasis on the nasal region and the encephalon. based on the degree of edema and congestion, the state of the epithelium and the inflammatory cells infiltrating the nasal mucosa, an irritability index was calculated as described elsewhere. the schedule proposed for human use conceives only there doses, but a further dose was administered to the rats in order to maximize their exposition to the test substance. animal housing and maintenance as well as the experimental groups were as described for the single - dose toxicity study. similarly, clinical symptoms, food and water intakes, and body weight were studied. additionally, hematology, blood biochemistry, and anatomopathological studies were conducted on groups of rats killed 3, 14, and 28 days after the last inoculation. hematology included quantification of hemoglobin, hematocrit, leukocytes, differential count of leukocytes, count of erythrocytes, and platelets. blood serum chemistry measured the levels of glucose, urea, creatinine, alkaline phosphatase, total proteins, triglycerides, cholesterol, direct, and total bilirubin (bil - d bil - t), creatine phosphokinase, transaminases, and urates. all the hemochemical determinations were carried out using commercial kits (centis, havana, cuba) following the procedures recommended by the producer. the anatomopathological studies included the necropsy of all the rats and tissue sampling for histological studies. the relative weight of the heart, thymus, lungs, kidneys, liver, and spleen was calculated. the irritation index caused by the product on the nasal mucosa (2003) ; statistica data analysis software system - version 6, www.statsoft.com) was used. body weights were compared by repeated measures analysis of covariance, using values at the beginning of the assay as covariate. the normality and homogeneity of variance assumptions were tested by means of shapiro - wilk 's w test and levene 's test, respectively, in order to decide whether to conduct parametric (analysis of variance and least significant difference tests) or nonparametric procedures (kruskal - wallis and distribution - free multiple comparisons tests). in the single dose test as well as in the repeated dose toxicity study all the animals increased their body weight (p0.1) among the experimental groups. in general, male rats consumed more water and food than female ones (p0.1). the relative irritability index calculated on the basis of histological changes found in the nasal mucosa of rats treated with afco1 or the placebo formulation at single dose were all under 1 [table 2 ] ; therefore, the test product was classified as nonirritant. frequency of histological changes in the nasal region and regional lymph nodes in the single - dose toxicity test calculation of nasal irritability indices in rats treated with a single dose anatomopathological, hematological, and hemochemical analysis carried out 72 hours after the last inoculation intended to reveal early side effects while those at 14 days were planed to assess recovery of damages or long - term effects. hematology and blood chemistry analysis did not show statistical differences among experimental groups [table 3 ]. relative organ weights were also statistically similar (data not shown), with the exception of the right kidney weight in females that received afco1 and placebo that were higher (p=0.025) to that of control rats. however, no histological changes were found in the kidneys of such animals that could support an association with toxic effects. p values refer to anova comparisons among treatment groups petechial hemorrhages in cervical lymph nodes in rats regardless of the sex, treatment group, or sacrifice time were found at necropsy again. due to their unspecific presentation mononuclear cells and sporadic suppurated focuses were also found at mucosal and submucosal tissues of the nasal septum as well as dorsal and ventral nasal cornets. besides, nasal congestion, edema, and degeneration of the superficial epithelium were found. however, statistical analysis did not evidence differences (p>0.05) among the histological changes in the three experimental groups. the irritability index calculated after repeated dosing (data not shown) also classified the test substance as nonirritant. repeated dose toxicity test : frequency of lesions in female rats 3 and 14 days after the last dose congestion and small hemorrhages of parotid, submaxillary, retropharyngeal, and cervical lymph nodes were sporadically observed at necropsy. these changes were also found in the histopathological analysis and were found in all groups [table 1 ], thus they were associated to the sacrifice method. in a general way, the histopathological changes were mild and similarly frequent in all the groups (p>0.1). the relative irritability index calculated on the basis of histological changes found in the nasal mucosa of rats treated with afco1 or the placebo formulation at single dose were all under 1 [table 2 ] ; therefore, the test product was classified as nonirritant. frequency of histological changes in the nasal region and regional lymph nodes in the single - dose toxicity test calculation of nasal irritability indices in rats treated with a single dose anatomopathological, hematological, and hemochemical analysis carried out 72 hours after the last inoculation intended to reveal early side effects while those at 14 days were planed to assess recovery of damages or long - term effects. hematology and blood chemistry analysis did not show statistical differences among experimental groups [table 3 ]. relative organ weights were also statistically similar (data not shown), with the exception of the right kidney weight in females that received afco1 and placebo that were higher (p=0.025) to that of control rats. however, no histological changes were found in the kidneys of such animals that could support an association with toxic effects. p values refer to anova comparisons among treatment groups petechial hemorrhages in cervical lymph nodes in rats regardless of the sex, treatment group, or sacrifice time were found at necropsy again. due to their unspecific presentation mononuclear cells and sporadic suppurated focuses were also found at mucosal and submucosal tissues of the nasal septum as well as dorsal and ventral nasal cornets. besides, nasal congestion, edema, and degeneration of the superficial epithelium were found. however, statistical analysis did not evidence differences (p>0.05) among the histological changes in the three experimental groups. the irritability index calculated after repeated dosing (data not shown) also classified the test substance as nonirritant. repeated dose toxicity test : frequency of lesions in female rats 3 and 14 days after the last dose a relevant animal model for the toxicological assessment of a vaccine or adjuvant must mount an immune response as expected to happen in the target species. thus, intrinsic toxicity due to the constituents, contaminants, the interaction among them and that related to the immune response elicited by the test product could be evaluated. sprague dawley is an outbred rat strain preferred for preclinical safety studies due to their heterogeneous response ; furthermore, a pilot study demonstrated that they respond immunologically to afco1 after nasal instillation. therefore, that strain was considered a relevant biomodel for the safety assessment of afco1. in order to increase rat exposure to afco1 and to test the potential risk of accidental overdosing, this design is in correspondence with current trends in the preclinical evaluation of preventive vaccines that suggest the administration of a further dose to that proposed for the clinical use. the growth curve, water and food intakes, blood chemistry values, and the relative organ weights of the rats in both tests conducted agreed with those observed for sprague dawley rats during the preclinical assessment of other candidate vaccines in our facilities[1215 ] and reference values reported elsewhere. histological changes were either part of physiologic reactions or incidental, but a connection with toxicity could not be established. focally aggregated or diffusely distributed lymphoid cells found at the lamina propria are part of the functional anatomy of mucosal tissues and are mainly represented by t cd4 + and b lymphocytes. the apoptotic lymphocytes and secondary follicles in the cortical and paracortical area of lymph nodes have also been observed by other authors and are associated to the response of secondary lymphoid tissues to the antigenic stimulus. finally, focal interstitial pneumonia is often observed as a consequence of viral infections in rats mainly found in animals like these which are maintained in a conventional sanitary environment.[2022 ] however, it is interesting that rats treated with afco1 developed interstitial pneumonia in a lower proportion (p<0.05) compared to placebo and control rats. detectable titers of specific igg have been found in saliva and blood serum 7 and 21 days after afco1 nasal instillation in rats, respectively. however, it has not been found in cerebro - spinal fluid, suggesting that afco1 does not reach the encephalic cavity, but instead is limited to the nasal region. these results support the idea that afco1 is capable of eliciting and modulating immune response after nasal instillation without significantly affecting the histology at the inoculation site as has been previously shown by pharmacology and local toxicity studies. nasal instillation of afco1 in sprague dawley rats at a high dose and frequency compared to the schedule proposed for human use was neither local nor systemically toxic. taking evidence presented here and previous pharmacology and toxicity tests conducted into account, afco1, the cochleate derived from neisseria meningitidis proteoliposome is considered potentially safe for human use.	background : the afco1 cochleate is a potential novel adjuvant derived from neisseria meningitidis b proteoliposome.aim:the aim was to assessing the safety of afco1 by single and repeated doses in sprague dawley rats.materials and methods : rats were grouped for treatment with afco1, placebo formulation or control. the first study was a single intranasal dose of 100 l and monitoring body weight, water, and food intakes as well as clinical symptoms. fourteen days later the rats were killed and anatomopathological studies were conducted. in a second study, four similar doses of the test substance were instilled every 5 days. clinical observations were carried out as for the single dose study and a number of rats from each group were killed 3 and 14 days after the last dose in order to conduct hematological, hemochemical, and anatomopathological studies.results:no variable showed differences of toxicological relevance ; the histological changes found were mild and similarly frequently in the three groups. according to the irritability index calculated form histology of the nasal region, afco1 was also classified as nonirritating.conclusion:afco1 is potentially safe for human use by nasal route as evidenced by the absence of local and systemic signs of toxicity in sprague dawley rats.
the two inflammatory bowel diseases (ibds) ulcerative colitis (uc) and crohn 's disease (cd) affect more than 3.6 million people in the western world, resulting in a marked decrease in the patients ' quality of life [1, 2 ]. the aetiology is poorly understood, but it has become clear that genetic, microbial, and environmental factors all play a role. a massive effort is taking place to develop new and better therapeutics, and the development of tumor necrosis factor- (tnf-) antagonists has ameliorated the disease in a large proportion of especially cd patients. however, about one third of the cd patients do not respond to anti - tnf- treatment and among the primary responders, about one third loose response or become intolerant to the treatment, thus leaving many ibd patients with inadequate therapeutic options. new ibd drugs and drug candidates include anti - interleukin (il)-12/-23 (e.g., ustekinumab, briakinumab), cytotoxic t - lymphocyte antigen 4 immunoglobulin (ctla4-ig, abatacept), anti - il-6r (tocilizumab), anti - interferon ((ifn-), fontolizumab), anti-47 (vedolizumab), anti-4 integrin (natalizumab), anti - il-2-r (daclizumab, basiliximab), antigranulocyte macrophage colony - stimulating factor (anti - gm - csf, sagramostim), anti - intercellular adhesion molecule 1 (anti - icam-1, alicaforsen), ril-18 binding protein (tadekinig-), ip-10/cxcl10 (mdx-1100), anti - cd3 (visilizumab), and anti - cd40l (tnx 100) [6, 7 ]. these compounds aim at targeting specific immunological mechanisms like cellular adhesion (anti-47, anti - icam-1) and costimulation (ctla4-ig, anti - cd40l), key cytokines (anti - il-12/-23, anti - il-6r, anti - ifn-) or cells (anti - il-2r/cd25, anti - cd3), or have specific immuno - stimulatory (gm - csf) or -inhibitory (ril-10) effects. animal models are essential for dissecting the role of the pathological mechanisms in ibd as well as for assessing the therapeutic effect of intervening with these pathways. to what extent the data from animal models can be translated into the clinic differs among the various models depending, for example, on the model 's etiopathogenesis and main drivers of disease. there is no single model which adequately mimics either uc or cd, and to be able to translate findings from a model to the human disease, it is important to know the model 's central pathological mechanisms and immunological pathways. adoptive transfer of a subset of cd4 t cells to syngeneic scid or rag - knock - out mice, results in the development of a chronic, progressive colitis and wasting disease as first described by morrissey. the colitis symptoms share several features with both cd and uc (e.g., chronic, progressive disease with diarrhoea and weight loss, heavily inflamed colon occasionally transmural damage, loss of mucus from goblet cells, th1/th17 dominated cytokine profile as found in cd (ifn-, tnf-, and il-23). the model has been extensively used for studying the immunologic background for the disease as well as testing new ibd drug candidates [1114 ]. we have previously described in detail the development of colitis following adoptive transfer of cd4cd25 t cells. briefly, in our hands, the adoptively transferred cells expand rapidly and the mice begin to develop colitis within the first two weeks. at week three, the disease is normally fully developed with weight loss, loose stools, increased white blood cell (wbc) count, and a both thickened and shortened colon. the disease progresses rapidly and by week 5 most mice have developed severe colitis requiring a termination of the study. this synchronized and predictable development of colitis makes it possible to conduct both prevention and intervention studies. the aim of this study was to analyze the model with respect to its usefulness in efficacy studies of new ibd drug candidates by evaluating the effect of known and potential ibd therapeutics in the model. we have decided to study a number of compounds, which each has a specific inhibitory effect on a central proinflammatory pathway. in addition, we have included some established ibd therapies, suggested to ameliorate ibd by a broad spectrum of mechanisms. human ctla4-ig (abatacept, orencia, bristol - myers squibb), human tumor necrosis factor receptor fc (tnfr - fc) (etanercept, enbrel, wyeth), enrofloxacin (baytril, bayer, equivalent to ciprofloxacin), metronidazole (flagyl, sanofi - aventis), cyclosporine (sandimmun, novartis). all surrogate antibodies, that is, anti - tnf- (clone xt3.11, rat igg1), anti - il-12p40 (clone c17.8, rat igg2a), anti - il-6 (mp5 - 20f3, rat igg1), anti-47 (clone datk32, rat igg2a), and isotype controls (cig) (rat igg2a clone 2a, rat igg1 clone hrpn and human igg1-fc) were from bioxcell, west lebanon, new hampshire, usa. dynabeads, mouse cd4 (l3t4), and detachabead mouse were from dynal, oslo, norway, and cd25 microbead kit from miltenyi biotech, bergisch gladbach, germany. the antibodies used for facs analysis were percp - conjugated anti - cd4 (l3t4) from bd pharmingen and fitc - conjugated anti - cd45.2 (104) from ebiosciences, ca, usa. c.b - igh-1b / icrtac - prkdcscid (c.b-17 scid) and balb / canntac female mice (810 weeks) bred under spf conditions (m&b taconic, denmark) were housed at novo nordisk a / s. for induction of colitis, cd4cd25 t cells were adoptively transferred from mhc - compatible balb / c mice to c.b-17 scid recipients as described previously in. in brief, balb / c splenocytes were positively selected for cd4 t cells using dynabeads and detachabead and depleted of cd4cd25 cells using the cd25 microbead kit. the purity of the cells was always analyzed by flow cytometry before reconstitution (> 98% of the cd4 cells were cd25). peripheral blood from all mice was subjected to flow cytometric analysis 2 or 3 weeks after transfer, and only mice with cd4 t cells (indicating successful transplantation of cells) were included in the study. the drugs tested, as well as the doses and dosing regimens are described in table 1. for prevention studies, the mice were treated from the day they were adoptively transferred with cd4cd25 t cells and until sacrifice when the disease was fully developed (three or four weeks after transfer, figure 1). for the intervention studies, the treatment was initiated at week three after adoptive transfer, when the cd4 t cells had expanded and caused colitis in the recipients. the control groups for the biologics (except for tnfr - fc) were treated with the relevant control immunoglobulin (cig), that is, rat igg1 for anti - tnf- and anti - il-6, rat igg2a for anti - il-12p40 and anti-47, and human igg1-fc for ctla4-ig. the vehicle groups for cyclosporine and antibiotics received sterile h2o (table 1). in the study with antibiotics, we also included a group, which was not reconstituted but received treatment, since we suspected that disturbance of the gut microflora in itself could have a marked effect on the measured disease parameters. we used the same doses and dosing frequencies for the various compounds in the prevention and intervention studies (table 1). the selection of doses and dosing frequencies were based on either literature describing efficacious treatment in various colitis models or based on our experience with efficacious treatment in the collagen induced arthritis model [1623 ]. body weight was determined three times weekly, and mice were sacrificed if they lost more than 20% of their initial body weight. fecal consistency was evaluated before the start of treatment and at the termination of the study using a semiquantitative score (normal stool = 0 ; slightly soft = 1 ; soft but formed = 2 ; not formed = 3 ; liquid stools or no feces in colon at sacrifice = 4) as previously described. the number of wbc per liter was analyzed in samples (20 l) of edta - stabilized peripheral whole blood, using a medonic ca 620 (boule nordic, denmark) blood analysis apparatus according to the manufacturer 's instructions. prior to sacrifice, the mice were anesthetized and blood from the periorbital venous plexus was collected in edta - containing tubes. after sacrifice, the colon was excised, rinsed gently with saline, and the weight and length recorded. the colonic weight - to - length ratio (w : l) was previously shown to correlate strongly with the clinical and histological severity of disease. the colon was opened longitudinally, mounted on a plastic plate, and fixed overnight in 4% paraformaldehyde. longitudinal segments of tissue representing essentially the entire length of the transverse and distal colon (where the inflammation is mainly located) were embedded in paraffin. a section (7 m) of the transverse and the distal colon from each animal was stained with hematoxylin and eosin / periodic acid schiff (h&e / pas) and analyzed by light microscopy. a total histological score was calculated for each animal as described previously and shown in figure 2. briefly, the samples were assigned a score (03 or 04) according to the severity (none, mild, moderate, severe) and extent (none, mucosal, submucosal, transmural) of inflammation, degree of crypt damage (basal 1/3 damaged, basal 2/3 damaged, crypts lost epithelium intact, crypts lost epithelium lost), and percentage of tissue affected (0, 125%, 2650%, 5175%, 76100%). the histological analyses were performed in a blinded fashion with respect to the treatment groups. fecal consistency score and histological score are shown as median (range) and analyzed using the mann - whitney u - test. wbc count, body weight at postmortem, and colonic weight : length ratio are shown as mean standard error of the mean (sem) and analyzed by student 's t - test using welch 's correction for unequal variances. in the following, the disease modifying effect of each of the investigated compounds in the adoptive transfer colitis model is presented. first, experimental data with the biologicals (i.e., monoclonal antibodies (mab) and receptor fusion proteins (r - fc)) are presented, followed by data from a number of compounds currently used to treat cd or uc. although broad - spectrum antibiotics and metronidazole are mainly used for subgroups of ibd patients or for complications like pouchitis, we have included this treatment regimen, since the influence of the microflora in the pathogenesis of ibd is a central topic. due to the large data material, readers are referred to the supplementary material for a complete presentation of data (figure sf1 and tables st1st10 available at doi : 10.1155/2012/412178). in the 28 day prevention study, mice treated with the isotype control began to loose weight after two weeks, while the weight curve for the rat anti - mouse tnf- mab - treated mice was comparable to that of healthy controls. at the end of the study, the anti - tnf--treated group had lost significantly less weight than the control group (p < 0.001, figure 4, tables 2 and st1). two mice in the control group were sacrificed due to extensive weight loss before the end of the study. the fecal score was increased in both groups but was significantly lower in the anti - tnf- group (p < 0.001, tables 2 and st2). the wbc count in the anti - tnf- group was almost as low as in the unreconstituted controls, while it was significantly higher in the isotype control group (p = 0.001, table 2 and st3). similarly, the colonic w : l ratio (p < 0.001) and histological score (p < 0.0001) were significantly lower in the anti - tnf- group compared to the isotype controls (tables 2, sf1, st4 - 5). in contrast to the prevention studies, intervention therapy with anti - tnf- did not consistently ameliorate colitis in this model. although the anti - tnf--treated group lost less weight than the control group (p < 0.05), none of the other clinical parameters were significantly affected by the treatment (table 2 and st610). both for the fecal score, colonic w : l ratio and histological score (sf1), the group seemed equally divided into responders and non responders, that is, having high or low scores and values, respectively, rather than being equally distributed around the mean or median. we first tested the human tnfr - fc fusion protein etanercept at a dose of 5 mg / kg in a 21 days prevention study and found no significant effect of the compound on any of the parameters analyzed (data not shown). in the subsequent 28 days prevention study with a dose of 50 mg / kg, this group had significantly less weight loss than the vehicle control group (p < 0.01), while the fecal score was slightly but not significantly lower (figure 4, tables 2 and st1 - 2). one mouse in the vehicle control group was sacrificed before week four, due to extensive weight loss. there was no difference in the mean wbc count or colonic w : l ratio between the two groups. since we found no effects of the compound (except from decreased weight loss) and since the colonic w : l ratio is highly predictable for the histological score (as described in), no histological scoring was made. in the intervention study, tnfr - fc (5 mg / kg) did not affect any of the measured parameters (table 2 and st610). since the prevention study using 50 mg / kg did not have any effect either, this dose was not tested in intervention studies. in the 28-day prevention study, mice treated with the isotype control began to loose weight after two weeks, while the weight curve for the rat anti - mouse il-12p40 mab - treated mice was comparable to that of healthy controls. at the termination of the study, mice treated with anti - il-12p40 mab had lost significantly less weight than the isotype control group (p < 0.001) (table 2 and st1). similarly, anti - il-12p40 mab treatment resulted in significantly lower wbc count (p < 0.05), colonic w : l ratio and histological score (sf1) (p < 0.001 for both parameters), while it did not significantly improve fecal score (tables 2 and st2-s5). intervention with anti - il-12p40 mab from day 21 reversed the progressive weight loss. at the end of the study, this group had lost significantly less weight than the control group (p < 0.0001) (table 2 and st6). the fecal score tended to be lower in the anti - il-12p40 mab group, and the colonic w : l ratio and histological score were significantly reduced compared to the isotype control (p < 0.01 and p < 0.05, resp., tables 2, sf1, and st7 - 10). preventive treatment with a rat anti - mouse il-6 mab reduced weight loss (p < 0.01) and fecal score (p < 0.01) but failed to significantly reduce wbc counts compared to the isotype control group (table 2 and st13). the colonic w : l ratio of the anti - il-6 mab treated group was significantly lower than that of the isotype treated group and was comparable to the unreconstituted healthy control group (table 2 and st4). similarly, the histological score was significantly less in the anti - il-6 mab - treated group compared to the isotype control (p < 0.01, tables 2, sf1, and st5). intervention with anti - il-6 mab from day 21 did not significantly affect any of the measured parameters (tables 2, sf1, and s6 - 10). preventive treatment with human ctla4-ig effectively inhibited development of colitis. the weight curves for ctla4-ig - treated mice were comparable to the weight curves for the unreconstituted healthy control mice. the fecal score at the termination of the study (p < 0.01), the wbc count (p < 0.01), and colonic w : l ratio (p < 0.001) were significantly lower in the ctla4-ig groups compared to the isotype control group (table 2 and st14). remarkably, no signs of inflammation were found in any of the animals, suggesting a very potent effect of the compound (tables 2, sf1, and st5). a profound effect of ctla4-ig treatment on all the measured parameters was likewise identified in the intervention study. shortly after initiation of treatment, the weight loss was reversed and the animals gained weight comparable to the unreconstituted. likewise, the fecal score (p < 0.01), wbc count (p < 0.001), colonic w : l ratio (p < 0.001), and histological score (p < 0.001) were significantly lower than for the isotype control group at the end of the study (tables 2, sf1, and st6 - 10). preventive treatment with a rat anti - mouse 47 mab diminished weight loss (p < 0.001) and fecal score (p < 0.05, table 2). however, the number of wbc was not significantly changed (tables 2 and st2 - 3). inhibition of t - cell homing to the gut by anti-47 mab treatment reduced colonic disease as indicated by the significantly lower colonic w : l ratio and histological score compared to the isotype control group (p < 0.01 for both parameters, tables 2, sf1, and st4 - 5). intervention with anti-47 mab at day 21 did not significantly affect any of the measured parameters (figure 4, tables 2, sf1, and st610). mice treated with antibiotics (unreconstituted and reconstituted) did not develop weight loss in a preventive setting. the fecal score was slightly increased by the treatment itself but was significantly lower in the reconstituted mice treated with antibiotics compared to the reconstituted vehicle group (p < 0.001) as was the wbc count (p < 0.05), the colonic w : l ratio (p < 0.001), and the histological score (p < 0.001, tables 2, sf1, and st2 - 5). intervention with antibiotics immediately reversed weight loss (figure 4). at the termination of the study, the mice treated with antibiotics had lost significantly less weight than the vehicle control group (p < 0.01). likewise, fecal score (p < 0.0001), wbc count (p < 0.001), colonic w : l ratio (p < 0.0001), and histological score (p < 0.001) were significantly lower than for the vehicle control group (figure 4, tables 2, sf1, and s710). cyclosporine had no effect on the degree of weight loss, fecal score, colonic w : l ratio or histological score, while the wbc count at necropsy was actually significantly higher in the cyclosporine group compared to the vehicle group (table 2, sf1, and st1 - 5). since there were no effects of cyclosporine in the prevention study, the compound was not tested in intervention studies. collectively, we found that ctla4-ig, anti - il-12p40 mab, and antibiotics prevented onset of colitis and cured established disease, while anti - tnf- mab, anti - il-6 mab, and anti-47 mab prevented onset of colitis but did not reverse established disease. neither tnfr - fc nor cyclosporine had any preventive or therapeutic effect in the current setup. the search for improved treatment opportunities against ibd is heavily dependent upon good animal models, both for efficacy studies and for understanding the underlying cause of the disease. to have any predictive value, the models must share central drivers of disease with the human disease they are representing. intervention with some of the known main mechanisms in autoimmune disease (i.e., costimulation, t - cell homing, effect of cytokines, etc.) can teach us more about which pathways are central for the model. this allows one to select a model appropriate for the inflammatory pathway, the test compound is supposed to act on. the aim of our study was to estimate the preventive and therapeutic effect of a number of established or potential ibd drugs, using the scid adoptive transfer colitis model. by using multiple drugs inhibiting potential or known pathogenic drivers in ibd, we sought after an increased understanding of the central drivers, in this specific model. several of these compounds or their surrogate antibodies have previously been tested in colitis models, but never in the same study with essentially similar experimental setup and with compounds which are all commercially available. this allows a more precise comparison of the different compounds and thereby a better assessment of the model 's predictive value. we chose the adoptive transfer colitis model not only because of its similarities with ibd (mainly cd), but also because the model has several practical advantages compared to other chronic colitis models in relation to pharmacological testing (e.g., the synchronized onset of disease, no generation of anti - drug antibodies and commercial availability of mice). the cytokine exerts its effects via activation of nfb and mapk pathways, and subsequently induction of il-6 and il-1b, inhibition of t - cell apoptosis, chemoattraction, and so forth. we found a significant preventive treatment effect of anti - tnf- mab, as has been previously described in the cd45rb model. our model setup suggests that tnf- is most important in the beginning of disease since anti - tnf- was largely effective in the prevention study. it is possible that the redundancy of the inflammatory cascades makes tnf- less important when the inflammation is already established and cd4 t cells have differentiated into a pathogenic effector phenotype. however, the human anti - tnf- mab 's (adalimumab and infliximab) can induce remission in the majority of cd patients. these mabs ' have been reported to neutralize tnf- as well as to induce apoptosis in t cells. whether the surrogate rat anti - mouse tnf- mab also has this dual function is unknown. as opposed to anti - tnf- mab treatment, there was no effect of tnfr - fc in our studies, which is in accordance to the findings in cd. it has a critical role in promoting the differentiation of nave cd4 t cells into mature t - helper effector cells. it is currently believed that il-12 (p23/p40) and il-23 (p19/p40) are central for the th1 and the th17 pathways, respectively, and both cytokines are inhibited by the anti - il-12p40 mab [16, 28 ]. we found that the anti - il-12p40 mab treatment was effective in our prevention as well as intervention setup, suggesting the importance of these pathways in the transfer model. our observation is in agreement with previous results [20, 29, 30 ]. significant clinical responses following treatment with an anti - il-12p40 mab have also been reported in cd patients [5, 31 ], and the drug is currently recruiting for a phase iii trial in cd. thus, our results suggest that the scid adoptive transfer model is suitable for studying compounds targeting the il-12p40 pathways. increased serum concentrations of il-6 were reported to correlate with clinical activity of cd, and antibodies targeting il-6 or il-6r were efficacious in several animal models [16, 32, 33 ]. in accord, a humanized mab against il-6r showed promising results in a phase ii study in active cd. we found a significant therapeutic effect of anti - il-6 in a preventive setting but not in the intervention study. as for the anti - tnf- mab treatment, this suggests that il-6 is most important in the beginning, while the redundancy of the inflammatory cascades may make il-6 less important when the inflammation is already established. ctla4-ig has shown therapeutic efficacy in several autoimmune diseases including rheumatoid arthritis, and in experimental models of autoimmune diseases [34, 35 ]. we found that ctla4-ig completely prevented colitis and very efficiently cured established disease, indicating a central role of t - cell costimulation in the model. however, shortly after completion of our experimental studies, clinical trials with ctla4-ig in uc and cd were terminated due to lack of efficacy. the lack of negative regulation through ctla-4 on, for example, tregs may yield a therapeutic window which is not present in humans. thus, the lack of important self - regulatory mechanisms must be taken into account when evaluating drugs targeting this specific pathway. neutralization of the integrin 47 on lymphocytes and monocytes inhibits homing of the cells to the gut via mucosal addressin cell adhesion molecule-1 (madcam-1) on endothelial cells. we found that this could prevent the onset of disease in the transfer model as demonstrated previously. however, once the colitis was already established, recruitment of leucocytes to the colon via this mechanism seemed no longer essential for maintenance of disease in our experimental setup. in contrast, resolution of established disease has been demonstrated in the cotton - top tamarin (spontaneous colitis). in phase ii studies, vedolizumab (targeting human 47) failed to meet the primary endpoint in cd patients with active disease, while there was a significant therapeutic effect in patients with uc [39, 40 ]. natalizumab, which neutralizes 4 in conjunction with 7 as well as with 1, was effective for cd and is approved by the fda to use in patients refractory to treatment with anti - tnf-. thus, in ibd as well as in the models, there is not a clear - cut effect of inhibiting leukocyte homing via anti-47, although it seems more effective in man than in the scid transfer model. this could be due to the slower progression of the human disease, that is, there is a larger window open for treatment where recruitment of cells to the gut is still important. an alternative explanation is the involvement of 47 in homing to the small bowel and payer 's patches in crohn 's disease compared to 47 involvement in colonic cd4 t cell infiltration in the scid adoptive transfer model. although anti-47 could not reverse established colitis in the model, the marked effect of the antibody in prevention studies suggests that the model is useful in studies of leukocyte homing to the colon via integrins and adhesion molecules. the compound is effective in patients with severe steroid - refractory uc [7, 42 ], while no controlled studies have shown effect of cyclosporine in cd. we did not find any effect of preventive treatment with cyclosporine in accord with previous studies in transfer models [35, 44 ]. in contrast, cyclosporine significantly ameliorated acute dss - induced colitis [19, 45 ]. considering that the dss model is mainly uc - like and the transfer model mainly cd - like (at least in terms of th1/th17 immunopathogenesis) it is not clear why cyclosporine lacks effect in cd and in our model, but it has previously been shown that t cells are able to proliferate and exert effector functions via cyclosporine - resistant mechanisms [4648 ]. in addition to its effect on t cells, cyclosporine has been shown to inhibit the effect of several other immune cells and proinflammatory mechanisms, which may account for its therapeutic effects in the acute dss model, where t cells are not required for development of disease. the normal intestinal microbial flora (microbiota) contributes significantly to the etiopathogenesis of ibd [3, 50 ], and antibiotics have in some studies been shown to induce and maintain remission in ibd patients. however, the side - effects and the risk of developing microbial resistance associated with long - term treatment with antibiotics prevent the use of this treatment strategy. instead, there is focus on developing microbial cultures, which can help bringing back the balance between the microbiota and the immune system. we found that the combination of enrofloxacin and metronidazole completely prevented onset of colitis and cured established colitis in the adoptive transfer model, emphasizing the significance of the microbiota in this model. thus, the experimental model and the human disease share this central factor in the immunopathogenesis, suggesting that the model may be useful in studies of the microflora 's impact on disease. combined treatment with metronidazole, which kills anaerobes and influences cell trafficking, was essential since enrofloxacin alone did not affect the disease (data not shown). we have evaluated the therapeutic effect of a number of ibd drugs and drug candidates in the scid adoptive transfer colitis model and compared this to the therapeutic effect in ibd patients, when this information was available. the study shows that certain drivers of inflammation are shared between the model and the human diseases, that is, the cytokines il-12p40, tnf- and il-6, the homing molecule 47, and the microbial flora. with regards to these drivers however, our studies indeed also show limitations of the adoptive transfer model in this respect, since not all drugs effective against ibd are effective in the model. following adoptive transfer, the development of disease is driven by the extreme expansion of the cd4 cells in a lymphopenic host, and neither b cells nor cd8 cells are present. thus, some treatment effects in the preventive studies might be due to interfering with homeostatic expansion, which is not relevant in ibd and conversely, a test compound 's effect on b and cd8 t cells will have no effect in the model, although it might be effective in ibd. moreover, compared to clinical trials where efficacy is evaluated, for example, 418 weeks post - initiation of treatment for some test compounds, an intervention period of two weeks may be too short to obtain a therapeutic effect in the model. this stresses the need of critically choosing for which types of experiments and compounds to use animal models and which of the models to use. it should be noted that there is a great variability in the disease pattern in ibd patients, and that patients respond differently to medication. the various experimental colitis models may represent different types and stages of severity of uc or cd and altogether this makes the translation of data from experimental models to humans even more challenging.	animal models are important tools in the development of new drug candidates against the inflammatory bowel diseases (ibds) crohn 's disease and ulcerative colitis. in order to increase the translational value of these models, it is important to increase knowledge relating to standard drugs. using the scid adoptive transfer colitis model, we have evaluated the effect of currently used ibd drugs and ibd drug candidates, that is, anti - tnf-, tnfr - fc, anti - il-12p40, anti - il-6, ctla4-ig, anti-47 integrin, enrofloxacin / metronidazole, and cyclosporine. we found that anti - tnf-, antibiotics, anti - il-12p40, anti-47 integrin, ctla4-ig, and anti - il-6 effectively prevented onset of colitis, whereas tnfr - fc and cyclosporine did not. in intervention studies, antibiotics, anti - il-12p40, and ctla4-ig induced remission, whereas the other compounds did not. the data suggest that the adoptive transfer model and the inflammatory bowel diseases have some main inflammatory pathways in common. the finding that some well - established ibd therapeutics do not have any effect in the model highlights important differences between the experimental model and the human disease.
polycystic ovary syndrome (pcos) is one of the most common female endocrine disorders, affecting 2%26% of women of reproductive age (1245 years old) in the uk,13 and is suggested to be responsible for up to 75% of cases of infertility caused by anovulation.4 while the exact etiology and pathophysiology of pcos is complicated, the resultant hormonal imbalance due to both elevated androgen levels and increased insulin secretion in combination with environmental (eg, lifestyle) and genetic factors collectively contribute to the etiology of this syndrome.5,6 pcos has been associated with a variety of reproductive and skin disorders and also with an adverse metabolic profile including systemic dysfunctions, such as insulin resistance with compensatory hyperinsulinemia, hypertension, and dyslipidemia.7,8 many studies have reported that there may be a genetic susceptibility to the development of pcos. for example, franks reported that a genetic polymorphism may be responsible for the upregulated insulin metabolism among pcos patients. moreover, between 50% and 87% of first - degree relatives of women with pcos will present with some symptoms to varying degrees,10 including but not limited to hypertriglyceridemia, hyperinsulinemia, and type 2 diabetes.11 increased weight, and central obesity in particular, play an important role in the development of pcos, and as such the majority of women with this syndrome are either overweight or obese.1214 although the exact pathophysiological interactions between excess weight and the clinical expression of pcos remain to be elucidated, it is generally acknowledged that obesity itself is an independent risk factor associated with disturbances in sex - steroid metabolism,15 insulin resistance,10 and menstrual irregularities.16 obesity in pcos, however, exacerbates the reproductive, metabolic, and psychological effects of excessive weight, and thus optimal weight - loss guidelines in the management of pcos are of crucial importance. obese women with pcos face additional barriers in achieving weight loss when compared to healthy women without this condition. this is postulated to be due to either the observed decrease in postprandial thermogenesis,17 which is also correlated with the degree of reduced insulin sensitivity,18 or to specific eating behaviors (eg, emotional eating) and snacking patterns (cravings for high - caloric sweet and savory snacks) that are observed among overweight / obese women with pcos.19 the first - line treatment for pcos includes diet and lifestyle interventions20 to promote a healthy weight, especially in cases of overweight and obese patients. in these cases, even a modest weight loss of 2%5% total body weight may restore ovulation,2125 improve the reproductive hormonal profile,2428 and achieve an improvement in insulin sensitivity.23,2629 there is still, however, much debate over what is considered to be the optimal weight - loss strategy in pcos, resulting in a lack of evidence - based advice given by dietitians.30 interestingly, jeanes investigated the dietary recommendations for pcos patients given by uk dietitians, and found little difference from that given to non - pcos subjects, despite the known difficulty among pcos patients to lose weight. in a systematic review by nicholson,32 the efficacy of long - term (12 months) nonsurgical weight - loss interventions were presented, and behavioral strategies for obese women with pcos in a systematic review were identified. it was concluded that there was not enough evidence to suggest an ideal weight - loss approach in patients with pcos. although many different dietary approaches have been investigated, data on the effectiveness of very low - calorie diets (vlcds) on pcos are still limited. a vlcd can be defined as a nutrition plan that provides 800 kcal per day,33 and it is comprised of either conventional meals or synthetic formulas, eg, shakes, soups, or bars, or a combination of both. however, it has been also mentioned by mulholland,34 modern vlcds should not be confused with the ones used in the 1970s, the latter being associated with several deaths, mainly due to the inclusion of poor - quality protein and the inadequate provision of vitamins and minerals.35,36 modern commercial vlcds take into consideration the maintenance of lean body mass by fortification of the formulae with high levels of good - quality protein and the inclusion of essential electrolytes (sodium, potassium, bicarbonate, chloride, calcium, phosphates), fatty acids, minerals, and vitamins.37 this approach is safe, and according to the national institute for health and care excellence,38 it can be used for up to 3 months continuously for obese patients who fail to meet their weight - loss target using a standard low - fat, calorie - deficient diet (cdd). a vlcd can lead to an average weekly weight loss of 1.52.5 kg vs the 0.40.5 kg loss achieved with the cdds.33 moreover, the average weight loss at 12 weeks for an obese individual is approximately 20 kg on a vlcd, whereas it is only 8 kg on a ccd.33 consequently, a vlcd could be a good alternative for obese pcos patients both in the short and longer term who may face difficulties losing weight despite following standard approaches. for example, tolino showed that weight loss achieved after 4 weeks of a vlcd may lead to a decrease of free testosterone and fasting insulin levels and induce further improvement in menstruation. van dam carried out an interesting study to investigate the effect of short - term dieting in luteinizing hormone (lh) homeostasis in obese patients with pcos. according to the observations, both basal and pulsatile secretion of lh were three times higher in pcos patients when compared to matched individual without pcos, implying that caloric restriction failed to normalize the lh secretion. on the contrary, total testosterone, glucose, and insulin levels were decreased. in another study, patients were studied at baseline (occasion 1), after 1 week of a vlcd (occasion 2), and then after achieving 10% weight loss via a vlcd (occasion 3). results suggested that after the weight loss achieved, the estradiol - dependent negative feedback on lh was normalized and led to resumption of ovulation. overall, although there is evidence supporting the efficacy of vlcds, for obese patients who fail to meet their weight - loss target using a standard cdd, this approach has not been adequately investigated in the area of pcos, and both short- and longer - term data are still needed. the aim of this retrospective analysis was to assess the change in weight in individuals with and without pcos during a 12-week course of a commercial vlcd complemented by a group - based behavior - change program (lighterlife total). for this retrospective analysis, data were included from women aged 1875 years with body mass index (bmi) 28 kg / m who followed the lighterlife total approach from january 2006 until december 2011. study participants were self - referred onto the vlcd program. prior to commencement of their weight loss, their health and fitness to undertake the weight - loss program was assessed using a standardized form provided by lighterlife. participants were excluded from taking part in the lighterlife total program, and consequently from the study as well, if they met any of the following criteria : type 1 diabetes ; porphyria ; total lactose intolerance ; major cardiovascular or cerebrovascular disease ; history of renal disorder or hepatic disease ; active cancer ; epilepsy, seizures, convulsions, major depressive disorder, psychotic episodes, schizophrenia, bipolar disorders, or delusional disorders ; current suffering from anorexia, bulimia, or undergoing treatment for any other eating disorder ; pregnant or breastfeeding ; had given birth or had a miscarriage in the last 3 months. from a total sample of 102,610 lighterlife participants, the pcos participants (n=508) were matched for age (age 1 unit) and bmi (bmi 1 unit) with the non - pcos participants (n=508) for analysis of 12-week weight - loss data. baseline demographics and 12-week changes in weight and blood pressure were compared for the two groups. for those individuals who were eligible, appointments and treatments were managed by lay (but trained behavior - change) facilitators. this is a tripartite approach for individuals with bmi $ 30 kg / m, comprising a vlcd and group support along with behavior therapy. the program aims to achieve weight loss and to identify personal psychological motivation for overconsumption, thereby enabling participants to develop robust strategies for more successful weight management in the future. the vlcd provides an average daily intake of 600 kcal (50 g protein, 50 g carbohydrate, mean 17 g fat, ie, 36% energy from protein, 36% carbohydrate, and 28% fat) in the form of food packs (soups, shakes, textured meals, and bars) that contain 100% recommended daily allowances for vitamins and minerals, including vitamins a, c, d, e, k, thiamine, riboflavin, niacin, b6, b12, folic acid, biotin, pantothenic acid, calcium, phosphorous, iron, zinc, magnesium, iodine, potassium, sodium, copper, manganese, selenium, molybdenum, chromium, chloride, and fluoride. clients were also able to purchase an ancillary fiber mix to add to their water, which contained inulin as the source of fiber. participants undertook the vlcd alongside a unique behavior - change program developed specifically for weight management in the obese. this is informed by concepts from cognitive behavioral therapy and transactional analysis (transactional cognitive behavioral therapy) and addiction / change theory.4143 it is delivered in small, single - sex, weekly groups by weight - management counselors who are specifically trained in the facilitation of behavior change for the treatment of obesity. measurements of height and weight took place in lighterlife centers, and were carried out by facilitators who had been trained and provided with protocols developed within lighterlife. measurements were taken during weekly meetings that occurred at the same location and time each week. analysis of the individual parameters was performed by the use of descriptive statistics, and all variables were assessed for normality. differences of continuous variables between groups were assessed using an unpaired two - tailed t - test, and differences within groups were assessed using a paired t - test. if more than two groups were compared, analysis of variance with a bonferroni post hoc test was used. all p - values were two - sided, and a p - value of 0.05 was considered statistically significant. chicago, il, usa). the sample size (508 per group) for this retrospective analysis provided a power greater than 88% to detect a minimum difference of 0.2 kg in weight change between the pcos and non - pcos groups. for this retrospective analysis, data were included from women aged 1875 years with body mass index (bmi) 28 kg / m who followed the lighterlife total approach from january 2006 until december 2011. study participants were self - referred onto the vlcd program. prior to commencement of their weight loss, their health and fitness to undertake the weight - loss program was assessed using a standardized form provided by lighterlife. participants were excluded from taking part in the lighterlife total program, and consequently from the study as well, if they met any of the following criteria : type 1 diabetes ; porphyria ; total lactose intolerance ; major cardiovascular or cerebrovascular disease ; history of renal disorder or hepatic disease ; active cancer ; epilepsy, seizures, convulsions, major depressive disorder, psychotic episodes, schizophrenia, bipolar disorders, or delusional disorders ; current suffering from anorexia, bulimia, or undergoing treatment for any other eating disorder ; pregnant or breastfeeding ; had given birth or had a miscarriage in the last 3 months. from a total sample of 102,610 lighterlife participants, the pcos participants (n=508) were matched for age (age 1 unit) and bmi (bmi 1 unit) with the non - pcos participants (n=508) for analysis of 12-week weight - loss data. baseline demographics and 12-week changes in weight and blood pressure were compared for the two groups. for those individuals who were eligible, the intervention was carried out within a community - based setting. appointments and treatments were managed by lay (but trained behavior - change) facilitators. this is a tripartite approach for individuals with bmi $ 30 kg / m, comprising a vlcd and group support along with behavior therapy. the program aims to achieve weight loss and to identify personal psychological motivation for overconsumption, thereby enabling participants to develop robust strategies for more successful weight management in the future. the vlcd provides an average daily intake of 600 kcal (50 g protein, 50 g carbohydrate, mean 17 g fat, ie, 36% energy from protein, 36% carbohydrate, and 28% fat) in the form of food packs (soups, shakes, textured meals, and bars) that contain 100% recommended daily allowances for vitamins and minerals, including vitamins a, c, d, e, k, thiamine, riboflavin, niacin, b6, b12, folic acid, biotin, pantothenic acid, calcium, phosphorous, iron, zinc, magnesium, iodine, potassium, sodium, copper, manganese, selenium, molybdenum, chromium, chloride, and fluoride. clients were also able to purchase an ancillary fiber mix to add to their water, which contained inulin as the source of fiber. participants undertook the vlcd alongside a unique behavior - change program developed specifically for weight management in the obese. this is informed by concepts from cognitive behavioral therapy and transactional analysis (transactional cognitive behavioral therapy) and addiction / change theory.4143 it is delivered in small, single - sex, weekly groups by weight - management counselors who are specifically trained in the facilitation of behavior change for the treatment of obesity. measurements of height and weight took place in lighterlife centers, and were carried out by facilitators who had been trained and provided with protocols developed within lighterlife. measurements were taken during weekly meetings that occurred at the same location and time each week. analysis of the individual parameters was performed by the use of descriptive statistics, and all variables were assessed for normality. differences of continuous variables between groups were assessed using an unpaired two - tailed t - test, and differences within groups were assessed using a paired t - test. if more than two groups were compared, analysis of variance with a bonferroni post hoc test was used. for categorical outcomes, all p - values were two - sided, and a p - value of 0.05 was considered statistically significant. chicago, il, usa). the sample size (508 per group) for this retrospective analysis provided a power greater than 88% to detect a minimum difference of 0.2 kg in weight change between the pcos and non - pcos groups. the sample size (508 per group) for this retrospective analysis provided a power greater than 88% to detect a minimum difference of 0.2 kg in weight change between the pcos and non - pcos groups. the 12-week analysis included both a baseline observation carried forward (bocf) (n=508 for pcos and n=508 for non - pcos) and a completers (n=137 for pcos and n=137 for non - pcos) analysis. there were no differences between the pcos and non - pcos women in age (34.58.2 vs 34.58.2 years, p=0.948), height (1.650.1 m vs 1.650.1 m, p=0.937), weight (105.418.9 kg vs 105.319.0 kg, p=0.892), and bmi (38.96.4 kg / m vs 38.86.4 kg / m, p=0.831), as they were all matched at baseline. weekly weight loss was similar for both groups, with no statistically significant differences (figure 1). after 12 weeks of the vlcd, there was a significant weight reduction within each group when compared to baseline pcos (105.418.9 kg vs 95.019.1 kg, p<0.001) and non - pcos (105.319.0 kg vs 94.919.5 kg, p<0.001). there was also a significant change in bmi compared to baseline pcos (38.96.4 kg / m vs 35.06.6 kg / m, p<0.001) and non - pcos : (38.86.4 kg / m vs 35.06.8 kg / m, p<0.001). total weight change did not differ significantly between the pcos group and the non - pcos group (10.410.6 vs 10.410.4 kg, p=0.938), and both of the groups achieved approximately 10% weight loss after the 12-week vlcd approach (pcos 9.7%9.4% vs non - pcos 9.7%9.7%, p=0.965). table 1 shows the changes in systolic and diastolic blood pressure between the pcos and non - pcos groups after the 12-week vlcd period. as can be seen from the table, although the baseline systolic blood pressure was similar for both pcos and non - pcos participants (pcos 127.412.5 mmhg vs non - pcos 127.014.0 mmhg, p=0.598), the reduction observed after 12 weeks of vlcd was greater in the pcos group (pcos 5.56.1 mmhg vs non - pcos 0.96.1 mmhg, p<0.001). with regard to diastolic blood pressure, although at baseline women with pcos had greater values than non - pcos (pcos 81.510.2 mmhg vs non - pcos 79.99.7 mmhg, p=0.014), after 12 weeks of vlcd both groups had similar values (pcos 80.816.1 mmhg vs non - pcos 79.49.9 mmhg, p=0.206). as can be seen in table 2, there were no differences between the pcos and non - pcos women in age (35.78.9 years vs 35.88.9 years, p=0.946), height (1.650.1 m vs 1.640.1 m, p=0.212), weight (108.318.1 kg vs 107.419.8 kg, p=0.713), or bmi (40.06.3 kg / m vs 40.06.3 kg / m, p=0.955). weekly weight loss was similar for both groups, with no significant statistical differences (figure 2). after 12 weeks of the vlcd, there was a significant weight reduction within both groups when compared to baseline pcos (108.318.1 kg vs 89.816.7 kg, p<0.001) and non - pcos (107.419.8 kg vs 88.017.6 kg, p<0.001). there was also a significant change in bmi compared to baseline pcos (40.06.4 kg / m vs 33.26.0 kg / m, p<0.001) and non - pcos (40.06.3 kg / m vs 32.85.7 kg / m, p<0.001). total weight change did not differ significantly between the pcos group and the non - pcos group (18.56.6 kg vs 19.45.7 kg, p=0.190), and the percentage of weight loss achieved by pcos women was 17.1%5.6% vs 18.2%4.4% by the non - pcos group (p=0.08). moreover, no significant differences in weekly weight change between the two groups were identified (figure 2). table 3 shows the blood pressure values at baseline and after 12 weeks of vlcd. as can be seen from the table, baseline systolic blood pressure was lower for the pcos group compared to the non - pcos group (pcos 127.212.4 mmhg vs non - pcos 130.513.9 mmhg, p=0.014) ; however, there were no differences after 12 weeks of the vlcd (pcos 119.113.6 mmhg vs non - pcos 119.413.8 mmhg, p=0.800). with regard to diastolic blood pressure, it was similar for the pcos and non - pcos groups, both at baseline (pcos 81.39.8 mmhg vs non - pcos 80.59.6 mmhg, p=0.598) and after 12 weeks of the vlcd (pcos 77.511.6 mmhg vs non - pcos 74.910.2 mmhg, p=0.237). there were no differences between the pcos and non - pcos women in age (34.58.2 vs 34.58.2 years, p=0.948), height (1.650.1 m vs 1.650.1 m, p=0.937), weight (105.418.9 kg vs 105.319.0 kg, p=0.892), and bmi (38.96.4 kg / m vs 38.86.4 kg / m, p=0.831), as they were all matched at baseline. weekly weight loss was similar for both groups, with no statistically significant differences (figure 1). after 12 weeks of the vlcd, there was a significant weight reduction within each group when compared to baseline pcos (105.418.9 kg vs 95.019.1 kg, p<0.001) and non - pcos (105.319.0 kg vs 94.919.5 kg, p<0.001). there was also a significant change in bmi compared to baseline pcos (38.96.4 kg / m vs 35.06.6 kg / m, p<0.001) and non - pcos : (38.86.4 kg / m vs 35.06.8 kg / m, p<0.001). total weight change did not differ significantly between the pcos group and the non - pcos group (10.410.6 vs 10.410.4 kg, p=0.938), and both of the groups achieved approximately 10% weight loss after the 12-week vlcd approach (pcos 9.7%9.4% vs non - pcos 9.7%9.7%, p=0.965). table 1 shows the changes in systolic and diastolic blood pressure between the pcos and non - pcos groups after the 12-week vlcd period. as can be seen from the table, although the baseline systolic blood pressure was similar for both pcos and non - pcos participants (pcos 127.412.5 mmhg vs non - pcos 127.014.0 mmhg, p=0.598), the reduction observed after 12 weeks of vlcd was greater in the pcos group (pcos 5.56.1 mmhg vs non - pcos 0.96.1 mmhg, p<0.001). with regard to diastolic blood pressure, although at baseline women with pcos had greater values than non - pcos (pcos 81.510.2 mmhg vs non - pcos 79.99.7 mmhg, p=0.014), after 12 weeks of vlcd both groups had similar values (pcos 80.816.1 mmhg vs non - pcos 79.49.9 mmhg, p=0.206). as can be seen in table 2, there were no differences between the pcos and non - pcos women in age (35.78.9 years vs 35.88.9 years, p=0.946), height (1.650.1 m vs 1.640.1 m, p=0.212), weight (108.318.1 kg vs 107.419.8 kg, p=0.713), or bmi (40.06.3 kg / m vs 40.06.3 kg / m, p=0.955). weekly weight loss was similar for both groups, with no significant statistical differences (figure 2). after 12 weeks of the vlcd, there was a significant weight reduction within both groups when compared to baseline pcos (108.318.1 kg vs 89.816.7 kg, p<0.001) and non - pcos (107.419.8 kg vs 88.017.6 kg, p<0.001). there was also a significant change in bmi compared to baseline pcos (40.06.4 kg / m vs 33.26.0 kg / m, p<0.001) and non - pcos (40.06.3 kg / m vs 32.85.7 kg / m, p<0.001). total weight change did not differ significantly between the pcos group and the non - pcos group (18.56.6 kg vs 19.45.7 kg, p=0.190), and the percentage of weight loss achieved by pcos women was 17.1%5.6% vs 18.2%4.4% by the non - pcos group (p=0.08). moreover, no significant differences in weekly weight change between the two groups were identified (figure 2). table 3 shows the blood pressure values at baseline and after 12 weeks of vlcd. as can be seen from the table, baseline systolic blood pressure was lower for the pcos group compared to the non - pcos group (pcos 127.212.4 mmhg vs non - pcos 130.513.9 mmhg, p=0.014) ; however, there were no differences after 12 weeks of the vlcd (pcos 119.113.6 mmhg vs non - pcos 119.413.8 mmhg, p=0.800). with regard to diastolic blood pressure, it was similar for the pcos and non - pcos groups, both at baseline (pcos 81.39.8 mmhg vs non - pcos 80.59.6 mmhg, p=0.598) and after 12 weeks of the vlcd (pcos 77.511.6 mmhg vs non - pcos 74.910.2 mmhg, p=0.237). one of the biggest health problems globally is the increasing prevalence of obesity. according to the national infertility group report,44 half of the women of reproductive age are either overweight or obese, and it is known that increased weight, and in particular central obesity, plays an important role in the development of pcos.13 although the first - line treatment in overweight and obese women with pcos is weight loss through lifestyle interventions, the complexity and heterogeneity of this syndrome in conjunction with the lack of robust large scale randomized clinical trials lead to lack of consensus over the optimal dietary guidelines to achieve that goal. in this paper, we attempted to investigate the effect of the lighterlife total vlcd program on weight loss in overweight and obese women with pcos compared to age - and bmi - matched non - pcos participants. it is generally proposed that as little as 5% weight loss can improve fertility outcomes.2128 in this study, pcos (completers) achieved similar weight reduction with the non - pcos (completers) group, which was more than three times higher than the minimum suggested weight loss : 17.1%5.6% by the pcos vs 18.2%4.4% by the non - pcos group (p=0.08) after 12 weeks of vlcd in conjunction with group counseling. the bocf analysis showed more conservative results (pcos 9.7%9.4% vs non - pcos 9.7%9.7%, p=0.965), although nevertheless promising. moreover, in both completers and bocf analysis, women with pcos lost weight at the same rate as non - pcos women. this is in contrast with a number of papers which mentioned that women with pcos may face difficulties in achieving weight loss either due to consequent metabolic issues18,19,44 or the emotional distress that accompanies this disorder, and predisposes towards increased energy consumption. for example, it has been suggested that women with pcos may have reduced postprandial thermogenesis associated with reduced insulin sensitivity, a factor that could influence the weight loss.18 in addition, adjusted basal metabolic rate appears to be statistically significantly lower in women with pcos, and is more evident in women with both pcos and insulin resistance.45 interestingly, similar observations have been made with regard to patients with type 2 diabetes.46 moreover, pcos patients very often have deranged appetite regulation due to reduced postprandial cholecystokinin (cck) secretion compared to control age - and bmi - matched individuals without pcos.47 however, in cases where weight reduction was achieved via a ketogenic diet, the levels of cck maintained similar concentrations with the ones before the weight loss.48 this could suggest that due to the anorexogenic effect of ketosis, the levels of cck were normalised among the pcos patients, thus similar weight loss was achieved between the pcos and non - pcos group, as also shown by our analysis. while improvements in weight and bmi have been reported by other researchers using variable dietary approaches, these lacked a control group,24,49,50 whereas in the present study, women without pcos acted as the control group and were also matched for age and bmi for further analysis. moreover, this is the only study in the area of pcos and vlcds that incorporated the counseling component. although the effect of counseling per se was not assessed, it is known that the element of behavioral change not only can enhance weight loss but may also improve the completion rates of weight - loss programs.51 this support is of extreme importance among pcos patients, where higher incidences of depressive and anxiety disorders may predispose them toward unhealthy eating behaviors. furthermore, several studies have suggested that there is an increased prevalence of hypertension among women with pcos compared to the general population, irrespective of their weight.5254 on the other hand, luque - ramrez support the view that high blood pressure is only observed among overweight and obese women with pcos. in our study, women with pcos presented with higher diastolic pressure at baseline, but after 12 weeks of the vlcd, both systolic and diastolic pressure were at similar levels with their non - pcos counterparts. this interesting finding could imply that in cases of obese women with pcos, weight loss could contribute to normalization of blood pressure levels. nevertheless, further research needs to be done, including 24-hour ambulatory blood pressure monitoring as well as assessment of medication intake and hormonal profile, both at baseline and after the end of the vlcd program. the main strength of the current study is that this is the largest investigation to date of a commercial vlcd program, lighterlife total, on weight loss in overweight / obese women with pcos compared to overweight / obese women without this syndrome. although the gold standard in evaluating health care interventions is carefully designed, conducted, and reported randomized controlled trials, our population - based retrospective analysis assessed the effectiveness of a commercial vlcd program in conjunction with weekly counseling group sessions, as it runs for real clients who joined the lighterlife total program. for example, there was no information on the criteria used for diagnosis of pcos, and it is highly possible that each of the pcos women had been diagnosed by a different doctor. however, rolland reported that there was no difference in weight loss between diabetes and nondiabetes participants when they followed the lighterlife total program, despite the lack of discrepancy in relation to diagnosis of type 2 diabetes. another potential bias could be financial status, as only women who can afford the weekly costs of this commercial program (approximately 65 per week) were included. moreover, due to the small numbers encountered in some of the ethnic groups, comparisons between the different ethnic categories encountered could not be performed. in addition, information on medication or potential changes in insulin sensitivity, glycemia, and reproductive phenotype was not assessed. pcos is a complex condition, with diverse implications that include reproductive, metabolic, and psychological comorbidities. its clinical management should focus on lifestyle changes with medical therapy when required, as well as providing patients with support and nutritional education. however, evidence - based guidelines are still needed to guide clinicians and patients in optimal dietary management of this syndrome. the present retrospective analysis suggests that women with pcos lose the same amount of weight and at the same rate as non - pcos women after following a commercial vlcd program for 12 weeks in conjunction with group - counseling sessions. consequently, this approach could be considered an effective alternative to standard lcds, especially in cases where difficulties in achieving weight loss are encountered.	backgroundpolycystic ovary syndrome (pcos) affects between 2% and 26% of reproductive - age women in the uk, and accounts for up to 75% of anovulatory infertility. the major symptoms include ovarian disruption, hyperandrogenism, insulin resistance, and polycystic ovaries. interestingly, at least half of the women with pcos are obese, with the excess weight playing a pathogenic role in the development and progress of the syndrome. the first - line treatment option for overweight / obese women with pcos is diet and lifestyle interventions ; however, optimal dietary guidelines are missing. although many different dietary approaches have been investigated, data on the effectiveness of very low - calorie diets on pcos are very limited.materials and methodsthe aim of this paper was to investigate how overweight / obese women with pcos responded to lighterlife total, a commercial very low - calorie diet, in conjunction with group behavioral change sessions when compared to women without pcos (non - pcos).resultspcos (n=508) and non - pcos (n=508) participants were matched for age (age 1 unit) and body mass index (body mass index 1 unit). a 12-week completers analysis showed that the total weight loss did not differ significantly between pcos (n=137) and non - pcos participants (n=137) (18.56.6 kg vs 19.45.7 kg, p=0.190). similarly, the percentage of weight loss achieved by both groups was not significantly different (pcos 17.1%5.6% vs non - pcos 18.2%4.4%, p=0.08).conclusionoverall, lighterlife total could be an effective weight - loss strategy in overweight / obese women with pcos. however, further investigations are needed to achieve a thorough way of understanding the physiology of weight loss in pcos.
	a beagle dog treated with saline as a control animal in a preclinical study was euthanized due to sudden systemic deterioration. on histopathological examination, contraction band necrosis of myocardial cells was observed widely in the left ventricular wall, including the papillary muscle and apex, and observed slightly in the ventricular septum and left atrium. in the brain, necrosis was observed in neurons and glia of the cerebral cortex, hippocampal pyramidal cells, glial cells of the rostral commissure and purkinje cells of the cerebellar vermis. it is highly probable that the marked systemic deterioration was caused by cardiac dysfunction due to the spontaneous contraction band necrosis of the myocardial cells, although the pathogenesis of the myocardial lesions remains unclear. given the distribution of neuronal necrosis in the brain, it is likely that these lesions resulted from the ischemia responsible for acute cardiac failure.
persistent hyperglycemic microenvironment in type 2 diabetes mellitus (t2 dm) leads to the development of complications like cardiovascular disease, neuropathy, nephropathy, and retinopathy. wound healing impairment is a serious complication of t2 dm which contributes to the high percentage of amputations performed worldwide. as per recent data, around 25% of t2 dm patients develop nonhealing wounds once in their life. the reason could be that hyperglycemia in t2 dm decreases the levels of cytokines and growth factors essential for wound healing. the immune system is also compromised in patients with t2 dm which generally leads to prolonged inflammation and unresolved infection in the wound which either takes a long time to heal or does not heal at all. toll - like receptors (tlrs) are a family of genetically conserved transmembrane receptors involved in the innate immunity and pathogen recognition. recognition of pathogen - associated molecular patterns by tlrs activates signaling events that induce the expression of effector molecules, such as cytokines and chemokines, controlling the adaptive immune responses. genetic variations within genes encoding these receptors have an important influence on the pathogenesis of inflammatory diseases. variations within genes of the family of innate immune receptors may account, in part, for the inherited differences in the susceptibility to autoimmune or inflammatory diseases. the ability of certain individuals to respond properly to tlr ligands may be impaired by single nucleotide polymorphisms within tlr genes, resulting in an altered susceptibility to the disease. considering the potential role of tlrs pathway in the overall immune reconstitution, we assessed tlr2 and 9 gene polymorphism in patients with t2 dm with (and without) diabetic foot (df) as a herald to complete studying of other members of the tlr family in such a common multisystemic inflammatory condition in further studies. group i included 30 patients (16 males and 14 females) with t2 dm and df, all patients were on insulin treatment and 13 of them were smokers. their mean (standard deviation [sd ]) age, diabetes mellitus (dm) duration, and body mass index (bmi) were 58 9.3 years, 14.6 7.2 years, and 29.7 5.5 kg / m, respectively. group ii included 30 patients (9 males and 21 females) with t2 dm with no evidence of df, 13 patients were on insulin treatment, and 7 patients were smokers. their mean (sd) age, dm duration, and bmi were 53.1 9.3 years, 11.9 6.7 years, and 33.3 5 kg / m, respectively. group iii included 30 volunteer normal control subjects (7 males and 23 females), their mean (sd) age and bmi were 38.7 12.6 and 30.2 5.2, respectively. the patients were recruited, from the internal medicine inpatient wards and outpatient clinics of cairo university hospitals during the period from january 2014 to january 2015. patients who had history of cerebrovascular events, renal failure, or were on renal replacement therapy were excluded. all participants underwent a complete screening panel, including medical history, clinical examination, and assessment of bmi. biochemical profile included fasting and 2-hours postprandial glucose, glycated hemoglobin, creatinine clearance, total cholesterol, and triglycerides (data available at table 1). tlr2 t / c genotyping were performed by polymerase chain reaction (pcr)restriction fragment length polymorphism technique. whole blood samples were collected in sterile vacutainer containing k3edta to prevent blood clotting (bd, becton, dickinson and company, south carolina) from the patients. for quality control, genomic dna extraction from peripheral blood leucocytes was performed using gene jet whole blood genomic dna purification mini kit (fermentas life sciences, canada) following the manufacturer 's instructions. tlr2 - 1350 t / c and tlr9 - 1237 t / c genotyping was performed by pcr all pcr reactions were performed in a total volume of 25 l containing 150 ng genomic dna, 2x dream taqgreen pcr master mix, 25pm of each forward and reverse primers (fermentas, lithuania). genotyping of tlr2 - 1350 t / c (rs3804100) was performed according to takahashi. the primer set used was forward : 5-tcatttggcatcattggaaa-3 and reverse : 5-gagttgcggcaaattcaaag-3. the thermocycler program conducted was initial denaturation at 95 c for 5 minutes followed by 35 cycles of denaturation at 95 c for 30 seconds, annealing at 58 c for 30 seconds, extension at 72 c for 30 seconds, and a final extension step at 72 c for 10 minutes. the generated amplicon is a 251 bp fragment, which was digested by mwoi enzyme (fermentas - lithuania). the wild type allele (t allele) produced a single band of 251 bp, while the polymorphic allele (c allele) produced 2 bands of 167 and 84 bp. for tlr9 1237t / c (rs5743836) genotyping, the primer set used was forward : 5-atgggagcagagacataatgga-3 and reverse : 5-ctgcttgcagttgactgtgt-3. the thermocycler program conducted was initial denaturation at 95 c for 5 minutes followed by 36 cycles of denaturation at 94 c for 40 seconds, annealing at 62 c for 40 seconds, extension at 72 c for 1 minute, and a final extension step at 72 c for 10 minutes. this generated a 135 bp fragment. the wild type allele (t allele) showed 2 bands of 108 and 27 bp, while the polymorphic allele (c allele) showed 3 bands of 60, 48, and 27 bp. for quality control, genotyping was repeated with respect to case / control status for 50 samples. results of genotyping were interpreted blindly by 2 different observers, and were 100% concordant. parametric data were expressed as mean, sd, and range, while nonparametric data were expressed as median and interquartile range. chi - square test or fisher exact test were used to examine the relation between qualitative variables. unconditional logistic regression analysis was used to calculate odds ratios (ors) and 95% confidence intervals (cis) for risk estimation. chi - square () test was performed to assess deviation from hardy weinberg equilibrium in controls. group i included 30 patients (16 males and 14 females) with t2 dm and df, all patients were on insulin treatment and 13 of them were smokers. their mean (standard deviation [sd ]) age, diabetes mellitus (dm) duration, and body mass index (bmi) were 58 9.3 years, 14.6 7.2 years, and 29.7 5.5 kg / m, respectively. group ii included 30 patients (9 males and 21 females) with t2 dm with no evidence of df, 13 patients were on insulin treatment, and 7 patients were smokers. their mean (sd) age, dm duration, and bmi were 53.1 9.3 years, 11.9 6.7 years, and 33.3 5 kg / m, respectively. group iii included 30 volunteer normal control subjects (7 males and 23 females), their mean (sd) age and bmi were 38.7 12.6 and 30.2 5.2, respectively. the patients were recruited, from the internal medicine inpatient wards and outpatient clinics of cairo university hospitals during the period from january 2014 to january 2015. patients who had history of cerebrovascular events, renal failure, or were on renal replacement therapy were excluded. all participants underwent a complete screening panel, including medical history, clinical examination, and assessment of bmi. biochemical profile included fasting and 2-hours postprandial glucose, glycated hemoglobin, creatinine clearance, total cholesterol, and triglycerides (data available at table 1). tlr2 t / c genotyping were performed by polymerase chain reaction (pcr)restriction fragment length polymorphism technique. whole blood samples were collected in sterile vacutainer containing k3edta to prevent blood clotting (bd, becton, dickinson and company, south carolina) from the patients. for quality control, genomic dna extraction from peripheral blood leucocytes was performed using gene jet whole blood genomic dna purification mini kit (fermentas life sciences, canada) following the manufacturer 's instructions. tlr2 - 1350 t / c and tlr9 - 1237 t / c genotyping was performed by pcr all pcr reactions were performed in a total volume of 25 l containing 150 ng genomic dna, 2x dream taqgreen pcr master mix, 25pm of each forward and reverse primers (fermentas, lithuania). genotyping of tlr2 - 1350 t / c (rs3804100) was performed according to takahashi. the primer set used was forward : 5-tcatttggcatcattggaaa-3 and reverse : 5-gagttgcggcaaattcaaag-3. the thermocycler program conducted was initial denaturation at 95 c for 5 minutes followed by 35 cycles of denaturation at 95 c for 30 seconds, annealing at 58 c for 30 seconds, extension at 72 c for 30 seconds, and a final extension step at 72 c for 10 minutes. the generated amplicon is a 251 bp fragment, which was digested by mwoi enzyme (fermentas - lithuania). the wild type allele (t allele) produced a single band of 251 bp, while the polymorphic allele (c allele) produced 2 bands of 167 and 84 bp. for tlr9 1237t / c (rs5743836) genotyping, the primer set used was forward : 5-atgggagcagagacataatgga-3 and reverse : 5-ctgcttgcagttgactgtgt-3. the thermocycler program conducted was initial denaturation at 95 c for 5 minutes followed by 36 cycles of denaturation at 94 c for 40 seconds, annealing at 62 c for 40 seconds, extension at 72 c for 1 minute, and a final extension step at 72 c for 10 minutes. the wild type allele (t allele) showed 2 bands of 108 and 27 bp, while the polymorphic allele (c allele) showed 3 bands of 60, 48, and 27 bp. for quality control results of genotyping were interpreted blindly by 2 different observers, and were 100% concordant. parametric data were expressed as mean, sd, and range, while nonparametric data were expressed as median and interquartile range. chi - square test or fisher exact test were used to examine the relation between qualitative variables. unconditional logistic regression analysis was used to calculate odds ratios (ors) and 95% confidence intervals (cis) for risk estimation. chi - square () test was performed to assess deviation from hardy weinberg equilibrium in controls. the mean - fasting blood glucose, 2 hours postprandial blood sugar, glycated hemoglobin, serum cholesterol, and triglycerides were significantly higher in group i in comparison to groups ii and iii (p <.001). the mean creatinine clearance was significantly lower in group i in comparison to groups ii and iii (p < there was no statistically significant difference noticed in the distribution of the wild type or the polymorphic tlr2 - 1350 t / c genotypes between the different study groups. tlr9 polymorphic variants (tc and cc) were detected in 24/30 (80%) of group i, 22/24 were heterozygous (tc), and (2/24) were homozygous (cc). in group ii, tlr9 polymorphic variants (tc and cc) were detected in 15/30 (50%), 15/15 were heterozygous (tc), and no patient was homozygous (cc). in group iii, tlr9 polymorphic variants (tc and cc) were detected in 5/30 (16.9%), 5/5 of patients were heterozygous (tc), and no patient was homozygous (cc). the frequency of tlr9 - 1237 polymorphism (tc + cc) was significantly higher in group i when compared to group ii (p <.029) and group iii (p < the frequency of tlr9 - 1237 polymorphism (tc + cc) was significantly higher in group ii when compared to group iii (p <.013). the frequency of tlr9 - 1237 (tc) heterozygous genotype was significantly higher in group i when compared to group iii (p <.001) ; however, it was nonsignificantly higher when compared to group ii. the frequency of tlr9 - 1237 (cc) homozygous genotype was nonsignificantly higher in group i when compared to groups ii and iii. calculated risk estimation revealed that tlr9 - 1237 polymorphism (tc + cc) conferred almost 20 times increased risk of df disorders in t2 dm (or = 20, 95% ci = 5.3874.30). tlrs are pivotal innate immune receptors that induce inflammatory responses and their expression and activation is increased in a plethora of inflammatory disorders including dm and its complications. persistent hyperglycemia in t2 dm elicits chronic low grade inflammation and the innate immune system.genetic susceptibility to secondary complications of t2 dm like df ulcer is multifactorial and risk may involve factors which are related to the activation of the immune system. previous studies showed that increased tlr expression, signaling, and activation may contribute to the increased inflammatory status in the human diabetic wounds, since tlr1, 2, 4, and 6 mrna expression were found to be significantly increased in wounds of diabetic patients compared with those in nondiabetic subjects (p <.05). it is known to be a signaling receptor for many microbial products including whole gram - positive bacteria and mycoplasma. antiinfectious property of tlr2 is evident from the fact that the tlr2 deficient mouse strain is more prone to infection with gram - positive bacteria staphylococcus aureus and shows defective clearance of spirochaetes after infection by borrelia burgdorferi as compared to their wild type counterparts. also it has been shown that tlr2 is a potent player in the wound - healing mechanism. the assessment of tlr2 - 1350 t / c gene polymorphism in our egyptian control subjects revealed that the frequency of wild genotype (tt) was 87.7% and the heteromutant genotype (tc) was 13.3%, while none of them had the homomutant genotype (cc). these results were close to those reported in caucasians being 10.2% to 15% and in norway being 15% for the tc purdue showed that the frequencies of the tc and cc genotypes in the united states of america were 12.9% and 0.5%, respectively. on the other hand, these frequencies were not in accordance with that reported in the far east being 47.4% for the heteromutant and 4.7% for the homomutant genotypes in chinese, 41.1 for the heteromutant and 7.9% for the homomutant genotypes in japanese, and 38% for the heteromutant and 10.6% for the homomutant genotypes in koreans. the difference in results could be attributed to the ethnic differences between the studied populations. our study revealed that the frequency of tlr2 - 1350 t / c polymorphism in both group i and group ii was 96.7% for the wild genotype (tt), 3.3% for the heteromutant genotype (tc), and no one was homomutant (cc). statistical analysis revealed that there was no significant difference encountered in the distribution of the polymorphic genotypes of tlr2 between the 3 groups. dasu and martin found that tlr2 mrna expression was increased significantly in wounds of patients with diabetes compared with nondiabetic wounds. mohammad reported an increased tlr2 expression in bone marrow derived macrophage of nonobese diabetic mice. creely showed increased tlr2 expression in the adipose tissue of type 2 diabetes (t2 dm) patients. tlr9, expressed primarily by the subsets of b cells and myeloid cells in humans, is involved in both systemic as well as local inflammatory responses. tlr9 is an important endosomal member of tlr family which causes wound necrosis by promoting inflammation. the increased expression of tlr9 with the increase in the wound grade suggests that the tlr9 expression directly modulates the severity of diabetic wounds. singh found that tlr9 message and protein were higher in diabetic wounds compared to control wounds. in our study, the frequency of tlr9 - 1237 t / c polymorphic genotypes in the control group was 16.7% for the heteromutant genotype (tc), while none of them had homomutant genotype (cc). these frequencies were close to that reported in americans (tc = 28.3% and cc = 2.4%), portuguese (tc = 18.7% and cc = 0.8%), and italians (tc = 17.3% and cc = 1.7%). on the other hand, our results were not in agreement with the chinese study reported by junjie being 46.9% and 9% for the polymorphic genotypes tc and cc, respectively. our study showed that in group i, the frequency of tlr9 heteromutant genotype (tc) was 73.3% and the homomutant genotype (cc) was 6.7% while in group ii the frequency of tlr9 heteromutant genotype (tc) was 50% and the homomutant genotype (cc) was 0%. the statistical analysis revealed that the frequency of tlr9 - 1237 t / c polymorphic variants (tc and cc) was significantly higher in group i compared to group iii (p <.001), but it was nonsignificantly higher in group i compared to group ii. the calculated risk estimation in our study revealed that tlr9 - 1237 polymorphism (tc + cc) conferred almost 20 times increased risk of df in patients with t2 dm (or = 20, 95% ci = 5.3874.30). liu studied the frequency of tlr2, 4, and 9 gene polymorphisms in chinese population and their susceptibility to type 2 diabetes and coronary artery disease. they examined several tlr family gene variants that have been commonly reported in western countries, 2 of them were tlr9 promoter-1237t / c polymorphism, and tlr2 arg677trp and arg753gln polymorphisms in all subjects. however, they did not detect the presence of any variant for these single nucleotide polymorphisms in their subjects. it was reported that the korean population had low frequency of tlr9 - 1237t / c (< 0.3%). in japanese population, no tlr9 - 1237t / c polymorphism was present in 183 patients with systemic lupus erythematosus (sle) and 198 controls. but high frequency of the variant allele was reported in the caucasian population (11%16%). lazarus showed that the genetic variation at position-1237 is associated with an increased risk of asthma in european americans. it is not clear whether the very low frequency of these tlr variants would be related to the low incidence of diseases such as diabetes and coronary artery disease in the oriental populations, compared with the western ones. it should be noted that the occurrence of diabetes and/or its complications depends on the interaction among multiple risk factors, like the presence of different risk alleles, environmental factors, and the lifestyle. the contribution of any single - gene polymorphism is rather small and the interactive effect of several factors may lead to an under- or overestimation of the role of a given gene polymorphism in determining the phenotype. therefore, the results might not apply to groups with different genetic or environmental backgrounds. moreover, studying the association between tlrs gene variant and susceptibility to t2 dm and/or its complication will strengthen our understanding of the link between innate immunity and t2 dm. the small number of studied subjects and lack of appropriate sample size quantification are among the limitations of our study. a multicenter trial could improve the credibility of the results and help in building a regression model. studying tlr2 and studying other tlr members could widen the scope of our understanding of the pathogenesis of such diabetic complication. in conclusion, our results suggest a potential role for the host genetic background in df susceptibility and demonstrate that there is an association between tlr9 - 1237 t / c polymorphism and the risk of df. ultimately, functional studies dissecting the role of this gene polymorphism may allow the identification of potential therapeutic targets.	abstracttoll - like receptors (tlrs) are innate immune receptors that mediate the inflammatory response in diabetes mellitus (dm). the aim of this study is to evaluate the association of tlr2 and tlr9 gene polymorphism in patients with type 2 dm (t2 dm) and diabetic foot (df).the study included 90 subjects divided into group i (30 patients with t2 dm and df), group ii (30 patients with t2 dm and no evidence of df), and group iii (normal control subjects). tlr2 (1350 t / c, rs3804100) and tlr9 (1237 t / c, rs5743836) genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (pcr - rflp) technique for all subjects.there was a statistically significant difference in the distribution of tlr9 - 1237 t / c genotypes between groups i and ii (p <.029) as well as between groups i and iii (p <.001). calculated risk estimation revealed that tlr9 - 1237 polymorphism conferred almost 20 times increased risk of df disorders in t2 dm (or = 20, 95% ci = 5.3874.30). there was no statistical difference in the distribution of tlr2 - 1350t / c genotypes between the 3 groups.tlr9-1237 t / c gene polymorphism may be considered as a molecular risk for df among patients with t2 dm.
a 34-year - old female (g2p2a0l2) visited our hospital with an eight - month history of a palpable abdominal mass. normal beta - human chorionic gonadotropin with increased alpha - fetoprotein (22.7 ng / ml), cancer antigen (ca) 125 (61.7 u / ml), and ca19 - 9 (553.7 u / ml) were observed. contrast - enhanced computed tomography showed a large solid and cystic mass in the left lower abdomen and pelvis. a huge left ovarian tumor filling the peritoneal cavity was found during laparotomy, and yellow - colored ascites (200 ml) was expelled. the external surface of the tumor was pinkish - tan, transparent, smooth, and intact. multiple firm, gray - white nodules measuring 0.1 to 0.3 cm were observed on the omentum, uterine serosa and posterior cul - de - sac peritoneum. left salpingo - oophorectomy, right wedge resection, partial omentectomy, and biopsy of the uterine serosal nodules were performed. under the impression of ovarian malignant tumor, for staging, pelvic lymph node sampling, partial omentectomy and posterior cul - de - sac peritoneal resection were performed. grossly, the excised left ovarian mass measured 21.0 cm in diameter and weighed 2,350 g. a predominantly solid mass with multicystic changes, which were filled with clear watery fluid, was observed on the cut surface (fig. thirty sections were taken from the ovarian tumor, and various mature tissues of tridermal lineage were demonstrated (fig. 1b) ; the mass was comprised predominantly of mature glial tissue (90%) with focal immature neuroepithelial cells forming rosette - like structures with few mitosis (fig. these immature neuroectodermal foci, including primitive mesenchyma, led to the diagnosis of an immature teratoma, grade 1, according to the classification of robboy and scully.3 there were no other germ cell tumor components, including a yolk sac tumor. the peritoneum, cul - de - sac and uterine serosa, as well as the opposite ovarian surface, showed involvement of glial peritonei (grade 0). nodules of the right ovary, peritoneum and omentum were composed of glial tissue (grade 0) (fig. 1d). out of 32 dissected lymph nodes, one left hypogastric lymph node showed one mature glial implant, i.e., nodal gliomatosis (grade 0) (fig. 1d, inset). some scattered psammoma bodies were observed in peritoneal and nodal gliomatosis, as well as brain tissue in the ovarian immature teratoma. immunohistochemically, glial fibrillary acidic protein (prediluted, polyclonal, dako, glostrup, denmark) (fig. the endometrial stroma of endometriosis was stained with cd10 (56c6, prediluted, dako) (fig. three cycles of chemotherapy of bleomycin, etoposide and cisplatin (bep) were performed. after the surgery, alpha - fetoprotein, ca125, and ca19 - 9 levels reached the normal range. during the nine - month post - operative follow - up period, gp is a rarely encountered complication of ovarian teratomas and is characterized by peritoneal implants of glial tissue usually of a low world health organization grade, although cases of malignant evolution or high grades have also been described.16 about 100 cases have been retrieved in the literature. to our best knowledge, nine cases of nodal gliomatosis (including the present case) were demonstrated in the pelvic or paraaortic lymph nodes;1 - 8 eight cases were associated with both gp and ovarian teratomatous tumors, while the remaining case was without gp.6 another interesting phenomenon is the rare combination of gp and endometriosis, as was seen in the present case. to date, seven cases of endometriosis combined with gp have been published in the world literature.9 - 15 a summary of peritoneal gliomatosis coexisting with nodal gliomatosis or endometriosis is shown in tables 1 and 2. lymphangeneous glial implants or direct spillage of the glial foci of an ovarian teratoma have been suggested by the previous articles.2 - 7 another viewpoint proposed that gp is a metastasis of glial tissue to a focus of pre - existing endometriosis because these endometrial foci are more vascular and therefore are more receptive. however, this concept contradicts the fact that endometriosis is rare in the pediatric population, and it is unlikely because despite extensive sampling, there were only mixed endometrial and glial cells but no pure endometriosis. originally, the presence of gp indicated that these lesions were shared by implantation metastases of an ovarian teratomatous tumor with varying maturation.7,10 - 12 metaplasia of pluripotent coelomic or mullerian stem cells has also been suggested, but the most scientifically supported and widely accepted mechanism of endometriosis is retrograde effluent flow through the lumen of the fallopian tubes into the pelvic - peritoneal cavities during menstruation.9 metaplasia may be induced by unknown stimuli in other uncommon peritoneal lesions, such as melanosis peritonei, etc.17 dworak.11 suggested that gp temporally predisposes a patient to the development of endometriosis. a recent assay for loss of heterozygosity revealed that normal tissues and gp showed a heterozygous pattern in the microsatellite loci, whereas the associated ovarian teratomas had a homozygous pattern.18 these findings suggest that gp with or without nodal involvement is genetically unrelated to the associated teratoma but may be stimulated by these teratomas. other rare locations such as intrathoracic gliomatosis, isolated gp independent to ovarian teratoma or one occurring after ventriculoperitoneal shunt operations also support this theory. the peritoneal mesothelium that modulates the cytoskeleton and shape is explained as subserosal multipotential subserosal cells that can replicate and differentiate diversely under unspecified stimuli.19 mesothelial hyperplasia is commonly found when it is stimulated by pleural and peritoneal effusion.19 this mesothelial hyperplasia may be rarely seen in lymph nodes, and in these cases it would be mimicking metastatic tumors.18 gliomatosis affecting the lymph node can be explained by this unusual location of mesothelial hyperplasia, although the present case showed no peritoneal mesothelial hyperplasia. a frequent association between gp and effusion was found ; about 35 cases of gp were accompanied by ascites, and 3 cases involved pleural effusion.1,4,6,8 the frequent association with ascites or pleural effusion and co - existing nodal or gp, as shown in table 1, may provide supporting evidence of this unusual location of gliomatosis.1,19 however, although the common association of gp with ovarian teratoma is indisputable, the reason why subperitoneal cells develop into glial islands in the presence of these ovarian tumors remains to be investigated. another explanation of the occasionally present ascites or pleural effusion is that the pressure on the lymphatics in the tumor of the peritoneum as well as the ovary causes the fluid to escape through the superficial lymphatic vessels of the tumor or induces increased vascular permeability.19 clinically, intraoperatively encountered widespread, grayish tan - colored, firm, tiny nodules can be mistaken as peritoneal carcinomatosis or disseminated tuberculosis, especially in cases showing calcification or psammoma bodies like the present patient.10,11 despite the rarity of ovarian immature teratomas with gp data and limitations, the prognosis of gp has long been regarded as good compared to that of immature teratoma without gp.14 however, the application of cisplatin - based chemotherapy regimens has dramatically improved the prognosis of immature teratomas of high grades and stages.16 recent studies suggested that the poor prognosis in immature teratoma is correlated with incomplete resection, tumor rupture, younger age, higher stage and grade, and gp, but not with cyst spillage, enucleation, nodal gliomatosis, or microfoci of yolk sac tumor, i.e., heifetz lesions.16 to the best of our knowledge, peritoneal and nodal gliomatosis as well as combined endometriosis are rare phenomena that have not yet been described in the english literature.	gliomatosis peritonei (gp) indicates the peritoneal implantation of mature neuroglial tissue and is usually accompanied by ovarian mature or immature teratoma. here, we report a case of ovarian immature teratoma associated with gliomatosis involving the peritoneum, lymph nodes and douglas ' pouch, where gliomatosis coexisted with endometriosis. as far as we know, only seven cases of gp have been reported as coexisting with endometriosis. eight cases with mature glial tissue in the lymph nodes, i.e., nodal gliomatosis, have been published either in association with gp or in its absence. metaplasia of pluripotent coelomic stem cells has been suggested to be responsible for the pathogenesis of endometriosis and gp rather than implantation metastases of ovarian teratomatous tumor with varying maturation. this theory is also applied to gp independently of ovarian teratomatous tumors. to the best of our knowledge, nodal gliomatosis coexisting with gp and also involving endometriosis has not yet been reported.
cancer stem cells are relatively resistant to conventional chemotherapy and radiotherapy. on the other hand, this population of cells might be the cells responsible for the relapse and progression of cancers after traditional cancer therapies. due to the lack of expression of differentiated tumor antigens on cancer stem cells, cancer stem cells may escape current immunologic interventions of therapy for cancer, which are mostly designed to target the antigens on the differentiated tumor cells. therefore, development of new strategies specifically targeting and destroying the cancer stem cells may hold promises to increase the therapeutic efficacy of current cancer treatment. to this end, we isolated cancer stem cell (csc)-enriched populations from two animal tumors (melanoma d5 and squamous cell cancer scc7), and used them as an antigen source to pulse antigen presenting cells (dendritic cells, dc) to prepare the csc - tpdc vaccine. we then evaluated the antitumor immunity induced by the csc - tpdc vaccine in the syngeneic immunocompetent hosts, b6 mice and c3h mice respectively. the csc - tpdc - induced antitumor efficacy was compared with the traditional dc vaccine pulsed with lysate from unsorted heterogeneous tumor cells (h - tpdc), which has previously been used by our group, as well as by other investigators both in preclinical studies and in clinical trials. cell sample preparation : prepare single cell suspension of tumor cells, either from cultured tumor cells or from freshly harvested tumor samples. count the cells and adjust cell suspension to a concentration of 1 x 10 cells / ml in aldefluor assay buffer. label four 12 mm x 75 mm polystyrene test tubes as control tubes as follows : # 1 : unstained, # 2 : aldefluor, # 3 : aldefluor plus deab, and # 4 : 7aad. for these control tubes, place 1ml cell suspension into # 1 and # 4, but 2 ml into tube # 2. add 5 l deab (aldh inhibitor) into tube # 3 and keep on ice. then add 10 l activated aldefluor substrate to tube # 2, mix and immediately transfer 1 ml of the mixture to tube # 3. at the same time, add 2 l activated aldefluor substrate per million cells to the rest of the cells (sample tubes) also at 1 x 10 cells / ml in aldefluor assay buffer. incubate both the 4 control tubes and the sample tubes for 30 min in 37 c water bath. following incubation, add 5 l 7aad into control tube # 4, and 1 l 7aad 1 x 10 cells to the sample tube. perform flow cytometry - based sorting. set the sorting gates using aldefluor - stained cells treated with deab as negative control and the propidium iodide (pi) 7aad- stained cells for viability control. based on these controls, a gate was established to distinguish the aldefluor+/aldh, d5 and scc7 cells. these gates will then be used to sort all the sample cells prepared above for the aldefluor+/aldh d5 and scc7 cells. euthanize mice (syngeneic b6 and c3h, respectively) using co2 and isolate femur and tibia bones. put the bones in 75% ethanol for 1 min at room temperature. cut head off bones and use a 21 g needle and 10 ml syringe to push cells into a dish. after centrifugation in 1,500 rpm for 5 min, discard the supernatant. then add 5 ml rbc lysis buffer to lysis the rbc for 1 min in 37 c water bath count cell number and adjust cell suspension to a concentration of 1 million cells / ml in complete medium (cm) containing 10 ng / ml gm - csf and 10 ng / ml il-4. culture the cells and compensate cm plus il-4 and gm - csf 3 days later. at the 5 day, harvest the immature dendritic cells (dcs) and prepare the dc isolation medium containing 4.2 ml solution c plus 1 ml optiprep density gradient medium. count cell number and adjust cell suspension to a concentration of 1 million cells / ml in culture medium containing 10 ng / ml gm - csf and 10 ng / ml il-4. prepare tumor lysates by suspending aldh or unsorted d5 or scc7 cells in culture medium and subject to rapid freeze - thaw exposures four times followed by spin at 100 x g for 5 min to collect the membrane portion of the lysates. to prepare csc - tpdc, pulse dcs with the lysate of autologous aldhcells. to prepare h - tpdc, pulse dcs with unsorted heterogeneous tumor cell lysate. vaccinate normal b6 mice respectively with 2,500 d5 csc - tpdc or d5 h - tpdc tumor cells subcutaneously (s.c.). vaccinate normal c3h animals s.c. with 5,000 scc7 csc - tpdc or scc7 h - tpdc tumor cells, respectively. after vaccination, challenge the b6 mice with the heterogeneous d5 tumor cells i.v. challenge the c3h mice with unsorted scc7 tumor cells s.c on the opposite side of the dc vaccine. monitor the tumor size. at the end of the experiments euthanize the b6 and c3h mice using co2. at day 34 after first vaccine, euthanize mice with co2, and at the same time collect the spleens of each group with an aseptic procedure. activate spleen t and/or b cells with immobilized anti - cd3 plus anti - cd28 mabs in cm containing hril-2 or lps plus anti - cd40 (fgk45) mab ascites. after activation, for elisa assay, coat plates with antibodies for ifn, gm - csf and igg at 4 c o / n. after the application of blocking buffer, add samples and standards and incubate at room temperature. wash the plate and add hrp detection antibody and tmb substrate for incubation. measure the absorbance on an elisa plate reader at 450 nm within 30 min after stopping the reaction. cell sample preparation : prepare single cell suspension of tumor cells, either from cultured tumor cells or from freshly harvested tumor samples. count the cells and adjust cell suspension to a concentration of 1 x 10 cells / ml in aldefluor assay buffer. label four 12 mm x 75 mm polystyrene test tubes as control tubes as follows : # 1 : unstained, # 2 : aldefluor, # 3 : aldefluor plus deab, and # 4 : 7aad. for these control tubes, place 1ml cell suspension into # 1 and # 4, but 2 ml into tube # 2. add 5 l deab (aldh inhibitor) into tube # 3 and keep on ice. then add 10 l activated aldefluor substrate to tube # 2, mix and immediately transfer 1 ml of the mixture to tube # 3. at the same time, add 2 l activated aldefluor substrate per million cells to the rest of the cells (sample tubes) also at 1 x 10 cells / ml in aldefluor assay buffer. incubate both the 4 control tubes and the sample tubes for 30 min in 37 c water bath. following incubation, add 5 l 7aad into control tube # 4, and 1 l 7aad 1 x 10 cells to the sample tube. perform flow cytometry - based sorting. set the sorting gates using aldefluor - stained cells treated with deab as negative control and the propidium iodide (pi) 7aad- stained cells for viability control. based on these controls, a gate was established to distinguish the aldefluor+/aldh, d5 and scc7 cells. these gates will then be used to sort all the sample cells prepared above for the aldefluor+/aldh d5 and scc7 cells. euthanize mice (syngeneic b6 and c3h, respectively) using co2 and isolate femur and tibia bones. put the bones in 75% ethanol for 1 min at room temperature. cut head off bones and use a 21 g needle and 10 ml syringe to push cells into a dish. after centrifugation in 1,500 rpm for 5 min, discard the supernatant. then add 5 ml rbc lysis buffer to lysis the rbc for 1 min in 37 c water bath count cell number and adjust cell suspension to a concentration of 1 million cells / ml in complete medium (cm) containing 10 ng / ml gm - csf and 10 ng / ml il-4. culture the cells and compensate cm plus il-4 and gm - csf 3 days later. at the 5 day, harvest the immature dendritic cells (dcs) and prepare the dc isolation medium containing 4.2 ml solution c plus 1 ml optiprep density gradient medium. count cell number and adjust cell suspension to a concentration of 1 million cells / ml in culture medium containing 10 ng / ml gm - csf and 10 ng / ml il-4. prepare tumor lysates by suspending aldh or unsorted d5 or scc7 cells in culture medium and subject to rapid freeze - thaw exposures four times followed by spin at 100 x g for 5 min to collect the membrane portion of the lysates. to prepare csc - tpdc, pulse dcs with the lysate of autologous aldhcells. to prepare h - tpdc, pulse dcs with unsorted heterogeneous tumor cell lysate. vaccinate normal b6 mice respectively with 2,500 d5 csc - tpdc or d5 h - tpdc tumor cells subcutaneously (s.c.). vaccinate normal c3h animals s.c. with 5,000 scc7 csc - tpdc or scc7 h - tpdc tumor cells, respectively. after vaccination, challenge the b6 mice with the heterogeneous d5 tumor cells i.v. euthanize the mice using co2 20 days later. challenge the c3h mice with unsorted scc7 tumor cells s.c on the opposite side of the dc vaccine. euthanize the b6 and c3h mice using co2. at day 34 after first vaccine, euthanize mice with co2, and at the same time collect the spleens of each group with an aseptic procedure. activate spleen t and/or b cells with immobilized anti - cd3 plus anti - cd28 mabs in cm containing hril-2 or lps plus anti - cd40 (fgk45) mab ascites. after activation, for elisa assay, coat plates with antibodies for ifn, gm - csf and igg at 4 c o / n. after the application of blocking buffer, add samples and standards and incubate at room temperature. wash the plate and add hrp detection antibody and tmb substrate for incubation. measure the absorbance on an elisa plate reader at 450 nm within 30 min after stopping the reaction. aldefluor / aldh has been used as a single marker to isolate stem cells in multiple malignancies. we identified cancer stem cell - enriched populations in two tumor models d5 and scc7 by using aldefluor as a marker. we found that aldefluor+ cells contribute approximately 0.5% and 5.2% in cultured d5 and scc7 tumor cell lines, respectively (figure 1). freshly harvested tumor cells from established tumors have been analyzed to confirm the existence of aldefluor+ cells. as also shown in figure 1, there were 2.5% and 4.2% of the aldefluor+ cells from in vivo established d5 and scc7 tumors, respectively. the tumorigenicity and the self - renewal capacity of these sorted d5 and scc7 aldefluor+/aldh populations were evaluated in the syngeneic immunocompetent host, the c57bl/6 and c3h mice, respectively. we have used heterogeneous unsorted tumor cell lysate to pulse dcs (h - tpdc) both in animal studies and in clinical trials. to examine the immunogenicity of cscs, we isolated aldh csc and pulsed dc with the lysate of the cscs to generated csc - tpdc (figure 2) and used h - tpdc as a conventional cancer vaccine control to test if csc - tpdc has any beneficial effect in preventing tumor growth. we evaluated the immunogenicity of cscs by examining protective antitumor immunity induced by csc - tpdc. in d5 model, nave immunocompetent mice were vaccinated s.c with csc - tpdc or h - tpdc (at the same lysate : dc ratio). one week after the last vaccine, the mice were challenged with unsorted d5 tumor cells intravenously (i.v.), and the lungs were harvested 3 weeks later to enumerate lung metastases. as shown in table 1, compared with pbs group, mice treated with the h - tpdc developed less lung metastases. importantly, mice treated with csc - tpdc had significantly fewer lung metastases than h - tpdc vaccine group in both experiments performed. in scc7 model, normal c3h animals were vaccinated s.c with scc7 csc - tpdc or h - tpdc respectively on the right flank, followed by challenging with unsorted scc7 tumor cells s.c into the left flank. compared with pbs group, h - tpdc induced modest anti - tumor immunity against tumor growth. however, there was significant inhibition of tumor growth in mice that were treated with csc - tpdc when compared with both the control group and h - tpdc group (p<0.05, figure 3). these results indicate that cscs could be used as an more effective antigen source to load dcs than traditional unsorted tumor cells in inducing protective immunity against the challenge of tumor cells. to further understand the mechanisms underlying the observed csc - induced protective antitumor immunity, we harvested the spleens from the animals subjected to dc vaccinations at the end of the experiments. the spleen cells were then activated by anti - cd3/cd28/il-2 or anti - cd3/cd28/il-2 + lps / anti - cd40. then the culture supernatants were collected to detect the expression of cytokines and antibody. there were significantly higher productions of ifn and gm - csf by the spleen cells from the animals vaccinated with d5 csc - tpdc or scc7 csc - tpdc (figure 4). furthermore, there was significantly (p<0.05) higher igg production by lps / anti - cd40 activated spleen cells collected from the animals vaccinated with d5 csc - tpdc or scc7 csc - tpdc compared with d5 h - tpdc or scc7 h - tpdc. these antibodies were found to bind to d5 and scc7 cscs respectively, and such binding could result in the csc lysis in the presence of complement. aldhfluor+/aldh populations were detected in cultured as well as newly harvested fresh murine d5 melanoma and scc7 squamous cell tumors. tumor cells treated with 50 mmol / l deab, a specific aldh inhibitor, were used as the negative control. click here to view larger image. generation of dendritic cell - based cancer stem cell vaccines. for the preparation of csc - tpdc and h - tpdc, bone marrow - derived dendritic cells were pulsed with aldh or unsorted tumor cell lysates, respectively. csc lysate pulsed dc (csc - tpdc) vaccine could induce more effective protective antitumor immunity in the s.c. more potent systemic cellular responses in immunocompetent host vaccinated with csc - tpdc. splenocytes were harvested from the animals subjected to h - tpdc or csc - tpdc vaccination, and were activated as indicated. the immunocompromised hosts, such as scid mice, preclude immunological assessments of cscs due to the lack of adaptive immunity within the hosts. in this study, we evaluated the immunogenicity of cscs in immunocompetent hosts, which could more closely mimic patient settings. enriched cscs are immunogenic and could induce more effective tumor protective immunity when their lysates are loaded to dcs as a vaccine compared with unselected tumor cell lysate - pulsed dcs. mechanistically, the protection was conferred by selective induction of csc - reactive antibodies and t cells as well as the production of type 1 cytokine, e.g. ifn and gm - csf. most of the current immunotherapies, including dendritic cell - based vaccines and the adoptive t cell transfer, are designed to target tumor- differentiated antigens. cscs, which may not express these differentiated antigens may therefore escape these immunological targeting. in contrast, csc vaccine designed to specifically target cancer stem cells may destroy this special population of the cancer cells, and thus improve the therapeutic efficacy of the vaccine by preventing tumor relapse and metastasis. in both cultured tumor cells and freshly harvested tumors, we identified csc - enriched population by flow cytometry based on high aldehyde dehydrogenase activity. such aldh cells could be isolated by flow sorting to be used as an antigen source to pulse dc to generate csc - tpdcs. comparison using aldh cells isolated from cultured tumor cells vs from freshly harvested tumors demonstrated no significantly difference in term of the induction of anti - csc immunity. these results revealed the potential to use cscs isolated either from cultured tumor cells or from freshly harvested tumors for clinical application. to be clinically relevant, a vaccine needs to be examined in a therapeutic setting.	we identified cancer stem cell (csc)-enriched populations from murine melanoma d5 syngeneic to c57bl/6 mice and the squamous cancer scc7 syngeneic to c3h mice using aldefluor / aldh as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (dcs). dcs pulsed with aldhhigh csc lysates induced significantly higher protective antitumor immunity than dcs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c. tumor growth setting, respectively. this phenomenon was due to csc vaccine - induced humoral as well as cellular anti - csc responses. in particular, splenocytes isolated from the host subjected to csc - dc vaccine produced significantly higher amount of ifn and gm - csf than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed - dc vaccine. these results support the efforts to develop an autologous csc - based therapeutic vaccine for clinical use in an adjuvant setting.
it is a common additive in agricultural antiseptics and also an intermediate in synthesis of organic chemicals. it has been used widely as a fungicide and an industrial synthetic material before its analogues were banned several decades ago. the chronic toxicity of hcb results in endocrine disruption, reproduction decrease, immune dysfunction, and neurobehavioral disorder [1, 2 ], which seriously damaged human health and attracted global concerns [3, 4 ], whereas hcb has been listed as one of the 12 priority persistent organic pollutants (pops) for a global phase - out according to the stockholm convention on persistent organic pollutants. due to its bioaccumulation, long - rang transport, and persistence, hcb has been observed all over the world in wastewater and sludge, water and sediment of rivers and lakes, biota, soil, and so forth. current treatments of hcb in the soil and sediment of contaminated sites mainly use surfactant (because surfactant can increase the solubility of hcb in water) solution for chemical washing, rendering hcb enter into the aqueous phase with the surfactants from the treated soil or sediment. it is therefore necessary to carry out subsequent treatment on the hcb - polluted effluent. so far, seldom reports were published on the treatment of the hcb - polluted effluent. at present, the treatments for hcb are performed by incineration, chemical degradation, biodegradation, photocatalytic oxidation, and the electrochemical method [69 ]. the electrochemical method is now emerging as a mild and environment - friendly process for destruction of pops, in which the hazardous material can be transformed into harmless products in a closed system, with no toxic emissions. the electrochemical destruction of chlorinated organic compounds has been extensively studied in aqueous solutions with various electrode materials, mainly metals, graphite, and composites [1115 ]. in some literature on electrochemical treatment of wastewater, researchers were concerned about the oxidation of organic pollutants on the anode, while the reduction behavior of cathode is often neglected. several studies [13, 1619 ] found that, under appropriate electrolysis conditions, organic substance can be reduced to small organic molecules on the cathode, suggesting that the cathodic reduction and degradation of organic matter should not be neglected. the cathode materials include metals, metal oxides, metals and composites of their oxides, and graphite. kumiko miyoshi. tried to use the electrode with titanium - based coatings as the cathode to perform the dechlorination treatment on 1,2,3-tcb. the results revealed that the dechlorination rate of electrode with ruo2/pt / pdo coatings was 91%, while that of ruo2 was 59%. additionally, pd and pt as well as some high hydrogen overvoltage metals such as ag, zn, cu, fe, pb, and ni were used as electrocatalytic cathodes for the reductive dehalogenation of chlorinated organic compounds. in this work, the simulated effluent of hcb contaminated soil was processed by an electrochemical approach. the optimal operation condition of electrochemical approach for degradation of hcb was studied. the degradation mechanism of hcb was investigated by using ruo2/ti as anode and zinc, stainless steel, graphite, and ruo2/ti as cathode, respectively, in a self - prepared electrolysis cell. the effect of different cathodes on electrochemical reduction of hcb and the degradation mechanism were also discussed. standard products of other series of chlorobenzenes were obtained from sigma - aldrich or fluka. the ph of solution was measured using a ph meter (shanghai, leici). all the other reagents were analytical grade and used without further purification. batch electrolysis was conducted in a self - made electrolytic cell of 200 ml with ruo2/ti as anode (42 cm) and different materials as cathode (42 cm). a laboratorial direct current power supply with current - voltage monitor was employed to provide electric power. the simulated wastewater was synthetic with an initial hcb concentration of 300 g / l, concentration of 1.0 g / l for the supporting electrolyte (na2so4), and an initial ph of 3.0. at regular time intervals, 5 ml of sample was drawn from the electrolysis cell to analyze the concentration of hcb and dechlorination products. hcb and dechlorination products in the supernatant were analyzed by a hewlett - packard 6890 gas chromatography (gc) equipped with an electron capture detector (ecd) and a zb-5 capillary column. the oven temperature was heated from 100 to 150c at the rate of 25c min, to 180c at 20c min, then to 190c at 40c min, and finally to 240c at 10c min. in order to achieve the maximal removal of hcb in the effluent by the electrochemical method, the operation conditions were first optimized with ruo2/ti as anode and stainless steel as cathode. the effects of the applied voltage, initial ph, initial hcb concentration, electrolysis time, and the concentration of electrolyte on the removal of hcb were investigated. the results showed that the best removal rate of hcb was up to 60.3% after 3 h with initial hcb concentration of 300 g / l, ph was at 3.0, with electrolyte concentration of 1.0%, voltage of 6 v, and tx-100 as a solubilizing agent. although the applied voltage, initial ph, hcb concentration, electrolysis time, and the concentration of electrolyte all have effects on the removal rate of hcb, the materials employed as anode and cathode play an important role. before a further study on the effects of electrode materials, a diaphragm was added in our self - made electrolysis cell to investigate whether the electrochemical process of hcb was mainly an anodic oxidation or a cathodic reduction process. in this process, the cathode compartment and the anode compartment of the electrolysis cell were separated by a cation - exchange membrane to prevent the generation of cl2 caused by the oxidation of cl under the hcb reduction on the anode surface. it was reported that if chloride ion was present in the solution, chlorine was produced on the anode and immediately reacted with water to form hypochlorite, which was a strong oxidizing agent and would react with ammonia during electrolysis. in our experiment, the results showed that the hcb concentration in cathode compartment was reduced gradually, while the hcb concentration in the anode one was nearly invariable after being electrolysed for 3 hours. the effect of different cathodes on the removal efficiencies of hcb was examined by using ruo2/ti as anode and zn, stainless steel, graphite, and ruo2/ti as cathode, respectively. figure 1 showed the removal efficiency of hcb with electrolysis time by using the four different cathode materials. as shown in figure 1, the removal efficiency of hcb all increased with electrolysis time in the cathodic compartment, respectively. this trend could be attributed to the fact that hydrogen atoms were a powerful reducing agent for dechlorination, which could enhance the removal efficiency of hcb. hcb exhibited the best effect with a removal efficiency of 53.2% after 3 h when zinc was used as the cathode, followed by stainless steel, of which removal efficiency of hcb was 43.4%. however, ruo2/ti was not found to be capable of accelerating the cathodic reduction during the electrochemical treatment process of hcb. instead, hcb showed the least effect when ruo2/ti was used as the cathode with an hcb removal efficiency of only 10.4% after 3 h. therefore, different cathode materials had different impact on the degradation of hcb. the descending order of removal efficiency was shown as follows : zinc > stainless steel > graphite > ruo2/ti. in all, it could be concluded that zinc cathode was more suitable for hcb reduction than stainless steel, graphite, and ruo2/ti cathodes because of its high hcb reduction rate. figures 2 and 3 showed all detectable byproducts with time varying during the hcb electrochemical reduction experiments by using zinc and stainless steel cathodes, respectively. distributions of different products were observed although the reductions were not complete till the end of these experiments. it was reported that nonchlorinated organic compounds were detected when dechlorination of hcb by a liquid potassium - sodium alloy, and the dechlorination rate achieved almost 100% after a 30 min reaction. however, none of nonchlorinated organic compounds was identified during our experiments. the reductive dechlorination by successive loss of chloride atoms was confirmed as the main pathway of the degradation of hcb using zinc and stainless steel cathodes. it was in agreement with the reduction studies of chlorinated compounds by the electrochemical treatment. in the experiments with zinc cathode, besides hcb, five other types of chlorobenzenes were also detected, whereas 1,2,4,5-tetrachlorobenzene was the dominant byproduct. the concentrations of pentachlorobenzene, tetrachlorobenzene, and trichlorobenzene increased with time and the increase of 1,2,4-tcb was the most obvious. for the stainless steel cathode, the concentration of pentachlorobenzene, 1,2,4,5-tetrachlorobenzene, 1,2,3,4-tetrachlorobenzene, and 1,2,4-trichlorobenzene increased with time and the increase of 1,2,3-tcb was the most obvious. these experiments showed the same trend with the byproducts that was observed when yang - hsin shih utilized pd / fe bimetallic nanoparticles to reduce hcb. figure 4 showed that the removal efficiency of hcb was 30.8% after 3 h when the graphite was utilized as the cathode, and pentachlorobenzene was the only detected degradation product. moreover, the removal efficiency of hcb was only 10.4% after 3 h when ruo2/ti electrodes were both used as the cathode and anode. it could be seen from figure 5, that the electrochemical reduction potential of zinc and stainless steel cathodes was higher than that of graphite and ruo2/ti cathodes. the oxidation process was reported in the treatment of chlorinated organic contaminants with zero - valent iron in oxygen - rich conditions and photocatalytic oxidation [24, 25 ]. in our experiment, however, none of the oxidation productions was tested during the electrochemical degradation of hcb. thus the degradation process of hcb was mainly reductive dechlorination and occurred on the cathode. the analysis on the intermediates of hcb dechlorination with different cathode materials indicated that, for the electrochemical degradation of hcb, different cathode materials may affect not only the removal efficiency of hcb, but also the degree of reductive dechlorination. it should be underlined that the mechanism of the reduction reaction could be different for the different electrodes, depending on the possible different reaction intermediates. the reason for electrochemical reductive dechlorination of chlorinated organic pollutants was that hydrogen atoms with chemical adsorption were generated on the electrode surface by water electrolysis and then the hydrodechlorination reaction followed. 2(h)adsm 2ohr cl + m (r cl)adsm(r cl)adsm + 2(h)adsm (r h)adsm + hcl(r h)adsm r h + m 2h2o + 2e + m 2(h)adsm 2oh r cl + m (r cl)adsm (r cl)adsm + 2(h)adsm (r h)adsm + hcl (r h)adsm r h + m hydrogenolysis depends on the first step, that is, whether the electrode can effectively adsorb the hydrogen atoms generated by electrolysis, and the third step was the key step of the dechlorination reaction. in the experimental process, ph value of the cathode chamber was observed to have a rapid increase to be alkaline, which was consistent with the above mechanism. regarding 1,2,3-tcb as a typical representative of persistent organic pollutants, kumiko miyoshi. selected different cathode materials including ruo2/pt / pdo, pt / iro2/ruo2, ruo2, pdo, pt, pdo / pt, pd / pt, pt, and pd to study the dechlorination process. by testing the intermediate dichlorobenzene of the step - by - step dechlorination, it was found that different cathode materials have different dechlorination paths ; it was mainly metadechlorination by ru, pd cathode, while it was mainly orthodechlorination by pt cathode. in terms of the chemical activities of the electrode materials, it was found that zn was the most active and then followed by stainless steel, graphite, and ruo2/ti. the electrons that reach the zn cathode in the electrolytic process can be release more quickly which are conducive for cathode materials to be more active. atomic hydrogen has strong reducibility, and it can replace the chlorine atoms on hcb, pentachlorobenzene, and tetrachlorobenzene. the possible reaction path when graphite was used as cathode is given in figure 6. analysis on the gas chromatography of hcb electrochemical products utilizing different cathode materials showed that cathode material can significantly affect the dechlorination extent of hcb. in this process, chlorine atoms were replaced one by one various intermediates such as pentachlorobenzene, tetrachlorobenzene, and trichlorobenzene occurred. the tetrachlorobenzene could be obtained when zinc and stainless steel are utilized as the cathode. electrochemical reduction of hcb using ruo2/ti anode and zn, stainless steel, graphite, and ruo2/ti cathodes in a self - made cell was studied. it was found that the degradation process of hcb was reductive dechlorination occurring only on the cathode. the removal efficiency of hcb was highly sensitive to the cathode materials and the descending order of removal efficiency was shown as follows : zinc > stainless steel > graphite > ruo2/ti. meanwhile, gas chromatography analysis for hcb electrochemical products showed that the type of cathode materials significantly affects the dechlorination extent of hcb. in the process, chlorine atoms were replaced one by one and various intermediates such as pentachlorobenzene, trichlorobenzene, tetrachlorobenzene were detected, and the tetrachlorobenzene was obtained when zinc and stainless steel were utilized as the cathode. rapid dechlorination of hcb suggests that the electrochemical method using zinc or stainless steel as cathode could be used for remediation of polychlorinated aromatic compounds in the environment.	hexachlorobenzene (hcb) is a persistent organic pollutant and poses great threat on ecosystem and human health. in order to investigate the degradation law of hcb, a ruo2/ti material was used as the anode, meanwhile, zinc, stainless steel, graphite, and ruo2/ti were used as the cathode, respectively. the gas chromatography (gc) was used to analyze the electrochemical products of hcb on different cathodes. the results showed that the cathode materials significantly affected the dechlorination efficiency of hcb, and the degradation of hcb was reductive dechlorination which occurred only on the cathode. during the reductive process, chlorine atoms were replaced one by one on various intermediates such as pentachlorobenzene, tetrachlorobenzene, and trichlorobenzene occurred ; the trichlorobenzene was obtained when zinc was used as cathode. the rapid dechlorination of hcb suggested that the electrochemical method using zinc or stainless steel as cathode could be used for remediation of polychlorinated aromatic compounds in the environment. the dechlorination approach of hcb by stainless steel cathode could be proposed.
nesidioblastosis is a condition with diffuse hyperplasia of the pancreatic islets, leading to hyperinsulinemic hypoglycemia. it is the most important differential diagnosis to insulinoma in the adult, but only 0.5% to 5.0% of the cases with hyperinsulinemic hypoglycemia can be attributed to noninsulinoma pancreatogenous hypoglycemia syndrome (niphs), which is currently more often seen in patients who have undergone bariatric surgery. to fulfill the diagnostic criteria, detection of endogenous hyperinsulinemic hypoglycemia, positive selective arterial calcium stimulation test (sacst), and negative imaging studies are required after exclusion of artificial causes of hypoglycemia such as inappropriate use of insulin or sulfonylurea. nonetheless, the results of the above - mentioned exams might be inconclusive, that is, small insulinomas might not be detected in imaging studies or large hyperplastic areas showing large gradients in sacst might be interpreted as insulinoma. thus, the final diagnosis can only be established after histopathologic examination. in mild cases, dietary modifications (low carbohydrate diet) pharmacological treatment options include diazoxide, acarbose, corticosteroids, verapamil, and octreotide. in patients developing severe symptoms partial or total we report a case of a patient with nesidioblastosis successfully treated with pasireotide, a somatostatin analog with high affinity for somatostatin receptor 5, originally developed for the treatment of cushing 's disease. for the herein presented case, the patient has provided written informed consent for publication. in 2009, a 46-year - old woman was admitted with a blood glucose level of 38 mg / dl (2.1 mmol / l). even upon intravenous glucose and glucose - rich diet, glucose levels did not exceed 90 mg / dl (5.0 mmol / l). the patient 's history revealed fatigue, sweating, craving for sweets over the last months and weight gain of 5 kg in 1 year. insulinoma was suspected. by performing an oral glucose tolerance test, reactive postprandial hypoglycemia could be ruled out. a consecutive 72 hours fast showed a decline in glucose to 34 mg / dl (1.9 mmol / l) after 14 hours of fasting. at that time the insulin level was inadequately in the normal range (13.0 u / ml, normal range : 2.025.0 u / ml) and c - peptide was elevated (8.2 ng / ml, normal range : 0.781.89 ng / ml), indicative of autonomous insulin secretion. imaging procedures including abdominal ultrasound, magnetic resonance imaging (mri), computed tomography scan (ct scan), fluorodeoxyglucose positron emission tomography (fdg - pet), dopamine receptor positron emission tomography (dopa - pet), octreotide receptor scintigraphy, and diagnostic laparotomy with palpation of the pancreas revealed no pathological findings. selective arterial calcium stimulation test (sacst) with hepatic venous sampling to determine the localization of hyperinsulinemia within the pancreas, showed a 2.1-fold, positive increase in insulin (> 2) measured in the hepatic vein after calcium injection in the great pancreatic artery. in patients with niphs, an increase in insulin is usually observed after injection of multiple arteries, in patients with insulinoma the response would be expected to be positive in 1 artery alone. in endoscopic ultrasound a hypoechogenic lesion in the pancreatic corpus at the height of the confluens could be located. as the results were inconclusive with regard to the localization of the lesion, portal venous sampling was additionally performed to differentiate localized (solitary insulinoma) from diffuse hyperinsulinism caused by adenomatosis, hyperplasia, and nesidioblastosis. this was confirmed by glucagon - like peptide-1 (glp-1) receptor scintigraphy showing an increased uptake not only in the head (physiologic) but also in the tail of the pancreas (figure 1 a and b). subsequently, distal pancreatectomy was performed, histopathologic examination of the pancreatic tail revealing hyperplasia of the islets of langerhans (figure 2). glucagon - like peptide-1 (glp-1) receptor scintigraphy showing an increased uptake both in the head and tail of the pancreas. microadenoma and enlarged lh islet : (a) h&e ; (b) synaptophysin ; (c) insulin ; (d) glucagon. (a) microadenoma in the vicinity of an enlarged lh islet (h&e). (b) the endocrine cells of the microadenoma and the lh islet stain with antibodies against synaptophysin. (c) the majority of the cells of the lh islet are positive, whereas most of the cells of the microadenoma are not labeled with insulin antibodies (d) but are positive with antibodies against glucagon. in contrast in the lh islet only the alpha cells are glucagon positive ; scale bars indicate 100 m. in addition, neuroendocrine microadenomas with immunohistochemical staining for glucagon in all of the adenoma cells were present (figure 3). co - occurrence of adult nesidioblastosis with microadenomas composed of alpha cells (figure 3) without elevation of serum glucagon, as measured on several occasions in the presented case, (figure 2c) has to the best of our knowledge not been described in the literature so far. nesidioblastosis with increased numbers enlarged of langerhans islets (a d) : (a) h&e ; (b) insulin ; (c) glucagon ; (d) srif (somatotropin release - inhibiting factor). (b) the islets consist of mainly of - cells (6070%) which are labeled with antibodies directed against insulin. (c) approximately 10% to 15% of the islet cells are decorated by antibodies against glucagon and (d) some islet cells are positive with antibodies against somatostatin. scale bars indicate 100 m. during the first 6 months following distal pancreatectomy, the patient was free of hypoglycemic symptoms. as the patient was hesitant toward any further surgical procedure, conservative medical therapy including administration of corticosteroids, everolimus, and octreotide was initiated ; however, all medical treatment approaches were futile. yearly diagnostic imaging follow - up including abdominal ultrasound, mri, octreotide receptor scintigraphy, and dopa - pet repeatedly showed no pathological finding. over time, hypoglycemia unawareness developed. subsequently, in the beginning of 2012, the patient was equipped with a continuous glucose monitoring (cgm) system (guardian rt, medtronic - minimed, northridge, ca) to alert her timely to prevent severe hypoglycemia. in the attempt to alleviate the burden of disease, pasireotide pasireotide is a somatostatin analog with a 40-fold increased affinity to somatostatin receptor 5 compared to other somatostatin analogs and is used in the treatment of cushing 's disease and acromegaly. it is known to suppress insulin secretion and thus to cause hyperglycemia as one of its most concerning side effects. pasireotide has been successfully applied in patients suffering from dumping syndrome with hypoglycemia. also, treatment with pasireotide in patients with severe post roux - en - y gastric bypass hyperinsulinemic hypoglycemia has been suggested. to the best of our knowledge after the first administration of pasireotide, glycemic levels started rising immediately, the carbohydrate requirements decreased, and the observed hypoglycemic levels were milder in their extent and mainly above the commonly accepted threshold for hypoglycemia (above 56 mg / dl [3.1 mmol / l ]) (table 1). the substance was well tolerated with only mild nausea that could be successfully treated with ondansetron. when comparing the cgm values prior to and 2 weeks following initiation of pasireotide therapy a significant increase in glycemia, reduced time in hypoglycemia, and increased time in eu- and even hyperglycemia could be observed. mean sensor glucose increased from 74 16 mg / dl (4.1 0.9 mmol / l) to 103 23 mg / dl (5.7 1.3 levels of hypoglycemia assessed by continuous glucose monitoring the last 14 days before initiation of pasireotide as compared to the first 14 days using pasireotide mean sensor glucose level over a 14-day period prior to (gray line) and immediately after (black line) initiation of pasireotide treatment. the patient has been successfully treated with pasireotide for 3 years now with marked improvement in quality of life. acth and cortisol were controlled regularly and have been in the normal range (table 2). imaging procedures (mri, somatostatin receptor scintigraphy, 18f - dopa - pet) were performed in yearly intervals and showed no abnormities over time. levels of acth (adrenocorticotropic hormone) and cortisol were monitored on a regular basis and did not show any clinically significant abnormalites in 2009, a 46-year - old woman was admitted with a blood glucose level of 38 mg / dl (2.1 mmol / l). even upon intravenous glucose and glucose - rich diet, glucose levels did not exceed 90 mg / dl (5.0 mmol / l). the patient 's history revealed fatigue, sweating, craving for sweets over the last months and weight gain of 5 kg in 1 year. a consecutive 72 hours fast showed a decline in glucose to 34 mg / dl (1.9 mmol / l) after 14 hours of fasting. at that time the insulin level was inadequately in the normal range (13.0 u / ml, normal range : 2.025.0 u / ml) and c - peptide was elevated (8.2 ng / ml, normal range : 0.781.89 ng / ml), indicative of autonomous insulin secretion. imaging procedures including abdominal ultrasound, magnetic resonance imaging (mri), computed tomography scan (ct scan), fluorodeoxyglucose positron emission tomography (fdg - pet), dopamine receptor positron emission tomography (dopa - pet), octreotide receptor scintigraphy, and diagnostic laparotomy with palpation of the pancreas revealed no pathological findings. selective arterial calcium stimulation test (sacst) with hepatic venous sampling to determine the localization of hyperinsulinemia within the pancreas, showed a 2.1-fold, positive increase in insulin (> 2) measured in the hepatic vein after calcium injection in the great pancreatic artery. in patients with niphs, an increase in insulin is usually observed after injection of multiple arteries, in patients with insulinoma the response would be expected to be positive in 1 artery alone. in endoscopic ultrasound a hypoechogenic lesion in the pancreatic corpus at the height of the confluens could be located. as the results were inconclusive with regard to the localization of the lesion, portal venous sampling was additionally performed to differentiate localized (solitary insulinoma) from diffuse hyperinsulinism caused by adenomatosis, hyperplasia, and nesidioblastosis. this was confirmed by glucagon - like peptide-1 (glp-1) receptor scintigraphy showing an increased uptake not only in the head (physiologic) but also in the tail of the pancreas (figure 1 a and b). subsequently, distal pancreatectomy was performed, histopathologic examination of the pancreatic tail revealing hyperplasia of the islets of langerhans (figure 2). glucagon - like peptide-1 (glp-1) receptor scintigraphy showing an increased uptake both in the head and tail of the pancreas. microadenoma and enlarged lh islet : (a) h&e ; (b) synaptophysin ; (c) insulin ; (d) glucagon. (a) microadenoma in the vicinity of an enlarged lh islet (h&e). (b) the endocrine cells of the microadenoma and the lh islet stain with antibodies against synaptophysin. (c) the majority of the cells of the lh islet are positive, whereas most of the cells of the microadenoma are not labeled with insulin antibodies (d) but are positive with antibodies against glucagon. in contrast in the lh islet only the alpha cells are glucagon positive ; scale bars indicate 100 m. in addition, neuroendocrine microadenomas with immunohistochemical staining for glucagon in all of the adenoma cells were present (figure 3). co - occurrence of adult nesidioblastosis with microadenomas composed of alpha cells (figure 3) without elevation of serum glucagon, as measured on several occasions in the presented case, (figure 2c) has to the best of our knowledge not been described in the literature so far. nesidioblastosis with increased numbers enlarged of langerhans islets (a d) : (a) h&e ; (b) insulin ; (c) glucagon ; (d) srif (somatotropin release - inhibiting factor). (a) increased number of enlarged langerhans islets (h&e). (b) the islets consist of mainly of - cells (6070%) which are labeled with antibodies directed against insulin. (c) approximately 10% to 15% of the islet cells are decorated by antibodies against glucagon and (d) some islet cells are positive with antibodies against somatostatin. scale bars indicate 100 m. during the first 6 months following distal pancreatectomy, the patient was free of hypoglycemic symptoms. as the patient was hesitant toward any further surgical procedure, conservative medical therapy including administration of corticosteroids, everolimus, and octreotide was initiated ; however, all medical treatment approaches were futile. yearly diagnostic imaging follow - up including abdominal ultrasound, mri, octreotide receptor scintigraphy, and dopa - pet repeatedly showed no pathological finding. over time, hypoglycemia unawareness developed. subsequently, in the beginning of 2012, the patient was equipped with a continuous glucose monitoring (cgm) system (guardian rt, medtronic - minimed, northridge, ca) to alert her timely to prevent severe hypoglycemia. in the attempt to alleviate the burden of disease, pasireotide pasireotide is a somatostatin analog with a 40-fold increased affinity to somatostatin receptor 5 compared to other somatostatin analogs and is used in the treatment of cushing 's disease and acromegaly. it is known to suppress insulin secretion and thus to cause hyperglycemia as one of its most concerning side effects. pasireotide has been successfully applied in patients suffering from dumping syndrome with hypoglycemia. also, treatment with pasireotide in patients with severe post roux - en - y gastric bypass hyperinsulinemic hypoglycemia has been suggested. to the best of our knowledge after the first administration of pasireotide, glycemic levels started rising immediately, the carbohydrate requirements decreased, and the observed hypoglycemic levels were milder in their extent and mainly above the commonly accepted threshold for hypoglycemia (above 56 mg / dl [3.1 mmol / l ]) (table 1). the substance was well tolerated with only mild nausea that could be successfully treated with ondansetron. when comparing the cgm values prior to and 2 weeks following initiation of pasireotide therapy a significant increase in glycemia, reduced time in hypoglycemia, and increased time in eu- and even hyperglycemia could be observed. mean sensor glucose increased from 74 16 mg / dl (4.1 0.9 mmol / l) to 103 23 mg / dl (5.7 1.3 levels of hypoglycemia assessed by continuous glucose monitoring the last 14 days before initiation of pasireotide as compared to the first 14 days using pasireotide mean sensor glucose level over a 14-day period prior to (gray line) and immediately after (black line) initiation of pasireotide treatment. the patient has been successfully treated with pasireotide for 3 years now with marked improvement in quality of life. acth and cortisol were controlled regularly and have been in the normal range (table 2). imaging procedures (mri, somatostatin receptor scintigraphy, 18f - dopa - pet) were performed in yearly intervals and showed no abnormities over time. levels of acth (adrenocorticotropic hormone) and cortisol were monitored on a regular basis and did not show any clinically significant abnormalites we present a case of nesidioblastosis with microadenomas expressing glucagon with recurrent hypoglycemic episodes after partial pancreatectomy unresponsive to usual therapeutic approaches. only initiation of therapy with pasireotide has re - established and maintained normoglycemia and a good quality of life for the affected patient for a total of 3 years. pasireotide is a somatostatin analog targeting 4 out of the 5 somatostatin receptor subtypes (sstr) with the highest affinity for sst1 and sst5. pasireotide has shown to effectively inhibit acth and corticosterone secretion both in vitro and in vivo and has proven effective in the treatment of cushing 's disease. although responders to pasireotide treatment showed decreased plasma acth and cortisol levels, increases in blood glucose and glycated haemoglobin levels were observed ; 46% of the treated patients in that specific study even required initiation of diabetes therapy. a study in healthy volunteers also reported elevated blood glucose levels 2 to 6 hours after single doses of pasireotide (2001200 g) which resolved within 23 hours after dosing. the described effect was more pronounced at higher doses of 600 to 1200 g pasireotide. in a mechanistic clamp study, henry demonstrated that hyperglycemia induced by pasireotide administration is caused by decreased insulin secretion and incretin hormone responses, without influencing the hepatic and/or peripheral insulin sensitivity. from the beginning we could observe the described effects of pasireotide on glycemia in our patient in support of previous studies showing effective application of pasiretoide in dumping syndrome and in a patient with postprandial hyperinsulinemic hypoglycemia following roux - en - y gastric bypass. in the latter patient, pasireotide inhibited insulin and glp-1 more efficiently than octreotide resulting in a better control of postprandial hyperinsulinemic hypoglycemia. inhibition of insulin secretion by islet beta cells is mediated by somatostatin receptor 2 (sstr2) and 5 (sstr5). as the affinity of pasireotide to sstr5 is much higher than to sstr2, pasireotide suppresses mostly insulin. another factor in the avoidance of severe hypoglycemia achieved by pasireotide might be its suppression of glp-1, as suggested by de heide. another effect of pasireotide rendering it successful in the treatment of neuroendocrine tumors could be its direct and indirect antitumor effects mediated via sstr, such as apoptosis and inhibition of cell proliferation. in patients with advanced neuroendocrine tumors refractory or resistant to octreotide lar therapy, pasireotide treatment was effective and well - tolerated in controlling symptoms of carcinoid syndrome in 27% of patients and resulted in stable disease in more than half of the patients. consistent with these data, pasireotide has been well tolerated by our patient for > 3 years now. according to the regularly monitored cgm signal the availability of which being one of the strengths of this case, hypoglycemia is well controlled and the patient is in euglycemia the majority of the time. further, as seen in the regularly performed imaging studies (mri, somatostatin receptor scintigraphy, dopa - pet), the discreet tracer uptake in dopa - pet, indicating that beta cell hyperplasia is still present, has remained stable. there has also been no growth of the pancreas following distal pancreatectomy, as seen in mri. another unusual feature in this case was the co - occurrence of nesidioblastosis and microadenomas consisting of alpha cells, which to the best of our knowledge has not yet been reported so far in adults. although genetic testing could rule out men1 syndrome, we can not exclude that other, so far unknown mutations could play a role in the pathogenesis of the microadenomas in our patient. finally, it may be speculated that regulatory mechanisms against the effects of nesidioblastosis could be involved in hyperplasia of alpha - cells and the formation of microadenomas ; these, however, are insufficient to counteract hyperinsulinism and hypoglycemia in the present case. data from the patient presented here and from the case report by de heide bring up the question as to the significance of pasireotide in the treatment of patients with niphs post - bariatric surgery in the future. the numbers of patients undergoing bariatric surgery are rising, consecutively causing higher rates of nesidioblastosis. especially in these patients pasireotide might be an interesting treatment option, as re - surgery might cause numerous complications including adhesions. however, further data are needed before pasireotide can be recommended as a treatment option in different scenarios of autonomous endogenous hyperinsulinemic hypoglycemia. expectations are raised, though, after successful application of pasireotide in postprandial hyperinsulinemic hypoglycemia after roux - en - y gastric bypass and upon the first successful long - term treatment with pasireotide of a patient with recurrent hypoglycemic episodes due to adult nesidioblastosis presented here.	abstractnesidioblastosis is a rare cause of endogenous hyperinsulinemic hypoglycemia in adults. diagnosis is often challenging and therapeutic options are scarce.in 2009, a 46-year - old female patient presented with recurrent severe hypoglycemia and immediate recovery after glucose ingestion. although 72-h - fasting test was positive, various imaging technologies (sonography, computed tomography, somatostatin receptor scintigraphy, dopamine receptor positron emission tomography [dopa - pet ]) were negative. endoscopic ultrasound revealed a lesion in the pancreatic corpus, whereas selective arterial calcium stimulation test, portal venous sampling and glp-1-receptor scintigraphy were indicative of a lesion in the pancreatic tail, which was surgically removed. the histopathologic examination revealed beta cell hyperplasia and microadenomas expressing glucagon. after surgery, the patient was free of symptoms for 6 months, after which hypoglycemic episodes recurred. after unsuccessful treatment with corticosteroids and somatostatin analogs, treatment with pasireotide, a novel somatostatin analog with high affinity to somatostatin receptor 5 and a possible side effect of hyperglycemia, was initiated (0.6 mg bid). to date, our patient has been free of severe hypoglycemic episodes ever since. yearly repeated imaging procedures have shown no abnormities over the last 3 years.we report for the first time that pasireotide was successfully used in the treatment of adult nesidioblastosis.
diagnosing vitreous changes, especially a posterior vitreous detachment (pvd), is important for predicting the prognosis and determining the indication for vitreoretinal surgery in many vitreoretinal diseases. in diabetic retinopathy, a partial posterior vitreous detachment (p - pvd) or the absence of a pvd is a risk factor for retinal neovascularization, but a complete posterior vitreous detachment (c - pvd) is a strong negative risk factor for retinal neovascularization.1,2 vitreous attachment to the retina is the scaffold for neovascularization arising from the retina. retinal neovascularization never arises from the retina in eyes with a c - pvd. in certain cases with diabetic macular edema (dme), vitreous traction or attachment to the macula worsens the macular edema.3 in these cases, vitreous surgery is indicated in addition to laser treatment or intravitreous injection of an anti - vascular endothelial growth factor drug or triamcinolone. rhegmatogenous retinal detachments due to retinal holes in the lattice degeneration in eyes without a pvd, usually found in young patients, progress slowly. however, because rhegmatogenous retinal detachments due to retinal tears associated with a pvd progress rapidly, emergency surgery is needed. optical coherence tomography (oct) can visualize perifoveal pvds with pinpoint vitreofoveal traction in cases with an idiopathic macular hole, but biomicroscopy cannot.4 a macular hole does not develop in the fellow eye of one with a macular hole when there is a shallow pvd over the fovea, even if there is no evidence of a pvd by biomicroscopy. there are three primary methods for detecting a pvd : biomicroscopy, ultrasonography, and oct. this article presents classifications of pvds by slit - lamp biomicroscopy and classifications of shallow pvds by oct. before slit - lamp biomicroscopy, the pupil should be dilated fully to obtain a wide illumination - observation angle for optical sections of the vitreous. to obtain a sharp optical section of the vitreous we usually use the sl 130 slit lamp (carl zeiss meditec ag, jena, germany) or slit lamp bm 900 with a stereo validator (haag - streit, bern, switzerland) slit - lamp biomicroscope for biomicroscopic vitreous examinations. three types of precorneal lens the goldmann three - mirror contact lens, the non - contact positive preset lens, and the wide - angle funduscopic contact lens should be available for a comprehensive biomicroscopic vitreous examination. in most cases, the posterior vitreous can be examined using a non - contact positive preset lens such as the superfield nc lens (volk, mentor, oh, usa). the technique of biomicroscopic vitreous examination has been previously published in detail.5 for oct vitreoretinal examinations, pupil dilation is preferred but not needed in all cases. time - domain (td) oct does not have sufficient resolution to evaluate the minute changes in the vitreoretinal interface. spectral - domain oct (sd - oct) is needed to evaluate the morphologic changes in the vitreoretinal interface. we usually use a cirrus hd oct (carl zeiss meditec) and select the 9-mm five - line raster mode including the optic disc and the macula. before slit - lamp biomicroscopy, the pupil should be dilated fully to obtain a wide illumination - observation angle for optical sections of the vitreous. to obtain a sharp optical section of the vitreous we usually use the sl 130 slit lamp (carl zeiss meditec ag, jena, germany) or slit lamp bm 900 with a stereo validator (haag - streit, bern, switzerland) slit - lamp biomicroscope for biomicroscopic vitreous examinations. three types of precorneal lens the goldmann three - mirror contact lens, the non - contact positive preset lens, and the wide - angle funduscopic contact lens should be available for a comprehensive biomicroscopic vitreous examination. in most cases, the posterior vitreous can be examined using a non - contact positive preset lens such as the superfield nc lens (volk, mentor, oh, usa). the technique of biomicroscopic vitreous examination has been previously published in detail.5 for oct vitreoretinal examinations, pupil dilation is preferred but not needed in all cases. time - domain (td) oct does not have sufficient resolution to evaluate the minute changes in the vitreoretinal interface. spectral - domain oct (sd - oct) is needed to evaluate the morphologic changes in the vitreoretinal interface. we usually use a cirrus hd oct (carl zeiss meditec) and select the 9-mm five - line raster mode including the optic disc and the macula. the classification of pvds by slit - lamp biomicroscopy is shown in figure 1 and table 1. first, the vitreous condition is classified according to the presence or absence of a pvd. the pvd then is subclassified as a c - pvd or a p - pvd. the c - pvd is further divided into a c - pvd with and without collapse, while a p - pvd is divided into a p - pvd with and without shrinkage of the posterior hyaloid membrane (p - pvd with shrinkage and p - pvd without shrinkage, respectively). a p - pvd without shrinkage has a subtype characterized by vitreous gel attachment through the premacular hole in a posterior hyaloid membrane to the macula (p - pvd without shrinkage[m ]). case 1 (figure 2, video 1 http://www.youtube.com/watch?v=gprcsxarbmy) shows asteroid hyalosis, which facilitates diagnosing the absence of a pvd. in this case, the posterior vitreous did not separate from the retina even after ocular movement. case 2 (figure 3, video 2 http://www.youtube.com/watch?v=h2hwj0ixoja) shows a normal eye without a pvd. although it was difficult to determine the absence of a pvd when compared with case 1, the tyndall phenomenon in the slit section of the vitreous and no evidence of a posterior hyaloid membrane with a prepapillary glial ring even after ocular movement established the absence of a pvd. case 3 (figure 4, video 3 http://www.youtube.com/watch?v=m0hl-sy_osq) is a normal eye with a c - pvd with collapse. a mobile, highly detached posterior hyaloid membrane was traced easily and a prepapillary glial ring was also observed on the posterior hyaloid membrane, making it easy to diagnose a c - pvd with collapse. case 4 (figure 5, video 4 http://www.youtube.com/watch?v=aaamakrd57m) had diabetic retinopathy after panretinal photocoagulation with a c - pvd without collapse. a shallowly detached posterior hyaloid membrane was traced just in front of the retina and then diagnosed as a c - pvd without collapse. the eyes of young patients with central retinal vein occlusion and uveitis also frequently have this type of pvd. case 5 (figure 6, video 5 http://www.youtube.com/watch?v=-rscnmdb7sc) had proliferative diabetic retinopathy with a p - pvd with shrinkage. a posterior hyaloid membrane, including the neovascular proliferative tissue, was not mobile even after ocular movement. this vitreous change caused strong vitreous traction to the retina along the retinal vascular arcade. case 6 (figure 7, video 6 http://www.youtube.com/watch?v=ftmpos5q2co) shows a normal eye with a p - pvd without shrinkage. however, the inferior vitreous did not detach from the inferior retina even after ocular movement. most cases with this type of a pvd are thought to be in a transient phase that will evolve into a c - pvd with collapse. case 7 (figure 8, video 7 http://www.youtube.com/watch?v=nrkupvfrwoy) had dme with a p - pvd without shrinkage. although most of the vitreous was detached from the retina, a detached posterior hyaloid membrane was traced except for over the macula. the vitreous gel was attached through a premacular hole in the posterior hyaloid membrane, and this vitreous attachment caused weak but chronic vitreous traction on the macula. although this vitreous pathology is somewhat difficult to detect by biomicroscopy, sebag and balazs clearly showed it using in vitro slit - lamp microscopy.6 td - oct does not have sufficient resolution to detect a shallow pvd and can not detect a highly detached pvd. a previous study showed the inferiority of td - oct for detecting a pvd compared with ultrasonography and slit - lamp - biomicroscopy.7 however, sd - oct can detect a shallow pvd that is undetectable by slit - lamp biomicroscopy. although some types of shallow pvds can be detected by td - oct,8 various types of shallow pvds and their associations with macular diseases have been reported9 more clearly since the development of sd - oct. slit - lamp biomicroscopy did not detect a shallow pvd that was observed by oct. a shallow pvd is initially classified as the absence or presence of a shallow pvd. the shallow pvd is then classified as a shallow pvd without shrinkage of the posterior vitreous cortex, a shallow pvd with shrinkage of the posterior vitreous cortex, or a peripheral shallow pvd. a shallow pvd without shrinkage of the posterior vitreous cortex has two subtypes : an age - related shallow pvd and a perifoveal pvd associated with a macular hole. the absence of a pvd can be diagnosed by oct when cloquet s canal or vitreous cortex and gel over the posterior retina are observed. however, there are many cases of the absence of a pvd without evidence of cloquet s canal or vitreous cortex and gel over the posterior retina (figure 9). to precisely diagnose the absence of a pvd, slit - lamp biomicroscopy should be performed in addition to the oct examination. in many cases, it is impossible to diagnose the absence of a pvd using only oct. at first, an age - related shallow pvd develops around the macula, which then progresses to one of three types : a shallow pvd without formation of a premacular hole in the posterior vitreous cortex, a shallow pvd with formation of a premacular hole in the posterior vitreous cortex with vitreous gel attachment to the macula, or a shallow pvd with formation of a premacular hole in the posterior vitreous cortex without vitreous gel attachment to the macula (figure 10). the primary type of age - related shallow pvd, a perimacular shallow pvd, is seen frequently ; the latter two types of shallow pvd are rarely observed because the liquefied vitreous enters the subhyaloid space through the premacular hole in the posterior vitreous cortex then induces the rapid formation of a high pvd, which can be detected by slit - lamp biomicroscopy but not oct (figure 11). a perifoveal pvd with pinpoint vitreous traction to the fovea the foveal retina is very thin and can be punched out easily with weak vitreous traction. during macular - hole progression, liquefied vitreous is initially trapped in the subhyaloid space and pushes the posterior vitreous cortex anteriorly to induce the pinpoint vitreous traction to the fovea (figure 12). a premacular hole in the posterior hyaloid membrane observed in an age - related shallow pvd seems to be protective of the thin fragile fovea from the vitreous traction. a full - thickness macular hole is observed with an operculum associated with a shallow pvd throughout the posterior pole in a late - stage macular hole (classic gass stage 3).10 in some cases, abortion of the macular hole can occur in association with vitreofoveal separation in the early stage11 (stage 1 or 2) (figure 13).12 therefore, a macular hole does not develop in the eye of patients with a macular hole in their other eye when there is a shallow pvd over the fovea, even if there is no evidence of pvd by biomicroscopy.4 a shallow pvd with shrinkage of the posterior vitreous cortex affects the macular function in many vitreoretinal diseases. most cases of epiretinal membrane (erm) have a shallow pvd with shrinkage of the posterior vitreous cortex (figure 14). in some erm cases, a shallow pvd is not present (figure 15). in advanced cases of erm, a tractional retinal detachment due to vitreomacular traction syndrome can develop (figure 16). in such cases we have previously reported that this pathology is observed by oct in some cases of retinitis pigmentosa.13 although posterior vitreoschisis can be confused with a shallow pvd with shrinkage of the posterior vitreous cortex, it is important that it be distinguished from this. the major difference can be seen by comparing the case with posterior vitreoschisis with the case with a shallow pvd with shrinkage of the posterior vitreous cortex shown in figure 17 ; in the former, some cortex can be seen on the retinal surface while this is not visible in the latter. in certain cases with dme, vitreous traction on the macula due to a shallow pvd with shrinkage of the posterior vitreous cortex vitreous surgery is more effective than laser treatment or intravitreous injection of an anti - vascular endothelial growth factor drug or triamcinolone to treat the dme. some cases of retinal tears have no evidence of a pvd on biomicroscopy. in such cases, a retinal tear can develop from a peripheral shallow pvd (figure 19), and scleral buckling is more appropriate than vitrectomy in such cases. the classification of pvds by slit - lamp biomicroscopy is shown in figure 1 and table 1. first, the vitreous condition is classified according to the presence or absence of a pvd. the pvd then is subclassified as a c - pvd or a p - pvd. the c - pvd is further divided into a c - pvd with and without collapse, while a p - pvd is divided into a p - pvd with and without shrinkage of the posterior hyaloid membrane (p - pvd with shrinkage and p - pvd without shrinkage, respectively). a p - pvd without shrinkage has a subtype characterized by vitreous gel attachment through the premacular hole in a posterior hyaloid membrane to the macula (p - pvd without shrinkage[m ]). case 1 (figure 2, video 1 http://www.youtube.com/watch?v=gprcsxarbmy) shows asteroid hyalosis, which facilitates diagnosing the absence of a pvd. in this case, the posterior vitreous did not separate from the retina even after ocular movement. case 2 (figure 3, video 2 http://www.youtube.com/watch?v=h2hwj0ixoja) shows a normal eye without a pvd. although it was difficult to determine the absence of a pvd when compared with case 1, the tyndall phenomenon in the slit section of the vitreous and no evidence of a posterior hyaloid membrane with a prepapillary glial ring even after ocular movement established the absence of a pvd. case 3 (figure 4, video 3 http://www.youtube.com/watch?v=m0hl-sy_osq) is a normal eye with a c - pvd with collapse. a mobile, highly detached posterior hyaloid membrane was traced easily and a prepapillary glial ring was also observed on the posterior hyaloid membrane, making it easy to diagnose a c - pvd with collapse. case 4 (figure 5, video 4 http://www.youtube.com/watch?v=aaamakrd57m) had diabetic retinopathy after panretinal photocoagulation with a c - pvd without collapse. a shallowly detached posterior hyaloid membrane was traced just in front of the retina and then diagnosed as a c - pvd without collapse. the eyes of young patients with central retinal vein occlusion and uveitis also frequently have this type of pvd. case 5 (figure 6, video 5 http://www.youtube.com/watch?v=-rscnmdb7sc) had proliferative diabetic retinopathy with a p - pvd with shrinkage. a posterior hyaloid membrane, including the neovascular proliferative tissue, was not mobile even after ocular movement. this vitreous change caused strong vitreous traction to the retina along the retinal vascular arcade. case 6 (figure 7, video 6 http://www.youtube.com/watch?v=ftmpos5q2co) shows a normal eye with a p - pvd without shrinkage. however, the inferior vitreous did not detach from the inferior retina even after ocular movement. most cases with this type of a pvd are thought to be in a transient phase that will evolve into a c - pvd with collapse. case 7 (figure 8, video 7 http://www.youtube.com/watch?v=nrkupvfrwoy) had dme with a p - pvd without shrinkage. although most of the vitreous was detached from the retina, a detached posterior hyaloid membrane was traced except for over the macula. the vitreous gel was attached through a premacular hole in the posterior hyaloid membrane, and this vitreous attachment caused weak but chronic vitreous traction on the macula. although this vitreous pathology is somewhat difficult to detect by biomicroscopy, sebag and balazs clearly showed it using in vitro slit - lamp microscopy.6 td - oct does not have sufficient resolution to detect a shallow pvd and can not detect a highly detached pvd. a previous study showed the inferiority of td - oct for detecting a pvd compared with ultrasonography and slit - lamp - biomicroscopy.7 however, sd - oct can detect a shallow pvd that is undetectable by slit - lamp biomicroscopy. although some types of shallow pvds can be detected by td - oct,8 various types of shallow pvds and their associations with macular diseases have been reported9 more clearly since the development of sd - oct. slit - lamp biomicroscopy did not detect a shallow pvd that was observed by oct. a shallow pvd is initially classified as the absence or presence of a shallow pvd. the shallow pvd is then classified as a shallow pvd without shrinkage of the posterior vitreous cortex, a shallow pvd with shrinkage of the posterior vitreous cortex, or a peripheral shallow pvd. a shallow pvd without shrinkage of the posterior vitreous cortex has two subtypes : an age - related shallow pvd and a perifoveal pvd associated with a macular hole. the natural course of an age - related pvd is as follows. the absence of a pvd can be diagnosed by oct when cloquet s canal or vitreous cortex and gel over the posterior retina are observed. however, there are many cases of the absence of a pvd without evidence of cloquet s canal or vitreous cortex and gel over the posterior retina (figure 9). to precisely diagnose the absence of a pvd, slit - lamp biomicroscopy should be performed in addition to the oct examination. in many cases, it is impossible to diagnose the absence of a pvd using only oct. at first, an age - related shallow pvd develops around the macula, which then progresses to one of three types : a shallow pvd without formation of a premacular hole in the posterior vitreous cortex, a shallow pvd with formation of a premacular hole in the posterior vitreous cortex with vitreous gel attachment to the macula, or a shallow pvd with formation of a premacular hole in the posterior vitreous cortex without vitreous gel attachment to the macula (figure 10). the primary type of age - related shallow pvd, a perimacular shallow pvd, is seen frequently ; the latter two types of shallow pvd are rarely observed because the liquefied vitreous enters the subhyaloid space through the premacular hole in the posterior vitreous cortex then induces the rapid formation of a high pvd, which can be detected by slit - lamp biomicroscopy but not oct (figure 11). a perifoveal pvd with pinpoint vitreous traction to the fovea is seen frequently in cases of idiopathic macular holes. the foveal retina is very thin and can be punched out easily with weak vitreous traction. during macular - hole progression, liquefied vitreous is initially trapped in the subhyaloid space and pushes the posterior vitreous cortex anteriorly to induce the pinpoint vitreous traction to the fovea (figure 12). a premacular hole in the posterior hyaloid membrane observed in an age - related shallow pvd seems to be protective of the thin fragile fovea from the vitreous traction. a full - thickness macular hole is observed with an operculum associated with a shallow pvd throughout the posterior pole in a late - stage macular hole (classic gass stage 3).10 in some cases, abortion of the macular hole can occur in association with vitreofoveal separation in the early stage11 (stage 1 or 2) (figure 13).12 therefore, a macular hole does not develop in the eye of patients with a macular hole in their other eye when there is a shallow pvd over the fovea, even if there is no evidence of pvd by biomicroscopy.4 a shallow pvd with shrinkage of the posterior vitreous cortex affects the macular function in many vitreoretinal diseases. most cases of epiretinal membrane (erm) have a shallow pvd with shrinkage of the posterior vitreous cortex (figure 14). in some erm cases, a shallow pvd is not present (figure 15). in advanced cases of erm, a tractional retinal detachment due to vitreomacular traction syndrome can develop (figure 16). in such cases, vitrectomy should be performed. we have previously reported that this pathology is observed by oct in some cases of retinitis pigmentosa.13 although posterior vitreoschisis can be confused with a shallow pvd with shrinkage of the posterior vitreous cortex, it is important that it be distinguished from this. the major difference can be seen by comparing the case with posterior vitreoschisis with the case with a shallow pvd with shrinkage of the posterior vitreous cortex shown in figure 17 ; in the former, some cortex can be seen on the retinal surface while this is not visible in the latter. in certain cases with dme, vitreous traction on the macula due to a shallow pvd with shrinkage of the posterior vitreous cortex vitreous surgery is more effective than laser treatment or intravitreous injection of an anti - vascular endothelial growth factor drug or triamcinolone to treat the dme. a retinal tear can develop from a peripheral shallow pvd (figure 19), and scleral buckling is more appropriate than vitrectomy in such cases. the classification of pvd by slit - lamp biomicroscopy and classification of shallow pvd by oct contribute to predicting the prognosis and determining the indication for vitreo - retinal surgery in many vitreoretinal diseases.	diagnosing a posterior vitreous detachment (pvd) is important for predicting the prognosis and determining the indication for vitreoretinal surgery in many vitreoretinal diseases. this article presents both classifications of a pvd by slit - lamp biomicroscopy and of a shallow pvd by optical coherence tomography (oct). by biomicroscopy, the vitreous condition is determined based on the presence or absence of a pvd. the pvd then is classified as either a complete posterior vitreous detachment (c - pvd) or a partial posterior vitreous detachment (p - pvd). a c - pvd is further divided into a c - pvd with collapse and a c - pvd without collapse, while a p - pvd is divided into a p - pvd with shrinkage of the posterior hyaloid membrane (p - pvd with shrinkage) and a p - pvd without shrinkage of the posterior hyaloid membrane (p - pvd without shrinkage). a p - pvd without shrinkage has a subtype characterized by vitreous gel attachment through the premacular hole in a posterior hyaloid membrane to the macula (p - pvd without shrinkage [m ]). by oct, a shallow pvd is classified as the absence of a shallow pvd or as a shallow pvd. a shallow pvd is then subclassified as a shallow pvd without shrinkage of the posterior vitreous cortex, a shallow pvd with shrinkage of the posterior vitreous cortex, and a peripheral shallow pvd. a shallow pvd without shrinkage of the posterior vitreous cortex has two subtypes : an age - related shallow pvd and a perifoveal pvd associated with a macular hole.
today, silage is the world 's largest fermentation process, with an estimated 287 million tons produced in eu alone. for dairy and beef cattle farmers, their purpose is to produce more high - quality silage, rich in energy and protein. the key factors influencing the feeding value of silages include crop characteristics, stage of crop development at ensiling, and the extent and type of fermentation achieved within the silo. it is widely accepted that silage additives can increase animal intake and animal performance through their effect on silage quality. however, the market became reluctant to use acid additives as they were considered corrosive to machinery and concrete, and dangerous to those farm operatives who had to use them. successful silage production depends on the promotion of fermentation by beneficial bacteria, and therefore bacterial inoculants have been very popular, especially over the last 10 years. microbial inoculants containing homofermentative lactic acid bacteria (lab), in most of the cases lactobacillus plantarum, are often added to silage because they very quickly produce large quantities of lactic acid, which lowers the ph of the silage [7, 8 ]. however, classic microbial inoculants often have no effect or can even make the aerobic stability of silages worse [7, 9, 10 ] because yeasts metabolize lactic acid to produce alcohol. recently, the aerobic stability of a variety of silage crops has been markedly improved through inoculation with a heterolactic acid bacterium. improvements in aerobic stability brought about by this organism have been reported in corn silage. a strain of heterofermentative lab lactobacillus brevis has been reported as a promising strain for improving the aerobic stability of silages. the aim of this trial was to study the effect of a silage inoculant on the nutrient content, the silage quality, the aerobic stability, and the nutritive value of whole plant ensiled corn, as well as on the feed intake and growth performance of fattening young cattle. a trial was carried out with whole plant corn harvested at the milk / dough stage of maturity (32.3% dm, see table 1) and used for ensiling, treated (bsm), or not (ct), with a silage inoculant. the corn was chopped using a conventional forage harvester massey ferguson 5130 and ensiled directly after harvest in 2 horizontal silos with a capacity of 200 tones. the inoculant application rate of the commercial product was the rate recommended by the manufacturers (blend of enterococcus faecium, lactobacillus plantarum, and lactobacillus brevis, dsm numbers 3530, 19457, and 23231 respectively ; 4 g of product / ton of silage diluted in 4 l of water) to guarantee a concentration of 1 10 cfu / g of material. the inoculant was applied uniformly using an applicator which was fixed on the harvester between the pick - up reel and cutting rollers. after weighing the untreated or inoculated chopped corn, five control bags (made from four layers of cheesecloth) filled with 1 kg of ensiling mass were put in each silo to determine dm losses. the silos were filled within 48 hours and were covered with polythene sheet and weighted down with tires. five representative samples of harvested and chopped whole - plant corn were taken throughout the harvesting - ensilaging period. silages were sampled every other week during the feeding experiment (14.12.2009 to 14.04.2010). at each sampling time two samples (approx. 500 g each) were taken 4050 cm deep from the cut surface by coring vertically to the full depth of the silo using a 50 mm - silage corer. volatile fatty acid and lactic acid, as well as alcohol concentrations, were determined by gas - liquid chromatography on aqueous silage extracts obtained from steeping 30 g of fresh silage in 150 ml of deionized water for 16 hours at 4c in a sealed container. this was followed by a preliminary filtering through 3 m filter paper. deionized water (3 ml) from an internal standard solution (0.5 g 3-methyl - n - valeric acid in 1000 ml 0.15 mol / l oxalic acid) was added to 1 ml of filtrate from the above, and the solution was filtered through a 0.45 m polyethersulfone membrane into a chromatographic sample vial for analysis. a gas - liquid chromatograph shimadzu 2010 was used with a wide - bore capillary column (stabilwax - da 30 m, 0.53 mm, i d, 0.5 m) according to the gas chromatography and biochemistry analyzer official methods. aerobic stability was measured using data loggers which recorded temperature readings once every 6 hours from thermocouple wires placed in three replicate 1.5002.000 g silage representative samples, which were aerated in open plastic bags and placed into open - top polystyrene boxes (volume about 3 liters). aerobic deterioration was denoted by days (or hours) until the start of a sustained increase in temperature by more than 2c above the ambient temperature. for the animal feeding trial, 40 young beef cattle (8 - 9 months old) with similar mean live weight were used and divided into two analogous groups (20 animals each)., all animals were fed with untreated silage (ct) and a diet (ct + 4 kg of a commercial compound feed + 1 kg of barley straw) as during the experiment. during the experiment, each group was divided again into four subgroups, of five bulls each, and placed in four separate pens. fresh silages were offered ad libitum twice daily, allowing for at least 10% orts (as - fed basis). every 10 days the amount of silage fed and the refused silage were weighed over 2 consecutive days in order to calculate the daily silage intake. silage dm intake was calculated per group as the difference between the amount of silage supplied and the amount of silage remaining. the compound feed was individually offered to all animals twice per day in a fixed amount. barley straw was included in the diet (1 kg / animal / day ; 88% of dm, energy value of 3.9 mj me / kg dm). the animals were individually weighed on the first day of the experimental period and then once per month, and on the final day of the experiment. the average weight gain and growth rates were calculated for each animal and for each group. feed conversion ratio was calculated as the ratio between feed intake and body weight gain. data was analyzed using variance analysis to test for the effect of silage treatments (genstat, 1987). for the feed intake and feed conversion rates, a subgroup, of 5 beef cattle, was considered as the experimental unit. for body weight and daily weight gain, respectively, each animal within a group was considered an experimental unit. the fisher 's least significant difference (lsd) procedure at the 5% significance level was used to determine statistical differences between treatments. a probability of 0.05 < p < 0.10 was considered a near - significant trend. the use of bsm significantly improved the silage quality compared to the ct (table 2). the silage treated with bsm showed statistically significant higher dm recovery and digestible protein, coinciding with merry., lower dm losses (p < 0.01 for all) and higher crude protein content (p < 0.05). its adf content was significantly lower, and the metabolizable energy was higher (p < 0.05), whereas the digestible energy content was highly significant in the treated silage compared to the untreated silage. there were no significant differences between untreated and treated silages in crude fiber, nfe, wsc, and ndf content. bsm treatment increased fermentation rates in whole - crop corn silages, resulting in a significant ph decrease (p < 0.01) and a significant increase in total organic acids concentration (p < 0.05) compared to the ct (table 3). the lactic acid content in the bsm treatment was also significantly higher (p < 0.01) since homofermentative lab were used. the acetic acid content of the bsm treatment was numerically higher than that of the ct. silage inoculation with bsm significantly decreased concentrations of butyric acid, ethanol, and ammonia - n (p < 0.01) of corn silage compared to the ct. homofermentative silage inoculants by improving silage fermentation can reduce wasteful end products such as ammonia - n and volatile fatty acids, which result in poorer feed conversion efficiency and higher in - silo dry matter losses [1618 ]. the use of silage inoculants containing homofermentative lactic acid bacteria, to increase lactic acid production and enhance the rate and extent of ph decline [1921 ], can also lead to a reduction in protein breakdown. as shown in table 2, the bsm silage treatment decreased dm losses by 3.0% (p < 0.01) and had higher digestible energy (de) and metabolic energy (me) concentrations by 2.3 and 1.00% (p < 0.01 and p < 0.05), respectively, compared to the untreated ct silage. during aerobic exposure after opening the silos, the ct (figure 1) started to heat after 66 hours, had a temperature increase of more than 2c above the ambient temperature after 84 hours, and reached a temperature of more than 14c above the ambient temperature after 186 hours. the temperature rise in the bsm treatment of more than 2c above the ambient temperature occurred after 156 hours and reached a maximum temperature (+ 6c above the ambient temperature) after 234 hours. therefore, bsm silage was more stable by 72 hours (3 days) compared to the ct. recently, silage studies with whole - crop corn silages using obligatory heterofermentative lab l. buchneri as an inoculant showed a 20-fold increase in the aerobic stability of the silage, which increased from approximately 40 hours for untreated silages to more than 790 hours for the inoculated silages. other studies [12, 23 ] provide more definitive evidence for the existence of certain lab strains with the power to inhibit yeast and molds growth, and to improve aerobic stability. some authors have described the positive aspect of the formation of acetic acid by heterofermentative lactic acid bacteria, which inhibits spoilage organisms [24, 25 ]. no statistical differences were found between animals fed with bsm or ct silages at 0, 31, and 63 trial days in the live weight (table 4). from the data presented it is obvious that, throughout the whole trial, animals fed with bsm silage achieved higher average weights compared to those from the ct. at the end of the experiment the difference in body weight reached 8.1 kg / animal, and this was considered a near - significant trend (0.05 < p < 0.10). average daily weight gains (adwgs) for bsm and ct are shown in table 5. between 0 and 31 trial days neither statistically, nor numerically marked, differences in adwg were found between the treatments ; however, in the trial period between 32 and 63 days the differences in adwg show a near - significant trend (0.05 < p < 0.10) with a p value of 0.055. the adwg in the last third of the feeding trial period (from 64 to 100 days), and throughout the whole trial period (0 to 100 days), showed a statistically significant difference (p < 0.01) of 138 and 80 g, respectively. in order to avoid differences due to different moisture contents, the intake is shown in table 6 on the dm basis. the silage dm intake for bsm was higher by 6.14% compared to the ct (3.97 versus 3.74 kg dm / animal / day) and had a near - significant trend (p = 0.065). as expected, because of the restricted feeding, no differences were found in compound feed dm intake. ; however, some researchers found that feeding microbial inoculated silage to cattle does not affect dry matter intake compared to noninoculated silage [26, 26 ]. a combination of increased dm intake and higher energy, in the silage treated with bsm, led to a significant increase (p < 0.05) in metabolizable energy intake compared to those animals fed with the ct. the animals receiving bsm had a better conversion of energy into body weight compared to that of the ct because they needed 2.37 mj of me (3.4%) less to increase the body weight by 1 kg the microbial silage inoculant which was tested, based on homo- and heterofermentative lactic bacteria, had a significant positive effect on whole - crop corn silage quality in terms of lowering ph and shifting fermentation towards lactic acid, suppressing butyric acid, ethanol, and ammonia - n formation, significantly reducing dm losses, statistically increasing digestible and metabolizable energy, statistical significant improvement of aerobic stability, and improvement of the silage intake and the performance of beef cattle, and a positive effect on the utilization of feed energy. therefore, it is concluded that using such a silage inoculant improves the whole - plant corn silage characteristics and the nutritive value for beef cattle.	the effect of inoculation on nutrient content, fermentation, aerobic stability, and beef cattle performance for whole - plant corn silage treated with a commercial product (blend of homo- and heterofermentative lactic acid bacteria, bsm, blend of enterococcus faecium, lactobacillus plantarum, and lactobacillus brevis, dsm numbers 3530, 19457, and 23231, resp.), was compared to a control treatment with no silage additives (ct). the material had a dm of 323 g / kg, crude protein, and water - soluble carbohydrate concentrations of 87.9 and 110.5 g / kg dm, respectively. bsm increased the fermentation rate with a significantly deeper ph (p < 0.01), a significant increase in the total organic acids concentration (p < 0.05), more lactic acid (p < 0.01), and numerically more acetic acid compared to ct. bsm significantly decreased the concentrations of butyric acid (p < 0.01), ethanol, and ammonia - n compared to the ct. bsm - treated silage decreased dm by 3.0 % (p < 0.01) and had a higher digestible energy and a higher metabolizable energy concentration by 2.3 (p < 0.01) and 1.00 % (p < 0.05), respectively, compared to untreated silage. aerobic stability improved by more than 2 days in bsm silage. the dm intake of silage treated with bsm increased by 6.14 %, and improved weight gain and the feed conversion by 8.0 (p < 0.01) and 3.4%.
intra - abdominal hypertension (iah) and abdominal compartment syndrome (acs) are increasingly common in critically ill patients following massive fluid resuscitation, severe abdominal trauma and extensive abdominal surgery.[13 ] these conditions left untreated have been associated with significant morbidity and mortality. increased intra - abdominal pressure (iap) monitoring and consensus definitions and recommendations have lead to greater recognition of this condition and patient survival. acs is defined as a sustained iap of > 20 mmhg that is associated with new organ dysfunction or failure. there have been some reports of temporary improvement in iap following neuromuscular blockade (nmb) in patients with iah, but all eventually required surgical management.[79 ] in this report, we describe a patient who was definitively treated with only nmb, with complete resolution of symptoms. a 48-year - old morbidly obese african - american male with a medical history of congestive heart failure, hypertension, obstructive sleep apnea on 2 l of home oxygen and non - complaint with continuous positive airway pressure at night presented to the emergency department (ed) with worsening shortness of breath and lower extremity oedema over one week. vital signs in the ed were temperature, 98.7 degrees fahrenheit ; blood pressure, 116/66 mmhg ; heart rate, 130 beats per minute ; respiratory rate, 30 ; oxygen saturation, 85% on 2 l nasal cannula ; weight, 162 kg and body mass index, 47. physical examination revealed a morbidly obese male in acute respiratory distress with an irregularly irregular pulse and one plus lower extremity pitting oedema. laboratory values in the ed include white blood cell count, 9 800/l ; haemoglobin, 14.2 g / dl ; platelet count, 230 000/l ; sodium, 140 meq / l ; potassium, 4.5 meq / l ; chloride, 104 meq / l ; bicarbonate, 29 meq / l ; blood urea nitrogen (bun), 38 mg / dl ; creatinine, 1.4 mg / dl ; glucose, 105 mg / dl ; aspartate aminotransferase (ast), 51 u / l ; alanine aminotransferase (alt), 40 u / l ; alkaline phosphatase, 68 u / l ; total bilirubin, 1.5 mg / dl ; albumin, 3.4 g / dl ; total protein, 7.1 g / dl ; international normalised ratio (inr), 1.2 ; myoglobin, 97 g / l ; troponin, 0.07 ng / ml and b - type natriuretic peptide, 934 pg / ml. arterial blood gas showed a ph 7.33, paco2 68.5 and pao2 88.6 on fio2 of 40%. echocardiogram revealed a myopathic severely enlarged left ventricular chamber with moderate concentric left ventricular hypertrophy and severe global hypokinesis with an ejection fraction of 8 to 10%. the patient was admitted to the step - down unit with a new diagnosis of atrial fibrillation. on hospital day number eight, his initial rhythm was ventricular fibrillation and he was shocked with 200 j twice before regaining pulses. when stabilised, he was transferred to the intensive care unit (icu) where he regained consciousness and full neurologic function within one hour of the event. intraoperatively, the patient had runs of ventricular tachycardia with hypotension to 80 mmhg systolic. postoperatively, the patient was found to have a right fixed mid - dilated pupil, flaccid paralysis of the left arm and leg with hyperreflexia throughout the left side. computed tomography (ct) of the head revealed a right midbrain and thalamic stroke thought to be secondary to transient arrhythmia - induced hypoperfusion, intraoperatively. on postoperative day number three, after aicd placement and stroke, the patient acutely developed a rigid distended board - like abdomen with dullness to percussion. vitals at that time were temperature, 98.4 degrees fahrenheit ; blood pressure, 112/67 mmhg ; heart rate range, 55 beats per minute, and respiratory rate 26 on ventilator settings of assist control volume control with a rate of 26 ; tidal volume, 500 ml ; fio2, 40% and positive end - expiratory pressure of 5. his peak inspiratory pressure on the ventilator had risen from 26 to 72 mmhg ; his plateau had risen from 15 to 48 mmhg with oxygen desaturation from 93 to 85%. the patient had received 1.5 l in and urinated 4.5 l over the 24 hours, leading up to this event. his laboratory values from that morning were unchanged from previous (white blood count, 10 400/l ; haemoglobin, 12.6 g / dl ; platelets, 200 000/l ; sodium, 139 meq / l ; potassium, 4.7 meq / l ; chloride, 100 meq / l ; bicarbonate, 34 meq / l ; bun, 36 mg / dl ; creatinine, 1.41 mg / dl ; glucose, 78 mg / dl ; calcium, 8.3 mg / dl ; magnesium, 2.0 meq / l ; phosphorus, 3.0 mg / dl ; ast, 77 u / l ; alt, 94 u / l ; alkaline phosphatase, 186 u / l ; total bilirubin, 1.3 mg / dl). this constellation of findings prompted the team to measure an abdominal compartment pressure which was elevated to 53 cm h2o (40 mmhg) with an abdominal perfusion pressure (app) of 42 mmhg. a ct scan of the abdomen and pelvis was performed which showed no dilated loops of bowel, no ileus, no evidence of free air, abscess or obstruction and minimal free fluid [figure 1 ]. pertinent labs from the event revealed a lactate of 1.0 mg / dl ; lipase, 159 u / l ; amylase, 76 u / l ; creatinine, 1.53 mg / dl. surgery was consulted and the fio2 was increased to 100%, which improved his oxygen saturation to 96% despite his elevated peak inspiratory pressures. computed tomography of the abdomen and pelvis showing no dilated loops of bowel, ileus, free air, abscess or obstruction surgical recommendations were for an emergent bedside surgical abdominal decompression. the patient was administered 10 mg of intravenous cisatracurium besylate by anaesthesia in preparation for surgical management. shortly following paralytic administration, his peak inspiratory pressure corrected from 72 to 23 mmhg, his plateau pressure from 48 to 16 mmhg and his oxygenation status tolerated a decrease in fio2 down to 40% with his oxygen saturation returning to baseline. measurement of his abdominal compartment pressure revealed a drop from 53 (40 mmhg) to 23 cm h2o (17 mmhg) and then normalised to 15 cm h20 (11 mmhg) over a three - hour period. during this entire event, the patient averaged 100 to 200 cc / hr of urine output and laboratory values from the following day showed a drop in creatinine from 1.53 to 1.29 mg / dl. to our knowledge, this is the first documented case of nmb definitively treating acs. a few case reports and one series discuss the use of nmb to temporarily ameliorate iah, but none have shown benefit in patients with acs.[79 ] de laet. performed the first prospective trial of nmb in the management of iah, reporting temporary reductions in iap in nine out of ten patients. the single patient who did not respond had an iap of 25 mmhg (grade iv iah), leading the authors to suggest nmb is not effective in severe iap or acs. the world society of the abdominal compartment syndrome state that nmb may be considered in selected patients with mild to moderate iah while other interventions are being performed (grade 2c). our patient met criteria for acs based on iap of 40 mmhg associated with worsening hypoxia and respiratory distress. his app (app = map iap) was 42 mmhg (82 40 mmhg). with this reduction in perfusion pressure, his creatinine increased from 1.41 to 1.53 mg / dl. his peak inspiratory pressures went as high as 72 mmhg, while his oxygen saturation dropped to 85% on a fio2 of 40%. numerous risk factors for the development of iah and acs have been suggested in an attempt to predict high - risk patients and explain the pathophysiology of this disorder. independent risk factors identified by three large prospective trials include large volume fluid resuscitation (> 3.5 l/24hours) ; pancreatitis, pulmonary, renal and liver dysfunction ; acidosis, hypothermia ; abdominal surgery ; coagulopathy and ileus.[25 ] despite the wide variety of risk factors, the final pathway remains the same ; hypoperfusion of the intra - abdominal organs leading to hypoxia and proinflammatory cytokine release. cytokines in concert with oxygen radicals promote capillary leakage causing oedema and the ever increasing swollen tissue further raises the iap leading to a feed - forward cascade. nmb is thought to improve iah by increasing abdominal wall compliance, thus decreasing the iap by increasing the total amount of abdominal compartment volume through muscle relaxation. although there is no consensus on how nmb ameliorates acs, it may be that early treatment of iah / acs stops the cycle of tissue abdominal organ hypoxia and oedema before it spirals out of control. this patient had no preceding acute volume resuscitation, ischaemia, acidosis, hypothermia or inflammation. this episode of acs was likely due to spastic contractions of his abdominal muscles decreasing abdominal wall compliance and increasing abdominal pressure. numerous causes have been reported, mostly secondary to intra - abdominal inflammatory conditions, which are not relevant to this case. it has also been reported with abdominal cutaneous nerve root entrapments which are more common in obese individuals like our patient. epilepsia partialis continua, secondary to an acute stroke, has also been reported to cause contraction of the abdominal wall musculature, and this is also remotely possible, given our patient 's recent large stroke. this includes evacuation of the intraluminal contents by nasogastric tubes, rectal tubes and prokinetics,[1618 ] removal of intraperitoneal fluid or ascites by paracentesis and improvement of abdominal wall compliance through adequate sedation, elevation of the head of bed and potentially nmb. in addition to removing intraluminal and extraluminal fluid, nmb should be attempted to ameliorate the pressure. in rare cases like ours	a 48 year old male admitted to the intensive care unit after a cardiac arrest complicated by a stroke intra - operatively during automatic implantable cardioverter defibrillator placement. he post - operatively developed a rigid abdomen, elevated peak and plateau pressures, hypoxia and renal insufficiency. he was diagnosed with abdominal compartment syndrome with an intra - abdominal compartment pressure of 40mmhg. the patient was administered 10 mg of intravenous cisatracuriumbesylate in preparation for bedside surgical abdominal decompression. cisatracurium eliminated the patients need for surgical intervention by reducing his abdominal compartment pressures to normal and improving his hypoxia and renal function. this case illustrates that neuromuscular blockade should be attempted in patients with abdominal compartment syndrome prior to surgical intervention.
unintentional poisonings, which mostly occur at home, are a major cause of nonfatal injuries in children aged 024 months. in 2003, 49.6% of all the reported poisonings exposures in the usa occurred among preschool children. the annual incidence of poisoning in dutch children aged 1 - 2 years is 3 per 1000 children, which is much higher compared to other age groups, for example, one - year - olds have a six - fold risk of poisoning compared to four - year - olds. most cases of poisoning in children aged 1 - 2 years occur due to unsafe storage of medicines and cleaning products (e.g., detergents, chloride, and other cleaners). examples of preventive action could include, according to guidelines, the use of child - resistant packaging, child - safe storage of potentially poisonous products, and extra attention and supervision when the possible hazardous products are in use [1, 48 ]. efforts should be made to promote poisoning preventive actions, including child - safe storage of potentially poisonous products (e.g., placed above adult eye level, or in a locked cabinet) [1, 48 ]. previous research showed a large variation in characteristics between parents who do and do not store medications or cleaning products child safe. in order to develop effective intervention strategies to improve parental safety behavior, more insight into the underlying psychosocial mechanisms and potential important and modifiable mediators health promoting behaviors are influenced by a complex, interrelated set of so - called mediators or determinants of behaviors, which include various cognitions and environmental factors. by using behavioral theories, like the protection motivation theory, one can go beyond basic unchangeable risk factors (e.g., gender, socioeconomic status) in order to explain why and how people change their behavior [10, 11 ]. until now, use of behavioral theories and models in unintentional injury prevention research is only marginally represented in the literature. the protection motivation theory (pmt) is a framework particularly suited for interventions of protective, precautionary behaviors. according to the pmt, the probability of health protective behavior or an adaptive responsein this case (child) safe storage is increased by four beliefs : (1) the threat is perceived as severe (severity), and (2) as of high personal relevance (vulnerability) ; (3) the adaptive response is perceived as effective for warding off the threat (response efficacy), and (4) the personal abilities and self - confidence to engage in the adaptive response is perceived as high (self - efficacy). according to the pmt, the probability of an adaptive response is decreased by the perceived rewards of a maladaptive response, for example, not using safety locks, and the perceived costs or barriers of the adaptive response (advantages and disadvantages of safe behavior) [13, 14 ]. we adopted this model for the present study to assess the influence of personal cognitions and attitudes on parental safety behavior and additionally included three social influence factors. in the present study, we assessed the psychosocial correlates of parental safety behaviors concerning unintentional poisoning from medicines and cleaning products, among parents of toddlers aged 1118 months in order to determine the most important changeable factors associated with safe storage of medications and cleaning products. a model based on the protection motivation theory with the inclusion of additional social variables was applied. in 2004 all parents (n = 2470) with at least one child aged 1118 months who were part of an opportunity sample of six preventive youth healthcare providers in the netherlands, both urban and rural, were invited by their preventive healthcare centers to complete a mailed questionnaire. the parents received a pre - survey letter from their healthcare provider informing them about the research and the survey was mailed to them by post. these six centers were chosen because of their ongoing collaboration with the erasmus university medical center in rotterdam. the preventive youth health care providers working in the centers have an average reach of parents of children in the age of 04 years old of 98%, indicating that all parents in the participating areas which fitted the selection criteria were invited to participate in the study. the parents were informed that the study was about home safety issues aiming to improve the safety information provided by preventive youth healthcare providers. up to two reminders were sent by mail (one after 10 days and the other after 21 days), no financial incentives were offered, parents were assured of confidentiality, and the results were processed anonymously. the questionnaire was developed and pilot tested among 25 parents, and refined based on interviews with these parents. these parents were requited through one of the participating centers. the questionnaire, with 160 questions, addressed child safe storage behavior of medication and cleaning products, standard socio - demographic variables as well as potential correlates of safe storage behavior (measured in pmt constructs). storage was measured by asking whether the respondents stored the various products, on the floor or in a drawer / cupboard without a lock, lower than 1.5 meter, in a drawer / cupboard without a lock, higher than 1.5 meter, or in a drawer / cupboard with a lock or safety catches. the first two answers were considered as unsafe storage, and the latter two as storing the products in a child safe manner. we adopted this model for the present study to assess the influence of personal cognitions and attitudes on parental safety behavior. in addition, we included three social influence factors in the explanatory model in order to assess the influence of these additional constructs on this behavior. the following social influence factors were assessed : perceived social support (or pressure), subjective norm, and descriptive norm. perceived social support can be considered as the direct perceived influence of significant others (e.g., by receiving mental support to perform the desired behavior). subjective norm is the perceived expectations of significant others (e.g., does my partner expect me to store medicines out of reach ?). descriptive norm refers to an individual 's perception of how much and how often others perform the behavior [1517 ]. potential correlates of storage of possibly poisonous products were measured within the domain of pmt factors (perceived vulnerability and severity of the potential accident, response efficacy of safety preventive behaviors, general self - efficacy to perform safety preventive behaviors, and perceived advantages and disadvantages of safe behavior), psychosocial factors (active encouragement from other parents (social support), subjective norm, and descriptive norm), and demographic variables. the demographic variables included in this study were chosen based on earlier studies indicating the influence of these variables on safety behavior [9, 18 ] (i.e., age / walking ability of the child, number of children in the family, ethnicity / employment status / educational level of the mother and educational level of the father). perceived vulnerability was measured by asking respondents their perception of the risk of their child unintentionally swallowing medicines or cleaning products (2 = low risk ; + 2 = high risk). perceived severity was measured with one item asking how seriously they perceived the consequences of this event (2 not serious ; + response efficacy was measured by asking parents if they thought that storing medications and cleaning products out of reach of children could help to prevent possible accidents (2 = not very helpful ; + 2 = very helpful). self - efficacy was assessed using items, which referred to the respondents ' perception of their ability to store medications and cleaning products out of reach of children (2 = very difficult ; + 2 = very easy). perceived advantages of the safe behavior were assessed and measured with two questions (spearman ranging from 0.70 to 0.77). perceived disadvantages of the safe behavior were also assessed on a two - sided five - point scale and measured with two questions (spearman ranging from 0.63 to 0.67). all items to assess pmt and other psychosocial constructs were measured on bipolar five - point scales. for constructs that were assessed with multiple items, the mean social support was measured by asking respondents if they received support from significant others to store medications and cleaning products out of reach of children (2 = no support ; + 2 = many support). subjective norm was assessed by asking if they perceived that their significant others thought storing medication and cleaning products out of reach of children is necessary, ranging from certainly not descriptive norm was measured by asking respondents to assess how many people they perceived in their direct social environment to store medications and cleaning products out of reach of children in the same age category as their children (2 = no body ; + 2 = every body). employment status of the parents was defined as employed when they had either a part - time or fulltime job. the educational level of the father and mother was divided into low and high (intermediate secondary education or less versus at least higher secondary education). walking ability of the child differences in the proportions and means of all potential correlates in the model were tested by chi - square for the dichotomous demographic variables and mann - whitney u test for the pmt and social factors. to determine significant correlates of safe storage of medicines and cleaning products, multiple hierarchical logistic regression analyses were performed, with safe behavior as the dependent variable (no / yes) and the various factors (demographic, pmt, and social) as independent variables. two sets of multiple logistic regression analyses were conducted for safe storage of medicines and cleaning products, respectively. in both models, demographic variables were entered as a first block, since these variables were considered to be the more distal, nonmodifiable potential correlates. subsequently blocks including the pmt (block 2) and social factors (block 3) were entered in the model. explained variance was calculated with nagelkerke r. effect sizes were used as indicator of the explanatory value of the model. nine questionnaires (0.5%) were excluded from the analyses because they had been incorrectly completed (n = 4) or because the questionnaire was not completed for the selected child but for an older sibling (n = 5) ; thus, 1722 questionnaires were used in the analyses. the mean age of the respondents (parent or guardian, no grandparents participated) was 32.4 years (range 1660 ; sd 4.5) ; 90.1% were mothers. in this study, 97.5% of the families included two parents ; 43.0% had one child (the child selected for the study). the age of the children ranged from 11 to 18 months (mean 13.5 ; sd 1.4) ; 47.0% were girls (table 1). medications and cleaning products were reported to be stored in a child - safe manner by, respectively, 74.4% (n = 1282) and 60.5% (n = 1042) of the respondents. respondents who stored their medications or their cleaning products in a child - safe manner had a significantly lower perceived vulnerability, lower perceived disadvantages, higher perceived severity, self - efficacy and advantages of the safe behavior, and more positive social influences (table 2). respondents who stored their cleaning products in a child - safe manner also had a higher perceived response efficacy (table 2). adding each block resulted in a significant increasing percentage of explained variance. in the first step, the number of children in the home was a significant variable, but this explained only 8% (nagelkerke r) of the variance in the safe storage behavior. more than one child living in the home increased the likelihood that medication was stored in a child - safe manner. in the second step when pmt factors were entered, perceived vulnerability, self - efficacy, and disadvantages of the safe behavior were significantly associated with safe storage of medication and the explained variance increased to 22%. in the third step, when social factors were included, both social support and descriptive norm proved to be additional significant correlates and the explained variance increased to 24% indicating a medium effect size. in the first step, the number of children in the family was a significant variable but explained only 6% of the variance in safe storage of cleaning products. more than one child living in the home increased the likelihood that cleaning products were stored in a child safe manner. in the second step, perceived vulnerability, self - efficacy, and disadvantage of the safe behavior were also significantly associated with safe storage of cleaning products and together explained 32% of the variance. in the third step, social factors were included and these explained 33% of the variance in safe storage of cleaning products, indicating a large effect size. in addition, descriptive norm proved to be significantly correlated to safe storage of cleaning products. this study shows that perceived vulnerability, self - efficacy, perceived disadvantages of safe behavior, descriptive norm, and number of children in the family were significantly associated with safe storage of the studied potentially poisonous substances in households with toddlers. from our study, it can be concluded that the pmt model is applicable to predict the safe storage of possible poisons, even more so for storage of cleaning products than for medication. the associations of some of the separate psychosocial correlates included in our study were similar to the results in previous studies. for example, earlier studies showed differences in parents who do and do not take injury preventive behaviors in their perceptions of the vulnerability to an injury [11, 20 ], beliefs about the response efficacy of taking preventive measures, and perceived social norms [20, 21 ]. furthermore, our results concerning the explained variance in safe storage (2433%) are in line with morrongiello & kiriakou (2004) who were able to explain 32% of the variance in safety behavior related to prevention of poisonings in general. the response rate was high, but we do not know whether families who refused to participate differed in demographic characteristics. (2001), it is unlikely that the children of nonresponders differed from the responders in this study. furthermore, the demographic characteristics of the participants in our study (age, employment status, and educational level) reflected those of the general dutch population and compare well with the distribution of these characteristics in a previous dutch random sample of parents with preschool children [23, 24 ]. the results might be different in other locations but do seem to be representative for the dutch situation, and comparable regions first, because our study relied on self - report of medication and cleaning products storage by parents, misclassification might have occurred, for example, parents might have given socially desirable answers (overstating safe storage and supervision of the child when products were stored unsafely) or might not have been fully aware of the identity of all poisonous products in their home [20, 2527 ]. this might result in an underestimation of households with unsafe product storage, and bias in the assessment of significant correlates. we were only able to assess the storage practices of parents related to potential poisonous substances (e.g., no information was available on child supervision during use of poisonous products). additional data on the use of possible hazardous products might indicate a higher exposure to possible poisonings among children than found in this study and would have provided more insight into parents ' injury preventive behavior concerning poisoning. on the other hand, this would have led to a more complex outcome measure and an increased probability of misclassification. the parents who do not store their medications and cleaning products in a child - safe manner perceived their child to be more vulnerable to possible unintentional poisoning than parents who do store their products in a safe manner. which is in practice a justified feeling of the parents, in their home these possible poisonous products are not stored safe, thus it is true that their child is probably more vulnerable to a poisoning injury. furthermore, the parents who do not store the products in a child - safe manner estimate the severity of a possible poisoning as being lower than parents who do store their products in a child - safe manner. this lower estimation of the severity of a possible poisoning may explain why a subgroup of parents does not store the medication and cleaning products in a child - safe manner. the strong significant contribution of both social influence (descriptive norm) and self - efficacy in the prediction of protection behavior indicates that parents are influenced by what they (perceive to) observe in their environment and what they perceive they themselves can do. our data also show differences in the mean perceived severity and response efficacy of safe storage of medications and cleaning products, respectively. this suggests that even within the different types of injuries, in this case poisoning, determinants of engaging in safety practices vary. this finding indicates that safety promoting messages should not be generalized to overall injury prevention message, but that poisoning prevention, fall prevention, and burn prevention should be approached in various ways according to the most important determinants of the behavior. further research should be executed to reject or support these findings, with that we recommend including a broader age and products range in future studies to give a better insight in possible poisonings in homes with young children. to increase parents ' safe storage behavior, insight into potentially important and changeable mediators the study findings yield some recommendations for developing programs to prevent unintentional poisonings due to unsafe storage. our study indicates that the promotion of safe storage of medication and cleaning products should address the family situation, and personal cognitive factors as well as social factors. for example, interventions focusing on behavioral change concerning prevention of poisoning should be open about the disadvantages of the safe behavior and with that point out that when performing the safe behavior the disadvantages will diminish. furthermore, interventions should focus particularly on parents ' self - efficacy, for example, show how can one store poisonous products safely, and perceived vulnerability of their child concerning a possible poisoning event, for example, point out hazardous situations in the home related to poisoning. this study shows the importance of the perceived vulnerability of the parents child, perceived severity of an injury occurring, and response efficacy of a safety measure in the reason why parents do or do not perform safety preventive behaviours concerning poisoning prevention. health promotion activities stimulating safe storage of these poisonous products should incorporate these constructs in their safety message in order to increase the effectiveness of the message.	unintentional poisoning is a major cause of nonfatal injuries in children aged 024 months. associations between self - reported habits on the child safe storage of medication and cleaning products and family, and psychosocial factors were assessed, using a model based on the protection motivation theory. by identifying correlates of safety behavior in this manner, more insight in factors which influence this behavior is obtained. health promotion activities in order to promote safety behavior should address these factors in order to increase the effectiveness of the health message. data were gathered from a cross - sectional survey using self - administered questionnaires, mailed to a population sample of 2470 parents with toddlers. the results indicate that the promotion of safe storage of medication and cleaning products should address the family situation, personal cognitive factors as well as social factors. interventions should particularly focus on parents ' self - efficacy of storing poisonous products in a child safe manner and on the vulnerability of their child in their home concerning an unintentional poisoning incident.
a 68-year - old man presented to a primary care skin cancer clinic in melbourne, australia. he was concerned about a skin lesion on his mid back which he reported having traumatised by scratching a week earlier (figure 1)., well - differentiated squamous cell carcinoma had been excised from his nose 3 years earlier. there was no history of excessive occupational or recreational sun exposure and he had never patronised solariums or used welding equipment. on examination the patient had fitzpatrick skin type 2 with significant actinic damage to the face, forearms and dorsum of the hands, with multiple solar lentigines and scattered small actinic keratoses. a whole body skin examination was undertaken with the aid of a heine delta 20 non - polarizing dermatoscope (heine optotechnik, herrshing, germany). digital clinical and dermatoscopic images were taken with a medicam 800 fotofinder non - polarized camera (fotofinder systems gmbh, aichner, birnbach, germany), the dermatoscopy images being at 20 magnification. the lesion of concern on the patient s central back was 6 mm in diameter (figure 2). it was non - pigmented, nodular and centrally ulcerated being covered in a yellow, dried, serous exudate. being non - pigmented, this lesion could not be assessed algorithmically for clues to melanoma. ulceration in the absence of trauma is described as a clue to malignancy, but in this case a history of prior trauma was present. dermatoscopically the lesion was a non - pigmented, structureless yellow with three terminal hairs emanating from it. central structureless red colour was evidence of ulceration as was a single thread of adherent fibre. vessels observed dermatoscopically were very sparse, there being just a few vessels as curved vessels and dots. although a pattern of polymorphous vessels including dots is a published clue to melanoma, there were insufficient vessels visible to constitute a clue to the specific diagnosis in this case. of diagnostic significance was the presence of three terminal hairs protruding from the lesion, visible both clinically and dermatoscopically. this caused the clinician to suspect that the lesion was melanocytic, and therefore amelanotic melanoma was considered as a possible diagnosis. an excisional biopsy was performed with the excision submitted for assessment by a specialist dermatopathologist. histologic sections (figures 311) showed a compound melanocytic proliferation with two components, the first consisting of bland nevus cells, which matured with descent and tracked down adnexal structures in a congenital pattern. the second component, by contrast, was comprised of centrally positioned atypical aggregates of grossly distended epithelioid melanocytes, exhibiting a pseudo - xanthomatous balloon cell change in their cytoplasm, and pleomorphic vesicular nuclei with nucleoli. these cells did not mature with descent, exhibited 4 mitoses per mm sq, and filled the dermis to a depth of 2 mm (level 4). the final diagnosis was : malignant melanoma of balloon cell type ; clark level 4 ; breslow thickness 2 mm, arising within a pre - existing congenital nevus. balloon cell malignant melanoma (bcmm) has been described as the rarest histological type of primary cutaneous melanoma. the first case was reported in 1970. only 7 out of 34 patients (21.2%) were alive without evidence of disease at last contact and 19 of 33 (57.5%) patients with adequate follow - up information died of disseminated tumours from 2 months to 12 years after the initial treatment. balloon cell melanoma has been reported arising in the dermal component of superficial spreading melanoma, and while one balloon cell nevus, which was described as an intradermal nevus, was noted to have rare focal junctional nests of balloon cells, a search of the literature has discovered no reports of in situ bcmm nor of bcmm with a junctional component of balloon cells. in fact, bcmm has been described as a vertical growth phase melanoma. the case herein reported had no apparent epidermal involvement and this, along with the lack of any examples of in situ bcmm reported in the literature, suggests that these melanomas may in fact have a dermal origin. the case we report arose in a pre - existing congenital type nevus as evidenced by both the presence of terminal hairs and the dermatopathologic findings. we speculate that in this case the melanoma may have arisen from a component of this nevus. there is one previous case report of a bcmm coinciding with a dermal nevus showing balloon cell changes. in another reported case a superficial spreading melanoma (level 4 breslow thickness 1.75 mm), arising in a giant congenital nevus which had balloon cell changes, histologically small foci of balloon cell change appearing as foamy cells with abundant cytoplasmic vacuoles are seen in up to 2% of melanocytic naevi. the melanocytic nature of balloon cells has been confirmed both by immunohistochemical studies and electron microscopy, which revealed that the ballooning change was due to progressive vacuolisation of melanosomes. bcmm is characterised by aggregations of balloon cells, and kao defined bcmm as a melanoma composed of more than 50% foamy cells. the dermatopathologic features differ from those of balloon cell nevus with respect to nuclear pleomorphism, atypia, mitoses and the absence of intervening stroma. an additional clue to bcmm is lack of maturation of melanocytes on descent into the dermis. sparse quantities of melanin granules were only found in 9 cases of the 34 reported by kao. the dermatopathologic diagnosis of bcmm is reportedly challenging both with respect to primary and meta - static lesions because the appearance may be cytologically bland and both careful clinico - pathologic correlation as well as correctly interpreted imunohistochemical stains can be important to achieve an accurate diagnosis. in the first reported case and several subsequent cases dermatopathologically the differential diagnosis includes balloon cell change in benign nevi, clear cell sarcoma, clear cell meta - static renal cell carcinoma, basal cell carcinoma, squamous cell carcinoma, malignant clear cell acrospiroma, sebaceous carcinoma and clear cell dermatofibroma. clinically balloon cell nevi can not be distinguished from other nevi, but a dermatoscopic feature of white globular structures in a balloon cell nevus has been described. in a review of the literature the various clinical appearances of bcmm were characterised as nodular, ulcerated, polypoid and papillomatous, and the common absence of pigmentation was noted. there are no discovered previous reports of the dermatoscopic features of bcmm. the case reported herein was clinically an ulcerated, nodular non - pigmented lesion with the presence of terminal hairs but without any specific dermatoscopic clues to melanoma or to the presence of balloon cells. nodular melanoma should always be considered in the differential diagnosis of a newly presenting raised lesion on the skin regardless of the absence of pigmentation or standard dermatoscopic clues to melanoma. in this case the presence of terminal hairs suggested that the lesion was melanocytic and excision biopsy led to the diagnosis of a rare melanoma subtype.	a case of balloon cell melanoma encountered in a primary care skin cancer practice in melbourne, australia is presented. the presenting lesion was 6 mm in diameter, ulcerated, non - pigmented and without any algorithmic clues to melanoma. however the presence of terminal hairs caused the clinician to suspect that it was melanocytic. the lesion was reported as a balloon cell melanoma, clark level 4, breslow thickness 2 mm with a mitotic index of 4 per square mm. this is an extremely rare melanoma subtype. author dw has encountered only two cases in a career involving in excess of one million signed out dermatopathology reports. a search of the literature has not discovered any previously published dermatoscopy images of a balloon cell melanoma.
a considerable proportion of the phosphorus in many soils occurs as stereoisomers of inositol hexakisphosphate (ip6). the most abundant of these is myo - ip6, which occurs as a phosphorus storage compound in seeds, although there is evidence that three additional stereoisomers also occur in the environment. in a remarkable series of papers published in the 1960s, dennis cosgrove identified scyllo-, neo-, and d - chiro - ip6 in soil extracts using a combination of alkaline extraction, column chromatography, dephosphorylation, and paper chromatography of the free inositols. subsequent studies have detected the stereoisomers of ip6 in a number of soils and sediments, yet they are extremely rare elsewhere in nature : scyllo - ip6 has never been identified in an organism, while neo- and d - chiro - ip6 have been identified only in human intestinal amoebae and velvet mesquite leaves, respectively. a report of muco - ip6 was criticized on analytical grounds, and this compound does not appear to exist in nature. the remaining four inositol stereoisomers (allo, l - chiro, cis, epi) do not appear to occur naturally in phosphorylated forms. given that the origins of the stereoisomeric forms of ip6 remain unknown, it is perhaps not surprising that the pathways to their synthesis, biological function, and behavior in the environment are also unknown. each of the three stereoisomers differs from the myo form in the orientation of a single phosphate group, so it is possible that they are synthesized by epimerization of myo - ip6 as well as by phosphorylation of the base isomer. it is well - known that myo - ip6 reacts strongly with metal ions to form insoluble complexes, and the other stereoisomeric forms presumably react in a similar manner. the inositol phosphates are therefore often considered to be relatively recalcitrant in the environment. despite this, a number of organisms can synthesize phytase enzymes that allow them to acquire phosphorus from myo - ip6, although scyllo-, neo-, and d - chiro - ip6 are all more resistant to phytase hydrolysis than the myo isomer. taken together, these properties suggest that possible functions of the stereoisomers might include protection against metal toxicity, conservation and protection of phosphorus from competitors, or an abiotic role in soil structure. despite the widespread occurrence of inositol phosphates in the environment, the stereoisomers of ip6 other than myo have been virtually unstudied for the previous 40 years in any field (but see refs (19 and 20)), including biomedicine, where the inositol phosphates are of particular interest for their role in cell signaling. this is unsatisfactory given the importance of the inositol phosphates in the phosphorus cycle in both terrestrial and aquatic ecosystems but is due in large part to analytical limitations inherent in the measurement of inositol phosphates in environmental samples. the study of organic phosphorus in such samples has been improved dramatically by the application of solution p nmr spectroscopy. this technique has been adopted widely because it provides information on phosphorus compounds in alkaline extracts of soils or sediments without the need for the complex extraction and sample clean up procedures involved in conventional chromatography. however, solution p nmr spectroscopy has been applied only recently to the quantification of ip6 in environmental samples : a technique using spectral deconvolution was used to quantify myo - ip6, while a procedure involving hypobromite oxidation, which destroys all organic matter except the inositol phosphates, and spiking with authentic compounds was used to identify scyllo - ip6. despite these advances, it has not been possible to identify neo- and d - chiro - ip6 by solution p nmr spectroscopy, due principally to the difficulty in obtaining authentic samples of these compounds. here we report the chemical synthesis of neo- and d - chiro - ip6, the first efficient and high - yielding route to the latter, and their identification in soil extracts by hypobromite oxidation and solution p nmr spectroscopy. our results confirm the presence of all four stereoisomers of ip6 in soils (i.e., myo, scyllo, neo, and d - chiro) and, by enabling a re - evaluation of the literature on solution p nmr spectroscopy of soils and sediments, reveal the widespread occurrence of neo- and d - chiro - ip6 in both terrestrial and aquatic ecosystems. bis(cyanoethyl)(n, n - diisopropylamino)phosphine (2.40 g, 9.00 mmol) was added to a stirred suspension of d - chiro - inositol (180.2 mg, 1.00 mmol) and 5-phenyltetrazole (1.75 g, 12.00 mmol) in anhydrous dichloromethane (10 ml) under an argon atmosphere. the reaction mixture was cooled to 40 c, and tert - butyl hydroperoxide (70%, 1.72 ml, 12.00 mmol) was added portion - wise while stirring. the mixture was allowed to warm to room temperature and stirred for a further 1 h. after removal of solvent under reduced pressure, the residue was redissolved in dichloromethane (100 ml) and washed with a 10% sodium sulfite solution (2 100 ml), the organic phase was dried (mgso4), and the solvent was concentrated under reduced pressure to afford the crude 1,2,3,4,5,6-hexakis - o-[bis(cyanoethyloxy)phosphoryl ] d - chiro - inositol. without further purification, the crude material was dissolved in concentrated aqueous ammonia solution (30 ml), and the mixture was heated at 60 c overnight in a pyrex pressure tube. after evaporation of the solution under vacuum, the residue was purified by ion exchange chromatography to afford the pure triethyl ammonium salt of d - chiro - inositol 1,2,3,4,5,6-hexakisphosphate (1.06 g, 91% from d - chiro - inositol) as a hygroscopic white solid (analytical information presented in the supporting information). authentic neo - ip6 was synthesized by a similar procedure to that described above for d - chiro - ip6 (see the supporting information). to determine chemical shifts of neo- and d - chiro - ip6 in naoh edta solution by solution p nmr spectroscopy, compounds (5 mg) were dissolved in deionized water (4 ml) to yield solutions containing 200 g p ml. of this, 3 ml was adjusted to ph > 13 by addition of 10 m naoh, while 1 ml was pretreated by hypobromite oxidation (see below). both samples were then analyzed by solution p nmr spectroscopy, and the unbrominated samples were used to prepare a spike solution (see below). we studied three soils from the falkland islands known to contain inositol phosphates (b. l. turner, unpublished data). the soils were from three distinct locations and were all under pasture consisting mainly of white grass (cortaderia pilosa) with some christmas bush (baccharis magellanica, an evergreen shrub). the soils were rich in organic matter (1123% total c), moderately acidic (ph in water 5.25.4), and contained relatively high phosphorus concentrations (7531107 mg p kg) (see table s1 in the supporting information). soils were extracted in a solution containing 0.25 m naoh and 50 mm disodium edta (ethylenediaminetetraacetate) for 16 h in a 1:20 solid / solution ratio at 22 c, centrifuged (8000 g, 30 min), lyophilized, and ground to a fine powder. an aliquot of each extract was taken prior to lyophilization for determination of total phosphorus by inductively coupled plasma optical - emission spectrometry (icp oes). solutions containing authentic inositol phosphates and soil naoh edta extracts were analyzed by solution p nmr spectroscopy before and after treatment by hypobromite oxidation (also known as alkaline bromination). the method was adapted from previous studies and is reported in full in the supporting information. briefly, samples were made strongly alkaline and treated with ice - cold pure bromine. after 1 h at room temperature the mixture was boiled (5 min), acidified to ph 13 with 1 m naoh. solutions containing authentic compounds were analyzed directly, while soil extracts were frozen and lyophilized for later analysis. the first spike solution contained myo- and scyllo - ip6, while a second spike solution contained neo- and d - chiro - ip6. spike solutions were prepared by combining 0.25 ml of each of the authentic compound solutions with 0.5 ml of a solution containing 1.0 m naoh and 100 mm edta. for analysis, 0.25 ml of spike solution was added to 0.75 ml of reconstituted soil extract (see below) and 1 ml of 1.0 m naoh and 100 mm edta solution. the spike was therefore added at relatively similar concentrations of both neo- and d - chiro - ip6 compared to soil extracts. samples were analyzed by solution p nmr spectroscopy using a procedure similar to that described previously, with details reported in the supporting information. briefly, compounds were redissolved in d2o and the 1 m naoh100 mm edta solution, vortexed (1 min), and transferred to a 10 mm nmr tube. soil extracts were filtered (1 m gf - b filter, whatman) prior to analysis. spectra were acquired on a bruker avance 500 using a 30 pulse, 0.58 s acquisition time, and a 2 s t1 delay. these parameters yield quantitative spectra based on literature reports (e.g., ref (29)). the number of scans varied from 1000 for authentic compounds to 30,000 for soil extracts to ensure acceptable signal - to - noise ratios. chemical shifts of signals were determined in parts per million (ppm) relative to an external orthophosphoric acid standard (85%) set to = 0 ppm. signals were subsequently adjusted using the chemical shift of scyllo - ip6 (= 4.03 ppm) to facilitate signal identification among spectra and assigned based on literature reports of model compounds spiked into naoh signal areas were calculated by integration, and deconvolution was performed on the region between = 3.0 and 7.5 ppm to separate orthophosphate from phosphomonoesters and to quantify signals from inositol phosphates (see below). finally, spectra were plotted with 1 hz line broadening to show fine resolution in the phosphomonoester region. the proportion of the total spectral area was determined for the four ip6 stereoisomers (myo, scyllo, neo, d - chiro). the proportion of scyllo - ip6 was determined from the strong signal at = 4.03 ppm. the proportion of myo - ip6 was determined by summing the four signals in a 1:2:2:1 pattern (see below for chemical shifts). the proportion of neo - ip6 was determined by multiplying the signal at = 6.67 ppm by 1.5 (i.e., the signal represents four of the six phosphates on the molecule). the proportion of d - chiro - ip6 was determined by multiplying the signal at = 6.48 ppm by three (i.e., it represents two of the six phosphates on the molecule). we assumed that all of the neo- and d - chiro - ip6 molecules were present as a single conformer in soil extracts, given the absence of detectable signals from the alternate conformers in spectra of brominated samples (see below). both neo- and d - chiro - ip6 can occur in two conformational forms in solution (figure 1), and both forms were present in preparations of the authentic samples in alkaline solution (figure 2). the authentic sample of neo - ip6 in naoh edta gave four signals in solution p nmr spectroscopy (figure 2a). two signals at = 6.61 and = 4.54 ppm in a 4:2 ratio was assigned to the 4-equatorial/2-axial conformation, based on h, c, and 2-dimensional nmr experiments (see the supporting information). two smaller signals at = 4.88 and = 5.14 ppm, also in a 4:2 ratio, were assigned to the 2-equatorial/4-axial conformation (figure 2a). the chemical shifts of the signals were essentially unchanged by hypobromite oxidation (figure 2b), but the ratio of the two conformers was reduced to 2:1. p, with the number of the associated carbon group indicated by the subscript numeral. at low ph both neo- and d - chiro - ip6 adopt the 4-equatorial/2-axial conformation. at high ph, the 2-equatorial/4-axial and 4-equatorial/2-axial forms exist in equilibrium, and the relative proportions of the two conformers might be influenced by factors other than ph (e.g., metal counterions). in p nmr spectra of alkaline soil extracts, neo - ip6 was observed (within the limits of detection) to be exclusively in the 4-equatorial/2-axial conformation, while d - chiro - ip6 occupied the 2-equatorial/4-axial conformation. solution p nmr spectra of authentic neo- and d - chiro - inositol hexakisphosphate dissolved in naoh edta. spectra show samples before (a, c) and after (b, d) pretreatment by hypobromite oxidation. spectra are plotted with 1 hz (a c) or 2 hz (d) line broadening. the authentic sample of d - chiro - ip6 gave six signals in solution p nmr spectroscopy (figure 2c). three large signals at = 4.62, 5.04, and 6.44 ppm in a 2:2:2 pattern were assigned to the 2-equatorial/4-axial conformation, based on h, c, and two - dimensional nmr experiments (see the supporting information). a second set of smaller signals at = 4.33, 5.61, and 6.85 ppm, also in a 2:2:2 pattern, were assigned to the 4-equatorial/2-axial conformation. the chemical shifts of signals from d - chiro - ip6 were essentially unchanged following hypobromite oxidation (figure 2d), although poor resolution in the brominated sample due to the low phosphorus concentration prevented clear quantification of signals from the 4-equatorial/2-axial conformer. in both cases, no inorganic phosphate was detected in brominated samples, confirming the resistance of neo- and d - chiro - ip6 to hypobromite oxidation. edta extracts of the three soils were similar in terms of their broad phosphorus composition (table 1, figure 3a). edta solution recovered 90% of the total phosphorus from the three soils (see table s1 in the supporting information), consistent with the efficacy of this extraction solution for high latitude soils rich in carbon and phosphorus. most of the extracted phosphorus was in organic forms, including phosphomonoesters (6670% extracted phosphorus), dna (dna ; 5.27.5%), alkali - stable phospholipids (1.84.4%), and phosphonates (1.22.4%). inorganic phosphorus included orthophosphate (1720% extracted phosphorus) and pyrophosphate (1.42.4%). edta extracts of a soil (east 50) from the falkland islands before (a) and after (b) pretreatment with hypobromite oxidation to destroy all organic phosphorus other than the higher - order inositol phosphates. the zoomed insets show the phosphonate signals between = 19 and 21 ppm. b) the spectrum is truncated vertically to show the scyllo - inositol hexakisphosphate signal at the same height in both spectra. signal assignments are as follows : a, neo- and d - chiro - inositol hexakisphosphate ; b, inorganic orthophosphate ; c, phosphomonoesters other than the signals from neo- and d - chiro - inositol hexakisphosphate between = 6.4 and 6.9 ppm ; d, alkali - stable phospholipids ; e, dna ; f, pyrophosphate ; g, phosphonates. values are the proportion (%) of the total spectral area assigned to functional groups of phosphorus compounds. hypobromite oxidation destroyed phospholipids, dna, phosphonates, and some phosphomonoesters (table 1, figure 3b). inorganic orthophosphate increased to 4247% of the total phosphorus following bromination, while phosphomonoesters declined to 5057% (table 1). all four stereoisomers of ip6 were identified in extracts of the three soils by spiking solutions with the respective stereoisomers (table 2, figure 4). the presence of myo- and scyllo - ip6, their chemical shifts, and their resistance to hypobromite oxidation was confirmed by spiking with authentic compounds (data not shown). the large stable signal from scyllo - ip6 was adjusted to = 4.03 ppm (the chemical shift of this compound in the unbrominated east 50 soil) in all spectra, to facilitate comparison among spectra with small differences in ph and salt content. based on this, signals from myo - ip6 occurred at = 4.38, 4.53, 4.89, and 5.79 ppm in a 1:2:2:1 pattern (figure 4, figure s1 in the supporting information). the chemical shift of inorganic orthophosphate varied slightly among samples, presumably due to variation in salt content, but was typically within 0.05 ppm of = 5.95 ppm (figure 4). chemical shifts of the remaining signals in the phosphomonoester region were remarkably consistent across soils and treatments. edta extracts of a soil from the falkland islands (east 50) showing the phosphomonoester region in detail (= 3.0 to 7.5 ppm). the following treatments are shown : untreated (a), untreated and spiked with a mixture of neo- and d - chiro - inositol hexakisphosphate (ip6) (b), pretreated by hypobromite oxidation (c), pretreated with hypobromite oxidation and spiked with a mixture of neo- and d - chiro - ip6 (d). signal assignments are from the untreated spiked sample and assigned to myo (m), scyllo (s), d - chiro (c), or neo (n) ip6 stereoisomers. inorganic orthophosphate (ortho - p) is the large signal close to = 6.00 ppm. spectra are plotted with 1 hz line broadening and referenced to the chemical shift of scyllo - ip6 in spectrum a (= 4.03 ppm). the signal at = 4.58 ppm from the two axial phosphates of the 4-equatorial/2-axial conformer of neo - ip6 were small in the spike solution and were not detectable in soil extracts. spectra for two additional soils showing the same treatments are in the supporting information (figure s1). signals are scaled relative to the large stable signal from scyllo - ip6 at = 4.03 ppm. values varied by 0.01 ppm or less among samples and treatments (unbrominated, brominated, spiked). the second spike solution contained neo - ip6 conformers in a 1:4 ratio (spectrum not shown), which did not appear to change after addition of the spike to the soils extracts. the ratio was different in the soil extracts themselves, because the largest neo - ip6 signal was at = 6.67 ppm (i.e., from the four equatorial phosphates of the 4-equatorial/2-axial conformer), with no similar signal detected at = 4.93 ppm (i.e., from the four axial phosphates of the 2-equatorial/4-axial conformer) in any of the brominated soil extracts. for quantification, we therefore assumed that all neo - ip6 in soil extracts was in the 4-equatorial/2-axial conformation. signals at = 6.48 and 6.67 ppm downfield of orthophosphate in all soils were identified as being from d - chiro- and neo - ip6, respectively. a signal at = 6.90 ppm from the 4-equatorial/2-axial conformer of d - chiro - ip6 present in the spike was not detected in any soil extract (untreated or brominated). other signals from d - chiro- and neo - ip6 were not identified clearly in soil extracts either with or without pretreatment by hypobromite oxidation. a signal at = 5.08 ppm in untreated extracts coincided with one of the three signals from the 2-equatorial/4-axial conformer of d - chiro - ip6, as shown by spiking, but was considerably reduced by hypobromite oxidation. this indicates that the signal was from an organic phosphorus compound other than an inositol phosphate, probably -glycerophosphate. it should be noted that the signals assigned to d - chiro - ip6 could also arise from its enantiomer l - chiro - ip6, although we consider this unlikely given that the latter has never been identified in nature (see discussion). a small signal at = 4.32 ppm increased in extracts of all soils after hypobromite oxidation. this remains unidentified but appears commonly following pretreatment by this procedure (b. l. turner, unpublished observations). total ip6 in the three soils was between 36.7 and 41.4% of the extracted phosphorus (mean 38.6 2.6%) with little variation between values calculated from untreated and brominated extracts (table 3). inositol hexakisphosphate therefore accounted for an average of 48.9 4.2% of the soil organic phosphorus and 57.0 4.7% of the phosphomonoesters (table s2 in supporting information). thus, about half of the organic phosphorus in the three soils was ip6. the remaining phosphomonoesters resisting hypobromite oxidation presumably represented lower - order inositol phosphates and constituted an additional 1821% of the soil organic phosphorus (see table s2 in the supporting information). values are expressed as both the proportion (%) of total spectral area and the proportion (%) of the total ip6. the overall average is the mean standard deviation of the three averages for each soil. most of the ip6 was in the form of myo - ip6 (55.9 1.3% of the total ip6) and scyllo - ip6 (32.8 3.3%), with smaller proportions of the neo (6.1 0.9%) and d - chiro (5.2 1.7%) stereoisomers (table 3). values were remarkably consistent across the three soils, and there was little variation between untreated and brominated extracts for all four stereoisomers in almost all cases. overall, the stereoisomers were present in the following order : myo > scyllo > solution p nmr spectroscopy is the most widely used procedure for determining the chemical nature of organic phosphorus in soils and sediments. our results extend the scope of this technique to include identification of all four stereoisomeric forms of ip6 present in nature. both neo- and d - chiro - ip6 can be identified readily by signals downfield of orthophosphate between = 6.40 and 6.70 ppm (relative to scyllo - ip6 at = 4.03 ppm) and are detectable at relatively low concentrations given that the signals are well - resolved from the large orthophosphate signal close to = 6.00 ppm. our results indicate that it is possible to quantify the ip6 in well - resolved spectra containing high concentrations of inositol phosphates without brominating samples. however, bromination is clearly a useful pretreatment for estimation of the ip6 in environmental samples by solution p nmr spectroscopy, as indicated by its ability to distinguish between -glycerophosphate and one of the three signals from the 2-equatorial/4-axial conformer of d - chiro - ip6 at = 5.08 ppm. the impact of this procedure on lower - order esters of all four stereoisomers remains unknown. both myo- and scyllo - ip6 can occur in two conformations depending on solution ph, although both appear in a single conformation at the high ph of the extracts used in solution p nmr spectroscopy of soils. in contrast, both conformers of neo- and d - chiro - ip6 can be present in strongly alkaline extracts, as indicated by spectra of authentic compounds shown here. in soil extracts, however, only a single conformer of each stereoisomer was detected. specifically, no signals were detected at = 4.93 or 5.18 ppm from the 2-equatorial/4-axial conformer of neo - ip6, nor at = 6.90 ppm from two equatorial phosphates of the 4-equatorial/2-axial conformer of d - chiro - ip6. this simplifies quantification of these compounds in soil extracts, which can be achieved using the two signals at = 6.67 and 6.48 ppm for neo- and d - chiro - ip6, respectively. the possible presence of other conformers is indicated by signals at = 6.90 ppm (d - chiro - ip6) or = 5.18 ppm (neo - ip6 ; the signal at = 4.92 ppm from this conformer may be obscured by a signal from myo - ip6). it remains unclear why both conformers were present in the authentic samples at high ph, especially as signals from these compounds in the spike solution were unchanged following addition to soil extracts. solution ph was strongly alkaline in all cases, so it seems possible that conformers might be stabilized by interaction with metals. further experiments are required to understand why the ratio of the two neo - ip6 conformers varies at strongly alkaline ph. a caveat to the identification of d - chiro - ip6 is that identical signals in solution p nmr spectroscopy would be expected from its enantiomer l - chiro - ip6, so the possibility remains that the form detected in soil extracts is the l-, rather than the d-, enantiomer. we assume that the signals are from d - chiro - ip6, however, given that chiro - ip6 in soils was previously identified as the d - enantiomer and that the l - enantiomer has never been detected in nature in phosphorylated form. very few studies have quantified neo- or d - chiro - ip6 in soils or sediments since the early work of cosgrove. for the soils studied here, both compounds were present in much lower concentrations than either the myo or scyllo stereoisomers, which is in agreement with the older literature. however, the concentration of the neo isomer appears to be greater than or similar to the d - chiro isomer in all three soils studied here. this contrasts with early reports that the d - chiro isomer occurred at greater concentration but is consistent with the suggestion that the neo isomer might have been underestimated in studies that did not include pretreatment by hypobromite oxidation. the identification of neo- and d - chiro - ip6 reported here allows a reappraisal of publications that have applied solution p nmr spectroscopy to soils and aquatic sediments. a number of studies reported signals downfield of orthophosphate in solution p nmr spectra of soil and sediment extracts but did not identify them as neo- or d - chiro - ip6. it was initially speculated that the signals represented aromatic phosphodiesters based on their similarity to compounds such as binaphthyl diylhydrogen phosphate, although the resistance of the signals to hypobromite oxidation subsequently indicated the likelihood that they represented inositol phosphates. a series of temperate pasture soils yielded signals that we can now assign to neo- and d - chiro - ip6 at concentrations of 851 mg p kg, which represented 25% of the extracted phosphorus. the same signals were also reported in madagascan rice soils at concentrations of 0.84.9 mg p kg (0.51.3% of extracted phosphorus) (note that these values are 6-fold lower than the erroneous values in the original article). similar signals were also reported in extracts of russian grassland soils, scottish mineral soil, sewage sludge, and humic acids extracted from an agricultural soil. however, only traces were detected in subarctic tundra and lowland temperate and tropical rain forests. the humic acid study also detected scyllo - ip6 but not the more widespread myo isomer, suggesting a difference in the mechanisms by which the various stereoisomeric forms of ip6 become stabilized in soils. importantly, we can now extend the identification of neo- and d - chiro - ip6 to aquatic systems. previously, the only report of either of these stereoisomeric forms was the measurement of d - chiro - ip6 by gas chromatography in riverine, estuarine, and marine sediments of tokyo bay and the surrounding catchment. in that study, most of the ip6 was in the myo form, with 1015% as scyllo and 15% as d - chiro (neo - ip6 was not detected). elsewhere, signals that can now be assigned to neo- and d - chiro - ip6 were reported in sediments from a wide range of danish lakes, settling seston and sediments from a eutrophic lake in china, and sediments from a brackish embayment in helsinki. in the latter study, the signal corresponding to neo - ip6 constituted 0.33.0% of the organic phosphate and was greatest in sediment layers dated to periods of high pollutant inputs into the embayment. no signals corresponding to neo- and d - chiro - ip6 were reported in nmr studies of marine sediments, although d - chiro - ip6 was detected in sediments from toyko bay. although it has been known for some time that inositol phosphate concentrations vary widely among soils (from none to all of the organic phosphorus, reviewed in ref (4)), it has been suggested recently that the amount of inositol phosphate in soil has been overestimated and is quantitatively unimportant. here, these results demonstrate unequivocally that ip6 can constitute a considerable proportion of the organic phosphorus in some soils. despite the apparent abundance of ip6 stereoisomers in terrestrial and aquatic ecosystems, their origins, dynamics, and biological function given the importance of phosphorus as both a nutrient and a pollutant, it is remarkable that so little is known about one of the most widespread groups of organic phosphates in the environment. results presented here confirm the prevalence of the ip6 stereoisomers in soils and lake sediments and support previous reports that the relative abundance of the four stereoisomeric forms of ip6 follows the pattern myo > scyllo > neo > d - chiro (although the scyllo isomer can be the dominant form in some environments ; e.g. ref (47)). the investment in energy and phosphorus involved in the synthesis of the stereoisomeric forms of ip6 suggests that they have some important yet currently unknown biological function. research on this topic has been restricted by the difficulty in identifying the inositol phosphate stereoisomers in environmental samples, but the methodology presented here allows the simultaneous determination of all four stereoisomeric forms of ip6 in a single analysis. this opens up new possibilities for studying the origins and ecological significance of these enigmatic compounds.	the inositol phosphates are an abundant but poorly understood group of organic phosphorus compounds found widely in the environment. four stereoisomers of inositol hexakisphosphate (ip6) occur, although for three of these (scyllo, neo, and d - chiro) the origins, dynamics, and biological function remain unknown, due in large part to analytical limitations in their measurement in environmental samples. we synthesized authentic neo- and d - chiro - ip6 and used them to identify signals from these compounds in three soils from the falkland islands. both compounds resisted hypobromite oxidation and gave quantifiable 31p nmr signals at = 6.67 ppm (equatorial phosphate groups of the 4-equatorial/2-axial conformer of neo - ip6) and = 6.48 ppm (equatorial phosphate groups of the 2-equatorial/4-axial conformer of d - chiro - ip6) in soil extracts. inositol hexakisphosphate accounted for 4654% of the soil organic phosphorus, of which the four stereoisomers constituted, on average, 55.9% (myo), 32.8% (scyllo), 6.1% (neo), and 5.2% (d - chiro). reappraisal of the literature based on the new signal assignments revealed that neo- and d - chiro - ip6 occur widely in both terrestrial and aquatic ecosystems. these results confirm that the inositol phosphates can constitute a considerable fraction of the organic phosphorus in soils and reveal the prevalence of neo- and d - chiro - ip6 in the environment. the hypobromite oxidation and solution 31p nmr spectroscopy procedure allows the simultaneous quantification of all four ip6 stereoisomers in environmental samples and provides a platform for research into the origins and ecological significance of these enigmatic compounds.
acute hyperglycemia is frequently seen in hospitalized patients and induced by stressors such as acute illness and surgical trauma. such transient increases in blood glucose concentrations may put patients at risk for adverse outcomes. hyperglycemia independent of preexisting diabetes mellitus is an established risk factor for increased mortality and morbidity after cardiac surgery.1 patients without a history of diabetes who were hyperglycemic at admission to the hospital had higher mortality and lower functional outcomes than normoglycemic and even hyperglycemic diabetic patients.2 van den berghe.3 showed that intensive insulin therapy (iit) reduces in - hospital mortality in surgical intensive care unit patients by 34% with subsequent investigations confirming that maintaining normoglycemia rather than glycemia - independent effects of insulin is responsible for the beneficial effects of iit.4,5 these findings emphasize the potential hazards of poor glucose control on patient outcome. animal models of acute hyperglycemia confirm the deleterious effects of even short episodes of hyperglycemia on cerebral6 and renal ischemia / reperfusion (i / r) injury.7 proposed mechanisms for the detrimental effects of acute hyperglycemia are increased oxidative stress, an enhanced inflammatory response with cytokine activation8,9 and impaired blood flow with reperfusion.10 diabetic mice have been shown to be more susceptible to liver ischemia,11,12 but so far, the effects of acute hyperglycemia on liver i / r injury have not been addressed. we therefore used a rat model of acute hyperglycemia to investigate its effects on hepatic i / r injury. all animal experiments were carried out with approval by the local committee on animal research. animal care was in agreement with the national institutes of health guidelines for ethical research (nih publication no. inbred male lewis rats (harlan, indianapolis, in, usa) were used for this study. animals weights on arrival at our facility were 250300 g. animals had access to standard laboratory diet and were maintained on a light dark cycle. prior to the study, animals spent several days in the animal care facility for acclimatization. the rats were divided into hyperglycemic and control group. in the hyperglycemic group (hg, n = 8), 2.5 g / kg glucose (25% solution) was injected intraperitoneally following the assessment of the baseline glucose serum concentration. the control group (con, n = 8) received 10 ml / kg 0.9% saline instead. thirty minutes later, rats were anesthetized with isoflurane. following liver exposure through a midline incision and collection of blood samples, the liver was mobilized, and vascular structures to the left and median lobe were identified and clamped for 45 min using a bulldog clamp. the unoccluded right and caudate lobe allow outflow from the splanchnic circulation, thus avoiding venous congestion. for the duration of hepatic ischemia, the abdominal cavity was closed with clamps. rectal temperature was continuously assessed using an electronic thermometer (rsp tm-200d, respiratory support products inc., santa ana, ca, usa using a mallinckrodt probe, cat no. louis, mo, usa) and held constant at 37c using a heating lamp. following reperfusion, the animals received 5 ml of normal saline intraperitoneally, and the incision was closed in two layers. all tissue was immediately frozen in liquid nitrogen and stored at 80c until further processing. sham experiments (sham, n = 5) served as reference for subsequent analysis. sham experiments were identical to control i / r experiments except that hepatic vessels were not clamped. hyperglycemic sham experiments (hg sham, n = 4) were added to the protocol to identify the effects of hyperglycemia alone. serum levels of aspartate aminotransferease (ast) and alanine aminotransferase (alt) were determined at baseline and following 4 h of reperfusion. the analysis was done in the general laboratory, san francisco general hospital, university of california, san francisco. five - micron paraffin - embedded tissue sections were stained with hematoxylin and eosin and examined using standard light microscopy by a pathologist (r.r.) who was blinded to the experimental condition of the animals. sections were scored from 04 for sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal necrosis as described by suzuki. (table 1).13table 1suzuki score for the assessment of liver damage following hepatic ischemia / reperfusionscorecongestionvacuolizationnecrosis0nonenonenone1minimalminimalsingle cell necrosis2mildmild30%3moderatemoderate60%4severesevere>60% suzuki score for the assessment of liver damage following hepatic ischemia / reperfusion activity of myeloperoxidase (mpo), an enzyme stored in the azurophilic granules of neutrophils, was used to measure tissue neutrophil sequestration. the assay is based on the oxidation of 3,3,5,5-tetramethyl benzydine by mpo in the presence of h2o2. units of mpo activity were calculated using a standard curve derived from a mpo standard sample (calbiochem, emd bioscience, la jolla, ca, usa). snap - frozen liver sections were homogenized in tissue protein extraction reagent (pierce biotechnology, rockford, il) containing 1 mm edta and 1:100 protease cocktail inhibitor (sigma, st. protein concentrations of liver homogenates were measured by the pierce bicinchoninic acid protein assay with bovine serum albumin as the standard. fifty micrograms of liver homogenates was separated on a novex - nupage 10% bis - tris sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (invitrogen) and transferred to nitrocellulose membrane using the xcell surelock system (invitrogen). a mouse anti - heat shock protein 70 (hsp70) monoclonal antibody (sc-24) and a goat anti - actin antibody (sc-1616) from santa cruz biotechnology (santa cruz, ca) were used. in addition, a mouse anti - heat shock protein 32 (hsp32) monoclonal antibody from stressgen (ann arbor, mi), a mouse anti - nitrotyrosine antibody from abcam inc. (cambridge, ma), and a rabbit anti - cleaved caspase-3 monoclonal antibody (cst 9661) from cell signaling technology (danvers, ma) were used for the western blots. the membranes were incubated with a 1:100 or 1:1,000 dilution of the primary antibody followed by a 1:10,000-fold dilution of a secondary anti - mouse or anti - goat immunoglobulin g from santa cruz biotechnology. immunoreactive proteins were developed using supersignal west dura (pierce biotechnology) and visualized on the fluorchem 5500 imaging system from alpha innotech (san leandro, ca). comparison between study groups was performed using analysis of variance with post hoc dunnett correction, with normoglycemic sham animals serving as controls. comparison of the two ischemic groups alone was done using a two - tailed unpaired t test. all animal experiments were carried out with approval by the local committee on animal research. animal care was in agreement with the national institutes of health guidelines for ethical research (nih publication no. inbred male lewis rats (harlan, indianapolis, in, usa) were used for this study. animals weights on arrival at our facility were 250300 g. animals had access to standard laboratory diet and were maintained on a light dark cycle. prior to the study, animals spent several days in the animal care facility for acclimatization. the rats were divided into hyperglycemic and control group. in the hyperglycemic group (hg, n = 8), 2.5 g / kg glucose (25% solution) was injected intraperitoneally following the assessment of the baseline glucose serum concentration. the control group (con, n = 8) received 10 ml / kg 0.9% saline instead. thirty minutes later, rats were anesthetized with isoflurane. following liver exposure through a midline incision and collection of blood samples, the liver was mobilized, and vascular structures to the left and median lobe were identified and clamped for 45 min using a bulldog clamp. the unoccluded right and caudate lobe allow outflow from the splanchnic circulation, thus avoiding venous congestion. for the duration of hepatic ischemia, the abdominal cavity was closed with clamps. rectal temperature was continuously assessed using an electronic thermometer (rsp tm-200d, respiratory support products inc., santa ana, ca, usa using a mallinckrodt probe, cat no. louis, mo, usa) and held constant at 37c using a heating lamp. following reperfusion, the animals received 5 ml of normal saline intraperitoneally, and the incision was closed in two layers. all tissue was immediately frozen in liquid nitrogen and stored at 80c until further processing. sham experiments (sham, n = 5) served as reference for subsequent analysis. sham experiments were identical to control i / r experiments except that hepatic vessels were not clamped. hyperglycemic sham experiments (hg sham, n = 4) were added to the protocol to identify the effects of hyperglycemia alone. serum levels of aspartate aminotransferease (ast) and alanine aminotransferase (alt) were determined at baseline and following 4 h of reperfusion. the analysis was done in the general laboratory, san francisco general hospital, university of california, san francisco. five - micron paraffin - embedded tissue sections were stained with hematoxylin and eosin and examined using standard light microscopy by a pathologist (r.r.) who was blinded to the experimental condition of the animals. sections were scored from 04 for sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal necrosis as described by suzuki. (table 1).13table 1suzuki score for the assessment of liver damage following hepatic ischemia / reperfusionscorecongestionvacuolizationnecrosis0nonenonenone1minimalminimalsingle cell necrosis2mildmild30%3moderatemoderate60%4severesevere>60% suzuki score for the assessment of liver damage following hepatic ischemia / reperfusion activity of myeloperoxidase (mpo), an enzyme stored in the azurophilic granules of neutrophils, was used to measure tissue neutrophil sequestration. we used a spectrophotometric method to assay tissue mpo activity. the assay is based on the oxidation of 3,3,5,5-tetramethyl benzydine by mpo in the presence of h2o2. units of mpo activity were calculated using a standard curve derived from a mpo standard sample (calbiochem, emd bioscience, la jolla, ca, usa). snap - frozen liver sections were homogenized in tissue protein extraction reagent (pierce biotechnology, rockford, il) containing 1 mm edta and 1:100 protease cocktail inhibitor (sigma, st. protein concentrations of liver homogenates were measured by the pierce bicinchoninic acid protein assay with bovine serum albumin as the standard. fifty micrograms of liver homogenates was separated on a novex - nupage 10% bis - tris sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (invitrogen) and transferred to nitrocellulose membrane using the xcell surelock system (invitrogen). a mouse anti - heat shock protein 70 (hsp70) monoclonal antibody (sc-24) and a goat anti - actin antibody (sc-1616) from santa cruz biotechnology (santa cruz, ca) were used. in addition, a mouse anti - heat shock protein 32 (hsp32) monoclonal antibody from stressgen (ann arbor, mi), a mouse anti - nitrotyrosine antibody from abcam inc. (cambridge, ma), and a rabbit anti - cleaved caspase-3 monoclonal antibody (cst 9661) from cell signaling technology (danvers, ma) were used for the western blots. the membranes were incubated with a 1:100 or 1:1,000 dilution of the primary antibody followed by a 1:10,000-fold dilution of a secondary anti - mouse or anti - goat immunoglobulin g from santa cruz biotechnology. immunoreactive proteins were developed using supersignal west dura (pierce biotechnology) and visualized on the fluorchem 5500 imaging system from alpha innotech (san leandro, ca). was performed using analysis of variance with post hoc dunnett correction, with normoglycemic sham animals serving as controls. comparison of the two ischemic groups alone was done using a two - tailed unpaired t test. intraperitoneal injection and surgery alone resulted in an increase in serum glucose concentrations from 89 19 baseline to 182 32 mg / dl in the saline pre - treated group. in the glucose - pretreated group, three animals were not considered for subsequent analysis due to an only moderate increase in serum glucose concentrations (< 250 mg / dl, 30 min after treatment). in the five remaining animals, serum glucose concentrations before ischemia increased from 107 32 to 360 32 mg / dl. at the end of the 4-h reperfusion period, serum glucose concentrations remained higher in the glucose pretreated group (fig. 1). figure 1intraperitoneal injection of saline and surgery alone increased serum glucose concentrations in control animals. intraperitoneal injection of 2.5 g glucose / kg resulted in significantly higher serum glucose concentrations. after 4 h of reperfusion, glucose concentrations were still significantly higher in hyperglycemic animals. p < 0.05 vs. control. intraperitoneal injection of saline and surgery alone increased serum glucose concentrations in control animals. intraperitoneal injection of 2.5 g glucose / kg resulted in significantly higher serum glucose concentrations. after 4 h of reperfusion, glucose concentrations were still significantly higher in hyperglycemic animals. serum marker of liver injury serum transaminase concentrations following i / r were higher in the glucose - pretreated animals : 7,832 3,374 vs. 3,245 2,009 u / l (alt, p < 0.05) and 10,677 4,119 vs. 5,385 3,393 u / l (ast, p < 0.05). transaminase concentrations after 4 h of reperfusion were correlated with glucose concentrations before ischemia of all animals that entered the study (fig. 2). figure 2correlation of serum glucose concentrations before the start of ischemia and serum concentrations of alt (a) and ast (b) after 4 h of reperfusion. liver injury, as assessed by transaminase concentrations and glucose concentrations, was correlated with correlation coefficients of r = 0.70 (alt) and r = 0.68 (ast). correlation of serum glucose concentrations before the start of ischemia and serum concentrations of alt (a) and ast (b) after 4 h of reperfusion. liver injury, as assessed by transaminase concentrations and glucose concentrations, was correlated with correlation coefficients of r = 0.70 (alt) and r = 0.68 (ast). histology both experimental groups showed liver damage including vacuolization and at least minimal congestion and single - cell necrosis (fig. there was no statistical difference between hyperglycemic animals and control animals in suzuki scores (6.0 2.2 vs. 6.1 1.8) or necrosis scores (2.0 0.8 vs. 2.0 0.8). whether individual cell death after 4 h of reperfusion was attributable to necrosis or apoptosis could not be determined by histology. using nuclear features to distinguish between types of cell death is not considered reliable,14 and both experimental groups demonstrated zonal as well as spotty areas of dead hepatocytes (fig. 3). figure 3sham - treated animals show no significant congestion at low power (a 100). vacuolization and cellular necrosis are not evident in periportal hepatocytes (b 200) or in centrizonal areas. hepatic architecture is unremarkable. in contrast, control animals following 45 min of ischemia and 4 h of reperfusion show diffuse, moderate sinusoidal congestion, with numerous sinusoidal channels distended by red blood cells in several areas of the liver section (c 200). two dying hepatocytes (likely evolving into councilman bodies) are visible in the center of the field (c). mild parenchymal vacuolization is visible in some hepatocytes, with several hepatocytes in this field showing irregular nuclear contours, chromatin condensation, pyknosis, and nuclear dust, histologic evidence of cell damage, and evolving cell death (d 400). moderate sinusoidal congestion also is seen in hyperglycemic animals treated with high levels of dextrose before ischemia (e 200). cell damage is seen in several adjacent hepatocytes containing vacuolated cytoplasm, pyknotic nuclei, and nuclear dust. these dying hepatocytes also show paler cytoplasm than their undamaged counterparts nearby (f 400). sham - treated animals show no significant congestion at low power (a 100). vacuolization and cellular necrosis are not evident in periportal hepatocytes (b 200) or in centrizonal areas. in contrast, control animals following 45 min of ischemia and 4 h of reperfusion show diffuse, moderate sinusoidal congestion, with numerous sinusoidal channels distended by red blood cells in several areas of the liver section (c 200). two dying hepatocytes (likely evolving into councilman bodies) are visible in the center of the field (c). mild parenchymal vacuolization is visible in some hepatocytes, with several hepatocytes in this field showing irregular nuclear contours, chromatin condensation, pyknosis, and nuclear dust, histologic evidence of cell damage, and evolving cell death (d 400). moderate sinusoidal congestion also is seen in hyperglycemic animals treated with high levels of dextrose before ischemia (e 200). cell damage is seen in several adjacent hepatocytes containing vacuolated cytoplasm, pyknotic nuclei, and nuclear dust. these dying hepatocytes also show paler cytoplasm than their undamaged counterparts nearby (f 400). apoptosis cleaved caspase-3 expression was higher in control animals (2.12 0.47) vs. hyperglycemic animals (1.49 0.42) when compared to sham animals (1.00 0.10), indicating more apoptotic cells in livers from control animals fig. (4) figure 4cleaved caspase-3 expression was highest in control animals, suggesting preferential apoptotic cell death in animals that were not pretreated with glucose. cleaved caspase-3 expression was highest in control animals, suggesting preferential apoptotic cell death in animals that were not pretreated with glucose. oxidative stress nitrotyrosine concentrations after 4 h of reperfusion were higher in hyperglycemic animals (1.63 0.54-fold when compared to control animals (1.00 0.30), p < 0.05) indicating increased oxidative stress resulting in nitration of tyrosine residues of proteins by peroxynitrite. inflammation mpo activity in the liver after 4 h of reperfusion was higher in glucose - pretreated animals (5,383 2,512 vs. 2,219 2,086 mu / mg protein min, p < 0.05), indicating increased neutrophil migration into the hepatic tissue of hyperglycemic animals (fig. 5). figure 5myeloperoxidase activity was increased in liver homogenates of hyperglycemic animals when compared to control animals, indicating increased neutrophil accumulation after 4 h of reperfusion. p < 0.05 vs. sham, p < 0.05 vs. control i / r. myeloperoxidase activity was increased in liver homogenates of hyperglycemic animals when compared to control animals, indicating increased neutrophil accumulation after 4 h of reperfusion. p < 0.05 vs. sham, p < 0.05 vs. control i / r. heat shock protein activation i / r increased hsp32 expression in control but suppressed hsp32 expression in hyperglycemic animals (1.97 0.89-fold vs. 0.46 0.13-fold when normalized to sham animals, p < 0.05 ; fig. hyperglycemia alone without i / r (hyperglycemic sham) did not affect hsp32 expression (0.92 0.20 vs. 1.00 0.16 when compared to control sham). i / r increased hsp70 expression in control animals more than in hyperglycemic animals (19.99 11.55-fold vs. 3.22 0.56-fold when normalized to sham, p < 0.05 ; fig. again, hyperglycemia alone without i / r did not affect hsp70 expression (0.94 0.08 vs. 1.00 0.15) when compared to control sham. figure 6heat shock protein expression as assessed by western blot for hsp32 (hemeoxygenase-1) (a) and hsp70 (b). i / r resulted in a distinct activation of both hsp32 and hsp70 expression in control animals. however, hyperglycemia ameliorated the activation of hsp70 by i / r and suppressed hsp32 expression. densitometric values were normalized to actin and are expressed as ratios of sham sd. p < 0.05 vs. sham, p < 0.05 vs. control i / r. heat shock protein expression as assessed by western blot for hsp32 (hemeoxygenase-1) (a) and hsp70 (b). i / r resulted in a distinct activation of both hsp32 and hsp70 expression in control animals. however, hyperglycemia ameliorated the activation of hsp70 by i / r and suppressed hsp32 expression. densitometric values were normalized to actin and are expressed as ratios of sham sd. p < 0.05 vs. sham, p < 0.05 vs. control i / r. acute hyperglycemia during hepatic ischemia amplified the inflammatory response and resulted in elevated transaminase concentrations following i / r. the elevated serum glucose concentration at the start of ischemia seemed to be responsible for the increase in injury, as there was a strong correlation between serum glucose concentrations before ischemia and transaminase concentration after 4 h of reperfusion. serum glucose concentrations were still higher in hyperglycemic animals at the end of the 4 h reperfusion period, albeit the graph (fig. hepatic i / r has been reported to result in hepatocyte death by two different pathways, necrosis and apoptosis. whether apoptotic or necrotic based on terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) staining, it was suggested that sinusoidal endothelial cells and then subsequently hepatocytes undergo apoptosis but rarely necrosis following 60 min of liver ischemia.15 however, a later study applying a very similar ischemia model found only few apoptotic cells and predominantly necrosis following 60 min of ischemia when combining tunel with morphological criteria.16 a subsequent review emphasized that apoptosis and necrosis share features and mechanisms that can make discrimination between both forms of cell death very challenging. in particular, the tunel assay is not suited to differentiate between necrosis and apoptosis, since dna fragmentation was reported in apoptosis as well as necrosis.17 in the present study, histological assessment of the liver samples after 4 h of reperfusion could not reliably distinguish between apoptotic and necrotic cell death. a longer reperfusion could potentially facilitate histological analysis, but 4 h of reperfusion was chosen to enable the detection of inflammatory mediators. as a result, the histological scores were basically identical in both experimental groups, in spite of serologic evidence for increased necrotic cell death in hyperglycemic animals. the higher transaminase concentrations measured in the hyperglycemic group after 4 h of reperfusion reflect increased cellular breakdown due to necrosis with release of intracellular enzymes. apoptosis maintains the barrier function of the cell membrane and would contribute only to a minor extent to elevated transaminase concentrations. caspase-3 activation is considered the most reliable method for the detection of apoptosis.14 we assessed caspase-3 activation to quantify the amount of apoptotic cell death and found higher apoptosis scores in the control group. the lower caspase-3 activation in the hyperglycemic group may be further evidence that necrosis, not apoptosis, is the preferential form of cell death in hyperglycemic conditions. hyperglycemia per se is known to increase oxidative stress and to cause a proinflammatory state.8 furthermore, hyperglycemia has been shown to amplify the inflammatory response caused by stressors such as lps administration.18 our results support the hypothesis that the mechanisms responsible for increased ischemic injury by hyperglycemia are the amplification of oxidative stress and of the inflammatory response normally seen with i / r. the increased concentration of nitrotyrosine containing protein is an established marker for severe oxidative stress. reactive oxygen species, such as the superoxide radical, react with no to form the more potent peroxynitrite species, which then subsequently nitrate tyrosine residues of proteins, leading to inactivation of key cellular proteins, dna damage, and eventually cell death.19 while kupffer cell - induced oxidative stress is considered the first step in i / r injury,20 it is followed by a profound inflammatory response that is largely responsible for the extent of i / r injury. this inflammatory response culminates in the hepatic accumulation of neutrophils, which directly damage hepatocytes by releasing oxidants and proteases. the mpo assay confirmed an increased neutrophil infiltration in the liver tissue of hyperglycemic animals. this neutrophil migration and infiltration is initiated by the production and release of cytokines such as tumor necrosis factor alpha and interleukin-6. earlier studies demonstrated that hyperglycemia enhances cytokine production in response to stress.21 a surprising finding of the present investigation was the downregulation of hsp32 and hsp70 in hyperglycemic animals undergoing i / r. hepatic i / r normally results in upregulation of hsps, and the observed effects in livers from hyperglycemic animals differ distinctly from the situation in kidneys7 and the brain,22 where hyperglycemic i / r injury is associated with an increased activation of hsps. we could demonstrate in sham experiments that hyperglycemia alone was not responsible for the downregulation of hsps (data not shown) but that the combination of hyperglycemia and i / r is required to block or even suppress hsp activation. since hsps are one of the most potent protective mechanisms against i / r injury, it can be assumed that their suppression in hyperglycemic i / r contributes to the increased injury during acute hyperglycemia. inhibition of hsp activation in response to ischemia has so far not been described in other organs and may represent a liver - specific (mal-)adaptation to hyperglycemia : it has been described before that diabetes does inhibit hepatic hsp70 activation by heat stress,23 although subsequent studies did not confirm this finding.24,25 the mechanism responsible for the downregulation of hsps remains to be defined. the expression of the heat shock genes encoding the different hsps is regulated by heat shock transcription factors (hsfs), which are normally bound to hsps within the cytosol. when cells are exposed to stress, hsfs are phosphorylated and form trimers that enter the nucleus and bind the heat shock elements located within the promoter of heat shock genes, thus initiating increased expression of hsps.26 it has been hypothesized that, in diabetes, the activation of hsf is inhibited in insulin - sensitive tissue.27 in type 2 diabetic primates, livers had reduced hsp70 and hsp90 tissue concentrations that were related to 50% lower levels of the transcription factor heat shock factor 1.28 but again, these results are challenged by a study that showed similar heat shock factor 1 content in livers from control and streptozotocin treated rats following heat stress.24 further interventional studies with activation of hsps are planned to show whether the suppression of hsp activation is responsible for the worsened injury during hyperglycemia and whether activation of hsps is capable of reversing such detrimental effects. acute hyperglycemia worsened liver injury as assessed by increased transaminase concentrations following hepatic i / r in rats. the effects of hyperglycemia on liver injury were associated with increased hepatic oxidative stress, an increased inflammatory response, and a suppression of hsp activation. these results, in spite of their descriptive nature, emphasize the need to better understand the role of hyperglycemia in organ injury, especially in clinical scenarios associated with a risk for ischemia. glucose concentrations in this study were overall high, suggesting that glucose control may not need to be very aggressive to have beneficial effects. preventing severe hyperglycemia alone may reduce i / r injury, thus avoiding the inherent risk of an iit to cause undesired hypoglycemia.	background / aimsacute hyperglycemia is known to worsen ischemia / reperfusion (i / r) injury following myocardial infarction and stroke. we investigated whether acute hyperglycemia worsens injury and amplifies the inflammatory response evoked by hepatic i / r.methodsrats were pretreated with an intraperitoneal injection of 25% glucose or 0.9% sodium chloride (10 ml / kg bw). subsequently, rats underwent partial (70%) hepatic ischemia for 45 min. after 4 h of reperfusion, hepatic injury, oxidative stress, inflammation, and heat shock protein expression were assessed.resultsliver injury was increased in the hyperglycemic group with alanine aminotransferase (alt) and aspartate aminotransferease (ast) serum concentrations of 7,832 3,374 and 10,677 4,110 u / l compared to 3,245 2,009 and 5,386 3,393 u / l (p < 0.05 vs. control). hyperglycemic i / r was associated with increased liver nitrotyrosine concentrations and increased neutrophil infiltration. i / r upregulated the protective heat shock proteins hsp32 and hsp70 in control animals, but this protective mechanism was inhibited by hyperglycemia : hsp32 expression decreased from 1.97 0.89 (control) to 0.46 0.13 (hyperglycemia), hsp70 expression decreased from 18.99 11.55 (control) to 3.22 0.56 (hyperglycemia), (expression normalized to sham, both p < 0.05 vs. control i / r).conclusionsacute hyperglycemia worsens hepatic i / r injury by amplifying oxidative stress and the inflammatory response to i / r. the increase in injury is associated with a downregulation of the protective heat shock proteins hsp32 and hsp70.
the antibiotic resistance provides a great therapeutical and economic burden in the treatment of infectious diseases and it may threaten the success of antimicrobial chemotherapy. the disproportion between the slow development of new drugs and the fast emergence of resistant strains necessitates the development and research of new antimicrobial agents or resistance modifiers. one strategy employed to overcome resistance mechanisms is a combination therapy such as using clavulanic acid as inhibitor of -lactamase in drugs sulbactam and tazobactam. however, the frequent use of clavulanate has led to the emergence of resistant bacterial strains. the promising strategy is the use of synergistic effects of natural compounds, products of plant secondary metabolism. traditionally, medicinal plants have been used throughout the world for centuries for a range of medicinal complications. plant drugs are considered to be less toxic and free of side effects than synthetic ones. some work demonstrated that plants either contain antimicrobials that can operate in synergy with antibiotics or possess compounds that have no intrinsic antibacterial activity but are able to sensitize the pathogen to a previously ineffective antibiotic [3, 4 ]. some plants can be sources of compounds that can potentiate the activity of antibiotics against resistant bacterial pathogens [59 ]. these compounds are believed to play a role in the plant 's defense against infection by working in synergy with intrinsic antimicrobials. it has been suggested recently that such compounds can potentially be used to improve the efficacy of antibiotics against bacterial pathogens. salvia species are used as traditional medicines all around the world, possessing antibacterial, antioxidant, anti - inflammatory, and analgesic properties. s. sclarea, popularly known as clary sage, is a biennial or perennial herb with diverse biological activities manifested by different components, mainly of eo. salvia and other plants such as thyme, lavender, sage, basil, coleus, hyssop, and skullcap belong to the large plant family lamiaceae. biological properties of eos and their antimicrobial activity have been attributed to their main compounds such as mono-, di-, and sesquiterpenes and a variety of low molecular weight aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones, coumarins, and homologues of phenylpropanoids. these compounds have hydrophobic characteristics and interact with different sites of microbial cell, namely, with cytoplasmic membrane. they affect the activities of membrane associated enzymes ; certain components of eos can act as uncouplers, which interfere with proton translocation over a membrane vesicle and subsequently interrupt adp phosphorylation. specific terpenoids with functional groups, for example, phenolic alcohols or aldehydes, also interfere with membrane integrated or associated proteins, stopping their production, or activity eos are also able to inhibit the synthesis of dna, rna, proteins, and polysaccharides in fungal and bacterial cells [13, 14 ]. the effect of eo may be associated with the cell envelope by interfering with the rigidity and integrity of the membrane, thereby changing the expression or function of cell wall - related genes such as penicillin - binding proteins (pbps). thymol, a p - cymene derived compound primarily found in some eo, suppresses the toxic shock syndrome toxin (tsst-1) secretion in s. aureus and decreases the production of -hemolysin, sea, and seb enterotoxins in s. aureus. diverse spectrum of eos (s. officinalis, r. officinalis, a. alba, and e. caryophyllata) as well as some of their major compounds (limonene, eugenol, and eucalyptol) inhibited qs gene expression in s. aureus. extract from rhus javanica showed the inhibition of the genetic expression of virulence factors such as sea, agra, and sara in methicillin resistant s. aureus, as well as meca gene, which is responsible for staphylococcal resistance to -lactam antibiotics. staphylococcus epidermidis was previously regarded as an innocuous commensal microorganism on the human skin and mucous membranes. however, nowadays it is seen as an important opportunistic pathogen, one of the most prevalent causes of nosocomial infections associated with newborn, severely ill, and immunocompromised patients, and is also frequently isolated from postsurgical infections, especially in association with indwelling prosthetic devices. it was found that approximately 70% of the s. epidermidis strains circulating in the hospital environment are resistant to methicillin and that the majority of them are also resistant to other antimicrobial classes. resistance to methicillin is at 7590% among hospital isolates of s. epidermidis, which is even higher than the corresponding rate for s. aureus (4060%). resistance to -lactam antibiotics in s. epidermidis, similarly as in s. aureus, is mediated by (i) production of -lactamase which hydrolytically destroys the -lactam antibiotics and (ii) the acquisition of meca gene that produces alternative pbp2, which have low affinity to -lactam antibiotics. gene meca is carried on a chromosomal genetic element designated as staphylococcal chromosomal cassette mec (sccmec). currently, there are several different types of sccmec elements, with several subtypes, characterized by a unique combination of the mec and the recombinase - encoding ccr gene complexes. some reports suggest that, in coagulase negative staphylococci, namely, in s. epidermidis, sccmec structures are more diverse and include either mec - ccr combinations not yet described for s. aureus or more than one ccr allotype. in s. epidermidis characterized 139 isolates of mrse and found that 41% of the isolates harbored sccmec type iv and 27% carried sccmec type iii, while sccmec types v, i, and ii were presented only in 6%, 4%, and 4% of the isolates, respectively. of the 44 mrse isolates recovered from the blood of patients with prosthetic valve endocarditis, 2% harbored sccmec type i, 34% harbored type ii, 28% harbored type iii, and 36% harbored type iv. expression of meca is inducible and can be controlled by either its cognate regulators meci (dna binding repressor protein) and mecr1 (sensor / signal transducer) or the structurally and functionally similar -lactamase regulators blai and blar1, respectively. the transcription of meca and blaz is corepressed by the regulators of the two regulons, meci and blai. these regulators are almost identical and can replace each other and both meci and blai can bind as homodimers to the promoter / operator region of both meca and blaz. in the presence of antibiotics, the sensing is mediated by signal transduction via the two transmembrane inducers, mecr1 or blar1, which will result in proteolytic autocleavage of the cytoplasmic domains of these proteins. to perform this autocleavage, autocleavage of the signal transducer is followed by cleavage of the cognate repressor, meci or blai, and by subsequent induction of the transcription of meca or blaz. in our previous work we showed synergistic effects of oxacillin with plant extracts and eos from several plant species of lamiaceae family. the purpose of the present study was to determine the effect of eo from s. sclarea on the expression of meca gene in strains s. epidermidis possessing different types of sccmec and determine synergistic effect of oxacillin and eo in these strains. the aerial parts of s. sclarea were harvested at the optimal growing and development stage. the plant material was air - dried and submitted to hydrodistillation for 4 h. isolated oil was diluted in n - hexane and dried over anhydrous sodium sulfate. mrse strains were obtained from clinical samples of patients with positive hemocultures from the university teaching hospital old town, bratislava, slovak republic, and were kindly provided by dr. slobodnkov from the institute of microbiology, faculty of medicine, comenius university, in bratislava, slovak republic. the isolates were screened for their susceptibility towards oxacillin using disc diffusion method on mueller - hinton agar (himedia, india) in accordance with the clinical and laboratory standard institute guidelines (clsi) standards. suspension of the tested bacteria (0.1 ml of 10 cells / ml) was spread onto solid media plates. antimicrobial susceptibility test discs (himedia, india) with ampicillin (10 g) and oxacillin (1 g) were placed on the incubated plates. these plates, after 2 h of maintenance at 4c, were incubated for 24 h at 37c and the diameters of the resulting zones of inhibition were measured in millimeters. extraction of genomic dna was performed by using dneasy blood and tissue kit (qiagen, germany) according to the manufacturer 's instructions. all the isolates were tested for genes of the mec and bla operon using the polymerase chain reaction method. each pcr sample contained 2.5 l of 10x star taq enzyme buffer (gene craft, germany), 0.2 mm of each deoxynucleoside triphosphate (gene craft, germany), 0.2 m of each of the forward and reverse primers, 1.5 u taq dna polymerase (gene craft, germany), and 2 l template dna. the pcr primers were designed by primer3 and primer - blast and synthesized by microsynth (switzerland). the cycling conditions were as follows : preheating for 5 minutes at 94c, followed by 30 cycles of denaturation at 94c/30 seconds, annealing at 55c/40 seconds, extension at 72c/30 seconds, and final extension for 5 minutes at 72c. the sccmec type was determined using the protocol and primers proposed by zhang.. multiplex pcr was performed in 25 l reactions with 2.5 l of 10x star taq enzyme buffer (gene craft, germany), 0.2 mm of each deoxynucleoside triphosphate (gene craft, germany), various concentrations of the respective primers, 1.0 u of taq dna polymerase (gene craft, germany), and 2 l of template dna. the amplification was performed using a biometra thermal cycler (germany) with an initial denaturation step at 94c/5 min followed by 30 cycles of denaturation at 94c/1 min, annealing at 55c/1 min, extension at 72c/2 min, and a final extension step at 72c/5 min. minimal inhibitory concentrations (mic) for oxacillin (sigma - aldrich) and eo (prepared as stated above) were determined by standard broth microdilution method using 96-well microtiter plates in accordance with clsi in mueller - hinton broth. the s. epidermidis suspension was adjusted to the 0.5 mcfarland standard and diluted to obtain a final turbidity in wells approximately 1 10 cfu / ml. 0.1 ml of bacterial suspension was mixed with an equal volume of each dilution of oxacillin or eo, and optical density (od600) values were determined after 24 hours of growth at 37c using a 96-well plate reader varioskan flash (thermo fisher scientific, finland). the mic was defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism. the study of the interaction between eo and oxacillin was done using the checkerboard method. twofold serial dilutions of oxacillin prepared in horizontal rows of 96-well microtiter plate were subsequently cross - diluted vertically by twofold serial dilutions of eo. microtiter plates were inoculated with test organism and incubated for 24 h at 37c. mic values of the combinations were determined as the lowest concentration that completely inhibited bacterial growth recorded as the optical density at 600 nm using varioskan flash (thermo fisher scientific, finland). interaction between eo and oxacillin was then determined by calculating the fractional inhibitory concentration (fic) indices. the fici is defined as follows : mic of substance a tested in combination / mic of substance a tested alone + mic of substance b tested in combination / mic of substance b tested alone. the fici is interpreted as follows : fici 4, antagonistic effect. the shape of the isobologram curve can be convex, linear, or concave, which is indicative of the synergistic, indifferent, and antagonistic interactions, respectively. the time - kill assay was carried out in order to determine antibacterial and potential synergistic effects of eo when used singly and in combination with oxacillin. bacteria (5 10 cfu / ml) were exposed to oxacillin and eo alone or in combination in concentrations 1/2 mic and incubated at 37c. aliquots (0.1 ml) were taken at 0, 6, 10, and 24 h and diluted in normal saline as needed to enumerate 30300 colonies. the diluted cultures were spread thoroughly on plates containing mueller - hinton agar. after incubating at 37c for 24 h, the growing colonies were counted. the experiment was performed in triplicate ; data are shown as mean standard deviation from three independent experiments. mrse strains were treated with various concentrations of oxacillin and eo alone or with their combination for 30 minutes. total rna was isolated by using rneasy mini kit (qiagen, germany) according to the manufacturer 's instructions. dna contamination from the total rna preparations was removed with on - column rnase - free dnase treatment (qiagen, germany). to generate cdna, total rna was reverse transcribed using the gotaq 2-step rt - qpcr system (promega) with specific primers (table 1). real - time pcr was performed at least three times for each examined gene with an applied biosystem 7500 fast real - time pcr system. the ct values and the qpcr were normalized to the housekeeping gene for glyceraldehyde-3-phosphate dehydrogenase (gapdh) using the 2 method. the experimental data are expressed as the mean standard deviation (sd) from three independent experiments. the essential oil obtained from aerial parts of s. sclarea was analyzed by gas chromatography (gc) and 12 compounds representing 75.2% of the essential oil were identified. the main components were linalyl acetate (38.67%), linalool (20.42%), germacrene (5.31%), geraniol (1.42%), and -caryophyllene (1.80%). in our previous work we showed that addition of plant extracts and eos from some species of salvia plants increases the susceptibility of mrse to oxacillin. eos and oxacillin in combination showed synergistic effect, inhibited growth, and even killed the bacteria. in this study we used 30 clinical isolates of mrse for study of potential synergistic effects of eo from s. sclarea and oxacillin, and by using checkerboard method we confirmed this effect in 23 isolates (76.7%) (fici from 0.125 to 0.381). the remaining 7 strains (23.3%) showed additive effect (fici from 0.531 to 0.793). based upon the phenotype analysis of methicillin resistance (mic of oxacillin, table 2), screening for presence or absence of meca, meci, mecr1, blaz, blai, and blar1 gene using pcr (figure 1), and analysis of mec class (figure 2) and on sccmec typization we selected four strains possessing different sccmec types i, ii, iii, and iva (figure 3), which cover all sccmec types and all variations of mec and bla operons present in collection of isolates. we have attempted to assess if eo alone or in combination with oxacillin influences the expression of meca gene. firstly, we characterize the meca / meci / mecr1 and blaz / blai / blar1 regions of our strains and determine the effect of presence or absence of mec and bla elements on the mic of oxacillin. isolates r17 and r12 which possess sccmec types i and iva, respectively, and class b mec have genotypes + blai/+blar1 and meci / mutmecr1 (absent meci and truncation of mecr1 by insertion of is1272). these strains showed high resistance, correlating with the fact that meci deletion can lead to increased resistance. isolate r8 (sccmec type ii) has both complete repressor / sensor systems + meci/+mecr1 and + blai/+blar1. isolate r22 (sccmec type iii) has + meci/+mecr1 system but this isolate was the only one in which we were unable to confirm the presence of the bla operon. we used real - time pcr to investigate the expression of genes from mec and bla operons and to determine the influence of eo on their expression. quantification data for all genes were normalized to the reference gene for glyceraldehyde-3-phosphate dehydrogenase (gapdh). in figure 4 regardless of constitutive or inducible expression of genes our data show that 30 min treatment of culture with subinhibitory concentrations of eo from s. sclarea leads to decreasing of expression of all genes from both operons. it is known that eos influence membranes and proteins in cytoplasmic membrane ; therefore we can assume that they can influence conformation of mecr1, or blar1, which can have impact on transcription of both operons. while eo showed similar and dose dependent effect on single genes from mec operon, in case of bla genes the effect of eo on blai and blar1 gene expression was weaker. similar results, the inhibition of expression of resistant genes meca, meci, and mecr1, against methicillin resistant s. aureus by hexane and chloroform fractions of salvia miltiorrhiza bunge, were described by lee.. one isolate from each sccmec type was exposed to increasing amount of eo and as is shown in figure 5, eo reduced the expression of meca gene in all strains. regarding sccmec type and genetic background of mec and bla operons, strains r8 and r22 have mec a class with all three mec genes, while strains r17 and r12 have mec b class (absent meci and truncation of mecr1 by insertion of is1272). as is obvious from figure 5, we did not find significant differences in expression of meca gene between strains with different mec class after treatment with eo at concentrations 1/4 and 1/2 of mic ; however in concentration 1/8 mic there was statistically significant (p < 0.05) difference between strains of both classes of mec complex, when eo decreased the expression of meca gene more in strains r17 and r12 in the comparison with strains r8 and r22. we estimated the expression of meca gene in the presence of eo also in three random selected strains with iva type of sccmec and the results were similar (data not shown). finally we examined the expression of meca gene after 30 min treatment with oxacillin and eo. the addition of eo not only inhibited the induction of expression, but also reduced the expression of meca gene in the comparison with untreated control (figure 6). the combination of 1/4 mic of both compounds was more effective, probably due to lower concentration of oxacillin and lower induction of expression of meca gene. we found similar trend as in the case of eo alone, that is, higher reduction of expression in strains r17 and r12 in the comparison with strains r8 and r22. the viable cell counts for four isolates of mrse differing in type of sccmec after exposure to 1/2 mic of oxacillin and eo alone and in combination at different times are shown in figure 7. the combination of 1/2 mic eo + 1/2 mic oxacillin completely inhibited the growth of all four strains after 24 h. the same combination caused an over 3 log10-fold reduction in the bacterial count yet after 10 h (strains r17 and r8) in comparison with the most active compound alone. in strain r12 the reduction was more than 5 log10-fold in 6 h and from 10 h the growth of this strain was completely inhibited. to get a better understanding of the modulatory effect of eo we examined the interaction between eo and oxacillin via the checkerboard method and described it in terms of fractional inhibitory concentration (fic) indices. the fic indices of eo in combination with oxacillin were 0.381 for strain r17, 0.156 for r8, 0.125 for r22, and 0.376 for r12, which according to pillai. the synergistic interaction can be read according to the curve indicating borderline synergy according to fic 0.5. isobolograms of all strains have convex shape beyond the borderline of synergisms and clearly show the potentiating effect of s. sclarea eo on oxacillin susceptibility in these strains. our data reveal the potential of eo from s. sclarea to be candidate for combination therapy against mrse, because it has synergistic effect with oxacillin in all tested strains. the killing effect of the combinatorial treatment is connected with reduction of expression of meca gene and other genes participating in staphylococcal resistance to -lactams antibiotics. lower expression of these genes reduces the two major bacterial defense mechanisms against -lactam antibiotics. although synergistic effect of eo may be caused by several mechanisms, observed different levels of the reduction of meca expression in strains with different types sccmec and different genetic background in mec and bla operons confirmed that reduction of meca gene expression can be one of major mechanisms.	the essential oil (eo) from salvia sclarea was shown to increase the susceptibility of methicillin resistant staphylococcus epidermidis (mrse) isolates to oxacillin. the purpose of this study was to investigate the effect of eo from s. sclarea on expression of meca gene of mrse carrying different types of staphylococcal chromosomal cassette (sccmec) and to evaluate potential synergistic effect of eo with oxacillin. using real - time pcr we found that eo alone inhibited the expression of the resistant genes meca, mecr1, and meci and blaz, blar1, and blai. the use of the combination of eo with oxacillin resulted in significantly inhibited expression of meca gene in all tested strains with different types of sccmec. using time - kill assay and checkerboard assay we confirmed synergistic effect of eo from s. sclarea and oxacillin in mrse.
the prevalence of hepatitis c virus (hcv) has been estimated to be 3% throughout the world. liver biopsy is currently considered as the gold standard for determination of the grade of fibrosis. presence of significant fibrosis (f24) is accepted as the treatment indication [2, 3 ]. liver biopsy is unfortunately associated with many complications for patients, including pain, bleeding, and rarely death, high costs, variations in diagnosis of histological grades, and sampling errors. furthermore, liver biopsy can provide only a view of a static or dynamic disease, and multiple biopsies are required to determine the disease progression or recurrence of fibrosis. this reveals the need for noninvasive, precise, and valid methods.considerable advances have occurred in identification of nonspecific fibrosis biomarkers. the nonspecific markers include age, gender, and laboratory markers of liver damage or dysfunction (ast, alt, -glutamyl transferase (ggt), bilirubin, hapatoglobin, platelet count, and prothrombin time), while metabolic markers are cholesterol, apoprotein a1 (apo a1), and 2-macroglobulin (a2 m). so far, the index of ratio of ast to platelet count (apri) has been the simplest test using nonspecific markers, which is valuable in predicting fibrosis [69 ]. the fibrometer test, which includes hyaluronic acid (ha), pt test, platelet count, ast, a2 m, urea, and age, is to some extent efficient. another achievement was the use of specific fibrosis biomarkers such as ha, matrix metalloproteinase 2, tissue inhibitor of matrix metalloproteinase 1, and amino - terminal peptide of type iii procollagen [1, 10 ]. when used in combination with each other, it is known that these markers are valid in determining the liver fibrosis score. recently, some attempts have been performed to improve the nonspecific indices by use of fibrosis - specific markers. adams. suggested hepascore, which is a combination of ha, total bilirubin, ggt, a2 m, age, and gender. hepascore was shown to be valid in the hcv population in australia and then in two other groups in a european country (france) [1214 ]. the aim of the current study is to compare the results of liver biopsy of patients with hepatitis c, genotype 1, in isfahan with their hepascore, and also to determine sensitivity, specificity, and positive and negative predictive values of hepascore. the participants were in the age range of 1865, with the mean age of 35.3 12.7. out of the participants, 68 patients (85%) were males. among the patients with hepatitis c, genotype 1, who referred to the gi clinic or ward of alzahra hospital from april to october 2010 and met the inclusion criteria of the study, 80 patients the inclusion criteria were the newly diagnosed patients of hepatitis c, genotype 1, who were candidates of liver biopsy. if the patients were not willing to participate in the study, they were excluded. all liver biopsies were taken under supervision of a hepatology subspecialist using an 18-gauge menghini needle or a 16-gauge trucut needle, with the size of 10 mm. the specimens were then evaluated by a single skilled pathologist for the degree of fibrosis according to the metavire classification. the degree of fibrosis was classified in a 04 scale as follows : f0 : no fibrosis, f1 : portal fibrosis alone, f2 : portal fibrosis with rare septae, f3 : portal fibrosis with many septae, and f4 : cirrhosis. grades f2, f3, and f4 indicate significant fibrosis, f3 and f4 show severe fibrosis, and f4 indicates cirrhosis. from all the participants, a 10 ml blood sample was obtained, and its serum was kept at 70c. determination of serum level of hyaluronic acid (corgenix, usa), ggt (biosystems, spain), and a2 m (immundiagnostik, german) was performed using enzyme - linked protein binding assay, and the level of total bilirubin was determined using biosystem a15 autoanalyzer with specific reagents. to calculate the hepascore, the values obtained for the four biomarkers, a2 m, ggt, total bilirubin, and ha, and the age and gender of the patient are set in the following formula, which was first issued by adam. in 2005 : (1)y = exp(4.185818(0.0249 age) + (0.7464 sex)+(1.0039 a2 m) + (0.0302 ha)+(0.0691 bil - t) (0.0012 ggt)),hepascore = y(1+y). the value for sex in the above formula is 1 for men and 0 for women. all data was analyzed in the spss software, using rho spearman and roc analysis. the range of variation, mean, and standard deviation values for ha, a2 m, ggt, and bilirubin is provided in table 1. the results of liver biopsy for the patients were as follows : 12 patients were f0, 25 were patients f1, 20 were patients f2, seven patients were f3, and 16 patients were f4. the frequency distribution for fibrosis severity according to the biopsy results is demonstrated in table 2. mean and median of hepascore in different degrees of fibrosis (according to biopsy) are shown in figures 1(a) and 1(b), respectively. correlation between hepascore and biopsy results according to the results obtained from spearman 's correlation test, there is a relatively strong correlation between severity of fibrosis estimated by hepascore and that determined by liver biopsy (r = 0.465, p = 0.003). according to the results obtained from spearman 's correlation test, there is a relatively strong correlation between severity of fibrosis estimated by hepascore and that determined by liver biopsy (r = 0.465, p = 0.003). sensitivity and specificity of hepascore in diagnosis of significant fibrosis (f2, f3, and f4 from f0 and f1) in different cut - off points are shown in figure 2. in cut - off point 0.34, sensitivity, specificity, ppv, and npv sensitivity and specificity of hepascore in diagnosis of severe fibrosis (f3 and f4 from f0, f1, and f2) in different cut - off points are demonstrated in figure 3. in point 0.61, sensitivity, specificity, ppv, and npv sensitivity and specificity of hepascore in diagnosis of cirrhosis (f4 from f0, f1, f2, and f3) in different cut - off points are shown in figure 4. in point 0.84, sensitivity, specificity, ppv, and npv were 100%, 97%, 89%, and 100%, respectively. prognosis of chronic liver diseases is strongly correlated with the degree of liver fibrosis. in chronic hepatitis c, besides having prognostic value, liver fibrosis is related to the therapeutic approach. so far, no fda - approved noninvasive method has been proposed for determination of liver fibrosis. a suggested and growing method is determination of hepascore of the patients on the basis of blood markers of fibrosis. the current study demonstrated that hepascore index has a reasonable sensitivity, specificity, npv, and ppv. according to the results obtained, from among the cut - off points between 0 and 1, the most appropriate point for diagnosis of significant fibrosis from mild fibrosis and no fibrosis (f0 and f1) was 0.34. in other studies, cut - off points from 0.32 to 0.55 were obtained [1, 12, 1416 ]. the authors who devised hepascore and becker. suggested point 0.55 as the most appropriate cut - off point, while leroy. considering the above - mentioned points, the sensitivity of hepascore in diagnosis of significant fibrosis was obtained to be point 67%. the value was reported to be from 54% to 82% [1, 12, 14 ]. moreover, the specificity of the method was determined to be 56%, which was reported to be from 63% to 89% in previously performed studies [1, 10, 12, 16, 17 ]. the ppv and npv of the test in diagnosis of significant fibrosis were determined to be 64% and 56%, respectively. in previous studies, the values were determined to be 59% to 89% and 64% to 80%, respectively [1, 14 ]. according to the results obtained, the most appropriate point for diagnosis of severe fibrosis from milder forms of fibrosis was determined to be 0.61, and the sensitivity and specificity were 82% and 86%, respectively. the appropriate cut - off points for this purpose were determined to be from 0.53 to 0.84 in previous studies. we obtained the most appropriate cut - off point for diagnosis of cirrhosis for milder forms of fibrosis to be 0.84. at this cut - off point, sensitivity, specificity, ppv, and npv were determined to be 100%, 97%, 89%, and 100%, respectively. determined sensitivity and specificity of hepascore in diagnosis of cirrhosis to be 71% and 84%, respectively. guchot. reported the sensitivity, specificity, ppv, and npv of the test in diagnosis of cirrhosis to be 86%, 74%, 37%, and 97%, respectively. in spite of the differences among the values obtained in different studies, the high sensitivity and npv were noteworthy in all the studies the values obtained for these items were 100% in the current study. therefore, using hepascore, one can surely make decision on performance of screening for hepatocellular carcinoma, as well as carrying out endoscopy for evaluation of esophageal varicosis, both of which are currently rather high - cost and invasive procedures. a factor that affects the above - mentioned elements is the difference in frequency distribution of severity of fibrosis in different studies. for instance, the prevalence of significant fibrosis in the current study was 53.8%, while the rate was reported to be 44% to 51% in other studies. nevertheless, since in clinical settings the aim of evaluation of these patients is detection of significant fibrosis to initiate the treatment, higher sensitivity of the test is of great importance. as can be observed in table 3, the findings of the current study are to a great extent consistent with other studies with regard to the relative weakness of hepascore in diagnosis of lower stages of fibrosis and its high power in diagnosis of high stages of fibrosis. however, compared with previously performed studies, the cut - off point determined in the current study has a higher sensitivity and specificity for diagnosis of severe fibrosis and cirrhosis. hepascore is highly valuable in diagnosis of the severity of liver fibrosis and particularly cirrhosis (f4) and can be used as a primary screening method for diagnosis of the need for carrying out liver biopsy, which is a method with high costs and complications. one of the limitations of the study was its small sample size, which was due to the economic constraints. with respect to the promising results obtained, similar study on a larger population can be performed.	introduction. liver biopsy is an invasive determinator for hepatic fibrosis. serum biomarkers can probably be used as an alternative to liver biopsy in assessment of the degree of fibrosis in patients with chronic hepatitis c. method. eighty patients with chronic hepatitis c were included in the study using simple nonrandom sampeling metod. after fulfillment of liver biopsy, the patients were categorized according to the metavir scoring system. the hepascore algorithm is computed based on age, sex, and the serum levels of total bilirubin, -glutamyl transferase, 2-macroglobulin, and hyaluronic acid. the spearman and roc tests were used. results. according to the liver biopsy results, 12, 25, 20, 7 and 16 patients had f0, f1, f2, f3, and f4, respectively. with regard to the 0.34 cut - off point hepascore had 67%, 56%, 64%, and 56% sensitivity, specificity, respectively, positive prediction value (ppv), and negative prediction value (npv), respectively, for diagnosis of significant fibrosis. for a hepascore cut - off point 0.61, sensitivity, specificity, respectively, ppv and npb 82%, 86%, 70%, and 92% in diagnosis of severe fibrosis. for a hepascore cut - off point 0.84, sensitivity, specificity, ppv and npb were respectively 100%, 97%, 89%, and 100% for diagnosis of cirrhosis. conclusion. hepascore has a high value in diagnosis of the level of fibrosis, particularly cirrhosis. therefore, it can be used for primary screening of patients to determine the need for liver biopsy.
desanto studied 238 cases with laryngeal cystic lesions and mainly classified them into two types as follows. the first type, laryngocele, contains air in the cyst, and the second, laryngeal cyst, has mucus. since both laryngoceles and laryngeal cysts are rare in japan, we present the results of physical examination, image findings and treatment. the patient was a 64-year - old japanese man who underwent digestive endoscopy at a clinic, where a laryngeal mass was pointed out. laryngeal fiberscope revealed a swelling of the left false cord (figure 1) that we diagnosed as a laryngocele. under general anesthesia, the laryngocele was removed by laryngomicrosurgery using an oral approach and the content of the specimen was confirmed to be air. the postoperative course was good. a 57-year - old japanese man presented with a one - month history of left laryngeal swelling with pain. laryngeal fiber - scope revealed a swelling of the left false cord and laryngeal ventricle. magnetic resonance imaging demonstrated a dumbbell - type cyst with mucus widely extending from the laryngeal lumen to the neck through the thyroid cartilage (figure 2). we performed fine needle aspiration and confirmed the presence of mucus (figure 3). the patient was a 64-year - old japanese man who underwent digestive endoscopy at a clinic, where a laryngeal mass was pointed out. laryngeal fiberscope revealed a swelling of the left false cord (figure 1) that we diagnosed as a laryngocele. under general anesthesia, the laryngocele was removed by laryngomicrosurgery using an oral approach and the content of the specimen was confirmed to be air. a 57-year - old japanese man presented with a one - month history of left laryngeal swelling with pain. laryngeal fiber - scope revealed a swelling of the left false cord and laryngeal ventricle. magnetic resonance imaging demonstrated a dumbbell - type cyst with mucus widely extending from the laryngeal lumen to the neck through the thyroid cartilage (figure 2). we performed fine needle aspiration and confirmed the presence of mucus (figure 3). small lesions localized at the laryngeal lumen are generally treated by laryngeal microsurgery using an oral approach. large cysts growing from the laryngeal lumen to the neck through the thyroid cartilage require extirpation by an external approach, often adding tracheotomy to maintain the air way. our case # 2 with a laryngeal cyst was under chemotherapy for advanced renal carcinoma with multiple metastases of both lung and bone. therefore, we performed only fine needle aspiration rather than aggressive surgery for extirpation of the cyst using an external approach. this fine needle aspiration could improve the quality of life by decreasing both the laryngeal swelling and pain.	we experienced two rare cases with laryngeal cystic lesions (laryngocele and laryngeal cyst). in the first case, the laryngocele case was removed by laryngomicrosurgery using an oral approach under general anesthesia. in the second case, the magnetic resonance imaging demonstrated a dumbbell - type cyst with mucus widely extending from the laryngeal lumen to the neck through the thyroid cartilage. the patient had undergone chemotherapy for renal carcinoma with multiple lung and bone metastases. therefore, we performed only fine needle aspiration rather than aggressive surgery for extirpation of the cyst using an external approach. this fine needle aspiration could improve the quality of life by decreasing both the left laryngeal swelling and the resulting pain, which were the chief complaints.
the most important arbovirus disease in humans, dengue, annually affects 80 million individuals in many countries, leading to 550,000 hospitalizations and 20,000 thousand deaths. the main vector is the mosquito aedes aegypti, an arthropod with an extremely high capacity to adapt to urban areas. since 1982, the reemergence of dengue has been reported in urban centers in all brazilian regions. the magnitude of this disease has led to high public federal, state, and municipal investments in vector control, epidemiological surveillance, and patient care. during the 1990s, the incidence of dengue increased greatly as a consequence of the dissemination of a. aegypti. dispersion of the vector was followed by the dissemination of dengue virus serotypes 1 and 2 in twenty of the 27 states of the country. between 1990 and 2000, several epidemics occurred, mainly in the largest urban areas of the southeast and the northeast, where the majority of notified cases were concentrated. the first great dengue epidemic occurred in 1998, with approximately 528,000 cases. in brazil, the increase in the incidence of dengue cases in 2002 and the emergence of a third serotype (denv-3) led to a prediction of an increased risk of dengue epidemics and an increase of the cases of dengue hemorrhagic fever (dhf). to face the expected risks for 2002, the brazil ministry of health, in collaboration with the pan - american health organization, carried out an international seminar in june 2000 to evaluate the dengue epidemic and to prepare a national dengue control program (pncd). however, the current epidemiological situation shows that these program measures have not achieved the expected results. epidemiological impact assessments of these interventions have shown that their effectiveness has been extremely limited. regardless of each local health system, even when these measures are well managed, their effectiveness is always low, given the intense viral circulation detected in the successive epidemics and the results of serological surveys conducted in several brazilian cities [4, 5 ]. the first dengue epidemic in belo horizonte (bh), the principal city of brazil 's third metropolitan area, occurred in 1996, and different from the subsequent epidemics, the 1996 epidemic started in the southern hemisphere 's fall. however, by the end of 1997, another epidemic of great intensity started, characterized by the simultaneous circulation of denv-1 and denv-2., denv-3 was identified for the first time in bh, and now the three serotypes coexist. the control measures, adopted in bh until the 1998 epidemic, had only a limited role, without much impact on the final numbers of cases. this situation was repeated in 1997 and only changed its stance in 1998, before the largest epidemic in the city when denv-1 and denv-2 virus serotypes were both circulating. in 2002, it was observed that the spread of serotype 3 from the state where it was originally detected presented a different pattern from that observed with serotypes 1 and 2. previously, the expansion of the new serotype (denv-3) occurred slowly and some years elapsed before autochthonous cases occurred in other states. during the first three months of 2002, the presence of the new serotype was detected in ten other states. in bh, it would be theoretically possible to attribute these results to the control measures proposed by the brazil ministry of health in 1996, the program of eradication of a. aegypti known as peaa which was only implemented in the municipality in 1998. this program took into account the difficulties of the previous control strategy and proposed an even more complex objective, predicated on the assumption that the vector could be eradicated. when compared to other large urban areas in southeast brazil, the dengue epidemic cycle in bh this epidemic behavior was probably only interrupted when the resistance to the larvicidal agent being used was detected in bh in 2006. currently, vector control is the only way to interrupt disease transmission, given that there is neither an effective vaccine nor specific therapy. vector control, however, is not a simple task, especially given the complexities of urban settings. the failure of dengue control programs has been pointed out by several authors [7, 1113 ]. spatial analyses are powerful tools in public health diagnosis and surveillance, allowing the identification of critical areas for intervention and the variables associated with the modulation of disease dynamics [14, 15 ]. dengue, whose pattern is well known to be clustered in certain areas, is a health - related event for which spatial analysis techniques may be useful. spatial analyses and statistics, such as spatial autocorrelation analysis, cluster analysis, and temporal analysis, are commonly used to highlight spatial patterns of dengue cases and to test whether there is a pattern of dengue incidence in a particular area [17, 18 ]. a geographic information system (gis) can be used to identify and assess potential compositional and contextual risk factors associated to disease transmission such as socioeconomic, climatic, demographic, and physical environment. gis technologies have been applied in epidemiologic and public health studies for many years [19, 20 ], providing information useful for studying and modeling the spatial - temporal dynamics of dengue [2123 ]. this paper aims to evaluate dengue dissemination in space and time, determining possible outbreak waves of dengue cases correlated with climatic data and presence of the vector. this study may contribute to implement interventions aimed at vector control and patient care, minimizing the collective and individual burden of this disease. this ecological study was conducted in belo horizonte (bh), the capital of the state of minas gerais, in the southeast region of brazil (1955s 4357w). occupying an area of 330.23 km with 2,375,151 inhabitants in approximately 600,000 households (figure 1), bh is brazil 's sixth most populous city. situated at altitudes ranging from 700 to 1,200 meters (mean 858 meters), bh has a tropical wet and dry climate with an average annual temperature of approximately 21c. each one of 147 primary care units is responsible for a geographic area known as a health services catchment area (hsca). the hscas are aggregated in nine sanitary districts (sds) named as north, northeast, northwest, east, south central, west, venda nova, pampulha, and barreiro. all dengue cases reported from 1996 to 2011 (partial) to the municipal surveillance system which in turn are forwarded to brazil 's national reporting system were used. the notification form contains, along with other information, each patient 's address and the date of onset of dengue symptoms. dengue larvae vectors foci data reported for years 1996 to 2011 (partial) and eggs collected in ovitraps from 2003 to 2010 were used in this study. the data was obtained from the municipality vector reporting system sczoo which contains the address for each larva focus and ovitrap and the dates of the survey. the ovitraps which cover a radius of 200 meters are installed every two weeks. the building larval index (bli) as proposed by connor and monroe measures the density of a. aegypti in urban areas and is estimated as the proportion of houses with a. aegypti larvae. rainfall (mm) and temperature (degrees celsius) for the years 20012010 were obtained from weather station of the 5th district of brazil 's meteorological institute (inmet). depending on the analysis (see below), dengue incidence was calculated on a monthly or annual basis from 1996 to 2011. then the dengue incidence in a given year for each sanitary district from 2005 to 2011 was correlated to september - october vector data (the mean number of eggs in the ovitraps of each sd and the bli in the larvae foci survey) from the previous year. we used the pearson correlation coefficient to estimate the correlation between the monthly incidence of dengue and climate data for the years 2001 to 2010. the vector data was geocoded using the address of the larvae foci building and the locations of the ovitraps. spatial statistical techniques used in this study included kernel 's estimation in order to determine the possible outbreaks of disease and specific patterns of distribution on the urban space. to find how dengue spread in space and time, we created map objects that change status with time. a hotspot moreover, cases clusters that occur randomly can also have an influence on the spread of an infectious disease. tabwin 3.5 was used to make brazil municipalities maps (http://www.datasus.gov.br/), and r (r development core team ; http://www.r-project.org/) was used to calculate the pearson correlations and kernel 's estimation. mapiinfo 8.5 was used to make bh hotspots maps, and excel 2003 was used to generate tables and figures. in this series of annual incident dengue cases, five distinct periods were identified : (1) between april 1996, the first epidemic in bh, and july 1998, the most important epidemic ; (2) between august 1998 and december 2000 with incidence rates not exceeding 10 cases per 100,000 inhabitants ; (3) between january 2001 and august 2002, during which two new epidemics occurred ; (4) between august 2002 and december 2005 again with low dengue incidence rates ; (5) the last period, between january 2006 and august 2010, during which the incidence rate was progressively higher (figure 2). the dengue temporal distribution with highest incidence in the rainy season presented a similar pattern during the period (figure 2). characteristically, dengue outbreaks generally occurred during the second part of the rainy season, when humidity was higher than average. in the period from 2005 to 2011, annual incidence rates of dengue showed a statistically significant correlation with the bli according to sanitary district (r = 0.60, p = 0.0000002). for the mean values of eggs captured in the ovitraps, the correlation was also statistically significant (r = 0.69, p = 0.00000005) (table 1). rainfall (rf) and temperature (temp) begin to increase in october, with dengue outbreaks occurring during the months of january to may, the period of highest rainfall and humidity. the number of cases then fall through june, a period when rf and temp also decrease (figure 2). analyzing the climatic data for the years 2001 to 2010, monthly dengue incidence rates showed a statistically significant correlation with the rf of the previous month (r = 0.36, p = 0.00006) and the monthly minimum temperature (r = 0.29, p = 0.001). the maps that comprise figure 3 illustrate the spatial and temporal evolution of dengue in cities of brazil and are accompanied by a comparative graph of annual incidence rates from 2001 to 2011 for bh, brazil. figures 4 and 5 demonstrate the spatial correlation between dengue cases hotspots and the location of aedes aegypti larvae foci in bh. figure 6 shows the same observation among dengue cases hotspots and the areas with the greatest presence of aedes aegypti eggs. the hotspot analysis also found a higher risk of dengue in areas of the city that are at lower elevations (figures 7 and 8). monitoring and planning control measures for dengue epidemics are vital for preventing or minimizing disease outbreaks. information based on notified cases only, however, is insufficient, because many people who are infected may either be asymptomatic or do not become part of the official statistics even if they present symptoms. the use of information on dengue incidences rates, mapping their patterns and dynamics of spread using spatial autocorrelation analysis, can be a valuable tool to analyze the spatial patterns change over time. therefore, instead of aiming to achieve a complete understanding of the transmission process, it may be more efficient to improve the surveillance system and optimize disease control. the heterogeneous intraurban distribution of dengue incidence according to sanitary districts for the years 2001 to 2011 suggests the importance of analyzing transmission at the sd level. the degree of acquired immunity to the dengue virus may vary across different areas of the municipality based on the spatial distribution of previous outbreaks. our results indicate that continuous vector surveillance using ovitraps and larvae foci is necessary, so that a greater number of areas with potential transmission can be identified, permitting the prioritization and scheduling of vector control measures. certainly, the identification of high - risk areas, in a process of surveillance and control of the disease and the mosquito, is an important step towards optimizing resources. once such areas have been identified, interventions may provide better results in decreasing incidences rather than through the traditional approach of a uniform control strategy for the city as a whole. determining whether greater vector presence or coefficients of dengue incidence predominant in certain intraurban areas may be operationalized through the use of the concept of persistence. for each sd, the number of months of uninterrupted vector presence would be calculated, thereby determining whether greater persistence occurs in specific sds over the various periods of the year. temporal analysis of climatic factors (rainfall, temperature, and humidity) revealed that dengue generally occurs when average temperatures increase, when the rainy season has started, and when the humidity is higher. previously, a report from bh showed that rainfall and relative humidity data from fifteen days before (t1) showed very high correlation with dengue vector incidence in time t. there are other studies in the literature reporting an important correlation between climate and dengue occurrences or dengue vector abundance [3537 ]. however, the occurrence of a residual vector population or the occurrence of dengue cases in distinct intraurban areas in the cold and dry months, with much lower dengue incidence than in january to may, should be taken into account for disease control. identifying locations and patterns of the vector population (species, density, and vector - control indices) should also be used to direct interventions with disease reduction as the preferred outcome measure demonstrating impact, and ovitraps index, house index, container index, and breteau index as proxy indicators of impact. with these strategies, information will be available in real time, which may uncover other aspects about the relationship between vector and the disease that could be revealed through spatial analyses [38, 39 ]. other tools such as the industrial control chart proposed by rich and terry, and adopted in several survey vigilance systems when applied to dengue require several improvements related to presentation and interpretation in order to enhance its usefulness. the ability to demonstrate trends, analyzing only notified dengue cases at a potentially earlier time point, is limited. heterogeneous internet access limits the use of query - based surveillance web tools to identify disease and location outbreaks as candidates for interventions. although this proposal is intriguing, so far the identification of a given outbreak is usually too late for control measures. our findings show that the strategies used in this study can help public health officials to visualize and understand the geographic distribution and trends of disease patterns and to prepare warnings and awareness campaigns. dengue spatial and temporal spread patterns and hotspot detection may constitute useful information for public health officials to control and predict dengue dissemination from critical hotspot areas. this may save time and cost and make public health department actions more efficient. public health officers may employ the model to plan a strategy to control dengue by analyzing the information received on distribution and hotspots for various months. some ancillary findings of the study such as influence of climate, which is seasonal and thus temporal, also contribute to knowledge regarding its significance. the methodology is based on principles of spatial statistics and has the potential to be applied to other epidemics. in the future, it will be important to have regular daily statistics accumulated over several years to permit faster recognition of outbreak locations and be prepared to promptly implement appropriate public health interventions.	this study considers the dengue occurrence in the city of belo horizonte over the last fifteen years. approximately 186,000 cases registered from 1996 to 2011 were analyzed. the home address of individuals whose dengue case was notified was used as a proxy for exposure location. for determining possible outbreaks of disease and the specific patterns of dengue cases, spatial statistics used included kernel 's estimation. the occurrence of waves of dengue outbreaks was correlated with climatic and vector presence data. outbreaks had different durations and intensities : case clustering, thinned out both spatially and temporally. these findings may be useful for public health professionals responsible for fighting the disease providing some tools for improving evaluation of interventions such as vector control and patient care, minimizing the collective and individual burden of the disease.
helicobacter pylori (h.pylori) affects approximately 50% of the world 's population and it is a leading cause of peptic ulcers and chronic gastritis. moreover h.pylori was defined as a possible risk factor for gastric adenocarcinoma and mucosa - associated lymphoid tissue lymphoma (1). it was classified as a type 1 carcinogen by the world health organization (who) in 1994 (2). treatment with amoxicillin, clarithromycin and a proton pump inhibitor (ppi) is the first - line triple therapy to eradicate h. pylori. however, due to high prevalence of treatment failure related to antibiotic resistance, frequent and uncontrolled use of antibioticand severe and frequent antibiotic adverse effects (3, 4) the clinicians have considered several alternatives therapies. these methods include : extended treatment duration, the use of new antibiotics, and quadruple therapy as the first option or recently the addition of probiotics to triple therapy (46). probiotics are live microorganisms that may have a positive effect on gastrointestinal microorganism and recover health conditions (7). these agents have become progressively popular in the world to treat a diverse type of gastrointestinal disease (810). in this regard numerous probiotic strains have been investigated for their clinical efficacy, including multiple bacterial strains and fungal strains as saccharomyces boulardii (s. boulardii) (1113). the probiotics inhibit h. pylori urease by lowering the gastric ph, adhesion of h. pylori to gastric epithelial cells, stabilize the gastric barrier function (14) and reduce the side effects of antibiotics (15, 16). however previous studies have provided conflicting information regarding the positive effect of probiotic on h.pylori eradication (17). in our knowledge not enough evidence are available on the beneficial effects of adding probiotics into the eradication regimen of h. pylori in iran. therefore this study aimed to evaluate the efficacy and safety of adding the probiotic saccharomyces boulardiito standard triple therapy for eradication of h.pylori. in this randomized controlled trial we evaluated 160 adult patients with biopsy confirmed h. pylori infection referred to gastroenterology ward in taleghani hospital (a tertiary academic center in tehran - iran) during 20112012. all adult patients who came to our hospital and were candidate for upper gi endoscopy within 12 months were evaluated in this trial. patients 95% of the medication had been taken. patients were asked to report any side effects of therapy during the treatment period (end of first, second, third and fourth weeks of treatment) and were given a possible side effect list, such as epigastric pain, diarrhea, taste disturbance, constipation, and stomatitis. eight weeks after treatment, urea breath test (ubt) was performed in all patients. successful eradication was defined as a negative 13c - urea breath test result (ubt) eight weeks after discontinuation of the therapy. non - parametric t - test, chi - square test were used to compare two independent groups. non - parametric t - test, chi - square test were used to compare two independent groups. in this clinical trial, 160 patients (58.7% female) with mean age 47.111.4 years were evaluated. regarding sex and age the difference between case and control the most frequent esophagogastroduodenoscopy (egd) finding group a and b was antral gastritis (51% and 44% respectively). the difference between two groups was not significant (p > 0.05) (figure 1). esophagogastroduodenoscopy findings in cases (group a) and control (group b) the success rate for h. pylori eradication was higher in group a 75(87.5%) than in group b 65 (81.2%), but the difference between two groups was not significant (p = 0.350) (table 1). distribution of h.pylori eradication and compliance in two groups group a (n : 80) were given amoxicillin (1000 mg, b.i.d), clarithromycin (500 mg b.i.d), omeprazole 20 mg bid and saccharomyces boulardii probiotic (250 mg, b.i.d) for 14 days ; group b (n:80) were given amoxicillin (1000 mg, b.i.d), clarithromycin (500 mg b.i.d) and omeprazole (20 mg b.i.d) for 14 days. regarding drug compliance 52(65.0%) in group the difference between case and control groups was not significant (p > 0.05) (table 1). the frequency of side effects as nausea, diarrhea, abdominal discomfort and bloating in group a, were significantly lower than group b in first and second weeks (p < 0.05), where as skin rash, insomnia in case group did not show remarkable difference compared to control group (table 2). recently, the alternative anti-h.pylori treatments have been extended and more studies have been steered to describe components that may effect on h.pylori infection. in general, probiotics can strengthen host systems and assist in recovery from certain diseases and in particular s. boulardii induces morphologic changes in h. pylori cells consistent with cellular damage (18) and in one trial by gottel. was shown s. boulardii reduce 12% of h. pylori colonization in infected children (19). furthermore de bortoli. indicated that the addition of probiotics to standard triple therapy (esomeprazole, clarithromycin, amoxicillin) could increase the eradication rate of h. pylori infection and reduce the side effects of antibiotic therapy (20). to evaluate this hypothesis, we designed a comparative clinical trial study to assess any positive effect of s.boulardii on h.pylori infection and side effects of therapy. the success rate for h.pylori eradication was higher in probiotic group (87.5%) than control (81.2%), but the difference between two groups was not significant. however, side effects as nausea, diarrhea, abdominal discomfort and bloating were significantly lower in group a than group b during the first and second weeks. study that indicated the efficacy of probiotics for eradication of h. pylori in 85 asymptomatic carriers. they randomized patients in three probiotic therapy groups (s. boulardii, l.rhamnosus gg or a mixture of l. acidophius and bifidobacterium lactis) and placebo for 14 days. after second week, the eradication rates were similar for all groups (81% for s. boulardiivs 80% forplacebo). but antibiotic - associated diarrhea in all the probiotic groups was significantly lower (5%) than placebo group (30%)21. moreover, the other study by cindoruk. in 2007 in turkey evaluated s. boulardii for both eradication of h. pylori and the reduction of side - effects of the standard triple treatment. they studied 124 carriers with dyspepsia who were receiving the triple therapy and randomized to either s.boulardii (1 g) or placebo group for two weeks. then patients were followed up for six weeks regarding side - effects and h. pylori eradication rate. the patients showed no significant difference in h.pylori eradication (71% in s. boulardiivs ; 60% in placebo), however epigastric distress and global dyspepsia symptom scores in s. boulardii group were significantly lower than control group (16). in addition, yasar. in another survey in turkey in 2010, studied the efficacy of probiotics in h.pylori eradication therapy and specified that the addition of probiotic - containing yogurt to the triple therapy did not significantly increase the h. pylori eradication rates, however, they informed that probiotics decreased the frequency of stomatitis and constipation (22). our results and these mentioned studies indicate that s. boulardii did not decrease eradication rate of h. pylori where as it is significantly reduce the side effects of the standard triple therapy. on the other hands, lesbros - pantoflickova reported that probiotics reduce the risk of developing disorders associated with high degrees of gastric inflammation (23). moreover wilhelm. reported probiotics reduce adverse effects and increase tolerability of h. pylori eradication regimens. they may especially be useful in patients with recurrent h. pylori infection and a history of gastrointestinal adverse effects with antibiotics (24). in addition kotowska signified that s. boulardii effectively reduces the risk of antibiotic - associated diarrhea in children (25). the most of reviewed studies signified treatment with probiotics is relatively safe (22, 23, 26), however it is not risk free and are potentially pathogenic (27) moreover a study described 3 patients with fungaemia after intake of s. boulardii (28). in contrast to these findings, we did not detect any remarkable adverse effect related to s. boulardiiin in group a of our study. in conclusion, we showed s. boulardii decreased the adverse effects associated triple therapy but did not significantly decrease the eradication rate of h.pylori. we suggest further trials with higher dose of s. boulardiior other types of probiotics in combination with standard therapy which may provide significant results.	aimevaluating the efficacy and safety of adding the probiotic saccharomyces boulardiito standard triple therapy for eradication of helicobacter pylori.backgroundseveral probiotics such as saccharomyces boulardii have been investigated for their clinical efficacy. this probiotic, inhibit h. pylori urease by lowering the gastric ph, adhesion of h. pylori to gastric epithelial cells, stabilize the gastric barrier function and reduce the side effects of antibiotics.patients and methodsin this randomized controlled trial we evaluated 160 adult patients with biopsy confirmed h. pylori infection referred to gastroenterology ward of taleghani hospital. the patients were randomized into two treatment regimens : patients in group a (n = 80) were given amoxicillin (1000 mg, b.i.d), clarithromycin (500 mg, b.i.d), omeprazole (20 mg, b.i.d) and probiotic of saccaromyces boularidi (yomogi) (250 mg, b.i.d) for 14 days, moreover patients in group b (n = 80) were given amoxicillin (1000 mg, b.i.d), clarithromycin (500 mg b.i.d) and omeprazole (20 mg, b.i.d) for 14 days.results160 patients (66 male 41.25%, 94female 58.75%) with the mean age of 47.111.4 years were evaluated. the success rate for h. pylori eradication in group a was higher 75(87.5%) than group b 65 (81.2%), but the difference between two groups was not significant (p = 0.350). moreover, in case group side effects as nausea, diarrhea, abdominal discomfort and bloating were significantly lower than control group in first and second weeks.conclusionthis study showed that saccaromyces boularidi decreased the adverse effects associated with h.pylori therapy but did not significantly decrease the eradication rate of h.pylori.
supercapacitors with their superior power density, high discharge / charge rates, and high cyclability compared to batteries are ideal energy storage devices for high - power delivery requirement. graphene with its high electrical conductivity, large surface area, and chemical inertness has been favored for supercapacitor electrodes. oxidation of graphite and formation of carbonyl functionalities result in an increased interlayer separation. two - dimensional graphene oxide exfoliated from oxidized graphite possesses an electronic structure similar to that of a wide bandgap semiconductor. the presence of sp- and sp - hybridized states in graphene oxide can be altered by either liquid phase or gas phase reduction processes. for supercapacitor applications, it is essential to reduce the graphene oxide to obtain electrical double - layer capacitance. the transformation of graphene oxide to reduced - graphene oxide is an important process, and understanding the chemistry of graphene oxide will provide insight into the reactivity and properties of both the graphene oxide and the reduced graphene oxide. the performance of supercapacitors can be enhanced by using a combination of electrical double - layer capacitance and faradaic capacitance arising through redox charge transfer reactions also called pseudocapacitance. the specific capacitance of reduced graphene oxide with 135 f / g could be increased to 480 f / g by incorporating pseudocapacitance materials in the electrode. reduced graphene oxide metal oxide nanocomposites as electrode material for supercapacitors has been the topic of intense research, due to the metal oxide pseudocapacitance capabilities that can increase the specific capacitance tremendously. graphene metal oxide nanocomposites have shown superior performance as anode materials in lithium ion batteries with high reversible capacities and good cycle performance. mno2 with its favorable redox properties results in high pseudocapacitance, and therefore is an excellent candidate for electrode material. cobalt oxide nanocomposite has also been demonstrated as a highly suitable material for supercapacitor applications exhibiting superior electrochemical behavior and large specific capacitance. however, these two materials when used in combination should retain good adhesion, uniform coverage, and good electrical contact with extremely low interface resistance. a robust electrophoretic deposition (epd) technique enabled uniform deposition of graphene oxide in the present work. in our earlier work, the epd process had been optimized to obtain a good adhering film, and also the electrical characteristics of the nanocomposite film were tested using cyclic voltammetry. it is essential to probe the morphology, electronic structure, and orientation of oxide materials to improve the electrode performance. carbon and oxygen core - level spectroscopy, in particular x - ray absorption spectroscopy (xas), is a very sensitive probe of the local electronic structure as well as chemical composition and local bonding environment, which has been applied in several studies of reduced graphene oxides. in this work, xas is employed to study the surface properties of the individual oxides and the nanocomposites, and the sensitivity of xas to the local environment is used to identify the various carbon bonding environments. angular - dependent xas provides further insight into molecular orientation of local structures in the graphene oxide and nanocomposite. chemicals used for go and metal oxide synthesis were purchased from sigma - aldrich ; 18 m-cm water was used for all experiments. the synthesis protocol is explained in detail in our previous works. for go, mno2, and co3o4 nanomaterial synthesis, the procedures in refs (2527)were followed. the produced go was suspended in water and then centrifuged at 3800 rpm for 40 min. the supernatant containing 0.75 mg / ml of graphene oxide flakes was reserved for film deposition. mno2 nanowires were synthesized solvothermally ; 1 mmol of potassium permanganate was mixed with 1 mmol of ammonium chloride and placed in a 45 ml acid digestion bomb (parr instruments). the vessel was placed in a 140 c furnace for 24 h. the synthesized mno2 nanowires were cleaned with di water and ethanol before drying. ammonium hydroxide, 2830%, was slowly added to the solution, stirred for 30 min, and poured into a petri dish. stainless steel discs were placed 1 mm horizontally above the bottom of the petri dish held by two custom - machined ptfe pieces. the petri dish was covered and placed in a 90 c furnace for 16 h to produce co3o4 nanorods. pieces of silicon wafers were cleaned in a 3:1 sulfuric acid / hydrogen peroxide mixture for 30 min. the cleaned, hydrophilic - rendered wafers were placed in di water until further use. mno2 nanowires and co3o4 nanorods were separately suspended in methanol at a concentration of 4.5 mg / ml. for the composite films, blodgett (lb) trough (102 m, ksv nima) was cleaned thoroughly with ethanol and filled with di water. the wafer was submerged in the trough prior to deposition of the material onto the subphase. the suspensions were slowly added dropwise using a 100 l pipet for up to 3 ml. barrier speeds were set at 20 cm / min until the target pressure was reached and the wafer pulled up at a rate of 2 mm / min for all films. graphene oxide samples were thermally reduced in a tube furnace (lindberg / blue minimite) by ramping to 300 c at a rate of 10 c / min and then held at the maximum temperature for 30 min before slowly cooling back to room temperature. the annealing temperature of 300 c was chosen as optimum after several trials of 600 c for 2 h, 400, 350, and 300 c. the nanocomposites were found to be damaged at temperatures 400 c and above, as confirmed by both visual inspection and raman spectroscopy. ex situ x - ray absorption spectroscopy (xas, nexafs) was performed on the 31-pole wiggler beamline 10 - 1 at the stanford synchrotron radiation lightsource (ssrl) using a ring current of 500 ma. the beamline is equipped with spherical grating monochromator operated with a 1000 linesmm grating at 40 m entrance and exit slits (o 1s xas, mn 2p xas, and co 2p xas), providing 10 photonss at about 0.20.35 ev resolution in a 1 mm beam spot. carbon xas was measured with 600l.mm grating using 30 m slits, corresponding to about 0.15 ev resolution. all data were acquired at room temperature and under ultrahigh vacuum (10 torr). both total electron yield (tey, measured via the sample drain current) and auger electron yield (aey, measured with a cylindrical mirror analyzer) were recorded simultaneously. all spectra were normalized by the incoming flux, measured from a thin grid with freshly evaporated gold, positioned upstream of the sample chamber. a reference sample with several transition metal oxides, intercepting a few percent of the beam upstream and recorded simultaneously, was used to calibrate the energy of the cobalt, manganese, and oxygen scans with a relative energy precision of 25 mev. carbon xas was referenced to the carbon dip which has been calibrated to 284.7 ev using hopg. the data was normalized to a linear background function at higher energy. when the background subtraction was associated with large error bars, and an additional constraint forcing the area to be the same across several samples sem imaging was performed using s4800, hitachi (pleasanton, ca) scanning electron microscope. the surface properties including electronic structure, molecular orientation, and surface morphology play a crucial role in determining the interaction of various materials within the composite and also the interaction of adsorbed molecules with the electrode. the morphology was investigated with sem imaging, and we have performed x - ray spectroscopy and vibrational spectroscopy to determine local bonding environments and molecular orientations for graphene oxide as well as the nanocomposites. figure 1a shows the sem image of planar graphene oxide prepared using electrophoretic deposition indicating a clear planar surface with no agglomeration, and the corresponding c and o k edge xas is shown in figure 2a and figure 2b, respectively. the graphene oxide blodgett deposition is shown in figure 1b in which the nanowires with varying diameters from 30 to 60 nm are clearly seen. co3o4 nanorods in figure 1c are over 10 m in length, and solvothermally prepared mno2 nanowires shown in figure 1d are 3060 nm in diameter and 35 m. the corresponding l edge xas plots for the mn and co metal oxides and the nanocomposites are in figure 4a and figure 4b. sem image of (top left, a) graphene oxide ; (top right. mangenese oxide nanocomposite ; (bottom left, c) cobalt oxide nanorods ; (bottom right, d) manganese oxide nanowires. (left, a) c 1s nexafs spectra of graphene oxide measured at x - ray incident angles 20 and 90 ; (right, b) o 1s nexafs spectra of graphene oxide measured at x - ray incident angle 54. the supercapacitance of the graphene material arises because it can store the electrostatic charges from the electrolytes by simple adsorption, which is reversible (electrical double - layer capacitance). graphene, with its large surface area and the ability to adsorb molecules reversibly on its surface, offers a unique advantage. as a large surface area of graphene oxide is essential for enhanced specific capacitance, it is essential to have nonagglomerated graphene layers. x - ray absorption spectroscopy is a powerful probe of the electronic structure in which a tunable x - ray source is used to excite core electrons into unoccupied bound or continuum states. apart from its elemental specificity, xas is particularly sensitive to the local electronic structure, including angular anisotropy in molecular orientation, bond length, oxidation state, and symmetry, as well as spin. xas analysis is highly relevant as the information obtained corresponds to a depth of few nanometers into the surface and not to the bulk. figure 2a shows the c k edge absorption spectra of graphene oxide taken at grazing and normal incidence, showing the sp - derived unoccupied and bands at 285.2 and 292.6 ev. the relatively broad resonance and the absence of a core - exciton at around 291.65 ev, indicate that the sp network in graphene is substantially defective, as expected. the resonances at 286.5, 288.7, and 289.6 ev correspond to states where the local sp bonding configuration has been interrupted by (mainly) oxygen functionalization. in particular, double - bonded oxygen functionalization gives rise to a intensity associated with the c = o bond. this carbonyl energy position will be shifted depending on the electronegativity of the local environment around the carbon (e.g., carbonyl c = o in cooh around 288.5, whereas c = o in a ketone environment is found at lower energies). also, sp or aromatic carbon with some single bond oxygen functionalization such as hydroxyl (phenol, oh) and epoxy groups (c o c) will shift the resonance from the aromatic core up by about 12 ev (for that particular carbon atom). in the spectra in figure 2a, the absorption edge at 285.2 ev corresponds to excitation of carbon in the sp network into the band, and the peak at 286.5 ev is assigned to excitations for carbon that are single bonded to oxygen (c o) either in hydroxyl (phenol) or epoxy configurations, consistent with previous studies on graphene oxide. the peak at 288.7 ev corresponds to c = o resonances in carboxyl groups (cooh). unfortunately, the literature on carbon xas can not give conclusive assignment of the 289.6 ev feature, but we hypothesize that it is associated with rydberg mixed oh - derived states from hydroxylated and ether - linked c - species (o c oh) based on xas of carbohydrates and amino sugars by solomon., as well as eels studies by hitchcock. the main resonance for the uninterrupted sp network is 292.6 ev, and there is a resonance attributed to single - bonded oxygen at 297.5 ev, but due to the strong functionalization and interference with many states, the c = o sigma feature from carboxyl expected at around 303 ev can not be well resolved. the high linearly polarized nature of synchrotron radiation is a powerful probe of molecular orientations, in particular for light element 1s 2p excitations, where the product of the electric dipole and the orbital symmetry axis gives rise to simple intensity relations between molecular orientation and incidence angle of the radiation (or e - vector). the nexafs spectrum in figure 2a is a c k edge absorption spectrum collected with a grazing incidence angle of 20 and 90 relative to surface, where for 90 the x - ray (e - vector) is parallel (perpendicular) to the surface normal (corresponding to in - plane excitation). in graphene, the band is oriented along the intermolecular bonding axes (in - plane) and the band is derived from pz orbitals oriented perpendicular to graphene plane (out - of - plane). for in - plane excitation (90) the resonance is stronger, and the resonance is suppressed, whereas for out - of - plane excitation the opposite is found, as expected. we note that the angular dependence is distinct but rather weak compared to high - quality pristine graphene due to defects and oxygen functionalization that interrupt the stoichiometric sp network. the c = o resonance shows the same angular dependence as the spnetwork, indicating that the c = o bond is primarily in - plane, e.g., pointing into the hollow space created by defects. in contrast, the c o resonance at around 297.5 ev shows opposite behavior to the sp - derived, indicating that those c o bonds are primarily directed out - of - plane, as expected for oh phenol groups on top of sp carbon. the absorption feature at 286.5 ev corresponding to excitations for carbons that are single bonded to oxygen (c o) either in hydroxyl (phenol) or epoxy configurations do not show observable angular dependency. a similar observation by jesus. on xas mapping of chemical domains on graphene oxide indicates that for the functional groups that decorate the basal plane and edges of the graphene oxide sheets, the resonances are observed in all due to elimination of angular dependence of intensity induced by the steric orientations of different functional groups. the o 1s xas spectra for graphene oxide in figure 2b display four main resonances at around 530.8, 531.8, 535.2 ev and a convolution of multiple peaks centered around 538.8 ev. the lower absorption edges 530.8 and 531.8 ev are resonances from oxygen double bonded to carbon (carbonyl, c = o). based on the c 1s xas, we associate this intensity mainly with carboxylic groups. there is an associated c = o intensity at around 539 ev which is not well resolved. the shoulder at 535 ev is a hydroxyl - derived state (o h), and the broad feature around 537.5 ev, which again is not well resolved, is attributed to single - bonded oxygen (e.g., c oh and c o c) based on the vast literature on various alcohols and esters by hitchcock., as well as the agreement with in - vacuum annealed graphene oxide, shown to be dominated by phenolic groups and with much reduced cooh functionality.both the c 1s and o 1s spectra are consistent with a hydroxide - rich surface with a mixture of carboxylic and other oh - derived functionalities. figure 3 displays the c k edge spectra of graphene oxide and the graphene oxide nanocomposites taken at grazing (20, figure 3a) and normal incidence (90, figure 3b) relative to the sample surface. the absorption edge at 285.2 ev corresponding to excitation of carbon in the sp network into the band shows significant variation in intensity with incidence angle of the radiation, for all the samples, demonstrating the out of plane orientation of band. overall, we find that the graphene in the nanocomposite mainly retains its structure and angular dependence with some subtle differences. there is some residual potassium l edge peaks at 297.5 and 300.2 ev corresponding to 2p3/2 and 2p1/2 transitions in figure 3b (top), indicating the incorporation of k ions in the graphene oxide the presence of k 2p peaks on electrophoretically deposited graphene oxide films reduced using koh / hydrazine has been reported by lee. c 1s xas spectra of graphene oxide, graphene oxide cobalt oxide nanocomposite, and graphene oxide manganese oxide nanocomposites measured at 20 incidence (left, a) and 90 incidence (right, b). in figure 3a, at 20 incident angle, the absorption edge intensity ratio 288.7 ev/289.6 ev is higher for graphene oxide manganese oxide nanocomposite compared to graphene oxide cobalt oxide nanocomposite and graphene oxide. this is accompanied by a suppression of the sigma star intensity at around 297 ev. oh) and also the intensity of the c o at 297 ev. the sonication in mno2/methanol suspension alters the go slightly to reduce the oh functionalization. to investigate the electronic structure of the mn oxide and co oxide nanoparticles, we acquired mn 2p (figure 4a) and co 2p (figure 4b) xas. l - edge xas of transition metal oxides is a sensitive probe of oxidation and symmetry through the direct dipole transition into the unoccupied frontier orbitals. cramer., in their xas analysis of manganese l edge, reported that the l2,3 edge features of transition metal complex that involve 2p3d transitions are sharper, richer, and much more sensitive in oxidation state determination than the k - edge features which are broad. further, it was well established that mn l edge absorptions are dominated by mn 3d states and hence an excellent indicator of oxidation state and coordination. in particular, all l - edge xas spectra are shown in an extended region covering both the l3 (2p3/2) and the l2 (2p1/2), displaying a large spin l - edge (2p) xas of (left, a) mn in manganese oxide nw (black curve) and graphene oxide mangenese oxide nanocomposite (red curve) and (right, b) co in cobalt oxide nr (red curve) and graphene oxide cobalt oxide nanocomposite (blue curve). the energy position, peak separation, and relative intensity of the peak in mn l - edge spectrum of the nanowires matches that of mno2. co l edge xas confirms that the co is found in a trivalent configuration resembling the spinel co3o4 structure with octahedrally coordinated co and co cations tetrahedrally coordinated with oxygen. the cobalt oxide nanostructures with varying oxidation states and crystal structures are known to exhibit different electrochemical behavior. moreover, the l - edge xas spectra of both oxides and their corresponding nanocomposite with graphene oxide are exactly the same with no observable difference in the absorption edges, demonstrating that the manganese oxide and cobalt oxide retain their crystal symmetry and electronic configuration in the nanocomposites. studies on the oxidation states of cobalt have demonstrated that the accumulation of chemisorbed oxygen and diffusion results in the formation of surface oxide. co3o4 is a stable form of oxide, and the observed similarity in the xas spectra of cobalt oxide and the composite shows that there is no variation in the electronic structure. in graphene composites, the stacking between individual graphene sheets is driven by strong interaction, and the metal oxide nanostructures are surface anchored to the graphene through van der waals force. the c k edge absorption spectra of reduced graphene oxide (figure 5a) and reduced graphene oxide cobalt oxide nanocomposite (figure 5b) taken at grazing and normal incidence show a distinct difference from that of graphene oxide shown in figure 2a. the absorption edge at 285.2 ev corresponding to excitation of carbon in the sp network into the band shows a distinct and strong angular dependency. a strong 289.6 ev feature observed for graphene oxide that we attributed to rydberg mixed oh - derived states from hydroxylated and ether - linked c - species (o c oh) is missing in the reduced graphene oxide, indicating that the hydroxylated species are removed upon heat treatment. this will invariably alter the electronic structure of the reduced graphene oxide, and the consequence of this change can be observed by comparing the peak position of the carbonyl (c = o) resonance before and after reduction. in graphene oxide, the peak at 288.7 ev corresponds to c = o resonances in carboxyl groups (cooh). the carbonyl resonance position has shifted by 0.3 ev to lower energy for reduced graphene oxide (from 288.7 to 288.4 ev). this peak position is consistent with trends in positions of c 1s to c = o transitions as a function of electronegativity of the substituents, studied in solid state and by computational methods by urquhart and ade. the removal of hydroxyl groups bonded to the carbonyl carbon actually results in the removal of electronegative oxygen, the presence of which increases the position of resonance of carbonyl carbon. another important aspect observed in the reduced graphene oxide is the ratio of intensities of excitation of carbon in the sp network to that of resonance around 292.6 ev and the ratio of excitation of carbon in the sp to that of resonance of carbonyl carbon. an enormous increase in intensity of sp - derived unoccupied resonance is observed for reduced graphene oxide suggesting substantial restoration of the conjugated network. unlike the graphene oxide, appearance of excitonic peak at 291.7 ev and the doublet nature of this excitonic peak with an additional peak around 292.8 ev further confirm the formation of extended conjugated network. c 1s nexafs spectra of (top, a) reduced graphene oxide and (bottom, b) reduced graphene oxide cobalt oxide nanocomposites measured at incident angles 20, 54, and 90. in our earlier work we studied the changes in chemical structure of graphene oxide upon reduction and observed the following : (i) both the d - band and g - band peaks were shifted to lower wavenumbers, and the shift of the d - band peak was noticeably larger than that of the g - band peak ; (ii) the d bandwidth decreased ; and (iii) the change in the relative intensity of the d and g band with d / g ratio calculated with integrated peak areas changed from 0.7:1 to 2.6:1 from go to rgo. the observed change in the c k edge xas spectra of graphene oxide upon reduction is similar to the changes observed in our raman studies. overall, the reduced graphene oxide has more graphene - like behavior. as the temperature used for reducing graphene oxide is 300 c, co3o4 is stable in this temperature region. the good contact between the graphene oxide and the nanostructured materials in the composite film produced by langmuir blodgett process ensures low contact resistance and good adhesion between them as evident from previous electrochemical studies. reported that the large surface area of graphene and the efficient use of the pseudocapacitive nanomaterial (mno2) for charge storage leads to remarkable electrochemical performance with high specific capacitance and extended cycling performance. graphene oxides and nanostructures of 3d transition metal oxides with various geometries (cobalt oxide nanorods, manganese oxide nanowire) were synthesized for using as electrode material in supercapacitors. the fundamental understanding on the coordination symmetry, oxidation station, and interactions of the neighboring compounds in a composite can lead to optimization of the material characteristics which in turn could result in an enhanced device performance. as most reactions take place at the surface, studying electronic structure and the surface chemical properties are crucial. xas spectroscopic measurements carried out to study the oxidation state and the bonding environments of the nanocomposites showed that the electronic structure and the oxidation state of the metal oxides remain the same in the nanocomposite. upon thermal reduction, the sp - hybridized framework of graphene is restored while removing the electronegative functionalities that reduce the electrical charge carrier transportation to a significant extent. thermal reduction of the graphene oxide and the nanocompsoite resulted in major changes like sharp increase in the angular dependency of the c k edge spectra, increased intensity of the sp - derived unoccupied band, removal of hydroxyl states, and the appearance of excitonic state, all of which confirm favorable change in electronic structure for supercapacitor applications.	the surface properties of the electrode materials play a crucial role in determining the performance and efficiency of energy storage devices. graphene oxide and nanostructures of 3d transition metal oxides were synthesized for construction of electrodes in supercapacitors, and the electronic structure and oxidation states were probed using near - edge x - ray absorption fine structure. understanding the chemistry of graphene oxide would provide valuable insight into its reactivity and properties as the graphene oxide transformation to reduced - graphene oxide is a key step in the synthesis of the electrode materials. polarized behavior of the synchrotron x - rays and the angular dependency of the near - edge x - ray absorption fine structures (nexafs) have been utilized to study the orientation of the and bonds of the graphene oxide and graphene oxide metal oxide nanocomposites. the core - level transitions of individual metal oxides and that of the graphene oxide nanocomposite showed that the interaction of graphene oxide with the metal oxide nanostructures has not altered the electronic structure of either of them. as the restoration of the network is important for good electrical conductivity, the c k edge nexafs spectra of reduced graphene oxide nanocomposites confirms the same through increased intensity of the sp2-derived unoccupied states band. a pronounced angular dependency of the reduced sample and the formation of excitonic peaks confirmed the formation of extended conjugated network.
phase ii studies of s-1 have noted responses of 4454% in patients with gastric cancer [13 ] and s-1 and cisplatin are used as one of the first - line treatments for this type of cancer in the country. s-1 is a novel 5-fluorouracil (5-fu) derivative, composed of tegafur, gimeracil and oteracil potassium. tegafur is slowly converted to 5-fu in vivo, mainly by the liver microsomal p-450 drug - metabolizing enzyme system. 5-fu is degraded by hepatic dihydropyrimidine dehydrogenase (dpd), converted to f--alanine, and excreted in the urine. 5-chloro-2,4-dihydroxypyridine (cdhp) is a reversible competitive inhibitor of dpd whereby 5-fu can be retained for longer at high blood concentrations. the plasma concentration of 5-fu is increased in the patient with renal dysfunction because 50% of cdhp is excreted in the urine. this case report describes a decrease of the adverse effects of 5-fu by haemodialysis (hd) along with providing plasma concentrations of 5-fu and gimeracil. a 35-year - old man suffered from immunoglobulin - a nephropathy 2 years before admission and was treated with steroid therapy. a mass on the greater curvature of his stomach was found by gastroscopy and gastric cancer (signet cells) was diagnosed by biopsy. the abdominal ct scan showed that there were tumours in the abdominal cavity with enlarged para - aortic lymph nodes. he started taking s-1 50 mg, twice daily for 3 consecutive weeks, followed by a 2-week rest period 8 months ago. he also started intravenous cisplatin every 5 weeks and took dipyridamole, lansoprazole, granisetron hydrochloride and naproxen orally every day. he received 60 mg cisplatin for the last time about 1 month before his admission, and his serum creatinine increased slightly (from 95.47 to 114.92 mol / l) 1 week after receiving cisplatin. he continued to report appetite loss and went to hospital 1 week before his admission. he also took a blood examination after the consultation and his serum creatinine had increased to 683.33 mol / l. he continued to take s-1 and the other drugs for about 1 week from the start of the renal insufficiency. his appetite loss and general fatigue were aggravated and he consulted the emergency room in our hospital. he was drowsy and on the glasgow coma scale his score was 13 of 15. he complained of a slight stomachache, and slight tenderness was found on physical examination. the serum creatinine and blood urea nitrogen were elevated to 1919.16 mol / l and 32.49 mmol / l, respectively. a nephrological consultation was requested ; the patient appeared to be dehydrated and there were no signs of hydronephrosis on abdominal ultrasonography. his acute kidney injury was ascribed to a combination of drug - induced kidney injury and dehydration. all drugs were withdrawn and hd was performed for 34 h with an aps-11md dialyzer (asahi kasei medical co. ltd) and with a blood flow between 120 and 150 ml / min on the second, third, fifth and sixth day of hospitalization ; the plasma concentrations of 5-fu and cdhp were measured. on the fifth day his urine output gradually increased and the serum creatinine decreased to the previous value (serum creatinine decreased to 85.75 the plasma concentration of 5-fu decreased from 840 ng/ ml before the first hd to 93.4 ng / ml after the second hd (figure 1). haemodialysis was performed on the second and the third day after his admission in the shaded region. elimination rate : [(plasma concentration before hd) (plasma concentration after hd)]/(plasma concentration before hd) 100 (%). however, on the fourth day, diarrhoea and stomatitis appeared till the sixth day. granulocyte colony stimulating factor (g - csf) and antibiotics were administrated, and on day 7 the neutrophil count decreased to approximately 50/mm. finally, the neutrophils increased on day 18 and the fever decreased, together with improvement of the fever, the stomatitis and diarrhoea. changes in serum creatinine, neutrophil and urine volume in the patient 's clinical course. this patient showed severe adverse effects (pancytopenia, diarrhoea and stomatitis) because of taking s-1 in the presence of acute kidney injury. usually s-1 should not be prescribed when the patient 's creatinine clearance is < 30 ml/ min, because of adverse reactions due to accumulation of 5-fu in the body. in japan, at least two patients with chronic kidney disease taking s-1 died from sepsis due to granulocytopenia because of this adverse effect. acute kidney injury is known to occur with s-1, but its mechanism is unknown and the incidence is < 0.1% according to the drug surveillance reports. we thought that this patient 's acute kidney injury could be ascribed to dehydration and the prescribed drugs, probably due to naproxen (nsaids) among others. the patient 's blood concentrations measured by liquid chromatography / mass spectrometry of 5-fu and cdhp, 24 h after taking 50 mg of s-1 were 840.0 ng / ml and 1596.0 ng / ml, respectively (figure 1). cmax levels of 5-fu and cdhp were reported to be 128.5 ng / ml and 284.6 ng/ ml, respectively, in individuals without renal insufficiency. when higher doses of 5-fu were taken, the number of neutrofils reaching the minimum nadir was decreased. moreover, the elimination rates of 5-fu and cdhp by a 4-h hd session were 71.087.6% and 53.163.4%, respectively. our patient 's first and second hd were performed for only 3 h because we wanted to avoid dialysis disequilibrium syndrome. we speculate that the elimination rate of 5-fu and cdhp by hd were only 50 and 45%, because of this short duration of hd. however, the plasma concentration of 5-fu decreased from 840 to 93.4 ng / ml after his second hd, which is under the cmax after taking a normal dose of s-1. the concentration of 5-fu was decreased from the end of the first hd to the start of the second hd while the concentration of cdhp did not decrease. the reason is that cdhp is a competitive inhibitor and that 5-fu was degraded by other pathways than the hepatic dihydropyrimidine dehydrogenase. but it was thought that we could decrease the risk of the adverse effects (especially shorten the period of bone marrow suppression) and enhance the recovery without sepsis by eliminating 5-fu by hd. in conclusion, when high plasma concentrations of 5-fu are suspected, the plasma levels of 5-fu and cdhp should be measured and the indication of hd for the removal of 5-fu and cdhp to avoid severe myelosuppression should be strongly considered.	s-1 and cisplatin are used as one of the first - line chemotherapies for gastric cancer in japan. the plasma concentration of 5-fluorouracil (5-fu) is increased in patients with renal dysfunction because gimeracil in s-1 inhibits the degradation of 5-fu and about 50% of gimeracil is excreted in the urine. we describe a 35-year - old man with acute kidney injury while taking s-1 and cisplatin for advanced gastric cancer and who presented severe adverse effects of 5-fu. this case report describes the evolution of the plasma concentrations of 5-fu with haemodialysis along with a decrease in the adverse drug effects.
psoriatic arthritis (psa) is an inflammatory arthropathy, associated with skin and/or nail psoriasis [13 ] and other possible systemic features [46 ]. in the last decade, the introduction of biologic drugs, in particular tumor necrosis factors (tnf)- blockers, has opened new horizons for patients and rheumatologists in the treatment of psa and other rheumatic diseases [710 ]. the use of these agents has outlined the importance of evaluating their safety, as it may expose patients to an increased risk of developing infections, in particular tuberculosis (tb). the global prevalence of tb is still close to 9 million, mainly in underdeveloped countries. this condition is believed to affect one third of the world s population, with a natural history showing a progression to active disease in only 5 % of cases. tnf- is an essential pro - inflammatory cytokine also involved in the host defense against mycobacterium tuberculosis (mtb) [13, 14 ]. therefore, patients eligible for anti - tnf- therapy require careful evaluation and need to be investigated about possible previous exposure to mtb, as its use may expose patients to an increased risk of developing active tb and reactivation of latent tuberculosis infection (ltbi). the aim of this study was to evaluate the efficacy and safety of tnf- blockers in patients with psa and concomitant ltbi comparing their outcome with non - infected psa patients. we performed a retrospective study, from january 2005 to december 2011, in 321 caucasian patients with psa, with no specific exposure risk to tb, attending the psoriatic arthritis clinic at the university federico ii of naples, who had an inadequate response to disease - modifying antirheumatic drugs (dmards) and started therapy with tnf- blockers. before starting anti - tnf- therapy, all patients, according to our screening protocol, undergo clinical medical history, physical examination, laboratory standard tests, chest x - ray, and tuberculin skin test (tst). therefore, they are not immediately eligible for tnf- blocker therapy and have to start treatment for ltbi before starting biologic therapy. negative tst patients, if not treated with immunosuppressive drugs or steroids and if immunocompetent, started tnf- blocker therapy at time of enrollment, performing an inf- release assay (igra) every 12 months. in case of negative patients under immunosuppressive drugs or prolonged steroid therapy or positive patients previously vaccinated, we perform in addition an igra test. treatment for ltbi consists of a 9-month therapy with isoniazid monitoring adverse events, in particular liver function tests, monthly. anti - tnf- treatment was always started after the first 45 days of antitubercular therapy. we identified 40 patients with ltbi, who were included in this study along with 40 not infected psa patients as control group, matched for age, sex, and disease duration. for all patients included in the study, we collected the following data at starting anti - tnf- therapy (t0) and every 3 months for the 2 years (t2) of follow up : physical examination, recording of vital signs, tender joint count (tjc ; 68 tender joints), swollen joint count (sjc ; 66 swollen joints), health assessment questionnaire (haq), psoriasis area and severity index (pasi), visual analogic scale (vas), erythrocyte sedimentation rate (esr), and c - reactive protein (crp). all patients underwent chest x - ray every 6 months (or 12 if appropriate). two units (0.1 ml) of standard preparation of ppd rt-23 (statens serum institut, copenhagen, denmark) were injected in the intradermal region of the forearm volar surface (mantoux method). the reaction was read at 72 h with the transverse diameter in millimeters of induration. the cutoff for a positive skin test was defined as an induration area greater than or equal to 5 mm in diameter. quantiferon - tb gold in - tube (qft - git) test used designed (1 ml) blood collection tubes that were coated with m. tuberculosis - specific antigens [esat-6, cfp-10, and tb 7.7(p4) ], along with a negative and a positive control tube. an enzyme - linked immunosorbent assay (elisa) was used to measure the amount of ifn- present in each of the three tubes (nil control, tb - antigen, mitogen control). descriptive statistics included mean values and standard deviation (sd) of the continuous variables and percentages and proportions of the categorical variables. for the variables of interest, a difference (delta) between t2 and t0 was computed, and student s test (for paired samples) was used for the analysis. to compare continuous variables and dichotomous ones, correlations between study variables have been calculated using analysis of covariance (ancova) and logistic regression. for logistic regression, different methods were used for the selection of variables (full model, backward, stepwise) to exclude the influence of the different methodologies. to check the effects of possible confounding variables, which can affect tst results, we computed the correlation coefficients, checking the specific effects of the different variables, as described successively. in the ancova, we compared values and changes (at t0 and t2) in crp, esr, vas, tjc, sjc, haq, and pasi between the two groups, adjusting for the following variables : type and duration of therapy with anti - tnf ; type, number, and duration of concomitant dmard ; use and duration of concomitant steroids ; and use and duration of isoniazid. two units (0.1 ml) of standard preparation of ppd rt-23 (statens serum institut, copenhagen, denmark) were injected in the intradermal region of the forearm volar surface (mantoux method). the reaction was read at 72 h with the transverse diameter in millimeters of induration. the cutoff for a positive skin test was defined as an induration area greater than or equal to 5 mm in diameter. quantiferon - tb gold in - tube (qft - git) test used designed (1 ml) blood collection tubes that were coated with m. tuberculosis - specific antigens [esat-6, cfp-10, and tb 7.7(p4) ], along with a negative and a positive control tube. an enzyme - linked immunosorbent assay (elisa) was used to measure the amount of ifn- present in each of the three tubes (nil control, tb - antigen, mitogen control). descriptive statistics included mean values and standard deviation (sd) of the continuous variables and percentages and proportions of the categorical variables. for the variables of interest, a difference (delta) between t2 and t0 was computed, and student s test (for paired samples) was used for the analysis. to compare continuous variables and dichotomous ones, correlations between study variables have been calculated using analysis of covariance (ancova) and logistic regression. for logistic regression, different methods were used for the selection of variables (full model, backward, stepwise) to exclude the influence of the different methodologies. to check the effects of possible confounding variables, which can affect tst results, we computed the correlation coefficients, checking the specific effects of the different variables, as described successively. in the ancova, we compared values and changes (at t0 and t2) in crp, esr, vas, tjc, sjc, haq, and pasi between the two groups, adjusting for the following variables : type and duration of therapy with anti - tnf ; type, number, and duration of concomitant dmard ; use and duration of concomitant steroids ; and use and duration of isoniazid. on the basis of our screening, 40 patients (12.5 %) (group 1) were diagnosed to have ltbi (12 males and 28 females, mean age 51.6 12 years). all patients had a negative history of contacts and chest x - ray negative for tb. among 281 not infected patients, we selected 40 consecutive patients (group 2) (12 males and 28 females, mean age 51.4 11.9 years), who used the tnf- blockers for the entire 2-year period of observation, matched for gender, age, disease duration, and type of tnf- blocker. demographic and clinical characteristics at baseline of both groups selected for this study were reported in table 1.table 1demographic and clinical characteristic of two groups of patients at baselinegroup 1group 2number4040female, n 2828age (years)51.6 1251.4 11.9disease duration (months)58.44 35.2859.4 33.6crp (mg / dl)0.9 1.20.4 0.2esr (mm / hr)23.1 20.315.7 11.5vas78.8 11.676.2 10.1haq1.5 0.61.9 0.5tjc12.9 4.717.1 7.6sjc2.7 22.6 3.1pasi0.77 0.890.68 0.76data expressed as mean standard deviation, unless otherwise indicated crp c - reactive protein, esr erythrocyte sedimentation rate, vas visual analogic scale, haq health assessment questionnaire, tjc tender joint count, sjc swollen joint count, pasi psoriasis area and severity index demographic and clinical characteristic of two groups of patients at baseline data expressed as mean standard deviation, unless otherwise indicated crp c - reactive protein, esr erythrocyte sedimentation rate, vas visual analogic scale, haq health assessment questionnaire, tjc tender joint count, sjc swollen joint count, pasi psoriasis area and severity index at recruitment, patients of group 1 had used steroids for a mean period of time greater than patients of group 2 (10.8 6.4 vs 6.3 4.8 months ; p = 0.32). in detail, steroid therapy had been used in 26 patients of group 1 and 24 of group 2. moreover, the mean duration of dmards treatment was greater in group 2 vs group 1 (41.7 40.8 vs 24.5 38.6 months ; p = 0.21). in group 1, a total of 11 (28 %) patients was taking immunosuppressive therapy (cyclosporine n = 1, methotrexate n = 7, leflunomide n = 1, salazopyrin n = 2). in group 2, a total of 15 (38 %) patients was taking immunosuppressive therapy (cyclosporine n = 1, methotrexate n = 11, leflunomide n = 1, salazopyrin n = 2). there was no significant correlation between the tst result and previous therapies with steroids and dmards (odds ratio 1.73, ic 0.536.57). patients of group 1 started anti - tnf- therapy after at least 45 days on prophylactic therapy with isoniazid. patients of group 2, who started anti - tnf- therapy, performed igra test every 12 months to detect a possible ltbi occurrence. in each group, 22 patients were on etanercept therapy, 14 on adalimumab, and 4 on infliximab. anti - tnf- therapy was effective in both groups of patients, and no statistically significant differences were found in the analysis of the study variables between the two groups from t0 to t2 (table 2).table 2comparison of variation of clinical characteristic in both groups of patients at t0 and t2group 1group 2 p crp (mg / dl)0.3060.0810.6085esr (mm / hr)0.3620.0340.9609vas0.00010.00010.2509haq0.00010.00010.2593tjc0.00010.00010.1772sjc0.0170.00010.8446pasi0.00010.00010.3531 crp c - reactive protein, esr erythrocyte sedimentation rate, vas visual analogic scale, haq health assessment questionnaire, tjc tender joint count, sjc swollen joint count, pasi psoriasis area and severity index comparison of variation of clinical characteristic in both groups of patients at t0 and t2 crp c - reactive protein, esr erythrocyte sedimentation rate, vas visual analogic scale, haq health assessment questionnaire, tjc tender joint count, sjc swollen joint count, pasi psoriasis area and severity index no serious adverse events occurred in both groups, and no patient was withdrawn from therapy. during the follow - up period, all patients on steroid reduced gradually and discontinued steroid within the first 3 months, while dmards therapy was continued for the entire 2-year period. among patients of group 1, no one developed active tb during follow - up, while two patients of group 2 showed positive igra results after 1 year of treatment. in those cases, after 45 days, no signs of active mtb infection occurred and biologic therapy was restarted. patients of group 1 started anti - tnf- therapy after at least 45 days on prophylactic therapy with isoniazid. patients of group 2, who started anti - tnf- therapy, performed igra test every 12 months to detect a possible ltbi occurrence. in each group, 22 patients were on etanercept therapy, 14 on adalimumab, and 4 on infliximab. anti - tnf- therapy was effective in both groups of patients, and no statistically significant differences were found in the analysis of the study variables between the two groups from t0 to t2 (table 2).table 2comparison of variation of clinical characteristic in both groups of patients at t0 and t2group 1group 2 p crp (mg / dl)0.3060.0810.6085esr (mm / hr)0.3620.0340.9609vas0.00010.00010.2509haq0.00010.00010.2593tjc0.00010.00010.1772sjc0.0170.00010.8446pasi0.00010.00010.3531 crp c - reactive protein, esr erythrocyte sedimentation rate, vas visual analogic scale, haq health assessment questionnaire, tjc tender joint count, sjc swollen joint count, pasi psoriasis area and severity index comparison of variation of clinical characteristic in both groups of patients at t0 and t2 crp c - reactive protein, esr erythrocyte sedimentation rate, vas visual analogic scale, haq health assessment questionnaire, tjc tender joint count, sjc swollen joint count, pasi psoriasis area and severity index no serious adverse events occurred in both groups, and no patient was withdrawn from therapy. during the follow - up period, all patients on steroid reduced gradually and discontinued steroid within the first 3 months, while dmards therapy was continued for the entire 2-year period. among patients of group 1, no one developed active tb during follow - up, while two patients of group 2 showed positive igra results after 1 year of treatment. in those cases, after 45 days, no signs of active mtb infection occurred and biologic therapy was restarted. this paper reports our experience in psa patients with concomitant ltbi under anti - tnf- drugs. however, given the epidemiological dynamics, it may represent an emerging question which we need to face. the results of this study show that the presence of concomitant ltbi in psa patients does not influence the effectiveness of anti - tnf- therapy during the follow - up period. in fact, tnf- blockers, as expected, improved signs of articular and cutaneous involvement and ameliorated function and quality of life in patients of both groups after 24 months of therapy. further important result concerns safety : there were no cases of active tuberculosis in both groups during the follow - up period. although anti - tnf- agents are effective in the treatment of psa, their use has been recognized as a risk factor for active tuberculosis development in patients with immune - mediated inflammatory diseases, including rheumatoid arthritis (ra), ankylosing spondylitis (as), crohn s disease, psa, and psoriasis [16, 17 ]. in fact, tnf- is an essential cytokine for the host s defense against infective pathogens. it has shown that during mtb infection, tnf- is produced transiently and focally by granulomas in response to mycobacterial challenge. it contributes to their containment and elimination, suggesting a pivotal role of tnf- in defense mechanisms against microorganisms. in animal models, tnf- plays a central role in mediating mycobacterial infections, and soluble tnf (especially membrane - bound tnf) expressed by activated macrophages and t lymphocytes is essential in protecting against tb infection. it has been observed that most cases of tb develop soon after treatment starting and correspond to reactivation of ltbi [19, 20 ]. several recommendations for screening patients with ltbi infection and for the treatment of such patients before initiating anti - tnf- have been proposed, even if at the present there are no international guidelines for monitoring of ltbi during biologic treatment and the data are poor. according to these recommendations, several studies have shown that isoniazid therapy for 6 and 12 months in patients with ltbi is more effective than placebo in preventing reactivation, and the best benefit can be obtained with 9 months of therapy. in our population, none of the patients treated with isoniazid developed serious toxicity in the liver, but it remains a threat that should be considered, especially in the event of concomitant use of hepatotoxic drugs other than isoniazid or methotrexate [22, 23 ]. effectiveness of chemoprophylaxis is limited not only by the efficacy of the drug but also by patient adherence to therapy. a recent open - label study tested the efficacy and the tolerability of a 3-month regimen with isoniazid plus rifampicin, showing that this therapy could be useful in patients with rheumatic conditions proposed for anti - tnf therapy. the french research axed on tolerance of biotherapies (ratio) registry records the annual incidence rate of tb in patients treated with anti - tnf- blockers, adjusted for age and sex, with the french population used as reference. data from this registry report 69 cases of active tb in patients with anti - tnf- for a period of 3 years for a total of 57,711 patient - years. tb cases were related to infliximab and adalimumab therapy, and the authors underline that none of the patients had received correct ltbi chemoprophylaxis according to the french recommendations. the british registry confirms these data, underlining a relative risk of tb three to four times higher for the monoclonal antibodies than for etanercept. in addition, the data show a greater increased risk of extrapulmonary disease with the monoclonal antibodies. in our study we did not observe any case of reactivation of tb in patients with ltbi treated with tnf- blockers. this result may be affected by the relative small sample size. on the other hand, it should be considered screening, chemoprophylaxis, and monitoring of patients, which may explain this optimal result. current evidence indicates that prophylaxis may be ineffective in up to 30 % of cases, when patient adherence to therapy is uncertain. recently, we have suggested that screening and constant monitoring of ltbi during tnf- blocker therapy are determinants for continued treatment of psa, in order to avoid risks of reactivation of tubercular disease. we have proposed that psa patients with ltbi under anti - tnf- therapy should undergo clinical and laboratory standard evaluation every 3 months and a chest radiograph every 6 months. even patients negative for tst and igra should undergo clinical and laboratory standard evaluation every 3 months, but they could perform chest radiograph and igra every 12 months. at any time, in case of fever and symptoms and/or signs of suspected tb reactivation, they should stop biologic therapy and perform a chest x - ray and other appropriate diagnostic and therapeutic measures. in conclusion, our experience suggests that anti - tnf- treatment is effective and safe in psa patients with concomitant ltbi. therefore, neither ltbi nor chemoprophylaxis seems to influence the course of anti - tnf- therapy. moreover, an accurate diagnosis and management of ltbi could reduce the risk of reactivation of tuberculosis. further studies are needed to clarify the controversial question of the monitoring of ltbi patients on biologic therapy who show tst / igra positive conversion. at present, our approach is to treat and monitor them as patients with ltbi at enrollment.	psoriatic arthritis (psa) is an inflammatory arthropathy associated with skin and/or nail psoriasis. tnf- is an essential cytokine for the host defense, and its depletion by treatment may facilitate the risk of infections or their reactivation. the aim of this study was to evaluate the efficacy and safety of tnf- blockers in patients with psa and concomitant latent tuberculosis infection (ltbi) comparing their outcome with non - infected psa patients. this is a retrospective study in 321 patients with psa, attending the psoriatic arthritis clinic at the university federico ii of naples, who had an inadequate response to dmards and started therapy with tnf- blockers. we identified 40 patients with ltbi, who were included in this study along with 40 not infected psa patients as control group. at baseline (t0) and every 3 months for 2 years (t2), data concerning psa activity were registered. all patients underwent chest x - ray every 6 months (or 12 if appropriate). in each group, 22 patients were on etanercept therapy, 14 on adalimumab, and 4 on infliximab. anti - tnf- therapy was effective in both group of patients, and no statistically significant differences were found in the analysis of the study variables between the two groups from t0 to t2. no serious adverse events occurred in both groups, and no patient was withdrawn from therapy. our experience suggests that anti - tnf- treatment is effective and safe in psa patients with concomitant ltbi. therefore, neither ltbi nor chemoprophylaxis seems to influence the course of anti - tnf- therapy.
a 29-year - old female was referred to the adult inherited metabolic disease clinic with a four - year history of episodic sensorimotor symptoms. the first episode began with numbness in the right foot, spreading to affect the right leg, trunk, arm and face, lasting six weeks before spontaneously resolving completely. subsequently, a further eight episodes of similar neurological disturbance occurred, each affecting her right side, lasting between twelve days and six weeks, and followed by recovery. the last attack was characterized by weakness and incoordination of the right leg, associated with dizziness and difficulty walking, lasting twelve days., she complained of residual numbness affecting her entire right hand, and struggled with prolonged writing. her only son was diagnosed with childhood cerebral ald (cc - ald) and died at the age of six years, despite bone marrow transplantation. her mother and sole sibling, a sister, are known x - linked adrenoleukodystrophy (x - ald) carriers, but her maternal grandmother does not have the mutation. the maternal grandfather died of a probable myocardial infarction in his late 60s and was not genotyped ; he did not have any gait disturbance. her ten - year - old nephew underwent bone marrow transplantation and is in remission. another nephew has magnetic resonance imaging (mri) changes consistent with ald, and was asymptomatic at age three years. at the time of presentation to the neurology service, visual acuity was 6/6 and colour vision 13/13 bilaterally, fundi were normal, and she had a full range of eye movements. there was no evidence of adrenal dysfunction, with normal serum cortisol and adrenocorticotrophic hormone (acth) levels. plasma very - long - chain fatty acids (vlcfa, sheffield children 's hospital) were elevated, c26:0 2.51 mol / l (reference range 0.331.50), c24:0 60 mol / l (14 - 80), c26/c22 0.045(0.005 - 0.030), c24/22 1.07(0.44 - 0.97). cranial mri demonstrated several areas of increased signal in a periventricular distribution, consistent with demyelination (figure 1). recovery magnetic resonance imaging sequences show periventricular hyperintense lesions in a typical pattern for multiple sclerosis. (a) axial t2-weighted and (b) sagittal fluid - attenuated inversion recovery magnetic resonance imaging sequences show periventricular hyperintense lesions in a typical pattern for multiple sclerosis. cerebrospinal fluid (csf) analysis was normal (glucose 3.1 mmol / l, protein 302 mg / l, white cell count 210/l) apart from evidence of oligoclonal band synthesis in csf (serum normal, type 2 pattern). visual evoked potentials (veps) based on the relapsing - remitting history, the examination findings, the mri appearances and the unmatched csf oligoclonal bands, a diagnosis of relapsing - remitting multiple sclerosis (ms) was made. as there had been three clinical episodes within the previous two years, treatment with subcutaneous interferon -1a (rebif 44 mg) was started. subsequently, this was changed to intramuscular interferon -1a (avonex), because of injection site reactions. initially, the frequency and severity of her relapses reduced over the ensuing three years, with two episodes of mild sensory symptoms at 6 months (paraesthesia affecting the right leg, lasting for a week) and 24 months (sensory symptoms affecting her left side). however, she then suffered two further attacks over the next 12 months, resulting in residual weakness in both legs and paraesthesia to the waist. there have been no further episodes of neurological symptoms to date, other than a mild episode of pins and needles in both feet, associated with difficulty climbing stairs in january 2010, which improved spontaneously. x - linked adrenoleukodystrophy (x - ald) is a clinically heterogeneous disorder with an overall incidence of 1:16,800. the disease is caused by mutations in the abcd1 gene on the x chromosome at q28, resulting in dysfunction of the peroxisomal membrane transporter protein, a member of the atp - binding cassette transporter superfamily. the function of this protein is unclear ; it may be involved in the transfer of vlcfa across the cell membrane. in x - ald, the clinical result is progressive organ dysfunction, predominantly affecting the adrenal glands and the white matter of the central nervous system. childhood - onset cerebral ald (cc - ald) is the most severe form, and manifests as early neurological dysfunction (including pseudobulbar palsy, dementia, personality change, cortical blindness, deafness, and ataxia) and adrenal insufficiency. adolescent - onset cerebral ald is much less common and usually occurs between the ages of 10 to 21. adult - onset cerebral ald resembles the childhood cerebral form, but psychiatric manifestations are more prevalent. in the adrenomyeloneuropathy variant (amn), patients usually present later, in the second to fourth decades, with spinal cord dysfunction, frequently spastic paraparesis. approximately 50% of these patients have changes on cranial mri, which may resemble the neuroradiological appearances of ms. female carriers, heterozygous for the x - ald mutation, are usually asymptomatic, but may present with mild cerebral or adrenal involvement, which progresses very slowly. the mean age of onset is 40 years and the clinical features may resemble the syndrome seen in males with amn. the pathology of cerebral x - ald and amn is thought to be distinct. in cerebral x - ald, lesions are characterized histologically by the presence of lymphocytes, macrophages and reactive astrocytes. in pure amn, distal axonopathy is seen, with loss of axons and myelin also commonly affecting the gracile and corticospinal tracts. fatemi and colleagues investigated the mri changes in heterozygous female carriers of x - ald and found that only 4% had changes in the brain attributable to the disease, with bilateral symmetrical white matter abnormalities. eighty - six percent had normal scans, and the remainder showed changes consistent with age, cerebrovascular disease or previous trauma. in another study, kumar and colleagues reported a prevalence of 20% brain mri abnormalities in female x - ald heterozygotes, most commonly a mild increase in signal intensity in the parieto - occipital or frontal white matter. an oligoclonal banding pattern in the csf is found in the majority of patients with ms and provides strong support for the diagnosis in the appropriate clinical context. however, oligoclonal bands may also be seen in ald (dr. geoffrey keir, personal written communication, 14/10/08) but the prevalence remains unclear. these have included restriction of dietary saturated fats rich in vlcfa, and a supplemented intake of monounsaturated fats, such as lorenzo 's oil, but the results so far have been disappointing. bone marrow transplantation is an effective therapy for childhood cerebral ald when performed promptly after onset of symptoms but is much less successful in adolescent or adult cerebral ald. an inflammatory response in the brain has been hypothesized to underlie the observed widespread white matter changes, especially in the cerebral forms of adrenoleukodystrophy, but a number of therapeutic trials with immunosuppressive drugs, such as cyclophosphamide, cyclosporin and -interferon have not resulted in any significant clinical benefits. there are published case reports of female heterozygous x - ald carriers presenting with neurological symptoms suggestive of ms, in whom a diagnosis of ald was subsequently established, based on clinical and biochemical findings. stockler and co - workers reported a young woman who presented with transient visual symptoms. visual evoked responses were delayed in the right eye, and cranial mri revealed two partially confluent periventricular lesions. the authors concluded that the neurological syndrome was more likely ald - related than ms. krenn reported a 40 year old female with spastic paraparesis, sensory and bladder symptoms and visual disturbance. the diagnosis of ms was established on the basis of the relapsing - remitting history, typical mri changes (including contrast enhancement, which does not appear to have been reported previously in ald), positive oligoclonal bands in csf, and favourable response to disease - modifying therapy. in particular, the predominance of sensory symptoms with complete resolution was considered more consistent with ms than ald. to the best of our knowledge, this is the first reported case of ms in an ald carrier, and serves as a reminder that neurological symptoms in carriers of rare diseases are not always related ; sometimes more common coexisting conditions explain the presentation.	x - linked adrenoleukodystrophy (x - ald) is a rare inherited metabolic disorder, in which accumulation of very long chain fatty acids (vlcfas) results in damage to the central nervous system. as the disease is x - linked, males are affected severely, but female carriers may also present with neurological symptoms. we report the case of a young adult female, who presented with episodic sensorimotor symptoms. although she was a heterozygous female carrier of x - ald, subsequent investigations confirmed a diagnosis of multiple sclerosis (ms). to the best of our knowledge, this is the first reported case of a female x - ald carrier in which the clinical features were more consistent with co - existent ms than ald - related pathology. the case serves as a reminder that alternative, more common diagnoses should also be considered in carriers of rare neurological syndromes.
between october 2003 and may 2009, we evaluated 33 patients (3 men and 30 women) who were treated with surgical treatment for refractory de quervain 's disease. all patients presented with clinical signs and symptoms of de quervain 's disease, which included positive finkelstein tests, localized tenderness over the first dorsal compartment, and positive sign on active epb extension test. in this active epb extension test, the patient was asked to abduct the thumb and extend the metacarpophalangeal joint, and induction or aggravation of pain over the first compartment was determined as positive. we defined the disease refractory when symptoms persisted for longer than 3 months after injection of 0.1ml betamethasone and 0.4 ml 1% (w / v) lidocaine once or twice, or when symptoms recurred after repeated injection more than three times. of the 33 patients, 31 had unilateral and 2 had bilateral de quervain 's disease. the mean age of the patients included in this study was 37.6 years (range, 26 to 44 years) for men and 51.6 years (range, 30 to 67 years) for women. four patients had associated diseases in the same upper extremity, which are one carpal tunnel syndrome, two cubital tunnel syndromes and one lateral epicondylitis. however de quervain 's disease at those cases was developed after the other diseases were cured. the mean duration of follow - up was 28.4 months (range, 18 to 39.5 months). all operations were performed by a single senior surgeon and intra - operative findings were recorded. we collected clinical data of finkelstein test, dash score,10) and overall and night visual analogue scale (vas) pain score before operation and at the final follow - up. all operations were performed in the day surgery unit, under local anesthesia using intravenous midazolam and pethidine. a mixture of 0.1% (w / v) lidocaine and 0.25% (w / v) bicarbonate was injected into the area of skin incision and a pneumatic tourniquet was inflated. a 2-cm long transverse skin incision was made over the first extensor compartment of the radial styloid process. after lifting the epb tendon, the extensor retinaculum covering the epb subcompartment was incised longitudinally along the epb tendon (fig. if there was a septum between the epb and apl tendons, the septum was incised longitudinally. any entanglement was double - checked by pulling and lifting up the tendon distally (fig. 2). when we found a ganglion from the tendon sheath, we removed the mass from the tendon sheath without excision of the tendon sheath. after meticulous hemostat control, the skin was closed and a compressive dressing was applied. paired t - test was used to compare the preoperative and postoperative clinical examination parameters with spss ver. 17.0 chicago, il, usa). a p - value of < 0.05 was considered to be statistically significant. between october 2003 and may 2009, we evaluated 33 patients (3 men and 30 women) who were treated with surgical treatment for refractory de quervain 's disease. all patients presented with clinical signs and symptoms of de quervain 's disease, which included positive finkelstein tests, localized tenderness over the first dorsal compartment, and positive sign on active epb extension test. in this active epb extension test, the patient was asked to abduct the thumb and extend the metacarpophalangeal joint, and induction or aggravation of pain over the first compartment was determined as positive. we defined the disease refractory when symptoms persisted for longer than 3 months after injection of 0.1ml betamethasone and 0.4 ml 1% (w / v) lidocaine once or twice, or when symptoms recurred after repeated injection more than three times. of the 33 patients, 31 had unilateral and 2 had bilateral de quervain 's disease. the mean age of the patients included in this study was 37.6 years (range, 26 to 44 years) for men and 51.6 years (range, 30 to 67 years) for women. four patients had associated diseases in the same upper extremity, which are one carpal tunnel syndrome, two cubital tunnel syndromes and one lateral epicondylitis. however de quervain 's disease at those cases was developed after the other diseases were cured. the mean duration of follow - up was 28.4 months (range, 18 to 39.5 months). all operations were performed by a single senior surgeon and intra - operative findings were recorded. we collected clinical data of finkelstein test, dash score,10) and overall and night visual analogue scale (vas) pain score before operation and at the final follow - up. all operations were performed in the day surgery unit, under local anesthesia using intravenous midazolam and pethidine. a mixture of 0.1% (w / v) lidocaine and 0.25% (w / v) bicarbonate was injected into the area of skin incision and a pneumatic tourniquet was inflated. a 2-cm long transverse skin incision was made over the first extensor compartment of the radial styloid process. after lifting the epb tendon, the extensor retinaculum covering the epb subcompartment was incised longitudinally along the epb tendon (fig. if there was a septum between the epb and apl tendons, the septum was incised longitudinally. any entanglement was double - checked by pulling and lifting up the tendon distally (fig. 2). when we found a ganglion from the tendon sheath, we removed the mass from the tendon sheath without excision of the tendon sheath. after meticulous hemostat control, the skin was closed and a compressive dressing was applied. paired t - test was used to compare the preoperative and postoperative clinical examination parameters with spss ver. chicago, il, usa). a p - value of < 0.05 was considered to be statistically significant. in 18 patients (55%), the epb tendon compartment was separated from the apl compartment. a thick retinaculum and neovascularization of the tendon sheath were noted. the epb tendon was easily identified by its dorsal location to the apl tendon and relatively distal musculotendinous junction compared to that of the apl tendon. only one patient showed a positive sign of finkelstein test at the last follow - up. we found that the average vas pain score significantly decreased, from 7.42 before surgery to 1.33 at the final follow - up (p < 0.05). individuals with pain during night showed a decrease in vas score from 5 preoperatively to 0.15 postoperatively (p < 0.05) (fig. the dash score also decreased from 53.2 to 3.45 (p < 0.05) (fig. there was no postoperative infection or subluxation of the tendon of the first extensor compartment when an active thumb extension or flexion was tested. no injury to the sensory branch of the radial nerve, adhesion between sensory branches and the fascia, scar hypertrophy or wound dehiscence was noted. in 18 patients (55%), the epb tendon compartment was separated from the apl compartment. a thick retinaculum and neovascularization of the tendon sheath were noted. the epb tendon was easily identified by its dorsal location to the apl tendon and relatively distal musculotendinous junction compared to that of the apl tendon. only one patient showed a positive sign of finkelstein test at the last follow - up. we found that the average vas pain score significantly decreased, from 7.42 before surgery to 1.33 at the final follow - up (p < 0.05). individuals with pain during night showed a decrease in vas score from 5 preoperatively to 0.15 postoperatively (p < 0.05) (fig. the dash score also decreased from 53.2 to 3.45 (p < 0.05) (fig. there was no postoperative infection or subluxation of the tendon of the first extensor compartment when an active thumb extension or flexion was tested. no injury to the sensory branch of the radial nerve, adhesion between sensory branches and the fascia, scar hypertrophy or wound dehiscence was noted. we achieved excellent clinical outcome in terms of pain relief with minimal complication using a dorsal release of the first extensor compartment for the treatment of refractory de quervain 's disease. because of troublesome complication of tendon subluxation,11) complete excision of the tendon sheath is not recommended anymore.12) instead of complete excision, several methods were reported to prevent tendon subluxation.12,13) but these methods can not warrant the release of apl tendon if the pathology is on the side of apl.14) because releasing the dorsal aspect of the first extensor compartment does not violate the volar side that contains the apl tendon, we also could prevent volar subluxation of the tendons. moreover, direct inspection is possible from the incision, we probably do not need preoperative ultrasonogram. several studies on the long - term results of surgical release of the first extensor compartment have been reported.15) however, those reports have limitations by their small number of patients. moreover, no study evaluated the results using validated scoring system, which was applied in our study. the dash developed by " upper extremity collaborative group"10) is considered as a reliable outcome measure in pathology of upper extremities16) and is widely accepted. regarding the pain relief, this procedure provided good pain relief, which was effective for both overall and night pain. there have been few reports in the literature reporting this ganglion.17) even though we can not know the cause - and - effect relationship between the ganglia and de quervain 's disease, we can speculate the association between the ganglion and de quervain 's disease by the incidence of ganglion. after resection of these ganglia, the symptoms disappeared and there was no recurrence case at the final clinical follow - up. the septum in the first extensor compartment we identified in this study was also of interest, because the epb tendon sheath was separated from the apl in over half of the patients ' cohort. second, we had four patients with other upper extremity disease that can affect dash and vas score. ganglia over the first extensor compartment and the separate septum of epb are often related to de quervain 's disease. dorsal release of first extensor compartment for refractory de quervain 's disease resulted in good clinical outcome with minimal morbidity.	backgroundfew studies have evaluated surgical outcomes in patients with refractory de quervain 's disease using validated outcome measures. we assessed the clinical outcomes of dorsal release of the first extensor compartment for the treatment of de quervain 's disease using the disabilities of the arm, shoulder and hand (dash) score.methodsfrom october 2003 to may 2009, we retrospectively evaluated 33 patients (3 men and 30 women) who underwent surgical treatment for de quervain 's disease. all patients had a positive finkelstein test and localized tenderness over the first dorsal compartment. all operations were performed under local anesthesia. a 2-cm - long transverse skin incision was made over the first extensor compartment and the dorsal retinaculum covering the extensor pollicis brevis was incised longitudinally. preoperative and postoperative clinical evaluation included the use of dash score, finkelstein test, and visual analogue scale (vas) score.resultsin 18 patients (55%), the extensor pollicis brevis tendon compartment was separated from the abductor pollicis longus compartment. eight patients had intracompartmental ganglia in the extensor pollicis brevis subcompartment. all patients except one had negative sign on finkelstein test at the last follow - up. the average vas score decreased from 7.42 preoperatively to 1.33 postoperatively (p < 0.05), and dash score was improved from 53.2 to 3.45 (p < 0.05). there were no postoperative complications such as subluxation of the tendon of the first dorsal compartment or injury to the sensory branch of the radial nerve.conclusionsintracompartment ganglia and the separate septum of extensor pollicis brevis are often related to de quervain 's disease. the release of the first extensor compartment for refractory de quervain 's disease resulted in good clinical outcomes with minimal morbidity.
the lifetime prevalence of bipolar disorder (bd) is approximately 3%5%, and 80%90% of all bd patients experience depressive symptoms.1,2 in patients with bd, depressive periods tend to last longer than manic periods.3 in addition, a study that examined the symptoms experienced by bd patients over a 6-month period reported that approximately 60% of bd patients experienced depressive symptoms, which is two - fold higher than the incidence of manic symptoms.4 in other words, the period of depressive symptoms lasts longer, and the incidence of depressive symptoms is higher than that of manic symptoms. however, bipolar depression does not respond well to treatment ; it frequently progresses to recurrent episodes of mood disorder, making it difficult to treat. the most widely used psychopharmacologic strategies for the treatment of bipolar depression include mood stabilizer monotherapy, a combination of mood stabilizers, and combination therapy with antidepressants. however, most recently, monotherapy with atypical antipsychotics, combination therapy with antidepressants, and combination therapy with mood stabilizers have been newly recommended.510 atypical antipsychotics are approved for the treatment of schizophrenia and psychotic disorders. however, due to their mechanism of action, they have been widely used to improve depressive symptoms in patients with psychotic disorders and bd.7 in particular, quetiapine has been known to exert antidepressant effects in patients with psychotic disorders and depressive symptoms.11 in two recent large - scale, double - blind, randomized controlled trials, quetiapine mono - therapy was proven to have significantly higher antidepressant effects than placebo on bipolar i and ii disorder (bd - i and bd - ii), rapid cycling bd, and depression with anxiety symptoms. moreover, the risk of inducing manic symptoms with quetiapine monotherapy was not higher than that with placebo. these results indicate that quetiapine could be used as a first - line drug for the treatment of bipolar depression.12,13 quetiapine was the first antipsychotic to be approved as a monotherapeutic agent for bipolar depression, not only by the us food and drug administration but also by the korean food and drug administration. however, the previous data were obtained using highly selective patients with many prescription restrictions, which could be different from actual clinical situations. in addition, no published large - scale data have evaluated the efficacy of quetiapine in bd patients in clinical settings. therefore, this observational study was conducted to evaluate the therapeutic efficacy of quetiapine, which was administered for 8 weeks to patients with bd - i, most recent episode depressed, and bd - ii, most recent episode depressed. this multicenter study was conducted at 98 sites including university hospitals and mental hospitals in korea from june 1, 2007 to february 28, 2008. the study subjects were older than 18 years, and all subjects were diagnosed with bd - i, most recent episode depressed, or bd - ii, most recent episode depressed, according to the diagnostic criteria of the diagnostic and statistical manual of mental disorder, fourth edition, text revision (dsm - iv - tr).1 the study procedure was explained to the patients who participated in this study, and only patients who submitted a written consent form were allowed to participate. because this study was an observational study conducted in an actual clinical setting, special exclusion criteria were absent, excluding rapid cycling, and even patients with chronic diseases were allowed to participate in the study, provided they were stable based on their past history and physical examination. however, patients with severe, unstable, or acute diseases as well as those with chronic physical illnesses were excluded from the study. furthermore, even medications that had been continuously administered under stable conditions without a change in dose for the treatment of physical illnesses were allowed during the study period. to evaluate therapeutic compliance, the clinical global impression bipolar (cgi - bp) scale and montgomery asberg depression rating scale (madrs) were used to evaluate therapeutic efficacy.14,15 therapeutic efficacy was evaluated prior to the administration of quetiapine and after 4 and 8 weeks of administration. as the main outcome measure of therapeutic efficacy, the cgi - bp and madrs scores were compared at baseline and after 8 weeks of drug administration. in addition, the response rate (50% reduction in the total madrs value) and remission rate (10 in the total madrs value) were examined at the 4- and 8-week time points. in addition, differences in therapeutic efficacy between the bd - i and bd - ii patients were examined. in addition, the numbers of patients in each cgi - bp subgroup at baseline and after 4 and 8 weeks were compared. to evaluate therapeutic compliance, the subjective level of therapeutic compliance was examined after 4 and 8 weeks of drug administration. the patients were divided by compliance rate as follows : > 75%, 50%75%, 25%50%, and 75%, 50%75%, 25%50%, and < 25%. of the patients who completed the baseline evaluation prior to drug administration, patients who took quetiapine more than once and patients who were evaluated at least once after drug administration were included in the intent - to - treat group. the data were analyzed by the last observation carried forward method. regarding the statistical methods, repeated measures of analysis of variance, haenszel test, and the multiple linear regression test were applied. the independent t - test and chi - squared test were used for comparisons of bd - i and bd - ii patients, and all statistical analyses were performed with spss 18.0 (ibm corporation, armonk, ny). a total of 1097 bd patients were recruited for participation in this study, and 1058 patients (96.4%) completed the entire study. to elucidate the true efficacy of quetiapine, patients who exhibited poor therapeutic compliance (< 75% compliance rate) after 4 or 8 weeks were excluded (n = 333). thus, the study finally included 764 bd patients (mean age : 42.7 15.6 years) consisting of 526 patients with bd - i (mean age : 41.1 14.7 years) and 238 patients with bd - ii (mean age : 46.3 16.9 years) ; the difference in age between the bd - i and bd - ii groups was significant (p < 0.001). the mean duration of current depressive episode was 6.3 8.8 weeks and duration of current episode was 7.9 12.1 weeks in bd - ii patients, which was significantly longer than in bd - i patients (5.6 6.7 weeks ; p = 0.006). psychiatric comorbidity was detected in 3.8% of the subjects (bd - i 2.7% versus bd - ii 6.3% ; p = 0.015). n = 250) were admitted (bd - i 35.7% versus bd - ii 26.1% ; p = 0.008), and mean number of admissions was 3.1 1.9 (bd - i 3.1 1.9 versus bd - ii 3.2 2.1 ; not significant) (table 1). the mean dose of quetiapine was initially 197.5 182.5 mg / day, and it was gradually increased to 349.0 231.5 mg / day by week eight. during all observation periods, the dose of quetiapine was significantly higher in the bd - i patients than in bd - ii patients (p < 0.001) (table 2). the combined administration of psychotropic agents was abundant in bd patients, and the difference in total concomitant medications administered between the bd - i and bd - ii groups was not significant. nonetheless, mood stabilizers (61.0% versus 35.7% ; p < 0.001) were administered more frequently in bd - i patients, but anxiolytics (22.8% versus 31.5% ; p = 0.001), antidepressants (12.4% versus 42.47% ; p < 0.001), and hypnotics / sedatives (8.0% versus 13.4% ; p = 0.018) were more frequently coadministered to bd - ii patients (table 3). the cgi - bp scores obtained at baseline, week four, and week eight were 4.3 0.9, 3.5 0.9, and 2.7 0.9, respectively. the values were significantly lower at week four and eight than at baseline (p < 0.001). however, the difference between bd - i and bd - ii patients was not significant (figure 1). moreover, when the entire patient group was divided according to disease severity and the subgroups were further divided according to cgi - bp score, the number of patients with a higher level of symptom severity was significantly decreased after 4 and 8 weeks compared to that at baseline (p < 0.001). such differences were also significant when the groups were compared after 4 and 8 weeks (p < 0.001 ; table 4). when comparing the severity of symptoms at baseline, week four, and week eight by madrs, there was a statistically significant decrease in severity for both the total madrs value and every subscale score (p < 0.001). in addition, the severity of symptoms was not significantly different between bd - i and bd - ii patients at baseline or after 4 and 8 weeks (table 5). the response rates (50% reduction in the total madrs value) at week four and eight were 25.5% and 58.9, respectively (p < 0.001), and the remission rates (10 in the total madrs value) were 14.9% and 42.1%, respectively (p < 0.001). however, there was no difference in the response and remission rates between bd - i and bd - ii patients (figure 2). to determine the factors independently associated with madrs reductions at week eight, age, illness duration, number of hospitalization, madrs score at baseline, number of concomitant medications, duration of current episode, presence of remission on previous episode, and use of antidepressants were added in the regression model. madrs score at baseline (= 0.612, p < 0.001), duration of current episode (= 0.152, p = 0.001), and the presence of remission on previous episode (= 0.111, p = 0.012) were significantly associated with improvements of depressive symptoms (table 6). to evaluate therapeutic compliance, the subjective level of therapeutic compliance was examined, and the results revealed compliance rates exceeding 75% after 4 weeks for 834 patients (76.0%). in addition, after 8 weeks, the compliance level exceeded 75% for 827 patients (75.4%). in excluded patients (n = 333), 30.6% of bd patients (n = 102) were admitted (bd - i 28.1% versus bd - ii 35.8% ; p = 0.108). in addition, there was no significant difference in compliance between the bd - i and bd - ii groups after 4 and 8 weeks (table 7). in total, 176 of 1097 bd patients (16.0%) felt fatigued, and 165 patients (14.9%) reported somnolence during the study period. manic / hypomanic episodes were observed in seven cases (three cases / four cases ; 0.6%) and three cases (three cases/ none ; 0.3%) at week four and eight, respectively. only four of the patients who exhibited manic / hypomanic episodes had symptom severities exceeding moderately ill according to the cgi - bp score after 4 weeks, and only one patient had to terminate quetiapine therapy. in addition, the symptoms could only be controlled by adjusting the dose of quetiapine and concomitant medications in all of the remaining cases. a total of 1097 bd patients were recruited for participation in this study, and 1058 patients (96.4%) completed the entire study. to elucidate the true efficacy of quetiapine, patients who exhibited poor therapeutic compliance (< 75% compliance rate) after 4 or 8 weeks were excluded (n = 333). thus, the study finally included 764 bd patients (mean age : 42.7 15.6 years) consisting of 526 patients with bd - i (mean age : 41.1 14.7 years) and 238 patients with bd - ii (mean age : 46.3 16.9 years) ; the difference in age between the bd - i and bd - ii groups was significant (p < 0.001). the mean duration of current depressive episode was 6.3 8.8 weeks and duration of current episode was 7.9 12.1 weeks in bd - ii patients, which was significantly longer than in bd - i patients (5.6 6.7 weeks ; p = 0.006). psychiatric comorbidity was detected in 3.8% of the subjects (bd - i 2.7% versus bd - ii 6.3% ; p = 0.015). n = 250) were admitted (bd - i 35.7% versus bd - ii 26.1% ; p = 0.008), and mean number of admissions was 3.1 1.9 (bd - i 3.1 1.9 versus bd - ii 3.2 2.1 ; not significant) (table 1). the mean dose of quetiapine was initially 197.5 182.5 mg / day, and it was gradually increased to 349.0 231.5 mg / day by week eight. during all observation periods, the dose of quetiapine was significantly higher in the bd - i patients than in bd - ii patients (p < 0.001) (table 2). the combined administration of psychotropic agents was abundant in bd patients, and the difference in total concomitant medications administered between the bd - i and bd - ii groups was not significant. nonetheless, mood stabilizers (61.0% versus 35.7% ; p < 0.001) were administered more frequently in bd - i patients, but anxiolytics (22.8% versus 31.5% ; p = 0.001), antidepressants (12.4% versus 42.47% ; p < 0.001), and hypnotics / sedatives (8.0% versus 13.4% ; p = 0.018) were more frequently coadministered to bd - ii patients (table 3). the cgi - bp scores obtained at baseline, week four, and week eight were 4.3 0.9, 3.5 0.9, and 2.7 0.9, respectively. the values were significantly lower at week four and eight than at baseline (p < 0.001). however, the difference between bd - i and bd - ii patients was not significant (figure 1). moreover, when the entire patient group was divided according to disease severity and the subgroups were further divided according to cgi - bp score, the number of patients with a higher level of symptom severity was significantly decreased after 4 and 8 weeks compared to that at baseline (p < 0.001). such differences were also significant when the groups were compared after 4 and 8 weeks (p < 0.001 ; table 4). when comparing the severity of symptoms at baseline, week four, and week eight by madrs, there was a statistically significant decrease in severity for both the total madrs value and every subscale score (p < 0.001). in addition, the severity of symptoms was not significantly different between bd - i and bd - ii patients at baseline or after 4 and 8 weeks (table 5). the response rates (50% reduction in the total madrs value) at week four and eight were 25.5% and 58.9, respectively (p < 0.001), and the remission rates (10 in the total madrs value) were 14.9% and 42.1%, respectively (p < 0.001). however, there was no difference in the response and remission rates between bd - i and bd - ii patients (figure 2). to determine the factors independently associated with madrs reductions at week eight, age, illness duration, number of hospitalization, madrs score at baseline, number of concomitant medications, duration of current episode, presence of remission on previous episode, and use of antidepressants were added in the regression model. madrs score at baseline (= 0.612, p < 0.001), duration of current episode (= 0.152, p = 0.001), and the presence of remission on previous episode (= 0.111, p = 0.012) were significantly associated with improvements of depressive symptoms (table 6). to evaluate therapeutic compliance, the subjective level of therapeutic compliance was examined, and the results revealed compliance rates exceeding 75% after 4 weeks for 834 patients (76.0%). in addition, after 8 weeks, the compliance level exceeded 75% for 827 patients (75.4%). in excluded patients (n = 333), 30.6% of bd patients (n = 102) were admitted (bd - i 28.1% versus bd - ii 35.8% ; p = 0.108). in addition, there was no significant difference in compliance between the bd - i and bd - ii groups after 4 and 8 weeks (table 7). in total, 176 of 1097 bd patients (16.0%) felt fatigued, and 165 patients (14.9%) reported somnolence during the study period. manic / hypomanic episodes were observed in seven cases (three cases / four cases ; 0.6%) and three cases (three cases/ none ; 0.3%) at week four and eight, respectively. only four of the patients who exhibited manic / hypomanic episodes had symptom severities exceeding moderately ill according to the cgi - bp score after 4 weeks, and only one patient had to terminate quetiapine therapy. in addition, the symptoms could only be controlled by adjusting the dose of quetiapine and concomitant medications in all of the remaining cases. active treatment of depressive symptoms may be very important in patients with bd because many bd patients attempt to commit suicide during the depressive episode or mixed episode including depressive symptoms, and the risk of committing suicide is 15-fold greater in bd patients than in the general population.16 generally, in patients with bipolar depression, the use of atypical antipsychotics was limited to patients exhibiting psychotic symptoms. however, the use of atypical antipsychotics has been accepted as a new treatment modality because recent studies have demonstrated the therapeutic efficacy of atypical antipsychotics in treating bipolar depression, regardless of the accompanying psychotic symptoms.12,13,1720 in addition, the korean medication algorithm project for bd 2006 (kmap - bp 2006) recommended the combined use of atypical antipsychotics and mood stabilizers in the treatment of bd, regardless of the presence or absence of psychotic symptoms.10 in the current study, treatment with the atypical antipsychotic quetiapine after 4 and 8 weeks resulted in noticeable improvements in depressive symptoms in both bd - i and bd - ii patients compared to the baseline findings. in addition, when disease severity was classified according to the cgi - bp score, the number of patients with more severe symptoms was decreased after both 4 and 8 weeks of treatment. in particular, such a change was observed between 4 and 8 weeks of treatment, which suggests that the therapeutic effectiveness of quetiapine continuously increased for at least 8 weeks. the improvement of depressive symptoms was consistently observed on every subscale of madrs, and this result is consistent with the results of previous double - blind, randomized controlled trials.12,13 in addition, the rates of response and remission continuously increased for 8 weeks, resulting in values of 58.9% and 42.1%, respectively. these results can be considered to support the results of previous studies and indicate that quetiapine may be effective in the treatment of bipolar depression.12,13,21 bd - ii patients were more chronically ill, they had a greater duration of illness and spent more time in partial remission and less time in full remission.22 in the current study, however, there were no differences in the response and remission rates measured by madrs between the bd - i and bd - ii groups throughout the entire observation period. these results were concurrent with the results of a previous naturalistic study indicating that the treatment outcome of depressive symptoms was not different between the bd - i and bd - ii patients.23 this suggested that quetiapine could be used effectively in both bd - i and bd - ii patients. the mean initial daily dose of quetiapine was 197.5 mg, and this was higher than the 50 mg / day dose of quetiapine monotherapy administered in previous studies.12,13 in addition, the percentage of patients treated with a combination of psychotropic agents was 73.2%, which was relatively high. this discrepancy is thought to be due to the fact that previous studies were conducted in outpatients, whereas 32.7% of the subjects in the current study were inpatients. in addition, 81.3% of the patients had a level of disease severity greater than moderately ill, and thus patients with relatively severe symptoms were more abundant in this study than in previous studies. furthermore, previous studies were conducted as randomized controlled trials, and the primary aim of the current study was to observe patients in an actual clinical setting. the mean daily dose of quetiapine was increased slowly from the beginning of administration to 327.7 mg and 349.0 mg after 4 and 8 weeks, respectively, which is in agreement with the results of previous studies demonstrating that therapeutic efficacy was significant with daily doses of 300 mg or 600 mg.12,13,21 on the other hand, throughout the entire period of observation in the current study, the dose of quetiapine was lower in the bd - ii patients than in the bd - i patients ; however, there was no difference in treatment outcomes. one reason may be that the ratio of combination therapy was higher in bd - ii patients than in bd - i patients, and this combination therapy might be countervailing the lower dose of quetiapine. in fact, it was found that with the exception of mood stabilizers combination therapy with antidepressants, anxiolytics, and hypnotics was administered more frequently to bd - ii patients. this is thought to be due to the fact that the diagnosis of psychiatric comorbidity is more common in bd - ii patients,22 and in the current study, psychiatric comorbidity was more prevalent in patients with bd - ii. however, there was no difference in the number of patients administered combination therapy between the two disease groups (71.7% versus 76.5% ; p = 0.186), and the number of concomitant medications (= 0.060, p = 0.165) and the use of antidepressants (= 0.041, p = 0.253) did not affect madrs changes. evaluation of therapeutic compliance in bd patients has been considered difficult.24 bd patients tend to evaluate their own therapeutic compliance as being high, and the pill count method or the measurement of drug concentration also has limitations in the accurate evaluation of therapeutic compliance.25 in the current study, the subjective level of therapeutic compliance was evaluated after 4 and 8 weeks of treatment, and a compliance rate exceeding 75% was observed for approximately 70% of the study population ; thus, the rate of therapeutic compliance was higher than in previous studies.26,27 this is thought to be attributable to several factors, including the fact that the current study was an observational study allowing for the dose of quetiapine to be adjusted freely, the lack of restriction on the use of concomitant medications, the large percentage of inpatients in the study population, and the relatively short period of observation. it has been reported that in the treatment of bd, quetiapine tends to exert antidepressant effects with mild adverse events. in the current study, the level of adverse events was not evaluated systemically, but the rates of fatigue, somnolence, and dropout were 16.0%, 14.9%, and 3.6%, respectively. this result was comparable to those of previous studies.11,28,29 however, the detected dropout rate was extremely low compared to the rate of 16.0% observed in the monotherapy group administered a comparable daily dose (300 mg). conversely, one of the difficulties encountered in the treatment of bipolar depression is manic / hypomanic switching. in the current study, the incidences of manic/ hypomanic episodes after 4 and 8 weeks were 0.6% and 0.3%, respectively, which were lower than those reported previously.12,13,21 although the current study was an open - label observational study, the research protocol was not strict, and the study was conducted with a relatively short period of observation ; these results suggest that quetiapine could be used safely for the treatment of bipolar depression from the viewpoint of adverse events. this was a large - scale study to examine the therapeutic efficacy of quetiapine in patients with bd - i and bd - ii depression in an actual clinical setting. it was an open - label observational study permitting the flexible adjustment of the dose of quetiapine and the use of concomitant medications, and thus the control of variables contributing to the improvement of symptoms was difficult. also, the investigation of adverse events was not systematic in this study. however, the use of a detailed checklist for adverse events was beyond the scope of this paper. therefore, additional studies for the safety of quetiapine will be needed. the results of this study demonstrated that the treatment of bd - i and bd - ii depression with quetiapine for 8 weeks had significant antidepressant effects in an actual clinical setting, and the therapeutic efficacy improved with time, with minimal manic switching. in addition, the clinical improvement was similar in both bd - i and bd - ii patients. however, the mean dosage of quetiapine was lower in bd - ii patients throughout the observational periods, and the combination therapy with antidepressants, anxiolytics, and hypnotics was more frequently administered to bd - ii patients.	purposeto evaluate and compare the therapeutic efficacy of quetiapine in bipolar i and ii depression patients in the clinical setting.patients and methodsthis was an 8-week, multicenter, open - label, observational study for bipolar depression. the dosage of quetiapine was flexible, and concomitant medications were permitted on clinician s judgments. a total of 1097 patients were enrolled, and 764 bipolar depression patients who exhibited good therapeutic compliance (> 75% compliance rate) were analyzed.resultsclinical global impression bipolar scale and montgomery asberg depression rating scale scores were significantly improved at weeks four and eight compared with the baseline scores. at the end of the 8-week study, the response rate was 58.9%, and the remission rate was 42.1%. however, there were no significant differences in the response and remission rates between bipolar i and ii disorder (bd - i and bd - ii) patients (response rate 60.1% versus 56.3% ; remission rate 44.5% versus 37.0%). montgomery asberg depression rating scale score at baseline (= 0.612, p < 0.001), duration of current episode (= 0.152, p = 0.001), and presence of remission on previous episode (= 0.111, p = 0.012) were significantly associated with improvements in depressive symptoms. fatigue (16.0%), somnolence (14.9%), and manic / hypomanic switching (0.6% at week four, 0.3% at week eight) were observed throughout the study period.conclusionthe results of this study suggest that quetiapine improves depressive symptoms in bd - i and bd - ii patients with a minimal incidence of manic switching. the therapeutic efficacy of quetiapine increased with time. quetiapine could be an effective and safe modality for the treatment of bd - i and bd - ii.
capsular tension rings are used to stabilize the capsular bag, reduce the tension on zonules and to provide safer conditions for phacoemulsification in patients, especially those with pseudoexfoliation or trauma - induced breakdown in zonular support. we present here an unusual case of retinal detachment secondary to a capsular tension ring - induced retinal tear. a 45-year - old man who had undergone cataract surgery elsewhere 2 years previously on the left eye presented to our clinic complaining of decreased vision of 2 months duration in the same eye. the patient was treated and followed up at the ophthalmology department of cukurova university. visual acuity was at hand motion level and the intraocular pressure was 14 mm hg in the left eye. slit lamp examination showed a well - located posterior chamber intraocular lens and intact posterior lens capsule. the retinal periphery was not optimally visualized because of peripheral posterior capsular opacification, but showed multiple retinal tears with the largest one located between 3 and 5 o'clock. ultrasonography of the left eye showed retinal detachment as well as the large retinal tear (fig. fundus examination during 20-gauge vitrectomy showed a large retinal tear located between 3 and 5 o'clock and multiple tears located peripherally, and also a dislocated capsular tension ring around the tear. one end of the ring was found under the retina just next to the large tear at the 3 o'clock position. the ring was freed from all vitreous adhesions, divided into 2 pieces with scissors and removed using vitrectomy forceps through the sclerotomy site. barrier photocoagulation was done around the tear, and the vitrectomy was terminated after perfluorocarbon - silicone oil exchange. on the first postoperative day, visual acuity in the left eye was 20/200. during follow - up, intraocular pressure in the left eye increased and was controlled with the use of topical brimonidine and fixed dorzolamide / timolol combination. capsular tension rings are mainly preferred to maintain the contour of the capsular bag in patients who have capsular instability in complicated cataracts. indicated in their large series including 9,528 cataract surgeries that the frequency of capsular tension ring use was 0.7%. the primary indications for the ring were found to be as mature cataract, traumatic cataract, pseudoexfoliation syndrome and lens subluxation due to marfan syndrome. in the present case, zonular weakness and phacodonesis probably led to the use of a tension ring, and dislocation of the capsular tension ring was thought to have occurred during its placement through a possible site of capsular tear ; however, the dislocation could have been caused by a late capsular contraction which resulted in capsular shrinkage and ring displacement. another possible explanation is the misplacement of the ring in the sulcus during cataract surgery. possible complications of intraocular lens dislocation are retinal tears, retinal detachment, cystoid macular edema and vitreous hemorrhages. spontaneous in - the - bag intraocular lens dislocations have also been reported in the literature [3, 4, 5, 6 ]. removal of the ring has been achieved through sclerotomy sites or limbal incisions [7, 8 ]. described a case of retinal detachment which occurred after removal of a dislocated capsular tension ring with pars plana vitrectomy. the present case differs from this case by the presence of an accompanying retinal detachment at presentation. to the best of our knowledge, this is the first case of isolated dislocation of a capsular tension ring into the vitreous, which possibly led to a retinal detachment with a large tear, while the posterior chamber lens was safely located in the capsular bag with an intact posterior lens capsule. the posterior capsule was thought to be intact during slit lamp examination before vitrectomy, but the integrity of the entire capsular bag could have been impaired by a small tear located at the equatorial region. the ring may have been placed in the sulcus or fallen into the vitreous through a tear. in the present case, the tear was considered to have occurred secondary to ring dislocation because of the critical localization of the ring around the tear area. whatever the underlying mechanism is, it should be kept in mind that the application of a capsular tension ring requires experience and a careful follow - up because it may be associated with unexpected and very rare posterior segment complications.	we aim to present a case of retinal detachment secondary to capsular tension ring dislocation following cataract surgery. a 45-year - old man who underwent cataract surgery 2 years previously presented with decreased vision in his left eye. the patient 's posterior capsule was intact with a well - located posterior chamber intraocular lens. fundus examination revealed retinal detachment with retinal tears, and a capsular tension ring located around the tear was noticed during pars plana vitrectomy ; it was removed through the sclerotomy site. in conclusion, dislocation of the capsular tension ring is a rare and unexpected complication of cataract surgery. although the integrity of the posterior lens capsule is partially preserved, the ring may shift through a small tear in the bag even during its placement, and may cause retinal tears, retinal detachment or just remain silently suspended in the vitreous.
retroperitoneal tumors represent a rare entity, most are malignant and sarcomas are most commonly. sarcomas are malignant tumors derived from embrionary mesoderm that arise from skeletal and extraskeletal connective tissues, including the peripheral nervous system. the majority of soft tissue sarcomas present in the extremities ; however, many other sites can be affected, including the retroperitoneum. leiomyosarcomas are second in frequency at this location, and also one of the most aggressive. the only effective treatment known at the time is surgical resection r0, since chemotherapy is used only in metastasis, and radiotherapy is a surgical complement to decrease locoregional recurrence risk. to obtain surgical safe margin, is often necessary the resection of another adjacent infiltrated organs. recurrence, which is the most important prognostic factor in these patients, is very common. we present a case of retroperitoneal leiomyosarcoma where was necessary multiorgan resection for its treatment. a 67 year old male was admitted for study of fever, back pain, constitutional symptoms and general illness with several months of evolution. during hospitalization the patient required repeated transfusions because of progressive anemia, needing more than twelve packed erythrocytes. physical examination revealed an abdominal mass in left upper quadrant, hard and fixed to deeper layers. ultrasound showed a enlarged spleen (14.5 cm) with difficult to access to splenic hilum due to a 15 cm diameter, heterogeneous and multi - lobed mass. located above and medial to left kidney upper pole and lateral to aorta, in intimate contact with posterior spleen face. the admission ct report a tumor in left upper quadrant of 17 cm in diameter, heterogeneous hypo - and hyperdense areas and gas in superoanterior portion. infiltrates spleen, left kidney and adrenal gland, colon and pancreas, with no clear dependence of any of these structures. the patient had gastrointestinal bleeding, thus we did oral endoscopy with biopsy, which evidenced ulceration of the gastric mucosa by extrinsic neoplasia. biopsies were informed as fusiform cell proliferation and mesenchymal neoplastic nature, unable to determine its origin. debating exposure the case at the digestive tumors interdisciplinary committee, it was decided practise an open mass biopsy, after which we had patologycal diagnosis : well differentiated leiomyosarcoma. we decided not to perform percutaneous biopsy because of the risk of spread and the low diagnostic sensitivity of this technique in our hospital. planned surgery is based on the ct image, showing a dissection plane starting laterally to the left side of the spleen, behind the pancreas neck, and including part of the colon with the transverse mesocolon and stomach. during surgery we confirmed this was possible and practiced block tumor removal, including full gastric resection, spleen, pancreas and 20 cm distal transverse colon. we obtained a diagnosis of well - differentiated retroperitoneal leiomyosarcoma that ulcerated stomach wall and infiltrated spleen, pancreas and transverse colon serosa. mitotic index of 15 mitoses per 50 high power fields and surgical margins were free of disease. postoperatively, the patient presented a left retroperitoneal abscess fistulized to preexisting renal cyst, which require percutaneous drainage for its resolution. as adjuvant treatment, the patient started radiotherapy. in subsequent image test, nine months later, there were evident liver lesions, and proceeded to fine needle puncture - aspiration ultrasound guided, confirming that those corresponded to metastasis. thus began chemotherapy treatment with gemcitabine and docetaxel, but lesions have not size reduction after six sessions, so the patient currently continues with palliative chemotherapy and is followed by oncology service. the retroperitoneum is defined anteriorly by the peritoneal extensions anchoring the transverse colon, the small bowel, and the ascending and descending colon. the posterior aspect of this space is a muscular wall comprising the psoas major and minor, the quadratus lumborum, obturator internus, pyriformis muscle and the tendinous portion of the transversus abdominis muscle. the retroperitoneum is bordered superiorly by the diaphragm, inferiorly by the levator ani muscles, and laterally by the ascending and descending colon in combination with the peripheral margin of the quadratus lumborum muscles. retroperitoneal tumors are defined as those that are formed from nerve, vascular, muscular, connective, supportive and fibroareolar tissue, excluding organs and great vessels that are in this space. at this level 85% of tumors the most common subtypes of sarcomas at this level are liposarcoma, with the best prognosis, and leiomyosarcomas, one of the most aggressive. the average size of these tumors at diagnosis is 16 cm, since they do not start giving symptoms until they reach 10 cm in diameter. the retroperitoneum has adaptability, allowing tumor growth without the rest of the body perceiving changes. however, size is not an impediment to total resection neither data for worse survival. tumor growth is centrifugal, being the most immature cells in the outer layer of the tumor, compressing surrounding structures. the symptoms presented by these tumors are nonspecific and varied, depending on the affected organ and its location.. usually also present gastrointestinal disorders, urinary discomfort, lower limb edema, sweating or constitutional symptoms. for the diagnosis, it can help determinate histological tumor type because of the indirect data (necrotic areas, fat or muscle tissue), helps to delineate the tumor and its relationship to other structures, distinguishing between invasion or contact ; staged the tumor, because it shows node and distant metastases, and sometimes provides information of the origin of the tumor. puncture / biopsy is commonly used for diagnosis, but is more reliable if it is guided by image tests, ct, as otherwise can be inconclusive. the use of adjuvant therapy has not demonstrated to increase survival neither provide a better quality of life. it needs a complete microscopic tumor resection r0 for an acceptable survival as locoregional recurrence is the most influential data in the prognosis. recurrences can occur more frequently in incomplete resections (macro or microscopic). for complete tumor resection is necessary removing neighbors organs by more than 50% of the time, the most frequently resected organs are kidney and adrenal gland, but pancreas, spleen, liver, colon, duodenum and cava are also affected on numerous occasions. these resections imply increased morbidity though to mortality, and longer and more complex surgeries. pancreatic manipulation (total sections and especially partial) increases the risk of complications especially postoperatively. resectability tumor can only be confirmed during surgery, and when it is not possible, is usually to do extensive infiltration of the mesenteric root, neurovascular extensive infiltration, distant metastasis or peritoneal sarcomatosis. nodal metastases and tumor size, have not prognostic significance, provided they can be removed in opposite to distant metastases, which are usually caused by hematogenous dissemination. the presence of distant metastasis should not suggest a palliative treatment, if it can be completely removed both the original tumor and metastasis.. a large percentage of patients with r0 resection require rescue surgery (between 45 and 82%). patients with complete resection, which do not require subsequent surgery have an average survival of 5 years. currently, surgery is the only curative option for retroperitoneal sarcomas. before surgery, image tests are needed to delineate the tumor and the affected organs to prepare a good surgical strategy that sometimes require more than one specialist. because the tumor at diagnosis is usually voluminous, wide approaches are preferred, especially if it requires multiple organ resection. aggressive surgical treatment is justified only in patients in which complete tumor resection is feasible. in our patient, the resection was carried out with curative intent to attempt to improve his quality of life severely limited by persistent anemia. chemotherapy and radiotherapy are in most cases a surgical complement although chemotherapy has not demonstrated a significant increase in survival. written informed consent was obtained from the patient for publication of this case report and accompanying images.	highlightsretroperitoneal tumors can have a large size and require complex excisions.surgery with curative intent is not always possible, and we must always evaluate the benefit risk when planning surgery for these tumors.currently only surgery has any chance in curing this disease. radiotherapy and chemotherapy are complementary to radical surgery.retroperitoneal tumors usually present as large tumors but pathological diagnosis can sometimes be difficult.
in its essence, the mammalian neuroendocrine reproductive axis is composed of three main components, which act in a coordinated manner to control the onset of puberty and to subsequently maintain fertility (silverman., 1994 ; ojeda., the pulsatile secretion of gonadotropin - releasing hormone (gnrh) from the hypothalamus stimulates the anterior pituitary gland to release luteinizing hormone (lh) and follicle - stimulating hormone (fsh). in turn, these two gonadotropins then act on the gonads to stimulate maturation of gametes and to synthesize and secrete sex - steroid hormones. for example, although sex steroids contribute to the development and maintenance of fertility and play a role in other physiological and behavioral functions, they also feedback onto the hypothalamo - pituitary unit to modulate gonadotropin release. there is, however, a conundrum regarding the mechanism by which sex steroids do this. on the one hand, the ovarian steroid estradiol usually exerts a negative feedback action on gnrh and gonadotropin release, which is epitomized by the marked increases in circulating lh and fsh levels that occur at menopause or after ovariectomy (i.e., when estradiol levels are markedly attenuated). on the other hand, around the time of ovulation estradiol appears to exert a positive feedback action, causing the production of a gonadotropin surge, which serves as the ovulatory trigger. the traditional explanation for these two radically different effects of estradiol is that gnrh neurons respond differentially to low and high levels of this sex steroid negatively when estradiol levels are low and positively when estradiol levels are high. inherent in this argument, however, is the assumption that the gnrh neurons are relatively homogenous, despite their diffuse distribution pattern, and that individual gnrh neurons have the capacity to show both negative and positive feedback responses. not only have morphologically distinct gnrh neurons been observed in discrete subpopulations, in some species these subpopulations have been shown to express different molecular forms of gnrh. importantly, different gnrh neuronal subpopulations respond differently to estradiol, suggesting that the negative and positive feedback actions are mediated by two distinctly different neuronal populations. this alternative view of the neuroendocrine reproductive axis lends itself to novel targeted approaches to fertility control and treatment of human reproductive disorders. it has generally been assumed that gnrh neurons are all essentially similar in their responses to neurotransmitters. indeed, this is a fundamental assumption that underlies the extensive use of immortalized gnrh neurons for in vitro studies by many researchers, including members of this laboratory (e.g., urbanski., 1996 ; olcese., 2003 ; garyfallou., however, this assumption is questionable because in humans gnrh neurons appear to show three distinct morphological types, based on cell size and gnrh cdna probe labeling density (rance., 1994 ; krajewski., 2003) : (1) small, heavily labeled, oval, or fusiform neurons, located primarily in the medial basal hypothalamus, ventral preoptic area, and periventricular zone ; (2) small, oval, sparsely labeled neurons located in the septum, and dorsal preoptic region and scattered from the bed nucleus of the stria terminalis to the amygdala (extended amygdala) ; and (3) large round neurons (> 500 m two sectional profile area), intermediate in labeling density, scattered within the magnocellular basal forebrain complex, extended amygdala, ventral pallidum, and putamen. the pronounced differences in morphology, labeling density, and location of the three subtypes suggest that distinct functional subgroups of gnrh neurons exist in the human brain. similar morphological subtypes have been observed in the brains of rhesus macaques (urbanski., 1996), and it has been shown that the type 1 and type 3 neurons have characteristically distinct biochemical properties (e.g., they differ in their capacity to express glutamate and estrogen receptors). currently, however, it is unclear whether the type 2 and 3 neuronal subtypes play any physiological role in the control of gonadotropin synthesis. a second basic assumption, and the one that is most relevant to this hypothesis, is that pituitary gland activity is influenced by a single molecular form of gnrh. although multiple molecular forms of gnrh have been identified in non - mammalian vertebrates (sherwood., 1993 ; fernald and white, 1999 ; dubois., 2002 ; roch., 2011), until recently only one form of this decapeptide was thought to exist in mammals. it now appears that at least a few eutherian mammals, including musk - shrews, tree - shrews, and humans express an additional form of gnrh (review : herbison, 2006). chicken gnrh - ii, or simply gnrh - ii, and it shows 70% similarity to mammalian gnrh (i.e., gnrh - i) at the amino acid level (figure 1). the genomic and mrna structures of gnrh - ii resemble those of gnrh - i, although significant differences exist within the gnrh - associated peptide (gap) regions of the respective genes (not shown) ; in addition, in humans the two molecules are encoded on different chromosomes (white., 1998). hplc and immunocytochemical studies have shown that gnrh - ii also exists in non - human primates (lescheid., 1997), and this has been corroborated through the cloning of gnrh - ii cdna from the monkey brain (genbank # 228312 ; urbanski., 1999). importantly, gnrh - ii gene expression has been demonstrated in the hypothalamus of monkeys, and shows a distribution pattern that is distinct from that of gnrh - i (figure 2). gnrh - i expression has a diffuse expression pattern in the hypothalamus, whereas gnrh - ii appears to be concentrated in specific nuclei such as the paraventricular, supraoptic, suprachiasmatic as well as the medial basal hypothalamus. subsequent studies used double histochemical labeling to show that gnrh - i and gnrh - ii are produced by two completely distinct populations of cells (latimer., 2000). a comparison between the amino acid sequences of gnrh - i and gnrh - ii. both decapeptides undergo similar post - translational modification, which includes conversion of gln to pglu at amino acid position 1 and amidation of the gly at position 10. differential distribution of gnrh - i and gnrh - ii mrna in the rhesus macaque hypothalamus, as revealed by in situ hybridization histochemistry. (b) autoradiographs showing gnrh expression in the rostral (upper panels) and caudal (lower panels) hypothalamic sections. son, supraoptic nucleus ; pvn, paraventricular nucleus ; scn, suprachiasmatic nucleus ; mbh, medial basal hypothalamus ; oc, optic chiasm ; ot, optic tract. (adapted from urbanski., 1999, with permission from the endocrine society). although several reviews of the mammalian gnrh system, have attempted to explain the physiological role of gnrh - ii (pawson., 2003 ; terasawa, 2003 ; cheng and leung, 2005 ; herbison, 2006 ; kah., 2007), the physiological function of this most ancient and highly conserved form of gnrh in primates is still unclear. there is some evidence from the musk - shrew and marmoset that gnrh - ii plays a role in coordinating reproductive behavior, although these behavioral effects are thought to be mediated by gnrh - ii neurons located in the central regions of the midbrain rather than in the forebrain (kauffman., 2005 ; barnett., rhesus macaques, however, show a high level of gnrh - ii expression in the hypothalamus as well as in the midbrain (urbanski., 1999 ; latimer., 2001), which raises the interesting possibility that gnrh - ii may contribute to the control of the primate reproductive neuroendocrine axis. indeed, it has already been shown that gnrh - ii is highly effective at stimulating lh and fsh release in rhesus monkeys in vivo, and from pituitary culture in vitro (lescheid., 1997 ; densmore and urbanski, 2003 ; kada., 2003 furthermore, this activation can be blocked by antide, a gnrh receptor-1 (gnrhr-1) specific antagonist (figure 3). taken together this suggests that both gnrh - i and gnrh - ii act through the same receptor to stimulate gonadotropin release. note that a second receptor for gnrh (gnrhr-2) has been cloned in monkeys and humans, but it probably does not have a specific role in controlling the reproductive neuroendocrine axis of primates ; in monkeys the gnrhr-2 has a ubiquitous distribution pattern, while in humans there is a stop codon in the middle of its gene sequence which precludes its translation into a functional protein (cheng and leung, 2005 ; herbison, 2006). gnrh - i or gnrh - ii (both at 1 g / kg body weight) were administered via an indwelling vascular catheter, and the effect of gnrh receptor-1 antagonist was determined by simultaneous administration of antide (100 g / kg body weight). the data demonstrate that (a) gnrh - i and (b) gnrh - ii are both potent stimulators of lh release and that they both act through the same gnrh receptor (i.e., gnrh receptor-1). (adapted from densmore and urbanski, 2003, with permission from the endocrine society). although gnrh - i and gnrh - ii can both stimulate gonadotropin release in the rhesus macaque (figure 3), the neurons that produce them show marked differences in their responsiveness to estradiol. firstly, the gnrh - ii gene promoter contains estrogen response elements and the gnrh - ii neurons express estradiol receptors (er), whereas gnrh - i neurons do not (sullivan., 1995 ; furthermore, semi - quantitative in situ hybridization histochemistry (densmore and urbanski, 2004) has shown that gnrh - ii gene expression increases in the monkey medial basal hypothalamus after exposure to estradiol, whereas gnrh - i gene expression decreases (figure 4). this observation is consistent with the central hypothesis that different gnrh neuronal subpopulations respond differentially to estradiol. importantly, the result suggests that gnrh - ii neurons are the primary mediators of positive estradiol feedback, whereas the gnrh - i neurons are the primary mediators of negative estradiol feedback. it is also consistent with the finding from other primate studies, showing that hypothalamic gnrh - i gene expression is elevated when estradiol levels are very low, such as after menopause or after ovariectomy (rance and uswandi, 1996 ; abel., 1999). additional support for the hypothesis comes from a microarray gene profiling study that examined hypothalamic gnrh - i and gnrh - ii gene expression across the monkey menstrual cycle (urbanski., 2009, 2010a). this study focused on three stages of the menstrual cycle that show three distinct sex - steroid profiles (figure 5). during the early follicular (ef) phase, both estradiol and progesterone concentrations are low, whereas in the late follicular (lf) phase estradiol is highly elevated ; during the mid - luteal (ml) phase, estradiol is moderately elevated and progesterone highly elevated (downs and urbanski, 2006). despite differences in circulating sex - steroid concentrations during these three phases of the cycle, gnrh - i gene expression showed no significant change (figure 5a) ; in marked contrast, gnrh - ii gene expression showed a marked increase during the lf phase, in association with the elevated estradiol levels. the positive relationship between estradiol and gnrh - ii gene expression, and the close temporal relationship between elevated gnrh - ii gene expression and the preovulatory lh surge, suggests that the gnrh - ii neurons play a dominant causal role in the preovulatory lh surge (figure 5b). further supportive evidence for this hypothesis comes from a recent study in which estradiol benzoate (eb ; 42 g / kg, s.c.) was administered to ovariectomized rhesus macaques (urbanski., 2010b). in this well - established experimental model plasma estradiol levels reached a peak within 4 h of eb injection, and this was associated with an expected suppression of plasma lh levels, followed by a surge approximately 2 days later. real - time - pcr showed that initial suppression of lh to be associated with a decrease in gnrh - i gene expression, which is consistent with estradiol exerting a negative influence on gnrh - i neurons. in marked contrast, gnrh - ii gene expression increased following the estradiol peak, and reached a maximum just before the plasma lh surge, which is consistent with estradiol exerting a positive influence on gnrh - ii neurons. differential regulation of gnrh - i and gnrh - ii gene expression by estradiol in ovariectomized rhesus macaques. (a) histogram depicting number of cells expressing gnrh - i mrna in the hypothalamus of ovariectomized (ovx) and ovariectomized / estradiol - treated (ovx + e) animals, as revealed by semi - quantitative in situ hybridization histochemistry. the number of detectable gnrh - i cells in the hypothalamus was markedly lower in the ovx + e animals than in the ovx animals. (b) histogram depicting number of cells expressing gnrh - ii mrna in the mbh of ovx and ovx + e animals, as revealed by in situ hybridization. the number of detectable gnrh - ii cells in the mbh was significantly greater in the ovx + e animals than in the ovx animals. the in situ hybridization was performed on a series of six coronal hypothalamic sections from each animal (n = 3/group) ; the sections were collected at 200-m intervals. p < 0.01, p < 0.05. (adapted from densmore and urbanski, 2004, with permission from the society for endocrinology). (a) mean expression of gnrh - i (upper panel) and gnrh - ii (lower panel) mrna levels in the rhesus monkey hypothalamus, as determined by affymetrix genechip microarray analysis (hu133a plus 2.0 ; n = 34/group). the following three characteristic stages of the menstrual cycle were examined : ef, early follicular ; lf, late follicular, ml, mid - luteal. the level of gnrh - i gene expression was similar across the menstrual cycle, whereas the level of gnrh - ii gene expression showed a significant mid - cycle increase (p < 0.05). (b) serum reproductive hormone profiles from a representative female rhesus macaque across a complete menstrual cycle. note the temporal relationship between the mid - cycle preovulatory lh surge and the elevated serum estradiol levels of the lf phase. together the data suggest that estradiol - mediated activation of gnrh - ii neurons may play a key role in triggering ovulation, whereas gnrh - i neurons are more likely to play a role in modulating tonic lh release and in follicular maturation. based on these recent findings, mainly from rhesus macaques, it is hypothesized that gnrh - ii neurons play a major role in the generation of the preovulatory lh surge in female primates, whereas gnrh - i neurons mediate the negative feedback influence of estradiol on tonic gonadotropin release (figure 6). it should be emphasized that this hypothesis does not invoke a special role for the gnrh - ii molecule, other than suggesting that gnrh - ii expression may be used to identify the estrogen - responsive neurons that are activated at the time of the lh surge. the hypothesis simply proposes that the menstrual cycle is orchestrated by the coordinated action of two separate subpopulations of gnrh neurons one subpopulation responds to estradiol in a negative manner and is involved in stimulating the ovary during the follicular phase of the cycle, while the other subpopulation responds to estradiol in a positive manner and hyper - stimulates the pituitary gland to produce a mid - cycle lh surge. a summary of the hypothesized mechanism by which estradiol (e2) modulates luteinizing hormone (lh) release in female primates. stylized representations of three distinct temporal patterns of lh release are depicted in the upper panels, while corresponding plasma e2 levels and gnrh neuronal activity are represented in the middle and lower panels, respectively. (a) low amplitude pulse of lh are typically observed during the follicular phase of the menstrual cycle ; this is associated with low - to - medium plasma e2 levels, which exert a moderate degree of negative feedback onto gnrh - i neurons (represented by the dashed green line) but cause little stimulation of gnrh - ii neurons (represented by the solid orange line). (b) an lh surge typically occurs in the middle of the menstrual cycle ; this is associated with high plasma e2 levels, which inhibit gnrh - i neuronal activity but markedly stimulate synthesis and release of gnrh - ii. (c) high amplitude pulses of lh are typically observed after ovariectomy and after menopause, as a result of the highly attenuated plasma e2 levels ; in the absence of significant e2 negative feedback, gnrh - i neurons show increased activity whereas gnrh - ii neurons lack positive feedback and so revert to a relatively quiescent state. although it is commonly assumed that the same gnrh neurons can mediate both negative and positive e2 feedback onto lh release, the data presented in this mini - review question this assumption. instead, it is hypothesized that tonic and surge modes of lh release are orchestrated by two distinct gnrh neuronal populations, which in primates can be distinguished by their capacity to produce different molecular forms of gnrh (i.e., gnrh - i and gnrh - ii, respectively) and to respond differentially to e2. in rodents it may be more difficult to distinguish between these two gnrh neuronal subpopulations because only one molecular form of gnrh (i.e., gnrh - i) has been equivocally shown to exist. despite attempts by several laboratories, gnrh - ii has still not been cloned in mice or rats, which is not surprising given that a blast search of the genome of these rodents reveals only one molecular form of gnrh (i.e., the traditional gnrh - i). nevertheless, there is evidence from female rodents that activation of a specific subpopulation of gnrh - i neurons is associated with the preovulatory lh surge (hiatt., 1992 ; porkka - heiskanen., 1994 ; rubin and king, 1994). therefore, it is plausible that the essence of the current hypothesis also applies to non - primate mammalian species. for many years there was much debate as to whether a gnrh surge even existed in primates, because the primate pituitary gland appeared capable of producing an lh surge in response to tonic pulsatile gnrh stimulation (knobil., 1980). although there is now good evidence to suggest that an estradiol - induced gnrh surge does occur in primates, as in rodents, the key studies (xia., 1992 ; pau., 1993) were performed before the existence of gnrh - ii was known in primates, and unfortunately relied on hormone immuno - assays that did not clearly distinguish between gnrh - i and the closely related gnrh - ii molecule. consequently, it is unclear if the assays measured gnrh - i exclusively or gnrh - i plus gnrh - ii. more recently, however, c - fos immunohistochemistry has been used to examine activation of gnrh - i neurons around the time of the primate preovulatory surge. in marked contrast to rodents (lee., 1990 ; hoffman., 1993 ; doan and urbanski, 1994), however, there was no obvious increase in the number of c - fos - expressing gnrh - i neurons at the time of the lh surge in primates (witkin. this negative finding gives further credence to the hypothesis that gnrh - i neurons do not play a dominant role in stimulating the preovulatory lh surge in primates, and implies that this role is more likely to be mediated by gnrh - ii neurons instead. like rhesus macaques, humans express two molecular forms of gnrh (i.e., gnrh - i and gnrh - ii). given that gnrh - i and gnrh - ii are produced by different neuronal populations, it is plausible that fertility in women is controlled by the coordinated action of two distinct gnrh neuronal subpopulations, rather than by a single homogenous population. if correct, this hypothesis has several implications, especially in primates where the distinct biochemical signatures of the two subpopulations lend themselves to targeted therapeutic interventions. for example, specific activation of the gnrh - ii neurons could help to treat amenorrhea and improve fertility. conversely, by specific silencing of the gnrh - ii neurons it may be possible to selectively block ovulation while retaining normal tonic lh secretion, a strategy that could open up novel approaches to contraception without negatively impacting ovarian steroidogenesis. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	in vertebrates, gonadotropin - releasing hormone (gnrh) represents the primary neuroendocrine link between the brain and the reproductive axis, and in some species up to three different forms of gnrh have been detected. until recently, it had been assumed that humans and non - human primates only express one form (gnrh - i), but it is now clear they also express a second form (gnrh - ii). gnrh - ii, like gnrh - i, is highly effective at stimulating gonadotropin release, both in vitro and in vivo, but the neurons that produce gnrh - ii are completely distinct from those producing gnrh - i. moreover, gnrh - ii and gnrh - i producing neurons respond very differently to estradiol ; specifically, estradiol stimulates gnrh - ii gene expression in the former and inhibit gnrh - i gene expression in the latter. consequently, the negative feedback action of estradiol may be mediated exclusively by the subpopulation of gnrh neurons that express gnrh - i, while the positive feedback action may be mediated exclusively by the subpopulation that expresses gnrh - ii. taken together, these findings raise the possibility that two completely different gnrh neuronal systems participate in the control of primate reproductive physiology. the primary role of gnrh - i neurons is likely to be focused on the maintenance and modulation of tonic pulsatile lh release, whereas the primary role of gnrh - ii neurons is likely to be focused on the generation of the preovulatory lh surge. this functional segregation of the primate neuroendocrine reproductive axis lends itself for novel targeted approaches to fertility control and for treatment of human reproductive disorders.
segmental neurofibromatosis (sn) is a rare variant of a common autosomal dominant neurocutaneous disorder wherein a postzygotic mutation in the nf1 gene is thought to cause lesions distributed in one area of the body. unilateral, bilateral, and late - onset cutaneous segmental neurofibromas have been described, as well as isolated deep - seated plexiform neurofibromas. there is also one case of multiple recurring schwannomas in deep and superficial locations, yet there have been no reported cases with deep schwannoma and clinically apparent multisegmental neurofibromas ; increased knowledge and awareness of this phenotype is important. a 70-year - old woman presented to dermatology outpatient clinic for evaluation of two clusters of small, fleshy tumors that appeared over the previous three years. twenty - five years prior, she had undergone a surgery for a deep nerve - based tumor lateral to her left knee. she had no other family members with similar lesions or a personal history of malignancy. on examination, several skin - colored to violaceous, fleshy, pedunculated papules and nodules were clustered over her left anterior shoulder and lateral left leg [figure 1 ]. biopsy confirmed that the cutaneous lesions were neurofibromas (figure 2, showing non - encapsulated peripheral nerve elements and schwann cells with wire - like collagen fibrils). given the clustered distribution of lesions and lack of other stereotypic findings of neurofibromatosis (caf - au - lait macules, musculoskeletal deformities, axillary freckling, lisch nodules of the iris), the patient was diagnosed with sn. cluster of fleshy, pedunculated papules on (a) left lateral leg and (b) left anterior shoulder (a and b) h and e staining of tissue consistent with neurofibroma (description of histopath image required) (a : 4 and b : 10) sn, classified by riccardi as neurofibromatosis type v (nfv), was traditionally described as caf - au - lait spots, freckling, and neurofibromas restricted to a discrete area. however, roth. realized that several patients with apparent sn did not meet these criteria and developed four subsets of nfv to better stratify and classify patients : true sn according to riccardi are localized deep neurofibromas only, hereditary segmental, and bilateral segmental. recently, hardin. reported that sn should not be regarded as a distinct entity from neurofibromatosis i. they believe cases previously referred to as unilateral or bilateral sn are now best referred to as mosaic generalized or mosaic localized neurofibromatosis. they found 55% had pigmentary changes only, 8.6% had neurofibromas only, 22.4% had neurofibromas and pigmentary changes, and 13.8% had plexiform neurofibromas only. the lesions often followed a dermatomal distribution, with the cervical areas being affected most commonly. sn has been reported to be associated with malignant melanoma, breast, colon, gastric, and lung cancer, affecting 5.3% of patients. as the current prevalence of cancer in the general population is lower, it seems that patients with sn are at an increased risk for certain malignancies. most patients were diagnosed with cancers after their diagnosis of neurofibromatosis ; therefore, a prudent review of systems and general examination for any neoplastic process could be of benefit in these patients. no report of late - onset multi - sn in two different dermatomes with associated deep schwannoma has been reported ; this appears to be a new nf phenotype. given the association of neurofibromatosis and plexiform lesions with various comorbidities, it is important to study the natural history of the disease to better counsel patients on risks, prevention, and family planning	an elderly patient presented with two clusters of asymptomatic fleshy and pedunculated papules. biopsy of the papules was consistent with neurofibromas. decades prior she had undergone a surgery for the excision of a large schwannoma. given her lack of other neurofibromatosis findings, the patient was diagnosed with multisegmental neurofibromatosis (multi - sn) with deep schwannoma, a possible new phenotype of sn. because this entity may be associated with internal malignancy, it is important to screen and educate these patients as well as to provide regular follow - up.
this protein is crucial for inhibiting bone formation, by decreasing proliferation and differentiation of osteoblasts, reducing apoptosis of mature osteoblasts, thus leading to higher bone formation and increased bone density. sclerostin levels are inhibited by mechanical loading and parathormone (pth) [1, 3 ] and stimulated by glucocorticoids and calcitriol. other determinants of sclerostin concentrations include bmi, sex, age, and several hormones, such as follicle stimulating hormone (fsh) and estradiol (e2) [3, 4 ]. sclerostin deficiency has been implicated in the pathogenesis of rare bone dysplasias, that is, sclerosteosis and van buchem disease, where mechanical strength of bone is extremely large. monoclonal antibodies to sclerostin, including romosozumab, blosozumab, and bps804, were demonstrated to increase bone mineral density and to stimulate bone formation together with reducing bone resorption. we have previously shown a decrease of sclerostin levels during successful treatment of hyperthyroidism in a preliminary study, involving 15 patients. in order to further elucidate the mechanism of that phenomenon we measured pth, calcium, and markers of bone metabolism in blood samples taken from the patients before and during treatment of hyperthyroidism. the study involved 33 patients (7 men), age 48 15 years, bmi 24.2 3.5 kg / m, with hyperthyroidism due to graves ' disease (n = 15) or toxic multinodular goitre hospitalized in the department of endocrinology and metabolic diseases. all patients received a thyrostatic drug, thiamazole, and beta - blocker, propranolol. serum sclerostin was measured by a quantitative sandwich elisa by biomedica (vienna, austria) at diagnosis of hyperthyroidism and after 610 weeks of treatment with thiamazole. thyroid - stimulating hormone (tsh), free t3 (ft3), free t4 (ft4), pth, osteocalcin (oc), marker of bone formation, and collagen type i cross - linked c - telopeptide i (ctx), marker of bone resorption, were determined by commercially available electrochemiluminescence immunoassays (eclia cobas e601, roche). a comparison of distributions of selected parameters before and during treatment was assessed by wilcoxon 's matched pairs test. analysis of relationship between sclerostin and demographic data (bmi and age) was done assessed by method of multiple correlation. in all analyses, after treatment of hyperthyroidism, a significant decrease in ft3 from 10.2 5.4 pg / ml to 3.2 1.0 pg / ml (p < 0.000001) and ft4 concentrations from 4.03 2.33 ng / ml to 1.10 0.82 ng / ml, respectively (p < 0.006), was accompanied by a marked decrease of serum sclerostin levels from 43.7 29.2 to 28.1 18.4 pmol / l (p = 0.000001). there was a simultaneous decrease of oc from 35.6 22.0 to 27.0 14.3 ng / ml, p = 0.00004, and ctx from 0.49 0.35 to 0.35 0.23 ng / dl, p = 0.0016. in contrast, pth concentrations increased from 29.3 14.9 pg / ml to 39.8 19.8 pg / ml, p = 0.0005 (table 2). during thyrotoxic phase sclerostin correlated positively with ctx (rs = 0.41, p < 0.05) (figure 1). sclerostin concentrations did not correlate with pth levels (r = 0.18, p = ns). in order to assess whether individuals with maximal changes in sclerostin also had maximal changes in pth concentrations, we analysed individual changes of sclerostin concentrations before and after treatment of thyrotoxicosis (i.e., sclerostin) and correlated these data with individual changes of pth levels before and after treatment of thyrotoxicosis (i.e., pth). here again we did not observe any significant correlation between sclerostin and pth (r = 0.10, p = ns). before treatment oc correlated positively with thyroid hormones, both ft4 (rs = 0.45, p < 0.05) and ft3 (rs = 0.42, p < 0.05), but after treatment we observed negative correlation between oc and ft3 (rs = 0.43, p < 0.05) (figure 2). moreover, before therapy we demonstrated a strong positive correlation between oc and ctx (rs = 0.76, p < 0.01) (figure 3) and negative correlation with age (rs = 0.43, p < 0.05). tables 3 and 4 we demonstrated that restoration of an euthyroid state after treatment of thyrotoxicosis is associated with a significant decrease of sclerostin, osteocalcin, and ctx serum concentrations coinciding with an increase of pth concentrations. the influence of thyroid hormones on bone metabolism is multifaceted and depends on age. during growth thyroid hormones have predominantly anabolic actions in bone, while in adulthood they predominantly exert catabolic effects on adult skeleton [7, 8 ]. hyperthyroidism accelerates bone metabolism, leading to osteoporosis or osteopenia [79 ], affecting more bone resorption than formation. population studies indicate that hyperthyroidism is associated with an increased risk of fractures. in some studies, even low - normal tsh values were associated with a high prevalence of vertebral fractures in women with postmenopausal osteoporosis or osteopenia, independently of thyroid hormones, age, and bmd. in our study mean pretreatment sclerostin levels were even higher than reference values presented by biomedica (43.7 29.2 pmol / l versus 19.3 pmol / l, range from 10.9 to 28.7 our results are, however, convergent with recently published study by tsourdi., performed on an animal model. in that research in hyperthyroid mice high sclerostin levels were observed, even when adjusted for bone mass. moreover, sclerostin mrna expression and the number of sclerostin - positive osteocytes were increased. it can not be ruled out that sclerostin is a mediator of thyroid hormone action on bone. we hypothesized that decrease in sclerostin levels in patients recovering from hyperthyroidism might be mediated by pth. in our study pth levels during hyperthyroidism, that is, state associated with high ft3, were significantly lower than after treatment. decreased pth concentrations in subjects with hyperthyroidism were also reported [15, 16 ], followed by an increase during treatment of hyperthyroidism. there are many sets of data about inhibition of sclerostin expression by pth both in vitro and in vivo. activation of pth receptor 1 expressed in osteocytes resulted in an increased bone remodelling with decreased osteoblast apoptosis and suppression of sost expression. in vivo were also shown in patients with high pth concentration caused by primary hyperparathyroidism ; on the contrary in hypoparathyroidism sclerostin levels were high. inhibition of sclerostin by pth is postulated to represent one of the mechanisms responsible for anabolic effect of pth. nevertheless, it should be mentioned that in our study we failed to observe any direct correlation between sclerostin concentrations and pth as well as between actual changes in sclerostin concentrations (i.e., sclerostin) versus pth. therefore, an issue of precise mechanisms that mediate changes in sclerostin concentrations during treatment of thyrotoxicosis requires further study. analyzing other reasons for sclerostin decrease during treatment of hyperthyroidism one must also consider influence of bone markers. in our study there were elevated concentrations of bone markers (oc and ctx) before treatment while they decreased after normalization of thyroid function. high concentrations of bone markers, during hyperthyroidism, and their improvement after therapy had been demonstrated before [9, 16 ]. in our study, before starting therapy oc levels correlated with both ft3 and ft4, but after therapy we observed a conversion of that relationship to negative one. this might suggest that the above mentioned relationship might be more pertinent to pathological condition, that is, thyrotoxic state, rather than to euthyroidism. the positive correlation between sclerostin concentrations and ctx is an argument in support of hypothesis about relationship between elevated levels of sclerostin and increased bone metabolism. similar relationship between sclerostin and ctx was shown in premenopausal but not in postmenopausal women, in whom a negative correlation was found [22, 23 ]. however, we did not observe any direct relationship between sclerostin and concentrations of free thyroid hormones or tsh. another factor, potentially responsible for a decrease in sclerostin levels, might be related to an increase in physical activity during treatment of hyperthyroidism. there is evidence that mechanical loading is responsible for stimulation of bone formation, due to inhibition of sclerostin expression. did not find any change of sclerostin levels after one year of exercise, despite an increase of thigh muscle volume. we note, however, that no formal assessment of physical activity was performed in our study, while duration of treatment was relatively short (610 weeks), so a marked change in physical activity was unlikely to occur over such a short time span. moreover, we can not exclude the influence of drugs used during therapy of hyperthyroidism on studied processes. all patients received a thyrostatic drug, thiamazole, and beta - blocker, propranolol. in literature there are no data on separate effect of such therapy on sclerostin or pth levels ; however in a follow - up study the reduction of fracture risk was observed in subjects that had received both radioactive iodine and thiamazole. in turn, the data on the influence of beta - blockers, and propranolol in particular, on fracture risk are inconclusive, both in terms of risk reduction and its increase. summing up, is still unclear and requires further study. limitations of our study included small number of treated patients and short time of observation. nevertheless, all patients achieved biochemical euthyroidism, so these data are in our opinion suitable for a preliminary study, given that changes of sclerostin during treatment of an acute phase of thyrotoxicosis had not been described before. we speculate that, in the context of high population prevalence of hyperthyroidism and its recurrent nature, our results might be clinically relevant in terms of potential benefits of prevention and treatment hyperthyroidism - induced osteoporosis with antibodies to sclerostin.	sclerostin, a protein expressed by osteocytes, is a negative regulator of bone formation. the aim of the study was to investigate the relationship between parathyroid hormone (pth) and markers of bone metabolism and changes of sclerostin concentrations before and after treatment of hyperthyroidism. patients and methods. the study involved 33 patients (26 women), age (mean sd) 48 15 years, with hyperthyroidism. serum sclerostin, pth, calcium, and bone markers [osteocalcin (oc) and collagen type i cross - linked c - telopeptide i (ctx) ] were measured at diagnosis of hyperthyroidism and after treatment with thiamazole. results. after treatment of hyperthyroidism a significant decrease in free t3 (ft3) and free t4 (ft4) concentrations was accompanied by marked decrease of serum sclerostin (from 43.7 29.3 to 28.1 18.4 pmol / l ; p < 0.001), oc (from 35.6 22.0 to 27.0 14.3 ng / ml ; p < 0.001), and ctx (from 0.49 0.35 to 0.35 0.23 ng / dl ; p < 0.005), accompanied by an increase of pth (from 29.3 14.9 to 39.8 19.8 ; p < 0.001). during hyperthyroidism there was a positive correlation between sclerostin and ctx (rs = 0.41, p < 0.05) and between oc and thyroid hormones (with ft3 rs = 0.42, with ft4 rs = 0.45, p < 0.05). conclusions. successful treatment of hyperthyroidism results in a significant decrease in serum sclerostin and bone markers concentrations, accompanied by an increase of pth.
cystic pancreatic lesions do not only have diverse histologic and imaging characteristics but also differ in clinical presentation and malignant potential. the prevalence and size of the cystic lesions increase with age, and about 60% of cystic pancreatic neoplasms are malignant in individuals older than 70 years. generally, surgery is indicated for mucinous cystic neoplasms, intraductal papillary mucinous neoplasms, and solid pseudopapillary neoplasms that have a malignant potential. since the risk - to - benefit ratio might be high in patients at a high risk for surgical resection, accurate preoperative risk stratification and decisions on operation versus observation require a precise characterization and diagnosis of the cystic lesion. however, different cystic pathologies may have overlapping presentations ; the sensitivity and specificity of imaging studies or cyst aspiration are insufficient to accurately differentiate among benign, premalignant, and malignant pancreatic cystic lesions. therefore, the discovery of tumor markers to differentiate benign, premalignant, and malignant cystic lesions would facilitate the diagnosis and decision - making process. an 87-year - old female presented with fever, abdominal discomfort, poor oral intake, vomiting, and generalized weakness lasting for 2 days in 2011. she was found to have paroxysmal supraventricular tachycardia with a heart rate ranging from 110 to 240 beats per minute and was hypotensive. her past medical history was significant for hypertension, hyperlipidemia, diabetes mellitus, coronary artery disease, congestive heart failure, multiple lacunar stroke, hypothyroidism, and a pancreatic mass that had been detected 4 years ago and left without further investigation because the patient had refused treatment. abdominal ct demonstrated a multicystic mass in the body and tail of the pancreas as well as a mesenteric venous thrombus. the cystic mass increased in size as shown on the ct scan, with the largest cyst growing from 2.5 cm in 2011 to 3.8 cm in 2013 (fig. 1b2, c2). the ct scan did not show any significant focal abnormality demonstrated in the abdominal and pelvic organs, except for the atrophic changes in the uterus and kidneys as well as a calculus in the left renal pelvis. the laboratory test showed an elevation in ca 19 - 9 in 2006 and erythropoietin (epo) in 2014 (table 1). cystic lesions of the pancreas present with diverse histological and imaging features, clinical presentations, and malignant potentials. pancreatic cysts may be broadly classified into nonneoplastic, cystic neoplasms, and necrotic degeneration of solid tumors. cystic neoplasms can be further categorized into serous cystadenoma (sca), mucinous cystic neoplasms, intraductal papillary mucinous neoplasms, and solid pseudopapillary neoplasms. we report a case of a slow - growing complex multicystic neoplasm that extensively occupied the body and tail of the pancreas and, more interestingly, was associated with an elevated epo level. in order to accurately categorize pancreatic cysts, information from a variety of sources, including history, radiography, and laboratory tests, is needed [5, 6 ]. as described above, the multicysts (> 6), lobulated outlines, and central calcification (fig. furthermore, the existence of a large cyst (> 2 cm) suggests a macrocystic variant of sca, which is very uncommon. although the malignant potential of sca is traditionally regarded as low, few cases of malignant sca have been reported. therefore, the potential for malignant growth of sca does exist [7, 8 ]. in addition, the less dramatic elevation in ca 19 - 9 in 2006 may indicate the development of a small pancreatic malignancy, although ca 19 - 9 may also increase in other benign or malignant conditions. furthermore, the progressive decrease in the lipase levels indicates a permanent loss of lipase - producing cells in the pancreas likely secondary to the expansion of the neoplasm (table 1). ectopic epo is produced in various neoplasms arising in the kidney, liver, and cerebellum, which are physiological sites of epo production. those neoplasms include renal cell carcinomas, nephroblastomas, liver cell carcinomas, cerebellar hemangioblastomas, uterine fibroids, pheochromocytomas, and ovarian and hepatic cancers. epo was reported to exert antiapoptotic, anti - inflammatory, proliferative, and angiogenic effects on islet cells. these features of epo and its receptors may contribute to the neoplastic growth of pancreatic tissue as well as the invasiveness of pancreatic tumors. in the absence of other possible resources of epo in this case, it is reasonable to presume that the elevated epo was due to the enlarging pancreatic neoplasm, especially the malignant transformation. the relationship between epo and pancreatic neoplasm is unclear, and further investigation is needed to clarify its function in the development and growth and its possible role as a biomarker of the malignant transformation of pancreatic neoplasms. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	cystic lesions of the pancreas are more frequently recognized due to the widespread use of improved imaging techniques. there are a variety of pancreatic cystic lesions with different clinical presentations and malignant potentials, and their management depends on the type of the cysts. although the early recognition of a cystic neoplasm with malignant potential provides an opportunity of early surgical treatment, the precise diagnosis of the cystic neoplasm can be a challenge, largely due to the lack of reliable biomarkers of malignant transformation. we report a case of a large, multicystic neoplasm within the body and tail of the pancreas complicated by elevated erythropoietin, which is likely related to the malignant transformation of the pancreatic neoplasm.
kctc-0041bp, have important clinical application in the treatment of both gram - positive and gram - negative bacterial infections (kim, 1996). the structure of the compound was speculated as to be hydrogenated analogue of chalcomycin antibiotic at c9-c10 double bonds (asolkar, 2002). similar to chalcomycin, the 8(s)-oh group and the 2,3-trans double bond presenting in geri compound may have overall potency for the contribution to the bioactivity of these compounds (woo, 1996). dihydrochalcomycin is integrated by macrocyclic lactone to which two deoxygar residues attached by the o - glycosydic linkages. the neutraral chalcose substituted at c5 of macrolactone ring and it is thought that 2'-oh group plays important role in the contribution to the binding of macrolide to domain v of bacterial ribosome (poehlsgaard, 2003). finally, the presence of sugar micynose substituted at c-14 of macrolide core and it was demonstrated that the micinose moiety makes contact with domain ii of the ribosome and contributes to enhanced binding of the macrolide (hansen, 2002). the gert1 is responsible to attachment of unusual sugar d - allose to the macrolactone at c-20 and requires a primary hydroxyl group at c20 of the macrolactone with the activity of a p450 enzyme and gert2 is responsible for the attachment of l - micynose moiety to the macrolactone core at c-5 position (jaishi, 2006). to better understand the biosynthesis pathway of dihydrochalcomycin and its applications, we have shown here the sequence analysis of two glycosyltransferase by using blast program (http://www.ncbi.nlm.nih.gov/blast/). geri-155) (kim at el, 1996) was used as host strain for sources of dna and for isolation of products. the isp2 media is to use as seed culture media, while r2ye and rich protein source media containing glucose 2%, soluble start 1%, meat extract 0.1%, yeast extract 0.4%, soybean meal 2.5%, nacl 0.2% and k2hpo4 0.005% (kim, 1996) was used for production media. e coli xl1-blue (mrf) (stratagene, usa) was used as a host cell for the preparation of the recombinant plasmids and dna manipulation. e. coli was grown at 37c in luria - bertani (lb) broth, or on an agar plate supplemented with the appropriate amount of antibiotics whenever necessary for the selection or maintenance of the recombinant plasmids (sambrook, 2001). well grown seed culture of s. sp kctc 0041bp was transferred in to production media and inoculate in the 2.5 liter jar fermentor which was operated for 74 days at 28oc (ph 7.4 and 250 rpm) with supplying oxygen (2.5 liters / min). the culture broth was extracted twice with ethylacetate and evaporated to dryness until remaining oil residue and finally result in methanol for further analysis. the crude extract was subjected to silica gel column chromatography using chcl3/ch3oh gradient from 2% to 10% methanol. the fraction eluted at 10% methanol was further applied to ptlc to provide geri compound and its derivatives. the solvent system for ptlc is chloroform and acetone (0.7:0.3) bioassay was carried out using paper disk method and agar overlay for testing the antibacterial of the bioactive compound. geri-155) (kim at el, 1996) was used as host strain for sources of dna and for isolation of products. the isp2 media is to use as seed culture media, while r2ye and rich protein source media containing glucose 2%, soluble start 1%, meat extract 0.1%, yeast extract 0.4%, soybean meal 2.5%, nacl 0.2% and k2hpo4 0.005% (kim, 1996) was used for production media. e coli xl1-blue (mrf) (stratagene, usa) was used as a host cell for the preparation of the recombinant plasmids and dna manipulation. e. coli was grown at 37c in luria - bertani (lb) broth, or on an agar plate supplemented with the appropriate amount of antibiotics whenever necessary for the selection or maintenance of the recombinant plasmids (sambrook, 2001). well grown seed culture of s. sp kctc 0041bp was transferred in to production media and inoculate in the 2.5 liter jar fermentor which was operated for 74 days at 28oc (ph 7.4 and 250 rpm) with supplying oxygen (2.5 liters / min). the culture broth was extracted twice with ethylacetate and evaporated to dryness until remaining oil residue and finally result in methanol for further analysis. the crude extract was subjected to silica gel column chromatography using chcl3/ch3oh gradient from 2% to 10% methanol. the fraction eluted at 10% methanol was further applied to ptlc to provide geri compound and its derivatives. the solvent system for ptlc is chloroform and acetone (0.7:0.3) bioassay was carried out using paper disk method and agar overlay for testing the antibacterial of the bioactive compound. open reading frames gert1 (1257 bp) and gert2 (1278 bp) are located within the dihydrochalcomycin biosynthesis gene cluster (figure 1a), the whole 75.5 kb region encoding the genes for dihydrochalcomycin biosynthesis has been deposited in the genbank with the accession number ay118081. gert1 is flanked downstream by gerr and upstream by germ3 and gert2 is flanked downstream by gerk2 and upstream by gery. using the clustal x program, the determined sequence analysis of the dihydrochalcomycin gene cluster revealed a deduced amino acid sequence (419 amino acids) encoded for allosyltransferase gert1, which displays a very high degree of similarity to a number of the known glycosyltransferase genes in the genebank database (figure 1a), including chmn from streptomyces bikiniensis (95% identity) (genebank accession no ay509120) (ward., 2004), tyln from streptomyces fradiae (66% identity) (genebank accession no af055922) (fouces., 1999 ; vanessa., 1998), mycd from micromonospora griseorubida (63% identity) (genebank accession no ab089954) (anzai., 2003), and orf 11 streptomyces neyagawaensis (47% identity) (genebank accession no dq149987). similarly, the deduced amino acid sequence (426 amino acid) encodes for the protein named as chalcosyltransferase gert2. this protein also displays similarities to glycosyltransferase from different sources, including chmciii from streptomyces bikiniensis (93 % identity) (genebank accession no ay509120) (ward., 2004), tylmii from streptomyces fradiae (61% identity) (genebank accession no x81885) (charles., 2004), desvii from streptomyces venezuelae (56% identity) (genebank accession no af079762) (xue., 2001), nbmd from streptomyces narbonensis (55% identity) (genebank accession no af521878). in addition, analysis of amino acid sequences of gert1, gert2 and several gtrs involved in the biosynthesis of poliketides showed a very well conserved domain including histidine residue rich region which depends on the gtf (figure 1a and figure 1b). it is suggested that the histidine amino acid present at this conserved region could play important role in the catalytic activity of the enzyme and histidine residues have been showed to be important active site of substrate binding and transition state stabilization in some oligosaccharide - independent gtfs (quiros, 2000a, b). three fractions containing active compounds were successively obtained from elution with 2% to 10% methanol as reported (kim, 1996). the third fraction was further purified by ptlc to afford dihydrochalcomycin along with its derivative. esi - mass analysis showed the peak at m / z=725 [m+na+ ] corresponding to the molecular weight of 702 da as the molecular weight of geri (figure 2) and the compound shows rf value at 0.3. in addition, the fraction with rf value at 0.2 containing unknown compound was isolated with m / z = 755 [m+na+ ] corresponding to the molecular weight 732 da as new compound that is not reported previous paper (figure 3). the compound was carried bioassay test and it also has same antibacterial activity as dihydrochalcomycin (data has not shown). from this results we found that the novel compound isolated has molecular weight equal with the dihydrochalcomycin derivative (rf = 0.2 and mw = 732) in which the macrolactone was attached by two mycinose sugar moieties. this can be suggested that gert2 may act as a flexible glycosyltransferase to different glycone. hence, it can accept either dtdp - allose or dtdp - chalcose as glycone donors during macrolactone glycosylation steps. for confirmation of this hypothesis, it is necessary to study more and carry out further analysis about gert2. open reading frames gert1 (1257 bp) and gert2 (1278 bp) are located within the dihydrochalcomycin biosynthesis gene cluster (figure 1a), the whole 75.5 kb region encoding the genes for dihydrochalcomycin biosynthesis has been deposited in the genbank with the accession number ay118081. gert1 is flanked downstream by gerr and upstream by germ3 and gert2 is flanked downstream by gerk2 and upstream by gery. using the clustal x program, the determined sequence analysis of the dihydrochalcomycin gene cluster revealed a deduced amino acid sequence (419 amino acids) encoded for allosyltransferase gert1, which displays a very high degree of similarity to a number of the known glycosyltransferase genes in the genebank database (figure 1a), including chmn from streptomyces bikiniensis (95% identity) (genebank accession no ay509120) (ward., 2004), tyln from streptomyces fradiae (66% identity) (genebank accession no af055922) (fouces., 1999 ; vanessa., 1998), mycd from micromonospora griseorubida (63% identity) (genebank accession no ab089954) (anzai., 2003), and orf 11 streptomyces neyagawaensis (47% identity) (genebank accession no dq149987). similarly, the deduced amino acid sequence (426 amino acid) encodes for the protein named as chalcosyltransferase gert2. this protein also displays similarities to glycosyltransferase from different sources, including chmciii from streptomyces bikiniensis (93 % identity) (genebank accession no ay509120) (ward., 2004), tylmii from streptomyces fradiae (61% identity) (genebank accession no x81885) (charles., 2004), desvii from streptomyces venezuelae (56% identity) (genebank accession no af079762) (xue., 2001), nbmd from streptomyces narbonensis (55% identity) (genebank accession no af521878). in addition, analysis of amino acid sequences of gert1, gert2 and several gtrs involved in the biosynthesis of poliketides showed a very well conserved domain including histidine residue rich region which depends on the gtf (figure 1a and figure 1b). it is suggested that the histidine amino acid present at this conserved region could play important role in the catalytic activity of the enzyme and histidine residues have been showed to be important active site of substrate binding and transition state stabilization in some oligosaccharide - independent gtfs (quiros, 2000a, b). three fractions containing active compounds were successively obtained from elution with 2% to 10% methanol as reported (kim, 1996). the third fraction was further purified by ptlc to afford dihydrochalcomycin along with its derivative. esi - mass analysis showed the peak at m / z=725 [m+na+ ] corresponding to the molecular weight of 702 da as the molecular weight of geri (figure 2) and the compound shows rf value at 0.3. in addition, the fraction with rf value at 0.2 containing unknown compound was isolated with m / z = 755 [m+na+ ] corresponding to the molecular weight 732 da as new compound that is not reported previous paper (figure 3). the compound was carried bioassay test and it also has same antibacterial activity as dihydrochalcomycin (data has not shown). from this results we found that the novel compound isolated has molecular weight equal with the dihydrochalcomycin derivative (rf = 0.2 and mw = 732) in which the macrolactone was attached by two mycinose sugar moieties. this can be suggested that gert2 may act as a flexible glycosyltransferase to different glycone. hence, it can accept either dtdp - allose or dtdp - chalcose as glycone donors during macrolactone glycosylation steps. for confirmation of this hypothesis, it is necessary to study more and carry out further analysis about gert2.	the dihydrochalcomycin (geri) synthetic gene cluster from streptomyces sp. kctc 0041bp has been isolated. two open reading frames (orfs), designated gert1 and gert2 as glycosyltransferase genes, has been identified by sequence analysis. gert1 encodes for the protein function as dtdp - deoxyallosyltransferase and it is responsible to the attachment of dtdp - allose to the macrolide ring. similarly, gert2 encodes for peptide named as dtdp - chacosyltransferase which can transfers the dtdp-4,6-dideoxyglucose to macrolactone core. during process of compound isolation, a new compound has been isolated with molecular weight m / z 755 [m+na+ ]. this compound could be the dihydrochalcomycin derivative. the compound has been shown the same antibacterial activity as geri compound.
the obtained crystal structures of tyrosine - phosphorylated stat1 and stat3 demonstrated that interaction of two nds within one stat dimer is unlikely. these observations suggested that the nds are free to promote other protein - protein interactions. in particular, two stat dimers bound to adjacent gas elements may form a stat tetramer via nd - nd interaction. such cooperation in dna binding via nds allows fine - tuning of transcriptional responses through selective binding of different stat proteins on the promoters containing multiple stat binding sites and through binding to weak stat - binding sites. so far, the nd of stat1, stat4, stat5 and somewhat stat3 were found to form tetrameric complexes, at least on selected promoters. crystallographic studies identified invariant w37 as essential for the nd dimerization. other amino acid residues (q36, t40 and e66) were predicted to be involved in interactions between -helices within the nd. however, subsequent mutational analysis of the stat1 and stat4 nds demonstrated that these residues are unlikely to mediate interactions at proposed interface, and suggested an alternative dimer interface that involves s12, l15, dr19 (-helix2) and f77 and l78 (-helix 7). deletion of the nd or the mutation in critical w37 residue responsible for nd dimerization resulted in abrogation of tetramer formation and transcriptional stimulation. the mutation of a single f77 residue in the nd of stat1 was recently found to preclude both the dephosphorylation and the oligomerization of stat1 dimers. vinkemeier and meyer have shown the influence of defective oligomerization on a complex phenotype such as the induction of an antiviral state. they found that the antiviral protection conferred by ifn was strongly reduced, whereas the ifn response was not measurably affected. nd - mediated stat5 tetramerization was found to be essential for il-2-induced regulation of the il-2 response element in the human il-2ra gene. the functional importance of tetramer formation was revealed by the decreased levels of transcriptional activation associated with hypomorphic mutations in n - terminal residues. in case of stat4, substitution of w37 with alanine unexpectedly prevented ifn-induced tyrosine phosphorylation of stat4 monomer, blocking both dimer and tetramer formation. the requirement of the stat4 nd for stat4 activation was confirmed for il-12 signaling using stat4-deficient transgenic mice that express human full - length stat4 or n - terminal deletion mutant. while full - length stat4 rescues il-12 responsiveness, the stat4 n - terminally truncated protein does not undergo phosphorylation and therefore t - cells expressing this mutant do not undergo proliferation. the requirement for stat tetramerization via nd may contribute to selective activation of certain genes expression. for example, tetramerization of stat3 is required for the formation of enhancesomes on the promoter of 2-macroglobulin, but it is dispensable for il-6-induced activation of socs3, which only requires stat3 dimer binding to the promoter. stat5 tetramerization is necessary for activation of il-2ra expression, but is dispensable for -casein. moreover, despite high homology between nds of various stats, each domain has specific functions that may, at least partially, define precise regulation of stat proteins functions. for example, substitution of stat4 nd with that of stat1 results in inability of chimera protein to undergo ifn-induced tyrosine phosphorylation and to bind dna probes in emsa assay. also, substitution of the stat1 nd with that of stat4 failed to restore ifn-induced mhc class i expression in u3a cells, despite the ability of this chimera to form emsa complexes similar to those of wild - type stat1. this data suggests that the nds of stat1 and stat4 are not fully interchangeable for gene - specific transactivation events. it is thus conceivable to disrupt functions of various stats using selective inhibitors of nds. recently, stat5a - stat5b double - knock - in nd mutant mice in which stat5 may form only dimers but not tetramers were generated. in contrast to stat5-deficient mice that exhibited perinatal lethality, nd mutant mice were viable but had fewer cd4cd25 t cells, nk cells, and cd8 t cells, with impaired cytokine - induced and homeostatic proliferation of cd8 t cells. the observation suggested that stat5 dimers were sufficient for survival and for regulation of some target genes, and that tetramerization of stat5 was only critical for cytokine responses and normal immune function. the data obtained from double - knock - in mouse model agreed with previous report on the critical role of the stat5 nd in human stem cells maintenance and repopulating activity. therefore, the stat5 nd has an essential function during normal physiological development of immune system. it has been found that stat5 exists as a tetramer in cancer cells of 25% patients with leukemia, while this was not observed in normal human bone marrow or peripheral blood cells. in addition, stat5 tetramer formation resulted in stronger and larger dna binding complexes compared with those formed by the dimers. mutations in the stat5a nd abolished tetramer formation and prevented induction of leukemia due to inability of nd - mutated stat5a to rescue stat5 t cell proliferation, despite the persistent activation of stat5n proteins. these observations have proven that the enhanced tetramer formation through nd is the essential feature responsible for leukemogenesis. one of the mechanisms by which tetramer formation may contribute to leukemogenesis is increased occupancy of weak sites to a threshold required for transcriptional activity, which together with the greater degree of flexibility in dna sequence tetramer recognition was suggested to widen target gene spectra. stat5 target genes that control apoptosis, cell cycle progression, and proliferation, such as cyclin d3, bcl - xl, bcl-2, osm, cd25, cis, socs-2, als and igf-1, contain at least two stat5 binding sites in their regulatory regions and their expression is controlled by stat5 tetramer. it has been established that stat5 lacking the nd (stat5a-n) can not protect c - kitlin - sca-1 cells from apoptosis or induce bcl-2 expression. signaling as an achilles heel of persistent stat5 activation and highlighted the potential therapeutic importance of targeting stat5 nd - mediated regulation of bcl-2 family members. although the significance of tetramerization vs. dimerization still remains to be established for other stat proteins, in particular for stat3, the importance of the nd in cancer cells was confirmed by a study in which peptides inhibitors targeting the nds of stat3 or stat5 caused growth inhibition in breast cancer cells. therefore, interference with stats tetramerization through the nd may be an effective therapeutic strategy for cancer treatment. the finding that stat4 nd is essential for activation by cytokine receptors led to an assumption that nd dimerization of unphosphorylated stat4 is a pre - requisite for stat4 phosphorylation and transcriptional function. the yeast two - hybrid analysis of nd interactions, where the nd of each stat protein was expressed in the pfbl23 and gadt7 vectors to explore nds as baits for all other nds (prey), demonstrated that all stat nds are involved in homotypic dimerization. interestingly, nds of stat5a and stat5b that differ only by 11 amino acid residues out of total 130 still showed only selective homotypic dimerization, and did not demonstrate any cross - reactivity. these data indicate that in addition to stabilizing tetramer formation, stat nds may have an important role in dimerization of non - phosphorylated stat proteins. however, the significance of this pre - association is not completely understood. in case of stat4, such dimer formation may enhance presentation to receptor - jak complexes favoring synchronized phosphorylation of the two monomers and allowing formation of active stat dimer by simple intramolecular rearrangement. dimerization of unphosphorylated stat1 strongly depends on the nd because its deletion increased the dissociation constant 100-fold, from 50 nm to 34 m. crystallographic studies of stat1 demonstrated that the structure of each nonphosphorylated monomer is identical to phosphorylated stat1 monomer, however, the monomers in the non - phosphorylated protein are arranged differently, and the nd interactions are essential for an antiparallel stat1 dimer structure. a deletions of nd or mutations disrupting the stat1 nd dimerization (m28, f77 and l78) did not affect stat1 ability to undergo phosphorylation in response to ifn or ifn and form tyrosine - phosphorylated dimers, although such stat1 mutants did not possess the transcriptional activity. stat1 nd appears to regulate association with the nuclear phosphatase tc45 and subsequent stat1 dephosphorylation. indeed, deletion of the n - terminal domain of stat3 abrogated dimer formation, as shown by bnpage and 2f - fcs. however, the homotypic interaction of the n - terminal domain of stat3 are of low affinity (3.7 mm) compared with that of stat1 (23 m) and stat4 (2.7 m). point mutations analogous to those that disturb homotypic interaction of the n - terminal domain of stat1 had no detrimental effect on the dimerization of stat3. therefore, the n - terminal domain of stat3 might not contribute to stat3 dimerization by homotypic interaction but by reciprocal interactions with another domain of stat3. the sh2-domain could be a candidate for an interaction with the n - terminal domain because it has been shown that mutation of the sh2-domain affects dimer formation of unphosphorylated stat3. such an interaction would lead to an antiparallel orientation of the latent stat3 dimer, in contrast to the parallel orientation of the activated stat3 dimer. however, it should be noted that concentration of unphosphorylated stat3 in jurkat cells stimulated with il-6 is about 100-times higher than stat1 ; therefore, it is possible that despite low affinity of the stat3 nd interactions they are biologically relevant. deletion of the stat3 nd does not impair il-6-dependent tyrosine phosphorylation, nuclear import or dephosphorylation kinetics, indicating that this region is not essential for stat3 recruitment to the il-6 receptor complex, translocation to the nuclear compartment or downregulation. however, the phosphorylated stat3 dimers lacking the n - terminal domain do not accumulate in the nucleus.a similar contribution of the n - terminal domain to nuclear accumulation has been shown for stat1. these findings point to a functional role of the n - terminal domain in nuclear import of activated stat3 that deserves further investigation. the deletion of the stat5a nd also does not abrogate cytokine - induced tyrosine phosphorylation, dimerization or dimer dna binding. however, such deletion appears to render constitutive activation, indicating nd s negative regulatory function. interestingly, the nd truncated stat5a / b are predominant isoforms binding to dna in prostate cancer cells. the authors convincingly demonstrate that processing takes place in vivo, but not in vitro during the sample preparation. however, the exact mechanisms of proteolytic stat5a / b cleavage in prostate cancer cells has not been deciphered and the enzymes responsible for it have not been identified. considering that pias3 interacts with stat5 nd to repress stat5-dependent transcription, this modification may represent a molecular mechanism by which stat5a is able to evade inhibition of signaling by pias3 in human prostate cancer cells. in contrast, breast cancer cells, like mcf-7 and t47d, contain full - sized stat5a / b only. prolactin - stimulated activation is efficiently inhibited by pias3, suggesting different mechanisms of regulation of stat5a / b activity in breast and prostate cancers. it is not known at present whether other stat proteins undergo the n - terminal proteolitic cleavage. identification of the proteases responsible for generation of the short form stat5a / b in prostate cancer may present new therapeutic targets. regulation of gene expression by unphosphorylated stats (u - stat) may constitute another potential role of the nd. the extensive investigation from stark s laboratory documented that both u - stat1 and u - stat3 play important roles in the regulation of gene expression. it has been proposed that u - stat1 binds to dna as a monomer by contacting one half of a palindromic gas motif, or forms dimers that also allows to bind gas sequences in vitro, though with much lower affinity, compared with the phosphorylated stat1 dimer. u - stat1 crystal structure suggests that unphosphorylated dimers bound to dna are likely to be formed via the nd interactions. most of the u - stat1 dimers exist in antiparallel conformation, but a small proportion of unphosphorylated stat1 adopts the parallel conformation of activated stat1. we recently observed that u - stat3 also can bind to gas sequences both as a dimer and as a monomer, consistent with previous observations for u - stat1. interestingly, atomic force microscopy allowed for detection of dimers of different shapes suggesting that u - stat3 dimers may bind dna in both parallel and anti - parallel conformation. the significance of the nd interactions for u - stat3 binding to dna and their role in regulation of gene expression remains to be investigated. if proven important for driving expression of genes regulated by u - stat3, the disruption of nd - based dimerization may be a powerful tool to inhibit stat3 functions for therapeutic purposes. despite the importance of tetramer formation for gene expression, and potentially for tumorigenesis, it is probable that nd is also involved in controlling gene expression through interaction with other binding partners. the human protein reference database (www.hprd.org) lists 102 binary interactions for stat3, 77 for stat1, 13 for stat2, 11 for stat4, 53 for stat5a, 42 for stat5b and 18 for stat6. the differences in the numbers of identified interactions reflect the level of popularity of a particular stat in research community rather than intricacy of its interactions. the diversity of interactions is a strong indicator of the complexity of their function s regulation. the list is far from being complete and includes only interactions for which interacting domains of stat3 have been identified. binary interactions of stats with other proteins are considered among the most biologically appealing yet chemically intractable targets for drug discovery. only for several proteins domains that are involved in interactions the nd of phosphorylated stat proteins is exposed on the surface of dimers or tetramers as suggested by crystallographic data and as recognized by native dna binding assays (emsa) or antibody supershift experiments. the location of the interacting stat3 domain is color - coded. in the cases where interacting domain is not localized precisely or interaction involves two domains, gradient coloring is used. nd, n - terminal doman ; cc, coiled - coil domain, dbd, dna - binding domain, ld, linker domain, sh2, sh2-domain, tad, transactivation domain. during last few years, it has been understood that the nds of stat proteins undergo post - translational modifications that control the repertoire of stat protein - protein interactions. it has been shown that stat nds may be phosphorylated, acetylated, methylated, and glycosylated. arginine 31 residue is known to undergo methylation and is conserved across stat family members. although the work describing this finding has been criticized, a recent review on jak - stat pathway raises a voice in support of the role of r31 methylation in regulation of stat1 interactions with pias1 which catalyzes sumoylation of lysine residues and stat1 transcriptional activity. several recent reports have shed the light on protein - protein interactions that involve stat3 nd, including interactions with hdac1 and ape1. two lys residues, 49 and 87 in the stat3 nd, are acetylated by p300. lys - to - arg point mutations (stat3 k49r / k87r) that blocked p300-mediated stat3 acetylation had no effect on inducible dna binding, but abrogated il-6-induced angiotensinogen expression. although stat3 k49r / k87r rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. stat3 was also found to interact with hdac1 through the nd, which resulted in deacetylation of the domain and repression of stat3 transcriptional activity. these findings indicated that acetylation - deacetylation of stat3 provides another signaling axis to control the il-6-stat3 pathway in addition to phosphorylation - dephosphorylation. the follow - up study from the same group demonstrated that only acetylated stat3 forms an inducible complex with the apurinic / apyrimidinic endonuclease 1 (ape1)/redox effector factor-1 (ape1/ref-1), an essential multifunctional protein in dna base excision repair in response to il-6. ape1 selectively binds nd, and this interaction is required for stat3 stable chromatin association with the promoters of suppressor of cytokine signaling-3 (socs3) and -fibrionogen. stat5 n - terminal domain interacts with the glucocorticoid receptor, which can control gene expression as either a coactivator or corepressor. the stat5 nd undergo glycosylation on t92 that is crucial for binding to the coactivator of transcription creb - binding protein and eventually p300 that are established coactivators of gene expression. in addition, phosphositeplus lists a number of post - translational modification of the stat nds that were only detected by mass spectrometry analysis. remarkably, no modifications were detected in the nds of stat2 and stat4, while stat1, stat3 and stat5a / b nds undergo phosphorylation, acetylation and ubiquitinylation. even without understanding the precise role of detected modifications, we may speculate that they regulate stat s protein - protein interactions that result in changes in stat functionality. it has been shown that stat3 can play opposing roles in cellular transformation depending on the genetic background of the tumor. one example includes induction of a highly aggressive t cell leukemia in mice by activated stat3 but prevention of c - myc - induced transformation of mouse embryonic fibroblasts deficient for p53. another example is tumor - suppressive function of stat3 in glioblastomas deficient in tumor suppressor pten, and oncogenic functions in glioblastomas that express nuclear epidermal growth factor receptor type iii variant (egfrviii). there is a substantial heterogeneity in genetic backgrounds of tested cell lines, therefore it is not surprising that there is also a heterogeneity in responses to inhibition of stat3 signaling. it is tempting to speculate that nd protein - protein interactions are responsible, at least partially, for switching of stat3 function from pro - apoptotic to pro - survival during cell transformation. we found that inhibition of the stat3 nd had little effect on normal epithelial cells, while it induced fulminate apoptosis in breast and prostate cancer cells. these data suggested that the stat3 nd performs different functions in cancer as compared with that observed under normal physiological conditions. one can speculate that different roles may be defined by involvement of the stat3 nd in various protein - protein interactions and possibly by differential post - translational modifications of the nd. the identification of differences in signaling events that underlie differential activity of the stat3 nd in normal and cancer cells may offer a potentially novel therapeutic target for cancer treatment. development of selective chemical probes and potential therapeutic agents for stat domains is challenging because the critical interacting surfaces appear to lack deep hydrophobic involutions that enable potent and selective targeting by small molecules. in addition, stats localization within the cell positions them beyond the reach of protein therapeutics. a considerable interest has therefore arisen in next - generation targeting molecules that combine the broad target recognition capabilities of protein therapeutics with the robust cell - penetrating ability of small molecules. we have been using successfully retro - inverso lipopeptides, one of novel classes of synthetic miniproteins with greatly improved pharmacologic performance, increased target affinity, proteolytic resistance and serum half - life while conferring on them high levels of cell penetration. it should be noted here that chemical biology offers very powerful tools in studying the function of certain parts of proteins that provide for much more solid conclusions than genetic methods, but only if the chemical probes are highly selective. experiments involving expression of stats mutants lacking certain domains or containing point mutation in stat - null cells are unlikely to generate the phenotype reflecting correctly the function of the mutant protein in stat - dependent cells. functions of stats are known to be cell - dependent and stat - null cells are unlikely to have the correct combination of partner proteins. we based the original design of stat nd inhibitors on the structure of stat4 n - terminal domain. dimerization of stat4 nd was well established and our original intention was development of inhibitors of dimerization. crystallography data suggested involvement of helixes 2 and 7, while nmr data detected helixes 2 and 8 in the dimer interface. we originally made peptides corresponding to both helixes 2 and 8 and tested them for ability to interact with stat4 nd by nmr. peptide corresponding to helix 2 produced well defined changes in chemical shifts of stat4 nd, while peptide corresponding to helix 8 caused protein to precipitate, most likely due to unfolding. published characterization of dimerization propensity for different stat nds has shown that they differ significantly and that stat3 nd dimer is significantly less stable than stat1 and stat4 dimers thus highlighting the mechanistic differences in the way different members of stat family function. however, it should be noted that the levels of expression of different stats also differ by 100-fold, as was demonstrated for leukemia cells. higher concentration of stat3 may compensate for low affinity and result in less pronounced structural differences between stats. the study by wenta. also suggested the existence of two modes of nd dimerization, at least for stat1. although the structural features of these two modes are unknown, it is possible that both models obtained from crystal structure and nmr studies of stat4 are correct and have indeed identified two naturally occurring interaction interfaces. nmr studies of nd inhibitors interaction with stat4 nd domain suggest that helix 2 analogs are likely to inhibit significantly more than just nd dimerization. changes in chemical shifts detected in the hsqc nmr spectrum of stat4 suggest that domain undergoes significant conformational changes upon binding of the peptide (fig. 3). it is interesting that the residues that are involved are localized mostly on one face of the domain, while the other half of it appears to be subjected to much lesser change (fig. however, the changes cover significant fraction of the domain structure and thus many binary interactions of the domain can be affected. figure 3. residues of stat4 nd (in red) with the most significant chemical shift perturbations detected upon binding of peptide corresponding to helix 2 (ac - eikfleqvdkfy - penetratin). the data generated for stat4 has been used for the design and development of nd inhibitors of stat1, stat3 and stat5. structural studies suggest that n - terminal domains of stat proteins have very similar folds. tertiary structures of stat1 and stat4 nds to select initial lead analog of stat3 helix 2 for optimization. during optimization of peptide length and structure, however, our later findings suggested that simple fusion of the peptides to fatty acids was as effective as attachment of cell - penetrating peptides for intracellular delivery of compounds. extensive studies of structural and biological properties of lipopeptide mimetics of the conserved region of several important but non - druggable molecular targets have revealed that membrane anchoring through the attachment of fatty acid chains can produce highly selective and potent inhibitors of the corresponding protein. membrane anchoring through lipidation contributes to high potency of compounds in three ways : (1) lipidation facilitates cell entry ; (2) fatty acid chain causes membrane insertion and concentrates the inhibitor near intracellular and plasma membrane, where practically all signaling events occur ; (3) membrane anchoring enables folding of otherwise unfolded protein fragment, which results in an increase in potency, frequently by 2 to 3 orders of magnitude. although stabilization is due to enhanced interaction of amino acid side chains with the lipid bilayer, it does nt interfere with peptide s ability to interact. thus, it increases the time the peptide spends in the active conformation, rather than freezes it leaving sufficient time for the folded peptide to stick out of the membrane. currently, four lipopeptides (daptomycin, liraglude, tesamorelin and caspofungin) are used in the clinic. however, many are being developed, and their pharmacological properties make them very convenient chemical biology tools. the remarkable advantage of the approach is that it can be used for rational design of the probes even in the absence of the structural data for the target protein. selection of the stretches of amino acid sequences for mimicking can be based on the conservation during the evolution. the assumption here is that the highly conserved regions are involved in functionally essential protein - protein interactions and thus compounds mimicking them can function as dominant negative inhibitors of the corresponding interactions. we have used the approach successfully for the development of lipopeptide inhibitors of receptors signaling upstream from stats and other non - druggable targets. the conservation - based selection however was nt applicable to stat proteins because entire primary structures are conserved.. however, this is a very unusual case that also has an important message in it : entire structure of stat3 is likely to be important for protein function and there are numerous opportunities in affecting stat function through the development of probes mimicking different parts of stat proteins. although not every member of the family was characterized structurally, available data allows speculating that overall fold is well preserved in entire family and thus stat1, stat3 and stat4 structural data can be used for identification of fragments suitable for development of potential dominant negative inhibitors of all stats. once the stretches for mimicking are identified, the design of the probes is straightforward. the major challenge is determination of optimal positions for fatty acids attachment and the optimal length of the mimicking sequence. here are some ground rules : the preferred lipid position is at the ends of secondary structure elements ; attachment of fatty acid to the side chains, such as -amino group of lys is more likely to result in active compounds. however, we did come across several exceptions, when derivatives with fatty acids attached to -amino group of the n - terminal amino acid were more active. for in vivo use, compounds can be converted into retro - inverso analogs composed on all - d amino acids. they tend to have a more rigid structure that is beneficial in majority but not all cases. however, shorter fatty acids are frequently sufficient and provide for better solubility of compounds. application of the approach to nds of stats allowed us to uncover previously underappreciated role of stat3 nd in tumor growth and stat1 nd in kidney development. the data shows that nds of stats are promising therapeutic targets and lipopeptide inhibitors have a potential to serve as effective therapeutic agents. the wealth of currently available data implies that targeting domains other than sh2 can be an effective way of modulating activity of stats for generation of chemical biology tools and potential therapeutic applications. the data generated for stat n - domains suggest that rationally designed lipopeptide mimetics of fragments of proteins involved in jak - stat signaling can serve as powerful tools in studying the molecular and cellular mechanisms of signaling. it is evident that the function of stat transcription factors is regulated by complex and thus far poorly understood mechanisms involving numerous protein - protein interactions. the latest data also indicate that members of stat family are likely to have multiple functions that they perform not only in the nucleus, but other cellular compartments. targeting protein - protein interactions with rationally designed probes will lead to significant advances in our understanding of molecular mechanisms of jak - stat signaling, pathological processes involving the pathway and consequently therapeutic approaches to controlling and redirecting the function of this important family of proteins.	most attempts to develop inhibitors of stat transcription factors target either activating phosphorylation of tyrosine residue or sh2 domains. however, all six domains of stats are highly conserved between the species and play important roles in the function of this family of transcription factors. stats are involved in numerous protein - protein interactions that are likely to regulate and fine tune transcriptional activity. targeting these interactions can provide plentiful opportunities for the discovery of novel drug candidates and powerful chemical biology tools. using n - terminal domains as an example we describe alternative rational approaches to the development of modulators of jak - stat signaling.
human, animal, and environmental health are inextricably linked in our modern, highly globalized society. we expect better health, secure food, clean water, and more comfort from limited natural resources. balancing competing demands of human health, animal health, and sustainable environmental health is a grand challenge of our time. as the distance between human, domestic animal, and wildlife populations narrows and global trade and travel amplify our connectivity, pathogens shared by humans and animals have concurrently emerged. since 1999, among many examples, humans experienced outbreaks of west nile virus in the united states and europe, severe acute respiratory syndrome (sars) virus in china, middle east respiratory syndrome coronavirus (mers - cov) in the middle east, and the emergence of the ebola virus in west africa. shared pathogen exchange is perhaps best illustrated in the case of influenza, where humans serve as the source of influenza a virus (iav) infection of pigs, and, after further evolution of the virus, it returns from pigs to humans, causing new outbreaks. the 2009 influenza outbreak was the most recent example of this cycle of human and animal infection. the failure to address interspecies transmission and ensuing pandemics as a one health issue has created the opportunity for continued emergence of novel iav like h3n2v that emerged as a human pathogen during the agricultural fair season. these events highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a scientific manner that considers all contributing factors. advancement of health at this level of complexity requires integration of medical disciplines spanning individual care to public health, with basic and applied sciences from atomic structure to sociology providing the knowledge base as shown in the figure. the concept of one medicine one science (comos) builds on the idea that the basic biological processes underlying health and disease share common features such that medical knowledge and expertise in one species is relevant and applicable to other species. the scientific approach that expands our understanding of biological processes and thus, comos provides common ground to assist the organization of interdisciplinary groups to seek solutions to health challenges at local and global scales. another aspect of comos is development of forums that, in addition to medical and scientific participants, includes industry, governmental and nongovernmental policy makers, and funders to assist in communicating the interconnectedness of human, animal and environmental health to a broad audience so that development of public policies that guide health priorities and investments at the national and international level are informed by the best scientific knowledge. icomos : a knowledge forum using science and medicine to find common ground and solutions to complex problems of animal, human, and environmental health. (a) process of policy development and implementation based on scientific and medical knowledge. (b) role of icomos in information sharing and networking among local and international stakeholders, partners, and policy makers. balancing competing priorities is a major public policy challenge as societies seek to maximize human health, animal health and welfare, and environmental integrity. abbreviations : dhhs, united states department of health and human services ; dod, united states department of defense ; epa, united states environmental protection agency ; ngo, nongovernmental organization ; usda, united states department of agriculture ; who, world health organization. the concept of one health emphasizes the interconnectedness of animal, environmental and human health, and has become a part of the health security and international development lexicon. the 2005 revised international health regulations and 2014 global health security agenda (ghsa) are 2 of many instruments that cement this connection / relationship / interdependence. at the international launch of the ghsa, the united states and 30 other countries, the world health organization (who), food and agriculture organization (fao), and world organisation for animal health (oie) agreed to work to prevent, detect, and respond to global infectious disease threats from the perspective of one health. years before the release of the ghsa, the us agency for international development established the emerging pandemic threats program to prevent, detect, and control animal and human pathogens. these activities, which promote one health approaches in the united states and globally, are an important beginning but fall short of providing the robust scientific and culturally informed approach that is needed to address real world challenges. much dialogue on one health has focused on emerging disease surveillance, public health preparedness, and policy issues without connecting these issues to the scientific foundations that underlie pathogen emergence, global health threats, food security, environmental health, social organization, communication, and implementation of health, security, and safety measures. this limitation has resulted in a perceived and apparent separation of science and policy, sometimes diluting the utility of the one health movement. this article builds on the discussions and dialogue about the science of one health and its connection to policy that were held at the international conference on one medicine one science (icomos ; www.icomos.umn.edu) in 2014 and to be held again in 2016. in particular, it was concluded that the key roles of food and medicine in addressing health needs of a changing world require the support of rigorous science that empowers informed decision - making and development of useful policies. the goal of icomos is to examine the connections between science and one health policy implementation by (1) focusing on case studies and research that demonstrate the successful integration of human, animal, plant, and environmental health and social and behavioral sciences ; (2) examining the science - policy connection by convening panels that include scientists, policy makers, and representatives from private industry ; and (3) conducting forward - looking workshops in areas of research, collaboration, and policy formulation. good policy must be based on objective scientific studies that integrate epidemiology, ecology, microbiology, social science, and economics to balance the expectation of safe and nutritious food with the need for efficient and profitable production, and sustained environmental health. given that an estimated 870 million people are currently suffering from malnutrition and that the combination of human population growth and consumer preference shifts associated with a rising global middle class are expected to exacerbate this problem, global production and delivery of food for protein, energy, and micronutrients must not just be maintained, it must be increased. in addition, existing land and water resources are already strained and their availability for agriculture will likely decrease, especially as the impacts of climate change shift traditional production regions. the obvious solution is to produce more efficiently, yet the very strategies that promote efficient production of food, such as concentrated farming systems, monoculture cropping, and chemical inputs of fertilizer, pesticides, and herbicides, have unintended consequences that threaten human, animal, and environmental health. we are faced with a paradox in which the demand for increased food production to improve human health today sows the seeds of resource depletion that limit health advances tomorrow. thus, new research on the direct and indirect impacts of food production on human, animal, and environmental health as well as social, organizational, and behavioral sustainability should be a priority. ensuring that sufficient food is not only safely and efficiently delivered to consumers but that it is produced in ways according to local preferences presents another paradox. consumer demand for local and organic production systems actually increases the greenhouse gas footprint of most developed and many developing country food systems, depending on the crop or animal product and also can increase food safety risks relative to today 's widely used production systems. those producers that remain local are often small in size, thus limiting benefits of scale, and many use organic methods, which can limit their ability to prevent disease through approaches such as constructing barriers and facilities for containment. the result is increased contact with wildlife, which increases opportunities for the transmission of infectious diseases to crops, livestock, and humans. for instance, interaction between domestic poultry and wild waterfowl reservoirs has resulted in the introduction of new iav, and the movement of h5 hpai from domestic poultry to free - flying bird populations demonstrates that the exchange can be bidirectional, which changes paradigms of transboundary disease spread. the interface between wildlife and agriculture will only increase as more land is converted for agricultural use to meet consumer demands, thus forcing wildlife into shrinking habitats or to adapt to increased contact with humans and livestock. housing food animals inside helps to protect animal health, prevent cross - species disease transmission, and improve production efficiency, but it does not always meet consumer preference for local needs and resources. nontherapeutic (nonclinical) antibiotic use in food animals is a tool that promotes production efficiency, but it has been associated with a rise in antibiotic resistance of human pathogens. however, epidemiological and ecological studies fail to support a causal link ; instead, these studies indicate the spread of antibiotic resistance from humans to pigs and chickens. in the future, the world food summit global food strategy of providing access for all people at all times to sufficient, safe, and nutritious food to maintain a healthy and active life must be expanded to include considerations for environmental sustainability, animal welfare and non - nutritional aspects of public health. progress toward these goals requires strong linkage of scientific knowledge and discovery that helps both to advance medical practice and health improvement as well as inform health policy development. human population expansion and exploration has increased as never before, and thus attention to the risk of disease emergence at the interface between humans, domesticated animals, and wildlife is increasingly important. humans and their companion animals have triggered epidemic plagues of canine distemper in serengeti lions and morbillivirus in pacific seals while spreading influenza a to food animal species. other dramatic changes include the explosive growth in global travel and trade patterns and legal and illegal trade in live animals, animal products, and wildlife as well as effects of climate change on all of the above. however, focusing on the processes and mechanisms that facilitate pathogen evolution and emergence will help us prepare for unpredictable new outbreaks, thus raising awareness of prevention and control policies that could reduce both the likelihood and magnitude of disease emergence and its resulting effects on animals and humans. single approaches to comprehend and anticipate disease emergence, especially using simplified disease models, are unlikely to produce informative insights involving environmental factors, multiple reservoirs, and complex human - animal - environment interactions. concepts of wildlife reservoirs and spill - over / cross - species transmission into food animal and human populations followed by expansion and outbreak potential have been developed into mathematical models. although not a predictor in itself, models of zoonotic transmission dynamics have made valuable contributions to inform public health recommendations for control of novel h1n1 influenza and sars and are crucial for understanding pathogen transmission patterns and changes in disease epidemiology. human orthopoxvirus infection is a highly relevant example in which modeling based on rigorously collected data provides prevention strategies for a highly plausible emergent disease. the global human population is increasingly susceptible to smallpox due to the cessation of vaccination programs following eradication. loss of crossreactive immunity has opened an ecological niche that can and has been replaced by related orthopoxviruses, including monkeypox in the congo and novel orthopoxvirus in the country of georgia. emergence of new orthopoxvirus outbreaks with pandemic potential is possible as human - infectious poxviruses circulate in natural reservoirs. the recent discovery of viable smallpox in a us food and drug administration refrigerator on the national institutes of health (nih) campus recently reinvigorated the discussion of both susceptibility and/or risks from infections to smallpox. the specter of a new pandemic, fueled by relatively recent experience with the emergence of aids and new influenza viruses, has driven the us centers for disease control and prevention, the nih - national institute of allergy and infectious diseases, the us department of agriculture, the us agency for international development, and others to develop programs focused on predictive modeling of and early detection and response to novel pathogens that have evolved under a complex set of pressures such as environmental pollution, land use changes, new trade patterns, climatic change, and other anthropogenic factors. the role of medicine in addressing individual and public health needs to be supported by rigorous science that empowers informed decision - making and development of useful policies in the face of unexpected disease threats. the rapid growth and persistence of the ongoing outbreak of ebola virus in west africa presents an unfortunate but perfect opportunity for implementing science - based policy at the crossroads of emerging infectious disease ecology and sustainable food security. experts hypothesize that ebola virus entered the west african population through consumption of infected fruit bats, a plausible scenario given local food needs and practices. the ongoing outbreak has stressed social organization and an already thin public health infrastructure to the breaking point in the affected countries. it threatens to exacerbate baseline food insecurity as disease control measures and fear shut down food and agriculture systems, further stressing local food security. this situation makes for the perfect storm of grand challenges in the one health arena. frighteningly, while more than 27 000 cases and 11 200 deaths have been recorded as of july 14, 2015, a new ecological niche risk model predicts that 22 million people in 22 central and west african countries are at potential risk for ebola. while the world seems to be stepping up with much - needed scientific, medical, and infrastructure support to this tragedy, broader policies and preparedness are needed for prevention, containment, and response if we are to avoid more extreme human suffering from the virus itself as well as malnutrition, community instability, and other social consequences. addressing the grand challenges of our time such as novel disease emergence and food security is difficult from any perspective. but the universal scientific discovery and exploration approaches that have been applied so effectively to resolve focused problems offer promise in addressing the complex health issues of modern life. a linear refocusing of health research away from disease surveillance and investigation resulting primarily in treatment toward environmental surveillance resulting in prediction and prevention is not enough to inform societal debates on these complex health issues. reducing challenges to simple scenarios allows us to find solutions that do not address the complex problems we face but instead bring on a myriad of unintended and undesirable consequences. rather, they represent wicked problems that are complex and difficult - to - balance dilemmas with fluid dynamics whose competing components change over time. the creation of forums for convergence on grand challenges where science and policy can be debated together, missing pieces identified, and science - based collaborations stimulated in the presence of industry, governmental and nongovernmental policy makers, and funders is an essential step. the underpinnings of comos ie, promoting team science that builds on the expertise of different disciplines, stakeholders, and research foci to support policy development is all the more relevant to the implementation of one health and the successful achievement of global health security goals (figure). universal scientific discovery and exploration approaches that have been applied so effectively to advance human society will need to be applied to complex health issues of modern life so that the resulting knowledge can inform public policy and decision - making at every level.	numerous interspecies disease transmission events, ebola virus being a recent and cogent example, highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a comprehensive scientific manner. the diversity of information gained from the natural, social, behavioral, and systems sciences is critical to developing and sustainably promoting integrated health approaches that can be implemented at the local, national, and international levels to meet grand challenges. the concept of one medicine one science (comos) as outlined herein describes the interplay between scientific knowledge that underpins health and medicine and efforts toward stabilizing local systems using 2 linked case studies : the food system and emerging infectious disease. forums such as the international conference of one medicine one science (icomos), where science and policy can be debated together, missing pieces identified, and science - based collaborations formed among industry, governmental, and nongovernmental policy makers and funders, is an essential step in addressing global health. the expertise of multiple disciplines and research foci to support policy development is critical to the implementation of one health and the successful achievement of global health security goals.
cortical blindness is a total or partial loss of vision in normal - appearing eyes caused by damage of the geniculocalcarine pathways. the incidence and etiology of cortical blindness seems to be changing with the development of effective means of cardiopulmonary resuscitation and better understanding of neurovascular physiopathology). the precise nature of the pathogenesis of this condition has not yet been understood. however, cerebrovascular disease is the most common cause, followed by cardiac surgery, cardiac arrest, head injury, pre - eclampsia and cerebral angiography [1, 4,5,6 ]. temporary blindness after coronary angiography has also been reported by many authors. other reported causes of cortical blindness are guillina barr syndrome, reversible posterior leucoencephalopathy syndrome. blue rubber bleb naevus syndrome, and injection of bleeding gastric varices with cyanoacrylate glue. it seems that the key to understanding cortical blindness following cardiac arrest is understanding the associated cerebral iscahemia. cardiac arrest usually leads to generalised brain anoxia or to focal neurological abnormalities, resulting in a variable spectrum of diffuse or multifocal cerebral atrophy due to neuronal loss and cortical and subcortical gliosis or even severe cortical damage [2, 3 ]. the global ischemic anoxia results in diffuse hypometabolism with preferential localisation of the metabolic alterations in the parieto - occipital cortex. although most patients recover from cortical blindness, the mechanisms of this recovery are poorly understood1. the posterior cerebral artery supplies the occipital lobe, and also seems to play an important role. all conditions that cause vascular impairment to the occipital cortexes may lead to cortical blindness. cortical blindness has not been described in the medical literature to follow surgical repair of stab wounds in the heart. in this report, we describe a 29-year old male patient who experienced compete loss of vision following successful surgical repair of stab wounds in the heart. to the best of our knowledge, this is the first case describing the association between cortical blindness and surgical repair of stab wound in the heart. cortical blindness followed successful surgical repair of two stab wounds in the heart in a 29-year old libyan man, who was brought in a state of shock to the accident and emergency unit of our hospital following a fight. the patient was semi comatosed, with cold extremities, and with capillary filling more than 4 seconds. he was in sinus bradycardia with weakening pulse of 45 per minute, and his blood pressure was 70/50 mmhg. the patient did n't arrest but was in a pre arrest condition with low cardiac output status and progressively collapsing pulse and dropping blood pressure. cross matching for 5 units of whole blood was ordered, while left chest tube was immediately inserted with 850 ml of fresh arterial blood gushed out, and continued to drain at an estimated rate of more than 200 ml per hour into the water - seal reservoir. the heart was reached via pericariotomy. during this time, iv fluid infusion therapy was instituted with 1000 ml 5% dextrose saline and 1000 ml ringers solution, and adrenaline 3 mg was given in the iv. two profusely bleeding wounds were determined ; one was at the ascending aortic root just above the aortic annulus, and the second was on the anterior surface of the left ventricle ; a few millimetres to the left side of the proximal left anterior descending artery. the bleeding was completely stopped after repairing the 2 wounds with 4/0 pledgeted polypropylene sutures. by then, the cross matched blood arrived to theatre and was immediately given. five units of whole blood were given over 24 hours, and were controlled by multiple complete blood counts. the patient did n't arrest during surgery, but had an episode to sinus tachycardia of 180 minutes per minute that did n't turn into ventricular fibrillation, and thus no dc shock was given. the patient recovered smoothly in the intensive care unit, and was discharged to the surgical ward after 24 hours with stable haemodynamics. three days later, he started to see again and eventually regained his vision completely. repeat eegs revealed regaining of alpha waves at this point. he was discharged with normal vision and in good condition on the 7th day of admission. one week, one month, and 3 month follow - up revealed no abnormalities. cortical blindness is not an uncommon clinical condition, but has not received due attention by the medical community. cardiac arrest usually leads to global brain anoxia and to lack of cortical functioning or to more focal neurological abnormalities, which may lead to cortical blindness, resulting in a variable spectrum of diffuse or multi - focal cerebral atrophy due to neuronal loss and cortical and subcortical gliosis or even severe cortical damage [2, 3 ]. it has been shown that the prognosis of cortical blindness was best in patients under the age of 40 years, in those without a history of hypertension or diabetes mellitus, and in those without associated cognitive, language, or memory impairments. it has been shown that eegs, which demonstrate alpha waves absence during the episodes of cortical blindness, are more specific than vep and ct scans for the diagnosis. some reports have shown that when bi - occipital abnormalities are demonstrated in the ct scans, a poor prognosis follows [1, 2 ]. this report describes a typical reversible cortical blindness with sudden loss of vision, absence of alpha waves in the eegs and complete regaining of vision. what is unique in our case is the association with the surgical repair of stab wound in the heart and the unreported cardiac arrest during the course of resuscitation ; before or during the surgical intervention. it seems that there were episodes of cerebral anoxia during the state of shock following the profuse bleeding and hypovolaemia that was caused by the multiple stab wounds in the chest. lack of cortical functioning would have caused this cortical blindness despite the un occurrence of cardiac arrest. the quick chest tube insertion improved the compromised respiratory function, and prevented cardiac tamponde, which provided an immediate improvement of circulatory functions and haemodynamics despite the continuing bleeding. the immediately given iv fluids and adrenaline have helped to support the failing circulation and correct the hypovolaemic shock. our quick and proper surgical intervention helped prevent the cardiac arrest and prevented undergoing progressive cortical and brain damage, and thus saved his vision and life. swift and proper patient transportation to advanced centres may prevent cardiac arrest in pre cardiac arrest cases. applying cardiac resuscitation guidelines is mandatory to stop pre cardiac arrest cases from developing cardiac arrest and to increase the survival rates of cardiac arrest resuscitation. sudden loss of vision may follow pre cardiac arrest cases and surgical intervention, even if cardiac arrest has not occurred, should lead to the suspicion of cortical blindness particularly when no gross ophthalmic and neurological abnormalities could be demonstrated. increased awareness of healthcare providers about this phenomenon will ensure avoiding confusing diagnosis, un - necessary investigations and improper treatment for cortical blindness.	this report describes a case of cortical blindness that followed successful surgical repair of two stab wounds in the heart in a 29-year old libyan man. the patient presented in a state of pre cardiac arrest (shock and low cardiac output status), following multiple chest stab wounds. chest tube was immediately inserted. surgery was urgently performed suturing the two wounds ; in the root of the aorta and in the left ventricle, and haemostasis was secured. cardiac arrest was successfully prevented. the patient recovered smoothly, but 24 hours later he declared total blindness. ophtalmic and neurological examinations and investigations that included fundoscopy, electroencephalograms (eegs) and computed tomography scans revealed no abnormalities, apart from absence of alpha waves in the eegs. we diagnosed the case as cortical blindness and continued caring for the patient conservatively. three days later, the patient regained his vision gradually and was discharged on the 7th postoperative day without any remarks.
the notion that an already consolidated memory could be destabilized and disrupted was first demonstrated in the late 1960s by lewis and colleagues (misanin., 1968) amidst an environment where the prevailing theory at the time, the consolidation theory, suggested that once a memory was consolidated, it was permanent or fixed. this was based on a dichotomy between short and long - term memory and it was assumed, though far from being proven, that amnesia was a permanent consolidation or retrieval deficit. it was against this backdrop that lewis argued, it was more parsimonious to consider that memory traces, regardless of their age, could shift between two states, an inactive and stable (consolidated) state corresponding to stored memories and an active and fragile state open to disruption by amnestic agents following the encoding of information and the readout of the trace during retrieval (lewis, 1979). the implication is that when the memory is converted again into an active state following reactivation, a further storage process is required for the trace to remain in long - term memory and be available once again for recall. this was based on his own studies and a number of others that showed a so - called consolidated memory could be disrupted when electroconvulsive shock (misanin., 1968), or interfering material (gordon and spear, 1973), lewis experiments and arguments went largely unheeded for some 2030 years, as the consolidation theory remained doggedly in place until przybyslawski and sara (1997) and nader. (2000) showed that a consolidated fear memory could be disrupted by the protein synthesis inhibitor, anisomycin if injected just after reactivation of the memory, 24 h following initial learning. importantly, they showed that if protein synthesis inhibition occurred in the absence of reactivation of the memory, it remained intact. subsequent to this initial resurrection of lewis results, there followed a slew experiments replicating nader and colleagues basic findings in a wide range of species from nematode (rose and rankin, 2006) to honey bees (stollhoff., 2005), sea slug (sangha., 2003), rodents (nader., 2000 ; kida., 2002 ; milekic and alberini, 2002 ; suzuki., 2004 ; see reviews by hardt., 2010 ; nader and einarsson, 2010) through to human studies (walker., 2003 ; tronson and taylor, 2007 ; hupbach., 2009 ; this reinforced the notion that memories were not fixed or permanent and when in an active state they were labile and susceptible to disruption by interfering events to a level where the scientific community accepted that this was now a bone fide phenomenon, common across species (see nader and einarsson, 2010 for details on supporting experimental data). despite the overwhelming support for reconsolidation there were the inevitable studies that showed an exception to the rule and a number of what was termed boundary conditions were applied in circumstances where a reactivated memory remained resistant to disruption, and therefore required no need for reconsolidation. to date the most commonly encountered boundary conditions that determine whether reactivated memories are vulnerable or immune to disruption are the age of the memory (milekic and alberini, 2002 ; suzuki., 2004 ; robinson and franklin, 2010), the strength of learning (suzuki., 2004 ; taylor., 2009),, 2004 ; power., 2006) and the degree of similarity between the environment and conditions in which the memory was consolidated and reactivated (bozon., 2003a ; debiec., 2006 ; artinian the fact that boundary conditions exist, itself raised the question of whether or not reconsolidation was truly a re - enactment of the consolidation process. this question has largely evolved from studies designed to understand the cellular and molecular mechanisms associated with consolidation and reconsolidation. a number of studies using inhibitors of de novo protein synthesis have shown that new proteins are necessary for both the initial encoding of a memory and its re - stabilization after reactivation, which would suggest reconsolidation is a faithful recapitulation of consolidation. it is not surprising that protein synthesis inhibitors impair both processes, as in order to have any effect on memory, nearly 90% of all proteins must be inhibited ; therefore, at least in terms of understanding the cellular and molecular mechanisms associated with both processes, the use of protein synthesis inhibitors lends little information about the specific genes and proteins that may be involved. in those studies targeting specific genes and proteins there are variations on whether activation of the same proteins and genes are necessary for both processes or whether they are involved in one and not the other. there are a number of studies that have shown a given gene or protein is expressed / activated in both processes or that selective knockdown of a gene or inhibition of a protein impairs both processes (reviewed in tronson and taylor, 2007). however, dissociations have also been shown ; for example, in a fear conditioning task, it was shown, using oligonucleotide antisense, that regulation of the transcription factor, zif268/egr1 in ca1 of the hippocampus was not necessary for consolidating fear memory, but was necessary for reconsolidation of the active memory (lee., 2004). conversely, other studies have shown the transcription factor ccaat / enhancer binding protein c / ebp is activated and necessary in the dorsal hippocampus in association with consolidation of fear memory but not its reconsolidation (taubenfeld., 2001). and yet another variation on the theme is the demonstration that consolidation and reconsolidation may activate the same genes and proteins but in different neural circuits (kelly., 2003 ; alberini, 2005). this suggests for certain memories, restabilizing the memory after reactivation is not a simple reiteration of the consolidation process, at least in terms of the underlying mechanisms. this is also supported by data showing that the memory trace takes a shorter time to re - stabilize than it does to consolidate (litvin and anokhin, 2000 ; milekic and alberini, 2002 ; suzuki., 2004 ; languille., however, there is controversy as to whether consolidation and reconsolidation are separate but overlapping processes (nader., 2000 ;, 2002 ; nader and hardt, 2009) or whether in fact it is merely part of an extended and dynamic form of consolidation (dudai and eisenberg, 2004 ; alberini, 2005) that has just not yet been experimentally demonstrated. this was an issue raised a number of years ago and appears to date not to be resolved. whether reconsolidation is an independent or even partially independent process or whether it is part of an ongoing consolidation process, whether the same molecules are required or not, whether the same circuitry is involved or not, are among the major issues debated in the field. as it is generally agreed that memories are encoded in widespread neural networks and neuropsychological evidence suggests that memories are encoded over lengthy periods of time, it would seem inevitable that when a memory is reactivated it is available for some form of modification and its content could well evolve over time depending on the environment and circumstances in which it is reactivated. it therefore may be fruitful to use the cellular and molecular mechanisms that have been associated with consolidation and reconsolidation to understand how memories evolve over time following repeated reactivation. a second, equally controversial debate that is intertwined with the dispute as to whether reconsolidation is a separate entity or part on a slow process of consolidation concerns the function of reconsolidation. in other words, why each time a memory is reactivated it is vulnerable to loss or disruption and requires reconstruction, as this is a costly and somewhat dangerous mechanism. currently there are two principle theories as to the functional role of reconsolidation that suggest it is either a means of strengthening the memory trace (alberini, 2005) or that it is a mechanism whereby the memory trace may be updated to incorporate new information (morris., 2006 ; hupbach., 2007 ; lee, 2009) neither of which seems to be mutually exclusive of each other. implicit in the theory that reconsolidation serves to strengthen memory is the notion that eventually memories should become immune to disruption. those that support this theory offer evidence that suggests newer memories are more vulnerable to disruption following reactivation than older memories (alberini, 2005 ; wang., furthermore, they suggest that strengthening the memory via a mechanism such as reconsolidation may be a means of explaining addictive behavior and pathological memories associated with post - traumatic stress disorder (ptsd) and phobias. indeed, at the clinical level, as behavioral therapy using extinction to attempt to eliminate or decrease episodes of stress associated with these pathological memories have not met with a great deal of success, the emphasis has changed to attempt to disrupt types of memories by reactivating them and administering drugs associated with mechanisms known to disrupt reconsolidation. at present, however, it is not clear what rate of success these treatments have had. a recent experiment in human subjects has shown that a form of fear conditioning with mild shock to the wrist can be erased if the memory is reactivated just prior to an extinction protocol that is successful a year later (schiller., 2010). however, given the strength of the emotional content and the complexity of pathological memories in phobias or drug addiction or ptsd, whether simple behavioral modification such as this would constitute an effective treatment for eliminating these types of memory or whether it could work successfully with older memories is not clear. opposed to the memory strengthening theory of reconsolidation is that its function is to update memories to integrate new information to confer adaptive benefit to the organism. it has been shown in both rodents and humans that subsequent additional learning can modify the original memory if it is in an active state. a number of studies using animals and humans have suggested that subsequent additional learning can modify the original memory (hupbach., 2009 ; lee, 2009 ; schiller., 2010). an important feature of this theory is the implication that regardless of the age of the memory, it can be altered and that at a systems level of consolidation or reconsolidation, memories can be linked to form a knowledge base in much the same manner of bartlett 's schemata (see hardt., 2010). although most of the experimental evidence to suggest the fluidity of memory and remembering has been within the domain of human studies, tse. (2007) have demonstrated that rats can rapidly incorporate new taste - spatial information into an existing memory. the importance of this theory and the evidence that supports it afford us an inroad into an area of research that has largely remained out with behavioral studies and the neurosciences to understand how memories can be built up and elaborated over time. to date, most rodent studies aimed at investigating the cellular and molecular mechanisms associated with reconsolidation have used negatively reinforced learning tasks such as fear conditioning or spatial learning in the water maze. relatively few studies have used non - emotive learning, and fear learning may constitute a highly specific form of learning in which important but subtle changes in the memory may not be detectable. in this review, we focus on recognition memory, a form of memory that can be based on incidental encoding, not relying on any explicit reinforcement. recognition memory is highly relevant to all species, it plays a key role in adaptive behaviors and is implicated in all forms of memory regardless of whether they are or not emotionally based, negatively, or positively reinforced. recognition memory is a prototypical form of event memory widely used in specific paradigms to probe episodic memory in humans. operationally, it refers to the ability to recognize previously encountered information and is commonly defined in humans as the conscious recollection of past personally experienced events (tulving, 2002). the most common paradigms used for testing recognition memory in human subjects involve verbal material or visuospatial recognition of faces, objects, or scenes. performance in recognition memory retrieval can however be based on at least two functionally distinct memory processes : it can rely on familiarity detection, a mental awareness that an event has been experienced in the past, leading to a sense of basic feeling of knowing or dj vu which is experienced rapidly, or on genuine recollection, the conscious remembering of the event incorporating detailed episodic features such as the content and spatio - temporal context within which the event occurred. these two processes are not mutually exclusive and boundaries between them are hazy, as on one side elements of familiarity are likely to occur in all recall tasks and on the other performance does not always provide insight into retrieval accuracy and confidence of recollection. several studies examining recognition memory performance in amnesic patients have revealed deficits in patients suffering medial temporal lobe lesion. although both recollection and familiarity detection can be affected by medial temporal lobe lesion, recollection is often more severely impaired than familiarity (reviewed in eichenbaum., 2007) and whereas several studies support the idea that recollection engages the hippocampus, the parahippocampal region can support familiarity (brown and aggleton, 2001 ; eichenbaum., 2007). attempts to delineate the neural circuits that underlie the two processes at retrieval using electrophysiological or neuroimaging techniques in humans also support the idea that these two processes engage at least partly distinct brain systems. for example, event - related potentials recordings in tasks that allow the dissociation between true recollection and levels of familiarity of retrieved items revealed a mid - frontal negativity which onsets at 300 ms and varies with familiarity strength, while recollected items are associated with a left parietal positivity, insensitive to familiarity, with a more delayed onset around 500 ms (woodruff. (2005) described two non - overlapping circuits involved in familiarity strength and recollection judged by recall of thoughts about the words and their shape on the screen. using fmri, they highlight a circuit involving areas of the lateral and medial (precuneus) parietal and of the anterior and lateral prefrontal cortices activated in relation to increased confidence in familiarity, whereas recollection - based recognition is associated with activation of distinct areas of the anterior medial prefrontal, posterior cingulate, lateral parietal and temporal cortices, and the hippocampus. as a further example, a study exploring the effect of sub - seizure electrical stimulation of the hippocampus during recognition memory tasks in humans reported deficits in word recognition after left hippocampal stimulation and deficits in face recognition after right hippocampal stimulation (coleshill., 2004). these and other studies thus support the notion that recollection engages a network of structures that includes the hippocampus, while familiarity detection even with a high level of confidence is less consistently associated with hippocampal activation. consistent with this, several other imaging studies have reported activation of the perirhinal cortex in association with familiarity and activation of the hippocampus and parahippocampal area in association with recollection - based recognition, although certain studies reported a decrease in activity in these two regions can be associated with familiarity while others suggested there is not a strict dichotomy between circuits of the medial temporal lobe engaged in recollection and familiarity (reviewed in eichenbaum., 2007 ; skinner and fernandes, 2007 ; squire., 2007). in rodents it is notoriously difficult to dissociate recollection from familiarity because it is impracticable to measure explicit recall of past experience in animals without language. recognition memory can however be tested in a task based on the spontaneous preference of rodents for novelty and their ability to remember previously encountered objects (ennaceur and delacour, 1988). in the standard protocol, animals are briefly exposed to two or three objects and a memory test can be given after a short or a long time interval by changing one object for a novel object or by changing the spatial position of one familiar object. during the test, preferential exploration of the novel or displaced object provides a measure of recognition memory. these tasks are based on incidental encoding during objects sampling, suggesting that performance at retrieval rely on both item familiarity and event recollection (nemanic., 2004). a large body of literature has examined the contribution of the hippocampus and medial temporal lobe structures in object recognition memory in rodents. in early behavioral studies, recognition memory at relatively short delays (4 h) was shown to be impaired by perirhinal lesions but spared by lesions of the hippocampus (see mumby, 2001 ; winters., 2008 for reviews). conversely, several lines of evidence have pointed to a delay - dependent impairment in long - term (24 h) recognition memory in rats with hippocampal lesions (vnek and rothblat, 1996 ; clark., 2000 ; hammond. 2006), depending on lesion size (broadbent., 2004), or in mice with lidocaine - induced inactivation of the hippocampus (hammond., 2004). as another example, post - training lesion of the hippocampus produces retrograde, although not anterograde, amnesia in recognition memory, suggesting the hippocampus, when functional, participates in the formation of recognition memory (broadbent., 2009). these studies, as well as others based on hippocampal specific gene inactivation (see below), lend support to the conclusion that while the perirhinal cortex is critically involved in object recognition (reviewed in winters., 2008), the contribution of the hippocampus becomes increasingly vital with increases in task demand. this depends on several factors such as the size and shape of the testing apparatus, the number and complexity of the objects in the sample phase, the retention delay (hammond., 2004) and the availability and richness of contextual information (see winters., 2008) and their consequences on the ability to form spatial configurations, associative relationships between items and objects scene relationships (jenkins., 2004). this structural double - dissociation effect seems to parallel the familiarity / recollection dual - process account of recognition memory studied in humans, and is also consistent with several electrophysiological recording studies showing perirhinal cortex neuron correlates of familiarity (reviewed in winters., 2008) and hippocampal neurons correlates of both objects location and identity (manns and eichenbaum, 2009). although there are few tangible means of experimentally testing this in rodents, a recent study using an odor - digging medium based associative recognition paradigm in rats provided striking support to the idea that familiarity detection and recollection are qualitatively dissociable processes and that the hippocampus supports recollection and not familiarity (sauvage., 2008). to date it is largely believed that memory formation involves long - lasting, activity - dependent changes in synaptic strength within the neural networks activated during learning, as exemplified by ltp - like mechanisms, and that this is mediated by molecular mechanisms in neurons underlying functional and structural remodeling of network connectivity. the prevailing model for the mechanisms underlying synaptic plasticity and the laying down of memories involves activation of specific cell - surface receptors and of several signaling cascades via kinase pathways that ultimately lead to post - translational modifications of numerous synaptic proteins and to the activation of nuclear transcription factors to trigger the expression of specific gene programs in neurons and the synthesis of proteins. in recent years, a wealth of experimental data has provided compelling evidence that canonical cellular and molecular mechanisms engaged in the formation of most forms of long - term memory are similarly implicated in the consolidation of recognition memory. the most familiar of such example is the requirement of protein synthesis for the stabilization of long - term memory, as demonstrated by the impairment of long - term, but not short - term recognition memory by protein synthesis inhibition in area ca1 of the hippocampus (rossato., 2007). other experiments however have shown a particular sensitivity to hippocampal protein synthesis inhibition when contextual information is a crucial cue (balderas., 2008). among the signaling cascades that have been consistently shown to be critical for synaptic plasticity and for the consolidation of several forms of memory, the map kinase (mapk / erk) cascade has attracted much attention as a key effector of the regulation of gene expression in response to neuronal activation (sweatt, 2001 ; davis and laroche, 2006 for reviews). examining whether the mapk / erk cascade is also required for the consolidation of object recognition memory, we used a paradigm that most probably places a high demand on hippocampal function by using complex three - dimensional objects in a wide open - field surrounded by rich contextual information and found that object exploration induces rapid phosphorylation of mapk / erk in the entorhinal cortex, dentate gyrus and to a lesser extent in ca1 (where novelty seems to be a more efficient activating factor), and that blocking mapk / erk phosphorylation during object exploration suppresses long - term, but not short - term, recognition memory (kelly., 2003). the mapk / erk cascade is known to convey signals from cell - surface receptors to the nucleus via transcription factors such as creb and elk-1 and to play a critical role in triggering gene expression by activating various inducible nuclear transcription factors (davis., 2000 ; one such transcription factor is zif268/egr1, a member of the egr family of transcriptional regulators that is rapidly activated in a structure dependent manner after different types of learning. examining key components of this pathway, we found that forebrain expression of a creb repressor in a transgenic mouse impairs both object and object - location recognition memory (bozon., 2003b). creb inactivation specifically in area ca1 of the hippocampus was also shown to impair object recognition memory (pittenger., 2002). similar results were obtained in zif268 mutant mice showing that zif268 is required for the consolidation of both object and object - location recognition memory (jones., 2001). zif268 protein is rapidly induced in the dentate gyrus of the hippocampus after object sampling (soul., 2008) and in mutant mice a gene - dosage effect was found as half the complement of zif268 in heterozygous mutant mice resulted in deficits in long - term object - location memory without impairment in novel object recognition (bozon., 2002). these findings support a model in which a mapk / erk - creb - zif268 cascade mediating gene regulation contributes critically to the consolidation of long - term object recognition memory. surprisingly, other members of the egr family of transcription factors, despite having high homology, conserved dna binding domains and similar brain localization, were found to affect differentially recognition memory (reviewed in poirier., 2008). while zif268/egr1 deletion affects long - term recognition memory, egr3 deletion affects short - term recognition memory (li., 2007) and we found that forebrain - specific deletion of egr2 in transgenic mice facilitates long - term recognition memory (poirier., 2007a), suggesting that the three egrs do not share similar function and can in contrast have opposite roles in recognition memory, egr2 acting as an inhibitory constrain on recognition memory. the mapk / erk cascade is not the only signaling cascade implicated in recognition memory and over the past several years a collection of studies has provided evidence that recognition memory involves rapid and coordinate regulation of several plasticity - related genes and molecules. for example, object exploration also increases arc, camkii and psd95 expression in the dentate gyrus (soul., 2008) and both arc and camkii inactivation in mutant mice have deficient object recognition memory (miller., 2002 ; plath., 2004) and via the pi3k - akt pathway (horwood., 2006) and one of its downstream target mtor involved in mrna translation (myskiw., 2008), as well as nmda and ampa receptors and several neuromodulators (reviewed in dere., 2007) have also been implicated in object recognition memory. conversely, forebrain expression of a calcineurin phosphatase inhibitor as well as nuclear protein phosphatase 1 inhibition in transgenic mice were both shown to enhance long - term object recognition memory (malleret., 2001 ; epigenetic regulation involving chromatin remodeling through histone post - translational modifications and dna methylation has also been implicated as a mechanism for gene transcription in recognition memory. for example, histone post - translational modifications at the creb promoter, were observed after object exploration (koshibu., 2009). the histone acetyltranferase creb binding protein cbp is required for recognition memory (barrett and wood, 2008 for a review) and histone deacetylase inhibition enhances long - term recognition memory (stefanko., 2009). finally, it is interesting to note that many, although not all (poirier., 2007b ; daoud., 2008), mice models of human x - linked mental retardation syndromes due to gene mutation have profound deficits in object recognition memory (alarcn., 2004 ; vaillend., 2004 ; ventura. 2008). while several, although not all, of the above experiments have directly targeted the hippocampus, either by monitoring protein or gene activation after learning or via the use of hippocampal - specific gene deletion, a wide range of these molecular mechanisms were also shown to be engaged for recognition memory in other structures such as the perirhinal cortex (reviewed in winters., 2008). remarkably, all the molecular mechanisms thus far demonstrated to be critical for the consolidation of recognition memory are known mediators of synaptic long - term potentiation (ltp). this suggests that recognition memory, like many other forms of memory, engages synaptic plasticity. clarke. (2010) recently reported evidence for this, showing that object exploration induces slow development of nmda - dependent synaptic potentiation at the schaffer collateral pyramidal cell synapse in mice, a phenomenon occluded by prior induction of ltp, resulting in memory deficits. a further point of interest concerns the potential role of hippocampal neurogenesis in recognition memory. it is now clearly established that new neurons are continuously generated in the dentate gyrus of the hippocampus and the past few years have witnesses accumulating evidence that young, newly generated dentate gyrus neurons play a significant role in several forms of hippocampal - dependent memories (e.g., ming and song, 2005 ; bruel - jungerman., 2007 ; indirect evidence for a role of neurogenesis in object recognition memory first came from a study demonstrating a neurogenesis - dependent effect of environmental enrichment on the enhancement of long - term recognition memory (bruel - jungerman., 2005). (2009) recently demonstrated that strong, but not partial, reduction of dentate gryus neurogenesis results in severe impairment in object recognition memory. the first evidence that an object recognition memory can undergo reconsolidation after recall came from two experiments that examined what happens after a recall test in conditions of blocking mapk / erk activation or of inactivating zif268. in both conditions, the reconsolidation protocol consisted, one or several days after training, in a brief reactivation episode during which animals were placed back in the training environment with the same objects as those they have experienced during the training phase, followed 1 day later by a standard test during which one object was changed for a novel one. in the first experiment it was found that icv injection of a mek inhibitor just prior to memory reactivation did not affect post - reactivation short - term memory but completely abolished the long - term post - reactivation memory that was observed in control rats (kelly., 2003). these results suggested that memory for objects can be destabilized upon reactivation and that a process of reconsolidation, in this case dependent on mapk / erk activation, is required for the memory to be available for further long - term recall. at the same time, similar conclusions were reached from analyzing the effect of memory reactivation in zif268 knockout mice. in this case, mice had first to be over - trained to override their consolidation deficit in the standard task so that they could form a long - term memory of objects. then, when a brief reactivation trial was interposed between training and testing, long - term, but not short - term, post - reactivation memory was completely abolished (bozon., 2003a). further, the impairment after a brief reactivation was found to be specific to reactivation with the previously memorized objects in the relevant context ; no impairment was observed when two novel objects were presented in the training context during the reactivation trial, or when the two previously experienced objects were presented in a novel and entirely different context. moreover, the training context alone, which can often serve as a reminder cue, was not in this case an effective cue to trigger reactivation of the target object memory, suggesting that a zif268-dependent reconsolidation process acts on an configural memory associating attributes of the objects and of the context. finally, we examined the temporal constraints on the requirement for zif268 in reconsolidation by varying the delay between training and reactivation, or between reactivation and test. both recent (1 day) and relatively remote (4 days) memories were found to be subject to zif268-dependent reconsolidation after recall and there was no apparent spontaneous recovery of the memory for at least several days (bozon., 2003a). the evidence that an object recognition memory can undergo reconsolidation after recall was recently confirmed using protein synthesis inhibition (romero - granados., 2010). in this study, systemic injection of anisomycin in mice just before or up to 4 h after memory reactivation using a similar test as above resulted in an impairment of post - reactivation long - term memory, leaving short - term memory intact. remote (21 days) object memory was also found to be susceptible to disruption by protein synthesis inhibition during recall. object memory reconsolidation can also be disrupted by systemic injection of an nmda receptor antagonist (winters., 2009). in this case, however, reconsolidation of older or stronger memories became resistant to disruption by the nmda antagonist unless a novel and salient contextual cue is present during reactivation. in all, these results provide evidence that at least part of the same transcriptional and translational mechanisms involved in consolidation of object recognition memory are also engaged after recall and required for that memory to be available again for a further recall. electrophysiological recordings of synaptic potentials at the schaffer collateral - ca1 synapse during and after a retention test consisting in presenting a familiar with a novel object revealed a transient depression of synaptic potentials, possibly reflecting a destabilization mechanisms, followed by the slow development of synaptic potentiation similar to that observed after training (clarke., 2010). this data suggest that at least this pathway undergo a similar form of synaptic change after both training and recall. the issue of whether or not both consolidation and reconsolidation of recognition memory engage the same brain circuits remains however debated. for example, protein synthesis inhibition in entorhinal cortex affects consolidation but not reconsolidation of object memory (lima., 2009), while in the ventromedial prefrontal cortex, protein synthesis inhibition, as well as nmda blockade or mapk / erk inhibition were shown to block both consolidation and reconsolidation of object memory (akirav and maroun, 2006 ; maroun and akirav, 2009). in our own mapk / erk studies, we found that learning about objects induces mapk / erk activation in the entorhinal cortex and dentate gyrus, while memory reactivation resulted in mapk / erk activation in entorhinal cortex and ca1, suggesting that cortico - hippocampal circuits engaged in consolidation and reconsolidation are at least in part distinct (kelly., bdnf and zif268 mrna expression were reported in the entorhinal and prefrontal cortices after object sampling, while in this experiment reactivation 9 days after training induced zif268 in entorhinal and somatosensory cortices and upon reactivation 21 days after training, bdnf and not zif268 was expressed in hippocampus and entorhinal and somatosensory cortices (romero - granados., 2010). thus, although certain circuits and mechanisms are common to both processes, there is not a strict match between the circuits engaged after training and recall of object memory and some of the signaling mechanisms seems to differ. it remain however difficult to construct a map of the mechanisms and circuits involved and several factors could account for some of the differences, such as details of the experimental procedure, strength of training, age of the memory, or the temporal dynamic of molecular changes. interestingly, recent evidence suggests reconsolidation of object memory can also be observed in humans. in an experiment where subjects were presented a first list of objects and then reminded or not before the presentation of a second list of objects, it was found that the reminder induced a misattribution of newly encoded objects (from list 2) into the reactivated list 1, suggesting that reactivation induced a reconsolidation process that served to modify the original object memory and update it with incorporation of objects belonging to list 2 (hupbach., 2009). experiments so far have demonstrated that object recognition memory can be destabilized after recall, requiring re - stabilization to re - enter a long - term store via a process that involves some but not all of the molecular mechanisms and brain circuits that are engaged in initial consolidation. here we report novel experiments that extend these findings and demonstrate that object - place recognition memory is also subject to reconsolidation after recall. the first experiment examined the potential role of the ribosomal s6 kinase rsk2 in consolidation and reconsolidation of object - place recognition memory. the rsk2 gene encodes a serine / threonine kinase that is activated by and acts downstream of mapk / erk via a dual function in cre - mediated transcriptional regulation and in chromating remodeling by phosphorylating histone h3. in humans, rsk2 gene mutations are responsible for a very handicapping x - linked form of syndromic mental retardation, the coffin - lowry syndrome (reviewed in hanauer and young, 2002 ; pereira., 2010). in a previous experiment, we found that rsk2 mutant mice have mild impairments in spatial working memory, delayed acquisition, and long - term memory deficits in spatial reference memory, but normal long - term object recognition memory (poirier., 2007b). thus, we examined whether rsk2 might have a more prominent role in the more demanding spatial version of recognition memory, object - place recognition. rsk2 and wild - type (wt) littermates were trained in a circular open - field covered with sawdust and containing three different objects constructed from assembling lego pieces. a cardboard cue was placed on the wall of the open - field to serve as a spatial landmark in addition to the multiple visual cues present in the environment. after habituation to the empty open - field for 2 days, rsk2 and wt mice were given three 5-min trials of exploration of the objects with an inter - trial interval of 5 min. retention was tested 2 days later during a single 5-min trial by moving one of the objects to a new position. in the reconsolidation experiment, 1 day after training the mice were briefly re - exposed for 5 min to the three objects placed as in the training phase, and retention was tested 1 day later by moving one object to a new location. analysis of the time spent exploring the displaced object revealed that rsk2 deficiency did not cause any observable impairment in long - term object - place memory (figure 1a). in the reconsolidation experiment, wt mice explored the displaced object significantly more than the two non - displaced objects (figure 1b), demonstrating a similar recognition performance to that when no reactivation was interposed. surprisingly, however, while post - reactivation short - term memory was intact in rsk2 deficient mice, post - reactivation long - term memory was completely abolished (figure 1b). these findings demonstrate that object - place memory is subject to a rsk2-dependent reconsolidation process following memory reactivation and provide an example of a divergence between mechanisms of consolidation and reconsolidation of recognition memory by showing that the signaling molecule rsk2 is at least more prominently implicated in object - place memory reconsolidation than in object - place memory consolidation. reconsolidation, but not consolidation of spatial, object - place recognition memory is impaired in rsk2 mutant mice. (a) rsk2 mutant mice showed no deficit in long - term spatial recognition memory (ltm) over 48 h as they showed preferential exploration of the displaced object (n = 13 ; t = 3.53 ; p = 0.0041) as did wild - type (wt) mice (n = 13 ; t = 9.83 ; p = 0.0001), with no significant difference in the amount of time spent exploring the displaced object between wt and mutant mice (f1,24 = 0.416 ; p = 0.525). (b) in contrast, 24 h after reactivation of the memory, rsk2 mutant mice showed a deficit as they displayed no preference for the displaced object (t = 1.43 ; p = 0.17) as opposed to the wt mice (t = 14.61 ; p = 0.0001) ; and the level of exploration of the displaced object was significantly greater in wt mice compared with mutant mice (f1,24 = 70.753 ; p ordinates : percent time spent exploring the displaced object over the mean of the time spent exploring the two non - displaced objects. in the second experiment to examine whether object - place recognition memory can become destabilized after recall and requires a process of reconsolidation to maintain the memory for further use, we tested zif268 mutant mice, which have been previously shown to be impaired in object memory reconsolidation (bozon., 2003a). as in the bozon and colleagues experiment, we first tested whether zif268 mutant mice could form a long - term object - place memory if given additional exposures to the objects in a distributed training paradigm, a precondition to examine the potential role of zif268 in reconsolidation. wt and zif268 mutant mice were given four blocks of two 5-min trials of exploration of two different objects with a within - block inter - trial interval of 5 min and a 90-min interval between blocks, and retention was measured 2 days later by moving one of the objects to a novel location. both wt and zif268 mutant mice showed preferential exploration of the displaced object (figure 2a), thus demonstrating the mice can form a long - term object - place memory in conditions of extended and distributed training. we were thus able to explore the effect of a brief reactivation trial (a single 5-min session with the objects in the same locations as during training), interposed at a 1-day interval between training and retention. when post - reactivation short - term memory was tested, both wt and zif268 mutant mice showed preferential exploration of the displaced object (figure 2b, pr - stm). one day after reactivation, wt mice also explored significantly more the displaced object (figure 2b, pr - ltm), demonstrating a similar recognition performance to that when no reactivation was interposed. in contrast, zif268 mutant mice showed equal exploration of the two objects (figure 2b). these findings demonstrate that a consolidated and stable object - place recognition memory can again become labile after brief reactivation and zif268 mutant mice can not in this case reconsolidate the object - place memory. thus a zif268-dependent reconsolidation process is similarly required after an object memory or an object - place memory is recalled. (a) the mice were exposed to a spatial configuration of two objects for eight consecutive sessions (overtraining) on day 1 to alleviate their consolidation deficit and retention was tested 2 days later. in this condition, zif268 mutant mice had normal object - place recognition long - term memory (ltm) as they showed preferential exploration of the displaced object (n = 5 ; p 0.05). (b) when zif268 mutant mice were briefly re - exposed to the familiar configuration of objects 24 h after training, post - reactivation short - term memory (pr - stm) was intact (left panel). both wt and zif268 mutant mice preferentially explored the displaced object (p 0.05). in contrast, post - reactivation long - term memory (pr - ltm) was impaired in zif268 mutant mice (right panel). while wt mice preferentially explored the displaced object (n = 5 ; p 0.05) and there was significant difference between groups (f1,8 = 6.52 ; p < 0.05). ordinates : percent time spent exploring the displaced object over the time spent exploring the non - displaced object. the demonstration that many forms of memory can be subjected to reconsolidation after recall has opened a new era in memory research, highlighting its dynamic and reconstructive nature. in a little over a decade, a considerable number of studies have been conducted on different aspects of reconsolidation in an attempt to understand the conditions under which this process occurs, what its function is and what the underlying mechanisms are. whereas a large number of these studies have used fear - associated or negatively reinforced memories, relatively few to date have addressed the issue of what happens after recall of forms of recognition memory that are based on incidental encoding with no explicit reinforcement. yet, recognition memory is a necessary component of nearly all forms of memories, be they pathological or normal. as reviewed in the preceding sections, it is now clearly established that both object and object - place recognition memory can become destabilized after recall and would then require reconsolidation to remain available for further recall. the evidence comes from several sources demonstrating that when signaling molecules or genes implicated in the type of synaptic plasticity believed to underlie memory stabilization are interfered with at the time of memory reactivation, the memory of a familiar object or of a familiar object - place association is lost. the findings thus extend to recognition memory the theoretical account put forth by lewis (1979), suggesting that memory of past encountered objects can return to a labile state and become vulnerable to disruptive factors after recall before eventually being re - stabilized as a result of their reactivation. what are the conditions under which recognition memories undergo reconsolidation after recall ? clearly, there is still a lack of behavioral experiments on recognition memory to come to a detailed account on the issue. conditions affect reconsolidation of both recent and relatively remote recognition memory, although this type of memory is usually far less enduring than negatively reinforced memories, which makes it impractical to test over a long time range. there is also some evidence to suggest that the apparent amnesia after reconsolidation blockade is not reversible. it is not known however whether this is a complete loss of all items of information that the episode includes or not. cueing experiments after reconsolidation blockade could provide valuable information as to whether or not some elements of the memory are spared, and could help attack the general issue of whether the deficit in performance reflects impaired re - stabilization of the memory or rather affects retrieval processes, which is not better resolved in the case of recognition memory than it is for other forms of memories. there is also indication that to be susceptible to disruption, reactivation of object recognition memory may have to be prompted by presenting the target memory (the objects) in the relevant context. a peculiarity of the experimental design used for reactivating recognition memory is that the reactivation session consists in presenting the same, and whole event that was present during the encoding phase, whereas in many other types of learning partial information alone (e.g., the conditioned stimulus, the context associated with a reinforcer) can reactivate the memory without the need for giving the reinforcer. this may be due to the importance of context in many paradigms of recognition memory in the absence of explicit reinforcement, apart for the natural, ecologically relevant tendency of rodents to attend to, and explore novel items in their environment. it does not preclude, however, the possibility that a past object memory can be reactivated in conditions of only partial similarity between initial training and reactivation, as shown in human studies (hupbach., 2007). inherent in recognition memory tests is the fact that during reactivation, as well as during testing with a novel object or a displaced object, memories of the objects or their location that are first encountered must be reactivated as a means of determining whether they are familiar or novel. evidence in humans and rodents suggest that the hippocampus plays a key role in novelty detection by acting as a comparator between past and current overlapping, or similar events (kumaran and maguire, 2007 ; lever., 2010). computational models suggest this is achieved by a match - mismatch comparator system to generate orthogonal memory representations via a pattern separation function, which may occur in the object recognition test when an object or location of an object is changed. in order to make comparisons between familiar and novel events, the comparator system also requires a mechanism whereby stored representations can be fully recalled in response to a minimal amount of input cue via pattern completion (lee and kesner, 2004 ; rolls, 2010). at present, given the procedure used for reconsolidation in rodents, it seems that a match condition is required for post - retrieval amnestic treatments to destabilize the memory, however, further experiments will be needed to explore whether a non - match condition either does not destabilize the past memory, rendering reconsolidation unnecessary, or does destabilize it in a manner that is less sensitive to treatments. consistent with the latter possibility, in a previous experiment investigating ca1 place cell representations in zif268 knockout mice (renaudineau., 2009), we found that the mice have deficits in stabilizing long - term place cell representations of a novel environment containing an object, which is consistent with their impaired long - term spatial memory, and also that an established place cell representation of a well - experienced environment (corresponding to an overtraining procedure) can be destabilized by placing the mice in a slightly different environment. thus, the formation of the new representation interfered with the ability to later reactivate the representation of a familiar environment. this did not result in a complete loss however, but lead to rotational remapping by the place cell system associated with rate change, indicating an incorrect orientation of the place cell map corresponding to the familiar representation, rather than creation of a totally new representation. these findings suggest the memory of the familiar environment was destabilized, although further successive exposures to the familiar and novel environments progressively suppressed this destabilizing effect of the now less novel environment (renaudineau., 2009). one possible explanation is that formation of the new representation triggers a form of reconsolidation that renders the previously formed representation labile and vulnerable to interference ; the absence of zif268 in this case preventing proper re - stabilization of the former representation. relevant to this, a recent fmri study in normal subjects submitted to object lists learning provided evidence for reactivation of a previously encoded memory during the encoding of a new, partially overlapping list and suggested the hippocampus takes part in reactivating the older memory when a new memory is formed, limiting the forgetting of the older memory (kuhl., 2010). within this framework, it is possible that the absence of zif268 in the hippocampus was deleterious to a process of reconsolidation of the reactivated older representation. the issue of whether or not consolidation and reconsolidation engage some of the same biochemical / molecular mechanisms has been raised since the start of research on reconsolidation, in particular because most treatments used in reconsolidation studies target molecular mechanisms known to be involved in consolidation of new memories. from the studies reviewed above first, there is evidence that consolidation and reconsolidation of recognition memory engage common mechanisms, including nmda receptor activation, mapk / erk phosphorylation, zif268 transcriptional regulation, and protein synthesis. there are also similarities in the mechanisms involved in reconsolidation of both object and object - place memories. however, there are also arguments to suggest that certain mechanisms may be more specific to one or the other process, as exemplified by the prominent requirement of rsk2, a mapk / erk substrate, in reconsolidation but not consolidation of object - place recognition memory. second, some of the studies directed at exploring the cellular and molecular mechanisms of consolidation and reconsolidation of recognition memory that were based on detecting activation of specific cell - signaling cascades in different neuronal populations or using structure specific genetic deletion further emphasizes the implication of the hippocampus in both object and object - place recognition memory. however, several examples indicate that the brain structures within which some of these molecular mechanisms take place, including within cortico - hippocampal circuits, do not completely overlap between consolidation and reconsolidation of recognition memory. admittedly, the available data on the mechanisms and brain circuits engaged in consolidation and reconsolidation of recognition memory remains too limited to draw firm conclusions. in addition, at this point these data do not tell us what specific function(s) the hippocampus may implement in recognition memory consolidation or reconsolidation, nor do they tell us whether reconsolidation strengthens or updates the memory. future research to achieve a more complete understanding of the cellular and molecular mechanisms and of the brain circuits engaged in reconsolidation of recognition memory will serve to further our knowledge of the function of these circuits in reconsolidation and of the function of reconsolidation in the makings of memories. determining how neuronal firing patterns in different brain regions encode independent aspects of recognition memory, such as individual items, ensembles of items, spatial, and non - spatial context, and the temporal order in which events occur, could be one possible route into gaining a better understanding of the dynamics of the processing, recall, and the circumstances in which memory for incidental learning may require a process of reconsolidation. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	the idea that an already consolidated memory can become destabilized after recall and requires a process of reconsolidation to maintain it for subsequent use has gained much credence over the past decade. experimental studies in rodents have shown pharmacological, genetic, or injurious manipulation at the time of memory reactivation can disrupt the already consolidated memory. despite the force of experimental data showing this phenomenon, a number of questions have remained unanswered and no consensus has emerged as to the conditions under which a memory can be disrupted following reactivation. to date most rodent studies of reconsolidation are based on negatively reinforced memories, in particular fear - associated memories, while the storage and stability of forms of memory that do not rely on explicit reinforcement have been less often studied. in this review, we focus on recognition memory, a paradigm widely used in humans to probe declarative memory. we briefly outline recent advances in our understanding of the processes and brain circuits involved in recognition memory and review the evidence that recognition memory can undergo reconsolidation upon reactivation. we also review recent findings suggesting that some molecular mechanisms underlying consolidation of recognition memory are similarly recruited after recall to ensure memory stability, while others are more specifically engaged in consolidation or reconsolidation. finally, we provide novel data on the role of rsk2, a mental retardation gene, and of the transcription factor zif268/egr1 in reconsolidation of object - location memory, and offer suggestions as to how assessing the activation of certain molecular mechanisms following recall in recognition memory may help understand the relative importance of different aspects of remodeling or updating long - lasting memories.
various epidemiological data suggests that malaria is still a major public health concern in an unindustrialized countries and is associated with high rates of morbidity and mortality throughout most of the tropics (1) and malaria also the second most frequent clinically suspected disease entity after acute respiratory tract infection (2). in pakistan, with an estimated 1.5 million cases annually reported and according to w.h.o malaria has re - emerged as a major cause of morbidity in pakistan (3). review of previous literatures showed that approximately 3 billion people living in 108 countries who are exposed, approximately 300 - 500 million people develop symptomatic malaria annually (4). in tropics plasmodium falciparum is the major cause of malaria associated mortality, ranging from 65% - 95% (5) as compare to p. vivax and p. knowlesi(6,7). many risk factors are associated in the transmission of malaria among them, mass population movements within the country and across international borders with iran and afghanistan, unpredictable transmission patterns, low immune status of the population, climatic changes, poor socioeconomic conditions, declining health infrastructure, resource constraints, poor access to preventive and curative services and mounting drug and insecticide resistance in parasites and vectors all contribute to this huge disease burden in pakistan (8). directorate of malaria control has reported that one person per thousand in the population is infected with malaria. active malarial transmission happens through - out the year, while aggressive out bursts of disease are seen mainly during and after the monsoon ' season. people residing in those areas are at an increased risk for malaria related mortality (9). in pakistan, severe flooding followed by heavy rains submerged approximately one - fifth of the total land area under water. this affects around 20 million people, mostly by destruction of property and with high death rates due to spread of diseases like dysentery and malaria. keeping in view the increased susceptibility of getting infected with malaria due to flood we aimed to conduct this study on determining the morbidity and mortality associated with malaria during monsoon flood at isra university hospital, hyderabad. this prospective observational study was conducted from july 2011 to october 2011 at isra university hospital (iuh), hyderabad, pakistan. iuh is a 300 bedded private, tertiary care academic teaching hospital that largely serve the residents of hyderabad (population 2 million) and surrounding 6 - 8 districts of sindh province. patients included in the study were adults (age 14 years) man and women diagnosed with malaria during the period of recent monsoon flood of 2011 (july 2011-october 2011). the study protocol was evaluated and approved by the hospital authorities, from where all the study participants were recruited and study was conducted in accordance with the declaration of helsinki guidelines. all the individuals provided informed consent before their participation. we recruited all patients with symptoms of malaria such as fever, vomiting, headache, and muscular pain etc. diagnosis of malaria was made on the basis of careful history and complete clinical examination. diagnosis was confirmed by performing rapid antigen testing i.e. immuno - chromatographic malaria parasite test (ict mp) and or identification of malaria parasite and its species on thick and thin film smear stained with giemsa stain. during the four months period which constituted 883 numbers of patients among them once the diagnosis of malaria made, investigations were performed (complete blood count, estimated sedimentation rate, serum creatinine, random blood sugar level, liver functions test, and serum electrolytes) to further evaluate the patients for malaria related complications. severity related variables such as degree of temperature, length of hospital stay, level of consciousness, associated complications such as pneumonia, seizures, and acute renal failure and associated co - morbid i.e. diabetes mellitus, hypertension, and pregnancy. diagnosed patients of malaria were treated with anti - malarial drugs, those who were able to took orally, givenchloroquine, artemether, and quinine and those who were unable to take orally (unconscious patients or severely distressed) treated parentally. patients were followed clinically as well as biochemically for clearance of parasite and complications during their hospital stay. data were recorded in standardized data sheet and analyzed in statistical package for social sciences 16.0 version. results are expressed as mean + standard deviation for continuous variables and number (percentage) for categorical variables. univariate analysis was performed by using the independent sample t - test corresponding to difference of means and pearson chi - square or fisher 's exact test corresponding to proportions whenever appropriate. odds ratio (or) and 95% ci (confidence interval) were estimated to identify the strength of association with independent factors. p - value 4 - mg / dl. the poor outcome with raised serum creatinine level malaria during pregnancy is a major contributor in increasing rate of mortality ranging from 0.5% - 23% (20, 21). in our study, there is an upward trend noticed with a very high percentage (35%) and significant (p = 0.005) relation between pregnancy and death due to malaria. malaria associated maternal death rate in an international data represent comparatively low percentage (8.5%) than ours (22). this low percentage in their setup could be because of early detection of the disease and start of empiric treatment on time. patients who develop complications due to malaria are at very high risk of having treatment failure and increase susceptibility of death. a substantial proportion in our study who develop septicemia, acute renal failure, and pneumonia, (64.3%, p = 0.001), (50%, p = 0.04), and (21.4%, p = 0.04) showed significant association between mortality due to complications of malaria these findings can be approved by the data published in the past as acute renal failure (23;24) septicemia, and pneumonia (25) are associated with poor outcome of the disease. most important limitations which can cause bias in this study are smaller sample size and hospital based study which may be unable to reflect actual incidence and mortality of malaria. since iuh is a private tertiary care hospital, strata belonging to lower socioeconomic could not have an easy access due to the cost factor. this study shows a significant increase in the morbidity and mortality in patients with malaria after flood. the probability of getting poor outcome is also associated when patient develop complications and patients with pregnancy. the data on malaria morbidity and mortality after flood on larger sample size is needed to validate the results of this study. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.	abstractbackgroundmalaria is the second most frequent clinically suspected disease entity after acute respiratory tract infection in developing countries. active malarial transmission occurs throughout the year, while aggressive out bursts of disease are seen mainly during and after the monsoon season. this study aimed to determine the morbidity and mortality associated with malaria during flood at isra university hospital, hyderabad.methodsthis prospective observational study was done at isra university hospital hyderabad during monsoon flooding from july 2011 to october 2011. all 883 patients presented with symptoms of malaria (fever, headache, and vomiting) were evaluated and diagnostic tool ict - mp was used for the detection of malaria parasite among them.resultsseventy four (8.38%) patients diagnosed for malaria. the mean age and sd was 30.11 1.67 years. overall mortality due to malaria observed (18.9%). mortality rate significantly observed high in pregnant women (0.005) and in those patients who developed complications such as, pneumonia (p = 0.04), renal failure (p = 0.04), unconsciousness (p = 0.001), and septicemia (p = 0.001).conclusiona significant increase in the morbidity and mortality in patients with malaria after flood noticed. the probability of getting poor outcome is also associated when patient develop complications.
sports and other exercise - related activities are a major cause of soft tissue and joint injuries, resulting in sprains, strains, and contusions. the ankle is frequently traumatized during sports activities, which account for an estimated 20% of all sports - related injuries.1 nonsurgical treatment of ankle sprains is predicated on prevention of further injury, reduction of inflammation, and management of pain.1,2 traditional oral nonsteroidal anti - inflammatory drugs (nsaids) are used to provide analgesia and relief from inflammation in patients with acute ankle sprain. however, use of these drugs can lead to adverse reactions associated with the upper gastrointestinal tract, and renal, cardiovascular, and respiratory systems, mainly by reduction of systemic prostaglandin production through direct inhibition of cyclooxygenase enzymes.3 in contrast, topical nsaid preparations permit direct application of drug at the site of pain. direct application results in continuous and localized drug delivery to the pain site, while minimizing systemic levels of drug (0.2% to 8% of the oral equivalent), thereby reducing the local inflammatory reaction while ostensibly avoiding systemic adverse events.4 the diclofenac epolamine topical patch (detp) is a topical nsaid patch with demonstrated analgesic activity.5 prior studies have shown that topical diclofenac can be transported across the skin into the injured site, maintaining measurable levels of the drug in synovial fluid, while providing a constant plasma level of diclofenac for up to 12 hours.6,7 because the efficacy of the detp has been demonstrated in patients treated for minor sports injuries in prior studies,810 this clinical trial was performed to establish its efficacy using a more rigorous methodology. the aim of this placebo - controlled clinical study was to demonstrate the efficacy and tolerability of the detp applied once per day for seven days in the treatment of minor acute ankle sprain. this phase iii, multicenter, double - blind, randomized, parallel - group, placebo - controlled trial, assessed the efficacy and tolerability of the detp in the treatment of minor ankle sprain. the study protocol was approved by the french ethical committee, and was conducted in accordance with the declaration of helsinki guidelines. male and female patients, aged 1865 years, who sustained an ankle sprain < 48 hours before study entry and had a pain score 50 mm on a 100 mm visual analog scale were included in this study if the injury justified use of a seven - day nsaid topical treatment. patients were excluded from the study if the ankle sprain required orthopedic or surgical treatment, the sprain had been treated prior to study entry, they had been treated for other sprains within 1 week of the study, or if they had a history of recurrent sprains. female patients who were pregnant, lactating, or of childbearing potential were excluded from this study. in addition, patients were excluded if they had : an allergy to aspirin, nsaids, or the excipients in the detp ; a history of skin allergy ; or an open skin lesion within the injured area (eg, eczema, psoriasis). finally, patients being treated by physiotherapy or an alternative medicine (eg, acupuncture, mesotherapy, homeopathy) or who had received oral or parenteral treatment with corticosteroids, nsaids, or aspirin seven days prior to study entry, or had received any analgesic within six hours of patient recruitment were also excluded. the study was discussed with each of the prospective patients, and those wishing to participate signed an informed consent form detailing the entire study. after signing an informed consent, each study patient was randomly assigned, in blocks of four and in increasing order, a study number corresponding to the preassigned number on a packet of blinded test articles (ie, the detp or placebo patch). all patients randomized to the active treatment received two boxes, each box containing five detps, sufficient for a seven - day treatment, with a surplus of three patches. patients who were randomized to the placebo group received two boxes, each containing five placebo patches. placebo patches were identical in appearance and used the same formula as the active patch, without the active ingredient, diclofenac epolamine. patients in both the detp and placebo groups applied a single daily patch to the site of injury in the morning, for seven days. the application of ice or protective wrap was allowed in both groups and recorded. if necessary, the intake of paracetamol was authorized as a rescue medication three hours after the first application of a patch ; the consumption in both groups was recorded. the primary efficacy variable was spontaneous pain (pain on active mobilization), which was assessed by the patient on a 100 mm huskisson - type visual analog scale ranging from 0 = no pain to 100 = severe pain. spontaneous pain, derived from the patient diary, was evaluated at the following times : on day 0 at the first six hours after application of the initial patch and at 8 pm, on days 1 and 2 at 8 am, 12 pm, and 8 pm, and on day 3 at 8 am. at the end of treatment, patients were considered improved and therapy efficient if the reduction in visual analog scale value between entry into the study and study termination was 20 mm or if the percentage change from baseline in visual analog scale value was 30%. an approximately 30% improvement in pain score has been considered to be a clinically important difference in treating acute pain.11 secondary efficacy variables included an evaluation of the analgesic effect, an assessment of the anti - inflammatory effect, and a patient assessment of treatment efficacy. the analgesic effect was assessed using the following four parameters : pain at rest according to a four - point verbal scale of 0 = no pain, 1 = slight pain, 2 = medium pain, and 3 = severe pain ; pain on passive stretch and pain on pressure (external lateral ligament, front and medium fascicle) according to the same pain at rest scale ; and possibility of single foot leaning on the affected foot according to a three - point scale of 0 = hold possible without any pain, 1 = hold possible but painful, and 2 = hold impossible. the anti - inflammatory effect was determined by assessing periarticular edema / swelling, which was evaluated by measuring, in mm, the difference in circumference between the healthy and impaired ankle, using a measuring tape. the safety measurements included an assessment of tolerability by the patient and physician, and a recording of adverse events. each patient who completed the protocol was exposed continuously to one patch applied to the painful area, with patch renewal once daily for a period of one week. a global tolerability assessment was done on day 3 and day 7 by both the patient and physician. the assessments were conducted using a four - point verbal scale, where none indicated little tolerability and excellent indicated the highest levels of tolerability. the patient and physician assessments on general tolerability were reported at the end of treatment on a four - point scale of excellent, good, fair, and bad. all patients who used a study patch were to be included in the intent - to - treat analysis. patients who stopped treatment for reasons of failure, worsening, healing, or disappearance of symptoms were part of the efficacy analysis, with data from the last observation carried forward. the measured parameter visual analog scale at the study visits was analyzed by a two - way analysis of variance (anova) with factors for treatment group and time, and an anova with one factor (time) within the groups. categorical variables (ie, single foot leaning ; pain on pressure, at rest, or on passive stretch ; and assessments by patient and physician on efficacy and tolerability) were analyzed using the mantel - haenszel chi - square or fisher s exact test. mean vas scores were summarized at each patch application using a last observation carried forward (locf) method for imputing missing data. this procedure assumes that the subject s response would have been constant from the last observation to the next, or to the end of the trial. in general, this is thought to be a highly conservative approach that may underestimate therapeutic benefits in a longitudinal study, such as this one. safety analyses included all patients enrolled in the study, and compared the number of patients with specific adverse events, total adverse events, and dropout due to treatment intolerability between groups. male and female patients, aged 1865 years, who sustained an ankle sprain < 48 hours before study entry and had a pain score 50 mm on a 100 mm visual analog scale were included in this study if the injury justified use of a seven - day nsaid topical treatment. patients were excluded from the study if the ankle sprain required orthopedic or surgical treatment, the sprain had been treated prior to study entry, they had been treated for other sprains within 1 week of the study, or if they had a history of recurrent sprains. female patients who were pregnant, lactating, or of childbearing potential were excluded from this study. in addition, patients were excluded if they had : an allergy to aspirin, nsaids, or the excipients in the detp ; a history of skin allergy ; or an open skin lesion within the injured area (eg, eczema, psoriasis). finally, patients being treated by physiotherapy or an alternative medicine (eg, acupuncture, mesotherapy, homeopathy) or who had received oral or parenteral treatment with corticosteroids, nsaids, or aspirin seven days prior to study entry, or had received any analgesic within six hours of patient recruitment were also excluded. the study was discussed with each of the prospective patients, and those wishing to participate signed an informed consent form detailing the entire study. after signing an informed consent, each study patient was randomly assigned, in blocks of four and in increasing order, a study number corresponding to the preassigned number on a packet of blinded test articles (ie, the detp or placebo patch). all patients randomized to the active treatment received two boxes, each box containing five detps, sufficient for a seven - day treatment, with a surplus of three patches. patients who were randomized to the placebo group received two boxes, each containing five placebo patches. placebo patches were identical in appearance and used the same formula as the active patch, without the active ingredient, diclofenac epolamine. patients in both the detp and placebo groups applied a single daily patch to the site of injury in the morning, for seven days. the application of ice or protective wrap was allowed in both groups and recorded. if necessary, the intake of paracetamol was authorized as a rescue medication three hours after the first application of a patch ; the consumption in both groups was recorded. the primary efficacy variable was spontaneous pain (pain on active mobilization), which was assessed by the patient on a 100 mm huskisson - type visual analog scale ranging from 0 = no pain to 100 = severe pain. spontaneous pain, derived from the patient diary, was evaluated at the following times : on day 0 at the first six hours after application of the initial patch and at 8 pm, on days 1 and 2 at 8 am, 12 pm, and 8 pm, and on day 3 at 8 am. at the end of treatment, patients were considered improved and therapy efficient if the reduction in visual analog scale value between entry into the study and study termination was 20 mm or if the percentage change from baseline in visual analog scale value was 30%. an approximately 30% improvement in pain score has been considered to be a clinically important difference in treating acute pain.11 secondary efficacy variables included an evaluation of the analgesic effect, an assessment of the anti - inflammatory effect, and a patient assessment of treatment efficacy. the analgesic effect was assessed using the following four parameters : pain at rest according to a four - point verbal scale of 0 = no pain, 1 = slight pain, 2 = medium pain, and 3 = severe pain ; pain on passive stretch and pain on pressure (external lateral ligament, front and medium fascicle) according to the same pain at rest scale ; and possibility of single foot leaning on the affected foot according to a three - point scale of 0 = hold possible without any pain, 1 = hold possible but painful, and 2 = hold impossible. the anti - inflammatory effect was determined by assessing periarticular edema / swelling, which was evaluated by measuring, in mm, the difference in circumference between the healthy and impaired ankle, using a measuring tape. the safety measurements included an assessment of tolerability by the patient and physician, and a recording of adverse events. each patient who completed the protocol was exposed continuously to one patch applied to the painful area, with patch renewal once daily for a period of one week. a global tolerability assessment was done on day 3 and day 7 by both the patient and physician. the assessments were conducted using a four - point verbal scale, where none indicated little tolerability and excellent indicated the highest levels of tolerability. the patient and physician assessments on general tolerability were reported at the end of treatment on a four - point scale of excellent, good, fair, and bad. all patients who used a study patch were to be included in the intent - to - treat analysis. patients who stopped treatment for reasons of failure, worsening, healing, or disappearance of symptoms were part of the efficacy analysis, with data from the last observation carried forward. the measured parameter visual analog scale at the study visits was analyzed by a two - way analysis of variance (anova) with factors for treatment group and time, and an anova with one factor (time) within the groups. categorical variables (ie, single foot leaning ; pain on pressure, at rest, or on passive stretch ; and assessments by patient and physician on efficacy and tolerability) were analyzed using the mantel - haenszel chi - square or fisher s exact test. mean vas scores were summarized at each patch application using a last observation carried forward (locf) method for imputing missing data. this procedure assumes that the subject s response would have been constant from the last observation to the next, or to the end of the trial. in general, this is thought to be a highly conservative approach that may underestimate therapeutic benefits in a longitudinal study, such as this one. safety analyses included all patients enrolled in the study, and compared the number of patients with specific adverse events, total adverse events, and dropout due to treatment intolerability between groups. a total of 134 patients were enrolled in the study by 24 investigators ; 68 patients were randomized to the detp group and 66 patients to the placebo group. all patients were included in the analysis of efficacy and safety (intent - to - treat analysis). a total of 127 patients completed the study as planned ; seven patients withdrew from the study before completion. these withdrawals were for pruritus, protocol violation, edema, or lack of efficacy. the mean age was 33.3 years in the detp group and 29.7 years in the placebo group (table 1). women in the placebo group were slightly younger than women in the detp group (p = 0.1), with a lower body mass index (23.6 kg / m detp versus 21.1 kg / m placebo, p = 0.05). a specific analysis that adjusted for bmi was performed and showed that there were no statistically significant differences between the treatment groups related to bmi. there were no differences between groups for application of ice by the patients before enrollment. in addition, no significant differences were found in initial measurements of pain or periarticular edema between the two groups. patients in both treatment groups demonstrated a prominent decrease in mean visual analog scale values over time. however, in the detp group the decrease in visual analog scale score from 66.9 on day 0 to 10.5 on day 7 was significantly greater compared with that in the placebo group, which decreased from 70.0 on day 0 to 18.4 on day 7 (p = 0.0008). additional analyses revealed that the mean decrease in visual analog score measured four hours after the first application was two - fold greater for the detp patients than for those given placebo (p = 0.02). the difference in relative change was significantly greater for the detp group at each time point, with the exception of day 1 at the 8 am time point, as shown by the median change from baseline (see figure 1). the success / failure analysis, with success defined as a visual analog score reduction of 20 mm between the day 0 consultation and the last known value, demonstrated that the rate of success was significantly higher in the detp group from four hours after the first application, and remained significant for six of the 10 measures until day 2 at the 8 pm time point. when success / failure was based on change in visual analog score (expressed as percent of baseline) and defined as (minimally) a 30% reduction in pain, the success rate was significantly higher in the detp group from four hours after the first application (36.8% detp versus 16.7% placebo, p = 0.01, n = 134), and remained significant for seven of the 10 measures until day 2 at the 8 pm time point. the secondary efficacy measures of pain at rest, on passive stretch, on palpation, and possibility of foot leaning were similar between the two groups on day 0. in contrast, on day 3 and day 7, the detp group showed a favorable response in analgesic effect that was statistically significant compared with the placebo group in each of the four analgesic efficacy measures (table 2). perimalleolar edema values did not show any significant difference between the treatment groups throughout the study. the detp and placebo patch were equally well tolerated on both day 3 and day 7, indicating consistent tolerance over time. no statistically significant difference was observed between the detp and placebo with either patient or physician assessment of tolerability on day 3 or day 7. two (3%) patients in the detp and three (4.5%) patients in the placebo group reported an adverse event (table 3). in both groups, patients in both treatment groups demonstrated a prominent decrease in mean visual analog scale values over time. however, in the detp group the decrease in visual analog scale score from 66.9 on day 0 to 10.5 on day 7 was significantly greater compared with that in the placebo group, which decreased from 70.0 on day 0 to 18.4 on day 7 (p = 0.0008). additional analyses revealed that the mean decrease in visual analog score measured four hours after the first application was two - fold greater for the detp patients than for those given placebo (p = 0.02). the difference in relative change was significantly greater for the detp group at each time point, with the exception of day 1 at the 8 am time point, as shown by the median change from baseline (see figure 1). the success / failure analysis, with success defined as a visual analog score reduction of 20 mm between the day 0 consultation and the last known value, demonstrated that the rate of success was significantly higher in the detp group from four hours after the first application, and remained significant for six of the 10 measures until day 2 at the 8 pm time point. when success / failure was based on change in visual analog score (expressed as percent of baseline) and defined as (minimally) a 30% reduction in pain, the success rate was significantly higher in the detp group from four hours after the first application (36.8% detp versus 16.7% placebo, p = 0.01, n = 134), and remained significant for seven of the 10 measures until day 2 at the 8 pm time point. the secondary efficacy measures of pain at rest, on passive stretch, on palpation, and possibility of foot leaning were similar between the two groups on day 0. in contrast, on day 3 and day 7, the detp group showed a favorable response in analgesic effect that was statistically significant compared with the placebo group in each of the four analgesic efficacy measures (table 2). perimalleolar edema values did not show any significant difference between the treatment groups throughout the study. the detp and placebo patch were equally well tolerated on both day 3 and day 7, indicating consistent tolerance over time. no statistically significant difference was observed between the detp and placebo with either patient or physician assessment of tolerability on day 3 or day 7. two (3%) patients in the detp and three (4.5%) patients in the placebo group reported an adverse event (table 3). in both groups, adverse events were of mild to moderate severity and generally involved the skin. the purpose of this study was to assess the analgesic efficacy of the detp in acute ankle injury. it is apparent from the efficacy data presented in this study that patients treated with the detp experienced a significantly greater reduction in pain associated with their ankle injury, determined from visual analog scale scores, beginning four hours after the first patch application and lasting (except for a single time point) for the remainder of the seven - day treatment period. in addition, patients in the detp treatment group experienced reduced pain at rest, on passive stretch, palpitation, and the possibility of single foot leaning on day 3 and day 7. the demonstrated benefits of topical therapy suggest that the origins of ankle pain in this case were primarily local. for patients who do not respond to local therapy, distant trigger points or other remote origins of pain analyses of safety data from this study are consistent with prior experience, with only five patients reporting adverse events of mild to moderate severity (four involving skin), and most of the patients in either treatment group having good / excellent product tolerability as judged by both the patient and investigator.8 an acknowledged shortcoming of this trial is that the comparison with placebo, which facilitated identification of treatment - related adverse events, precluded the use of an active analgesic comparator. additional studies should include comparison with other active analgesics. by comparison, in both seven - day and 14-day studies involving the detp for the treatment of acute pain, the incidence of adverse events related to the gastrointestinal system was similar for both the detp - treated and placebo - treated patients (0%10%).810 in contrast, studies comparing celecoxib with naproxen or ibuprofen for the management of acute pain reported gastrointestinal - related adverse events in up to 20% of patients in the celecoxib group and 25% of patients in the naproxen group.12,13 additional topical products approved in the us at this time include the lidocaine patch 5%, diclofenac sodium topical gel 1%, and diclofenac sodium topical solution 1.5%. significant improvements compared with baseline were seen for the lidocaine patch in several studies of osteoarthritis and low back pain.1416 because all of these studies were open label, the results should be interpreted with caution. the diclofenac sodium gel 1% is a prescribed topical gel that can be applied directly for osteoarthritis pain of the knee and hand, producing efficacious pain reduction.17,18 however, a study comparing diclofenac gel and a ketoprofen patch in sports - related soft tissue injuries revealed that patients preferred the patch application to the gel despite equivalent pain reduction.19 this study assessed the superior analgesic efficacy of the detp compared with placebo in minor sports injuries with a four - hour onset, while maintaining superior pain relief for the treatment duration. therefore, the detp has an important role as an alternative to traditional oral nsaids in the treatment of these injuries, without the adverse event profile of the oral agents.	background : sports - related injuries, such as sprains and strains, commonly occur during exercise and athletic events. current therapy includes nonsteroidal anti - inflammatory drugs (nsaids), which have a high incidence of upper gastrointestinal side effects. the present study assessed the efficacy and safety of the diclofenac epolamine topical patch (detp, 1.3%), a topical nsaid for the treatment of acute minor sprains and strains.methods:this multicenter, randomized, placebo - controlled clinical study enrolled adult patients (n = 134) with acute ankle pain (due to a minor sprain) occurring less than 48 hours prior to entering the study. patients were treated with either the detp or a placebo topical patch daily for seven days. pain intensity was evaluated during the first six hours after application of the patch, and on treatment days 1, 2, 3, and 7.results:patients treated with the detp experienced a significantly greater reduction in pain associated with their ankle injury compared with placebo, beginning four hours after the first patch application (p = 0.02). the detp was well tolerated and was comparable with placebo in terms of safety.conclusion:overall, the results of this study demonstrate that the detp is an effective analgesic for local treatment of pain in mild acute ankle sprain.
by presenting this case we aimed to describe an uncommon complication of generalized peritonitis following spontaneous pyometra perforation in untreated cervical carcinoma. this report describes a 60-year - old postmenopausal woman presenting with clinical features mimicking intestinal perforation who was later diagnosed as cervical carcinoma with pyometra perforation at exploratory laparotomy. spontaneous pyometra perforation in a case of untreated carcinoma of cervix is a rare condition, yet it should be suspected and kept in the differential diagnosis of acute abdomen in elderly women. generalized peritonitis in elderly women is a serious condition and finding an etiology is usually a challenge for surgeons. spontaneous perforation of pyometra is a rare cause of acute abdomen with only 28 cases reported and indexed in the english literature (16). furthermore, only six cases (including the present one) have been due to cervical cancer (1). in the case the etiology was diagnosed only intraoperatively. a high index of suspicion is required to make a correct preoperative diagnosis to allow early intervention, thereby reducing morbidity and mortality. a 60-year - old postmenopausal, para 2 woman presented with acute central abdominal pain for the past 12 hours along with abdominal distension. she also gave a history of mild fever for 3 days associated with chills but no rigors. there was no history of prolonged illness, medication use or intervention in the past. on examination, vitals were stable. abdominal x - rays showed free air under diaphragm with no distended bowel loops or air fluid levels. being an endemic region, a provisional clinical diagnosis of enteric perforation with subsequent peritonitis was made and the patient was planned for urgent exploratory laparotomy by surgeons. preoperatively, there was about 100 ml of pus with soft bulky uterus perforated at fundus which had sealed. bilateral thickened uterosacral and a short pelvic wall raised strong suspicion of cervical cancer and accompanying pyometra. on per speculum examination, contrast enhanced computerized tomography was done post operatively to assess tumor extent (figure 1) in consultation with a gyneco - oncologist to plan further treatment according to its staging. the patient received radiotherapy in view of advanced stage of cervical cancer and was placed under follow up. pyometra is an uncommon condition, but the incidence of associated malignancies is considerable, and the risk of spontaneous perforation is relatively high (2). pyometra represented 0.038% of gynecologic admissions in a retrospective study between 1993 and 1999 in two regional hospitals in a developing country (2). of these, 22.2% of the cases were associated with malignancy, 3.7% with genital tract abnormality and 74.1% were idiopathic (2). a preoperative diagnosis was correctly made in 77.3% of the patients without spontaneous perforation in their series. thus, preoperative diagnosis of malignancy can be often missed in cases presenting perforation later in the course of the disease as in our case. the common causes of pyometra in postmenopausal women are malignancies of genital tract and the consequence of their treatment (radiotherapy). pyometra was associated in 0.89% of cervical cancer patients in a large study of 3041 patients (7). other postmenopausal causes are benign tumor - like leiomyomas, endometrial polyps, senile cervicitis, cervical occlusion after surgery or idiopathic conditions (3). spontaneous rupture of carcinoma cervix associated pyometra resulting in peritonitis is an extremely rare complication with only 5 cases reported till date (8). the classic triad of symptoms in patients with pyometra consists of purulent vaginal discharge, postmenopausal bleeding and lower abdominal pain. symptoms being nonspecific, the diagnosis of pyometra is difficult unless suspected and specifically looked for. once diagnosed, a careful history and a detailed pelvic examination should be performed to rule out associated malignancies. our case illustrated one of the truly rare and unsuspected complications of carcinoma cervix associated pyometra : a presentation of a spontaneous perforation and subsequent peritonitis. most of the times, surgeons perform an exploratory laparotomy with provisional diagnosis other than pyometra perforation. on reviewing the indexed english literature, table 1 describes these cases grouped according to the etiology along with presenting symptoms and their outcomes. all cases were postmenopausal elderly females, mostly in the sixth to tenth decades of life. an analysis of preoperative diagnosis in the above cases hardly shows spontaneous perforation of pyometra in carcinoma cervix. the common preoperative diagnosis kept were generalized peritonitis (50%), perforation of gastrointestinal tract (40%) and pneumoperitoneum (30%). only 5 out of 28 cases including our case had untreated carcinoma cervix as the cause leading to spontaneous pyometra perforation. all the 5 described cases did not have a preoperative diagnosis of malignancy and were missed cases of carcinoma cervix as in our case (8). review of literature : the etiology of 28 cases of pyometra perforation along with their provisional diagnosis and outcome (16) abbreviations : yrs : years ; gp : generalized peritonitis ; pit : perforated gastrointestinal tract ; ppu : perforated peptic ulcer ; pp : pneumo - peritoneum ; drain & irrigation : drainage & irrigation ; subtotal hyst : subtotal hysterectomy ; tah : total abdominal hysterectomy ; panhyst : total abdominal hysterectomy & bilateral salpingo - opherectomy the diagnosis of pyometra perforation is usually one of exclusion. abdominal x - ray may show gas under diaphragm. confirmation is aided by ultra - sonography of the abdomen with fluid collection in the uterine cavity. urgent laparotomy should be performed. total hysterectomy along with bilateral salpingo - oophorectomy and thorough drain - age and irrigation of abdominal cavity remain the preferred immediate treatment in emergency. postoperatively, broad spectrum antibiotics and intensive care can help in good recovery followed by definitive management according to the etiology. cervical cancer is managed according to the cancer stage in consultation with gyneco - oncologists. radiation in combination with cisplatin - based chemotherapy has become the standard of care for patients with locally advanced cervical cancer (9). pyometra is a serious medical condition, because of both its association with malignant diseases and the danger of spontaneous perforation. although rare, ruptured pyometra should be considered in the differential diagnosis of acute abdomen in elderly women, especially those with malignant disorders of the genital tract. the treatment of pyometra rupture is immediate peritoneal lavage and drainage, or simple hysterectomy.	introductionby presenting this case we aimed to describe an uncommon complication of generalized peritonitis following spontaneous pyometra perforation in untreated cervical carcinoma.case presentationthis report describes a 60-year - old postmenopausal woman presenting with clinical features mimicking intestinal perforation who was later diagnosed as cervical carcinoma with pyometra perforation at exploratory laparotomy. the patient had good post - operative recovery following drainage and peritoneal lavage.conclusionspontaneous pyometra perforation in a case of untreated carcinoma of cervix is a rare condition, yet it should be suspected and kept in the differential diagnosis of acute abdomen in elderly women.
hiv / aids is the leading cause of death among adults in sub - saharan africa (ssa), but the burden of noncommunicable chronic diseases (ncd) is high and growing. the regional prevalence of diabetes mellitus (dm), for example, is expected to double between 2010 and 2030, when 28 million people in ssa are projected to be living with dm. in addition to dm - specific morbidity and mortality, diabetes contributes to the burden of other noncommunicable diseases (e.g., renal and cardiovascular disease) as well as communicable diseases (e.g., pneumonia and tuberculosis), further increasing its impact on public health. in 2010, 6% of total mortality in ssa was attributable to dm. unfortunately, access to prevention, care, and treatment services for ncd like dm remains out of reach for most in ssa, and health systems in lower - income countries are rarely designed to provide the continuity services required to effectively identify patients at risk, engage them in care, and retain them for the course of what is usually life - long treatment. the international diabetes federation estimates that 78% of those with dm in ssa remain undiagnosed, a consequence of limited access to trained health workers and laboratory testing as well as limited awareness of dm and its risk factors. although there have been several promising pilot studies of nurse - led dm management and other innovations [69 ], glycemic control tends to be suboptimal for those enrolled in care, even at specialized treatment centers [10, 11 ]. out - of - pocket costs for medicines, laboratory tests, and transportation create formidable barriers to adherence, as do stock - outs of drugs and supplies and the absence of effective systems to support chronic care [4, 12 ]. there is a pressing need to expand the coverage, quality, and equity of services for dm and other ncd in ssa. although often overlooked in this context, hiv programs are the first large - scale chronic disease initiatives in the region and, as such, an important resource for those hoping to expand ncd prevention, care, and treatment. in country after country, ministries of health with support from donors and partners have developed locally owned, contextually appropriate chronic care programs for hiv. with the expansion of hiv care and treatment programs, health systems that had previously delivered only episodic acute care services have been redesigned to provide longitudinal services and lifetime care for people living with hiv (plwh). in some cases, these changes represent innovations and new approaches, while in others they represent the availability of unprecedented levels of funding to implement time - tested strategies. from the health system and program management perspectives, chronic diseases have much in common with one another, whether they are communicable or noncommunicable. for example, both dm and hiv require laboratory diagnosis, daily medication (in some stages), and life - long self - management, including behavior changes. symptoms of both diseases wax and wane over time, requiring ongoing clinical and laboratory monitoring, patient education, and adherence support. in addition, both hiv and dm may cluster within families and households, the former due to sexual and perinatal transmission and the latter due to shared genetic and environmental risk factors in some settings [4, 13 ]. there are also key differences, including the characteristic age groups affected, dissimilar stigma attached to the two conditions, and disease - specific mortality rates. nonetheless, based on the key similarities, our hypothesis is that the systems, tools, and implementation strategies developed to provide continuity care for hiv in ssa can be rapidly, efficiently, and effectively utilized to support services for dm and other chronic ncd [1417 ]. icap at columbia university supports ministries of health and other local organizations in 21 countries, including 16 in sub - saharan africa. icap provides a wide range of hiv - related technical and infrastructure support to more than 2,500 health facilities, enabling the provision of quality comprehensive hiv / aids prevention, care, and treatment services. recognizing the potential to build upon icap 's experience to support continuity care programs for a range of chronic diseases, we embarked on two pilot studies to further the understanding of the status of ncd services and the feasibility and effectiveness of adapting hiv program - related tools and systems for patients with dm. in swaziland, we compared systems and services for hiv and dm at 15 health facilities in order to identify opportunities for experience sharing and diffusion of innovations. in ethiopia, a multi - component intervention adapted the approaches used in hiv clinic to enhance diabetes services at an urban referral hospital (see table 1 for additional context). in consultation with the swaziland ministry of health (moh), an analysis of ncd services was developed, using dm as an example. the study had three components : site assessments, chart review, and health care worker questionnaires. the study was approved by the moh, and ethical approvals were obtained from the columbia university institutional review board and the swaziland scientific and ethics committee. data were entered into a microsoft access database, which was used for the relevant analyses. site assessmentsan existing hiv - specific site assessment tool was adapted for dm, feedback was obtained from local clinicians, and the revised tool was piloted at a rural health facility. the resultant site survey tool used a checklist and rating scales to consistently and objectively describe site - level implementation of dm - specific health systems and services. fifty - nine questions enabled the systematic description of six domains, including (1) program management ; (2) organization of services and clinical care ; (3) monitoring, evaluation, and medical records systems ; (4) human resources ; (5) laboratory capacity ; and (6) pharmacy capacity. a convenience sample of 15 icap - supported health facilities from three of the four regions in swaziland was selected for the survey : three of the country 's six hospitals, three of five health centers, and nine of 202 health clinics. eleven of the facilities were public and four were supported by not - for - profit nongovernmental organizations. all of the facilities had functioning hiv care and treatment clinics at the time of the site assessment. with the permission of moh and the appropriate facility leadership, icap research staff completed the structured survey for each of the 15 facilities between june and august 2010, visiting relevant wards and clinics, interviewing site - level staff, and observing continuity care services for adults with dm. an existing hiv - specific site assessment tool was adapted for dm, feedback was obtained from local clinicians, and the revised tool was piloted at a rural health facility. the resultant site survey tool used a checklist and rating scales to consistently and objectively describe site - level implementation of dm - specific health systems and services. fifty - nine questions enabled the systematic description of six domains, including (1) program management ; (2) organization of services and clinical care ; (3) monitoring, evaluation, and medical records systems ; (4) human resources ; (5) laboratory capacity ; and (6) pharmacy capacity. a convenience sample of 15 icap - supported health facilities from three of the four regions in swaziland was selected for the survey : three of the country 's six hospitals, three of five health centers, and nine of 202 health clinics. eleven of the facilities were public and four were supported by not - for - profit nongovernmental organizations. all of the facilities had functioning hiv care and treatment clinics at the time of the site assessment. with the permission of moh and the appropriate facility leadership, icap research staff completed the structured survey for each of the 15 facilities between june and august 2010, visiting relevant wards and clinics, interviewing site - level staff, and observing continuity care services for adults with dm. chart reviewa chart abstraction tool was developed to collect information from outpatient records, including clinical, laboratory, pharmacy, and counseling services recognized to be important for dm management. only four of the 15 facilities maintained on - site medical records for outpatients with ncd, so the decision was made to abstract charts at the study site with the most available charts mbabane government hospital recognizing that it is not representative of health facilities in swaziland. the charts of 100 diabetic patients were sampled using a random number generator and reviewed by study staff. a chart abstraction tool was developed to collect information from outpatient records, including clinical, laboratory, pharmacy, and counseling services recognized to be important for dm management. only four of the 15 facilities maintained on - site medical records for outpatients with ncd, so the decision was made to abstract charts at the study site with the most available charts mbabane government hospital recognizing that it is not representative of health facilities in swaziland. the charts of 100 diabetic patients were sampled using a random number generator and reviewed by study staff. clinician surveysa questionnaire was developed to elicit the attitudes and practices of health care workers regarding programs and systems for dm. the written survey was self - administered by 72 clinicians (11 doctors, 43 nurses, 15 nurse assistants, and 3 individuals from other cadres) at three health facilities. this nonrandom sample represents approximately 17% of the physicians, 13% of the nurses, and 11% of the nursing assistants working at the three sites in 2010. a questionnaire was developed to elicit the attitudes and practices of health care workers regarding programs and systems for dm. the written survey was self - administered by 72 clinicians (11 doctors, 43 nurses, 15 nurse assistants, and 3 individuals from other cadres) at three health facilities. this nonrandom sample represents approximately 17% of the physicians, 13% of the nurses, and 11% of the nursing assistants working at the three sites in 2010. in consultation with the federal ministry of health, the ethiopian diabetes association (eda), and other stakeholders, ethical approvals were obtained from the columbia university institutional review board and the oromiya regional health bureau in ethiopia. the study design was a single time - series (a baseline assessment, a multicomponent intervention, and a six - month follow - up assessment) conducted at adama hospital, an urban referral hospital 100 km southeast of addis ababa. at the time of the study, adama hospital provided hiv care to more than 17,000 adults and children, of whom 10,600 had initiated art. consistent with other ethiopian health facilities, adama hospital provides outpatient dm services within the general outpatient department (opd) rather than at a separate diabetes clinic. the intervention package included strategies, systems, and tools adapted from adama hospital 's hiv program and applied to dm services in the opd. strategies included the introduction of an essential package of key services, the use of step - by - step protocols, emphasis on family - focused care and point - of - service diagnosis, and identification of simple, useful monitoring and evaluation (m&e) indicators. systems included appointment systems, clinical mentoring approaches, and the use of peer educators. tools included appointment books, charting tools and flow sheets, job aids, and logbooks / registers. no new or experimental clinical services were introduced : all protocols were consistent with local guidelines and best practices. no additional support was provided for medications, laboratory testing, or transportation, and no new staff were engaged for implementation ; services were provided by existing adama hospital clinicians supported by existing icap ethiopia clinical advisors who also provided ongoing support for the adama hospital 's hiv clinic. baseline assessment and six - month follow - upthe baseline assessment, conducted in the second quarter of 2010, included systematic observation of dm services in the opd. patient flow was mapped, patient encounters were observed, and key informant interviews were conducted with hospital leadership, clinicians, and patients. a chart abstraction tool was developed and piloted, and the baseline chart review was conducted for the 261 adult dm patients who had visited the opd for dm services within the prior three months. data were entered into a microsoft access database, which was used for the relevant analyses. the baseline assessment, conducted in the second quarter of 2010, included systematic observation of dm services in the opd. patient flow was mapped, patient encounters were observed, and key informant interviews were conducted with hospital leadership, clinicians, and patients. a chart abstraction tool was developed and piloted, and the baseline chart review was conducted for the 261 adult dm patients who had visited the opd for dm services within the prior three months. data were entered into a microsoft access database, which was used for the relevant analyses. intervention packagefollowing the approach used to support adama hospital 's hiv clinic, an intervention package was adapted and implemented to support dm services in the opd. clinical equipment was procured (ophthalmoscopes, sphygmomanometers, and scales) ; ongoing training and clinical mentoring of nurses and doctors were provided in partnership with the eda ; hiv - specific standard operating protocols and provider support tools (wall posters, pocket guides, and desktop references) were adapted for dm, as were hiv - specific charting and health management information system tools (checklists, flow sheets, standardized forms) ; dm appointment books and an appointment system were introduced ; a dm peer educator curriculum was developed, and training and logistical support for volunteer dm peer educators was provided ; family - focused care and point - of - service dm testing for patient family members were initiated ; simple standardized data were collected with ongoing feedback to clinicians. following the approach used to support adama hospital 's hiv clinic, an intervention package was adapted and implemented to support dm services in the opd. clinical equipment was procured (ophthalmoscopes, sphygmomanometers, and scales) ; ongoing training and clinical mentoring of nurses and doctors were provided in partnership with the eda ; hiv - specific standard operating protocols and provider support tools (wall posters, pocket guides, and desktop references) were adapted for dm, as were hiv - specific charting and health management information system tools (checklists, flow sheets, standardized forms) ; dm appointment books and an appointment system were introduced ; a dm peer educator curriculum was developed, and training and logistical support for volunteer dm peer educators was provided ; family - focused care and point - of - service dm testing for patient family members were initiated ; simple standardized data were collected with ongoing feedback to clinicians. none of the facilities utilized formal treatment algorithms or identified a basic package of care for dm. in contrast, hiv care and treatment were supported by national guidelines and step - by - step algorithms. in dm clinics, on - site medical records were the exception, not the rule : 4/15 (27%) of the sites had on - site medical records for dm patients, while all hiv clinics had on - site medical records and charting tools. none of the facilities had established appointment systems, defaulter tracking, adherence support programs, educational materials, or peer educators for dm, despite the fact that these services were available for plwh. hiv clinics used structured appointment registers printed by moh and available nationwide ; the dm clinics that kept any appointment records used ad hoc handwritten notes. availability of equipment at dm clinics and drugs for dm patients was variable. at the time of the assessment, all the facilities had sphygmomanometers and scales, 80% had stadiometers / height measures, and only 40% had ophthalmoscopes. 100% of facilities reported that aspirin and thiazide diuretics were usually or always available at the pharmacy, but oral hypoglycemics were usually or always available at only 70% of facilities and less than half of the facilities usually or always had insulin, beta blockers, or ace - inhibitors in stock. urine glucose testing was available at 93% of facilities, but blood glucose levels, creatinine, and total cholesterol tests were available at only 73%, 53%, and 26% of facilities, respectively. dm patients paid out of pocket for all medications and laboratory tests, in contrast to patients attending hiv clinic for whom drugs and diagnostic tests were provided at no cost. the median age of patients randomly selected for chart review was 56 years ; 66% were female. the patients attended dm clinic fairly regularly, with 60% of patients making more than 15 documented visits since their initial enrollment. most of the patients (81%) had been attending clinic for more than two years, and 75% had been seen at dm clinic within the past 6 months. however, only 26% of patients had optimal dm control, as defined by the international diabetes federation (figure 1). in addition, 72% of the diabetic patients had htn, defined as either a most recent blood pressure (bp) measurement of systolic bp > 140 mmhg and/or diastolic bp > 90 mmhg or at least three documented prior elevated bp measurements. within this subgroup, none of the facilities used flow sheets, checklists, or structured note templates for dm management, in contrast to many of the hiv clinics in swaziland. at mbabane government hospital, all charts had records of at least one fasting blood glucose measurement and at least one blood pressure measurement ; 68% had weight documented at least once. other documentation was scanty ; current medications were documented in 1% of charts, foot exam in 7%, and fundoscopic exam in 1%. none of the charts reviewed had documentation of smoking status, medication adherence, or presence / absence of diabetic complications. the health care workers who completed the questionnaire indicated that barriers to provision of appropriate dm care included lack of guidelines and standard operating protocols, shortages of drugs and equipment, and inadequate staffing. health care workers also recognized substantial barriers to adherence and retention of patients in care, including the costs of medications and laboratory tests, transportation barriers, and limited access to patient education and counseling services. of note, 35% of respondents said that they or a family member had dm. the median age of dm patients at adama hospital was 47 years (range 1883 years), 51% were male, 60% had type 2 dm, and 57% were currently receiving insulin. patients had been diagnosed with dm for a median of 3 years (range 123 years) and had been followed at adama hospital for a median of 2 years (range 022 years). 21% had been previously hospitalized for dm, 5% had a history of foot ulcer, 20% had been diagnosed with peripheral neuropathy, 8% had reported visual impairment, and 2% had at least one amputation. at baseline, no algorithms or standard operating protocols were used to support dm care ; clinic staff reported that each individual clinician drew upon his / her training to determine optimal clinical management on a case - by - case basis. of 45 clinicians surveyed (5 doctors, 37 nurses, and 3 other), 47% had received their degree or certification more than 10 years ago, and only 16% reported additional training in dm management since graduation. (of note, 22% of clinicians reported having at least one family member with dm). in the 260 charts reviewed, although 80% of patients had a blood pressure (bp) measurement documented at least once, only 21% had ever had a documented fundoscopic exam. foot exam had ever been documented in 10% of patients, and only 1% had a documented weight in their chart. when review was confined to the patients ' three most recent visits, figure 2 shows baseline and follow - up data on documentation of services within the patients ' past three visits. the percentage of charts in which patient weight was documented at least once in the past three visits increased from 2% to 82%. documentation of fundoscopic exam, foot exam, and neurologic exam rose from 1% to 50%, 3% to 81%, and 3% to 56%, respectively. adherence assessment was documented in 2% of charts prior to the intervention and 77% after the intervention ; record of the next appointment date rose from 17% to 81%. the program assessment identified significant gaps in the strategies, systems, and tools used to support dm services at 15 health facilities in swaziland, as well as suboptimal control of hyperglycemia and hypertension amongst patients with dm at mbabane government hospital. although effective dm care requires longitudinal services and chronic care systems, diabetic patients in swaziland did not have access to the types of interventions needed for appropriate management of their disease. this contrasted with the presence of functional chronic care programs developed for hiv situated within the same health care facilities. in ethiopia, the rapid proof - of - concept study found similar gaps in dm services at baseline and illustrated the potential to swiftly improve the quality of care and treatment for dm by adapting hiv - specific strategies, systems, and tools. there was a marked increase in documented service delivery and significant improvements in standards of care with no added staff. the ethiopia pilot study measured process indicators rather than clinical outcomes and did not include a comparison site ; it is possible that alternate interventions not based on hiv programs could have achieved a similar effect. although clinicians reported that the introduction of new systems was facilitated by the fact of local ownership that the same systems and tools were already being used in the hospital 's hiv clinic the magnitude of this effect was not quantified. in swaziland, site assessments and clinician surveys used nonrandom samples ; the results may not be representative of health facilities and health providers nationwide. there are multiple ways in which to build upon the lessons and resources of hiv programs when working to expand and enhance dm and ncd services, and no single approach is likely to work in all settings. in some countries, integration of chronic disease services for hiv and ncds at the point of care in other settings, as in ethiopia, this may be neither feasible nor desirable. a continuum of approaches is available to implementers and policy makers, ranging from parallel side - by - side services to complete integration of chronic disease care and treatment. the intermediate approach is one in which clinical services are not merged or integrated, but the systems behind them are shared, including guidelines, training, procurement of drugs and supplies, laboratory systems, and monitoring and evaluation strategies. in summary, the pilot studies described in this paper are among the few to document the potential of leveraging lessons learned from hiv programs to support ncd care and treatment services in sub - saharan africa. the findings suggest that countries which have successfully scaled up hiv services have already learned profound lessons about the delivery of chronic care. using these locally owned and contextually appropriate resources may be an efficient and effective way to jumpstart no one approach will be right for all countries and contexts, and funds are needed to support larger, controlled studies to elucidate the impact and costs of using this strategy.	the scale - up of hiv services in sub - saharan africa has catalyzed the development of highly effective chronic care systems. the strategies, systems, and tools developed to support life - long hiv care and treatment are locally owned contextually appropriate resources, many of which could be adapted to support continuity care for noncommunicable chronic diseases (ncd), such as diabetes mellitus (dm). we conducted two proof - of - concept studies to further the understanding of the status of ncd programs and the feasibility and effectiveness of adapting hiv program - related tools and systems for patients with dm. in swaziland, a rapid assessment illustrated gaps in the approaches used to support dm services at 15 health facilities, despite the existence of chronic care systems at hiv clinics in the same hospitals, health centers, and clinics. in ethiopia, a pilot study found similar gaps in dm services at baseline and illustrated the potential to rapidly improve the quality of care and treatment for dm by adapting hiv - specific policies, systems, and tools.
in accordance with the study protocol, possible cases detected by general physicians and emergency department physicians had to be referred within 24 hours to the closest unit of infectious or tropical diseases. serum samples from persons with possible cases were sent to the regional reference laboratory (padua, italy) for confirmation. if neuroinvasive disease was present, the specific protocol for wnnd was followed (1). we defined a possible case of denv or chikv infection as fever > 38c during the past 7 days in a traveler who had returned within the previous 15 days from countries to which these viruses are endemic, absence of leucocytosis (leukocyte count 38c for 15 years, no recent travel history, rash, and absence of other obvious causes of fever (figure 1). algorithm for detection of possible cases of west nile fever, veneto region, italy, 2010. n, no ; y, yes ; wnnd, west nile neuroinvasive disease ; neg, negative ; pos, positive. in instances of high clinical suspicion for denv and chikv in patients with autochthonous fever, laboratory tests for these 2 diseases also were performed. of 79 possible cases, we detected 14 cases of denv infection and 1 case of chikv infection among travelers with fever (table ; table a1). all were positive for wnv igm and/or igg and confirmed by plaque - reduction neutralization test, but none were wnv rna positive. denv, chikv, and wnv infections are arboviral diseases that find potentially suitable vectors in italy, particularly in veneto. no autochtonous case of fever caused by denv has been documented in italy, but the possible role of the aedes albopictus mosquito as a vector has been demonstrated by recent cases in france (2) and croatia (3). chikv caused the well - known outbreak in emilia romagna region (northern italy) in 2007, which was detected, by coincidence a few days after the imported cases in italy had been reported (4) ; the published report concluded that the possibility of introducing chikv into italy can not be ruled out on the basis of current evidence. the index case had occurred 2 months before the first case was diagnosed (5). the recent occurrence of 2 locally transmitted cases of chikungunya in france, despite a low number of imported cases (6), shows that the risk remains high. since summer 2008, wnv has caused wnnd in humans, first in emilia romagna region (7), then in veneto region (8). in contrast, the more common presentation, wnf, has been detected in only 1 patient ; the casewas identified retrospectively (9,10), despite the expected wnf : wnnd ratio of 20:1 (11). because we were concerned about being overwhelmed by an unmanageable number of case reports of unspecific fevers, we chose a selective case definition, particularly for wnf, with the obligatory presence of a rash, and thereby lowered the sensitivity of the surveillance. however, the proportion of virus - positive patients was strikingly high : 20% of persons tested who had imported fever were positive for denv or chikv, as were 10% of persons with locally acquired fevers for wnv. compared with the 2 previous years, the special surveillance enabled detection of substantially more cases, showing that you only find what you are looking for (table). wnv circulation has now been documented in many areas of italy, from north to south, through retrospective screening of solid organ donors (12) and through entomologic (13) and animal surveillance (14) ; nevertheless, in 2010, no human clinical cases were detected outside veneto. the success of this pilot phase prompted regional authorities to propose a 3-year plan, which the ministry of health has approved and funded, as part of the integrated surveillance of arboviral diseases, along with animal and entomologic surveillance. relying only on the latter 2 would not be sensible. however, mosquito surveillance was able to predict cases in animals and humans (figure 2). expected rates of wnv infection in mosquitoes at the only site with repeated positivity in animals, humans, and vectors (venice province) are shown together with the time of exposure of animals and humans in the same province. time of exposure was estimated as 1 week before onset of symptoms (incubation range 214 days) (15). when the expected rate of mosquito infection was low (i.e., 0.06%), no clinical cases were recorded ; when the expected rate of infection was higher (> 0.24%), clinical cases were observed in animals and humans. expected rates of infection (eri) in mosquitoes in the west nile virus positive site and hypothetical time from exposure to infected mosquitoes to clinical cases in animals and humans (calculated 1 week before symptom onset) recorded in the same province, venice province, italy, 2010. concerning the new plan for human surveillance of summer fevers, the case definition, particularly for wnf, has been modified by removing the compulsory presence of rash, to enhance sensitivity. on the basis of a predefined threshold of vector intensity in an area where a new case has been identified, immediate vector control measures will be started when necessary.	in 2010, in veneto region, italy, surveillance of summer fevers was conducted to promptly identify autochthonous cases of west nile fever and increase detection of imported dengue and chikungunya in travelers. surveillance highlighted the need to modify case definitions, train physicians, and when a case is identified, implement vector control measures
recently, several measures for the evaluation of upper extremity function have been developed, including the disability of the arm, shoulder, and hand scale (dash), the shoulder pain and disability index (spadi), and the american shoulder and elbow surgeons (ases). there are a large number of upper limb assessments available, and selecting the most appropriate outcome measure to predict the effectiveness of clinical interventions can be challenging1,2,3,4. the choice of the most appropriate questionnaire to use may be based on the specific study group, the purpose of the questionnaire, its clinimetric quality as shown by validity, reproducibility, responsiveness, or practical considerations. 3) questionnaire was devised as a region - specific, subjective (self - report) measure by the japanese orthopaedic association and the japan shoulder society in 2010. the rationale for the creation of this measure is that the upper extremity is a functional unit or kinetic chain. 3) is suitable for measuring health status outcomes because it is primarily a measure of disability. recently, the shoulder36 questionnaire has been used extensively throughout, however, it has not been validated against other questionnaires for its functional evaluation of the shoulder, such as the quickdash, which was developed for patients with any disorders in any joints of the upper limbs. 3) has been introduced as a new evaluation system, and the similarities and differences between the shoulder36 and other questionnaires have not been assessed by a novel evaluation system in the literature. thus, the purpose of this study was to demonstrate the reliability, validity, and responsiveness of the shoulder36 (v 1. 3) has six domains ; 1) the severity of activity - related pain (sh36-p) ; 2) the restrictions to range of motion (sh36-rom) ; 3) the effects of the muscle strength on activities (sh36-ms) ; 4) the effects of the injury related problems on the patient s sense of well - being (sh36-gh) ; 5) the effect of the injury on activities of daily living (sh36-adl) ; and 6) the degree of determent on athletic ability (sh36-s). each scale concerns the function of the patient s upper extremity, and each item has five possible responses, ranging from can not do it to no difficulty the items ask about pain (sh36-p, 6 items : numbers 3, 16, 22, 24, 28 and 32) ; range of motion (sh36-rom, 9 items : numbers 2, 4, 5, 7, 8, 9, 11, 12 and 18) ; muscle strength (sh36-ms, 6 items : numbers 13, 20, 23, 27, 29 and 34) ; general health (sh36-gh, 6 items : numbers 1, 17, 25, 26, 31 and 33) ; activities of daily living (sh36-adl, 7 item : number 6, 10, 14, 15, 19, 21 and 30) ; and the ability to play sports (sh36-s, 2 items : numbers 35 and 36). 3) with six domains ranging from 0 (the severest disability) to 4 (no disability), after totaling the average value of the scores for each domain. a series of 46 patients with upper extremity disorders were seen on an inpatient or outpatient basis at orthopedic surgery department in fuefuki central hospital. exclusion criteria included, age younger than 19 years and an existing comorbidity. the study was conducted on 46 patients (25 males, 21 females) who were suffering from rotator cuff disease (33 patients), osteoarthritis of the shoulder (6 patients), or other problems (7 patients). the mean age was 66.2 years (sd : 14.5 years ; range : 2885 years). all patients underwent therapy during the consecutive inpatient or outpatient visits, and filled out both the shoulder36 (v 1. 3) and the subjects provided written informed consent to participate in the study prior to its commencement, and the study conformed to the principles of the declaration of helsinki. this study was conducted with the approval of the research ethics committee of health science university (approval number : 31). a principal component analysis was conducted to examine the construct validity and the unidimensionality of the six domains of the shoulder36 (v 1. correlation coefficients between the shoulder36 (v 1. 3) and the quickdash - jssh were obtained, and the following hypotheses were examined to investigate concurrent validity : 1) the sh36-rom, sh36-adl, and quickdash - jssh disability / symptom (quickdash - jssh - ds) would exhibit the strongest associations to each other ; 2) the sh36-p, the sh36-ms, and dash - jssh work (quickdash - jssh - w) would exhibit the next strongest association to each other ; 3) the sh36-gh and the sport / music (dash - jssh - sm) would exhibit the weakest association. correlation coefficients between the shoulder36 (v 1. 3) domains (sh36-p, sh36-rom, sh36-ms, sh36-gh, sh36-adl, and sh36-s) were also obtained. the interval measurements of the sh36-p, sh36-rom, sh36-ms, sh36-gh, sh36-adl, dash - jssh - w, and dash - jssh - sm were normally distributed ; therefore, the correlation was assessed using a parametric test (pearson s correlation). the other interval measurements (sh36-s, dash - jssh - ds) were not normally distributed, and thus, the correlation was assessed using a nonparametric test (spearman s correlation). statistical analyses were conducted using the statistical package for social science (spss) software and statcel3 (oms, saitama, japan). 3) questionnaire. the total answer rate for the shoulder36 (v 1. moreover, the sh36-p, sh36-rom, and sh36-adl answer rates were 100%, as shown in table 1table 1.scores for the shoulder36 and quickdashinstrumentnquestion answer ratescoremean (sd)medianrangecronbach s rangesh36-total46992.4 (1.5)30.04.00.98sh36-p461002.7 (0.9)2.90.24.00.86sh36-rom461002.6 (1.0)2.80.04.00.94sh36-ms46991.9 (1.2)1.70.04.00.94sh36-gh46982.7 (0.8)2.80.84.00.82sh36-adl461002.5 (1.0)2.90.34.00.92sh36-s46951.5 (1.3)1.50.04.00.81quickdash - ds469931.8 (17.8)320720.84quickdash - w3010041.8 (28.1)2801000.96quickdash - sm1110058.5 (30.5)6301000.95sh36-total : functioning subscale of the 6-domains of the shoulder36 (v 1. 3) ; sh36-p : the severity of activity - related pain ; sh36-rom : the restrictions to range of motion ; sh36-ms : the effects of muscle strength on activities ; sh36-gh : the effects of the problems on the patient s sense of well - being ; sh36-adl : the effect on activities of daily living ; sh36-s : the degree of determent on athletic ability ; quickdash - ds : disability / symptom scale of the quickdash - jssh ; quickdash - w : work module of the quickdash - jssh ; quickdash - sm : sport / music module of the quickdash - jssh. however, the answer rate for the quickdash - ds, quickdash - w, and quickdash - sm were 99%, 100% and 100%, respectively. four patients did not respond to items 31 or 33 regarding general health, and the majority of the patients considered all of the items of the shoulder36 (v 1. the mean age (69 12 years) of the nonrespondent group (n=5) was higher than the mean age (66 15 years) of the respondent group (n=41) who completed all of the items (p=0.694) (data not shown). 3) ; sh36-p : the severity of activity - related pain ; sh36-rom : the restrictions to range of motion ; sh36-ms : the effects of muscle strength on activities ; sh36-gh : the effects of the problems on the patient s sense of well - being ; sh36-adl : the effect on activities of daily living ; sh36-s : the degree of determent on athletic ability ; quickdash - ds : disability / symptom scale of the quickdash - jssh ; quickdash - w : work module of the quickdash - jssh ; quickdash - sm : sport / music module of the quickdash - jssh internal consistency was assessed using the cronbach s alpha coefficient. the total cronbach s alpha coefficient for the 36 items of the shoulder36 (v 1. the alpha coefficients for the six individual domains were also high (0.810.94), especially sh36-rom and sh36-ms which were both 0.94. the alpha coefficients for the quickdash - ds, quickdas - w, and quickdash - sm were 0.84, 0.96, and 0.95, respectively. 3) questionnaire and quickdash - jssh are shown in table 2table 2.ceiling and floor scores for the shoulder36 (v 1.3) and dash - jsshinstrument scaleno.no. floor scoressh36-p465(11%)0sh36-rom466(13%)1(2%)sh36-ms463(7%)7(15%)sh36-gh464(9%)0sh36-adl465(11%)0sh36-s462(4%)15(33%)dash - jssh - ds461(2%)0dash - jssh - w304(13%)2(7%)dash - jssh - sm112(18%)2(18%)maximum health status scores, minimum health status scores. no patients reported the maximum disability score of 0 (floor) on the sh36-p, sh36-gh, or sh36-adl. fifteen patients reported the minimum score and six patients had the maximum score for the sh36-s and the sh36-rom respectively, as shown in table 2. maximum health status scores, minimum health status scores a principal component analysis was conducted to confirm the unidimensionality of the shoulder36 (v 1. the first factor had an eigenvalue (amount of variation in the total sample accounted for by that factor) of 41.23, which explained the 61% of the total variance of the patients shoulder36 (v 1. the unidimensionality was found to be strong due to a substantial difference between the first and the second factors (eigenvalue 3.57). when looking at the first - factor loading for each item, all items had a loading of 0.4 or higher, as shown in table 3table 3.factor loading (unrotated) of the principal components of the shoulder36 (v 1. 3)question no.itemloading1daily activities at home0.762reading a newspaper at a shoulder high0.843reaching a back pocket of your trousers using your affected side 0.694putting your arm through a jacket 0.655wearing a sweater jacket over your head 0.806taking off clothes 0.577placing a jacket on a hanger 0.838knotting your hands together behind your head 0.829washing your face with hands 0.6210combing your hair 0.7711washing your armpit opposite to your affected shoulder, using your affected shoulder 0.7912rinsing your whole body by holding a shower head with your affected side 0.8413washing your back with a towel by holding both ends of the towel with an affected side holding the top of the towel0.8214squeezing out the water from a towel using both hands 0.7315carrying a bowl of soup on the tray 0.7616reaching into a condiment on a table (soy sauce, salt, pepper, etc.) using your affected shoulder 0.8417eating0.6518tying an apron behind your back0.8119washing plates with a sponge 0.8020placing plates on a shelf above your head height using the affected shoulder 0.8521holding a filled up kettle with your affected side 0.8522clapping your hands 10 times0.4523stretching your body with your hands held up 0.8324sleeping side ways with your affected shoulder lying on the floor0.7225getting a good sleep 0.5226feeling less fatigue than usual getting through the week 0.4827keeping both your arms horizontal for a minute 0.8228walking with your arms swinging back and forth0.7629managing daily tasks using your affected shoulder, without help of another shoulder0.9230wiping windows with your affected shoulder at a head high 0.8831going shopping close to your house 0.8032opening an umbrella (with exception to push - button umbrella) with your affected shoulder 0.7633getting on a bus or train0.7434holding on a strap in a bus or train with your affected shoulder 0.8435moving your shoulders at a recreational level of activities 0.8336moving your shoulders at a competitive level of activities 0.73. 3) the correlation coefficients between the six domains of the shoulder36 (v 1. 3) ranged from 0.71 to 0.89 (p<0.01), as shown in table 4table 4.the correlations for the shoulder36 (v 1. 3) and quickdash - jsshsh36-psh36-romsh36-mssh36-ghsh36-adlsh36-ssh36-p------sh36-rom 0.89-----sh36-ms 0.83 0.84----sh36-gh 0.84 0.780.83---sh36-adl 0.83 0.870.85 0.81--sh36-s # 0.73 0.710.78 0.73 0.73-quickdash - ds # 0.75 0.710.78 0.77 0.77 0.56quickdash - w 0.80 0.620.68 0.65 0.74 0.55quickdash - sm0.600.430.760.640.520.77p<0.05, p<0.01 ; pearson s correlation ; spearman s correlation. these results indicate strong correlations between the sh36-p and sh36-rom, as well as between the sh36-rom and the sh36-adl. the subscales of the quickdash - jssh ranged from 0.43 to 0.80 as presented in table 4. the strongest correlation was observed for the sh36-p followed by the quickdash - jssh - w. therefore, these strong correlations support the hypotheses stated earlier with the exception that, despite the high correlation coefficient between the sh36-s and quickdash - jssh - sm, it was not significant. p<0.05, p<0.01 ; pearson s correlation ; spearman s correlation clinicians and researchers are confounded by the various outcome measures used for the assessment of injury of the upper limbs5,6,7. in addition to rating the clinimetric properties of a questionnaire, an easy scoring method and information regarding acceptable levels of missing data sever to enhance applicability. in this study our data indicates that the questionnaire was easy to understand as none of the patients left more than four items unanswered, which were thought to pertain to specific recreational or competitive level activities not performed by these individuals. for the respondent burden, a positive rating was assigned when the questionnaire could be completed within 10 minutes (data not shown). the lack of floor and ceiling effects assured the authors that this version of the shoulder36 (v 1. however, large, high - quality studies are still required to confirm these findings. moreover, other studies have confirmed that the relative reliability of questionnaires is excellent when the intraclass correlation coefficients are 0.85 or higher8, 9. 3) should, be used with caution for the daily assessment of the status of individual patients. the validation process of the shoulder36 (v 1. sh36-ms, sh36-gh, sh36-adl, and the quickdash - jssh support its validity, and demonstrates results similar to those of validation papers for the other questionnaire10. similarly, the strong correlations between sh36-p, sh36-adl, and the quickdash - jssh - w support this validity and demonstrate results similar to those of a published validation paper11. additionally, the correlations between sh36-p, sh36-rom, sh36-gh, sh36-adl, sh36-s, and the quickdash - jssh - sm were weak due to the small sample size. these results indicate that the shoulder36 (i.e., sh36-s and quickdash - jssh - sm) measures the important elements that describes health factors related to quality of life. one merit of this study was the finding that the shoulder36 demonstrated good consistency and validity. on the other hand, a limitation of this present study is that we were unable to successfully validate the relationship between the sh36-s and the quickdash - jssh - sm since the sample size was relatively small and the patient s response rate was low. in addition, the subjects in this study are not representative of the general population. self - report measures are regarded to be simple, inexpensive ways of obtaining good clinical data, and in many instances they are easy to score and interpret for the clinician, thus adding valuable information to the clinical picture12, 13. the dash, spadi, and ases have been evaluated most often and the dash has received the best overall ratings for its clinimetric properties. in addition, the dash has been previously recommended for evaluative purposes in outpatient clinics14. similar to the quickdash, we have demonstrated that the shoulder36 questionnaire has good reproducibility, consistency, and validity. interest in using patient - based instruments during clinical practice for the assessment and monitoring of individual patients receiving treatment continues to grow. therefore, these tools assist clinicians in the detection and treatment of functional and psychological problems that may have previously been missed. furthermore, they promote shared decision - making and facilitate doctor - patient communication. furthermore, questionnaires with fewer items and shorter administration times may be more practical for routine use in clinical practice. some limitations include that the shoulder36 has not been used by anyone except its developers and it measures only the physical components of health. in addition, the study population consisted of a non - random sample ; the sample size was low with no mention of patient characteristics and no cross - cultural validation was performed. furthermore, there are no standardized criteria for evaluating the quality of the shoulder36, and the specific criteria we used to evaluate the shoulder36 may be disputed. in the future, more consolidated guidelines are required to set standards and define the instruments by which patients with shoulder disorders should be assessed. in addition, the continued accumulation of research data regarding the clinimetric properties of the evaluation questionnaire is important for demonstrating how the scale might be used in both clinical practice and research applications.	[purpose ] the shoulder36 (v 1. 3) is a regional questionnaire in japan that has not been validated as a functional evaluation of the shoulder via a thorough comparison with other questionnaires (e.g., quickdash). the purpose of this study was to test the reliability, validity, and responsiveness of the shoulder36 (v 1. 3). [subjects and methods ] a series of 46 patients with upper extremity disorders completed the shoulder36 (v 1. 3) and the quickdash japanese version (quickdash - jssh). the reliability of the shoulder36 was assessed for consistency and validity. the correlation coefficients between the shoulder36 (v 1. 3) and the quickdash - jssh were obtained. [results ] the total of the cronbach s alpha coefficients for the shoulder36 (v 1. 3) was 0.98. the intraclass correlation coefficients for the six domains of the shoulder36 (v 1. 3) were similarly high, ranging from 0.81 to 0.94. the correlations between the six domains of the shoulder36 (v 1. 3) and the three domains of the quickdash subscales ranged from 0.43 to 0.78. [conclusion ] the shoulder36 (v 1. 3) was able to evaluate the relationship between activities of daily living and shoulder joint function with the same degree of accuracy but in more detail than quickdash - jssh. therefore, it should prove to be a valuable asset in physiotherapy plans and have multiple research applications.
type 1 diabetes mellitus (t1 dm) is the result of autoimmune destruction of beta cells in pancreas, which usually result in insulin deficiency. in two recent decades, the worldwide prevalence of diabetes mellitus (dm) has risen significantly from an estimated 30 million cases in 1985 to 177 million in 2000. it is estimated that 7.7% of iranian adults aged 2564 years, or 2 million adults, have dm, one - half of whom are undiagnosed. there is a genetic predisposition toward t1 dm, which also predisposes patients to other autoimmune diseases such as thyroid disease, celiac disease, adrenal insufficiency, vitiligo, alopecia, and gastric autoimmunity[3, 4 ]. according to studies, 15 - 30% of t1 dm patients have autoimmunity of thyroid (ait)[58 ], 4 - 9% have celiac disease, and 0.5% have addison 's disease. ait as a more prevalent autoimmune disorder associating with t1 dm is often clinically silent but it may progress to autoimmune thyroid disease (aitd), recognized as overt or subclinical hypothyroidism and hyperthyroidism. hypothyroidism can lead to growth delay, weight gain, menstrual abnormality, hyperlipid - emia, and cardiovascular complications in diabetic patients. hyperthyroidism can worsen metabolic control of diabetes and increase its liability, often with a need for increased insulin dosage and increased chance of diabetic keto - acidosis[10, 11 ]. aitd is detected most easily by measuring circulating antibodies against thyroid peroxidase (anti - tpo ab) and thyroglobolin (anti - tg ab). studies have measured varying prevalence of between 3 and 50%, depending on the methodo - logy of the study and patient 's characteristics (age, sex, ethnicity, and genetic background)[4, 12 ]. it should be noted that the prevalence of thyroid dysfunction in diabetic people is higher than in the general population, too. studies have estimated a prevalence of 1 - 5% for overt hypothyroidism and 0.5 - 7% for thyrotoxicosis in young diabetic patients[4, 13 ]. because of the high prevalence of ait and thyroid dysfunction in patients with t1 dm and probable effects of thyroid disorders in metabolic control of these patients, there is a general agreement about screening of diabetics for thyroid antibodies and dysfunction. despite this fact, there is still no consensus regarding screening of ait and thyroid function in t1 dm patients. there are limited studies in iran estimating the prevalence of thyroid disorders and ait in patients with t1 dm and most of them have been done in adults. the aim of this study was to determine the prevalence of thyroid disorders such as ait, thyroid dysfunction, and goiter in children and adolescents with t1 dm compared with age and sex matched healthy controls in isfahan. patients with t1 dm who were under 18 years old and had a health profile in isfahan endocrine and metabolism research centre with regular attending to the centre were enrolled into the study consecutively. as a control group, healthy subjects matched for age and sex were selected with a multistage cluster random sampling from isfahan school children. we excluded individuals with history of recent or acute illness and history of taking drugs affecting thyroid function or size. a questionnaire was filled out for diabetic patients, which included information on the date of birth, sex, duration of diabetes, age at onset of dm, history of thyroid disease, drug history, and mean hba1c level during the past one year. blood samples were taken for determination of serum level of thyroid stimulating hormone (tsh), total thyroxin (t4), anti - tpo ab, and anti - tg ab. all samples were measured at the reference laboratory of the isfahan endocrine and metabolism research center by the same person using the same method. serum t4 concentrations were measured using radio - immunoassay (iran kavoshyar co., tehran, iran). serum tsh concentrations were determined with immunoradiometric assay (iran kavoshyar co., tehran, iran). overt hypothyroidism was defined as elevated tsh and low t4, subclinical hypothyroidism as elevated tsh and normal t4, overt hyperthyroidism as low tsh and elevated t4 and subclinical hyperthyroidism as low tsh and normal t4. serum anti - tpo ab and anti - tg ab were measured by rapid elisa (genesis diagnostics, littleport, uk). anti - tg ab and anti - tpo ab concentrations more than 100 iu / ml and 75 iu / ml, respectively, were considered positive. independent sample t - test was used to compare normally distributed data in different groups. mann - whitney u - test was used when quantitative variables were not normally distributed. all analysis was performed using spss version 15 (spss corp, chicago, il, usa). written informed consent the study was approved by the local ethics committee of the endocrine and metabolism research center. one hundred children and adolescents with t1 dm and 284 controls were enrolled into the study. demographic characteristics of the individuals with and without t1 dm quantitative variables are presented as mean (standard deviation) and range / ns : not significant serum tsh concentration was abnormal in 19 (19%) children with t1 dm and 53 (18.7%) children of the control group (p=0.90). eighteen (18%) patients with t1 dm and 51 (51%) individuals in the control group had subclinical hypothyroidism. subclinical hyperthyroidism was detected in one (1%) patient with t1 dm and two (0.7%) children in the control group (p=0.35). there were no significant differences between diabetic patients and non - diabetic ones regarding mean serum tsh (2.591.32 vs. 2.781.55 mu / l, p=0.30) and t4 (10.8816 vs. 8.551.52 g / dl, p=0.10) con - centrations. the prevalence of goiter was higher in the control group (38% vs. 21%, p=0.001). there was no significant difference between the prevalence of goiter and thyroid dysfunction in t1 dm patients and non - diabetic participants based on different sex group. the mean serum anti - tpo ab and anti - tg ab in diabetic children were three times higher than those in the control groups (table 2). thyroid antibodies concentration and thyroid autoimmunity in individuals with and without t1 dm sd : standard deviation / ns : not significant anti - tpo ab was positive in 16 out of 83 (19.3%) patients and 15 out of 284 (5.3%) non - diabetic participants (p=0.000). this difference was not significant according to positivity for anti - tg ab (11% in t1 dm children vs. 6.4% in the control group, p=0.10). the prevalence of ait in diabetic children was 2.5 times higher than that in the control group (p=0.001) (table 2). dividing diabetic children into two groups according to the presence of thyroid antibodies, there were no significant differences between the groups in age, sex, age at onset of diabetes, duration of diabetes, hba1c, serum tsh, and t4 concentration (p>0.05). the chance for having thyroid dysfunction in diabetic children who had ait was five times higher than those who had not ait(odds ratio : 5, ci 95% : 1.5 - 15.6%, p=0.008). clinical characteristics of diabetic children with and without ait sd : standard deviation / ns : not significant the prevalence of goiter in diabetic children with ait was higher than that in diabetics without ait. however, this difference was not statistically significant (table 3). there was no correlation between age, duration of diabetes, and age at onset of diabetes on the one hand, and anti - tpo ab and anti - tg ab, on the other hand, in diabetics. a positive correlation was found between anti - tpo ab and anti - tg ab concentrations in these patients (r=0.5, p<0.001). in the present study, we showed that children with t1 dm had higher levels of both anti - tpo ab and anti - tg ab compared with healthy ones. also, t1 dm children had higher prevalence of positive anti - tpo ab than non - diabetic individuals. it has been shown that t1 dm has strong relationship with autoimmune disorders such as pernicious anemia, celiac disease, and idiopathic adrenal insufficiency. ait is the most prevalent autoimmune disorders associated with t1 dm [13, 17 ]. the reason for the high prevalence of some autoimmune disorders in these patients still remains undetermined. it may be due to a generally increased tendency to react against certain antigens, or a genetically impaired ability to acquire tolerance to some autoantigens, or certain common antigens present in the tissues of individuals prone to autoimmune diseases. according to some studies, common genetic determinants, mainly human leukocyte antigen (hla) risk alleles[19, 20 ] or other genes outside the hla region (i.e., ctla4 gene and ptpn22 gene), could play a role[21, 22 ] in the occurrence of ait in t1 dm patients. moreover, environmental factors such as stress, infection, trauma, smoking, drugs, and nutrition (especially increased iodine intake) seem to be involved. both t1 dm and ait are organ - specific t - cell mediated diseases, and have similar patho - genesis, which involves t - cell infiltration resulting in dysfunction of the target organ. in the present study, the prevalence of positivity for anti - tpo ab, anti - tg ab, and the prevalence of positivity for both antibodies and ait (at least one positive ab) in children with t1 dm was 19, 11, 8.4, 22%, respectively, which was higher than those in non - diabetic individuals. in other studies, the prevalence of positive anti - tpo ab in t1 dm patients was reported to be 5.5 - 46.2%. the prevalence of high anti - tg ab in these patients ranged from 2.1 to 40%. in those studies, the prevalence of ait in t1 dm and healthy individuals was reported to be 11 - 46% and 1.4 - 11%, respectively. the wide range of these data can be explained by the difference in genetic factors, age, and sex of the studied population, as well as the different methods for measurement of antibodies. most studies that reported the low prevalence of ait were conducted 1 - 2 decades ago, showing the lower sensitivity of the laboratory tests. meanwhile, this finding might be a result of a real increase in the prevalence of ait during the recent decades. epidemiologic studies have shown higher incidence of ait after elimination of iodine deficiency in endemic areas. in previous studies in iran, the prevalence of positive anti - tpo ab and anti - tg ab in t1 dm patients were reported to be 27 - 39.6% and 27 - 34%, respectively[17, 2527 ]. the lower prevalence of ait in our study could be explained by the different age group of studied individuals in our study. the previous studies in iran were conducted in adult population or had recruited some adults. however, the present study was conducted on children and showed comparable results with other studies performed in similar age group in northwestern part of iran and other countries. the prevalence of clinical and subclinical thyroid dysfunction in t1 dm patients is suggested to be 13.4 - 20%, whilst the prevalence of hypothyroidism and hyper - thyroidism in the normal population is 5 - 10% and 1%, respectively. in the present study, there was no case of clinical thyroid dysfunction. however, subclinical hypothyroidism was present in 18% of both t1 dm patients and the control group. in our study, the prevalence of subclinical hyperthyroidism in t1 dm patients and non - diabetic subjects was 1% and 0.7%, respectively, which is consistent with the findings of previous studies. we found that diabetic patients with ait had higher prevalence of subclinical hypothyroidism than diabetic patients without ait (38.8% vs. 13.8%), which was statistically significant. also, there was a positive correlation between thyroid dysfunction and ait (data not shown) therefore, the higher prevalence of subclinical hypothyroidism in t1 dm patients could be explained by the high prevalence of ait (22%) in these patients. as well as ait, unfortunately, we did not evaluate iodine status of the studied population and as a result, we could not investigate the role of iodine deficiency in the high prevalence of subclinical hypothyroidism. in the present study, the prevalence of goiter in t1 dm and non - diabetic individuals was 21% and 38%, respectively. the lower prevalence of goiter in t1 dm patients in comparison with that in non - diabetic ones is in contrast with previous studies, which showed no difference in goiter prevalence between t1 dm patients and control group[2931 ] or higher prevalence of goiter in patients with t1dm[32, 33 ]. studies that showed higher thyroid volume and goiter prevalence in t1 dm patients attributed this finding to the higher incidence of ait in t1 dm. however, we showed the higher prevalence of goiter in t1 dm patients with ait (38.8%) than in patients without ait (17%). as stated before, we did not evaluate the iodine deficiency as a possible contributor of goiter. the sensitivity and specificity of palpation is poor and it would be more accurate if we use thyroid ultrasonography to determine goiter size. in our study, the prevalence of ait in female patients with t1 dm was higher than that in male t1 dm patients. in the control group, many studies found higher prevalence of positive thyroid autoantibodies in females[12, 35, 36 ] and some studies reported similar prevalence in both genders[37, 38 ]. in agreement with previous studies[6, 39, 40 ], in the current study, we found no relationship between hba1c, as a measure of metabolic control in diabetic patients, and ait or thyroid dysfunction. children and adolescents with t1 dm had higher levels of thyroid autoantibodies, higher prevalence of ait, and subclinical hypothyroidism than non - diabetic ones. it seems beneficial to measure thyroid function indices and thyroid antibodies in patients with t1 dm, regularly.	objectivestudies in different populations have shown great variation in the prevalence of thyroid diseases in patients with type 1 diabetes mellitus (t1 dm). our aim was to study the prevalence of thyroid disorders such as autoimmunity of thyroid (ait), thyroid dysfunction, and goiter in children and adolescents with t1 dm, compared with age- and sex - matched healthy controls in isfahan.methodsone hundred patients with t1 dm who were referred to isfahan endocrine and metabolism research center and 184 healthy schoolchildren matched for age and sex were included. they were examined for goiter by two endocrinologists. thyroid function test and serum thyroid antibodies (anti - tpo ab and anti - tg ab) were measured.findingsthe prevalence of subclinical hypothyroidism was high in both groups (18%). t1 dm patients had lower frequency of goiter (21% vs. 38%, p=0.001), and higher prevalence of positive ait (22% vs. 8%, p=0.001), anti - tpo ab positivity (19.3% vs. 5.3%, p=0.000), and anti - tg ab (11.1% vs. 6.4%, p=0.1) in comparison with the control group. being positive for ait in diabetic patients meant an odds ratio of 5 (ci 95% : 1.5 - 15.6) for thyroid dysfunction. there was no association between age, sex, duration of diabetes and hba1c with serum anti - tpo ab and anti - tg ab concentrations in this group.conclusionour results demonstrated the high prevalence of ait and thyroid dysfunction in patients with t1 dm. we suggest regular thyroid function and antibody testing in these patients.
the most common etiology of spontaneous excess cortisol production is cushing 's disease due to hypersecretion of pituitary adrenocorticotropic hormone (acth) by a corticotroph adenoma. cushing 's disease is a rare disorder, with an annual incidence of 13 per million and a female : male ratio of 3 : 1, that is responsible for significant morbidity and mortality resulting from cardiovascular complications, infections, and psychiatric disturbances. it is now recognized that, in a small proportion of cases, cushing 's disease is accompanied by an elevated prolactin (prl) level. for example, lado - abeal and colleagues identified a mildly elevated serum prl in 20% of women with cushing 's disease and menstrual abnormalities, and both mahler. and sherry. have presented a patient with cushing 's disease and symptomatic hyperprolactinemia. although cushing 's disease with hyperprolactinemia due to mixed acth- and prl - secreting adenomas occurs rarely, elevated preoperative prl levels in cushing 's disease are of diagnostic significance. despite the abundance of published data concerning various aspects of the clinical course and treatment of cushing 's disease, very few investigations have focused on the characteristics of patients with cushing 's disease and hyperprolactinemia, and there is a paucity of studies comparing the clinical features of patients with cushing 's disease and hyperprolactinemia and those with cushing 's disease but no hyperprolactinemia. this is particularly the case for patients in china, about whom very little information is available concerning clinical outcomes following transsphenoidal surgery. the aims of our investigation were to describe the prevalence and clinical features of cushing 's disease in patients treated with transsphenoidal surgery at our hospital in china and to evaluate the differences between patients with cushing 's disease and hyperprolactinemia and those with cushing 's disease alone. we therefore performed a retrospective analysis of patients with cushing 's disease, with or without concomitant hyperprolactinemia, to clarify the clinical features and confirm the efficacy of transsphenoidal surgery for treatment of the disease. this study provides novel insights that extend our current knowledge and will help to improve the diagnosis and treatment of patients with cushing 's disease and hyperprolactinemia. this was a retrospective study of patients diagnosed with cushing 's disease, who had been treated by primary transsphenoidal surgery between january 2002 and june 2011, at the department of neurosurgery, shandong provincial hospital affiliated to shandong university, jinan, shandong, china. the diagnosis of cushing 's disease was made on the basis of the medical history, symptoms, the levels of serum hormones, immunohistochemical staining, computer - aided tomographic (ct) imaging, and magnetic resonance imaging (mri). patients were divided into two groups, according to the preoperative hormonal levels and immunohistochemical results : a cd group (patients with elevated cortisol only) and a cd + prl group (patients with both elevated cortisol and prl levels). due to the retrospective nature of this study, written informed consent oral informed consent was obtained from each participant, and the data were analyzed anonymously. the ethics committee of the shandong provincial hospital affiliated to shandong university approved the research protocols. patients were included in the study if all the following criteria were satisfied : classical signs and symptoms of cushing 's disease ; increased serum cortisol levels ; loss of diurnal rhythm of serum cortisol ; increased 24-hour urinary free cortisol levels ; normal or slightly elevated plasma acth levels ; lack of suppression of urinary free cortisol and serum cortisol following oral 2 mg dexamethasone loading ; suppression of urinary free cortisol and serum cortisol after oral 16 mg dexamethasone (urinary free cortisol and/or serum cortisol suppressed by > 50% of the baseline value following oral administration of 2 mg dexamethasone every 6 hours for 48 hours) ; an adenoma staining positively for acth ; the presence of a pituitary mass on mri ; treatment by surgery alone ; and availability of a complete set of medical records. an additional inclusion criterion required for the exclusion criteria for the cd group were patients with hypersecretion of two or more hormones ; adenomas with positive immunohistochemical staining for two or more hormones ; adrenal - derived cushing 's syndrome ; ectopic cushing 's syndrome ; and other potential causes of an elevated cortisol level. the exclusion criteria for the cd + prl group were patients with hypersecretion of two or more hormones (except for cortisol and prl) ; adenomas with positive immunohistochemical staining for two or more hormones (except for acth and prl) ; and other causes of elevated cortisol and prl levels. microscopically assisted transsphenoidal pituitary tumor resection surgery was performed by two experienced neurosurgeons (yuan - ming qu and guang - ming xu), and the resection was considered subtotal or complete in all enrolled patients. in order to accurately assess the effects of surgery, only patients treated by surgery alone were included in this study. the data collected included the age and gender of the patient, past medical history, clinical presentation, preoperative neurological, endocrine, and ophthalmological status, and postoperative outcomes. the time from the onset of symptoms and signs to the date of diagnosis (time to diagnosis) was noted. a microadenoma was defined as a tumor with a diameter of 10 mm or less, whereas a macroadenoma was defined as a tumor with a diameter of more than 10 mm. representative t1-weighted coronal mri scans showing an example of a microadenoma and a macroadenoma are shown in figure 1. cavernous sinus invasion was suspected when adenoma tissue was located lateral to the cavernous portion of the internal carotid artery on mri. definite diagnosis could be made when the medial wall of the cavernous sinus was found to be destroyed during surgery by invasion. endocrinological assessment was carried out in all patients, including measurements of the serum levels of cortisol, prl, growth hormone (gh), thyroid stimulating hormone (tsh), luteinizing hormone (lh), follicle stimulating hormone (fsh), testosterone, estradiol, thyroxine, and progesterone. in this study, cortisol was measured using an electrochemiluminescent immunoassay kit (roche diagnostics gmbh, mannheim, germany) with a functional sensitivity of < 8.5 nmol / l and intra - assay and interassay variation coefficients of < 10% and < 15%, respectively. the serum level of prl was measured using a commercially available chemiluminescence kit (beckman coulter, inc., the normal range for prl is 2.6413.13 ng / ml in males, 3.3426.72 ng / ml in premenopausal females, and 2.7419.64 ng / ml in postmenopausal females. the analytical sensitivity for prl was 0.25 ng / ml, and the intra - assay and interassay variation coefficients were < 10% and < 15%, respectively. serum levels of cortisol and prl were evaluated in all patients on three occasions : 3 days after surgery, approximately 3 months after surgery, and 12 months after surgery. the resected tumor fragments were fixed in 10% buffered formalin, stained with hematoxylin and eosin (h&e) using routine histochemical methods, and embedded in paraffin blocks. immunohistochemical analysis was also performed on specimens, using specific antibodies against acth (rabbit polyclonal antibody ; maxin co. ltd., fujian, china), gh (rabbit polyclonal antibody ; maxin co. ltd., fujian, china), prl (rabbit polyclonal antibody ; maxin co. ltd., fujian, china), fsh (mouse monoclonal antibody ; maxin co. ltd., fujian, china), lh (mouse monoclonal antibody ; maxin co. ltd., fujian, china), and tsh (mouse monoclonal antibody ; maxin co. ltd., examples of images obtained using these pathological techniques are shown in figure 2 (original magnification 400). a major complication was considered to be a disease or injury diagnosed after surgery that was potentially fatal and/or resulted in a permanent defect. less serious adverse events, including diabetes insipidus (di) and hyponatremia, were counted as minor complications. serum levels of sodium were measured immediately before surgery and on days 1, 3, and 7 after surgery ; hyponatremia was diagnosed when the serum sodium level was 135 mmol / l, and, when present, its onset, frequency, and time course were monitored. central di includes both transient di and permanent di ; transient di is diagnosed when hypotonic polyuria (40 ml / kg body weight daily) ensues soon after surgery and is usually self - remitting within a few days. since permanent di requires assessment of endocrine function several months after surgery, the incidence of di in this study may only represent transient di. all patients were followed up as outpatients for three months after surgery and then at annual or biannual intervals. follow - up information for patients not attending our department was obtained by contacting the patients or their relatives by telephone or mail. normalization was defined as a return of hormone levels to within the normal range without the requirement of any further treatment. recurrence was diagnosed from the clinical features, hormonal function, or identification of residual tumor, based on the results of hormone and mri examinations performed two years after surgery. quantitative data are reported as the mean standard deviation (sd), and qualitative data are expressed as percentages. quantitative and nonparametric data, such as mean age, time to diagnosis, and tumor size, were compared by student 's t - tests or mann - whitney u tests. chi - square tests were used to compare qualitative data, including gender, invasiveness, the presence of pituitary apoplexy, tumor classification, remission rate, and recurrence rate. statistical comparisons of endocrine outcomes at different follow - up times were adjusted for multiple testing using a bonferroni correction. differences between groups in endocrine outcome at the same follow - up time were assessed by student 's t - test. analyses were performed using statistical package for social sciences version 16.0 (chicago, il, usa). all statistical tests were two - sided, and p < 0.05 was considered statistically significant. a total of 84 patients were enrolled in the study, with a mean age of 33.60 12.70 years (range : 1775 years) ; more than half the patients were female (n = 59 ; 70.2%). the demographics and tumor characteristics of the two groups of patients are summarized in tables 1 and 2. of the 84 patients enrolled, 48 (15 males and 33 females) were categorized into the cd group and 36 (10 males and 26 females) into the cd + prl group. at the time of diagnosis, the patients in the cd + prl group (mean age : 30.28 14.23 years ; range : 1775 years) were significantly younger than those in the cd group (mean age : 36.08 10.91 years ; range : 2365 years) (p = 0.037). furthermore, maximal adenoma size was larger for patients in the cd + prl group (mean diameter : 2.44 1.32 cm ; range : 0.66.0 cm) than for those in the cd group (mean diameter : 1.44 1.05 ; range : 0.54.0 cm) (p < 0.001). the mean time to diagnosis did not differ significantly between the cd group (25.32 37.98 months ; range : 0.1180 months) and the cd + prl group (33.68 34.96 months ; range : 0.3120 months), and there were also no significant differences in the incidences of invasiveness or pituitary apoplexy between the two groups. the patients were classified according to the degree of invasion of the cavernous sinus according to knosp classification, and the two groups showed significant differences with the cd + prl group showing a tendency for higher grades reflecting higher rates of invasion (p = 0.005). in the cd group, the corresponding values for patients in the cd + prl group were 5.6% and 94.4%, indicating that macroadenomas were more prevalent in the cd + prl group than in the cd group (p < 0.001). the most commonly presenting clinical manifestations in the two groups were menstrual disorders, headaches, and dizziness (table 3). menstrual disorders and visual field defects were more common in patients in the cd + prl group than in patients in the cd group (p = 0.027 and 0.021, resp.), whereas the incidences of progressive obesity and hypertension were much lower in patients in the cd + prl group than in patients in the cd group (p = 0.009 and p < 0.001, resp.). preoperative and postoperative serum cortisol and prl levels were available for all patients (table 4). elevated preoperative levels of serum cortisol and prl were detected in patients in the cd + prl group, and these patients had lower cortisol levels and higher prl levels than those in the cd group (p < 0.05 for both comparisons). the cortisol level of patients in the cd group decreased from 833.87 235.75 the preoperative and postoperative prl levels in the cd group were all within the normal range. in the cd + prl group, furthermore, the prl levels of this group decreased from 233.63 188.06 ng / ml before surgery to 72.63 66.94 ng / ml at 3 days, 74.51 62.58 ng / ml at 3 months, and 77.43 there were no significant differences in the postoperative cortisol levels between the cd and cd + prl groups, but the postoperative prl levels in the cd + prl group remained markedly higher than those in the cd group. among the patients with microadenomas, the normalization rates (based on the postoperative hormone levels at three months) in the cd and cd + prl groups were 95.7% and 100%, respectively (figure 3(a)). for patients with macroadenomas, overall, normalization of hormonal levels was achieved in 44 patients (91.7%) in the cd group, but only 15 patients (41.7%) in the cd + prl group (p < 0.001). in the cd + prl group, normalization of both cortisol and prl levels occurred in 41.7% of the patients (figure 3(b)). no deaths or major complications were observed, although some minor complications did occur, including transient di and hyponatremia. transient di was observed in 26 of 48 patients (54.2%) in the cd group and in 18 of 36 patients (50.0%) in the cd + prl group. hyponatremia occurred in 50% of the patients in both groups, with nadir serum sodium levels in the cd group (113134 mmol / l) similar to those in the cd + prl group (109130 mmol / l). there were no significant differences in the incidences of transient di and hyponatremia between the two groups. the median postoperative duration of hospitalization was 5 days (range : 315 days). the median duration of follow - up for the entire group was 45 months (range : 13121 months). tumor recurrence was significantly less frequent in the cd group (4 patients, 8.3%) than in the cd + prl group (13 patients ; 36.1%) (p < 0.001). cushing 's disease, caused by excessive acth secretion from tumorous pituitary corticotrophs, is a potentially life - threatening endocrine condition. the first - line treatment for cushing 's disease is pituitary surgery to control excessive hormone production and improve pituitary function. cases of pituitary adenoma associated with increased production of both acth and prl, causing apparent cushing 's disease and hyperprolactinemia, are extremely rare [5, 11 ]. the present study has evaluated the clinical features and endocrine and surgical outcomes, over a relatively long follow - up period, of a large series of patients who underwent transsphenoidal surgery for cushing 's disease with or without hyperprolactinemia. the majority of our patients were diagnosed in their thirties and forties, with those in the cd + prl group significantly younger at diagnosis than those in the cd group. in addition, our results revealed that the mean maximal diameter of the adenoma varied between the two groups, consistent with a previous report that patients with cushing 's disease alone usually present with small tumors. the cd + prl group also had a tendency for higher knosp grades so the adenoma was more likely to have invaded the cavernous sinus space. approximately half of the patients in the cd group were found to have microadenomas, and the other half were found to have macroadenomas, a finding similar to that of others. although recent advances in imaging technologies, including mri, can detect relatively small lesions within the pituitary gland, these methods can localize microadenomas in only 6080% of patients with cushing 's disease, since most of the lesions are very small [8, 14 ]. in our study, the average diameter of the adenoma was less than 5 to 6 mm, in agreement with previous investigations [8, 15 ]. we also found that nearly 95% of patients in the cd + prl group had macroadenomas ; this higher incidence may be related to the higher levels of prl. menstrual disorders and visual field defects were more common in patients in the cd + prl group (compared with the cd group), whereas the incidences of progressive obesity and hypertension were much lower. hypersecretion of prl has a variety of manifestations, including menstrual disorders in women, and our findings concurred with those of previous investigations. pituitary compression by a tumor may present with visual field defects ; furthermore, patients in the cd + prl group were much more likely to have macroadenomas, which are more commonly associated with the development of neurological deficits and visual disturbances. acth has antagonistic effects on protein and lipid metabolism that can lead to uncontrolled hypertension and other serious complications. overall, the clinical symptoms observed in our study agreed with those of previous reports in the literature [1921 ]. of the patients in the cd + prl group who had hormonal symptoms, elevated preoperative cortisol and prl levels were detected in patients in the cd + prl group, with prl levels improving after surgery ; this is consistent with the findings of ratliff and oldfield and wynne.. we also found that patients in the cd + prl group with high prl levels often had a relatively low serum cortisol level. yamaji and colleagues reported prl elevation in 23% of patients with cushing 's disease, while caufriez and coworkers found that 91% of patients with cushing 's disease had preoperative elevation of prl levels that normalized after transsphenoidal surgery. although wynne. reported that the predictive value of preoperative prl levels in cushing 's disease was limited, we found that patients in the cd group were more likely to achieve normalization of endocrine function. it was notable that, in the cd + prl group, the normalization rate for patients with macroprolactinomas was significantly lower than that for patients with microprolactinomas. since complete resection of microadenomas was possible, it is perhaps not surprising that the normalization rates for patients with microadenomas exceeded 95% for both groups. our results, together with those of previous investigations, indicate that surgery is a highly effective treatment for cushing 's disease, and transsphenoidal surgery is considered the definitive treatment method. thus, the discovery of patients with a plurihormonal tumor depends on the hormonal levels as well as pathological identification [27, 28 ]. plurihormonal adenomas are defined as those that secrete two or more hormones that differ in chemical composition, immunoreactivity, and biologic effects [29, 30 ]. in every patient in the cd + prl group, a mixed tumor with acth- and. the occurrences of postoperative transient di and hyponatremia correlate with intraoperative manipulation of the pituitary stalk and posterior lobe, multiple incisions in the gland, and partial hypophysectomy. since pituitary exploration may damage the normal gland, gland manipulation is always kept to the minimum necessary to identify and remove the adenoma. the rate of postoperative transient di reported in the literature varies widely [32, 33 ]. although there was no significant difference in the incidence of di between the two groups in our study, relatively higher rates of di were observed in our patients overall, likely due to an increased awareness regarding this potential complication. there was also no significant difference in the incidence of hyponatremia between the two groups. future prospective studies of the impact of transsphenoidal surgery on transient di and electrolyte abnormalities are merited. wide variations in surgical outcomes and recurrence rates have been reported, depending on the tumor characteristics, the surgeon 's experience, and the duration of the follow - up. the recurrence rate in the cd group was 8.3%, lower than that reported by others [34, 35 ], whereas it was 36.1% in the cd + prl group. the elevated rate of recurrence in the cd + prl group is an important finding, which suggests that careful long - term follow - up is needed in this subset of patients, even if surgery is considered successful. mixed acth and prl adenomas often manifest themselves as coexisting cushing 's disease and prolactinoma. several hypotheses have been proposed to explain the coexistence of two or more hormones within an adenoma. second, different hormones could be derived from distinct cell types within the borders of a single adenoma [4, 37 ] ; for example, two separate tumors growing in a small space could become conjoined as a single mass. first, this was a retrospective cohort study in a single institution ; prospective, multicenter studies are merited to extend our observations. second, the follow - up period was relatively short for some patients, limiting the evaluation of the real recurrence rates. hence, long - term postoperative follow - up of a similar cohort of patients is needed to better assess the effects of surgery and treatment outcomes. in summary, the current study has compared characteristics between patients with both cushing 's disease and hyperprolactinemia and those with cushing 's disease alone. we found that patients in the cd + prl group were characterized by younger age, larger tumor size, more clinical manifestations (including a higher incidence of menstrual disorders caused by prl secretion), lower endocrine normalization rate, and more frequent recurrence rate, compared with patients with cushing 's disease alone. our observations extend our knowledge of the characteristics of patients with cushing 's disease and hyperprolactinemia and may provide insights that will help to improve the diagnosis and treatment of the disease.	objective. we compared the characteristics of patients with cushing 's disease alone with those of patients with cushing 's disease and hyperprolactinemia. methods. eighty - four patients were enrolled between 2002 and 2011, in a hospital in china. clinical, endocrinological, and histopathological data, mri scans, and surgical outcomes were reviewed throughout the follow - up period. results. patients with cushing 's disease and hyperprolactinemia had a younger age at diagnosis (30.28 14.23 versus 36.08 10.91 years ; p = 0.037) and a larger adenoma maximal diameter (2.44 1.32 versus 1.44 1.05 cm ; p < 0.001) than patients with cushing 's disease alone. menstrual disorders (p = 0.027) and visual field defects (p = 0.021) were more common and progressive obesity (p = 0.009) and hypertension (p < 0.001) were less common in patients with cushing 's disease and hyperprolactinemia. the rate of normalization of hormonal levels was lower (41.7% versus 91.7% ; p < 0.001) and the recurrence rate was higher (36.1% versus 8.3% ; p < 0.001) in patients with cushing 's disease and hyperprolactinemia. conclusions. careful long - term follow - up is needed of patients with cushing 's disease and hyperprolactinemia.
amantadine hydrochloride (amd), scheme 1, is an antiviral agent used against infection with influenza type a virus and to ameliorate symptoms when administered during the early stages of infection as well as in the management of herpes zoster. it has mild anti - parkinsonism activity and thus it has been used in the management of parkinsonism, mainly in the early disease stage and when the symptoms are mild. spectrophotometry and spectrofluorometry are considered as the most convenient analytical techniques in pharmaceutical analysis because of their inherent simplicity and availability in most quality control and clinical laboratories [39 ]. however, amd does not possess any chromophore or fluorophore in its molecule, which are the essential requirements for the direct analysis by either spectrophotometric or spectrofluorometric techniques. therefore, derivatization of amd was necessary for its determination by either of the two techniques. for spectrophotometric determination of amd the involved derivatization reactions that have been published prior to 1983 have been reviewed by kirschbaum. the derivatizing reagents used thereafter included iodine, acetaldehyde / chloranil,,-diphenyl--picrylhydrazyl, bromocresol green, tetracyanoethylene, iron(iii), and cyclodextrin. these methods were based on its oxidation with cerium(iv) or its derivatization with 2,3-diphenylquinolizinium bromide, fluorescamine, and 9-isothiocynatoacridine. as well, many derivatization techniques coupled with chromatography have been established for the determination of amd in the dosage forms and biological matrices : tlc, hplc [2123 ], gc, and capillary electrophoresis. 1,2-naphthoquinone-4-sulphonate (nqs) has been used for the determination of many compounds [2628 ]. therefore, the present study was devoted to explore nqs as a derivatizing reagent in the development of selective and sensitive spectrophotometric and spectrofluorometric methods for the determination of amd in capsules and plasma. uv-1601 pc (shimadzu, kyoto, japan) ultraviolet - visible spectrophotometer with matched 1 cm quartz cells was used for all spectrophotometric measurements. spectrofluorimeter, kontron sfm 25 equipped with a 150 w xenon high - pressure lamp was used for measuring the fluorescence intensity. mlw type thermostatically controlled water bath (memmert gmbh, co. schwa bach, germany). biofuge pico centrifuge (heraeus instruments, germany). amantadine hydrochloride (amd ; sigma - aldrich chemie gmbh, steinheim, germany) was obtained and used as received, its purity was 100.02 1.25%. 1,2-naphthoquinone-4-sulphonate ; (nqs ; el - nasr pharmaceutical chemical co., abo - zaabal, egypt). potassium borohydride (sigma - aldrich chemie gmbh, steinheim, germany). adamine capsules (rameda co. for pharmaceutical industries & diagnostic reagents, cairo, egypt) are labeled to contain 100 mg of amd per capsule. human plasma samples were collected from normal healthy volunteer at king khaled university hospital (riyadh, kingdom of saudi arabia), and they were stored at 20c until analysis. double distilled water was obtained through wsc-85 water purification system (hamilton laboratory glass ltd., an accurately weighed amount (100 mg) of amd was quantitatively transferred into a 50 ml calibrated flask, dissolved in 30 ml distilled water, completed to volume with the same solvent to obtain a stock solution of 2 mg ml. this stock solution was further diluted with water to obtain working solutions in the ranges of 50800 and 0.5100 g ml for the spectrophotometric and spectrofluorometric methods, respectively. accurately weighed amount of nqs (150 mg) was transferred into a 25 ml calibrated flask, dissolved in 5 ml distilled water, completed to volume with water to obtain a solution of 0.6% (w / v). an accurately weighed quantity of the capsule contents equivalent to 100 mg of amd was transferred into a 100 ml calibrated flask, and dissolved in about 40 ml of distilled water. the contents of the flask were swirled, sonicated for 5 minutes, and then completed to volume with water. the contents were mixed well and filtered rejecting the first portion of the filtrate. the prepared solution was diluted quantitatively with distilled water to obtain a suitable concentration for the analysis. aliquots of 1.0 ml of plasma were spiked with different concentration levels of amd. the spiked plasma samples were treated with 0.1 ml of 70% perchloric acid and vortexed for 1 minute. the supernatants were transferred into test tubes and neutralized with 1 m naoh solution. one milliliter of the standard or sample solution (50800 g ml) was transferred into a test tube. one milliliter of 0.01 m naoh and 1 ml of nqs reagent (0.6%, w / v) were added. the contents of the tubes were heated in a water bath at 80 5c for 45 minutes and then cooled in ice water for 2 minutes. the contents of the test tubes were transferred quantitatively into a separating funnel containing anhydrous sodium sulphate and extracted with two portions (5 ml) of chloroform. the combined chloroformic extracts were transferred into 10 ml calibrated flasks and the solutions were completed to volume with chloroform if necessary. the absorbances of the resulting solutions were measured at 460 nm against reagent blanks treated similarly. one milliliter of the standard or sample solution (0.5100 g ml) was transferred into a test tube. one milliliter of 0.01 m naoh and 1 ml of nqs reagent (0.6%, w / v) were added. the contents of the test tubes were heated in a water bath at 80 5c for 45 minutes and then cooled in ice water for 2 minutes. the contents of the test tubes were transferred quantitatively into a separating funnel and extracted with two portions (5 ml) of chloroform. the combined chloroformic extracts were evaporated under stream of air and the residues were reconstituted in 2 ml of methanol and quantitatively transferred into 10 ml calibrated flask. a one milliliter of kbh4 solution (0.03%, w / v in methanol) was added and the reaction was allowed to proceed for 5 minutes at room temperature (25 5c). the solution was diluted to volume with 0.025 m ethanolic hcl and the fluorescence intensity of the resulting solution was measured at 382 nm after excitation at 293 nm against reagent blanks treated similarly. master equimolar (2.5 10 m) aqueous solutions of amd and nqs were prepared. series of 5 ml portions of the master solutions of amd and nqs were made up comprising different complementary proportions (0 : 10, 1:9, one milliliter of 0.01 m naoh was added to each tube, and the tubes were further manipulated as described under the general recommended procedure for the spectrophotometric method. because of the absence of any chromophoric group in the amd molecule, it has no absorption in the ultraviolet - visible region above 200 nm, and it has no native fluorescence as well. therefore, derivatization of amd was attempted in the present study for the development of both spectrophotometric and spectrofluorometric methods for its determination. nqs has been used as chromogenic and fluorogenic reagent for primary and secondary amines [2628, 30 ], however, its reaction with amd has not been investigated yet. therefore, the present study was devoted to explore nqs as a derivatizing reagent in the development of spectrophotometric and spectrofluorometric methods for the determination of amd in capsules and plasma. our preliminary experiments in investigating the reaction between amd and nqs revealed that nqs - amd product is orange colored exhibiting a maximum absorption at 460 nm and it is insoluble in water, but soluble in organic solvents. since the present work was directed to involve plasma samples, the interference of endogenous amines was a major concern. it is well known that nqs reacts with the endogenous amines (e.g., amino acids) and yields water - soluble products. for this reason, an extraction step was necessary for the development of selective methods for the determination of amd in the presence of the endogenous amines. as well, the reduced amd - nqs derivative was found to be fluorescent and exhibited one emission maximum at 382 nm and three excitation maxima at 293, 325, and 344 nm. the highest fluorescence intensity was obtained after excitation 293 nm, thus the excitation in the present study was performed at this wavelength. scheme 2 shows the reaction pathway between amd and nqs, and figure 1 shows the absorption, excitation, and emission spectra of the reaction product. the following sections describe the optimization of the assay variables and validation for the performance of both spectrophotometric and spectrofluorometric methods. the factors affecting the derivatization reaction (the concentrations of nqs and naoh, reaction time, temperature, diluting solvent, and the extracting solvent) were investigated by altering each variable in a turn while keeping the others constant. the studying of nqs concentrations revealed that the reaction was dependent on nqs reagent (figure 2). the highest absorption intensity was attained when the concentration of nqs was 0.060.075% (w / v) in the final solution ; further experiments were carried out at nqs concentration of 0.06% (w / v). the results of investigating the effect of naoh concentration on the reaction revealed that the optimum naoh concentration was 0.01 m (figure 2). the effect of temperature on the derivatization reaction was studied by carrying out the reaction at different temperatures (25100c) and the maximum readings were obtained at 70100c (figure 3). for more precise readings, the effect of heating time on the formation of the reaction product was investigated by carrying out the reaction at different times. the maximum absorbance intensity was attained after 40 minutes, and longer reaction time did not affect the absorbance intensity (figure 3). for more precise results, it was found that the colored amd - nqs product is insoluble in the aqueous reaction medium. for measurements, the reaction product might be either dissolved in a miscible organic solvent of lower polarity than water or extracted with an immiscible extractive solvent. different solvents were tested for dilution ; methanol, ethanol, acetonitrile, dimethylsulphoxide, isopropanol, 1,4-dioxane, and acetone. the highest readings were obtained when dioxane was used for dilution (data not shown). as well, different nonmiscible solvents were tested for the extraction of the amd - nqs product : carbon tetrachloride, chloroform, dichloromethane, ethyl acetate, toluene, and benzene. the highest readings were obtained when chloroform was used for extraction (table 1). the results revealed that the extractive procedure is more sensitive (1.5 fold) than the nonextractive procedure. this was attributed to the effective decrease in the blank readings and consequently enhanced the sensitivity of the assay. for developing the spectrofluorometric procedure, a reduction step for amd - nqs product however, the reduced nqs reagent itself is also fluorescent and it had the same excitation and emission maxima of the amd - nqs product, therefore, a selective extraction step for the amd - nqs product from the remaining nqs reagent was necessary before carrying out the reduction step. furthermore, the extraction step is essential to provide the required selectivity for analyzing the plasma samples as the nqs products with endogenous amines were water soluble. based on the reported efficiency, potassium borohydride as a reducing reagent was selected for nqs - derivatives. in order to investigate the effect of potassium borohydride concentration on the reduction, the reaction was performed using varying concentrations (0.00050.01%, w / v). the highest fluorescence intensity was obtained when the concentration was 0.003% in the final solution (1 ml of 0.03%, w / v). concentrations more than 0.003% did not affect the fluorescence intensity (figure 4). the effect of ph on the fluorescence intensity was also studied and the results showed that the highest fluorescence intensity was obtained at ph 2.0 (figure 5). this ph could be attained by diluting the reaction mixture with 0.025 m ethanolic hcl solution. under the optimum conditions, the stoichiometry of the reaction between amd and nqs was investigated by job 's method and was found to be 1:1 because amd molecule contains only one center (primary amino group) available for this condensation reaction. based on this ratio, the reaction pathway was postulated to be proceeded as shown in scheme 2. in the proposed methods, linear plots (n = 6) with good correlation coefficients (0.9974 and 0.9972) were obtained in the concentration ranges of 580 g ml for and 0.0510 g ml for the spectrophotometric and the spectrofluorometric methods, respectively (table 2). the limits of detection (lod) and quantitation (loq) were determined using the formula lod or loq = sda / b, where = 3.3 for lod and 10 for loq, sda is the standard deviation of the intercept, and b is the slope. the lod values were 1.39 and 0.013 g ml for the spectrophotometric and spectrofluorometric methods, respectively (table 2). the precision of the proposed methods was determined by replicate analysis of five separate sample solutions at three concentration levels of amd. the relative standard deviations (rsds) were 0.830.96 and 0.461.01% for the spectrophotometric and spectrofluorometric methods, respectively (table 3), indicating the good reproducibility of the proposed methods. furthermore, the inter- and intra - assay precisions of the proposed spectrofluorometric method were determined from the recovery studies of spiked human plasma samples. the rsd values of the recovery were 0.572.04 and 0.724.20% for the intra- and inter - assay determinations, respectively (table 4). the obtained recovery values were 98.8100.2 1.041.54% indicating the high accuracy of the proposed methods. moreover, the accuracy of the proposed spectrofluorometric method was evaluated by the recovery studies of spiked human plasma samples. these recovery results of the spiked human plasma indicate the suitability of the proposed spectrofluorometric method for the analysis of amd in human plasma. the results of the interferences study showed that no interferences were found from any of the excipients studied ; lactose, sucrose, starch, talc, gum acacia, glucose, and magnesium stearate ; the recovery of amd was 98.15100.72%. moreover, the interferences from the amino acids with the assay procedures were also studied using glycine as an example for the amino acids. the results of this study revealed that the amino acids could interfere with the spectrophotometric procedures. however, there is no any interference coming from the amino acids after an extraction step for the derivatized amd product because the derivatized amino acids products are water soluble. therefore, the extraction step increased both the sensitivity and selectivity by removing the interferences caused by both amino acids and proteins in the plasma samples. robustness was examined by evaluating the influence of small variation of method variables, including concentration of analytical reagents and reaction time on the performance of the proposed methods. in these experiments, one parameter was changed whereas the others were kept unchanged, and the recovery percentage was calculated each time. it was found that small variation of method variables did not significantly affect the procedures. this provided an indication for the reliability of the proposed method during its routine application for the analysis of amd. ruggedness was also tested by applying the proposed methods to the assay of amd using the same operational conditions but using two different instruments at two different laboratories and different elapsed time. results obtained from lab - to - lab and day - to - day variations were found to be reproducible, the full range of recovery values was 98.4101.3% and the rsd was 0.73 and 1.06% for the spectrophotometric and spectrofluorometric methods, respectively. it is evident from the above - mentioned results that the proposed methods gave satisfactory results with amd in bulk. thus, its capsules were subjected to the analysis of their contents from the active ingredient by the proposed methods and the official (nonaqueous titration) method. the capsule contents, as percentage, were 98.70 1.79 and 98.91 1.93% for the spectrophotometric and spectrofluorometric methods, respectively (table 5). these results were compared with those obtained from the official method by statistical analysis with respect to the accuracy (t - test) and precision (f - test). no significant differences were found between the calculated and theoretical values of t- and f - tests at 95% confidence limit proving similar accuracy and precision in the analysis of amd in its dosage form. the present study described the use of nqs reagent for the development of selective, sensitive, and accurate spectrophotometric and spectrofluorometric methods for the determination of amd in bulk, capsules, and plasma. the described methods are superior to the previously reported spectrophotometric or spectrofluorometric methods for analysis of amd in terms of their selectivity and sensitivity. the linear ranges of the proposed spectrophotometric and spectrofluorometric methods were 580 and 0.0510 g / ml, respectively, which are much less than some reported methods 1001300, 1590, 2575, 94940 g / ml [11, 14, 15 ] which were based on either the nonselective oxidation or charge - transfer complex formation of amd base. although the sensitivity of the proposed spectrofluorimetric method is comparable to that described by darwish., which was based on the nonselective oxidation of amd with cerric sulphate, however, our proposed methods are more selective. also, the proposed methods involved spectrophotometric and spectrofluorometric measurements with comparable analytical performance devoid from any potential interference. this gives the advantage of flexibility in performing the analysis on any available instrument. furthermore, all the analytical reagents are inexpensive, have excellent shelf life, and are available in any analytical laboratory. therefore, these methods can be recommended for the routine analysis of amd in quality control and clinical laboratories.	new selective and sensitive spectrophotometric and spectrofluorometric methods have been developed and validated for the determination of amantadine hydrochloride (amd) in capsules and plasma. the methods were based on the condensation of amd with 1,2-naphthoquinone-4-sulphonate (nqs) in an alkaline medium to form an orange - colored product. the spectrophotometric method involved the measurement of the colored product at 460 nm. the spectrofluorometric method involved the reduction of the product with potassium borohydride, and the subsequent measurement of the formed fluorescent reduced amd - nqs product at 382 nm after excitation at 293 nm. the variables that affected the reaction were carefully studied and optimized. under the optimum conditions, linear relationships with good correlation coefficients (0.99720.9974) and low lod (1.39 and 0.013 g ml1) were obtained in the ranges of 580 and 0.0510 g ml1 for the spectrophotometric and spectrofluorometric methods, respectively. the precisions of the methods were satisfactory ; rsd 2.04%. both methods were successfully applied to the determination of amd in capsules. as its higher sensitivity, the spectrofluorometric method was applied to the determination of amd in plasma ; the recovery was 96.3101.2 0.574.2%. the results obtained by the proposed methods were comparable with those obtained by the official method
wear resistance of denture teeth has been considered as one of the most important requirements for oral rehabilitation of edentulous patients with removable dentures, in order to maintain a stable occlusal support over time13. wear of the occlusal surfaces may result in insufficient posterior tooth support25, loss of chewing efficiency1,21 and nonfunctional activities1,2. although wear of acrylic resin teeth has also been related to the loss of vertical dimension of occlusion with complete dentures, the major factor affecting it is the reduction of residual ridges by resorption22. initially, denture teeth were made of ceramic material. with the advent of polymethyl methacrylate (pmma) in the 1940s, denture teeth are currently made of either methacrylate - based resins (acrylic resin) or ceramics, although resin teeth have almost eliminated ceramic teeth from the market25 due to a number of advantages, such as the chemical bond to denture base5,6,16, lower susceptibility to fracture6 and decrease of clicking4,24. nonetheless, the wear resistance of acrylic resin teeth has been questioned for being lower than that of ceramic teeth10 - 12,18,20,22,28. manufacturers have then tried to develop acrylic resins designed to offer improved wear resistance for resin denture teeth25. pmma has been reported to have advanced with the advent of cross - linked agents27, which are bifunctional monomeric molecules that are added to the polymeric material to allow crossing between linear polymeric chains11. the cross - linked polymer was later modified by increasing the quantity of cross - linked agents to ensure a higher degree of cross - linking between the polymers22, or by blending special polymers and co - polymers.3 other modification of cross - linked polymer was the appearance of resins with interpenetrating polymer network (ipn) 22,28. ipn resin is formed when a polymer network is crossed inside another network occupied by a second crossed polymer. the crossed networks coexist in the same volume of the space (one being physically retained within the other) and can not be separated without chemical bond rupture22,29. these factors may influence the final quality for the acrylic resin and produce denture teeth with better mechanical and physical properties26. the search for a more wear - resistant denture tooth material resulted in the development of modified resin teeth that display acceptable wear resistance21. in present days, acrylic resin denture teeth can be classified into two main categories : conventional (with crossed - linked polymeric chains) and high - strength (with various polymer technologies that provide different chemical compositions). some studies22,27,28 have found a higher wear resistance for the high - strength acrylic resin denture teeth with modified polymers in comparison to conventional pmma, while others14,15,17,19,29 have found no advantages of such materials with regard to wear properties. composite resin denture teeth were developed in the 1980s as an effort to achieve greater wear resistance and bond strength to denture bases30. tooth materials made with microparticle inorganic fillers immersed in a bis - gma (bisphenol a glycidyl methacrylate) matrix1,28, or nanometric inorganic fillers immersed in a pmma matrix26 have been used for fabrication of composite denture teeth. it has been reported that composite denture teeth show a higher wear resistance than acrylic resin denture teeth3,26, although data vary depending on the experimental design3. moreover, some authors found denture teeth with inorganic fillers to be 40 - 50% more abrasion - resistant than ipn teeth27. despite the evolution of the mechanical properties of pmma, wear of this material continues to occur with clinical use10,12. the wear resistance of acrylic resin denture teeth has been investigated22,27,28 ; however, the constant appearance of resins with modified polymers used for fabrication of denture teeth manufacturing, impose the need for further studies in this context. furthermore, the results are not clear because the wide variety of wear - testing devices have hindered the comparison of the results19,26, which reflect the lack of a standardized and reproducible methodology for testing the wear resistance of acrylic resin denture teeth28. this in vitro study tested the null hypotheses that there is no difference among the wear resistance of one conventional (trubyte biotone) and two high - strength pmma denture teeth (trilux and vivodent) when opposed to glazed or airborne particle abraded ceramic. the two different antagonist ceramic surfaces were used to allow the test to be carried out under conditions that would cause minimal and maximal wear of the denture teeth. both right and left maxillary canines (n=10) of 3 types of pmma denture teeth were purchased : trubyte biotone (a conventional denture tooth composed by cross - linked pmma) ; trilux (a high - strength tooth composed by highly cross - linked ipn - pmma) ; and vivodent (a high - strength tooth composed by highly cross - linked pmma) (table 1). all teeth were stored in distilled water at 37c for 24 h before wear testing to allow water absorption. the in vitro two - body wear - testing apparatus used in this study was a simulated brushing machine. the denture tooth was fixed on the top of a tooth brush using a screw and composite resin to allow the tooth be fixed on the wear - testing apparatus. the tooth was securely mounted on the upper stainless steel holder of the apparatus (movable part), whereas the antagonist plate was mounted on the fixed bottom part so that they contact tightly on each surface (figure 1). glazed or airborne particle abraded metalloceramic plates (2.5 x 1.2 x 0.15 cm) were used as antagonist surfaces and replaced after each change of denture tooth. the opposing plates were cast in ni - cr alloy (durabond universal, odonto comercial importadora ltda) and trimmed with aluminum oxide abrasive burs at low rotation. thereafter the surface was sandblasted with 100 m aluminum oxide particles (trijato odonto larcon, maring, pr, brazil) and the conventional application of porcelain was made, including the glaze final process (duceram plus, degudent ind. the antagonist ceramic was planed and polished using silicon carbide paper up to 600 (extec corp., enfield, usa) and then sandblasted with 100 m aluminum oxide particles. at each change of paper, the metalloceramic plates were cleaned in an ultrasonic cleaner at 40 hz for 2 min. wear testing was performed by repeated sliding contact at 4.5 cycles / s with 20 mm sliding distance per cycle in the buccolingual direction with a load of 300 g. during the wear test, continuous rinsing with demineralized water (37c) was used to remove abraded particles from the sample surface and to simulate the wet environment of the oral cavity. the height (mm) of each tooth was measured using a digital measuring microscope accurate to 0.001 mm (tm-505, mitutoyo corporation, japan) before and after the wear test. the wear resistance assessed as height loss (mm) was calculated as the decrease in height after 100,000 cycles. the mean wear values were analyzed individually by two - way analysis of variance (anova) and the differences between the denture teeth were determined by the tukey 's test (=0.05). the mean wear values obtained after 100,000 cycles of all denture teeth tested against the glazed or airborne particle abraded ceramic plates are summarized in table 2. there were no statistically significant differences (p>0.05) in wear among the 3 denture teeth evaluated against glazed ceramic. there were significant differences among the teeth tested against abraded ceramic, with trubyte biotone presenting significantly more wear compared to the high - strength denture teeth (p0.05) when tested against either glazed or airborne particle abraded ceramic. all teeth showed significantly more wear against airborne particle abraded than against glazed ceramic (p0.05). nonetheless, with regard to the data obtained from the airborne particle abraded ceramic, trubyte biotone (a conventional denture tooth made of cross - linked pmma) showed significantly more wear than trilux (a high - strength denture tooth made of highly cross - linked ipn - pmma) and vivodent (a high - strength denture tooth made of highly cross - linked pmma). since higher mean wear values were found against the abraded ceramic compared to the glazed ceramic, these results appear to be more consistent in this current wear test series. it is thus assumed that a connection between the chemical composition and the wear resistance of these denture teeth may rather exist. these data are consistent with those other studies that have also found a lower wear resistance for conventional pmma denture teeth evaluated in comparison to high - strength denture teeth8,21,24,28,27,26. however, regarding the high - strength teeth evaluated in the present study (trilux and vivodent), no significant differences were found in wear for either of the opposing ceramics. this finding was in accordance with some authors15,19,21 and had previously been reported by other study3. in spite of that, differences in the wear of high - strength denture teeth formulations have been reported27. therefore, the results of previous in vitro studies on wear resistance of acrylic resin denture teeth seem to be rather inconsistent25. the findings of whitman,.28, hirano,.13, and suzuki26 have suggested that denture teeth made of ipn resin, highly cross - linked polymers or composite resin are more wear - resistant than the conventional acrylic resin denture teeth made of cross - linked pmma. 25, on the other hand, did not find any connection between the chemical composition and the wear resistance of the tested denture teeth. other investigations also failed to show differences among denture teeth made of polymers with a high degree of cross - linking or polymers with inorganic fillers and teeth made of conventional pmma3,14,15,19,22,28,29. these evidences put in question the superiority of high - strength denture teeth and raises doubts about the advantageous clinical use of these products over time. indeed, recent clinical investigations have not revealed any statistically proven difference in wear behavior between polymers with a high degree of cross - linking or with inorganic fillers and conventional polymethyl methacrylate15,19,22. these conflicting data may be due to the large variety of experimental designs, measuring instruments and wear - testing methods used in these investigations. the large number of denture tooth brands with different chemical compositions has created additional difficulties to the analysis of these data. in spite of the controversy the results of this study may assist dentists in selecting pmma denture teeth from the standpoint of wear resistance. nonetheless, it is important to consider that there is not a determinant factor for predicting the wear of pmma denture teeth. on the contrary, besides the chemical composition of the acrylic resin, several other factors should be taken into account on the abrasive process to allow the scientific understanding of the complex phenomenon of wear, namely the chewing pattern10,18, chewing frequency14, occlusion force, food abrasion10,18, non - functional tooth - grinding habits11, abrasive cleansers3, materials ' mechanical properties1,5,20 and dusty atmosphere12. under the experimental conditions and within limitations of this study, the null hypothesis was rejected, since the wear resistance of the three types of polymethyl methacrylate denture teeth was different against glazed or airborne particle abraded ceramic.	the wear resistance of denture teeth is important to the longevity of removable prostheses of edentulous patients. the ability of denture teeth to maintain a stable occlusal relationship over time may be influenced by this property. the purpose of this in vitro study was to evaluate the wear resistance of polymethyl methacrylate (pmma) denture teeth based on their chemical composition when opposed by a ceramic antagonist. the maxillary canines (n=10) of 3 pmma denture teeth (trubyte biotone, cross - linked pmma ; trilux, highly cross - linked ipn (interpenetrating polymer network)-pmma ; and vivodent, highly cross - linked pmma) were secured in an in vitro 2-body wear - testing apparatus that produced sliding contact of the specimens (4.5 cycles / s, sliding distance of 20 mm, under 37c running water) against glazed or airborne particle abraded ceramic. wear resistance was measured as height loss (mm) under 300 g (sliding force) after 100,000 cycles, using a digital measuring microscope. mean values were analyzed by 2-way anova and tukey 's test (=0.05). the wear of trubyte biotone (0.93 0.14 mm) was significantly higher than that of both other types of teeth tested against abraded ceramic (p0.05) in wear among the 3 denture teeth evaluated against glazed ceramic. trilux and vivodent teeth tested against either glazed or airborne particle abraded ceramic did not differ significantly from each other (p<0.05). all teeth showed significantly more wear against airborne particle abraded ceramic than against glazed ceramic (p<0.05). in conclusion, the three types of pmma denture teeth presented significantly different wear resistance against the abraded ceramic. the high - strength pmma denture teeth were more wear - resistant than the conventional pmma denture tooth.
injection is a main source of fear, pain, anxiety and may lead to different levels of distress among children (1, 2) particularly who should have aggressive treatment like frequent injections resulting from chronic conditions (3, 4). type 1 diabetes (t1d) is one of the most common chronic diseases in childhood (5). children diagnosed with t1d should be treated with multiple daily insulin injections (6, 7). rzeszut (2011) reported that fear and anxiety due to insulin injection are common in children with t1d (8). howe. have shown that 40.9% of children with t1d had fear of insulin injection and 75% of children below 9 years old experienced higher fear in the starting of insulin therapy (9). a similar study by antal. (10) showed that half of parents had a history of diabetes - related injection distress in their children such as verbal resistance, whine, refuse to come into the room or get proper position for the injection, squirm to escape. also common child s behaviors during first month after diagnosis were upset, angry, and unhappy. if children with t1d can not cope with these daily injections, the distress and anxiety due to injections will affect their daily life. this distress may influence children s cooperation while injecting, hence they will experience poor treatment, higher hba1c level, negative attitude toward diabetes (8, 9). study of sewell (2004) have shown that education of deep breathings and relaxation techniques are effective to decrease anxiety - related self - injections in adolescents with t1d (11). some studies have also indicated that show films like " peer modeling film " may affect insulin self - injection learning but no effect on reducing anxiety (12). showed that parent - training interventions such as use of electronic toy, verbal and written descriptions, modeling, role playing, and other behavioral strategies are not effective to decrease children distress of needle procedures (13). in recent years, computer game has become an attractive and enjoying entertainment among children and adolescents (14 - 16). some researchers believe that computer and video games can be effective to improve health - related outcomes (17). there are many studies on positive effective of video games on improvement of physical rehabilitation in children with cerebral palsy (18) and cystic fibrosis (19), facilitating spirometry in younger children (20), increasing the level of disease - related knowledge in children with cancer (21), improvement of self - care in adolescents with diabetes (22). also, the researchers have found significant findings about the effectiveness of video games on pain relief during burn dressing (23, 24) and anxiety reduction in pediatric surgery (25). many authors claim video games that are related to the reason of fear and anxiety of children and their developmental have been more effective in adaptation with medical procedures (23). there is a question here whereas new technologies such as computer games can reduce the behavioral distress due to insulin injection in children with type 1 diabetes, to find the answer ; we did nt find any especial computer game for reducing behavioral distress of diabetes children. so, we designed and programmed an interactive computer game about insulin injection and have studied its effect on the behavioral distress induced by insulin injection in children with type 1 diabetes in isfahan, iran. this randomized controlled clinical trial was conducted on t1d children who referred to isfahan endocrine and metabolism research center, iran in 2013. inclusion criteria were age between 3 - 12 year, administering insulin by mothers by use of syringe or insulin pen, as well as observed behavioral distress in children, or what their mothers stated as children s distress while injecting insulin, willing to participate in the study, no morbidity of specific mental and physical disorders, and home availability of computer. exclusion criteria were uncooperative mothers or children in follow - up, and who had acute recent problems (acute illnesses, hospitalization, and death of close family members). demographic form : data were gathered by demographic questionnaire include child 's age, gender, the number of daily insulin injections, injection tools and illnesses history that completed by the mothers. observational tool : child behavioral distress was measured by observational scale of behavioral distress revised (osbd r consists of eight behaviors that are indicative for children distress result from therapeutic procedures. these behaviors and their weights in this tool are crying (1.5), screaming (4.0), restraining (4.0), verbal resistance (2.5), and information - seeking (1.5), solicitation of emotional support (2.0), verbal pain expression (2.5) and flail (4.0). a total score 22 is considered as higher level of behavioral distress (26). in this study, we thought mothers how to complete the osbd - r at home, they have been requested to observe their child behavior before injection to two minutes after the injection, and then mark observed behaviors in the checklist. total scores were calculated by researchers. the iranian version of the osbd - r was found to be good valid and reliable (27). we used test - retest (r = 0.79) and cronbach 's alpha (r = 0.85) to verify reliability of osbd - r. game design : koodak - e - tavana is an interactive computer game that programmed for first time by ebrahimpour. (2013), in this study this computer game utilized for teaching diabetic children about diabetes and insulin injections to decrease their anxiety and distress (28). game content : the computer game has seven parts including:1) paired game (familiarize with equipments needed for insulin injection) ; 2) puzzle game (observing another child that has no fear of insulin injection) ; 3) question and answer game (some educational topics on insulin injection) ; 4) insulin kit game (preparing insulin kit) ; 5) painting room ; 6) story game (narration a short story) ; 7) insulin injection - room (creating a simulated environment for insulin injection) (28). stage 1 : the participants in this study were thirty children with type 1 diabetes and their mothers who randomly allocated study (n = 15) and control (n = 15). mothers were asked to complete osbd - r for three times injection during first week at home. stage 2 : in the second week, both groups came to the isfahan endocrine and metabolism research center in two different days. children in study group received a cd of koodak - e - tavana computer game. they were asked to play at least once a week with the cd at home. in both groups, mothers were requested to fill osbd - r during one of the daily insulin injections for a week. stage 3 : during third and fifth week (no play with the game) the checklists completed as the same way in first week. finally, osbd - r checklists collected and calculated the mean of distress score in each stage. this study was approved by the research and ethic committee of isfahan university of medical sciences in iran to number 12/4/0.5/162. repeated measure anova and mann - whitney tests were used for comparing of behavioral distress score in two groups. pearson correlation and mann - whitney tests were used to evaluate associations between behavioral distress and demographic characteristics. this randomized controlled clinical trial was conducted on t1d children who referred to isfahan endocrine and metabolism research center, iran in 2013. inclusion criteria were age between 3 - 12 year, administering insulin by mothers by use of syringe or insulin pen, as well as observed behavioral distress in children, or what their mothers stated as children s distress while injecting insulin, willing to participate in the study, no morbidity of specific mental and physical disorders, and home availability of computer. exclusion criteria were uncooperative mothers or children in follow - up, and who had acute recent problems (acute illnesses, hospitalization, and death of close family members). demographic form : data were gathered by demographic questionnaire include child 's age, gender, the number of daily insulin injections, injection tools and illnesses history that completed by the mothers. observational tool : child behavioral distress was measured by observational scale of behavioral distress revised (osbd r consists of eight behaviors that are indicative for children distress result from therapeutic procedures. these behaviors and their weights in this tool are crying (1.5), screaming (4.0), restraining (4.0), verbal resistance (2.5), and information - seeking (1.5), solicitation of emotional support (2.0), verbal pain expression (2.5) and flail (4.0). a total score 22 is considered as higher level of behavioral distress (26). in this study, osbd - r we thought mothers how to complete the osbd - r at home, they have been requested to observe their child behavior before injection to two minutes after the injection, and then mark observed behaviors in the checklist. total scores were calculated by researchers. the iranian version of the osbd - r was found to be good valid and reliable (27). we used test - retest (r = 0.79) and cronbach 's alpha (r = 0.85) to verify reliability of osbd - r. game design : koodak - e - tavana is an interactive computer game that programmed for first time by ebrahimpour. (2013), in this study this computer game utilized for teaching diabetic children about diabetes and insulin injections to decrease their anxiety and distress (28). game content : the computer game has seven parts including:1) paired game (familiarize with equipments needed for insulin injection) ; 2) puzzle game (observing another child that has no fear of insulin injection) ; 3) question and answer game (some educational topics on insulin injection) ; 4) insulin kit game (preparing insulin kit) ; 5) painting room ; 6) story game (narration a short story) ; 7) insulin injection - room (creating a simulated environment for insulin injection) (28). stage 1 : the participants in this study were thirty children with type 1 diabetes and their mothers who randomly allocated study (n = 15) and control (n = 15). mothers were asked to complete osbd - r for three times injection during first week at home. stage 2 : in the second week, both groups came to the isfahan endocrine and metabolism research center in two different days. children in study group received a cd of koodak - e - tavana computer game. they were asked to play at least once a week with the cd at home. mothers were requested to fill osbd - r during one of the daily insulin injections for a week. stage 3 : during third and fifth week (no play with the game) the checklists completed as the same way in first week. finally, osbd - r checklists collected and calculated the mean of distress score in each stage. this study was approved by the research and ethic committee of isfahan university of medical sciences in iran to number 12/4/0.5/162. chi square and t - test were used for controlling demographic characteristics in groups. repeated measure anova and mann - whitney tests were used for comparing of behavioral distress score in two groups. pearson correlation and mann - whitney tests were used to evaluate associations between behavioral distress and demographic characteristics. there was no significant difference between the two groups in this regard (p > 0.05). table 2 contains the mean scores of distress by group over time, and figure 1 shows a graph of those means. the results of the repeated measure anova showed no signification difference of osbd - r mean score in over time for control group (p = 0.08), but this changes is signification in the study group (p = 0.001). the results indicate signification decreases in distress over time in the study group. also, results of the repeated measure anova showed comparison mean score of the behavioral distress over time significant statistical differences between two groups (p = 0.033). diabetic children who played with computer game were significantly lower behavioral distress during insulin injection than the control group. result of repeated measure anova analysis of variance : f= 7.61, df = 3. in addition, the result of mann - whitney tests showed no significant differences between these groups in the comparison of the mean ranks of behavioral distress before (p = 0.87), during (p = 0.17), after (p = 0.19) and two weeks after the intervention (p = 0.18). pearson correlation coefficient showed significant association between of age, illnesses history and number of daily insulin injections with behavioral distress of before intervention. in the present study, younger children (p = 0.045, r = -0.43), with lower duration illnesses (p = 0.035 r = -0.47) and the high number of daily insulin injections (p = 0.04 r = 0.44) presented more behavioral distress in insulin injection. the mean playing time was 23.20 minutes (sd = 15.8 rang = 5 - 60), and the mean number of computer game played was 5.2 (sd = 2.75 rang = 1 - 11). pearson correlation coefficient showed no significant correlation between playing time and number of computer game played with decrease osbd - r mean score in study group (p > 0.05). also, the results show that 40% of children in the computer game group (odds ratio = 6, 95%cl) have experienced insulin self - injection during the intervention for the first time which this rate was 10% in the control group (figure 2). this study showed that interactive computer game is effective in decreasing children s behavioral distress during mother - administering insulin injection. other researchers have shown positive effects of video games on reducing behavioral distress of burnt children and increasing their cooperation while change dressing (29), as well as pain relief in preschool children (30 - 32). indicated that computer games as affective cognitive interventions can reduce behavioral distress and fear of venipuncture and venous port access among children and adolescents who suffer from cancer (33). in the past, most computer or video games applied as a distraction method, and they were not necessarily relevant with the specific procedures. this study showed positive effect during use of computer game and even during two weeks after end of the intervention. therefore, it is necessary to use efficient strategies for reducing children s fear, anxiety and behavioral distress. because these strategies may be useful for children to achieve more positive attitude toward injection, this interactive computer game (koodak - e - tavana) has remarkable characteristics in structure and its content such as pay attention to the reasons of children s fear and concerns toward injection, use of cognitive and behavioral theories for designing the games, special considerations about computer programming and developmental levels of children, as well as applying various entertaining games along with role playing, all aforementioned attributes have contributed in successful results of this inventory computer game. the game could provide suitable information to children in a simulated safe, joyful and attractive environment so that fear and distress of diabetic children about their daily injections came effectively down. so many researches that have pointed to positive impacts of video and computer games in field of health education (34, 35). one of the considerable strengths of computer games is their highly motivating effect on specific behaviors (36). in fact, if we could promote our clients participation in their self - care we would achieve higher knowledge and performance regarding to follow treatment regimen particularly when we are facing to repetitive daily treatments like insulin injection. one of the surprising findings in this study was performing insulin self - injection by children. it may be inferred that playing with interactive computer game can help children to control their fear and anxiety result from injection, in addition they achieved higher motivation for insulin self - injection. in the present study osbd - r mean score were 2.5 and 2.4 in groups in baseline. slifer. (2009) reported that most children (61%) with chronic diseases including diabetes mellitus, pointed to no distress to low distress (osbd = 0 - 1) (13). some studies have reported different level of distress because of the kind of procedure studied, variety of age groups and distinct assessment tools (10, 37 - 39). there are so many influencing factors on level of distress that children experience due to insulin therapy. younger children usually suffer from higher level of distress - related behaviors in compare to older children and adolescents ; the shorter history of diabetes in children, the higher level of distress might be manifested during insulin injection (10). mohr. have shown direct relation between the numbers of injection and anxiety and fear of injection among chronically ill children (40). consistency between this research and present emphasizes that during first months of diagnosis of diabetes and insulin therapy for younger children should be thoroughly monitored by medical staffs, because they may be experience high level distress. there were a few limitations in this study such as small sample size and no comparative game for control group, hence further studies are recommended to conduct with three groups, and or perform comparison between this computer games with other educational methods. also, it is recommended that the future study to be conducted to assess the impact of this interactive game on self - injection anxiety in diabetic children. according to results of the study, computer games can be an enjoying, easy and affordable availability for reducing behavioral distress of children resulting from insulin injection, particularly among newly - diagnosed younger children.	background : diabetic children and their families experience high level stress because of daily insulin injection.objectives:this study was conducted to investigate the impact of an interactive computer game on behavioral distress due to insulin injection among diabetic children.patients and methods : in this clinical trial, thirty children (3 - 12 years) with type 1 diabetes who needed daily insulin injection were recruited and allocated randomly into two groups. children in intervention groups received an interactive computer game and asked to play at home for a week. no special intervention was done for control group. the behavioral distress of groups was assessed before, during and after the intervention by observational scale of behavioral distress revised (osbd - r).results : repeated measure anova test showed no significantly difference of osbd - r over time for control group (p = 0.08), but this changes is signification in the study group (p = 0.001). comparison mean score of distress were significantly different between two groups (p = 0.03).conclusions : according to the findings, playing interactive computer game can decrease behavioral distress induced by insulin injection in type 1 diabetic children. it seems this game can be beneficial to be used alongside other interventions.
when the adaptive immune system arose, approximately 500 million years ago, it provided ancient vertebrates with the ability to recognise sets of amino acids and respond specifically to them. before the development of the adaptive system, organisms were fated to dealing with environmental molecules in a much simpler and less specific manner ; that is, by recognising structural patterns and responding to them in a pre - scripted manner. the inauguration of the adaptive immune system allowed the ancestors of jawed vertebrates to mount sophisticated responses to a varied panel of molecules -- from environmental particles, such as allergens, to intracellular parasites and from viruses to intestinal helminthes. moreover, the adaptive immune system provided these animals with immunological memory. after mounting an effective response against a certain microorganism, the immune system retains the ability rapidly to recognise and neutralise the same microorganism at the onset of a second encounter. the ability to identify a set of amino acids depends on the presence of antigen - specific receptors. antigen - specific receptors comprise t cell receptors (tcrs) and immunoglobulins (igs or antibodies). an organism 's antigen - specific receptors comprise a panel of receptors bearing different specificities being clonally expressed. the higher the variability of the receptors that an organism is capable of expressing, the larger the number of different sets of amino acids that its cells will be able to recognise. the antigen - specific receptor binding sites are the products of the germline recombination of gene segments named v (variable), d (diversity) and j (joining) within the precursors of t and b cells. in humans, these segments are organised in a translocon configuration -- that is, a large number of gene segments are grouped together. several v gene segments lie upstream of grouped d gene segments, which are localised upstream of j gene segments. recombination of one segment from each group will result in an antigen - specific receptor. the combination of these gene segments is known as v(d)j recombination, and results in a variety of receptors that recognise different peptides, providing the system with a repertoire of possible responses. each gene segment is flanked by elements known as recombination signal sequences, which are conserved heptamer and nonamer sequences, separated by a 12- or 23-base pair spacer. when two segments are brought together for joining, further combinatorial variation might occur by addition of nucleotides between the fragments by the terminal deoxynucleotidyl transferase, a template - independent polymerase. the v(d)j recombination is performed by a complex containing the product of the recombination - activating genes (rags). rag-1 and rag-2 were first discovered when these authors attempted to identify the ' recombinase ' responsible for v(d)j recombination. the origin of rag is considered to be a foundation hallmark for adaptive immunity, and rag homologues were identified in many jawed vertebrates. nevertheless, no rag homologues have been found to date in jawless vertebrates and invertebrates. the sudden acquisition of rags, which allowed the immune system to create and deal with diversity, has been described by some as the immunological ' big bang '. in the past few decades, a lot of work has been carried out to elucidate the mechanisms coordinating the adaptive immune system ; however, some questions remain unanswered, including the origin of rag proteins, which ultimately gave rise to the adaptive immune system. a series of excellent reviews on several aspects of rag biology and v(d)j recombination was recently published. the present review is not the forum for discussing these aspects ; instead, it will focus on recent studies on rag genes and proteins in humans and other vertebrates for which the genome is completely sequenced, and will discuss these in the context of evolutionary genomics, addressing the role of current and future genome projects in understanding the evolution and diversity of the adaptive immune system. the rag-1 and rag-2 genes are mapped in the same chromosome, separated by only a few kilobases and placed in opposite orientation to each other. in all mammals, birds and amphibians characterised so far, they have a compact organisation, with no introns ; however, interrupted genes have been described in most fish for which rags have been identified. gene organisation of rag-1 in human (m29474), mouse (m29475) and zebrafish (u71093). rag proteins are functionally annotated as ' recombination - activating protein ', ' recombinase - activating protein ', ' recombinase ' or even ' v(d)j recombination - activating protein '. rag-1 is over 1,000 amino acids in length (1,040 amino acids in the mouse, 1,057 amino acids in zebrafish) and rag-2 is over 500 amino acids in length (520 amino acids in frog, 528 amino acids in chicken). many studies on rag proteins are conducted in the so - called core proteins, mainly because of the difficulties encountered during protein purification. these core proteins correspond to truncated portions of the rag proteins, and have catalytic activity. in humans, the core proteins correspond, approximately, to 63 per cent and 73 per cent of rag-1 (residues 384 - 1040) and rag-2 (1 - 387), respectively. comparisons between full - length and core proteins have revealed important aspects of their function and regulation. non - core portions of the rag proteins seem to play important roles in v(d)j recombination that do not influence the specificity of the recombinase site. catalytic residues were identified in the rag proteins using different approaches, including sequence analyses, site - directed mutagenesis and functional characterisation of mutant proteins, among others [19 - 21 ]. the rag-1 protein contains a dde motif (d600, d708 and e962), which is critical for both dna cleavage and hairpin formation. proteins containing the dde motif are members of the retroviral integrase superfamily, found in bacterial transposases and retroviral integrases. therefore, rag proteins, bacterial transposases and retroviral integrases are functionally related, despite the low sequence similarity shared by them. occurrence of rag homologues across major animal groups. tree topology is based on the phylogenetic analysis and time - scale is based on fossil records published elsewhere. most rag genes and proteins currently listed in the databases have not been genetically or biochemically characterised, but have been assigned a putative function based on standard sequence similarity methods. this common practice in computational functional genomics seems to have worked reasonably well in the case of the rag datasets, mainly because their high level of sequence similarity, combined with full coverage of the alignments and the absence of paralogues (originated by gene duplication). the great majority of rag genes and proteins found in databases corresponds to partial sequences. in order to find out more about the conserved motifs, critical residues, diversity and potential regulation of rag-1 and rag-2, it will be necessary to increase the number of full - length genes and proteins in the databases. the complete or near - complete sequences of the nuclear genomes of several vertebrates are available in public databases, and many more are underway (http://www.ensembl.org, http://www.genomesonline.org). this extraordinary amount of data creates a platform for comparative analysis and contributes to the understanding of complex biological systems. rag-1 and rag-2 have been identified in a wide range of jawed vertebrates, including human, chimpanzee, mouse, rat, dog, chicken, zebrafish, fugu and tetraodon (http://www.ensembl.org, http://www.genomesonline.org). rag homologues show high levels of sequence similarity across organisms -- for example, 90 per cent between human and mouse. nevertheless, no rag homologues have been found to date in jawless vertebrates and invertebrates. rag genes have proved to be excellent targets for phylogenetic reconstructions and, in fact, have been extensively applied in studies in a wide range of organisms [23 - 25 ]. some of the main reasons to choose rag genes are that : (i) they are composed by long sequences of dna and protein (providing more sites for statistical analysis) ; (ii) they show high levels of sequence conservation (helping primer design and use in samples from diverse taxonomic groups) ; and (iii) there is an absence of introns, as discussed earlier. moreover, nuclear - encoded genes are under the rules of a different evolutionary context compared with mitochondrial genes and gene products. like mitochondrial genes, rag-1 and rag-2 can be (at least for now) considered as ' perfect ' orthologues, which adds another advantage of using them as molecular markers in phylogenetic studies. all together, these features have promoted rag genes as suitable markers for phylogenetic reconstruction. furthermore, rags were selected as targets in the tree of life project, a huge initiative that claims to decode the relationships between all living organisms. in the search for the tree of life, rag-1 and rag-2 figure 2 shows the proposed phylogeny of the major animal lineages, based on molecular data and fossil records, and indicates the occurrence of the rag genes and proteins in mammalia, reptilia (including birds), amphibia and actinopterigii (fish), which correspond to the jawed vertebrates no significant sequence similarity has been detected between rag-1 and rag-2 that could suggest a possible event of gene duplication of a common ancestral rag gene. experimental scientists use loss - of - function point mutations widely to assess the function of genes, as well as to evaluate their permissiveness to selective pressures. in humans, rag-1 and rag-2 are mapped to chromosome 11p13-p12, and extensively studied spontaneous mutations in both genes cause a spectrum of severe combined immunodeficiencies. loss - of - function mutations of rag-1 and rag-2 lead to disruption of the v(d)j recombination, disturbing t- and b cell development and causing severe combined immune deficiency (scid). a patient with scid does not have mature functional t- and b cells (t - b - scid), disabling this patient 's ability to recognise and respond to specific antigens and to develop immunological memory after infections. mutations of either one of the rag genes are associated with another immune deficiency, omenn syndrome (os), which is an autosomal dominant disease with variable expressivity and two causative loci. it is frequently lethal in the homozygous condition. os patients presenting with partial loss of function of rag lack circulating b cells, but show a small and highly activated population of circulating t cells. it is not clear what other factors are involved in the development of one or other immune deficiencies, since they both require mutations of the rag-1 and/or rag-2 genes. interestingly, the analysed t - b - scid patients had nonsense, frameshift or null mutations resulting in biochemical changes that abrogated the recombination ability of rag-1/rag-2. in patients with os, the authors found a predominance of missense mutations that allowed partial activity of rag-1/rag-2. one of these studies has also identified three homozygous missense mutations in rag-1 in one patient and in rag-2 in three patients, located outside the rag core regions. taken together with recent results from group, another which showed that removal of regions outside the core impaired the processing of recombination substrates, these results point to the possibility that the core region alone accounts for the whole recombination activity. up until now, there have been no spontaneous mutations of rag-1/rag-2 described in animals other than humans. the closest phylogenetic model available for studies on t - b - scid is the rag-1/rag-2 knockout mouse. these mice have their rag-1/rag-2 genes disrupted and do not have circulating t- and b cells. more recently, mutations of rag-1 in zebrafish have become a second model of choice. nevertheless, other forms of scid have been described in other animals, but all of these have been due to defects in proteins downstream of rag-1/rag-2 in the v(d)j recombination cascade. scid in foals is limited to the arabian breed and is an auto - somal recessive disorder due to an absence of dna - dependent protein kinases (dna - pkcs) in the cells of these animals. in addition, spontaneous scid due to dna - pkcs defects was described in c.b-17 mice of balb / c origin and in jack russell terrier dogs. a third class of spontaneous scid is found in dogs from the basset and cardigan welsh corgi breeds and in humans [43 - 45 ]. this form of scid is linked to the x chromosome and is thus called xscid or scid - x1, and involves a mutation on the gene that encodes the common chain of interleukin-2 (il-2) receptor (also named chain or c). as this chain is common to receptors of several interleukins involved in t- and b cell maturation (il-2, -4, -7, -9, -15 and -21), a defect on this chain disrupts the signalling pathway downstream of these receptors, preventing cell maturation. regardless of whether their defect affects rag genes or downstream components of the v(d)j recombination cascade, these animal models teach us how crucial the adaptive immune system -- or, in other words, the acquisition of functional rag genes -- is to the survival of complex organisms. the world surrounding us poses constant challenges that must be identified and neutralised, not to mention our own internal challenges, such as the development of cancers. in order to have a better understanding of the function, interaction, regulation and evolution of rag genes and proteins, it is very important to access their full - length sequences and protein structures. primary sequence analysis may fail to detect distant relationships among proteins, while secondary and tertiary structures might add to the analyses. most sequences available are partial sequences, and so far no crystal structures have been published for whole rag proteins. dense sampling across diverse taxonomic groups, defining genomic diversity (or biodiversity), can improve phylogenetic reconstruction considerably and give us insights into evolutionary patterns and novelties in the molecular world. as mentioned previously, there is a strong bias in databases towards partial sequences of rag genes and proteins and also concerning mammals and birds. evolutionary approaches would benefit tremendously from a rational choice of targets for future genome sequencing projects, especially if the aspects discussed here are taken into consideration. the study of rag proteins and other signature proteins of the immune system is relevant for immunomics, an interdisciplinary field that integrates immunology, genomics, proteomics, bioinformatics and related areas. in the ' omics ' era, this important approach aims to unravel aspects of the origin, function, regulation and generation of diversity of the immune system. understanding the acquisition and evolution of the v(d)j recombination machinery will enlighten us on how the challenges posed by a changing ancient world selected the earliest immune systems. moreover, these studies will disentangle the present pressures shaping our fellow animals and our own immune systems. grasping the diversity of the adaptive immune system, including the function and regulation of its key elements -- such as rag proteins -- is crucial for the design and development of new therapies to modulate immune responses in humans. as researchers continue to study key components of the adaptive immune system, their interaction and regulation, they will be contributing to a better understanding of this important system and for the development of alternative new therapies. while this manuscript was being reviewed, another work was published, describing the evolution of duplicated rag-1 genes in the polyploid clawed frogs, xenopus and silurana. this work was supported by fapesp -- fundao de amparo pesquisa do estado de so paulo, brazil (grant 01/02584 - 2), mbl associates, frederick b. bang and catherine f. shouse fellowships and was partly supported by grants to david d. pollock from the national institutes of health (gm065612 - 01 and gm065580 - 01), the national science foundation (eps-0346411) and the state of louisiana board of regents (support fund and the millennium research program 's biological computation and visualization center). is an assistant professor at the university of so paulo (usp), brazil, and is a recipient of a young investigators grant from fapesp. is currently a postdoctoral associate with david d. pollock at louisiana state university, usa. special thanks to mitchell l. sogin (marine biological laboratory) for hosting m.m.d.c. in 2002 ; to auro nomizo (usp), jos gustavo amarante - mendes (usp) and srgio luiz pereira (royal ontario museum) for valuable suggestions to this manuscript ; and jos adriano de sousa for providing artwork.	the origin of the recombination - activating genes (rags) is considered to be a foundation hallmark for adaptive immunity, characterised by the presence of antigen receptor genes that provide the ability to recognise and respond to specific peptide antigens. in vertebrates, a diverse repertoire of antigen - specific receptors, t cell receptors and immunoglobulins is generated by v(d)j recombination performed by the rag-1 and rag-2 protein complex. rag homologues were identified in many jawed vertebrates. despite their crucial importance, no homologues have been found in jawless vertebrates and invertebrates. this paper focuses on the rag homologues in humans and other vertebrates for which the genome is completely sequenced, and also discuses the main contribution of the use of rag homologues in phylogenetics and vertebrate evolution. since mutations in both genes cause a spectrum of severe combined immunodeficiencies, including the omenn syndrome (os), these topics are discussed in detail. finally, the relevance to genomic diversity and implications to immunomics are addressed. the search for homologues could enlighten us about the evolutionary processes that shaped the adaptive immune system. understanding the diversity of the adaptive immune system is crucially important for the design and development of new therapies to modulate the immune responses in humans and/or animal models.
the shiba goat (capra hircus), a japanese miniature goat, is small in size with less food consumption. the shiba goat is a nonseasonal breeder under natural daylight, reaches puberty at 3.5 months and is considered a useful model animal for studying the physiology of ruminants. animals and management : eight 10- to 12- month- old postpubertal male shiba bucks (capra hircus), weighing 18 to 25 kg and housed under natural daylight, were used in this study during may and december 2013. each animal received a maintenance diet of 400 g of hay cubes twice a day. each male underwent complete andrological and ultrasonographic examinations to ensure absence of any abnormalities related to the reproductive tract before beginning the research. the animals were vaccinated and dewormed, and none of them had evidence of disease. all procedures were carried out in accordance with guidelines established by the tokyo university of agriculture and technology, japan, for the use of animals. gnrh and hcg administrations : male shiba goats were exposed to a single intramuscular injection of either gnrh (gnrh group ; 1 g / kg bw ; fertirelin acetate 100 g ; conceral ; nagase pharmaceuticals co., ltd., osaka, japan) or human chorionic gonadotropin (hcg group ; 25 iu / kg bw ; pubergen ; yell pharmaceutical co., ltd., the dose of gnrh or hcg was chosen depending on previous studies in goats [30, 32 ]. both hcg and gnrh administrations were started at a fixed time (between 08:00 and 09:00 hr) to avoid any effects of circadian rhythmicity on blood flow. additionally, to minimize individual variation, animals that had been treated with hcg in the first trial were subjected to gnrh treatment in the second trial and vice versa. so, all goats (n=8) were injected either with gnrh or hcg in 2 trials with a 3- week interval. the time interval between the 2 trials was based on previous reports [26, 40 ]. blood sampling : a venous blood sample was collected from a jugular vein into an evacuated heparinized tube (venoject ii, terumo, tokyo, japan) just before injection of either hcg or gnrh (basal or 0 hr) and at 1, 2, 3, 6, 24, 48, 72, 96, 120, 144 and 168 hr after injection in both groups. the blood samples were centrifuged at 3,200 rpm for 15 min at 4c and stored at 20c until hormonal analysis. these times were selected based on previous studies and the pharmacokinetics of hcg in goats [2, 32, 40 ]. hormonal analysis : all hormonal assays were performed in triplicates by a double antibody radioimmunoassay system using i labeled radioligands. plasma concentrations of testosterone (t) and estradiol (e2) were measured as described by taya., and immunoreactive inhibin (inh) was measured as described by hamada.. ultrasound examinations : all ultrasonographic measurements were carried out by the same ultrasonographer just after blood sampling. all examinations were performed using ultrasound scanner (eub-7500, hitachi medical corporation, tokyo, japan) equipped with a linear array transducer (6.5 mhz ; model eup - l65 ; hitachi medical corporation). doppler examination : preparation for examination : the bucks were simply restrained without tranquilization or sedation. to eliminate the presence of air spaces, the hairs on both sides of the scrotum were shaved well, and the transducer was covered with a copious amount of gel to facilitate ultrasonographic imaging. in goats, the testicular artery on approaching the testis convolutes in order to form a convoluted or coiled part known as the supratesticular artery (sta) (fig. 1.testis of a male shiba goat imaged by computerized tomography (a) showing the supratesticular artery (white arrows) and by b - mode ultrasonography (b) for measurement of testis volume (tv) using the following formula : tv = length (l) width (w) height (h).). however, for distinguishing between a testicular artery and vein by doppler analysis, an artery, for example, will typically have a waveform on the spectral graph corresponding to the arterial pulse in each cardiac cycle (systole and diastole), while the flow in veins be almost constant, that is, without a pulse. testis of a male shiba goat imaged by computerized tomography (a) showing the supratesticular artery (white arrows) and by b - mode ultrasonography (b) for measurement of testis volume (tv) using the following formula : tv = length (l) width (w) height (h). doppler analysis was performed by identifying all the vascular structures using gray - scale ultrasonography and locating the largest and possibly a longitudinal or oblique section of the supratesticular artery (sta). the angle between the doppler beam and the long axis of the vessel never was over 60 in the direction of blood flow with the high - pass filter set at 50 hz. doppler parameters of the sta : after appearance of the spectral pattern of the sta, the doppler indices studied were resistive index (ri = (maximum velocity minimum velocity) maximum velocity) and pulsatility index (pi = (maximum velocity minimum velocity) mean velocity). three to 5 measurements were taken for each parameter in different locations along the path of the sta. furthermore, the waveforms of sta were characterized as either resistive or non - resistive. resistive waveforms are characterized by great differences between systolic and diastolic velocity of blood flow with high values of ri, while non - resistive waveforms are the opposites. to minimize the variations in recording, the ultrasound settings (focus, gains, brightness and contrast) were standardized, fixed and used equally for all examinations. in this study, all examinations were recorded digitally on videotape for subsequent analysis. testicular volume (tv) measurement by b - mode ultrasonography : at least 3 separate transverse and longitudinal images of each testis were recorded, and the testicular length (l), width (w) and height (h) were measured using electronic calipers without inclusion of the epididymis (fig. the testis volume was measured using this formula : tv (ml)=l w h 0.71. the statistical significance of hormonal changes and ultrasonic findings in both groups was evaluated by repeated measures analysis of variance (anova). a students t- test was used to determine the significance at each time point in each group considering 0 hr as the control. an unpaired t - test was used for comparisons between the 2 groups at each time point. moreover, the relationships between plasma concentrations of hormones and ultrasonographic results were evaluated using pearson s correlation coefficient. hormonal results : testosterone (t) : plasma concentrations of t significantly (p0.05). testicular blood flow by color doppler ultrasonography : color pulsed doppler ultrasonography of the sta in the spermatic cord was easily performed (fig. 3fig. 3.ultrasonographic scan of the goat testis using color pulsed - wave doppler ultrasonography showing blood flow within the supratesticular artery (convoluted part of the testicular artery) (left side with red colors). blood flow within the supratesticular artery revealed a spectral pattern with a wave - like pattern that appeared as monophasic non - resistive waveforms (right side). ; left side). arterial pulsation of blood in this vessel was clearly visible during real - time scanning. doppler ultrasonography of the sta revealed a spectral pattern with a wave - like pattern. ultrasonographic scan of the goat testis using color pulsed - wave doppler ultrasonography showing blood flow within the supratesticular artery (convoluted part of the testicular artery) (left side with red colors). blood flow within the supratesticular artery revealed a spectral pattern with a wave - like pattern that appeared as monophasic non - resistive waveforms (right side). although the resistive index (ri) of the sta significantly decreased (p0.05) between the groups at any time during the period of a study (fig. testicular volume (tv) measurements by b - mode ultrasonography : both the gnrh and hcg groups showed significant changes in tv compared with 0 hr. however, we did not find any significant difference (p>0.05) between them at any time during the period of study. similar to the ri changes, a faster increase of tv was observed in the hcg group (i hr) compared with in the gnrh group (2 hr) (fig. 5fig. 5.the results of ultrasonographic examination of male shiba goats showed changes in testis volume (tv) after injection of gnrh or hcg. values are significantly different (p<0.05) compared with 0 hr in the gnrh and hcg groups, respectively. (note : no significant difference was found between the groups at any time during the study.)). the results of ultrasonographic examination of male shiba goats showed changes in testis volume (tv) after injection of gnrh or hcg. values are significantly different (p<0.05) compared with 0 hr in the gnrh and hcg groups, respectively. (note : no significant difference was found between the groups at any time during the study.) correlations between hormonal changes, doppler parameters and tv changes : in the gnrh group, a strong negative correlation was found between the t level and both the ri (r= 0.862, p=0.001) and pi (r= 0.707, p=0.007) of the sta. additionally, this group showed a high positive correlation between inh and both the ri (r=0.661, p=0.014) and pi (r=0.701, p=0.008) of the sta. controversially, high negative correlations were found between e2 and both the ri (r= 0.610, p=0.027) and pi (r= 0.763, p=0.002) of the sta in the hcg group. moreover, high negative correlations were found between tv and the ri of the sta in the gnrh group (r= 0.607, p=0.028) as well as the hcg group (r= 0.799, p=0.001). a single injection of either gnrh or hcg in male shiba goats evoked series of changes in three different parameters : hormonal, testicular blood flow, and tv changes. although the dose of gnrh analogue was the same, the maximum t response recorded in the current study (6 hr) did not occur as earlier as those reported in 3- to 6- month - old male shiba goats (2 hr). the different response might be attributed to the different route of injection and/or the age effect. in the current study, gnrh injected intramuscularly resulted in lower absorption than when injected intravenously in the previous study. the biphasic t response observed in the hcg group (at 2 hr and 96 hr) was in agreement with those reported in equine (13 hr and 2472 hr) [2, 29 ]. unlike the gnrh group, the faster t response and biphasic peaks recorded in hcg group might be attributed to various factors. firstly, t response was secondary to lh release in the gnrh group, while hcg had a direct effect. secondly, the hormones have different half - lives : that of lh is short, while that of hcg is prolonged [41, 43 ]. finally, binding to the receptor is reversible for lh, while it is not for hcg [3, 20 ]. the nonsignificant changes in the level of e2 observed in the gnrh group were consistent with a previous published study in stallions. however, the significant increase of e2 observed in the hcg group might be related to the pattern of the plasma levels : pulsatile for lh - induced gnrh and sustained for hcg, which resulted in a prolonged stimulation of leydig cell steroidogenesis [3, 39 ]. moreover, the strong positive correlation found between t and e2 responses after hcg injection in the current study was consistent with those reported in stallions and might be attributable to transformation of t into e2 by the aromatase enzyme in leydig cells [2, 29, 45 ]. blood flow is important to testis function, because any defect in the arterial blood flow to the testis could cause impaired spermatogenesis secondary to defective energy metabolism at the mitochondrial level. the non - resistive monophasic waveforms of the testicular blood flow observed in the current study were consistent with those reported in the human testis and dog testis [7, 8 ]. on the other hand, pozor and mcdonnell found high resistive waveforms of testicular blood flow in stallions. although color doppler ultrasonography has been used to evaluate testicular blood flow after hcg administration [2, 26 ], the present study was the first to evaluate the effect of gnrh compared with hcg on testicular hemodynamic. an earlier study in bulls and a recent study in rams found an increase of the scrotal surface temperature measured with a laser thermometer, representing changes of testicular blood flow after gnrh administration. the ri of the testicular artery is considered a valuable indicator of the sperm production rate score in the human testis and semen quality in dogs. it was reported that ri has a negative correlation with vascular perfusion of tissue downstream from the sample gate. in the current study, because the ri and pi of the supratesticular artery significantly (p<0.05) decreased after the injections in both groups, it can be said that hcg or gnrh injection could induce an improvement of testicular blood flow. however, this improvement occurred significantly earlier and was greater in the hcg group (1 hr) than in the gnrh group (2 hr). a possible explanation for this may be related to the direct effect of hcg compared with the indirect effect of gnrh. in addition, one of the main objectives of this study was to evaluate how testicular hormone levels affect testicular blood flow. in the current study, although an increase in testicular blood flow (represented by decreases in the ri and pi of the sta) was observed in both groups, there were discrepancies in terms of t and e2 correlations with doppler indices. in the hcg group, e2 was highly negatively correlated with the ri and pi of the sta without any correlation with t, and the opposite was observed in the gnrh group. these results were consistent with previously published studies revealing that hcg treatment induces an increase in testicular blood flow in parallel with an increase in e2 level without any correlation with t levels [2, 6 ]. however, another study found a strong correlation between testicular blood flow and t after hcg treatment. indeed, the strong correlations between e2 and doppler indices in the current study might be related to the strong vasodilatory effect of estrogen and its role in testicular perfusion [2, 28 ]. importantly, the involvement of t and e2 in the present study only indicated an associative relationship between these hormones and doppler indices and did not elucidate the mechanism ; however, verification of this hypothesis requires a further study at the testicular level. interestingly, the strong positive correlations found between inh and the ri and pi of the sta post gnrh injection were novel data obtained in the present study. in humans, both the peripheral inh level and the ri of the testicular artery were significantly related to the sperm production rate score and good predictors of spermatogenesis. therefore, a further study is needed to clarify the role of inhibin in testicular blood flow in male shiba goats. b - mode ultrasonography is considered to be a more accurate, precise and reproducible method for measurement of tv in assessing testicular function and pubertal development [21, 31, 37 ]. an increase of tv recorded in this study after hcg injection was similar to those observed in men as well as in rats. in both groups, the strong negative correlations found between tv and the ri and pi of the sta might provide us with an explanation for the tv increases. decreased values of the ri and pi of the sta might result in an increase of testicular blood perfusion and leads to an increase in testicular vascular permeability and interstitial fluid (if). similarly, in hcg - treated rats, dose- and time - dependent covariation was found between the increase in testicular if volume and the increase in testicular microcirculation. moreover, it was recently reported that administration of gnrh triggers a rapid increase in testicular fluid content in rams. collectively, the increase in tv observed in this study might be attributed to increases in testicular blood flow resulting in increases in if. in conclusion, the results of the present study show that the administration of a single dose of gnrh or hcg may have a benecial effect on testicular blood flow in male shiba goats as well as on tv, but that hcg has a faster effect on testicular blood flow compared with gnrh. these beneficial effects may result in an increase in male fertility through positive inuences on testicular blood flow and, in turn, on spermatogenesis. moreover, the involvement of t, e2 and inh in testicular hemodynamics in male shiba goats requires a further study to test this hypothesis.	although color doppler ultrasonography has been used to evaluate testicular blood flow in many species, very little has been done in goat. eight male shiba goats were exposed to a single intramuscular injection of either gonadotropin - releasing hormone (gnrh group ; 1 g / kg bw) or human chorionic gonadotropin (hcg group ; 25 iu / kg bw). plasma testosterone (t), estradiol (e2) and inhibin (inh) were measured just before (0 hr) and at different intervals post injection by radioimmunoassay. testis volume (tv) and doppler indices, such as resistive index (ri) and pulsatility index (pi) of the supratesticular artery, were measured by b - mode and color doppler ultrasonography, respectively. the results indicated an increase in testicular blood flow in both groups, as ri and pi decreased significantly (p<0.05), but this increase was significant higher and earlier in hcg group (1 hr) than in the gnrh group (2 hr). a high correlation was found for ri and pi with both t (ri, r= 0.862 ; pi, r= 0.707) and inh in the gnrh group (ri, r=0.661 ; pi, r=0.701). however, a significant (p<0.05) correlation was found between e2 and both ri (r= 0.610) and pi (r= 0.763) in hcg group. in addition, tv significantly increased and was highly correlated with ri in both groups (gnrh, r= 0.718 ; hcg, r= 0.779). in conclusion, hcg and gnrh may improve testicular blood flow and tv in shiba goats.
the first ever case - control studies which revealed a strong association between active smoking and lung cancer were conducted in nazi germany in 1939 and 1943. many other studies, thereafter, have explored the effects of tobacco smoke on human health. it is now an accepted fact that smoking is responsible for the development of chronic diseases as well as increased morbidity and mortality. during the last decades research has also expanded in the area of passive smoking (ps) and its effects on aspects of human health. the first conclusive evidence on the danger of ps arose in 1981 from a study showing that nonsmoking japanese women married to men who smoked had an increased risk for lung cancer. since then, a vast number of studies have appeared investigating the unfavorable effects of ps. in line with active smoking, it is now generally accepted that ps leads to increased prevalence of various cardiovascular diseases and increases the risk of death by at least 20%. more importantly, recent methodologically robust data from nonsmoking adults have shown that ps compromises health not only when individuals are exposed frequently for prolonged periods of time as initially thought but also after a single brief exposure [610 ]. this novel evidence clearly shows that ps may have a substantive role in the development of chronic diseases. despite the recent measures adopted in different countries to eliminate indoor smoking, 700 million children globally are still exposed to environmental tobacco smoke while the smoking epidemic continues to increase worldwide. given that tobacco smoke contains chemicals characterized as carcinogenic (e.g., benzene, chlorinated dioxins, and benzo[a]pyrene) and have adverse health consequences on the cardiovascular (e.g., arsenic) and respiratory (e.g., acetaldehyde) systems in adults, the physiologically immature children may be more vulnerable to damage as a result of ps exposure because of their partially developed or compromised cardiovascular, endocrine, and immune systems. for these reasons, we conducted a systematic review of the available literature to investigate the evidence regarding ps and its association with factors that influence the development of cardiovascular disease (cvd) in children. five databases [medline, cochrane library, cumulative index to nursing & allied health (cinahl) research database, google scholar, excerpta medica database (embase) ], the 2006 report on second hand smoke by the office of the surgeon general, and the 2005 report by the california environmental protection agency were searched to identify publications from 1975 to april 2009 in english regarding ps and cardiovascular disease in children (< 18 years old). second hand smoking, environmental tobacco smoking, maternal smoking, and parental smoking, were searched in combination with obesity, diabetes, hypertension, blood pressure, cholesterol, lipids, and cardiovascular disease. if the abstract did not provide sufficient information for this process, then the full - text manuscript was examined. the cited articles of the selected papers were also searched manually in order to identify relevant studies not identified in the original search. a flow diagram of the studies identified from databases and the combination of key words used to identify the observational studies included in the present paper in figure 1. we identified a total of 77 articles, from which only 42 were relevant ; of which 30 were reviews and 12 were observational studies and thus included in the present paper. table 1 depicts the results of each study included in the present paper (n = 12). from the results of the present paper it appears that newborn children exposed to ps at home are more likely to be overweight and obese, particularly when exposed during the first three years of their life. it was also found that ps is associated with deteriorated blood lipids profiles in children, particularly lower high - density lipoprotein which has been a consistent finding in most studies [1721 ]. a randomized controlled trial (rct) also revealed that in passive smokers the rate of factors associated with the development of cvd, such as body weight, cholesterol, and triglycerides, was higher than that in non - ps smokers. ps appears to directly affect endothelial function in children via a dose - dependent decrease in the bioavailability of nitric oxide. two randomized controlled studies (rcts) found that children exposed to ps have also reduced aortic elasticity possibly via the direct effects of ps on the mechanical properties of the arteries (e.g., impaired nitric oxide production, platelet activation, or adrenalin levels) [23, 24 ]. the effects of exposure to ps during pregnancy with regards to risks factors associated with cvd were investigated in two separate studies. results revealed a significantly higher concentration of cotinine in children due to exposure to ps from the mother during pregnancy and that ps increased oxidative stress in cord blood. the effects of maternal ps exposure on absolute red blood counts (rbc) were assessed in newborn infants of 55 mothers exposed and 31 not exposed to ps during the last trimester ; data revealed that the counts of absolute nucleated rbcs were significantly elevated in the ps exposed group. table 1 depicts the results of all studies identified in relation to ps and cvd in children. the present systematic review investigated the effects of ps on cvd (classical risk factors and vascular function) in children. the results reveal that ps may be implicated in deteriorating cardiovascular status in terms of unfavorable high - density lipoprotein levels and deteriorated vascular function, whereas the mechanisms by which ps negatively affect the cardiovascular status of children (in terms of classical cvd risk factors) have yet to be elucidated. perhaps, the significant decrease in cardiorespiratory function, as a result of smoking / ps, leads to a decrease in physical activity in children which may then have a significant unfavorable impact on body fat and cholesterol levels. this is supported by a vast amount of evidence demonstrating that physical inactivity results in worse profile in classical cvd risk factors in both healthy and diseased populations [2830 ]. two rct studies [23, 24 ] have consistently revealed that children exposed to ps have reduced aortic elasticity. the significant alterations in vascular biological mechanisms from an early age may lead to a deteriorated health status of children exposed to ps, given that vascular dysfunction precedes atherosclerosis and leads to increased cvd mortality [31, 32 ]. ps appears to directly affect endothelial function in children via a dose - dependent decrease in nitric oxide, a mechanism preceding atherosclerosis. vascular endothelial dysfunction has been implicated in the progression of atherosclerosis and cardiovascular event rates ; hence, its assessment may provide pivotal information, particularly given that it may detect this is the reason why this assessment is consistently used in research and clinical setting for both prognostic and diagnostic means. nitric oxide is a vasoactive factor responsible for dilation of the vessels ; the drop in exhaled nitric oxide levels within the first minutes of ps may be caused by decreased production of nitric oxide synthase through a negative feedback mechanism, given the high concentrations of nitrogen oxides inherent in tobacco smoke. possible other mechanisms comprise an increased breakdown of nitric oxide by ps oxidants or a ps - induced accelerated uptake of nitric oxide. data from adults have shown that the ps - induced changes in lipoprotein oxidization generate various effects in the vessel wall. animal experiments have shown a synergistic effect between ps and lipoprotein that influence the oxidation of lipoprotein in the vessel wall. it is also important to note that polycyclic aromatic hydrocarbons byproducts of the incomplete combustion of organic material inherent in ps bind to lipoprotein subfractions and can be integrated into the atheromatic plaques promoting the proliferation of vascular cells and plaque progression. elevated absolute nucleated rbcs count in the neonate, as observed by cochran - black. periods of hypoxia may stimulate bone marrow to increase the hematocrit possibly in concert with a more rapid smoke - induced rbc turnover. the findings from this study suggest that maternal ps exposure has similar effects on the fetus as active maternal smoking. it is of great clinical importance to explore the associations between ps and either pregnancy or early childhood. clearly, ps has well - established adverse health effects in children [37, 38 ] whereas the mechanisms by which ps affects the fetus may be different to those that affect the child or adolescent ; the deterioration in cardiorespiratory and immune system as well as the toxic substances in tobacco smoke are currently the main suggested mechanisms. through this systematic review of the current available data, it appears that paternal ps promotes the development of cvd. however, we have previously demonstrated by using a robust methodological design that in young adults, even the normal ps exposures (similar to those seen in public places and houses) may significantly compromise health parameters [68 ]. minimizing children 's exposure to ps should therefore be amongst the main aims of interventional studies in order to raise public awareness and improve parent education and counseling. there are several limitations in the methodological designs of studies conducting research on the effects of ps on children 's health. most of the studies do not take into account very important confounding factors such as the environmental ones (e.g., exposure to other carcinogenic substances) or maternal diet and obesity that may indeed result in increasing cvd prevalence in children. most importantly, the adopted designs allow only for cross - sectional comparisons, in which the directionality and causality of associations can not be clearly justified. also, the vast majority of published studies justify ps on self - reports without an objective measurement of exposure (e.g., cotinine levels). moreover, it may be possible that ps has indirect effects on cvd outcomes in children, via its immunosuppressive capabilities. for example, it is estimated that ps is equivalent to smoking ~100 cigarettes per year. it has been found that even 30 minutes of ps significantly deteriorate cardiovascular outcomes similarly to smoking. hence, it seems reasonable to suggest that the physiologically undeveloped systems of fetus / children may be more susceptible to damage from the toxic effects of ps, particularly given the fact that the effects of ps may occur at very low levels of exposure. the fact that the majority of the available published paper on this research area were reviews rather than original research investigations demonstrates the difficulty of performing research on ps and cvd in children. we have performed a systematic review of the literature in order to be objective rather than assume causality and report only results that favor directionality of associations, which appears to be the case in many published papers. based on the available findings, interventions that target the cessation of parental smoking at home and preconception parental smoking, are necessary. despite that indoor ps has been banned in many countries, ps at home is the most difficult to control and at the moment pulled data from systematic study reveal that interventions to reduce exposure in children are ineffective. the findings of this systematic paper are particularly timely given the emerging experimental evidence on the adverse effects of passive smoking on health [68 ], as well as the shifting global smoking patterns, with an estimated 930 million of the world 's 1.1 billion smokers living in developing countries, that also demonstrate increasing smoking rates amongst young individuals [30, 43 ]. the findings of this paper indicate that public health preventive actions toward minimizing the exposure of children to ps should aim not only at suppressing tobacco use, but should also target family influence as attitudes that reinforce smoking behaviors and increase the exposure of children to ps. we conclude that the current available data reveal that ps in children is linked with deteriorated lipid profile and vascular function whereas data for maternal smoking (during pregnancy and ps after birth) are still inconclusive mainly due to design constrains and lack of adjusting because of important confounding factors.	passive smoking may be implicated in the development of cardiovascular disease (cvd) in children because of their partially developed physiological systems. the aim of the present systematic paper is to investigate whether passive smoking is associated with factors that influence the development of cvd in children. data sources included medline, cochrane library, cumulative index to nursing & allied health (cinahl) research database, google scholar, excerpta medica database (embase), the 2006 office of the surgeon general 's report, and the 2005 report from the california environmental protection agency. we identified a total of 42 relevant articles (i.e., 30 reviews and 12 observational). results revealed that passive smoking may be implicated in deteriorating cardiovascular status in children in terms of unfavorable high - density lipoprotein levels and deteriorated vascular function.
in 2014, there were an estimated 2.6 million children younger than 15 years living with hiv and 190,000 children became infected. hiv - infected children and adolescents have unique social and psychological issues that could affect their adherence to antiretroviral treatment (art) and health outcomes. one particular issue that may influence pediatric outcomes is knowledge of their own hiv status. timely and supportive disclosure may improve treatment adherence, retention in care, psychological adjustment, family relationships, and morbidity and mortality in hiv - infected children and adolescents. however, disclosing an hiv - positive status to a child remains a global challenge. in high hiv prevalence settings, most perinatally hiv - infected children and adolescents are unaware of their diagnosis, including those who attend regular clinic visits and take art. there are several barriers to pediatric hiv disclosure, including caregiver fears and lack of health care worker (hcw) knowledge and tools for disclosure. caregivers are reluctant to disclose because of potential to experience hiv stigma, guilt regarding transmission, uncertainty in how to disclose, and fears of negative child reactions or questions the child may ask. in addition, high - volume pediatric hiv clinics often lack systematized processes or standardized materials for disclosure, making disclosure a challenging task for overburdened hcws. interventions that address caregiver fears, as well as provide more training and standardized materials to hcws, may help to improve disclosure rates and experiences and improve child outcomes. to date, limited peer - reviewed literature describes disclosure interventions and their associated outcomes. after a rapid expansion in art access for children, hcws in namibia noted that they were unprepared for dealing with complex issues associated with telling an hiv - infected child their diagnosis. to address these concerns, the namibian ministry of health and social services (mohss) hcws who were providing pediatric hiv services, and the international education and training center for health (i - tech), collaboratively and iteratively developed a pediatric hiv disclosure intervention. in 2010, we have previously published evaluation data describing how the intervention improved hcw and caregiver 's confidence and communication skills for pediatric disclosure. in this retrospective study, we evaluated the impact of the intervention on child knowledge of their hiv status, adherence to art, and viral suppression, using the most complete routine service delivery data available. the evaluation was conducted at 4 high - volume hiv clinics in namibia : onandjokwe, oshakati, engela, and katutura. evaluation sites were selected based on the timing of intervention roll - out and pediatric hiv patient volumes. briefly, the disclosure intervention is intended to be used with children aged 618 years. the centerpiece of the intervention is a 5-chapter cartoon book which uses empowering language and metaphors of body soldiers being strengthened by medicine [antiretroviral medications (arvs) ] and keeping the bad guys (hiv virus) asleep. the further the child progresses in reading the book, the more information about his or her disease and the role of medications is revealed. it is not until chapter 5 that the words hiv or a portion of the book is read, or reread, at each visit by a hcw until the caregiver and child are ready to read chapter 5 in which full disclosure occurs. the chapters are read in a highly interactive manner with each one taking approximately 510 minutes to complete the first time it is read. a disclosure form is attached to the patient care booklet on which the hcw notes how far in the disclosure book the child has gone at each visit and why the child thinks they are taking medicine. a readiness assessment form helps hcws assess the child 's and family 's readiness to engage in the full disclosure process. there is variation in how the intervention is implemented at each site because of site - specific contexts. for example, in facilities where children are unaccompanied by caregivers at their clinic visit, book chapters 14 are frequently used in group education settings for children. although the intervention is implemented in all sites, the completeness of routine documentation associated with the intervention varies widely. given that the disclosure intervention had been implemented nationally by the namibian mohss as part of routinely offered pediatric hiv treatment services, the university of washington institutional review board determined that the evaluation of this program was not human subjects research. data for this evaluation was abstracted between september and december of 2013 from routinely collected programmatic data. data sources included patient charts and 3 national electronic databases : (1) the national institute of pathology database that contains all hiv viral load (vl) test results performed in the country, (2) the electronic patient management system (epms) that stores general contact and demographic information on all children enrolled in hiv care, and (3) the electronic dispensing tool (edt) which contains prescription and medication information for all hiv - infected children receiving medications. initial participant lists for each of the 4 target clinics were generated by searching the epms database and identifying all children with birth dates within the appropriate date range (age 715 years at the time of data abstraction) who had been on art for at least one year. data abstractors pulled patient charts and verified and abstracted demographic data for all children identified through the epms database. we abstracted data for 2 components of evaluation : (1) a disclosure process evaluation to determine disclosure outcomes and changes in medication knowledge and (2) a clinical outcome evaluation to assess the impact of partial and full disclosure on cd4 count, vl, and adherence to art. children were included in the disclosure process evaluation if they had at least one hiv vl in the national institute of pathology database within the previous 6 months and documentation of initiating the disclosure intervention at least 13 months before the date of abstraction. of the children included in the disclosure process evaluation, the clinical outcome analysis was limited to children enrolled in the intervention during 2011 who had preintervention and postintervention initiation vl, cd4, and/or adherence data. the 2011 enrollment cutoff was selected so that children had at least 2 years of follow - up postintervention initiation at the time of data abstraction (fig. detailed flow chart of inclusion and exclusion criteria for participants included in disclosure evaluations. dashed line boxes include potential populations while solid - lined boxes describe actual populations included in the evaluation. adherence scores were calculated using pill pick - up and dispensing information found in edt and the following formula provided by the mohss : ppc = previous pill count pc = current pill count qd = quantity dispensed d = days since last visit cnppd = number of pills per day children in namibia begin receiving tablets at ages 34 years or earlier. a select few reported visits in the edt database included syrups for enrolled children, and those visits were removed before adherence analysis. for children documented as initiating the disclosure intervention, disclosure status was classified as full or partial. children who specifically mentioned that they took medication for hiv or had the full disclosure box checked and a corresponding date listed that was at or before enrollment in the intervention program were considered fully disclosed at baseline. children participating in the program were asked if they knew why they took their medicine at each visit, and responses were recorded. children characterized as having reached full disclosure during the intervention period had the full disclosure box checked and an appropriate date listed, had a record of reading the intervention booklet chapter where hiv is named, or a recorded response to the question why do you take your medicine ? that included the word hiv. data abstracted from patient charts and electronic medical record databases were analyzed using intercooled stata version 13.0 (college station, tx). correlates of partial vs. full disclosure were determined using univariate logistic regression, and variables statistically significant (p 0.05) in univariate analyses were included in a multivariate logistic regression model. variables were assessed for collinearity before being included in the multivariate model, and only one variable from collinear groupings was selected to be included in the multivariate model. for the subset of children enrolled in the disclosure program during 2011, paired t - tests and mcnemar tests were used to compare mean differences in adherence scores, cd4 counts, cd4 percent, and log vls or the proportion of children virally suppressed or considered adherent before versus after enrollment into the intervention. we evaluated virologic success using 2 categories of clinical significance : 100 copies per milliliter and 1000 copies per milliliter. these categories reflect the who threshold for virologic suppression (1000 copies / ml) and good viral suppression (100 copies / ml). children were considered adherent if they had a mean adherence percentage at or above 80% during the time period described. the evaluation was conducted at 4 high - volume hiv clinics in namibia : onandjokwe, oshakati, engela, and katutura. evaluation sites were selected based on the timing of intervention roll - out and pediatric hiv patient volumes. briefly, the disclosure intervention is intended to be used with children aged 618 years. the centerpiece of the intervention is a 5-chapter cartoon book which uses empowering language and metaphors of body soldiers being strengthened by medicine [antiretroviral medications (arvs) ] and keeping the bad guys (hiv virus) asleep. the further the child progresses in reading the book, the more information about his or her disease and the role of medications is revealed. it is not until chapter 5 that the words hiv or a portion of the book is read, or reread, at each visit by a hcw until the caregiver and child are ready to read chapter 5 in which full disclosure occurs. the chapters are read in a highly interactive manner with each one taking approximately 510 minutes to complete the first time it is read. a disclosure form is attached to the patient care booklet on which the hcw notes how far in the disclosure book the child has gone at each visit and why the child thinks they are taking medicine. a readiness assessment form helps hcws assess the child 's and family 's readiness to engage in the full disclosure process. there is variation in how the intervention is implemented at each site because of site - specific contexts. for example, in facilities where children are unaccompanied by caregivers at their clinic visit, book chapters 14 are frequently used in group education settings for children. although the intervention is implemented in all sites, the completeness of routine documentation associated with the intervention varies widely. the namibian mohss ethics review committee reviewed and approved the study. given that the disclosure intervention had been implemented nationally by the namibian mohss as part of routinely offered pediatric hiv treatment services, the university of washington institutional review board determined that the evaluation of this program was not human subjects research. data for this evaluation was abstracted between september and december of 2013 from routinely collected programmatic data. data sources included patient charts and 3 national electronic databases : (1) the national institute of pathology database that contains all hiv viral load (vl) test results performed in the country, (2) the electronic patient management system (epms) that stores general contact and demographic information on all children enrolled in hiv care, and (3) the electronic dispensing tool (edt) which contains prescription and medication information for all hiv - infected children receiving medications. initial participant lists for each of the 4 target clinics were generated by searching the epms database and identifying all children with birth dates within the appropriate date range (age 715 years at the time of data abstraction) who had been on art for at least one year. data abstractors pulled patient charts and verified and abstracted demographic data for all children identified through the epms database. we abstracted data for 2 components of evaluation : (1) a disclosure process evaluation to determine disclosure outcomes and changes in medication knowledge and (2) a clinical outcome evaluation to assess the impact of partial and full disclosure on cd4 count, vl, and adherence to art. children were included in the disclosure process evaluation if they had at least one hiv vl in the national institute of pathology database within the previous 6 months and documentation of initiating the disclosure intervention at least 13 months before the date of abstraction. of the children included in the disclosure process evaluation, children included in the clinical outcomes evaluation met additional inclusion criteria. the clinical outcome analysis was limited to children enrolled in the intervention during 2011 who had preintervention and postintervention initiation vl, cd4, and/or adherence data. the 2011 enrollment cutoff was selected so that children had at least 2 years of follow - up postintervention initiation at the time of data abstraction (fig. detailed flow chart of inclusion and exclusion criteria for participants included in disclosure evaluations. dashed line boxes include potential populations while solid - lined boxes describe actual populations included in the evaluation. adherence scores were calculated using pill pick - up and dispensing information found in edt and the following formula provided by the mohss : ppc = previous pill count pc = current pill count qd = quantity dispensed d = days since last visit cnppd = number of pills per day children in namibia begin receiving tablets at ages 34 years or earlier. a select few reported visits in the edt database included syrups for enrolled children, and those visits were removed before adherence analysis. for children documented as initiating the disclosure intervention, children who specifically mentioned that they took medication for hiv or had the full disclosure box checked and a corresponding date listed that was at or before enrollment in the intervention program were considered fully disclosed at baseline. children participating in the program were asked if they knew why they took their medicine at each visit, and responses were recorded. children characterized as having reached full disclosure during the intervention period had the full disclosure box checked and an appropriate date listed, had a record of reading the intervention booklet chapter where hiv is named, or a recorded response to the question why do you take your medicine ? that included the word hiv. data abstracted from patient charts and electronic medical record databases were analyzed using intercooled stata version 13.0 (college station, tx). correlates of partial vs. full disclosure were determined using univariate logistic regression, and variables statistically significant (p 0.05) in univariate analyses were included in a multivariate logistic regression model. variables were assessed for collinearity before being included in the multivariate model, and only one variable from collinear groupings was selected to be included in the multivariate model. for the subset of children enrolled in the disclosure program during 2011, paired t - tests and mcnemar tests were used to compare mean differences in adherence scores, cd4 counts, cd4 percent, and log vls or the proportion of children virally suppressed or considered adherent before versus after enrollment into the intervention. we evaluated virologic success using 2 categories of clinical significance : 100 copies per milliliter and 1000 copies per milliliter. these categories reflect the who threshold for virologic suppression (1000 copies / ml) and good viral suppression (100 copies / ml). children were considered adherent if they had a mean adherence percentage at or above 80% during the time period described. of the 1466 children screened for inclusion in the evaluation, only 314 satisfied all inclusion criteria (fig. the median age of children was 12 years, and approximately half (47%) were female (table 1). more than 50% of the children had been on art for more than 6 years. only 64% of the study participants had a cd4 count or percent recorded within 1 year before data abstraction. almost half (46%) of children had vls at or below 100 copies per milliliter, and an additional 18% were suppressed at or below 1000 copies per milliliter. population description at the time of data abstraction, the median time of enrollment in the intervention was just below 3 years (table 1). most children (89%) had more than 1 visit recorded in the disclosure tracking form. for children with multiple entries, the median number of entries tracking responses to the question was 5, and the average time between entries was 4.6 months. at enrollment, only 34 children (11%) knew their hiv status. during their time enrolled in the program, there was documented full disclosure to 120 (43%) children. when stratified by age, only 20% of children aged 710 years were fully disclosed during the course of the intervention compared with 57% of children aged 1115 years (p < 0.001). however, among those who reached full disclosure, the average time to full disclosure was not significantly different between younger and older children (29 vs. 31 months, respectively ; p = 0.40). just below half (48%) of the children who were disclosed after enrollment into the intervention had a record of reading chapter 5 of the intervention booklet, suggesting that many caregivers may have decided to disclose to their children outside the clinic setting, which is one of the options discussed with individual caregivers as part of the intervention. children who reached full disclosure during the course of the intervention were similar to children who remained only partially disclosed with respect to sex and cd4 count measurements (table 2). children who reached full disclosure were almost 1.5 years older at enrollment and at the time of data abstraction (p < 0.001 for both) and had been in hiv care [odds ratio (or) : 1.33, p < 0.001 ] and on art for longer (or : 1.36, p < 0.001). more children from the clinic at katutura were fully disclosed compared with those from the other 3 clinics. whether evaluated continuously or as clinical cutoffs below 100 copies per milliliter or 1000 copies per milliliter, children with lower vls were more likely to have been fully disclosed during the course of the intervention. children enrolled in the intervention longer (or : 1.08, p < 0.001) and who had more intervention visits (or : 1.26, p < correlates of disclosure at their first visit, more than half (61%) of children had no knowledge or incorrect knowledge of why they take their medications (fig. this included responses in which the child reported that they did not know why they took medicines or reported taking medication for another ailment such as cough or malaria. initially, only 10% of children used hiv - specific terms to describe why they take medications, and 16% used basic health and wellness descriptions. by the last visit recorded before data abstraction, the number of children who had no knowledge or incorrect knowledge of fwhy they take their medicine dropped to 18%. of 153 children who did not know why they took medications initially, 42% became fully disclosed and used hiv - specific language, while 34% adopted language specific to the disclosure program. changes in children 's responses to the question why do you take your medicine ? from enrollment to the time of data abstraction among 278 children with more than 1 entry in the disclosure tracking form. the 92 children included in the preanalysis or postanalysis of vl and adherence measures had been on arvs for at least 18 months at the time of enrollment into the intervention and had at least one vl or adherence measurement during 2 periods of assessment time : (1) 12 months before enrollment in the intervention and (2) 012 months after intervention enrollment or 1224 months after intervention enrollment. although we found no significant difference between pre - enrollment and postenrollment vl measurements 012 months after enrollment (p = 0.896), we did find that vl measurements significantly decreased from pre - enrollment measurements by 0.5 log10 copies per milliliter (p = 0.004) by 1224 months after enrollment into the disclosure program. we also observed a decrease in vl measurements between 012 months after enrollment and 1224 months after enrollment (p = 0.053). when evaluating sustained viral suppression at or below 1000 copies per milliliter during the 12-month period before and after enrollment, we saw no effect of enrollment in the intervention on virologic failure. although not statistically significant, we observed slightly improved viral suppression at or below 100 copies per milliliter when comparing viral suppression before enrollment to 1224 months after enrollment (p = 0.103). by 12 months after intervention, 18 (30%) children had reached full disclosure, and 8 more children reached full disclosure by 24 months. we saw no statistically significant association between full disclosure status at 12 months and mean vl values or proportion of children virally suppressed during the 1224-month period after intervention enrollment. 83 of these children were on first line regimens and 6 were on second line regimens. when evaluating calculated adherence percentage measurements before and after enrollment, we observed a significant adherence percentage increase over pre - enrollment measurements by 8% (p < 0.001) by 012 months after enrollment into the disclosure program and by 10% (p < 0.001) by 1224 months after enrollment (fig. 3). there was no significant difference between adherence percentages 012 months and 1224 months after enrollment. when evaluating adherence measures categorically (mean adherence 80%), we also observed a significant increase in the proportion of children adherent to medications during the 12 months (p < 0.001) and 24 months (p < 0.001) after enrollment. by 12 months and 24 months after intervention, 22 and 31 children, respectively, we did not observe statistically significant differences between full disclosure status and adherence measurements between the 12 months before enrollment and 12 months or 24 months after enrollment into the disclosure program. panel b, percent of children adherent (defined as mean adherence 80%) or with sustained viral suppression (defined as all vl measurements 1000 copies / ml or 100 copies / ml) during the period specified. panel c, paired t - tests and mcnemar tests comparing adherence and vl measurements during the periods specified for all children with data recorded during the 2 periods specified. of the 1466 children screened for inclusion in the evaluation, only 314 satisfied all inclusion criteria (fig. the median age of children was 12 years, and approximately half (47%) were female (table 1). more than 50% of the children had been on art for more than 6 years. only 64% of the study participants had a cd4 count or percent recorded within 1 year before data abstraction. almost half (46%) of children had vls at or below 100 copies per milliliter, and an additional 18% were suppressed at or below 1000 copies per milliliter. at the time of data abstraction, the median time of enrollment in the intervention was just below 3 years (table 1). most children (89%) had more than 1 visit recorded in the disclosure tracking form. for children with multiple entries, the median number of entries tracking responses to the question was 5, and the average time between entries was 4.6 months. at enrollment, enrolled in the program, there was documented full disclosure to 120 (43%) children. when stratified by age, only 20% of children aged 710 years were fully disclosed during the course of the intervention compared with 57% of children aged 1115 years (p < 0.001). however, among those who reached full disclosure, the average time to full disclosure was not significantly different between younger and older children (29 vs. 31 months, respectively ; p = 0.40). just below half (48%) of the children who were disclosed after enrollment into the intervention had a record of reading chapter 5 of the intervention booklet, suggesting that many caregivers may have decided to disclose to their children outside the clinic setting, which is one of the options discussed with individual caregivers as part of the intervention children who reached full disclosure during the course of the intervention were similar to children who remained only partially disclosed with respect to sex and cd4 count measurements (table 2). children who reached full disclosure were almost 1.5 years older at enrollment and at the time of data abstraction (p < 0.001 for both) and had been in hiv care [odds ratio (or) : 1.33, p < 0.001 ] and on art for longer (or : 1.36, p < 0.001). more children from the clinic at katutura were fully disclosed compared with those from the other 3 clinics. whether evaluated continuously or as clinical cutoffs below 100 copies per milliliter or 1000 copies per milliliter, children with lower vls were more likely to have been fully disclosed during the course of the intervention. children enrolled in the intervention longer (or : 1.08, p < 0.001) and who had more intervention visits (or : 1.26, p < at their first visit, more than half (61%) of children had no knowledge or incorrect knowledge of why they take their medications (fig. this included responses in which the child reported that they did not know why they took medicines or reported taking medication for another ailment such as cough or malaria. initially, only 10% of children used hiv - specific terms to describe why they take medications, and 16% used basic health and wellness descriptions. by the last visit recorded before data abstraction, the number of children who had no knowledge or incorrect knowledge of fwhy they take their medicine dropped to 18%. of 153 children who did not know why they took medications initially, 42% became fully disclosed and used hiv - specific language, while 34% adopted language specific to the disclosure program. changes in children 's responses to the question why do you take your medicine ? from enrollment to the time of data abstraction among 278 children with more than 1 entry in the disclosure tracking form. the 92 children included in the preanalysis or postanalysis of vl and adherence measures had been on arvs for at least 18 months at the time of enrollment into the intervention and had at least one vl or adherence measurement during 2 periods of assessment time : (1) 12 months before enrollment in the intervention and (2) 012 months after intervention enrollment or 1224 months after intervention enrollment. although we found no significant difference between pre - enrollment and postenrollment vl measurements 012 months after enrollment (p = 0.896), we did find that vl measurements significantly decreased from pre - enrollment measurements by 0.5 log10 copies per milliliter (p = 0.004) by 1224 months after enrollment into the disclosure program. we also observed a decrease in vl measurements between 012 months after enrollment and 1224 months after enrollment (p = 0.053). when evaluating sustained viral suppression at or below 1000 copies per milliliter during the 12-month period before and after enrollment, we saw no effect of enrollment in the intervention on virologic failure. although not statistically significant, we observed slightly improved viral suppression at or below 100 copies per milliliter when comparing viral suppression before enrollment to 1224 months after enrollment (p = 0.103). by 12 months after intervention, 18 (30%) children had reached full disclosure, and 8 more children reached full disclosure by 24 months. we saw no statistically significant association between full disclosure status at 12 months and mean vl values or proportion of children virally suppressed during the 1224-month period after intervention enrollment. 83 of these children were on first line regimens and 6 were on second line regimens. when evaluating calculated adherence percentage measurements before and after enrollment, we observed a significant adherence percentage increase over pre - enrollment measurements by 8% (p < 0.001) by 012 months after enrollment into the disclosure program and by 10% (p < 0.001) by 1224 months after enrollment (fig. there was no significant difference between adherence percentages 012 months and 1224 months after enrollment. when evaluating adherence measures categorically (mean adherence 80%), we also observed a significant increase in the proportion of children adherent to medications during the 12 months (p < 0.001) and 24 months (p < we did not observe statistically significant differences between full disclosure status and adherence measurements between the 12 months before enrollment and 12 months or 24 months after enrollment into the disclosure program. panel a, mean summary of adherence or vl measurements during the period specified. includes only children who have measurements collected during all 3 periods. panel b, percent of children adherent (defined as mean adherence 80%) or with sustained viral suppression (defined as all vl measurements 1000 copies / ml or 100 copies / ml) during the period specified. panel c, paired t - tests and mcnemar tests comparing adherence and vl measurements during the periods specified for all children with data recorded during the 2 periods specified. this evaluation provides additional support to previously published qualitative results indicating that this hiv disclosure intervention was beneficial to pediatric patients, their primary caregivers, and health providers. previous publications cited caregiver and hcw descriptions of how the disclosure intervention improved their care of hiv - infected children and reports of children 's improved adherence to care and treatment. the analysis presented here contributes clinical outcome, quantitative data describing statistically significant improvements in adherence measurements before and after enrollment into the intervention. children exhibited better adherence by 12 and 24 months after the initial exposure to the intervention. children also showed improved vl measurements between pre - enrollment and postenrollment into the intervention, although these changes were not statistically significant when evaluated at a threshold of 1000 copies per milliliter or 100 copies per milliliter, measuring virologic success. interestingly, when evaluating continuous measures of vl and adherence, we saw improved adherence measurements preceding improvements in vl. although nonintervention studies have also shown that disclosure of hiv status is associated with improved adherence to art regimens among children and adolescents, our study is the first to evaluate changes in adherence longitudinally before and after the introduction of a disclosure intervention. in evaluating knowledge of why they take their medicine over time, we found that there was a dramatic decrease in the number of children who did not know why they took their medicines. our data, captured from routine tracking of pediatric patient knowledge which is a component of the intervention, depict the evolution of patient thinking about adherence and can directly relate it to the intervention 's communication and education strategy, thus unpacking the black box of programmatic interventions. after exposure to the intervention, most children changed responses and either related their partial understanding to terminology described in the disclosure book (to keep bad guys asleep and/or soldiers strong) or had documented full disclosure. we found that almost half of the children enrolled in the program had reached full disclosure by the time of data abstraction. this is almost 4.5 times the number of children who knew their status at enrollment. similarly to other studies, we saw that younger children were fully disclosed less frequently than older children. guidelines and current literature suggest that disclosure should be a guided step - by - step process, progressing from partial to full disclosure depending on caregiver readiness and child 's maturity. however, this is the first study of a disclosure intervention implemented at scale (nationally) to provide a description of the length and steps in that process. our study found that the average length of time from partial to full disclosure was almost 2.5 years. interestingly, our study did not find that the time to full disclosure differed by age. rather, the time required to reach full disclosure may instead reflect the frequency that children attend clinic visits, the need to overcome caregiver barriers to disclosure, and variable child readiness for full disclosure, regardless of age. the data on which the findings presented in this article are based were drawn from routinely collected patient information, based on health care service delivery as routinely implemented in busy practice settings. thus, we were limited on the variables we were able to evaluate, the time when variables were collected, and the number of participants we were able to include. unfortunately, no data on who performed disclosure was collected, and we were unable to assess the location where disclosure happened. however, despite these limitations, the results of this study are promising and demonstrate that disclosure can impact clinical outcomes and improve hiv knowledge in children and adolescents. analysis of each of 3 key variables indicates a consistent picture of a disclosure intervention that facilitates the disclosure process in such a way as to improve adherence and decrease vl. the fact that the intervention was successful in nonresearch settings suggests that while some specific intervention content adaptation would be necessary for different cultural contexts, major adaptations for real world implementation would not. there is an urgent need to develop interventions to assist hcws with the challenging but crucial process of hiv disclosure to children and adolescents. throughout sub - saharan africa the namibia hiv disclosure intervention seems to have improved disclosure rates, child knowledge of why they take their medicine, vl, and adherence measurements for children enrolled in the disclosure program. the namibian disclosure intervention may provide a helpful example of what could be adapted and used in other settings.	objectives : using routinely collected data, we evaluated a nationally implemented intervention to assist health care workers and caregivers with hiv disclosure to children. we assessed the impact of the intervention on child 's knowledge and health outcomes.methods:data were abstracted from national databases and patient charts for hiv - infected children aged 715 years attending 4 high - volume hiv clinics in namibia. disclosure rates, time to disclosure, and hiv knowledge in 314 children participating in the intervention were analyzed. logistic regression was used to identify correlates of partial vs. full disclosure. paired t - tests and mcnemar tests were used to compare adherence and viral load (vl) before versus after intervention enrollment.results:among children who participated in the disclosure intervention, 11% knew their hiv status at enrollment and an additional 38% reached full disclosure after enrollment. the average time to full disclosure was 2.5 years (interquartile range : 1.23 years). children who achieved full disclosure were more likely to be older, have lower vls, and have been enrolled in the intervention longer. among children who reported incorrect knowledge regarding why they take their medicine, 83% showed improved knowledge after the intervention, defined as knowledge of hiv status or adopting intervention - specific language. on comparing 012 months before vs. 1224 months after enrollment in the intervention, vl decreased by 0.5 log10 copies per milliliter (n = 42, p = 0.004), whereas mean adherence scores increased by 10% (n = 88, p value < 0.001).conclusions : this hiv disclosure intervention demonstrated improved viral suppression, adherence, and hiv knowledge and should be considered for translation to other settings.
attention deficit / hyperactivity disorder (adhd) is a common chronic psychiatric disorder, characterized by a pattern of developmentally inappropriate inattention, motor restlessness, and impulsivity, which affects between 3% and 7% of school - age children.1 prospective follow - up studies found that approximately 50% of children with adhd show symptoms that continue into adulthood, and when left untreated are associated with substance abuse, depression, unemployment, and criminal offenses.2,3 clinical guidelines on the pharmacological treatment of adhd recommend psychostimulants (eg, methylphenidate, dexamphetamine, mixed amphetamine salts, lisdexamphetamine) and selective norepinephrine - reuptake inhibitors, such as atomoxetine.4 psychostimulants are known to increase synaptic dopamine concentrations through inhibition of the dopamine transporter, a mechanism that facilitates dopamine reuptake into presynaptic neurons.5 selective norepinephrine - reuptake inhibitors increase extracellular levels of norepinephrine and dopamine in the prefrontal cortex.6 this accumulating evidence suggests that behavioral problems associated with adhd may be related to cognitive dysfunction and early disturbances in dopaminergic innervation of basal ganglia and the frontal lobe.7 genetic and molecular studies of adhd also demonstrate an association between this disease and dopamine - related genes.712 however, psychostimulants induce a variety of side effects, including anorexia, headaches, stomach aches, insomnia, pyrexia, and tics.13 moreover, the frequency of suicidal ideation was greater among atomoxetine - treated patients compared with placebo groups.14 these results highlight the need to identify new therapies for adhd, particularly treatment with fewer side effects than currently available drugs. tipepidine (3-[di-2-thienylmethylene]-1-methylpiperidine) has been used as a nonnarcotic antitussive in japan since 1959. it is reported that tipepidine inhibits g - protein - coupled inwardly rectifying potassium (girk)-channel currents.15 the activation of the girk channels causes membrane hyperpolarization through potassium efflux. the inhibition of girk channels by tipepidine is expected to modulate the level of monoamines in the brain, since girk channels are coupled with g - protein - coupled receptors, including 5-hydroxytryptamine (5-ht)1a, adrenaline 2 and dopamine d2 receptors.15 using in vivo microdialysis, kawaura demonstrated that tipepidine increases the levels of 5-ht and catecholamines, including dopamine, in the prefrontal cortex of rats.16 furthermore, fujieda showed that tipepidine could attenuate the hyperactivity induced in methamphetamine - treated mice (an adhd model) by modulating these monoamine systems. given these results, we put forward the hypothesis that tipepidine can improve adhd symptoms by modulating monoaminergic neurotransmission through the inhibition of girk channels coupled with monoamine receptors in the brain. the purpose of this open trial was to confirm whether treatment with tipepidine could improve symptoms in pediatric patients with adhd. the ethics committee of chiba university graduate school of medicine approved the study protocol (g24061), and all subjects provided written informed consent for participation in the study. we registered this trial on the official database of clinical research (clinicaltrials.gov) on april 16, 2013 (nct01835093).18 this was a 4-week, open - label, proof - of - efficacy pilot study for pediatric subjects with adhd. the baseline demographic, clinical, and treatment characteristics of adhd are shown in table 1. tipepidine was taken orally at 30 mg / day (10 mg after breakfast, 10 mg after supper, and 10 mg before bedtime), for 4 weeks. all subjects were diagnosed according to the diagnostic and statistical manual of mental disorders (dsm)-iv criteria for adhd,19 and were classified into three subtypes : combined (inattentive and hyperactive / impulsive), n=7 ; inattentive, n=3 ; and hyperimpulsive, n=0. seven subjects had received some drugs before entering the trial, namely methylphenidate (1854 mg / day, n=2), atomoxetine (75 mg / day, n=1), aripiprazole (15 mg / day, n=1), a combination of methylphenidate and atomoxetine (18 and 60 mg / day, respectively, n=1), a combination of atomoxetine and aripiprazole (35 and 9 mg / day, respectively, n=1), and a combination of methylphenidate and risperidone (18 and 6 mg / day, respectively, n=1), while three subjects were drug - nave. treatment with these drugs was stable for the 4 weeks prior to enrollment, and was stable during the trial. the mini - international neuropsychiatric interview for children and adolescents (mini - kid)20 was conducted to exclude any current, past, personal, or familial history of mental illness. two subjects were also diagnosed as having a learning disorder : one subject was diagnosed with tic disorders, and one with learning and tic disorders, according to the dsm - iv criteria.19 all patients were assessed using the adhd rating scale iv (adhd - rs), japanese version.21 the adhd - rs is a reliable and easy - to - administer instrument both for diagnosing adhd in children and adolescents and for assessing treatment response. it consists of 18 items, with the scale being linked directly to dsm - iv diagnostic criteria for adhd.22 the das naglieri cognitive assessment system (dn - cas), japanese version,23 was used to assess cognitive function. it was developed to integrate theoretical and applied psychological knowledge, using cognitive processing theory and tests, designed to measure planning, attention, and simultaneous and successive processing (pass) in individuals aged 517 years. the wechsler intelligence scale for children third / fourth editions (wisc - iii / iv),24,25 japanese versions, were used to assess the full intelligent quotient of all patients. statistical analyses were performed using the software package spss version 21.0 for macintosh (ibm armonk, ny, usa). the wilcoxon signed - rank test was used as a post hoc test to compare changes from baseline to 4 weeks. the ethics committee of chiba university graduate school of medicine approved the study protocol (g24061), and all subjects provided written informed consent for participation in the study. we registered this trial on the official database of clinical research (clinicaltrials.gov) on april 16, 2013 (nct01835093).18 this was a 4-week, open - label, proof - of - efficacy pilot study for pediatric subjects with adhd. the baseline demographic, clinical, and treatment characteristics of adhd are shown in table 1. tipepidine was taken orally at 30 mg / day (10 mg after breakfast, 10 mg after supper, and 10 mg before bedtime), for 4 weeks. all subjects were diagnosed according to the diagnostic and statistical manual of mental disorders (dsm)-iv criteria for adhd,19 and were classified into three subtypes : combined (inattentive and hyperactive / impulsive), n=7 ; inattentive, n=3 ; and hyperimpulsive, n=0. seven subjects had received some drugs before entering the trial, namely methylphenidate (1854 mg / day, n=2), atomoxetine (75 mg / day, n=1), aripiprazole (15 mg / day, n=1), a combination of methylphenidate and atomoxetine (18 and 60 mg / day, respectively, n=1), a combination of atomoxetine and aripiprazole (35 and 9 mg / day, respectively, n=1), and a combination of methylphenidate and risperidone (18 and 6 mg / day, respectively, n=1), while three subjects were drug - nave. treatment with these drugs was stable for the 4 weeks prior to enrollment, and was stable during the trial. the mini - international neuropsychiatric interview for children and adolescents (mini - kid)20 was conducted to exclude any current, past, personal, or familial history of mental illness. two subjects were also diagnosed as having a learning disorder : one subject was diagnosed with tic disorders, and one with learning and tic disorders, according to the dsm - iv criteria.19 all patients were assessed using the adhd rating scale iv (adhd - rs), japanese version.21 the adhd - rs is a reliable and easy - to - administer instrument both for diagnosing adhd in children and adolescents and for assessing treatment response. it consists of 18 items, with the scale being linked directly to dsm - iv diagnostic criteria for adhd.22 the das naglieri cognitive assessment system (dn - cas), japanese version,23 was used to assess cognitive function. it was developed to integrate theoretical and applied psychological knowledge, using cognitive processing theory and tests, designed to measure planning, attention, and simultaneous and successive processing (pass) in individuals aged 517 years. the wechsler intelligence scale for children third / fourth editions (wisc - iii / iv),24,25 japanese versions, were used to assess the full intelligent quotient of all patients. statistical analyses were performed using the software package spss version 21.0 for macintosh (ibm armonk, ny, usa). student s paired t - test was used to compare changes from baseline to 4 weeks. the wilcoxon signed - rank test was used as a post hoc test to compare changes from baseline to 4 weeks. the baseline scores and mean changes of primary and secondary outcomes from the 4-week trial of tipepidine in adhd subjects are shown in table 2. a comparison of baseline scores and 4-week end - point scores showed that all the adhd - rs scores (total scores, hyperimpulsive subscores, and inattentive subscores) improved significantly (p<0.001). the wilcoxon signed - rank test also detected statistical significance in all adhd - rs scores (p<0.005). however, a comparison of baseline scores and 4-week end - point scores found that none of the dn - cas scores (total scores and planning, attention, simultaneous, and successive subscores) showed significant change. the wilcoxon signed - rank test also failed to detect statistical significance in any dn - cas score changes. no significant effects were revealed in blood parameters, urine analysis, weight, height, blood pressure, or cardiac frequency during the 4-week follow - up period. tipepidine improved the adhd symptoms of inattention and hyperimpulsivity, as shown by adhd - rs scores. to our knowledge, this is the first report demonstrating the beneficial effect of tipepidine in treating pediatric adhd subjects. comparisons of all baseline dn - cas scores (total scores and planning, attention, simultaneous, and successive subscores) and 4-week end - point scores detected no significant differences. however, a comparison of baseline dn - cas total scores and 4-week end - point scores did show a mild trend of improvement (p=0.093). the lower improvement in dn - cas symptomatology may have been partly due to the relatively low dosage of tipepidine used in this study (1.2880.349 mg / kg / day, table 1), compared with that of the fujieda trial in mice (20 mg / kg).17 higher dosages may be more beneficial for adhd symptoms, as they are associated with higher monoaminergic neurotransmission through girk channels. at present, the optimal dosage of tipepidine for adhd is unknown, and defining this dosage should be the primary focus in the treatment of these patients. the inhibition of girk channels by tipepidine is predicted to modulate brain monoamine levels in a similar manner to psychostimulants and selective norepinephrine - reuptake inhibitors. however, this trial found none of the side effects typically associated with psychostimulants and selective norepinephrine - reuptake inhibitors. tipepidine has been used safely as an over - the - counter antitussive for children and adults in japan since 1959. therefore, safety issues will be no of no concern if this is used as a new treatment for adhd. very recently, hamasaki showed that tipepidine activated dopamine neurons in the ventral tegmental area through the inhibition of girk channel - activated currents, and their preliminary microdialysis study showed that tipepidine dramatically increased dopamine levels in the shell of the nucleus accumbens (nac).26 in addition, costa dias identified the possible involvement of nac connections in the pathophysiology of impulsive decision making in adhd, using functional connectivity magnetic resonance imaging.27 therefore, further detailed studies of tipepidine use in adhd are needed to investigate dopamine activation in the nac and its neural pathways. further studies with greater analytical power, larger sample sizes, and more drug - nave subjects will be necessary. in conclusion, our pilot study suggests that tipepidine therapy may prove to be an effective alternative treatment for pediatric patients with adhd, and since this drug is already in wide clinical use for other conditions, there should be no ensuing safety issues. however, the safety of long - term tipepidine use needs to be evaluated carefully, since many antitussive medications are completed within 1 week. nonetheless, more detailed randomized, double - blind studies are needed to confirm tipepidine s efficacy and safety.	backgroundtipepidine (3-[di-2-thienylmethylene]-1-methylpiperidine) has been used solely as a nonnarcotic antitussive in japan since 1959. the safety of tipepidine in children and adults has already been established. it is reported that tipepidine inhibits g - protein - coupled inwardly rectifying potassium (girk)-channel currents. the inhibition of girk channels by tipepidine is expected to modulate the level of monoamines in the brain. we put forward the hypothesis that tipepidine can improve attention deficit / hyperactivity disorder (adhd) symptoms by modulating monoaminergic neurotransmission through the inhibition of girk channels. the purpose of this open - label trial was to confirm whether treatment with tipepidine can improve symptoms in pediatric patients with adhd.subjects and methodsthis was a 4-week, open - label, proof - of - efficacy pilot study for pediatric subjects with adhd. ten pediatric adhd subjects (70% male ; mean age, 9.9 years ; combined [inattentive and hyperactive / impulsive ] subtype, n=7 ; inattentive subtype, n=3 ; hyperimpulsive subtype, n=0) received tipepidine hibenzate taken orally at 30 mg / day for 4 weeks. all subjects were assessed using the adhd rating scale iv (adhd - rs), japanese version, and the das naglieri cognitive assessment system (dn - cas), japanese version.resultsa comparison of baseline scores and 4-week end - point scores showed that all the adhd - rs scores (total scores, hyperimpulsive subscores, and inattentive subscores) improved significantly (p<0.001). furthermore, a comparison of baseline dn - cas total scores and 4-week end - point scores showed a mild trend of improvement (p=0.093). tipepidine was well tolerated, with no patients discontinuing medication because of side effects.conclusionour pilot study suggests that tipepidine therapy may prove to be an effective alternative treatment for pediatric patients with adhd. nonetheless, more detailed randomized, double - blind trials are needed to confirm tipepidine s efficacy.
over the years, the proportion of hiv patients older than 50 years of age is constantly increasing. it is estimated that by the end of 2015, more than half of hiv patients in the united states will be older than 50 years of age. currently, about 15% of hiv infected adults in sub - saharan africa are older than 50 but the prevalence of older hiv patients in that area is expecting to increase in the coming decades. the main reason for the increasing age of patients living with hiv is the improvement of specific antiretroviral treatment (highly active antiretroviral treatment (haart)) and its expanding accessibility. better treatment of hiv complications and comorbidities such as diabetes mellitus (dm) and hypertension also contributes to the improving survival of hiv - infected patients. thus, hiv patients grow older and live longer with a life expectancy approaching that of the general population. currently, the majority of the older (50 years of age) hiv patients were infected and diagnosed with hiv in youth or in middle age. in the united states, 10% to 15% of all patients newly diagnosed with hiv diagnosed patients are above the age of 50. a similar prevalence (11%) it is still unknown whether hiv diagnosed at older age represents delayed diagnosis or a real new (recent) hiv infection. several studies had demonstrated differences between older and younger patients with new hiv diagnosis regarding their demographic, risk behavior, clinical and immunological characteristics. some studies suggested worse prognosis with a rapid disease progression, despite usage of haart, in patients diagnosed with hiv at older age as compared to patients who were diagnosed with hiv at a younger age, however, other studies failed to show such a difference. the aim of the present study is to characterize the demographic, risk behavior, clinical, virological, and immunological status at the time of hiv diagnosis as well as the prognosis and outcome of patients with new hiv diagnosis at older age (50) compared with young patients (hiv diagnosis 1 year ; ambiguity > 0.43). student t test, fisher exact test, and were used for statistical analysis. all patients with a new hiv diagnosis during the years 2004 to 2013 in our hiv - aids center were recruited to the study. patients with a prior positive hiv elisa test, patients 1 year ; ambiguity > 0.43). student t test, fisher exact test, and were used for statistical analysis. four hundred eighteen hiv patients (men 243 (58%) ; women 175 (42%)) were included in our retrospective study. the mean age of the patients at the time of hiv diagnosis was 40.4 13.5 (range 1888) years. the mean age of the women (39 12.6 years), at the time of hiv diagnosis, was significantly lower compared with the age of the men (41.3 14.2 years ; p = 0.01). the mean follow - up period (53.6 33.6 (range 13126) months) was similar for the men and women. eighty nine (21%) patients were first diagnosed with hiv at older age fifty - two of them (59%) were men and 37 (41%) were women. the number and the percentage of the older patients with new hiv diagnosis varied (nonsignificantly) along the study period (15%34%). it peaked at 2008 (34%) and then decreased gradually (16% at 2013). table 1 summarizes the demographic, clinical, immunological, and virological characteristics at the time of hiv diagnosis, of our older and younger patients. as can be seen in the table, the proportion of men and women was similar between the younger and the older patients. the prevalence of men who have sex with man (msm) and intravenous drug users (ivdu) was significantly higher among the younger compared with the older patients. in contrast, 70% of our older patients were heterosexual immigrants from ethiopia (an hiv endemic country) as compared with only 50% in the younger patients. the hiv subtype was c in the ethiopian immigrants, b in the msm (few infected msm had subtype a), and a in the ivdu patients, regardless of their age. demographic, clinical, immunological, and virological characteristics of older " vs younger " patients with a new hiv diagnosis the immune system at the time of hiv diagnosis was significantly impaired in the older compared with the younger group of patients (table 1). moreover, 31% of the older patients had cd4 cell counts of less than 50 cells/l at hiv diagnosis (compared with only 15% in the younger patients ; p = 0.001). significantly more older than younger patients had vl above 100,000 copies / ml at the time of diagnosis (table 1). comorbidities were more commonly observed in our older patients (25%) as compared with only 3.3% in the younger group of patients. aids defining illnesses were observed in 55 of our patients at the time of hiv diagnosis, with a similar prevalence in both age groups. the main aids defining illnesses were tb (mycobacterium tuberculosis ; 34%), and pneumocystis jirovecii pneumonia (pcp, 23%). lymphoma, brain toxoplasmosis, esophageal candidiasis, kaposi sarcoma, and cryptococal meningitis occurred less frequently. haart was initiated according to the aids info guidelines regardless of the age of the patients. the harrt regiments were based on 2 reverse transcriptase inhibitors (mainly combivir, truvada, or kivexa) and either a nonnucleoside reverse transcriptase inhibitor (efaviranze) or a boosted protease inhibitor (mainly kaletra, boosted atazanavir, or boosted darunavir) or an integrase inhibitor (isentress). there were no differences in the recommended haart regimes between the older and the younger patients. in the years of the study, we did not recommend haart to patients with cd4 > 350 cells/l. the prevalence of such patients was significantly higher among the younger group of patients (33% vs 19% in younger and older patients, respectively ; p = 0.03). adherence to haart medications during the follow - up period was similar in the 2 groups of patients (about 85%90%). the mean follow - up period (53 33 months) was similar in both age groups (table 2). during that period, the mortality rate was significantly higher in the older compared with the younger group of patients (table 2). the mortality rate among older men (28%) was significantly higher than that of the older women (11% ; p = 0.04). no such sex difference was observed in the younger group of patients (4.1% and 3.5% mortality for younger men and women ; respectively ; p = ns). as was observed at the time of hiv diagnosis (table 1), at the end of the study comorbidities were more often observed in the older group of patients (table 2). immunological, virological, and clinical characteristics of older vs younger hiv patients at the end of follow - up period at the end of the follow - up period, the cd4 cell counts were significantly lower in the older, compared with the younger, group of patients. furthermore, half of the older patients had cd4 cell counts below 350 cells/l as compared with 33% of the younger patients (table 2). as can be seen in figure 1 the mean change in cd4 cell counts (cd4) was lower in the older (183 cells/l) compared with the younger patients (200 cells/l) though without statistical significance. however, in the subgroups of patients with initial cd4 cell counts of less than 50 cells/l, and in patients with initial cd4 cell counts between 201 and 350 cells/l, cd4 was significantly lower (p = 0.05 and p = 0.02 ; respectively) in the older compared with the younger group of patients (figure 1). cd4 cell counts increments (cd4) of older and younger patients during the study period. patients were stratified into 4 subgroups according to their initial cd4 cell counts at the time of hiv diagnosis. cd4 was determined as the mean sd differences between cd4 cell counts at the end of the study (last visit) and the initial cd4 (at hiv diagnosis) cell counts for each patient. the older patients revealed, at the end of the study, significantly higher vl levels as compared with the younger group. in addition, significantly, fewer older patients achieved a good virological response (ldl). however, among the patients who were still alive at the end of the study, ldl rates were similar in both age groups (table 2). to rule out the possibility that the unfavorable outcome of the older patients resulted, mainly, from their initially worse virological and immunological status (table 1) rather than from their age, we further stratified our older and younger patients into 4 subgroups according to their initial cd4 cell counts at the time of hiv diagnosis. group i patients with initial cd4 50 cells/l ; group ii with initial cd4 51 to 200 cells/l ; group iii with initial cd4 201 to 350 cells/l, and group iv with initial cd4 cell counts above 350 cells/l. table 3 summarizes the immunological, virological, and clinical outcomes in those older and younger subgroups of patients. as can be seen in the table, in each subgroup the outcome of the older patients was less favorable than that of the younger patients. thus, at the end of the study, the older group of patients demonstrated higher vl, lower cd4 counts, higher mortality rates, and higher rates of aids defining illnesses as compared with the younger subgroup of patients who were presented with a similar cd4 cell counts at the time of hiv diagnosis. similarly, the older patients with vl > 100,000 copies / ml at diagnosis demonstrated worse prognosis compared with the younger patients who had a similar high initial vl levels. thus, end of study comparison between older and younger patients who were presented with a similar initial high vl (> 100,000 copies / ml) demonstrated significantly lower cd4 cell counts (360 259 vs 437 288 cells/l for older and younger patients respectively ; p = 0.04), higher vl (343,834 1009,060 vs 100,537 579,618 copies / ml for older and younger patients respectively ; p = 0.04) and a higher mortality rate (32% vs 6% for older and younger patients respectively ; p 1 year) disease (delayed hiv diagnosis). demographic, immunological, virological, and clinical outcome (end of study) of older vs younger hiv patients as related to the time of their hiv infection in both age groups, patients with delayed diagnosis were diagnosed at an older age compared with those with a recent hiv infection. in the younger group of patients, significantly more msm (p = 0.009) and fewer immigrants from ethiopia (p = 0.01) were estimated to have a recent hiv infection at the time of diagnosis. younger patients with delayed diagnosis demonstrated significantly higher vl (p = 0.002) and lower cd4 cell counts (p 50 years). active sex life with a risky sexual behavior of older men and women is probably the major cause for hiv infection at older age. hiv education and prevention programs should be implanted in older populations and not exclusively among young populations. as was reported by others, the older patients in our study presented with lower cd4 cell counts and higher vl compared with the younger patients (tables 1, 3, 4). previous studies suggested that this is probably due to delayed hiv diagnosis of the older patients. using the sequence ambiguity assay, we were able to show, for the first time, similar rates (40%) of recent (1 year) hiv infection in our older and younger patients (table 4) excluding the role of late diagnosis as the main cause, for the worse immunological, virological, and clinical status of older patients with a new hiv diagnosis. furthermore, among the subgroup of patients with a recent hiv infection, the older patients revealed lower cd4 cell counts and higher vl at the time of hiv diagnosis as well as less favorable outcome compared with the younger patients (table 4). moreover, the prognosis of older patients was worse (mortality, immunological impairment, aids defining illnesses) than that of the younger patients who presented with similar vl and cd4 cell counts at the time of hiv diagnosis (table 3), further supporting the effect of age, at the time of hiv diagnosis, on the clinical, immunological and virological course of hiv infection. despite good haart adherence (about 85%90%), the prognosis of the older hiv patients was less favorable compared with young patients. the older patients demonstrated higher mortality rate (table 2) and less immunological improvement (figure 1) compared with the younger patients. our results agree with other studies, which reported high mortality rate in patients diagnosed with hiv above the age of 50, especially in men. similar to our observations, some studies reported a blunted immunological response of older hiv patients despite good virological response. several factors, not mutually exclusive, may contribute to the worse prognosis of patients infected with hiv at older age. the level of physician 's clinical suspicions of hiv in old age is relatively low. this may lead to delayed diagnosis of hiv in older patients. in our study, this was not a significant factor since the prevalence of patients with delayed hiv diagnosis was similar in our younger and older patients (table 4). older patients have more comorbidities compared with young patients (tables 24) which may affect their general clinical condition. the therapeutic effects, the pharmacokinetics (including drug drug interactions), and the tolerability of haart may differ in different ages although previous studies had demonstrated similar response to haart in younger and older patients. indeed, as was previously reported, we observed good virological suppression (ldl) in our older patients, who had good adherence to haart, despite a blunted cd4 cells recovery (tables 24). immunosenescence (ageing of the immune system) may be the main cause for the worse course of older hiv patients. indeed, older patients, regardless of their hiv status, revealed malfunction of b cells, cd4 and cd8 cells (especially memory b / t cells) and regulatory t cells. in agreement, we observed immune impairment at the time of hiv diagnosis (tables 1, 3) with a blunted t - cell recovery (figure 1 ; tables 24) in the older patients compared with the younger hiv patients. thus, old age (50 years) at the time of hiv diagnosis affects the course of the disease by several mechanisms. second, the number of the older patients with new hiv diagnosis (89) is relatively small. on the other hand, our strict inclusion and exclusion criteria ascertained that all participants were, indeed, patients with a new hiv diagnosis. to the best of our knowledge, systematic analysis of different subgroups of patients, according to their initial virological and immunological status and to the time of their hiv infection (recent infection vs delayed diagnosis), was not reported previously. our study enabled us to highlight the significant role of age on the course of hiv infection, overcoming several confounding factors. to conclude, one - fifth of patients with a new hiv diagnosis are above the age of 50. those older patients have less favorable outcome compared with patients newly diagnosed with hiv at younger age. this highlights the urgent need for educational and screening programs in older populations, as well as the mandatory need for close follow - up and early haart initiation in patients diagnosed with hiv at older (50 years) age.	abstractto characterize the clinical, virological, and immunological status at presentation as well as the outcome of patients diagnosed with hiv above the age of 50.a retrospective study of 418 patients newly diagnosed with hiv in 1 israeli center, between the years 2004 and 2013.patients with new hiv diagnosis 50 years of age defined as older " and < 50 defined as younger. " patients were evaluated every 1 to 3 months (mean follow - up 53 33 months). patients with < 2 cd4/viral - load measurements or with < 1 year of follow - up were excluded. time of hiv infection was estimated by hiv sequence ambiguity assay. ambiguity index 0.43 indicated recent (1 year) hiv infection.eighty nine (21%) patients were diagnosed with hiv at an older age. those older patients presented with significant lower cd4 cell counts and higher viral - load compared with the younger patients. at the end of the study, the older patients had higher mortality rate (21% vs 3.5% ; p < 0.001) and lower cd4 cell counts (381 228 vs 483 261cells/l ; p < 0.001) compared with the younger patients. this difference was also observed between older and younger patients with similar cd4 cell counts and viral load at the time of hiv diagnosis and among patients with a recent (1 year) hiv infection.one-fifth of hiv patients are diagnosed at older age (50 years). those older patients have less favorable outcome compared with the younger patients. this point to the need of educational and screening programs within older populations and for a closer follow - up of older hiv patients.
in recent years there has been a broadening in focus in the measurement of health beyond traditional health indicators such as mortality and morbidity, and quality of life (qol) has turned into an important outcome in clinical and interventional studies. different definitions of qol have been proposed by different researchers or organizations. the world health organization (who) has defined qol as an individual 's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns. recently, many general instruments have been used to measure qol in different groups (e.g., patients, workers, population and so on). one of these instruments is the world health organization qol - bref (whoqol - bref) questionnaire which captures many subjective aspects of qol. this questionnaire is one of the best known instruments that has been developed for cross - cultural comparisons of qol and is available in many languages. this instrument, by focusing on individuals own views of their well - being, provides a new perspective on life. the whoqol - bref questionnaire has translated into persian and then validated in iran by dr. most studies were performed to evaluate quality of life in patients people, but there are few studies that evaluated quality of life of health - care staff, which provide services for patients. health - care staff provides higher quality services for their customers when they are healthy and have good quality of life. the objective of this study was to evaluate the quality of life of neyshabur health - care staff and some factors associated with it with use of whoqol - bref scale. the data were collected between may and july 2011, at the health centers in the city of neyshabur (a city in northeastern of iran). of all neyshabur health - centers staff (n = 583), forty eight persons exclude from study because of disagreement of them. all participating subjects provided informed consent after being acquainted with the purpose of study. in this study, questionnaires have been filled by participants and for enhance accuracy ; all participants were informed that their responses would remain confidential. we used the brief version of the who 's qol scale (whoqol - bref) in this study. the whoqol - bref questionnaire contains two items from the overall qol and general health and 24 items of satisfaction that divided into four domains : physical health with 7 items (dom1), psychological health with 6 items (dom2), social relationships with 3 items (dom3) and environmental health with 8 items (dom4). five hundred and thirty five of neyshabur health - care staff filled out the iranian version of the whoqol - bref questionnaire. each item of the whoqol - bref is scored from 1 to 5 on a response scale. raw domain scores for the whoqol were transformed to a 4 - 20 score according to guidelines. domain scores are scaled in a positive direction (i.e., higher scores denote higher qol). the mean score of items within each domain is used to calculate the domain score. the other data collected were included sex, age, education years, marital status, employment type, income level (per month), job background, chronic disease existence and local residence as independent variables. the age of participants was represented by two categories of 35 year and > 35 year. education years was categorized into two groups : 0 - 12 year and > 12 year. income level was divided into two categories including 5 million rial and > 5 million rial per month. job background was divided into two categories including 35 year. education years was categorized into two groups : 0 - 12 year and > 12 year. income level was divided into two categories including 5 million rial and > 5 million rial per month. job background was divided into two categories including < 10 year and year 10 year. descriptive analyses performed including frequencies, percentages, ranges, means, and standard deviations (sd). cronbach 's alpha (internal consistency index) was used to estimate the reliability of the whoqol - bref (cronbachs alpha values of 0.70 and over were deemed acceptable). pearson 's correlation coefficient was used to determine the level of agreement between four domains of whoqol - bref. paired t - test was used to compare difference between score means of different domains of whoqol - bref. to investigate the association between participants characteristics and their qol, t - independent test was used. at the end multiple linear regression (with backward method) transformed scores were used for statistical analyses in four domains. in this study, the level of significance was set at p < 0.05 for all analyses. in total, 535 individuals filled out the whoqol - bref questionnaire in this study. thirteen questionnaires had more than 20% missing data and thus were excluded from the study. the mean age of study population was 35.1 7.7 year (rang : 21 - 65 yr). of all participants who completed whoqol - bref questionnaire 318 persons (60.9%) were female, with a mean age of 33.38 6.77 and 204 persons (39.1%) were male with a mean age of 37.76 8.29. there was a significant difference between them in terms of age (p < 0.001). the percentage of respondents scoring at the lowest level (floor effect) ranged from 1 to 16.3 while the percentage of respondents scoring at the highest level (ceiling effect) ranged from 2.9 to 38.5. in this study cronbach 's alpha coefficient was applied to examine the internal consistency of whoqol - bref scale (26 items) as well as the four domains of it. the cronbach 's alpha coefficient of whoqol - bref was adequate (0.925) for all 26 questions and for each domain the values are : physical health domain (0.813), psychological health domain (0.811), social relationship domain (0.65) and environmental health domain (0.772). table 3 present correlations between four domains of whoqol - bref ; as observed, there are statistically significant correlations between all domains. there is also statistically significant correlation between overall qol (q1) and scores obtained from different domains. in this study in order to compare the significant difference between score means of different domain ratings, the paired t - tests were used. as table 4 shows, significant differences were found between all four different domains of whoqol - bref. as seen in table 5 and figure 1, among the different domains, the highest and the lowest mean and percentage of satisfaction were found for dom1 (mean = 15.26 ; percentage = 70.49) and dom4 (mean = 13.09 ; percentage = 56.94) respectively. the mean score of four domains and total of whoqol - bref according to sex, age, education years, marital status, employment type, income level, job background, chronic disease existence and local residence separately are presented in table 5. mean and percentage of satisfaction rating in dom1, dom2 and dom3 was higher in males than females but this is reversed in the dom4 [table 5 and figure 1 ]. as table 5 shows, there were significant differences between different states of some variables in four domains and total of whoqol (p < 0.05). table 6 illustrates the results of backward multiple linear regression, it is apparent that existence chronic disease and education years are significantly associated with total whoqol. is associated with four domains of whoqol, education years associated with dom2 and dom4, sex associated with dom2. in the dom1, the confounder was employment type ; in the dom2 and dom3, age, employment type and job background were recognized as confounders and in dom4, employment type, income level and local residence were confounders. characteristics of study population (n=522) response pattern and missing items for each item (n=522) correlation coefficients in two overall questions and four domains of whoqol - bref paired t - test for the four domains of whoqol - bref comparison of the whoqol - bref mean scores in four domains according to sex, age, education years, marital status, employment type, income level, job background, chronic disease existence and local residence comparison transformed scores (0 - 100-scale) of the whoqol - bref in four domains according to sex backward multiple linear regression analyses of significant factors associated with qol one of the major objective of this study was to evaluate the reliability (internal consistency) of whoqol - bref questionnaire in health- care staff. reliability analysis in this study indicated an acceptable internal consistency of whoqol - bref scale (= 0.925) and for each of its domains were high, except for social relationships domain that is partly low (= 0.65) which is similar to asnani (=0.66), skevington (= 0.68) and mazaheri (= 0.62) studies. lower internal consistency can be attributed to the small number of questions (3 items) in social relationships domain. other purpose of this study was to evaluate the qol of neyshabur health - care staff with use of the iranian version of the whoqol - bref questionnaire. to our knowledge, this is one of the first studies assessing qol among the health - care centers staff in iran. qol as a measurement can identify groups with physical or mental health problems and provide a guide to intervention and follow up evaluation. in this study, among the four domains of whoqol - bref, the highest mean satisfaction rating was found for dom1 (physical health, mean = 15.26), implying good activities of daily living, less dependence on medicinal substances and medical aids, enough energy and mobility, less pain and discomfort, sufficient sleep and rest and good work capacity. moreover, the lowest mean score was shown for dom4 (environmental support, mean = 13.09), indicating not very good financial resources, opportunities for acquiring new information and skills and leisure activities. most sd from mean (sd = 2.82) was observed in dom3 (social relationships). greater sd of mean obtained from dom3 might be associated with different interpretations of the questions used in this domain and also small number of questions. as table 4 shows, mean scores of four domains were different and statistically significant. observed that mean scores of four domains were different and the most difference was between dom1 and dom4. as seen in table 5, the mean score of satisfaction rating in dom1, dom2 and dom3 was higher in males than females but this difference was only statistically significant in dom2 (psychological health). some factors may be associated with lower psychological health in women as well as their job (e.g., pregnancy, delivery, milking, homemaking and so on) that need to do more investigation. in this study, item 4 in responses had upper mean score (3.97) and item 14 had lower mean score (2.44). in nedjat study observed that items 3 and 4 in responses had upper mean score and item 14 had lower mean score. in this study after use of multiple linear regression (as shows in table 6) observed that chronic disease existence is most important factor that affects qol of study population in total and four domains of whoqol. sex and education years were other factors that affect qol of study population. in abdollahpour study, the influential factors affecting each domain were : educational level and current disease in the physical health domain, employment status in the psychological health domain, status of house ownership and number of years employed in the environmental health domain and marital status in the social relationship domain. the findings from this study confirm that the whoqol - bref questionnaire is a reliable instrument to measure quality of life in health - care staff. from the data it appears that neyshabur health - care staffs have whoqol - bref scores that might be considered to indicate a relatively moderate quality of life. in this study observed that chronic disease in health - care staff is important health issue influencing quality of life of them.	background : the objective of this study was to evaluate the quality of life of neyshabur health - care staff and some factors associated with it with use of whoqol - bref scale.methods:this cross - sectional study was conducted on 522 staff of neyshabur health - care centers from may to july 2011. cronbach 's alpha coefficient was applied to examine the internal consistency of whoqol - bref scale ; pearson 's correlation coefficient was used to determine the level of agreement between different domains of whoqol - bref. paired t - test was used to compare difference between score means of different domains. t - independent test was performed for group analysis and multiple linear regression was used to control confounding effects.results:in this study, a good internal consistency (= 0.925) for whoqol - bref and its four domains was observed. the highest and the lowest mean scores of whoqol - bref domains was found for physical health domain (mean = 15.26) and environmental health domain (mean = 13.09) respectively. backward multiple linear regression revealed that existence chronic disease in staff was significantly associated with four domains of whoqol - bref, education years was associated with two domains (psychological and environmental) and sex was associated with psychological domain (p < 0.05).conclusions : the findings from this study confirm that the whoqol - bref questionnaire is a reliable instrument to measure quality of life in health - care staff. from the data, it appears that neyshabur health - care staff has whoqol - bref scores that might be considered to indicate a relatively moderate quality of life.
hamartomas are non - neoplastic malformations or inborn errors of tissue development first described in 1904. hamartomas can occur in any area of the body, but commonly originate from the spleen, intestine, and lung. birt and knight - jones reported the fisrt case of a harmatoma in the upper aerodigestive tract. in 1995, wenig and heffner documented the clinicopathologic features of 31 cases of hamartoma and introduced the term respiratory epithelial adenomatoid hamartoma (reah) as one particular type of hamartoma. approximately 70% of reahs occur in the nasal cavity, most often localized to the posterior part of the nasal septum. we experienced two cases of reah originating from the nasal septum, which were treated by endoscopic surgery, and present their clinical findings and histopathologic features. a 50-year - old woman presented with left nasal obstruction for 3 years and had no other associated sinonasal symptoms. the patient denied any past history including chronic rhinosinusitis, alleric rhinitis, and sinonasal operation. examination with a nasal endoscope revealed a smooth, mucosa - covered, and polypoid mass on the posterior aspect of nasal septum in the left nasal cavity ; the mass originated from the posterior part of the septum (fig. computed tomography (ct) coronal view scans of paranasal sinuses revealed a left nasopharyngeal mass (fig. axial view ct revealed that the mass was attached to the posterior aspect of the nasal septum (fig. a pre - operative punch biopsy of the mass suggested the diagnosis of reah. under general anesthesia, intranasal endoscopic excision was performed. the excised specimen measured 23 mm16 mm13 mm. on histological analysis, the surface of the lesion was found to comprise pseudostratified, ciliated, columnar epithelium and was composed of proliferated glands lined by ciliated respiratory epithelium. the patient tolerated the operation well and there were no complications during the postoperative period. at 1-year follow - up a 23-year - old man with a history of obstruction of both nasal passages visited our office. a ct scan of the paranasal sinus showed soft tissue density in all sinus cavity and both middle meatus (fig. 2c, a pre - operative punch biopsy of the mass suggested the diagnosis of reah. under general anesthesia, left nasal cavity mass originated from posterior aspect of nasal septum and mass was removed without remnant. pathology of left nasal cavity mass revealed reah associated with nasal polyposis and right nasal cavity polyp revealed chronic inflammation. a 50-year - old woman presented with left nasal obstruction for 3 years and had no other associated sinonasal symptoms. the patient denied any past history including chronic rhinosinusitis, alleric rhinitis, and sinonasal operation. examination with a nasal endoscope revealed a smooth, mucosa - covered, and polypoid mass on the posterior aspect of nasal septum in the left nasal cavity ; the mass originated from the posterior part of the septum (fig. computed tomography (ct) coronal view scans of paranasal sinuses revealed a left nasopharyngeal mass (fig. axial view ct revealed that the mass was attached to the posterior aspect of the nasal septum (fig. a pre - operative punch biopsy of the mass suggested the diagnosis of reah. under general anesthesia, intranasal endoscopic excision was performed. the excised specimen measured 23 mm16 mm13 mm. on histological analysis, the surface of the lesion was found to comprise pseudostratified, ciliated, columnar epithelium and was composed of proliferated glands lined by ciliated respiratory epithelium. the patient tolerated the operation well and there were no complications during the postoperative period. at 1-year follow - up a 23-year - old man with a history of obstruction of both nasal passages visited our office. 1c, d). a ct scan of the paranasal sinus showed soft tissue density in all sinus cavity and both middle meatus (fig. 2c, d). a pre - operative punch biopsy of the mass suggested the diagnosis of reah. under general anesthesia, left nasal cavity mass originated from posterior aspect of nasal septum and mass was removed without remnant. pathology of left nasal cavity mass revealed reah associated with nasal polyposis and right nasal cavity polyp revealed chronic inflammation. reahs are very rare in the sinonasal tract and were first recognized as a distinctive entity by wenig and heffner in 1995. to date, a very limited number of cases have been reported. in the report by the aforementioned authors, the patients included 27 men and four women aged 27 to 81 years (mean age, 58 years). symptoms varied depending on the location of the lesions and included nasal obstruction, rhinorrhea, headache, hyposmia, anosmia, epistaxis, facial pain, and proptosis. another report described a case in which the lesion was discovered as a periapical radiolucency during the course of a dental examination, without the typical sinonasal symptoms being apparent. approximately 70% of reahs occur in the nasal cavity, the most common site being the posterior nasal septum. less frequently, the origin is the lateral nasal wall, middle meatus, inferior turbinate, ethmoid and maxillary sinuses, and nasopharynx. one of the present patient featured nasal polyposis and had a past medical history of chronic rhinosinusitis. reah has clinical presentations, histologic changes, treatment, and behavior similar to inflammatory polyps, suggesting that the development of reah is induced secondary to the inflammatory process. however, a recent report on the molecular profile of reah chronicled a mean fractional allelic loss of 31%, a finding that raises the possibility of reah being a benign neoplasm rather than a hamartoma. ct and magnetic resonance imaging shows no characteristic signals because they vary according to the main element of the hamartoma. one study reported that, compared to normal and nasal polyposis ct scan, reah significantly enlarges the olfactory cleft width. on gross evaluation, histologically, the main morphological aspect is a proliferation and accumulation of glands and ducts covered by pseudostratified, ciliated epithelial cells with no metaplastic or atypical changes. the characteristic glandular components consist of respiratory epithelium originating from the surface epithelium, with an absence of seromucous glands. in contrast, seromucous glands are characterized by the absence of ciliated epithelial cell proliferation, which represents a reactive response often seen in inflammatory nasal polyps. a misdiagnosis of reah as either low - grade adenocarcinoma or inverted papilloma would likely result in more aggressive surgical intervention than is needed for the reah.	respiratory epithelial adenomatoid hamartoma (reah) is a rare benign lesion in nasal cavity. we report two cases of reah of the nasal cavity arising from nasal septum. the etiology of reah is unknown although inflammation may induce gland proliferation observed in hamartomas. one of our cases was associated with nasal polyposis. reah is a self - limiting disease, so it is important to differentiate reah from other pathologic process, including inverted papilloma and low - grade adenocarcinoma. the treatment of choice is complete excision through a conservative approach.
in 2014, obokata., reported a cellular reprogramming phenomenon with acid treatment called stimulus - triggered acquisition of pluripotency (stap). unfortunately, their article was retracted because of misconduct, and the trial to replicate of the stap cell phenomenon by other groups failed. nevertheless, the idea that strong environmental stimuli may reprogram differentiated somatic cells into less - differentiated ones is still worth pursuing. the pou family transcription factor oct4 (octamer - binding transcription factor 4) is an essential regulator of pluripotency and is of central significance in nuclear reprogramming. mesenchymal stromal cells (mscs) are a rare population of non - hematopoietic stromal cells that reside in the bone marrow and connective tissues. they have the potential to differentiate into mesenchymal tissues such as bone, cartilage, muscle, and adipose tissues and, therefore, can be significant in tissue repair. cd90 (thymus cell antigen 1) and cd73 (ecto-5-nucleotidase) are surrogate positive markers of mscs, while mscs lack cd45 (leukocyte common antigen) expression. transurethral electro - resection of intravesical tumors (tur - bt) is the first step in the treatment of bladder cancer. to confirm the completeness of endoscopic resection of bladder tumors, secondary resection of bladder tissue at the site of the preceding resection when no tumors remain in the resected specimens, the bladder is often preserved in situ. in the patients, the bladder tissue after primary electro - resection undergoes tissue repair. we hypothesized that the stap phenomenon can be introduced by the stimulus of electro - resection in the bladder tissue during regeneration and that various levels of stem cell markers such as oct4, cd90, and cd73 would appear in the bladder tissue resected by the secondary electro - resection. following endoscopic primary bipolar electro - resection of bladder tumors in saline (the turis system, olympus, tokyo, japan), secondary resection of bladder tissue at the primary resection site was performed. eight bladder paraffin - embedded specimens of secondary resections without remaining bladder tumors on examination with hematoxylin - eosin staining were immunohistochemically stained using anti - human oct4 rabbit monoclonal antibody raised against amino acids 250 - 350 of human origin (epr2054, abcam, cambridge, uk), anti - human oct4 mouse monoclonal antibody raised against amino acids 1 - 134 of human origin (sc-5279, santa cruz biothchnology, inc., dallas, texas, usa), anti - human cd90/thy1 rabbit monoclonal antibody (epr3132, abcam, cambridge, uk), anti - human cd73 mouse monoclonal antibody (sc-32299, santa cruz biotchnology, inc., dallas, texas, usa), and anti - human cd45 mouse monoclonal antibody (ir751, dako japan, tokyo, japan). the mean interval between the primary and secondary electro - resection for the eight patients was 58 days. epr2054 antibody detects both oct4a and oct4b proteins, while sc-5279 identifies oct4a protein only. the pathological diagnoses of corresponding primary tumors were all urothelial cancer and those specimens were also stained for oct4 and cd90 as above. two paraffin - embedded specimens of bladders excised at autopsy (died of cardiac infarction and pancreatic cancer), two paraffin - embedded specimens of testicular embryonal cancers, and paraffin - embedded specimens of primary tur - bts were similarly stained. as summarized in table 1, the pt stages of primary electro - resection were pt1 or possibly more than pt1, and tumor grades were g2 to g3 in the eight cases with no tumors in the secondary electro - resected specimens. both epr2054 and sc-5279 detected oct4-expressing cells in the nucleus of embryonal cell cancer cells (figure 1). control autopsy bladder tissues did not show oct4-expressing cells at all (figure 2) by either epr2054 or sc-5279. in four out of the eight cases, oct4 protein was sporadically stained with epr2054 in the cytoplasm of interstitial cells located in the specimens of second tur - bt (figure 3 and figure 4), but sc-5279 did not detect oct4a protein in the specimens, revealing that oct4 detected by epr2054 is oct4b. in bladder tumors resected by the primary tur - bt, oct4 was sporadically demonstrated in five out of the eight bladder tumors in the cytoplasm of interstitial cells, but not in tumor cells by epr2054, showing that the detected oct4 was oct4b (data not shown). cd73 was negative in embryonal cell cancer, while cd45 was expressed in lymphocytes infiltrating cancer. in the stroma surrounding embryonal cell carcinomas, upper middle : oct4 (oct4b) staining with epr2054 (a portion of positive cells is magnified at the corner). cd90 expression was weakly positive in the vascular endothelial cells and cd73 was basically negative in control autopsy bladders and embryonal cell carcinomas, while cd45 was detected in lymphocytes there. in the stroma surrounding embryonal cell carcinomas, cd90 and cd73 were markedly up - regulated in the interstitial cells in addition to showing enhanced expression in vascular endothelial cells in all electro - resected specimens by the second tur - bt (p=0.0016 by the chi - square test, compared with autopsy controls). cd45 was not detected in either interstitial or endothelial cells, being expressed only in lymphocytes infiltrating the electro - resected specimens. in bladder tumors resected by the primary tur - bt, cd90 was stained in vascular endothelial and interstitial cells, and possibly smooth muscle cells (data not shown). one case has shown no tumor recurrence without additional therapy since second tur - bt and another has undergone total cystectomy without any remaining tumor in the cystectomy specimen. oct4b : oct4b protein expression in the 2nd tur - bt specimens stained with epr2054 antibody, but not with sc-5279 antibody ; oct4a : oct4a protein expression in the 2nd tur - bt specimens stained with sc-5279 antibody ; cd90, cd73 : cd90 and cd73 protein expressions in the 2nd tur - bt specimens, cd45 : cd45 protein expression except in infiltrating lymphocytes in the 2nd tur - bt specimens, cx : cystectomy ; bcg : intravesical bcg (bacille de calmette et gurin) instillation, cis : carcinoma in situ. in the present study, oct4-positive cells were observed in electro - stimulated bladder tissue. however, it is important to identify which isoform of oct4 is expressed and to take the function of expressed oct4 into consideration. there are three splice variants of oct4 : oct4a, oct4b, and oct4b1, caused by alternative splicing of pou5f1 mrna. oct4b mrna produces three isoforms : oct4b-164, oct4b-190, and oct4b-265, by alternative translation. only oct4a has been shown to be a definite marker of pluripotency and self - renewal of cells. in applying immunohistochemistry to specimens, an antibody against exon 1a of the oct4 gene uniquely stains oct4a protein, but not oct4b and oct4b1 isoforms because oct4b and oct4b1 mrnas are missing exon 1a. additionally, oct4a is generally located in the nucleus, while oct4b and oct4b1 are in the cytoplasm. in our study, oct4 protein expression was detected in the cytoplasm with epr3132 antibody against exons 4 - 5, but not with that against exon 1a (sc-5279). this indicates that oct4b - positive cells appeared in the tissue following electro - resection of the bladder. epr3112 will not detect oct4b1, as the oct4b1 protein is truncated due to the stop codon tga located in exon 2b harbored as a cryptic exon. oct4b1 was originally regarded as a putative marker of stemness and is expressed in human colorectal cancer. additionally, two isoforms of oct4b, oct4b-190 and oct4b-265, as well as oct4b1 have been reported to be upregulated in response to various kinds of stress. electro - resection of bladder tissue and the subsequent reactions in the present study may have caused the stress - triggered appearance / infiltration of oct4b protein - expressing cells. cd90 and cd73, markers of mscs, were enhanced in the tissue resected by the second tur - bt, while cd45 expression was limited to infiltrating lymphocytes. then, electric stimulation administered to the bladder tissue in the primary tur - bt may have induced the recruitment and activation of mscs that participate in the repair of bladder tissues. cd90 has been reported to be overexpressed in activated tumor endothelial cells compared with normal endothelial cells. electro - resection of bladder tissue induced the appearance of oct4b protein - expressing interstitial cells as well as enhanced cd90 and cd73 expression in the vascular endothelial and interstitial cells. not pluripotent stem cells, but the more downstream mscs may be mainly involved in tissue repair of the bladder.	aim : we tested the hypothesis that stimulation by electro - resection of bladder tissue induces stem cells in the tissue repair process. materials & methods : after primary transurethral resection of a bladder tumor and surrounding tissue (tur - bt), second tur - bt was performed. tissues excised by second tur - bt were immunohistochemically stained for oct4, a marker of pluripotency, and for cd90 and cd73, markers of mesenchymal stromal cells, when no bladder tumor cells remained. results and conclusions : oct4b protein was sporadically stained in the cytoplasm of interstitial cells in four out of eight cases. cd90 and cd73 are upregulated in interstitial and vascular endothelial cells without cd45 expression. mesenchymal stromal cells, but not pluripotent stem cells, may be mainly involved in bladder tissue repair.
although obesity disproportionately affects minorities and the socioeconomically disadvantaged, prior research has shown that clinician diagnosis and treatment of obesity is not consistent with underlying population prevalence [25 ]. health care providers are more likely to diagnose younger adults and women as obese and more likely to encourage weight loss activities among individuals who are white, are middle aged [810 ], or who have a higher socioeconomic status (ses) [8, 11, 12 ]. the relationship between residence in a socioeconomically deprived neighborhood and obesity diagnosis has not been studied. research in other disease areas has identified a relationship between neighborhood characteristics and disease diagnosis. for example, residence in low ses neighborhoods has been associated with an increased likelihood of late - stage cancer diagnosis, particularly among black and hispanic women. social ecological theory forms the theoretical basis for this study and conceptualizes individuals as nested within broad social / environmental networks that influence their perceptions, outlooks, and behaviors. it suggests that neighborhood racial characteristics and ses may be important influences to consider in addition to individual - level characteristics [13, 14 ]. according to social ecological theory, individuals ' decisions and actions depend not only on their characteristics but also on the social forces that shape the contexts in which they live. in the case of physicians, these forces could originate from social institutions (e.g., health care systems, medical school), media, peer groups, or cultural norms. each of these factors may serve as an intervening or moderating factor in the relationship between neighborhood characteristics and provider diagnosis of obesity. for example, the location of residence may serve as a proxy for the availability of health system resources (e.g., health professional supply, health systems infrastructure, nutrition / fitness services). in areas where healthcare resources are limited, the diagnosis of obesity may be prioritized lower than acute medical problems resulting from excess body weight such as hypertension, type 2 diabetes, or hyperlipidemia. social norms about body weight in a given neighborhood of residence may influence providers ' diagnosis of obesity. for example, blacks, who are more accepting of excess weight, have less body weight dissatisfaction, and have higher body weight ideals compared to whites [15, 16 ], may be less likely to cite body weight as a chief complaint. as a result, health care providers working in areas serving predominantly black members may be less likely to diagnose their patients as obese. underlying population prevalence of obesity in a given neighborhood may negatively or positively impact physician diagnosis of obesity. on the one hand, increased exposure to obese patients, particularly in areas with smaller healthcare facilities servicing a more homogenous population, may increase the likelihood of a physician diagnosis. on the other hand, negative physician attitudes towards patient body weight and recent research which suggests that higher patient body mass index is associated with lower physician respect may reduce the likelihood of an obesity diagnosis claim in areas characterized by a higher population prevalence provider recognition of obesity is one of the biggest predictors of patient receipt of weight - related counseling and is associated with receipt of obesity - related services and patient self - efficacy [19, 20 ]. also, patients who are told by their physicians that they are overweight are more likely to lose weight compared to those who are not told [21, 22 ]. moreover, patients who are counseled about their weight or weight - related behaviors are more likely to report working on those areas [8, 10, 23 ], and patients who are advised by their physician to modify their behavior are generally more confident and motivated to engage in lifestyle modifications (e.g., dietary changes, increased physical activity) [8, 20, 24 ]. the purpose of this paper was to identify variation in provider identification of obesity in claims data by both neighborhood- and individual - level characteristics among obese, insured individuals. we hypothesized that plan members living in lower ses neighborhoods or neighborhoods with higher percentages of minority residents would be less likely to be diagnosed with obesity by their providers. we further hypothesized that obese plan members who are female, younger, and who had higher body mass indices (bmis) would be more likely to receive an obesity diagnosis code in their claims. a key contribution of this study to the existing literature is its inclusion of neighborhood characteristics in the analyses, which have received little attention to date. in light of current federal priorities to reduce obesity and eliminate health disparities [2527 ], there is a pressing need to better understand how neighborhood characteristics contribute to providers ' diagnosis of obesity. to examine whether neighborhood- and member - level characteristics affect whether providers include an obesity diagnosis code in their obese patients ' claims, we used 20022005 claims data linked to health risk assessment (hra) data for a sample of health plan members enrolled in one of three blue cross blue shield (bcbs) plans from different sections of the country. the hras were administered through an in - person clinical encounter, an online survey tool on the bcbs plan 's website or direct mailing to eligible plan members (two health plan sites). for plan members completing an online or paper - based survey (two health plan sites), height and body weight were self - reported ; members who completed an hra during an in - person clinical encounter (one health plan site) had their height and body weight measured directly by a health care provider. census and linked to the members ' claims and hra data based on their zip code listed in the enrollment file. bcbs members were excluded from the study sample if they were enrolled less than 6 months in the year in which they completed an hra, were less than 18 years old or their age was missing, had a pregnancy and/or delivery claim during 20022005, had a body mass index (bmi) less than 30 kg / m or greater than 100 kg / m, or were missing height or body weight data needed to calculate their bmi. some members eligible for the study were found to have multiple hra records ; in such cases, the most recent record was selected. the final study sample consisted of 16,151 obese adult plan members. during the study period, all three bcbs plans covered weight loss medications, nutritional counseling, and bariatric surgery as part of their benefits. the data used in this study met health insurance portability and accountability act 's (hipaa) definition of a limited data set. the data were secured and used in accordance with federal standards for protecting the confidentiality of the personal health information of the enrollee. the johns hopkins university office of research subjects deemed the study to be exempt from federal regulations because the research activities were considered to be of minimal risk to subjects. the outcome variable of interest was an obesity claim, specifically, whether the provider included an obesity diagnosis code in their obese patients ' claims. an obesity claim was indicated by international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes in the claims data. in particular, an obesity claim was defined by the presence of icd-9 codes for obesity (icd-9 codes : 278, 278.0, 278.00, 278.01, 278.1, v77.8, or v85.3-v85.4). the main independent neighborhood - level variables of interest (measured at the zip code level) were the proportion of black residents, median household income adjusted for inflation, percentage of residents with a high school diploma, and percentage of residents living in an urban area (each measured in tertiles) as well as the percentage of people living above the federal poverty line (based on a poverty income ratio at or above 1.00). the following member - level variables also were included in the models for their moderating or confounding influence on the outcome variable : age, sex, mean bmi obesity class (class i : bmi 30.034.9 kg / m, class ii : bmi 35.039.9 kg / m, and class iii : bmi 40 kg / m), and diagnosis of type 2 diabetes, hypertension, or dyslipidemia. the comorbidities were measured as binary outcomes based on the presence of relevant icd-9-cm codes in the claims data (see tables 3 and 4 for codes). we also controlled for the distinct number of providers and the number of specialists seen by the members in the year in which the hra data were collected. we used a multilevel logistic regression model to measure the associations between the outcome variable obesity claim and the neighborhood member characteristics. analyses were performed using the stata, version 9.2, software package (statacorp lp, college station, tx). table 1 reports the characteristics of the study sample overall and by the presence or absence of an obesity diagnosis code in their claims data. with respect to neighborhood characteristics (measured at the zip code - level), the mean proportion of black residents was 5.3%, the median household income was $ 46,759, the mean percentage of high school graduates was 70.5%, the mean proportion of members living in urban zip codes was 81.7%, and the mean proportion of residents above the federal poverty threshold was 87.6%. census estimates of 12.3% black, $ 41,994 median household income, 80.4% high school graduates, 79.0% urban, and 88.7% above the federal poverty threshold. with respect to member - level characteristics, 48.0% of the sample was female, the mean age was 48 years, 29.2% were hypertensive, 10.3% had type 2 diabetes, 26.8% had dyslipidemia, the mean bmi was 34.7 kg / m, and 64.9% were class i obese. the mean number of distinct providers seen in the year they completed the hra was 2.0 and the mean number of specialists seen in that same year was 8.2. nearly 8% of the sample had an obesity diagnosis code recorded in their claims records. the mean bmi of obese members with an obesity claim was significantly higher than the mean bmi for members without an obesity claim (38.4 kg / m versus 34.5 kg / m, p <.01). table 2 shows the multilevel logistic regression results relating neighborhood- and member - level characteristics to whether the provider included an obesity diagnosis code in the patients ' claims. tertile ranges for the neighborhood - level variables in the model can be found in table 4. after controlling for individual - level characteristics, bcbs members living in neighborhoods with the largest proportion of blacks were 29 percent less likely to receive an obesity claim (or = 0.71 ; 95% ci : 0.57, 0.89). bcbs members living in neighborhoods with the second largest proportion of black residents were 21 percent less likely to receive an obesity claim (or = 0.79 ; 95% ci : 0.64, 0.96). we also observed a significant association between provider identification of obesity in patient claims data and urbanity ; bcbs members living in neighborhoods with the second largest proportion of urban residents were 62 percent more likely to receive an obesity claim (or = 1.62 ; 95% ci : 0.31, 2.01). while we did not observe associations between household income and obesity diagnosis code in the patients ' claims, the direction of the effect was consistent with our study hypothesis that plan members living in lower ses neighborhoods would be less likely to be diagnosed with obesity by their providers. at the individual - level, the odds of having an obesity claim were greater among plan members who were female (or = 1.47 ; 95% ci : 1.27, 1.96), age 44 or below (or = 1.70 ; 95% ci : 1.48, 1.96), with hypertension (or = 1.56 ; 95% ci : 1.29, 1.87), with dyslipidemia (or = 1.56 ; 95% ci : 1.30, 1.88), with class ii or iii obesity (class ii : or = 2.02 ; 95% ci : 1.71, 2.38 ; class iii : or = 4.02 ; 95% ci : 3.38, 4.75) or who had a greater number of distinct provider visits (or = 1.20 ; 95% ci : 1.16, 1.23). the central aim of this study was to identify variation in provider identification of obesity (as indicated by claims data) by neighborhood- and member - level characteristics among an insured population of obese blue cross blue shield plan members. overall, we found that the proportion of black residents in the members ' neighborhood was moderately associated with whether a provider included an obesity diagnosis code in their obese patients ' claims and that individual - level characteristics (female gender, younger age, obesity - related comorbidities, and bmi 35 our results also suggest that most providers do not record obesity as a reason for clinical visits when billing private insurers. while all bcbs plans included in this study covered weight loss medications, nutritional counseling, and bariatric surgery during the study period, the widespread perception among providers that obesity is not a reimbursable medical condition [2931 ] may have contributed to this low diagnosis reporting rate., we observed differences in rates of obesity diagnosis codes by patients ' demographic characteristics [6, 32, 33 ]. also similar to prior research, we observed higher rates of obesity claims among members with obesity - related comorbidities and higher bmis. also like previous studies [35, 36 ], we observed low rates of obesity diagnosis codes. this study makes four important contributions to the evidence base. to our knowledge, this is the first paper to examine associations between neighborhood characteristics and receipt of an obesity claim. second, because our study population is insured, our analyses are not confounded by financial barriers to access. third, bcbs is the largest health insurer which likely makes the plans included in this study typical of the ones used by a large proportion of the u.s. fourth, the bcbs data used in this analysis are the largest and most current database that has been used to examine provider practice pattern of adult obesity care. first, the cross - sectional nature of the study design prevents conclusions about causation. second, the measure of obesity was based primarily on self - reported height and body weight. because women tend to underestimate their true body weight and men tend to overestimate their actual height, some members may have been included or excluded from the sample erroneously and/or their bmi may have been misclassified. likewise, individuals ' obesity classification may have changed over time depending on the timing of the hra administration and medical encounters. third, some providers may have verbally indicated that their patients were obese, or recorded obesity in the medical records, but did not submit the diagnoses in the insurance claims. we are unable to determine the extent to which patients were diagnosed with obesity but their diagnoses were not recorded in the insurance claims. fourth, factors other than those included in the models (e.g., member race ; provider race, age, gender, location of training ; type of hospital) may influence providers ' diagnosis of obesity. fifth, there may have been changes in sociodemographic characteristics between 2000, the year of the u.s. census data used for measuring neighborhood characteristics, and 20022005, the years of the claims and hra data used for measuring the outcome and member variables. if sociodemographic changes did occur, the study population may have been mischaracterized. however, it is unlikely that neighborhood characteristics changed significantly during the 3- to 5-year period and the study 's use of tertiles minimizes the effect of any changes that did occur on the results. sixth, for some study participants, the zip code of residence may not be the same as the location of care. because the members ' zip codes were obtained from the administrative data rather than the clinical data, providers may not be aware of the zip code where members live, which may explain why most of the neighborhood characteristics were not statistically significant. however, even in the absence of knowledge about members ' zip code of enrollment, providers may have a general sense of the neighborhood characteristics of their members ' place of residence. seventh, three modes were used to collect the hra data an online survey tool on the bcbs plan 's website, a direct mailing to eligible plan members, or an in - person clinical encounter. differences in the mode of data collection may introduce some bias to the results. finally, it is possible that plan members had an obesity diagnosis code reported in a claim earlier or later than the year in which they completed an hra. going forward, more research is needed to better understand the impact of neighborhood context on the likelihood that a physician provides an obesity diagnosis code in their obese patients ' claims. in particular, why do individuals living in neighborhoods with the highest proportion of black residents have a lower likelihood of having an obesity claim ? and why do individuals living in more urbanized areas have a higher likelihood of having an obesity claim ? are there other, potentially more precise, neighborhood characteristics (not included in this analysis) that affect provider obesity claims ? what is the appropriate unit of analysis to examine the relationship between neighborhood context and provider identification of obesity (e.g., zip code, census block) ? a better understanding of the association between neighborhood context and provider obesity claims could help better target local, state, and national efforts aimed at reducing obesity rates and eliminating health disparities. given that receipt of an obesity diagnosis is an important gateway to receipt of obesity care [8, 10, 2024 ] and given the considerable costs associated with obesity and its comorbidities, insurers may consider increasing their coverage of obesity management and treatment as a way of reducing obesity - related costs in the long run. such a change in member benefits would need to be clearly communicated since many physicians identify lack of payment by insurance companies as a key barrier to providing obesity care [2931 ]. physicians and other health care professionals are uniquely positioned to have an impact on the obesity epidemic in the u.s. the results of this study suggest, however, that they may be missing opportunities to educate and treat their obese patients. given the considerable health and economic consequences of obesity and its related comorbidities, providers who consistently diagnose their obese patients may facilitate modest weight loss at the individual - level which, in turn, may encourage significant health benefits and reduced costs at the population - level [40, 41 ]. finally, the low level of obesity diagnoses reported in claims suggests that claims data may be a poor source of data for understanding trends in obesity diagnosis and treatment. we found very low rates of obesity claims among an insured, obese population, particularly for members who were morbidly obese or living in neighborhoods with a higher proportion of black residents. these findings indicate the need for better systems or incentive structures to encourage more appropriate diagnosis of obese patients in claims data.	objective. the purpose of this study was to examine whether neighborhood- and individual - level characteristics affect providers ' likelihood of providing an obesity diagnosis code in their obese patients ' claims. methods. logistic regressions were performed with obesity diagnosis code serving as the outcome variable and neighborhood characteristics and member characteristics serving as the independent variables (n = 16,151 obese plan members). results. only 7.7 percent of obese plan members had an obesity diagnosis code listed in their claims. members living in neighborhoods with the largest proportions of blacks were 29 percent less likely to receive an obesity diagnosis (p <.05). the odds of having an obesity diagnosis code were greater among members who were female, aged 44 or below, hypertensive, dyslipidemic, bmi 35 kg / m2, had a larger number of provider visits, or who lived in an urban area (all p <.05). conclusions. most health care providers do not include an obesity diagnosis code in their obese patients ' claims. rates of obesity identification were strongly related to individual characteristics and somewhat associated with neighborhood characteristics.
the flatworm schistosoma mansoni is one of the major etiological agents of human intestinal schistosomiasis mansoni. the disease affects over 200 million individuals in 74 developing countries and causes high morbidity in infected populations (1). current strategies of disease control depend heavily on the use of the sole drug available for mass treatment, praziquantel (1). treatment is effective in single dose and has resulted in decreased morbidity at endemic areas. however, it is highly desirable that control strategies include other countermeasures such as vaccines and new drugs. in addition, praziquantel is not efficacious against all life cycle forms present in the human host and there is evidence that drug resistance may arise in schistosomes (2). the s. mansoni genome is 270 mb contained in eight pairs of chromosomes (3). schistodb contains several different s. mansoni data sets and the results of different computational analyses. one highlight of the database is its integration to the metabolic pathway prediction generated using the sri pathwaytools software (4). the database also contains all drugs available on kegg drug database (5), thus enabling us to indicate enzymes known to be targeted in other organisms. protein topology and cellular location predictions are important tools for the selection of vaccine candidates. we expect that schistodb will contribute to efforts towards the identification of drug and vaccine candidates in addition to a more comprehensive analysis of genes. schistodb provides access to the latest draft genome sequence and annotation of s. mansoni (6,7) (puerto rico strain) obtained from the wellcome trust sanger institute and the mitochondrial genome (8) (nmri strain). the current database version (release 2.0) also contains oligonucleotides (9) used in the agilent 44 k element array widely used by the community and ests mapped to the genome. the database provides the results of computational analyses including open reading frames (orfs) > 50 aa and protein feature predictions such as signal peptides, transmembrane domains, hydrophobicity plots and interpro domains (10), gene ontology (11) function predictions, ec number assignment and blast similarities to the ncbi non - redundant protein database and protein data bank database (12). we also loaded the orthomcl (13) group of genes from s. mansoni with orthologous genes from 86 other eukaryotic and prokaryotic genomes. in addition, drugs provided by kegg (5) were loaded and their targets were associated to s. mansoni genes that have matching ec numbers. users are able to visualize all data types in record pages and by queries using the query interface (see data - mining tools section) (table 1). table 1.data types and sources that have been integrated into schistodb and the number of genes that are impacteddata typedata sourcegene numberprotein coding genessanger13 339orthologsorthomcl9516go gene ontology termsinterproscan5667ec ezyme commission numbersschistocyc712estsgenbank9534pdb protein data bankrscb pdb2713 data types and sources that have been integrated into schistodb and the number of genes that are impacted schistodb uses gus 3.5 to systematically load data into an underlying oracle database. the open source database schema (gus genomics unified schema) uses controlled vocabularies and ontologies to provide wide relations between the different data types and analyses. online access to schistodb occurs via the gus wdk (web development kit, www.gusdb.org/wdk) which facilitated the creation of the website. the use of gus significantly facilitates the data loading and analysis process, enabling future and frequent release cycles. gus and wdk have been used for the development of other databases such as plasmodb (14). schistodb provides access to the latest draft genome sequence and annotation of s. mansoni (6,7) (puerto rico strain) obtained from the wellcome trust sanger institute and the mitochondrial genome (8) (nmri strain). the current database version (release 2.0) also contains oligonucleotides (9) used in the agilent 44 k element array widely used by the community and ests mapped to the genome. the database provides the results of computational analyses including open reading frames (orfs) > 50 aa and protein feature predictions such as signal peptides, transmembrane domains, hydrophobicity plots and interpro domains (10), gene ontology (11) function predictions, ec number assignment and blast similarities to the ncbi non - redundant protein database and protein data bank database (12). we also loaded the orthomcl (13) group of genes from s. mansoni with orthologous genes from 86 other eukaryotic and prokaryotic genomes. in addition, drugs provided by kegg (5) were loaded and their targets were associated to s. mansoni genes that have matching ec numbers. users are able to visualize all data types in record pages and by queries using the query interface (see data - mining tools section) (table 1). table 1.data types and sources that have been integrated into schistodb and the number of genes that are impacteddata typedata sourcegene numberprotein coding genessanger13 339orthologsorthomcl9516go gene ontology termsinterproscan5667ec ezyme commission numbersschistocyc712estsgenbank9534pdb protein data bankrscb pdb2713 data types and sources that have been integrated into schistodb and the number of genes that are impacted the open source database schema (gus genomics unified schema) uses controlled vocabularies and ontologies to provide wide relations between the different data types and analyses. online access to schistodb occurs via the gus wdk (web development kit, www.gusdb.org/wdk) which facilitated the creation of the website. the use of gus significantly facilitates the data loading and analysis process, enabling future and frequent release cycles. gus and wdk have been used for the development of other databases such as plasmodb (14). schistodb currently provides approximately 30 different queries of the data and several tools for analyzing, retrieving or viewing the data such as blast, pathway tools and gmod genome browser (15). once the appropriate selection of data types to display has been achieved, users can integrate different search results using the query history page. refining the original query iteratively until a narrow list of genes of interest is obtained, providing a manageable number of targets to validate, a time consuming and expensive process. gbrowse genome browser (www.gmod.org) is used in schistodb to display gene models, est alignments, blast results, etc. gbrowse enables visualization of the parasite genome and gene models, orf identification, and facilitates downloading of data in various formats. schistosoma mansoni metabolic pathways are available through pathway tools web interface where several queries provide access to pathways, reactions, enzymes, compounds and other elements. the graphical overview allows the user to visualize the complete set of pathways and highlight specific reactions or perform organism comparison and expression analyses. mining for candidate drug and vaccine targets will benefit from many of the analyses available. schistodb integrates different datasets in a relational database that has permitted us to apply a technique known as genomic filtering (16). genomic filtering allows the identification of gene products that might be of interest for drug targeting based on several criteria e.g. absence of alternative pathways that consume or produce a given compound, presence or similarity to the host molecule to avoid toxicity, est evidence, cellular location, known drugs that target the same gene product in other organisms or 3d models of the protein. the presence of signal peptides and transmembrane domains will be important for the identification of vaccine candidates. est evidence permits the verification if the putative target is expressed in the relevant life cycle stages. the identification of similar proteins with structure information permits homology modeling of s. mansoni proteins which will contribute to the design of new chemicals and the identification of exposed antigenic peptides. the user could perform complex operations with the results, such as use boolean operators (and, not, or) to search for proteins that, for example, have a signal peptide, do not have transmembrane domains and are expressed in the schistosomula life cycle stage according to est evidence, to identify secreted proteins. figure 1 shows an example where the combination of the queries genes by pdb similarity, genes by drug evidence and genes by est evidence generates a narrow list of 56 genes from a total of 13 339. that means, 56 genes have similar 3d structures in pdb database, drugs known to target the same gene product in other organims and also overlaping ests. clicking on any of the gene identifiers opens a page with information on that gene. figure 1.screenshots from schistodb displaying the flow of a query. from the initial page users select from the various query choices for identifying genes, contigs, orfs or ests. from each query individual genes are displayed in the results page and it links to the gene page. in the example, the gene for ribokinase is displayed. the gene results page includes : annotation, links to schistocyc and genedb, the gene model, blast hits, est clusters, microarray oligonucleotides, orfs, ec, gene ontology, kegg drugs, orthology, protein domains, the predicted protein, mrna and coding sequences. screenshots from schistodb displaying the flow of a query. from the initial page users select from the various query choices for identifying genes, contigs, orfs or ests. from each query individual genes are displayed in the results page and it links to the gene page. in the example, the gene for ribokinase is displayed. the gene results page includes : annotation, links to schistocyc and genedb, the gene model, blast hits, est clusters, microarray oligonucleotides, orfs, ec, gene ontology, kegg drugs, orthology, protein domains, the predicted protein, mrna and coding sequences. the current version contains only s. mansoni data, so the expansion of the database will start with the integration of data sets from other schistosome species. we also expect to load and integrate other data types such as snps, microarray and sage. as new data are added, we will include additional queries and tools to view these data. national institutes of health fogarty international center (5d43tw007012 - 03 to a.z., r.l.v.m. and	schistodb (http://schistodb.net/) is a genomic database for the parasitic organism schistosoma mansoni, one of the major causative agents of schistosomiasis worldwide. it currently incorporates sequences and annotation for s. mansoni in a single user - friendly database. several genomic scale analyses are available as well as ests, oligonucleotides, metabolic pathways and drugs. in this article, we describe the data sets and its analyses, how to query the database and tools available in the website.
the studies participating in the fnih sarcopenia project are described in the first manuscript in this series (6). they include : age, gene and environment susceptibility - reykjavik study (ages) (7) ; boston puerto rican health study (bprhs) (8) ; six clinical trials at university of connecticut (uconn) (914) ; framingham heart study (fhs) original cohort (15) and the offspring cohort (16) ; health, aging, and body composition study (habc) (17) ; invecchiare in chianti (inchianti) (18) ; osteoporotic fractures in men study (mros) (19,20) ; rancho bernardo study (rbs) (21) ; and study of osteoporotic fractures (sof) (22,23). to be included in these analyses, participants must be aged 65 and older and must have completed, at a single time point, the following measures : objectively measured body mass index (bmi), appendicular lean mass (alm : sum of lean mass in the arms and legs), grip strength, and gait speed. participants from rbs and ages were excluded because rbs did not measure walking speed and ages measured body composition with bioelectrical impedance (bia). a total of 7,113 men and 2,950 women were included in the analyses presented here. gait speed was measured as the length of the walking course (4 or 6 m) divided by the time it took participants to walk the course at their usual pace. walking courses that were longer or shorter were converted to a speed that would have been achieved on a 4- or 6-m course using previously published equations (24). if more than one test was administered, the average gait speed (m / s) was used. grip strength was measured by a handheld dynamometer, and the maximum value of either hand was analyzed. total body fat mass and total bone - free lean mass (kg) were acquired using dual energy x - ray absorptiometry (dxa) on hologic (waltham, ma) or lunar / ge healthcare (madison, wi) machines. the fnih cutpoints for grip strength and lean mass, derived from classification and regression tree analysis, were reported in two accompanying articles (25,26). participants with gait speed less than or equal to 0.8 m / s were classified as having slow walking speed. men with grip strength less than 26 kg and women with a grip strength less than 16 kg were defined as weak (25). we used alm divided by body size (almbmi) to determine lean mass ; men with almbmi less than 0.789 and women with almbmi less than 0.512 were classified as low lean mass (26). we also examined our alternative definitions for low lean mass using absolute alm (not corrected for body size) ; men with alm less than 19.75 kg and women with alm less than 15.02 were classified as low lean mass (data not shown). using these cutpoints, we examined two possible fnih definitions : (i) clinically relevant weakness and low lean mass (low grip strength + low almbmi) or (ii) clinically relevant slowness with weakness and low lean mass (slow gait speed + low grip strength + low almbmi). several groups have previously published operational criteria to define sarcopenia, including : (i) international working group (iwg) (4) ; (ii) european working group on sarcopenia older persons (ewgsop) (3) ; (iii) european society for clinical nutrition and metabolism special interest group on cachexia - anorexia in chronic wasting diseases (espen) (2) ; and (iv) society of sarcopenia, cachexia, and wasting disorders (scwd) (5). the ewgsop suggested that sarcopenia be defined as low lean mass + low strength and/or low performance. the ewgsop differentiated presarcopenia (low mass) from sarcopenia (low mass + low strength or low performance) and severe sarcopenia (low mass + low strength + low performance). several possible performance measures (grip strength, chair stand, gait speed), lean mass assessment methods (dxa, bioimpedance, computed tomography, and magnetic resonance imaging), and different cutpoints were suggested by the ewgsop. for the fnih analyses, we used alm by dxa, grip strength, and gait speed as measures of muscle mass, strength, and physical performance (3) and used similar cutpoints that were recently published to operationalize the ewgsop criteria (2729). the iwg recommended gait speed as a measure of physical performance and defined gait speed less than 1 m / s as slow (4). the recommendations for espen and scwd were similar to ewgsop and iwg, thus were not analyzed separately. summary of operational definitions for sarcopenia and prevalence by gender note : almbmi = ratio of appendicular lean mass over body mass index ; alm / ht = ratio of appendicular lean mass over height squared. sensitivity and specificity were not determined because they are not applicable in the absence of a gold standard criterion to define sarcopenia. furthermore, positive predictive value, negative predictive value, and likelihood ratios can not be computed because a participant s status (as determined by a reference standard) is unknown. therefore, we described the agreement between the fnih criteria with other proposed criteria using several different statistical measures (30), including : (i) positive percent agreement : the proportion of participants who were categorized as having the condition by both the fnih criteria and a second set of criteria divided by the number of participants who were categorized as having the condition by the second set of criteria. this is analogous to a sensitivity calculation ; (ii) negative percent agreement : the proportion of participants who were categorized as not having the condition by both the fnih criteria and a second set of criteria divided by the number of participants who were categorized as not having the condition by the second set of criteria. this is analogous to a specificity calculation ; and (iii) cohen s kappa (). kappa () values less than 0.40 are considered poor reliability, 0.400.75 are considered fair - to - good reliability, and greater than 0.75 are considered excellent reliability (30). the studies participating in the fnih sarcopenia project are described in the first manuscript in this series (6). they include : age, gene and environment susceptibility - reykjavik study (ages) (7) ; boston puerto rican health study (bprhs) (8) ; six clinical trials at university of connecticut (uconn) (914) ; framingham heart study (fhs) original cohort (15) and the offspring cohort (16) ; health, aging, and body composition study (habc) (17) ; invecchiare in chianti (inchianti) (18) ; osteoporotic fractures in men study (mros) (19,20) ; rancho bernardo study (rbs) (21) ; and study of osteoporotic fractures (sof) (22,23). to be included in these analyses, participants must be aged 65 and older and must have completed, at a single time point, the following measures : objectively measured body mass index (bmi), appendicular lean mass (alm : sum of lean mass in the arms and legs), grip strength, and gait speed. participants from rbs and ages were excluded because rbs did not measure walking speed and ages measured body composition with bioelectrical impedance (bia). a total of 7,113 men and 2,950 women were included in the analyses presented here. gait speed was measured as the length of the walking course (4 or 6 m) divided by the time it took participants to walk the course at their usual pace. walking courses that were longer or shorter were converted to a speed that would have been achieved on a 4- or 6-m course using previously published equations (24). if more than one test was administered, the average gait speed (m / s) was used. grip strength was measured by a handheld dynamometer, and the maximum value of either hand was analyzed. total body fat mass and total bone - free lean mass (kg) were acquired using dual energy x - ray absorptiometry (dxa) on hologic (waltham, ma) or lunar / ge healthcare (madison, wi) machines. the fnih cutpoints for grip strength and lean mass, derived from classification and regression tree analysis, were reported in two accompanying articles (25,26). participants with gait speed less than or equal to 0.8 m / s were classified as having slow walking speed. men with grip strength less than 26 kg and women with a grip strength less than 16 kg were defined as weak (25). we used alm divided by body size (almbmi) to determine lean mass ; men with almbmi less than 0.789 and women with almbmi less than 0.512 were classified as low lean mass (26). we also examined our alternative definitions for low lean mass using absolute alm (not corrected for body size) ; men with alm less than 19.75 kg and women with alm less than 15.02 were classified as low lean mass (data not shown). using these cutpoints, we examined two possible fnih definitions : (i) clinically relevant weakness and low lean mass (low grip strength + low almbmi) or (ii) clinically relevant slowness with weakness and low lean mass (slow gait speed + low grip strength + low almbmi). several groups have previously published operational criteria to define sarcopenia, including : (i) international working group (iwg) (4) ; (ii) european working group on sarcopenia older persons (ewgsop) (3) ; (iii) european society for clinical nutrition and metabolism special interest group on cachexia - anorexia in chronic wasting diseases (espen) (2) ; and (iv) society of sarcopenia, cachexia, and wasting disorders (scwd) (5). the ewgsop suggested that sarcopenia be defined as low lean mass + low strength and/or low performance. the ewgsop differentiated presarcopenia (low mass) from sarcopenia (low mass + low strength or low performance) and severe sarcopenia (low mass + low strength + low performance). several possible performance measures (grip strength, chair stand, gait speed), lean mass assessment methods (dxa, bioimpedance, computed tomography, and magnetic resonance imaging), and different cutpoints were suggested by the ewgsop. for the fnih analyses, we used alm by dxa, grip strength, and gait speed as measures of muscle mass, strength, and physical performance (3) and used similar cutpoints that were recently published to operationalize the ewgsop criteria (2729). the iwg recommended gait speed as a measure of physical performance and defined gait speed less than 1 m / s as slow (4). the recommendations for espen and scwd were similar to ewgsop and iwg, thus were not analyzed separately. summary of operational definitions for sarcopenia and prevalence by gender note : almbmi = ratio of appendicular lean mass over body mass index ; alm / ht = ratio of appendicular lean mass over height squared. sensitivity and specificity were not determined because they are not applicable in the absence of a gold standard criterion to define sarcopenia. furthermore, positive predictive value, negative predictive value, and likelihood ratios can not be computed because a participant s status (as determined by a reference standard) is unknown. therefore, we described the agreement between the fnih criteria with other proposed criteria using several different statistical measures (30), including : (i) positive percent agreement : the proportion of participants who were categorized as having the condition by both the fnih criteria and a second set of criteria divided by the number of participants who were categorized as having the condition by the second set of criteria. this is analogous to a sensitivity calculation ; (ii) negative percent agreement : the proportion of participants who were categorized as not having the condition by both the fnih criteria and a second set of criteria divided by the number of participants who were categorized as not having the condition by the second set of criteria. this is analogous to a specificity calculation ; and (iii) cohen s kappa (). kappa () values less than 0.40 are considered poor reliability, 0.400.75 are considered fair - to - good reliability, and greater than 0.75 are considered excellent reliability (30). mean sd for gait speeds were 1.230.24 m / s and 0.970.24 m / s ; grip strengths were 40.78.8 kg and 21.15.9 kg ; almbmi were 0.880.11 kg and 0.600.10 kg ; and alm were 40.238.92 kg and 20.585.81 kg, for men and women, respectively. the proportion of participants who fell below the fnih cutpoints for gait speed, grip strength, lean mass, and the multiple combinations are presented in figure 1. compared with alm divided by height squared (alm / ht), participants with low lean mass by the fnih criteria (almbmi) were heavier with higher bmi and alm ; more participants were overweight or obese and reported a history of diabetes and heart failure (supplementary table 1). despite their larger body size, adults with low lean mass by the fnih almbmi cutpoint had poorer physical function including weaker grip strength, slower mean walking speed, and a higher percentage with walking speed less than or equal to 0.8 m / s compared with participants with low lean mass defined by alm / ht. independence and overlap of prevalent fnih criterion (slowness, weakness, and low lean mass) among men (n = 7,113) and women (n = 2,950) in the pooled data set. numbers in the circle represent the percentage of the sample with each of these criteria. numbers in the areas of overlap indicate the percentage of the sample that has more than criterion. only 0.5% men and 1.8% women share all criteria. the various proposed operational definitions and prevalence of sarcopenia are presented in table 1. note that primary indicator of lean mass for the fnih project was almbmi, whereas all other proposed criteria used alm / ht. these sets of candidate definitions largely differed from each other in regards to the cutpoint for slow gait speed and whether or not to include a measure of weakness. the prevalence of sarcopenia was higher in women than men. in men, the prevalence was 1.3% for the fnih criteria, 5.1% for iwg, and 5.3% for ewgsop. in women, the prevalence was 2.3% for fnih, 11.8% for iwg, and 13.3% for ewgsop. when we included gait speed with grip strength and lean mass in the fnih definition, the proportion of participants who three was lower : 0.5% in men and 1.8% in women. furthermore, the prevalence of severe sarcopenia by the ewgsop was 0.7% in men and 2.9% in women. in general, the fnih criteria, compared with other proposed definitions, identified participants who were older with higher bmi and higher lean mass, but were functionally more impaired, including a higher proportion with slow gait and inability to rise from a chair (supplementary tables 2 and 3). table 2 presents agreement proportions between the various criteria. in general, agreement was higher in men than in women and higher between the fnih and ewgsop criteria. the positive percent agreements between the fnih criteria and other criteria were low, ranging from 4.3% to 32% in men and 4.0% to 19.9% in women. our results demonstrated that kappa values were modest, with a range of 0.110.53 in men and 0.040.17 in women. between the iwg and ewgsop definitions, the positive percent agreement for sarcopenia was 52.1% and 61.7% in men and women, respectively. the negative percent agreement for sarcopenia was 97.1% and 96.4% in men and women, respectively. agreement comparing the fnih criteria with other operational criteria for sarcopenia note : ewgsop = european working group on sarcopenia older persons ; fnih = foundation of the national institute of health ; iwg = international working group on sarcopenia ; npa = negative percent agreement : the proportion of participants who were categorized as not having the condition by both the fnih criteria and a second set of criteria divided by the number of participants who were categorized as not having the condition by the second set of criteria ; ppa = positive percent agreement : the proportion of participants who were categorized as having the condition by both the fnih criteria and a second set of criteria divided by the number of participants who were categorized as having the condition by the second set of criteria. the prevalence of sarcopenia varies greatly depending on the criteria used for diagnosis. based on the presence of lean mass alone, initial prevalence of sarcopenia the large range was due to differences in the criterion used to diagnose sarcopenia, including (i) definitions that only included lean mass with or without correction for height (1,31), body mass (32), or body height and body fat (33) ; (ii) methodological differences to measure muscle mass (dxa or bia) (1,3235) ; and (iii) differences in the reference population used to establish normative data. in this study, the prevalence of sarcopenia incorporating both low lean mass and poor function were much lower between 0.5% and 5.3% in men and 1.8% and 13.3% in women compared with definitions based on muscle mass alone. when comparing the fnih, ewgsop, and iwg criteria in this pooled sample, the positive percent agreements were low, but the negative percent agreements were high (all > 95%), suggesting that there was good agreement on the absence of the condition. the prevalence of sarcopenia was highest with the ewgsop criteria in both men and women (5.3% and 13.3%, respectively). given that the fnih criteria were more restrictive, it was not surprising that the prevalence of sarcopenia was lower than either the ewgsop or iwg criteria. adults considered sarcopenic by both the fnih and ewgsop or by both fnih and iwg criteria had little overlap as evidenced by the modest positive percent agreement and kappa values. this was because the fnih and ewgsop had similar conceptual frameworks and both included measures of lean mass, strength, and/or performance. we believe the lack of agreement between these two criteria is explained by differences in the participants categorized as having low lean mass : the fnih criteria used almbmi, whereas both ewgsop and iwg criteria used alm / ht. although adults with alm / ht less than 5.67kg / m in women and less than 7.23kg / m in men did have low lean mass relative to a young reference population, this amount of lean mass was associated with faster gait speeds, stronger grip strength, and lower rates of obesity compared with participants who had low lean mass by almbmi. on the other hand, adults who met the fnih criteria for low lean mass (almbmi) were slightly more impaired with slower walking speeds and lower grip strength even though they had higher bmi and alm, and proportionately more obesity. these data suggests that almbmi, lean mass corrected for body size, is a good discriminator for low lean mass and is likely capturing adults who were unable to generate enough strength or function relative to their body size (sarcopenic obesity), and almbmi may be a good measure for low muscle quality or efficiency. in this study, there were several limitations. in particular, the prevalence and agreement rates may have been affected by several factors, including (i) the conceptual model, (ii) the strength or performance measure and cutpoints used, (iii) the method of assessment, and (iv) the study population. therefore, our conceptual framework and statistical approach was based on using mobility impairment as the clinically relevant functional state to determine meaningful weakness and low lean mass. in this series, we provided two different possible fnih criteria : (i) weakness + low lean mass or (ii) slowness + weakness + low lean mass. although this framework resulted in a low prevalence of sarcopenia, we chose to combine gait speed with grip strength and lean mass because the goal of the fnih sarcopenia project was to develop criteria that were conservative with few false positives in order to identify individuals who were clearly abnormal. second, there were many different measures of strength or performance that could have been used (eg, grip strength, leg power, dynamic leg strength, short physical performance, gait speed, and chair stand). however, we chose gait speed as a measure of mobility because it was available in all but one of the pooled studies, it has been reliably measured in clinical studies, and has been closely linked to function. additionally, we chose gait speed less than or equal to 0.8 m / s because it has been associated with survival (36), and the prevalence of gait speed less than or equal to 0.6 m / s was very rare in our pooled sample. in one study, among 70- to 80-year - old finnish women, the prevalence of sarcopenia was 2.7% vs. 0.9% when gait speed less than 1.0 m / s was used instead of gait speed less than 0.8 m / s (28). we chose grip strength as a measure for muscle strength because it is easy to use in both clinical and community settings and was available across all of the studies participating. selection of other measures like short physical performance battery or chair stand could have increased or decreased the prevalence. however, among the participating studies that had chair stands in our pooled data set, the proportion of participants unable to complete a chair stand was similar to the proportion with gait speed less than or equal to 0.8 m / s. since the ewgsop consensus statement recommended several different cutpoints for different measures and did not mandate specific measures or cutpoints, the prevalence of sarcopenia using the ewgsop criteria could have been influenced by our operational decisions. in fact, recent publications operationalizing the ewgsop and iwg criteria have reported different prevalence rates. for example, landi and coworkers used the lowest tertile of skin fold thickness for low lean mass, the prevalence was 21.8% among italians aged 8085 years (29). in another study, among 103 community - dwelling men in the uk hertfordshire sarcopenia study, the prevalence for sarcopenia was 6.8% and 7.8% when the lowest tertile of dxa - based lean mass was used instead of the lowest tertile of skin fold based fat free mass, respectively (27). furthermore, the positive percent agreement between the fnih and ewgsop are likely overestimated because we used similar gait speeds (< 0.8 m / s) and grip strength cutpoints (< 26 vs < 30 kg in men and < 16 vs < 20 kg in women). third, different brands / methods of dxa were used in the studies participating in the fnih sarcopenia project, and therefore potential bias may lead to different results. however, we tried to account for these differences between studies by using a random effects term in all our analyses that evaluated the association between our definitions and outcomes. finally, the fnih sarcopenia project pooled data sets from nine different studies for pooled analyses the largest to ever be studied in this area and are generalizable because the data set had broad representation of community - dwelling older adults. however, the pooled data set included primarily healthy community - dwelling populations with few comorbidities. this prevalence may be lower compared with more vulnerable populations (eg, assisted living, nursing home, or hospitalized adults), where disability rates are higher. however, these vulnerable populations are more heterogenous, and the factors contributing to slow gait are more numerous including cognition, osteoarthritis, pain, disuse atrophy, and cachexia. whether the relationship between lean mass, strength, and mobility are the same in more vulnerable populations is not known. this large variation in the prevalence may lead to different conclusions and implications for treatment. in particular, it is not clear whether treatment of weakness and low lean mass, especially with interventions that only target improving muscle strength and mass, in different populations is beneficial. thus, the work presented in this series is a work in progress, and many more questions and studies are needed. however, the fnih sarcopenia project provides evidence - based and data - driven cutpoints that will help the field come to a consensus on a diagnostic criteria. future studies will need to address whether : (i) the prevalence of low lean mass, weakness, and poor physical performance with the fnih criteria is higher among different populations ; (ii) the associations between mass, strength, and disability are similar or stronger in more disabled or sick populations ; (iii) the fnih criteria is useful in identifying participants for clinical trials ; and (iv) these criteria allow clinicians to recognize and potentially treat this disabling condition. we envision that the fnih criteria for clinically relevant weakness and low lean mass might be used to plan prevention studies in which older persons with weakness and low lean mass, but have not yet developed mobility limitations, would receive interventions designed to reduce the incidence or increase the time to onset of mobility impairment. alternatively, the fnih criteria for clinically relevant slowness with weakness and low lean mass may be used to identify candidates who already have mobility impairment, weakness, and low lean mass for recruitment in treatment studies that may look at outcomes like maintaining independence, preventing disability, delaying transitions from home to long - term care, quality of life, and/or survival. in conclusion, the current work taps into the expertise in the field and utilizes the largest sample of community - dwelling older adults to build upon and validate prior recommendations. these data provide comparison of the different definitions and suggest that the definitions have good negative, but poor positive agreement. thus, future studies should examine the predictive validity of these different definitions with important clinical outcomes (eg, disability, mortality) among different populations that may benefit from the diagnoses of sarcopenia. funding support for the conference and the work of the consortium was provided by the national institute on aging (u13ag04158 and p30 ag024827), the food and drug administration, and through grants from the foundation of the national institute of health (fnih) made possible by funding from abbot nutrition, amgen, eli lilly, merck, novartis, and the dairy research institute. this research was supported in part by the intramural program of the nih, national institute on aging. additional acknowledgments for each contributing cohort and members of the fnih sarcopenia project can be found in an online supplement.	background.several consensus groups have previously published operational criteria for sarcopenia, incorporating lean mass with strength and/or physical performance. the purpose of this manuscript is to describe the prevalence, agreement, and discrepancies between the foundation for the national institutes of health (fnih) criteria with other operational definitions for sarcopenia.methods.the fnih sarcopenia project used data from nine studies including : age, gene and environment susceptibility - reykjavik study ; boston puerto rican health study ; a series of six clinical trials from the university of connecticut ; framingham heart study ; health, aging, and body composition study ; invecchiare in chianti ; osteoporotic fractures in men study ; rancho bernardo study ; and study of osteoporotic fractures. participants included in these analyses were aged 65 and older and had measures of body mass index, appendicular lean mass, grip strength, and gait speed.results.the prevalence of sarcopenia and agreement proportions was higher in women than men. the lowest prevalence was observed with the fnih criteria (1.3% men and 2.3% women) compared with the international working group and the european working group for sarcopenia in older persons (5.1% and 5.3% in men and 11.8% and 13.3% in women, respectively). the positive percent agreements between the fnih criteria and other criteria were low, ranging from 7% to 32% in men and 5% to 19% in women. however, the negative percent agreement were high (all > 95%).conclusions.the fnih criteria result in a more conservative operational definition of sarcopenia, and the prevalence was lower compared with other proposed criteria. agreement for diagnosing sarcopenia was low, but agreement for ruling out sarcopenia was very high. consensus on the operational criteria for the diagnosis of sarcopenia is much needed to characterize populations for study and to identify adults for treatment.
improved medical technology and improved standard of living are accelerating the increase of the senior citizens population1. according to the data of the national statistics office of korea2, the ratio of senior citizens population stood at 13.1% in 2015, and is forecast to rise to 20.8% in 2016 and 30.1% in 2037. the negative effect is that there is a large number of elderly who are suffering from the increase of chronic disease, which results in a lower quality of life3. therefore there has been increasing interest in maintaining good health and improving the quality of life1. this is particularly significant according to the research about mortality from respiratory disease. among people aged 60 and over, 22.6 per 100,000 have chronic respiratory disease, which was the fifth major cause of death, and it has been on the rise4. as a result, research shows that for the elderly who have copd, as breathing difficulty increases, fear of death and symptoms of depression increase, which causes a decline in the quality of life5. breathing difficulty is the most serious symptom for patients with copd, which is especially life - threatening6. the decrease of functional capacity from breathing difficulties7 and motor disturbance8 lower the quality of life drastically. additional research comparing healthy people to patients with copd has also demonstrated a lower standard of life related to health among those suffering from impaired lung function9. in addition, social and economic expenses caused by copd are forecasted to rise steadily10, demonstrating the need to take additional measures to improve lung function. shin11 examined the effects of walking exercise programs on cardiopulmonary function in female seniors. as a result, maximum oxygen intake, heart rate, relaxed blood pressure, and maximum forced vital capacity have improved as they were stabilized. more recently, kang.12 demonstrated how a 12 week complex exercise training program affects cardiopulmonary function and the risk factor of metabolic syndrome of elderly persons who live in the countryside. in their study, maximal voluntary ventilation (mvv) has increased significantly in the group who performed a resistant workout and walking program. vo2max and mvv both increased meaningfully in the group that performed aerobic and yoga exercises. accordingly, kang said that complex exercise must include a resistant workout, walking, aerobics, and yoga in order to improve pulmonary function in the elderly. based on respiratory rehabilitation exercise programs improving pulmonary function on patients who have copd (such as chronic bronchial trouble and pulmonary emphysema), research has demonstrated how exercise can be productive. patients with general lung diseases such as interstitial lung disease, fibrous cystoma, bronchial expansion syndrome, thorax malformation, lung transplantation, pneumonectomy, and neuromuscular disease, can all benefit from exercise treatments13. however, results of research that has developed and applied various types of respiration rehabilitation for copd have not been consistent. one of the reasons for the discrepancies is that the age of the participants was not taken into account when exercise programs were developed and applied. therefore most research programs have overlooked the fact that exercise intensity depends on age. therefore, this study was designed to find out how much pulmonary function would vary as age increases. on the basis of the results of this study, an effort was made to provide basic information on developing an exercise program that takes into consideration the age of the individual. they were fifty - five female senior citizens who agreed in writing to take part in this study after listening to the purpose of the study. they were all non - smokers who had no history of lung disease or lung infection, and who have clear cognitive abilities on lung capacity measurement. age on average was 64.6 2.7 years ; height on average was 155.8 3.1 cm ; and weight on average was 57.3 7.2 kg. in the 7079 years group, age on average was 74.2 3.1 years ; height on average was 153.5 5.0 cm ; and weight on average was 57.0 6.8 kg. in the 8089 years group, age on average was 81.8 2.0 years ; height on average was 147.2 5.5 cm ; and weight on average was 54.6 6.1 kg. this study complied with the ethical standards of the declaration of helsinki, and written informed consent was received from each participant. the measuring instrument of lung capacity used in this study was a digital pulmonary function measurement (pony fx, cosmed inc. this measuring instrument can check the amount and the speed of air exiting from the lungs. this mse measurement was taken while the subject was in a straight sitting posture in a chair. legs were opened to the width of the subject s shoulders, and feet were placed vertically on the floor. the measuring instrument was held with one hand and inserted between the teeth with the lips closed over the measuring instrument. after 34 normal breaths, the subject inhaled and exhaled quickly and deeply while the measuring instrument was in place. three measurements were taken for each subject14 unless dizziness was experienced, in which case only two measurements were taken. this study was designed to demonstrate the effect on the variation of lung capacity as elderly women progress in age. an independent variable was divided into three groups : age 60 to 69, age 70 to 79, and age 80 to 89. a dependent variable was set up as seven factors from the pulmonary function tests. to examine the difference among dependent variables based on age if a significant difference among variables was demonstrated, post - hoc test by using scheffe was used. the result of pulmonary function based on the age of normal elderly women is shown in (table 1table 1.the changes of lung capacity in elderly women by life span (unit)variables6069 years (n=22)7079 years (n=22)8089 years (n=11)fvc (l)2.43 0.322.11 0.381.57 0.32fev1 (l)1.98 0.281.69 0.351.31 0.27pef (l)4.75 1.194.05 1.43 3.07 0.85 mef 75%4.37 1.053.68 1.352.75 0.74 mef 50%2.59 0.72.23 0.88 1.75 0.49 mef 25%0.98 0.470.76 0.29 0.78 0.37 ic (l)1.84 0.311.57 0.421.14 0.38 mean sd. p 7079 year, 6069 year > 8089 year, 7079 year > 8089 yearfvc : forced vital capacity ; fev1 : forced vital capacity in one second ; pef : peak expiratory flow ; mef : maximum expiratory flow ; ic : inspiratory capacity). a statistical difference was demonstrated among the three groups. there was a statistical difference among the groups as forced vital capacity (fvc) (p 7079 year, 6069 year > 8089 year, 7079 year > 8089 year fvc : forced vital capacity ; fev1 : forced vital capacity in one second ; pef : peak expiratory flow ; mef : maximum expiratory flow ; ic : inspiratory capacity lung aging can cause lung problems that are exacerbated by toxins from the external environment, smoking, and respiratory infections. as the lung ages, it decreases in physiological capacity, and breathing mechanism problems occur due to limitations in chest wall movement and decreases in lung elasticity15. furthermore, fat content and elastin in lungs decrease as age increases, breathing muscle strength weakens, and pleural elasticity and fibers decrease in length and diameter, further weakening the lungs elastic contractility16. as a result, lung tissues become stretched. changes in age - related breathing mechanisms cause limitations in respiratory airflow, and those limitations are known to reduce the rates of fev1 and fev1/fvc in breathing function tests, which show the occurrence of air trapping and hyperventilation as well as increases in residual volume and functional residual volume17. song.18 investigated vital capacity (vc) in accordance with age among participants between the ages of 20 to 70, and they reported that as age increases, age and vc correlate negatively. kim.19 also measured changes in lung function among participants divided into four groups according to age (60 to 64, 65 to 69, 70 to 75, and over 75 years of age), and reported that fev1, fvc, and vc decrease as age increases in all participants, both male and female, in all four groups. abe.20 measured vc, fev1 and assessed their physical function and mobility in 1,022 women aged 75 years who were living in an urban environment, and reported older women exhibited inferior pulmonary function as well as reduced physical function and mobility. the results of this study also confirmed that fev1, fvc, and fev1/fvc decrease as age increases in participants in their 60s, 70s, and 80s, which corresponds with the results of previous studies. while previous studies did not consider measurement variables for peripheral bronchi, this study tested the numbers of mef75%, mef50%, and mef25% in order to check the changes in peripheral bronchi as age increases. the study confirmed that peripheral bronchi also decrease as age increases. in these decreases in peripheral bronchi, closing volume speeds up, that is, the air flow closes particularly rapidly in small airways due to the decrease in lung - supporting elastic fibers. the increase in closing capacity causes a decrease in oxygen saturation because of the decrease in alveolar ventilation for lung perfusion, which is triggered by air flow closure21. likewise, potential to develop breathing problems increases as age increases because of vc decrease and closure of peripheral bronchi. as before mentioned, such vc decreases in the elderly are considered to be due to decreases in breathing mechanism and to limitations in chest - wall movement. although the lung - protective structure of the thoracic cavity is essential for normal lung functioning, it more greatly affects normal lung function as age increases due to changes in the structure of the thoracic cavity (i.e., spines, muscles, and ribs)22. lombardi,.23 confirmed vc changes in various angles of the thoracic kyphosis in 55 non - smoking women, and reported that fev1 and vc decreased as angles increased in pulmonary function test. aging is known to be highly related to decreases in inspiratory and expiratory muscle strength24. according to previous studies on breathing muscle correlation, maximal inspiratory and expiratory pressures (assessments of maximal inspiratory and expiratory muscle strength) decreased in the elderly. when the mean heights and weights were compared among participants in their 50s to 80s, the mean maximal inspiratory pressure of participants in their 50s was 111 cm h2o, and that of participants in their 80s was 70 cm h2o, confirming a significant decrease25. age - related decreases in maximal inspiratory and expiratory pressures relate to problems in breathing mechanism and to elderly people s sarcopenia26. the results of this study confirmed the overall vc decrease in elderly people as age increases. this age - related vc change is caused by changes in the thoracic structure and breathing muscle weakness in elderly people, as confirmed by previous studies. therefore, if exercise programs for the elderly can be developed in accordance with age, they will help to prevent vc decreases in the elderly, and it will lead to successful aging. additional studies are needed to develop exercise programs that are fit for the elderly in each age group and to confirm vc changes after doing the exercises.	[purpose ] although lung capacity in the elderly is affected by age, little research has been performed studying decreasing lung capacity in relation to increasing life expectancy. the aim of this study was to examine the effects of increased life span on the lung capacity of women. [subjects and methods ] the subjects of this study were 55 healthy elderly women over 60 years of age who were living in busan. subjects were classified in the following age categories : 60s, 70s and 80s. for the pulmonary function test, a spirometry (pony fx, cosmed inc., italy) was used. the item for measurement of pulmonary function in elderly women was maximum - effort expiratory spirogram (mes). the pulmonary function test was performed 3 times, and its mean value was used for analysis. [results ] among items of maximum - effort expiratory spirogram, a significant difference according to age was demonstrated in forced vital capacity, forced expiratory volume in 1 second, peak expiratory flow, maximum expiratory flow 75%, maximum expiratory flow 50%, and inspiratory capacity. [conclusion ] according to this study, lung capacity decreases remarkably as age increases. in conclusion, a continuous exercise program beginning at an early age is essential to prevent decrease in lung capacity as age progresses.
handedness, or the innate dominance of one hand 's dexterity over another, is generally not regarded as an impairment during open surgery, because the surgeon can adjust their body positioning to optimize intracorporeal maneuverability. interestingly, although the choice of instrument can be changed according to hand preference, this accommodative capability does not exist in robotic surgery. although this difference may initially seem to be a deficit, no such hindrance is routinely reported or perceived when using the robotic platform. it would then follow that the advantage of this system, namely the greater degree of freedom for rotational and/or fine motion, could obviate the surgeon 's inherent hand dominance, thus eliminating another constraint encountered in a traditional surgical setting. to examine this hypothesis, 10 residents who were relative novices at robotics were asked to complete various established skill sets with each hand, using both the open and the robotic technique. the relative performance of each hand for a given approach and task was then directly compared. in theory, if robotics eliminates the role of hand dominance, outcomes in this arm of the study would show similar hand performance distinct from open counterparts. residents from the urology and obstetrics and gynecology residency programs at one tertiary care institution were recruited for voluntary participation in the study protocol. those who self - reported as ambidextrous or left - hand dominant were excluded. participants completed a questionnaire at enrollment pertaining to their level of training as well as their experience / comfort level using the robotic technique. subjects were then asked to perform three tasks modified from the fundamentals of laparoscopic surgery curriculum, namely (1) peg transfer, (2) precision cutting, and (3) intracorporeal suturing, as previously described. the precision cutting involved excising a circle along a dotted line traced on paper, and the suture exercise focused on knot tying. each of these tasks was first performed by hand and then using the da vinci surgical system (intuitive surgical, sunnyvale, california). subjects were randomized to begin with the left or right hand and then asked to repeat the task with the opposite hand using both the open and robotic approaches for a given skill. the primary end points used to assess the interchangeability of each hand first involved the raw time for exercise completion. second, left : right (l : r) ratios were calculated by directly comparing the time score to complete a task with the left to the right hand : the greater the divergence of this number from 1, the larger the difference between hand performances. linear mixed model analysis presented in analysis of variance format was used with raw time and l : r ratio data to assess the relative impact of handedness for the robotic and open techniques. the mixed model analysis controlled for independent variables reflecting hand use, task performed, and residency robotic training characteristics to determine whether the open versus the robotic approach afforded a significant differential in hand performance. residents from the urology and obstetrics and gynecology residency programs at one tertiary care institution were recruited for voluntary participation in the study protocol. those who self - reported as ambidextrous or left - hand dominant were excluded. participants completed a questionnaire at enrollment pertaining to their level of training as well as their experience / comfort level using the robotic technique. subjects were then asked to perform three tasks modified from the fundamentals of laparoscopic surgery curriculum, namely (1) peg transfer, (2) precision cutting, and (3) intracorporeal suturing, as previously described. the precision cutting involved excising a circle along a dotted line traced on paper, and the suture exercise focused on knot tying. each of these tasks was first performed by hand and then using the da vinci surgical system (intuitive surgical, sunnyvale, california). subjects were randomized to begin with the left or right hand and then asked to repeat the task with the opposite hand using both the open and robotic approaches for a given skill. the primary end points used to assess the interchangeability of each hand first involved the raw time for exercise completion. second, left : right (l : r) ratios were calculated by directly comparing the time score to complete a task with the left to the right hand : the greater the divergence of this number from 1, the larger the difference between hand performances. linear mixed model analysis presented in analysis of variance format was used with raw time and l : r ratio data to assess the relative impact of handedness for the robotic and open techniques. the mixed model analysis controlled for independent variables reflecting hand use, task performed, and residency robotic training characteristics to determine whether the open versus the robotic approach afforded a significant differential in hand performance. ten subjects were enrolled, nine from the institutional urology program and one from the obstetrics / gynecology training program. most were at a junior level of training (postgraduate years 13), reported nil to minimal robotics console experience, and described a mixed / low level of comfort using the robotic technique (table 1). table 2 displays the technical tasks and skill sets the subjects completed using variable open and robotic approaches, comparing time with completion and l : r ratio, respectively. all of the tasks were completed faster using the open compared with the robotic approach for both hands. knot - tying was the exercise in which the performance of the right and left hands was most disparate for open surgery (right 30.2 s, left 51 s), whereas the times to complete this exercise with the robot were marginally longer, with less of a difference in mean times between both hands (right 60.2 s, left 58.6 s). cumulatively, the difference in raw time score between both hands for all three tasks was significantly smaller using the robot, with the mean difference in performance scores being 12.5 seconds for the open and 8 seconds for the robotic modules (p <.05). similarly, the overall l : r ratios were 1.45 versus 1.12 (p <.05), statistically favoring the robotic technique for dual - hand maneuverability. technical tasks and skill sets completed by subjects (peg transfer, circle cutting, knot tying) the linear mixed model analysis presented as analysis of variance format for both raw time and the l : r ratio are displayed in table 3. subset a demonstrates that handedness remained significantly different between the robotic and open approaches in terms of raw time scores (p <.0001), even after controlling for task completed, starting hand, prior robotic experience, or comfort level. these results were corroborated with the l : r ratio analysis (table 3) showing that the relative hand performance was different between the two techniques (p =.03) after controlling for similar variables. linear mixed model in analysis of variance format for raw - time analysis and l : r ratio analysis the scope of robotics continues to exhibit expanding applications as it pertains to the field of urology as well as to all other surgical disciplines. these benefits are well described and involve not only the technical aspects of refined optics, wristed - instrumentation, dexterity, and ergonomics, but also the diminished physical demands and constraints on the surgeon, who can operate from a seated position largely apart from the sterile field. this pilot investigation adds to these known benefits by corroborating emerging data that handedness is not a limitation in robotic surgery. of note, this lack of chirality may be particularly important in tasks that involve the interchange of hands, such as suturing and knot tying. prior work has examined the impact of robotics on dexterity and skill acquisition compared with pure laparoscopic techniques. although there is a general dearth of literature on this topic, small studies have demonstrated that task achievement in terms of knot tying is significantly faster using robotics as opposed to the pure laparoscopic approach, irrespective of existing laparoscopic experience. in a similar vein, moorthy and colleagues performed an investigation with 10 surgeons having variable laparoscopic experience to examine relative hand dexterity during suturing tasks. when an exercise was undertaken robotically, the subjects demonstrated a 40% relative reduction in the total time taken (p =.01) and had significantly reduced economy of motion for both hands (p <.05). although these studies did not compare relative hand performance, they establish that robotics affords a level of agility distinct from laparoscopy, and that this advantage is conferred to both hands of the operator. the role of handedness in robotic and mucksavage evaluated manual dexterity measurements among 19 robotic novices who performed the purdue pegboard test and needle - targeting exercises. although the performance scores for each hand were statistically disparate for the open approach, this difference was nullified for both tasks when they were done robotically. although this work substantiates the conclusions laid forth herein, the design of the current investigation augments and furthers these findings. in fact, the present study examined outcomes in a greater number of simulated exercises and focused on endpoints apart from performance scores, such as time for task completion and the novel concept of l : r ratio that directly correlates the performance of each hand. most importantly, this project controlled for known confounders to the univariate outcomes reported in the aforementioned investigation by the incorporation of a mixed model analysis. therefore, this report cumulatively extrapolates and strengthens precedents that have been set by prior published work. despite the relative merits of these conclusions, several limitations and considerations warrant discussion for future work on this subject. first, this pilot investigation needs validation in a larger group of subjects, including left - handed subjects and those with greater surgical experience, to make the results applicable beyond relatively novice, right - handed surgeons. to that point, follow - up studies should incorporate study arms evaluating residents as they perform the same set of skills using laparoscopic instruments and experienced surgeons completing tasks in the open, laparoscopic, and robotic settings. the data generated between these different study arms could provide clinically useful information about handedness and surgical learning curves between novice and experienced surgeons. scheduling constraints with the residents enrolled in this study and the limited availability of the robot may have affected our analysis with regard to the number of repetitions of tasks completed per subject. to make future results more robust and to define a mechanism for the elimination of hand dominance by the surgical robot, follow - up studies could require subjects to not only repeat tasks multiple times but to also repeat tasks at different levels of motion scaling. future studies should also record the number of errors each subject commits to detail the accuracy and precision of surgical task completion. certainly, adding these components to the foundation set forth by this pilot study would enable future studies to further describe the effects of hand dominance as they relate to different surgical modalities. indeed, one prior report looking at the impact of handedness on clinical outcomes showed that during robotic prostatectomy, a greater number of lymph nodes (right 3.26 vs left 2.76, p =.010) and a closer neurovascular bundle dissection (right 1.99 vs left 2.64 mm, p <.001) were routinely achieved on the right compared with the left. as the authors comment, this finding may be attributable to the durable effect of surgeon handedness, in addition to the contribution of instrument laterality or assistant instrument positioning. regardless, these are factors that can only be accounted for during in vivo surgery and can not be capitulated in the training models typically used in these types of investigations. despite these factors, however, the conclusions from this pilot study bring forth significant considerations for ensuing robotic training programs. handedness, or the innate dominance of one hand 's dexterity over the other 's, appears to be diminished in robotic surgery, in distinction to the clear impact of this factor on open surgical technique. this finding adds to the relative merits of the robotic approach, because it may have a significant role in the learning curve of tasks such as suturing and knot tying, which involve hand interchange. further investigation of this topic is warranted to focus on the import of this finding on intraoperative performance measures and clinical outcomes.	background and objectives : handedness, or the inherent dominance of one hand 's dexterity over the other 's, is a factor in open surgery but has an unknown importance in robot - assisted surgery. we sought to examine whether the robotic surgery platform could eliminate the effect of inherent hand preference.methods:residents from the urology and obstetrics / gynecology departments were enrolled. ambidextrous and left - handed subjects were excluded. after completing a questionnaire, subjects performed three tasks modified from the fundamentals of laparoscopic surgery curriculum. tasks were performed by hand and then with the da vinci robotic surgical system (intuitive surgical, sunnyvale, california). participants were randomized to begin with using either the left or the right hand, and then switch. left : right ratios were calculated from scores based on time to task completion. linear regression analysis was used to determine the significance of the impact of surgical technique on hand dominance.results:ten subjects were enrolled. the mean difference in raw score performance between the right and left hands was 12.5 seconds for open tasks and 8 seconds for robotic tasks (p <.05). overall left - right ratios were found to be 1.45 versus 1.12 for the open and robot tasks, respectively (p <.05). handedness significantly differed between robotic and open approaches for raw time scores (p <.0001) and left - right ratio (p =.03) when controlling for the prior tasks completed, starting hand, prior robotic experience, and comfort level. these findings remain to be validated in larger cohorts.conclusion:the robotic technique reduces hand dominance in surgical trainees across all task domains. this finding contributes to the known advantages of robotic surgery.
the viralzone database (http://viralzone.expasy.org) is an online resource that brings together viral molecular biology knowledge with viral genomic and protein sequences (1). viralzone was created in 2009 and is updated regularly on a bi - monthly basis. the resource contains two main types of information : virus description pages and lists of relevant uniprotkb proteins (which are generated automatically for each virus). the core data in viralzone are the virus description pages, which provide information on all viral genera referenced by the international committee for taxonomy of viruses (2). curators combine data from recent publications and textbook knowledge to create the tables, pictures, textual annotations and links to original publications that are found in each virus page. these provide an accessible summary of the available information on a viral genus, including illustrations of the virion and genome schematics, descriptions of the replication cycle, links to many databases (38), epidemiology data and lists of manually annotated proteins in uniprotkb (4). viral description pages are virus - centric and describe the processes and biology that are relevant to each viral genus. to complement these descriptions we have now added another layer of information to viralzone in the form of a viral ontology. this describes common replication steps or characteristics that are shared between multiple viral genera and is organized in the form of 133 ontology pages. the ontology is used to link common processes in the viral description pages each of these linking back to the ontology pages. viruses use a variety of unique molecular mechanisms during replication in hosts (9). these often circumvent or exploit cellular processes, and their study affords a greater understanding of the cellular functions concerned. viral mechanisms are also widely exploited as tools for biological research and biotechnology ; examples include the reverse transcriptase (10) and t7 rna polymerase (11) enzymes, internal ribosome entry site (12) and lentiviral vectors (13). most of these replication mechanisms are described in viralzone fact sheets for the viral genus that uses them. however, these are designed to provide a short overview of the biology of a virus and do not contain detailed explanations of the molecular events that occur. moreover, information disseminated in fact sheets is not easily extracted and does not offer a means to group viruses sharing a common process. for example, all viruses using ribosomal read - through (14) are annotated as such, but there is no way to list them all in viralzone. to address this need the information is structured with a vocabulary that is used both in virus fact sheets and molecular mechanisms pages, and represents a basis to develop virus ontology. the long - term goal is to link viralzone page, uniprot keywords and gene ontology terms. the concept of a central ontology was chosen because it has proven to be efficient for managing large data sets and analysis generated by transcriptomic and proteomic studies (15). in viralzone, the ontology is divided in five parts that describe the main steps in the viral life cycle : 18 pages linked to viral entry (figure 1), 29 pages linked to viral replication, 13 pages linked to viral exit, 11 pages linked to the virion structure and 62 pages linked to host virus interactions. each of these pages contains a description of the viral process associated with the term, a picture describing the molecular events and pathways, the list of associated viruses and links to original publications. they provide an overview of viruses using a common mechanism and improve the level of detail in virus fact sheets. for instance, the hepatitis b virus (hbv) fact sheet did not specify in what way the viral genome exits the cell nucleus : through nuclear lysis, nuclear egress or nuclear pore. using the viral ontology, we have now classified the hbv in the nuclear pore export pathway (16). this picture is effectively a graphical menu that provides links to 18 pages describing possible mechanisms of viral entry into host cells and events occurring subsequent to host - cell entry. this picture is effectively a graphical menu that provides links to 18 pages describing possible mechanisms of viral entry into host cells and events occurring subsequent to host - cell entry. this same principle applies to the virus ontology : the same terms described in viralzone pages are used to annotate uniprotkb virus entries. the 120 viral terms created for the ontology correspond to 120 new uniprot keywords that have been assigned to relevant viral entries. for example, the term inhibition of host stat1 by virus (17) is linked to 1024 viral protein entries in uniprot release 2012_07. viralzone virus fact sheets are virus - centric and display specific information on molecular virology, taxonomy, hosts and epidemiology. gene expression and viral replication cycle sections describe every specific process used by the virus to enter, replicate and exit the host cell. these sections have been updated to link directly to the ontology pages (figure 2). the advantages of these new links are multiple : first, the amount of available information is increased without overloading the fact sheet page that remains short and concise ; second, users can have in - depth insight of the virus molecular biology put into the broader context of all viruses sharing the same feature. figure 2.improved fact sheets. each step of the viral cycle is explained in detail and users can return to the influenza a page from any of the ontology pages it appears in. each step of the viral cycle is explained in detail and users can return to the influenza a page from any of the ontology pages it appears in. for example, description of the simplex herpes virus replication cycle now contains a total of nine processes that are clickable and lead to specific pages explaining these steps in detail. among these, the page viral penetration into host nucleus (18) depicts the various mechanisms by which viral genomes enter the host nuclear pores, with a short explanatory text and a schema representing the different strategies employed by the virus. in addition, all viruses penetrating the host nucleus are listed on the right part of the page with a link to original publications. viruses are obligate parasites and have evolved to have many ways of interacting with or hijacking host cell mechanisms. most of these interactions prevent antiviral cell defense or facilitate viral replication and transmission (19).. often viruses interact with key cellular regulatory elements, resulting in many complex phenotypes. that is why among the 133 ontology pages in viralzone, 62 are devoted to host virus interactions. in virus fact sheets, a new tab termed host this new section briefly describes in a few sentences the molecular mechanism by which specific viral proteins interfere with key cellular pathways, including hijacking of the host immune response, perturbation of the cell cycle, apoptosis or autophagy. key host virus interactions are clickable and link to the corresponding ontology pages for further details. although only around a dozen cellular pathways are commonly targeted by viruses, each of these pathways can be modulated in a variety of ways by different viruses. in viralzone, the host virus interaction tab provides information organized according to the main pathways that are most commonly hijacked. viral and host proteins involved are described and commented in details with links to publications. as an example, many vertebrate viruses contain information relating to the innate immune response inhibition in their host virus interaction tab. this section is also linked to the viral ontology pages, therefore users can switch between views that are specific to a given virus or mechanism. it has been estimated that 350 million people worldwide are chronic hbv carriers (20). the virus genome is only 3.2 kb long and encodes seven proteins (21). despite the apparent simplicity of hbv, the life cycle of this virus is complex. in partnership with hoffmann - la roche, an interactive hbv life cycle resource has been created in viralzone with 28 new pages linking to 180 publications from pubmed. the entry point to the hbv resource is an illustration depicting the virus replication cycle in a hepatocyte host. the cycle has been divided into 27 steps and molecular events that are each clickable and link to hbv specific description pages. the replication cycle pages describe the current knowledge on the topic with all major publications and some comments. many hbv pages are linked to the viralzone ontology section in such a way that users have access to knowledge that is specific to hbv or that concerns shared viral mechanisms. this activity of the swiss - prot group is supported by the swiss federal government through the federal office of education and science.	viralzone (http://viralzone.expasy.org) is a knowledge repository that allows users to learn about viruses including their virion structure, replication cycle and host virus interactions. the information is divided into viral fact sheets that describe virion shape, molecular biology and epidemiology for each viral genus, with links to the corresponding annotated proteomes of uniprotkb. each viral genus page contains detailed illustrations, text and pubmed references. this new update provides a linked view of viral molecular biology through 133 new viral ontology pages that describe common steps of viral replication cycles shared by several viral genera. this viral cell - cycle ontology is also represented in uniprotkb in the form of annotated keywords. in this way, users can navigate from the description of a replication - cycle event, to the viral genus concerned, and the associated uniprotkb protein records.
a 42-year - old male patient (height 170 cm, weight 63.7 kg) visited our pain clinic with complaint of severe right groin pain. two years earlier, the patient had been diagnosed with bilateral cam type fai and underwent arthroscopic surgery in the right hip after hip magnetic resonance imaging which showed fibrocystic change of the right femur and superior labral tear and minimal joint effusion on both hips. the recurrent pain began about 1 year prior to his visit and walking was impossible. the degree of pain was 10/10 on the visual analogue score system (vas, ranging from 0 = no pain to 10 = absolutely intolerable pain) and the oxford hip score (ohs, function of hip joint, excellent = below 19, good function = 19 - 26, fair = 27 - 33, poor = 33 or more) was 47/60. during the physical examination, the straight leg raising test (slr) was right 45 and left 80. the faber test and anterior impingement test (flexion and internal rotation of knee) were all positive at the right hip. the frog lateral view of the x - ray showed left superior labral calcification and an osteophyte at the right femur neck. in addition, the head - neck offset of the left femur was decreased (fig. the patient had no previous past medical history and took tramadol 200 mg, nsaids 200 mg and gabapentin 600 mg per day. written informed consent was received after sufficient explanation about the procedure and related complications. with the patient in the supine position, after insertion of a 22 gauge spinal needle, 0.75% ropivacaine 5 ml and triamcinolone 40 mg injection was achieved in the right hip joint (fig. the patient visited our clinic again checking his vas, ohs and satisfaction scores (5-point likert scale ; 5 = very satisfied, 4 = somewhat satisfied, 3 = neither satisfied nor dissatisfied, 2 = somewhat dissatisfied, 1 = very dissatisfied) at 2, 4, 8 and 12 weeks after injection (table 1). although moderate right hip pain remained, the patient could walk and return to work. a 59-year - old female patient (height 161 cm, weight 59 kg) the patient had been diagnosed with pincer type fai about two years prior to her visit. the hip ap (anteroposterior) view of the x - ray showed suspicious fai, with labral calcification and excessive coverage by the superior margin of both acetabula (fig. the pain site was in the lateral and subgluteal area of only the left hip joint. the degree of pain was 7/10 on the vas and the ohs was 26/60. during the physical examination, the slr was right 90 and left 90. faber test was positive on the left side. written informed consent was received after sufficient explanation of the procedure and related complications. with the patient in the supine position, the hip was internally rotated about 15 - 20. an ultrasound convex probe (2 - 5 mhz, micromaxxtm, sonosite, usa) was aligned with the long axis of the femoral neck, including the acetabulum and the femoral head. a 22 gauge spinal needle was then advanced under direct ultrasonographic guidance into the anterior synovial recess at the junction of the femoral head and neck (fig. 4b). injection of 0.75% ropivacaine 5 ml and triamcinolone 40 mg in the left hip joint was performed. the patient visited our clinic again checking her vas, ohs and satisfaction scores at 2, 4, 8 and 12 weeks after injection (table 1). the patient took her previous medications for rheumatoid arthritis during the 12 weeks and additional analgesics were not prescribed. three months after the injection, the pain in the trochanteric area was partially reduced. a 50-year - old male patient (height 175 cm, weight 73 kg) visited our pain clinic with complaint of pain in both hip joints. the patient had been prescribed nsaids and muscle relaxants at a local pain clinic whenever he felt pain. the pain in the right anterior groin area was more severe than that of the left side. because of the hip pain the degree of pain was 3/10 on the vas scale and the ohs was 21/60. during the physical examination, the slr was right 90 and left 90. the faber test and anterior impingement test were positive on both sides. the frog lateral view of the x - ray showed the possibility of mixed type fai of the right hip (fig. 2c). the head - neck offset of the right femur was decreased and there was excessive coverage by the superior margin of the right acetabulum. the alpha angle in the translateral view was 75.3. no abnormality was found in the ultrasound image. we performed c - arm fluoroscopy guided injection of 0.75% ropivacaine 5 ml and triamcinolone 40 mg in the right hip joint just like case 1. the patient visited our clinic again checking his vas, ohs and satisfaction scores at 2, 4, 8 and 12 weeks after injection (table 1). the patient took nsaids 200 mg during the first month after injection and thereafter, was discontinued. the patient could sit crossed - legged on the floor 2 weeks after the injection although mild right hip pain remained. a 42-year - old male patient (height 170 cm, weight 63.7 kg) visited our pain clinic with complaint of severe right groin pain. two years earlier, the patient had been diagnosed with bilateral cam type fai and underwent arthroscopic surgery in the right hip after hip magnetic resonance imaging which showed fibrocystic change of the right femur and superior labral tear and minimal joint effusion on both hips. the recurrent pain began about 1 year prior to his visit and walking was impossible. the degree of pain was 10/10 on the visual analogue score system (vas, ranging from 0 = no pain to 10 = absolutely intolerable pain) and the oxford hip score (ohs, function of hip joint, excellent = below 19, good function = 19 - 26, fair = 27 - 33, poor = 33 or more) was 47/60. during the physical examination, the straight leg raising test (slr) was right 45 and left 80. the faber test and anterior impingement test (flexion and internal rotation of knee) were all positive at the right hip. the frog lateral view of the x - ray showed left superior labral calcification and an osteophyte at the right femur neck. in addition, the head - neck offset of the left femur was decreased (fig. the patient had no previous past medical history and took tramadol 200 mg, nsaids 200 mg and gabapentin 600 mg per day. written informed consent was received after sufficient explanation about the procedure and related complications. with the patient in the supine position, after insertion of a 22 gauge spinal needle, 0.75% ropivacaine 5 ml and triamcinolone 40 mg injection was achieved in the right hip joint (fig. the patient visited our clinic again checking his vas, ohs and satisfaction scores (5-point likert scale ; 5 = very satisfied, 4 = somewhat satisfied, 3 = neither satisfied nor dissatisfied, 2 = somewhat dissatisfied, 1 = very dissatisfied) at 2, 4, 8 and 12 weeks after injection (table 1). although moderate right hip pain remained, the patient could walk and return to work. a 59-year - old female patient (height 161 cm, weight 59 kg) visited our pain clinic with left hip joint pain. the patient had been diagnosed with pincer type fai about two years prior to her visit. the hip ap (anteroposterior) view of the x - ray showed suspicious fai, with labral calcification and excessive coverage by the superior margin of both acetabula (fig. the pain site was in the lateral and subgluteal area of only the left hip joint. the degree of pain was 7/10 on the vas and the ohs was 26/60. during the physical examination, the slr was right 90 and left 90. faber test was positive on the left side. written informed consent was received after sufficient explanation of the procedure and related complications. with the patient in the supine position, the hip was internally rotated about 15 - 20. an ultrasound convex probe (2 - 5 mhz, micromaxxtm, sonosite, usa) was aligned with the long axis of the femoral neck, including the acetabulum and the femoral head. a 22 gauge spinal needle was then advanced under direct ultrasonographic guidance into the anterior synovial recess at the junction of the femoral head and neck (fig. 4b). injection of 0.75% ropivacaine 5 ml and triamcinolone 40 mg in the left hip joint was performed. the patient visited our clinic again checking her vas, ohs and satisfaction scores at 2, 4, 8 and 12 weeks after injection (table 1). the patient took her previous medications for rheumatoid arthritis during the 12 weeks and additional analgesics were not prescribed. three months after the injection, the pain in the trochanteric area was partially reduced. a 50-year - old male patient (height 175 cm, weight 73 kg) visited our pain clinic with complaint of pain in both hip joints. the patient had been prescribed nsaids and muscle relaxants at a local pain clinic whenever he felt pain. the pain in the right anterior groin area was more severe than that of the left side. because of the hip pain the degree of pain was 3/10 on the vas scale and the ohs was 21/60. during the physical examination, the slr was right 90 and left 90. the faber test and anterior impingement test were positive on both sides. the frog lateral view of the x - ray showed the possibility of mixed type fai of the right hip (fig. 2c). the head - neck offset of the right femur was decreased and there was excessive coverage by the superior margin of the right acetabulum. the alpha angle in the translateral view was 75.3. no abnormality was found in the ultrasound image. we performed c - arm fluoroscopy guided injection of 0.75% ropivacaine 5 ml and triamcinolone 40 mg in the right hip joint just like case 1. the patient visited our clinic again checking his vas, ohs and satisfaction scores at 2, 4, 8 and 12 weeks after injection (table 1). the patient took nsaids 200 mg during the first month after injection and thereafter, was discontinued. the patient could sit crossed - legged on the floor 2 weeks after the injection although mild right hip pain remained. in this case series report, the first case was a typical cam type in a young patient and the second case was a pincer type in a rheumatoid arthritis patient. three months after ia steroid injection, the patient in the first case complained of moderate pain although function of the hip joint was improved. on the other hand, the patient in the second case complained of partially decreased pain (change from vas 7 to 5) and improved hip joint function. the patient in the second case did not take additional analgesics after ia injection and had high satisfaction. the patient in the first case had advanced hip oa although the labral tear had been removed through arthroscopic surgery. therefore, the morphological abnormalities resulted in the recurrence of fai and impingement leading to an early degenerative change of the hip joint which could cause injuries to the labrum in young patients. early diagnosis of fai is important ; however, the risk factors of fai and the current prevalence rate in the general population are not well known. the pain begins insidiously with or without trauma and impingement occurs by flexion of the hip during exercises. the patient complains of sharp pain with flexion and internal rotation of the hip joint. although the pain depends on the location and size of the lesion, the pain is mainly located in the groin. limited terminal hip motion is a typical feature in fai patients and the pain can worsen while sitting for a long time or from climbing stairs. fai patients have a difficult time to squat and a snapping or clicking sensation is common. on the other hand, the physical exams induce impingement between the acetabular rim and femur head - neck junction. the anterior impingement test consists of passive hip flexion and internal rotation, and adduction causes deep anterior pain and decreased motion. the faber or patrick 's test is performed by flexing, abducting and externally rotating the tested leg. however, the positive results for those tests are not specific to fai ; abnormalities of intra - articular, psoas, or sacroiliac lesions can show positive results, too. ap pelvic, 45 dunn, and frog lateral radiographs could be used as diagnostic method for most fai patients without ct. the cam type in cases 1 and 3 showed a decreasing head - neck offset in the frog lateral view. the pincer type in case 2 showed excessive bony coverage by the acetabular rim in the hip ap view. additionally, plain radiography of the femoral head alpha angle for the cam type had higher sensitivity than that of ct. the alpha angle is the angle between two lines ; one line is up to the point of no sphericity of the femoral head from the center of the femoral head and the other line is up to the center of the femoral head from the center of the femoral neck at the narrowest point (fig. 3). if an alpha angle greater than 50 was measured, the camtype fai deformity was considered. the alpha angles in cases 1 and 3 in the translateral view were 78.2 and 75.3 (fig. first line treatment of femoroacetabular impingement is physiotherapy or anti - inflammatory therapy, but there have been many questions about their effectiveness. surgical treatment of the morphological changes of the femoral head and acetabulum is also done. ganz argued that a delay in surgical correction of symptomatic patients may lead to disease progression and that fai requires surgery. fai patients with early primary oa who received hip arthroscopic labrectomy had relative improvement in pain score and showed higher satisfaction. however, hip arthroscopic surgery of fai patients with severe oa showed relatively less improvement and only a temporary pain reduction. surgical dislocation was effective in patients with early degenerative changes but had no benefit in patients with advanced degenerative changes or extensive cartilage damage. on the other hand, intra - articular steroid injection as a treatment for oa is known to be effective. it is effective in reducing pain and synovial hypertrophy and could be a relatively safe treatment. therefore, we assumed that ia steroid injection for fai with secondary oa would be effective. ia steroid injection has an important role in the treatment of fai, which is an anti - inflammatory effect. this effect was explained such that patients with hip joint effusion show a better response to ia steroid injection. according to the american college of rheumatology (acr) recommendations for ia steroid injection, we chose a steroid (triamcinolone) dose of 40 mg mixed with local anesthetics. ohs is simple, has a high follow - up rate, and can be easily analyzed. it consists of twelve questions, and each question is composed of five scores from 1 (none) to 5 (extreme). the total score is calculated by summing each score and the minimum is 12 and the maximum is 60. the ohs scores for all 3 patients decreased meaning the patients were gradually getting a better quality of life. as a result, the two main problems of fai, disability and pain, were improved through ia steroid injection. the limitation of ia steroid injection is a low response to fai with severe oa. another author reported corticosteroid injections in fifty - two patients with symptomatic hip osteoarthritis were effective for 3 months. although it showed good efficacy in moderate and mild oa, it showed only a modest effect in severe oa. treatment for fai due to severe oa has low effectiveness for not only ia steroid injection but also for surgery. we think that the good responses to the ia steroid injections in these cases were associated with the severity of oa. the patient with severe oa (case 1) showed good functional improvement, but moderate pain remained after injection. whereas the patients with mild oa showed both good functional improvement (case 3) and pain relief (cases 2 and 3). therefore, fai, a leading cause of secondary hip oa in young adults, should be detected early to prevent disease progression. moreover, it is important to be aware of the symptoms and signs associated with fai. however, complications from ia steroid injection such as infectious arthritis, osteonecrosis and necrotizing fasciitis have been reported in previous studies. ia steroid injection in weight - bearing joints based on the recommendation of the acr should not be performed more than once per month or more than 4 times per year. in conclusion, ia steroid injection reduced the pain and improved function in the 3 cases with fai, and it could be an effective treatment for mild hip oa more than for severe hip oa. however, it is important to be aware of complications from ia steroid injection. a large number of studies have not been conducted and there are limited long - term studies for the treatment of fai. further research for novel treatments and evidence - based algorithm for fai should be conducted in the future.	femoroacetabular impingement (fai) arises from morphological abnormalities between the proximal femur and acetabulum. impingement caused by these morphologic abnormalities induces early degenerative changes in the hip joint. furthermore, fai patients complain of severe pain and limited range of motion in the hip, but a guideline for treatment of fai has not yet been established. medication, supportive physical treatment and surgical procedures have been used in the treatment of the fai patients ; however, the efficacies of these treatments have been limited. here, we report the diagnosis and treatment for 3 cases of fai patients. intra - articular (ia) steroid injection of the hip joint was performed in all three patients. after ia injection, pain was reduced and function had improved for up to three months.
amplification of human epidermal growth factor receptor 2 (erbb2 or her2) occurs in approximately 20 % of breast cancers and is associated with poor prognosis. trastuzumab, a monoclonal antibody toward the extracellular domain of her2 receptor, combined with chemotherapy, increases time to progression and overall survival in advanced breast cancer patients. lapatinib, an orally available small molecule reversible tyrosine kinase inhibitor and an in vitro and in vivo potent selective dual inhibitor of erbb1 (egfr) and her2 receptor, has been approved since 2007 in combination with capecitabine for treatment of metastatic breast cancer overexpressing her2 and previously treated with anthracycline, taxane and trastuzumab. lapatinib inhibits erbb1 and her2 intracellular kinase domains ; it is known to be active in erbb1 mutants and truncated forms of her2 receptor (p95), and can overcome the resistance to trastuzumab (blackwell. 2009 ; burstein. 2008 ; gomez. 2008 ; iwata. the association of lapatinib and capecitabine was evaluated in a phase iii randomized trial (geyer. 2010), showing the efficacy of the combination treatment after trastuzumab failure, and the superiority over capecitabine alone in anthracycline and taxane pretreated advanced breast cancer patients. among the most common adverse events observed in the registrative trial was diarrhea, representing the main limiting toxicity, occurring in more than a half of the patients in the combination arm (60 %), and partially reducing treatment compliance. moreover, it was the most serious g34 adverse event [together with hand - foot syndrome, (hfs) ], occurring in 12 % (g3) and 1 % (g4) of the patients, respectively (geyer. 2006). in the expanded access program (leap), diarrhea was the most frequently reported drug - related serious adverse event (9.7 %) (capri. other clinical studies reported similar results, confirming diarrhea as the most common side effect occurring with lapatinib plus capecitabine regimen, requiring drug dose modification or treatment interruption in some cases (crown. the frequency and severity of diarrhea, along with the not unusual long duration of the toxic effect, may limit full dosing and optimal treatment duration, possibly having an impact on treatment efficacy. in clinical practice, outside of clinical studies, diarrhea in relation to lapatinib plus capecitabine treatment is a well - known side effect and, even if often of low grade, it is a common knowledge how it may lead to a reduced treatment compliance and a lower quality of life in treated patients. treatment guidelines for the management of lapatinib - associated toxicities (primarily diarrhea) are available (crown. 2008 ; benson. 2004 ; moy and goss 2007), and clinicians are now more capable of managing this toxic adverse event effectively in clinical practice, but diarrhea still represents an important limitation to the optimal regimen delivery in many patients. in order to reduce the incidence and severity of the frequent gastrointestinal toxicity observed in patients treated with conventional schedule of lapatinib capecitabine regimen, and to increase treatment compliance, this consisted in administering capecitabine from day 11 instead of day 1 for the first cycle, then in subsequent cycles from day 8, and permanently dividing the planned capecitabine dose in three daily doses as a chronomodulated schedule as suggested by santini. moreover, lapatinib was dissolved in water, and cholestyramine was administered twice a day. we recruited patients from nine italian cancer centers, all treated with the above described modified administration schedule. our analysis comprises of her2-positive advanced or metastatic breast cancer patient candidates for treatment with lapatinib capecitabine. the her2 status was considered positive if the local institution reported grade 3 + staining intensity (on a scale of 03) by immunohistochemical analysis, or grade 2 + staining intensity with gene amplification on fluorescence or chromogenic in situ hybridization. patients recruited had an eastern cooperative oncology group (ecog) performance status of 2 or less, a life expectancy of at least 12 weeks, a left ventricular ejection fraction (lvef) within the institution s normal ranges, and adequate organs and hematological functions. the treatment schedule consisted of lapatinib, at a dose of 1,250 mg daily, 1 h before or after breakfast, administered as single agent for the first 10 days, then continuously, in combination with capecitabine, which was given at a dose of 2,000 mg / m, starting on day 11 (for the first cycle), and then from day 8, for 14 days out of a 21-day cycle, and with a chronomodulated schedule (25 % of the dose at 8.00 a.m., 25 % at 12.00 a.m., and 50 % at 22.00 p.m.). according to the third amendment of allto trial, lapatinib was always dissolved in water ; furthermore, cholestyramine was administered, twice a day on a continuous basis, long after capecitabine and lapatinib intake. standard efficacy and toxicity evaluations were performed in all the patients treated, paying special attention to diarrhea incidence and severity. the number and duration of diarrhea episodes were reported by patients in a personal diary. standard recommendations for capecitabine and lapatinib dosage modifications were followed for the management of adverse events. the primary endpoint of the analysis was the evaluation of the tolerability of treatment in terms of diarrhea g 2 incidence and of the patients compliance ; secondary end points were treatment efficacy evaluation, in terms of overall response rate (orr), clinical benefit (cb, responses and stable disease for at least 6 months), response duration, progression - free survival (pfs), and overall survival (os). the association between variables was tested by the pearson s chi - square test or fisher s exact test, when appropriate. meier product - limit method from the treatment starting date until the time of death (os), progression (pfs), or last visit (os and pfs), whichever applicable. the association between variables was tested by the pearson s chi - square test or fisher s exact test, when appropriate. meier product - limit method from the treatment starting date until the time of death (os), progression (pfs), or last visit (os and pfs), whichever applicable. from november 2010 to december 2012, 38 her2-overexpressing advanced or metastatic breast cancer patients were treated with the modified schedule of lapatinib capecitabine regimen in 9 italian cancer centers. twenty - one patients received adjuvant chemotherapy, in 13 patients combined or followed by adjuvant trastuzumab ; the remaining 8 patients received chemotherapy without trastuzumab as adjuvant treatment. ten patients received neoadjuvant treatment, in 9 patients including trastuzumab (one patient chemotherapy only). eight patients received adjuvant endocrine treatment for 5 years, concomitantly to trastuzumab for 6 months ; 15 patients received endocrine treatment in combination with trastuzumab for advanced disease. in regard to chemotherapy, the median number of previous chemotherapy lines was 1 (76.3 %), ranging from 1 to 4 overall. all of the patients had previously been treated with trastuzumab, ranging from 1 to 4 previous lines, including neoadjuvant and adjuvant setting, but most of the patients (78.9 %) had received only one prior chemotherapy regimen containing trastuzumab for advanced disease. the median number of cycles of lapatinib capecitabine modified schedule delivered was 7 (range 221). ten women (26.3 %) received more than 10 cycles of therapy. table 1patient characteristics (n = 38)characteristics n (%) median age, years (range)55 (4184)ecog ps, median (range)1 (01)er / pr status, n (%) er+ and/or pr+15 (39 %) prior chemotherapy regimens, median (range)1 (14)prior trastuzumab therapy, n (%) neoadjuvant9 (23.6 %) adjuvant13 (34.2 %) metastatic38 (100 %) number of prior trastuzumab lines, median (range)1 (14)prior adjuvant hormonal therapy, n (%) 8 (21 %) prior hormonal therapy for advanced disease, n (%) 15 (39 %) cycles of lapatinib and capecitabine delivered, median (range)7 (221) patient characteristics (n = 38) overall, diarrhea events were low grade, neither requiring lapatinib nor capecitabine dose modification nor interruption. none of the patients discontinued or reduced lapatinib dose for any reason when lapatinib was given as single agent in the first 10 days of treatment. overall, in 312/330 (94.5 %) cycles administered and in 28/38 patients, no treatment - related diarrhea was observed ; 5 patients (13.2 %) experienced g1 diarrhea (11/330 cycles, 3.3 %), and 4 patients (10.5 %) experienced g2 diarrhea (6/330 cycles, 1.8 %), while g3 diarrhea was observed in only 1 (2.6 %) patient in the second cycle (1/330 cycles, 0.3 %). overall, the incidence of g2 or more severe diarrhea in our analysis was 13.2 %. no capecitabine or lapatinib diarrhea - related discontinuation was observed, and in the only one patient experiencing a single episode of g3 diarrhea, a 25 % capecitabine dose - reduction was performed for 2 weeks, rapidly improving the symptom. in regard to other toxicities, cutaneous toxicity was the only side effect leading to dose reduction and/or treatment discontinuation (table 3). three patients had to reduce capecitabine dose use due to hfs, after 7, 9, and 10 cycles, respectively, and in 1 patient, capecitabine was definitively discontinued due to g3 hfs, while lapatinib was continued as monotherapy until disease progression. other relevant toxicities observed were rash and ungueal alterations, with no cases of rash g3/g4, 2 (5.3 %) patients experiencing transient g1 rash, and 7 (18.4 %) patients g2 rash, after more than 8 cycles, with only 1 patient discontinuing capecitabine for 2 weeks, with a prompt resolution of the symptom. ungueal alterations, usually mild, occurred in 6 patients, and were g1 in 1 patient and g2 in 5 patients. table 2diarrhea incidence and severitygrade n (%) 15 (13.2 %) 24 (10.5 %) 31 (2.6 %) 4table 3cutaneous toxicity in 38 patients (%) toxicitiesg1g2g3g4rash2 (5.3 %) 7 (18.4 %) hand - foot syndrome (hfs)1 (2.6 %) 3 (7.9 %) 1 (2.6 %) ungueal alterations1 (2.6 %) 5 (13.1 %) diarrhea incidence and severity cutaneous toxicity in 38 patients (%) as treatment efficacy concern (table 4), 2 patients (5.3 %) achieved a complete response (cr) and 11 patients (28.9 %) a partial response (pr), for an orr of 34.2 % (95 % ci 19.149.3) ; 20 patients (52.6 %) had stable disease (sd) and 5 patients (13.2 %) progressive disease (pd). the median duration of response was 16 months (95 % ci 421). the median pfs was 10 months (95 % ci 316), and 1-year pfs was 45.0 % (fig. after a median follow - up of 10 months, (range 320), 1-year os was 71.2 %, with 17 (44.7 %) patients surviving more than 12 months (fig. table 4summary of clinical efficacypatients (n = 38)orr cr or pr confirmed, % (95 % ci)34.2 % (19.149.3)cbr cr or pr or sd 24 weeks, % (95 % ci)55.3 % overall pfs progressions n (%) 31 (81.5) censored n (%) 7 (18.5) median pfs, months, % (95 % ci)10 (316) 1-year pfs, % 451-year overall survival, % 71.2 orr overall response rate, cr complete response, pr partial response, cbr clinical benefit rate, pfs progression - free survival patients who did not die or progress until the clinical cutoff for these data (may 31, 2013)fig. 1 a 1-year progression - free survival (pfs). b 1-year overall survival (os) summary of clinical efficacy orr overall response rate, cr complete response, pr partial response, cbr clinical benefit rate, pfs progression - free survival patients who did not die or progress until the clinical cutoff for these data (may 31, 2013) a 1-year progression - free survival (pfs). despite the existence of treatment guidelines for the management of lapatinib capecitabine - associated diarrhea, it still represents a significant limitation in the optimal regimen administration in many patients. this frequently has a negative impact on patients quality of life, where the dose reduction, interruption, and discontinuation of treatment may negatively influence the efficacy in daily clinical practice. in the present analysis, we intended to assess whether a change in the timing and mode of administration of lapatinib capecitabine regimen, in combination with support medication, could lead to a reduction in treatment - related main toxicities, with a particular focus on diarrhea. our results satisfy a post hoc statistical hypothesis according to ahern exact single - stage phase ii design (2001), considering a percentage of diarrhea of grade 2 higher than 35 % (10) unacceptable, and assuming levels 15 %, with a significance of 5 % and a power of 90 %, interesting. although lapatinib provides a new treatment option for the management of her2 positive breast cancer patients, and the combination with capecitabine represents an active and consolidated treatment choice in trastuzumab - resistant disease, clinicians and patients still face a number of clinical challenges, including minimizing toxicity. in fact, toxic effects of cancer treatments are one of the main limitations among planned dose - intensity maintenance, as well as having a relevant impact on costs, and on quality of life, which is of paramount importance in the advanced stages of the disease, where a satisfactory quality of life represents one of the major goals of the treatments. moreover, patient non - adherence to oral antineoplastic therapy, especially if not well tolerated, is a well - known barrier to treatment effect. the search for alternative drug administration schedules that allow to reduce the toxic effects of standard therapy is a major challenge to make the patients to be able to assume adequately therapy, and to improve their quality of life. gastrointestinal side effects, mainly diarrhea of any grade, are described in the literature data in more than a half of the patients with lapatinib as single agent (crown. 2008). the data from the trial of gomez show, for lapatinib 1,500 mg daily as monotherapy, an incidence of diarrhea of 46 %, usually mild, being of grade 3 only in 1 % of patients (2008). in the allto adjuvant trial, diarrhea of any grade was reported in 61 % of patients, being of grade 34 in 6 % of patients in the lapatinib arm (goss. 2013). in regard to the use of lapatinib in combination with antineoplastic agents other than capecitabine, phase iii trials in neoadjuvant setting showed a higher incidence of treatment interruptions in the arms containing lapatinib, mainly due to diarrhea. the neoaltto trial reports 21 % of grade 3 diarrhea in the lapatinib arm, whereas in the cherlob trial, grade 34 diarrhea was recorded in 37 % of patients (in association with chemotherapy) (baselga. neoadjuvant trial, diarrhea of any grade was observed in 75 % of the patients, being of grade 34 in 11.7 % of the patients (untch. the combination of lapatinib and capecitabine in the phase iii registrative trial determined grade 3 diarrhea in 12 % of patients, with 1 % of patients experiencing grade 4 diarrhea, while mild (grade 12) diarrhea occurred in 47 % of patients. these effects led to treatment discontinuation in 3 % of patients, while delays or dose reductions have been reported in 11 % and 5 % of patients, respectively (geyer. phase i ii iii trials evaluating diarrhea, in which lapatinib was administered at doses ranging from 1,000 to 1,500 mg daily as a single agent or in combination with capecitabine or taxanes, diarrhea occurred in 54 % of lapatinib - treated patients and in 24 % of patients not receiving lapatinib. in more detail, diarrhea of any grade was reported in 51 % of the patients treated with lapatinib as a single agent, in 65 % of patients treated with lapatinib plus capecitabine, and in 48 % of patients treated with lapatinib plus a taxane. the symptom was usually mild to moderate, being of grade 3 in less than 10 % of patients, and of grade 4 in less than 1 % of patients. moreover, grade 3 diarrhea in 1017 % of the patients treated with 1,500 mg once daily was reported, whereas this percentage increased to 21 % in patients treated with 750 mg bid, suggesting an association with dose and schedule. in regard to timing of symptom development, approximately 40 % of patients developed diarrhea within the first 6 days of treatment. in addition, each diarrhea episode lasted a mean of 79 days, and most (81 % in the lapatinib capecitabine group) diarrhea events were resolved without dose - modification and with conventional approaches, even if 16 % of patients had to reduce doses or delay treatment, with 3 % discontinuing treatment (crown. capecitabine regimen in advanced breast cancer, other sporadic experiences with treatment schedule modification were carried out. a recently published report on lapatinib capecitabine schedule modification, consisting of a 7 day capecitabine intake followed by a 7 day rest, reported satisfactory activity and an improvement in gastrointestinal toxicity, with no cases experiencing g34 diarrhea, with g2 diarrhea occurring in 26 % of patients (gajria. 2012). in our experience, the difficulty in successfully managing of diarrhea often limiting patients compliance in terms of treatment adherence and a decreased quality of life has been addressed by delaying the administration of capecitabine in the first cycle of treatment, which was introduced in a chronomodulated manner when lapatinib was administered as a single agent after a 10-day time frame. at the same time, lapatinib was dissolved in water, and cholestyramine was given after lapatinib consumption to counter possible drug interference. in fact, we observed a very high adherence to therapy administered, with a median of 7 cycles delivered and 10 patients (26.3 %) receiving more than 10 cycles, observing a particularly low incidence of grade 2 diarrhea (10.5 %), with only one episode of grade 3, and no episodes of grade 4 diarrhea. the incidence of diarrhea of grade 2 in the present experience (13.2 %) compares favorably with conventional lapatinib plus capecitabine regimens in the literature data, reporting an incidence of approximately 33 % (geyer. moreover, the efficacy of lapatinib capecitabine treatment observed in our patient population was very encouraging, with an orr of 34.2 %, a cb of 55.3 %, a median duration of response of 16 months, and a median pfs of 10 months. these compare favorably with other results of lapatinib capecitabine regimens administered with standard schedules, reporting response rates ranging from 22 to 30.8 %, a median response duration of about 6.5 months, and a median pfs ranging from 5.0 to 8.4 months (geyer. moreover, in our experience, treatment was safely tolerated for a long period in many patients, confirming its optimal tolerability. whether this consistent improvement in tolerability and the related toxicities increased patients compliance may have favorably influenced regimen efficacy is difficult to demonstrate, but the characteristics of patients in the present analysis do not differ from those enrolled in other published trials, and the possible effect on treatment efficacy can not be excluded either. on the whole, even taking into consideration the limitations of a post hoc analysis of a subgroup of patients treated across different cancer centers, our experience with the above reported modified schedule of lapatinib capecitabine regimen in her2 positive advanced breast cancer patients can be considered a very satisfactory experience, with encouraging treatment efficacy results and, above all, an optimal quality of life in the treated patients. our experience of applying the modified schedule of lapatinib capecitabine regimen led to a significant reduction in the incidence and severity of treatment - related diarrhea, allowing optimal delivery of chemotherapy with encouraging efficacy data, while maintaining a good quality of life in the majority of patients treated.	purposediarrhea in relation to the lapatinib capecitabine regimen is a common and debilitating side effect which may interfere with optimal treatment delivery. we performed a post hoc analysis in human epidermal growth factor receptor 2-positive advanced breast cancer patients treated with a modified schedule in its administration, aimed primarily to evaluate grade (g) 2 diarrhea incidence and, secondarily, treatment efficacy.patients and methodstreatment schedule consisted of lapatinib 1,250 mg daily for the first 10 days, then in combination with capecitabine, 2,000 mg / m2, starting day 11 for the first cycle, and thereafter from day 8, for 14 days of a 21-day cycle, in 3 daily administrations. lapatinib was dissolved in water, and cholestyramine was continuously given twice a day.resultsamong 38 patients treated and analyzed, the incidence of g 2 diarrhea was 13.2 %. in 28 patients diarrhea was not observed, while g12 diarrhea was reported in 9 (23.7 %) patients ; a single episode of g3 diarrhea was observed in 1 (2.6 %) patient. overall response rate was 34.2 %, clinical benefit 55.3 %, and median progression - free survival 10 months.conclusionthe results of the present post hoc analysis are very encouraging, both in terms of tolerability and treatment efficacy, and all data compare favorably with previous reports of conventional administration of the lapatinib capecitabine regimen.
despite the fact that the relative burden of chronic obstructive pulmonary disease (copd) in women is high in both the developing and developed countries, the occurrence of copd in women is not widely appreciated. the historical perception that copd is principally a disease occurring in men has been challenged by the fact that, copd - related events including the prevalence, mortality and number of emergency hospital admissions have been swiftly increasing among women over the past two decades.12 the recent data suggests that copd causes more than 3 million deaths every year, making it the 4 leading cause of death in the world.3 it has been estimated that by the year 2030, copd will become the third major cause of death. in the south east asian region, as per the world health organization estimations, copd is responsible for more deaths than that of people with hiv - aids, malaria and tuberculosis all put together. the more disturbing fact that needs to be addressed, is that the death rates due to copd are expected to surge by over 160% in the next two decades.4 half a million people die every year due to copd in india, which is over four times the number of people who die due to copd in the usa and europe.5 this area also represents a large population of women. there is a wide discrepancy in the prevalence of copd across different studies, ranging 1.2%-19% in women in our country.6 these studies have used respiratory health questionnaires, which have not been validated for the diagnosis of copd. it is worth mentioning that all studies conducted thus far did not use a spirometer as diagnostic tool, and therefore, the prevalence of copd in indian women has been grossly underestimated. since the prevalence of smoking in indian women is negligible, health care providers are more likely to misdiagnose them as showing bronchial asthma or attribute their symptoms to cardiac diseases and/or age related issues. tobacco smoking is the most common and best - known cause of copd, but it is not the only cause. the wealth of knowledge available from various studies suggest that 30% of copd patients have never smoked and that 80% of these non - smokers are women. there is limited knowledge of the risk factors associated with spirometrically confirmed copd in non - smokers in the general population as well as in women.7 it has been demonstrated that the exposure to high levels of indoor air pollution due to the burning of biomass fuels such as animal dung, crop residues, or wood substantially enhances the risk of copd relative to smoking tobacco.7 in india, around 60 - 70 % of the population resides in rural areas. about 80% of rural homes in india still continue to use biomass fuel as their primary cooking and heating energy source. another factor that describes its high usage rate among these residents is its easy availability. almost all the kitchens in these dwellings are unventilated without any modern protective amenities. in indian rural settings, as women in the early stages of copd are relatively less symptomatic, they tend to ignore and do not bring it to the notice of healthcare provider. nevertheless, even if they do complain, measurements of airflow limitation by spirometer that is essential for the diagnosis are often not accessible.8910 early detection of airflow limitation is crucial as many women, by the time they have been diagnosed as copd, would be suffering from advanced symptoms of the disease. hence we undertook this study to find the true prevalence of copd in women exposed to biomass fuel using sa pirometer in both rural and urban dwellings. the study also aimed to examine the determinants of under - diagnosis of copd in these participants. this is a cross sectional study conducted at a tertiary care hospital with 3 rural and 3 urban health centers attached to it under a govt. of india 's public - private partnership that ranged between november 2013 and november 2014. women aged 40 years and above with a history of exposure to biomass fuel for more than 10 years were enrolled in the study after obtaining informed consent. biomass fuel exposure was defined as a lifetime exposure of 10 or more years from the use of indoor fire using (1) coal or coke ; (2) wood, crop residues or dung as the primary means of cooking or heating. subjects with cardiac illness, active or past history of pulmonary tuberculosis, hiv infections, chest wall deformities, other chronic pulmonary diseases like ild, bronchiectasis, bronchial asthma etc and both current and ex smokers were excluded from the study. all women attending six health centers and tertiary care hospital were first screened for the inclusion criteria. women who met the criteria of 10 or more years of exposure to biomass fuel as defined above were enrolled in the study. then they were asked what was the source of energy and type of stove they used for cooking and heating. detailed inquiry for causes of delayed diagnosis including the availability of spirometry were done and recorded. previous prescriptions and investigations along with treating doctor 's advice was noted. a thorough assessment of chest radiographs and medications used by the women currently or previously was carried out. baseline data was documented which included level of education, socioeconomic status, age, years of exposure to biomass fuel, year at which the exposure started, bmi and respiratory symptoms. the body mass index (bmi) was calculated as the weight in kg divided by height in m. then these subjects underwent screening for copd. copd definition derived from the global initiative for chronic obstructive lung disease (gold) based on postbronchodilator fev1/fvc 25 years, p 40 - 50 years.22 this chronic insult, beginning in childhood, may act as an early stimulus for airway damage and impaired pulmonary functions. our study highlights observations of some studies that suggest increased lower respiratory tract infection during childhood due to exposure to biofuels is a risk factor for developing chronic airway obstruction in adulthood.23 our results indicate that copd underdiagnosis was frequent in women in the study cohorts. determinants of copd under - diagnosis in our study cohorts were younger, never having smoked, had a lower level of education, an absence of reported symptoms, an unavailability of spirometry, a lack of knowledge of hazards of biomass fuel and a milder severity of airflow limitation. in india, smoking among women furthermore, the age - old perception that copd is principally a disease of men who smoke tobacco still remains embeded in the minds of the health care providers. spirometry, which is the gold standard diagnostic test, remains poorly utilized in clinical practice. clinicians are not aware that relying on history and clinical examinations only to diagnose copd under - estimates the disease in over 50% of cases.24 clinicians need to accept the fact that spirometry is the most important diagnostic test for copd. women should be made aware of the consequences of biomass fuel both by clinicians and environmental experts to a larger extent. women should be educated about use of " clean " fuel (e.g., liquid propane gas or natural gas) or electricity. other interventions such as outdoor relocation of cooking using biomass fuel, addition of windows to the kitchen and using improved cook stoves with better ventilation systems such as a chimney can be implemented. 25 these preventive measures with early diagnosis of copd in these subjects which could lead to improvement in the quality of life, reduce their symptoms, prevent exacerbations and hospitalizations and even improve survival. to our knowledge, it is the first and largest assessment of copd in women exposed to biomass fuel in india using spirometry, unlike many questionnaire - based evaluations. both spirometry and bronchodilator testing were conducted with similar standard protocols, which was likely to reduce bias due to methodological issues. availability of thorough exposure details of all subjects made it possible to demonstrate a clear relationship between biomass fuel and copd risk in a larger number of subjects representing both rural and urban houses. mainly, it is single center study and women who were current or ex - smokers remained excluded from the study even though some of these smokers had concomitant exposure to biomass fuel as well., this will not affect the outcome of the study, as the primary objective of the trial was to assess the impact of biomass fuel on copd diagnosis in non - tobacco smoking women. there is strong correlation between the risk of copd and duration of exposure along with the age at which the exposure to biomass fuel begins. under - diagnosis of copd was frequent in women due to the unavailability of spirometry, a lack knowledge of hazards of biomass fuel, the low level of education of the subjects, and the ignorance of the health care providers being the important determinants of under - diagnosis.	background and objectivestobacco smoking has been established as a major risk factor for chronic obstructive pulmonary disease (copd) in women of developing countries, but emerging evidence suggests that biomass fuel is an important risk factor as well. the primary objective of the study was to find the true prevalence of copd in indian women exposed to biomass fuel using spirometry. we also aimed to find the determinants of underdiagnosis of copd in these participants.methodswomen with a history of exposure to biomass fuel for > 10 years were screened for copd using spirometry following all standard protocols as per gold / ats / ers definitions.resultsof the 2868 women screened, a total of 529 (18.4%) women were confirmed to have copd in which 123 (4.2%) were " women with known copd " and 406 (14.2%) " women with new copd ". the mean age at the time of diagnosis was 615.2 and 473.6 respectively. the duration of exposure to biomass fuel had a great impact on the risk of copd with or 1.2, 95% ci (1.1 - 1.9) for patients with 10 - 15 years exposure and or 2.9, 95% ci (2.5 - 3.1) for exposure > 25 years, p<0.001.conclusionthe prevalence of copd among women exposed to biomass fuel is very high. a strong correlation was found between the risk of copd and the duration of exposure along with the age at which the exposure to biomass fuel begins. underdiagnosis of copd was frequent in women due to the lack of the availability of spirometry, lack knowledge of hazards of biomass fuel, a low level of education and the ignorance of the health care provider being the important determinants of underdiagnosis.
menopause is one of the most critical periods in women 's life. although being a natural biological process that occurs with aging, physiological alterations observed during this period can be challenging. caused by a reduced secretion of ovarian hormones estrogen and progesterone after depletion of the storage of ovarian follicles, menopause defines the end of women menstrual cycle and their natural fertility. on average, spontaneous or natural menopause occurs around the early 50s and is confirmed after 12 months of nonpathological amenorrhoea. however, when premature ovarian failure (pof) occurs before the 40s due to pathological causes, an early or premature menopause can be induced, which is thus disconnected from the aging process properly said. when a bilateral oophorectomy is necessary, menopause occurs immediately without women experiencing the gradual transition of perimenopause. women who experience an early menopause are more susceptible to certain health problems, such as osteoporosis and heart diseases, since they spend more time in their lives without the benefits of estrogens. pof can also be temporary (temporary menopause) induced by high levels of stress, excessive exercising and/or dieting, and by medications used to treat fibroids and endometriosis. however, as soon as women adopt a healthier life style or stop medication, the ovaries may resume normal production of hormones. normally, menopausal transition or perimenopause starts around mid - to - late 40s and persists several years before the last menstrual period, normally for 4 - 5 years (figure 1). smoking and genetic background are two factors that can influence the timing of spontaneous menopause. levels of estrogen and progesterone start gradually to decline and menstrual periods become irregular. since sex hormones are physiologically important to maintain the health and normal functioning of several organs, such as the heart, liver, brain, and bone, hormonal changes observed during this menopausal transition may induce several chronic medical conditions. the variation of menopause phenotypes around the world and in different ethnic groups suggests both cultural and genetic influences [6, 7 ]. menstrual irregularities, vaginal atrophy, and vasomotor instability are the most frequent menopausal symptoms that have been directly related with the decreased levels of female sex hormones. menopause - associated vasomotor symptoms (also known as hot flashes) include spontaneous feeling of warmth, usually on face, neck, and chest and are usually associated with perspiration, palpitations, and anxiety, being variable in frequency, duration, and severity, and can be the cause for fatigue, difficulty concentrating, and memory lapses, symptoms that have also been observed during menopause transition. the cause for menopause - associated vasomotor symptoms is not completely understood, although some theories have been proposed [8, 9 ]. vaginal atrophy is also a common symptom during menopause transition. due to loss of estrogens, vagina lining may become thinner and dryer, and the ph also changes, making the vagina more susceptible to infections. others menopause - associated complications include increased cardiovascular risk (see below), osteoporosis and body weight gain, which can all be a combination of changes in hormone levels and aging. although it is known that the metabolic rate decreases with aging, the increase in body weight and visceral adipose tissue accumulation after menopause have been associated with ovarian hormone withdrawal. it has been shown that, in abdominal adipocytes, estrogen regulates the expression of lipoprotein lipase (lpl) and hormone - sensitive lipase (hsl). in hepatocytes, estrogen regulates the synthesis of structural apolipoproteins for very low - density lipoproteins (vldls) and high - density lipoproteins (hdls) and decreases the synthesis of hepatic lipases. by regulating lipidogenesis in adipocytes and hepatocytes, estrogen modulates lipid concentration in plasma. the withdrawal of estrogens during induced or natural menopause leads to several lipid metabolism disorders. abdominal accumulation of adipose tissue and associated dyslipidemia are important components of a group of metabolic irregularities strongly related with increased cardiovascular risk in the menopausal woman. cardiovascular disease (cvd) is a multifactorial disease. both bad lifestyle including inappropriate diet, sedentary life, smoking and drinking, and determined factors (e.g., aging, sex, genotype, and menopause) influence cvd [16, 17 ]. the impact of cvd on overall mortality in westernized countries is enormous, accounting for up to 30% of all deaths worldwide. the definition of cvd includes four major groups of diseases : coronary heart disease (chd) disclosed by angina pectoris, myocardial infarction, heart failure, and coronary death, cerebrovascular disease such as stroke or transient ischemic attack, clinically evident peripheral artery disease, aortic atherosclerosis, and thoracic or abdominal aortic aneurysm. what is less known is that cvd is the leading cause of death in women, with more deaths than all other causes combined yearly. various studies showed a growing risk for cvd in menopausal women due to negative changes in metabolism and hemodynamic parameters. according to the guidelines of the national cholesterol education program (ncep), the american heart association (aha), and the american college of cardiology (acc) [18, 20 ], evaluation of cvd risk factors in women must include a personal chd history, age over 55, family history of premature chd, diabetes mellitus, dyslipidemia, hypertension, personal history of peripheral artery disease, and smoking. guidelines for prevention of cvd in women were first published in 1999 by the american heart association (aha). one consequence of such increased attention to gender - related health problems, is awareness of cvd as the leading cause of death among women has nearly doubled since 1997. the impact of menopause should be taken into account when discussing cvd, and this aspect has been the matter of debate. premenopausal women have a lower incidence of cvd when compared to men with the same age - range. whereas chd is sporadic in premenopausal women, the incidence of myocardial infarction increases with age in both sexes, but occurs later and after menopause. estrogen loss during menopause causes negative effects on metabolism and cardiovascular function, and the progression to menopause with the changes in estrogen levels decreases or cancels the women advantage versus men [2629 ]. postmenopausal women have a higher risk of coronary artery disease, atherosclerosis, and all causes of mortality. a consequence of this gender - related trend is that the postmenopausal state is acknowledged as a risk factor for chd, with a weight similar to that of male sex. furthermore, an early natural menopause appears to be associated with increased risk of cvd [31, 32 ], even in non - smokers. indeed, menopause is associated with increased total serum cholesterol, triglycerides, and fibrinogen, as well as with a decrease in high - density lipoprotein (hdl) cholesterol. a plausible explanation is that menopause is believed to be a result of fluctuations in hormonal status, primarily a deficiency in estrogen. whether other contributing factors may have a role on cvd after menopause, is less clear and difficult to demonstrate. the transition from premenopausal phase to menopause, for example, may induce a weight gain responsible for increased in blood pressure, total cholesterol, low - density lipoprotein (ldl), triglycerides, and fasting insulin. what should be mentioned is that aging per se can be more important than menopause itself for a number of chd risk factors. in the swan study (study of women 's health across the nation), changes in traditional risk markers of chd were evaluated in three different stages : before, within a year, and after the final menstrual period within a multiethnic group (african, american, hispanic, japanese, or chinese and caucasian women). changes due to menopause were only represented by total cholesterol, low - density lipoprotein cholesterol, and apolipoprotein b. by contrast, chronological aging was responsible for changes in the other risk factors with a linear model. many other potential factors might be also implicated in the sex differences in coronary heart disease. the possibility that heart disease risk determines menopausal age rather than the inverse has already been proposed. oxidative stress plays a role in hypertension, hypercholesterolemia, diabetes, and promoting cvd. the formation of free radicals leads to cellular oxidative stress with a contribution to the first step of endothelial damage and the progression to atherosclerotic lesion. the perpetuation of the process induces the final events of cvd, which appears to be linked to some oxidative stress biomarkers [38, 39 ]. oxidative stress appears to be an emerging factor also in the pathophysiology of cvd in menopausal women. studies have shown that during menopause the risk of cvd increases at the same time of a rise in oxidative status [40, 41 ]. it is still unclear if the type of menopause (surgical or natural) can have a role on cardiovascular risk. the nurses ' health study (1987) demonstrated that the risk of chd was higher in patients undergoing bilateral oophorectomy compared with natural menopause. carotid artery intima - media thickness showed a positively association with years elapsed since menopause ; however, according to this marker of subclinical atherosclerosis, women with natural menopause presented no difference compared with those who had surgical menopause. indeed, men with the common estrogen receptor alpha (esr1) c.454 - 397cc genotype have a major risk of myocardial infarction, suggesting the potential linkage between estrogen receptors and cvd susceptibility. in this respect, a variation in estrogen receptor could clarify the contrasting results of hormone therapy on cvd susceptibility in women. the apparent protective effect of hormone replacement therapy (hrt) has been a matter of debate for several years [4547 ]. prevention of chd and osteoporosis in menopausal women was originally achieved by exogenous estrogen plus progestin, assuming a protective effect of estrogen on the heart. additional effects included a protective effect on the bone and on colon cancer [4852 ], despite increasing incidence of breast cancer [53, 54 ]. the women 's health initiative (whi) estrogen plus progestin (e+p) trial in 2002 showed no protection for chd and confirmed the increased risk in breast cancer and thromboembolic disease. two years later the whi estrogen alone trial confirmed the lack of effect on chd while suggesting a trend for decreased breast cancer, with a rise in stroke and venous thromboembolic disease. a nonsignificant protective effect on chd was seen in the younger women (ages 50 to 59). the public consequence was that hormone therapy was abandoned or was conducted with lower doses. the possibility that chd risk is lowered by earlier hormone therapy after menopause should also be considered, although results are not conclusive. whether hormone replacement therapy results in either increased or unchanged risk for stroke, is also a matter of debate. of note, recent guidelines do not identify estrogen therapy for the primary or secondary prevention of chd [59, 60 ]. although the whi and the heart and estrogen / progestin replacement studies (hers) showed no cvd protection resulting from hrt, several animal studies have suggested an important cardioprotective role for estrogens against heart failure, mediated by a genomic or a nongenomic estrogen - receptor - mediated signaling pathway (see for a review). tumor necrosis factor - alpha (tnf-) has been reported as an important factor during i / r injury and ischemia preconditioning. in a langendorff - perfused rat heart model, several evidences have been demonstrated that stromal cell - derived factor 1 (sdf-1) is increased in ischemic hearts and induced cardioprotection. a higher expression of myocardial sdf-1 was observed in female rats in response to i / r and the increased myocardial sdf-1 production in female hearts was due to estrogen - estrogen receptor (er) interactions. in c57bl/6j male mice, estrogen also induced cardioprotection after acute myocardial infarction through a decreased activity of matrix metalloproteinase-9 and increased akt - bcl-2 antiapoptotic signaling. in a langendorff isolated perfused rat heart model, estrogen increased the perfusion pressure and coronary resistance through activation of l - type calcium channels. estrogen - related receptor alpha (err) is a transcription factor for some myocardial mitochondrial enzymes, essential to maintain cardiac energy reserves. a decrease in myocardial err, regulated by the metabolic sensor amp - activated protein kinase alpha 2 (ampk2), was recently reported during congestive heart failure. it was reported that lipin 1 is the principal protein of this family in myocardium and is also regulated by err. the lack of cvd protection observed during hrt has been proposed to be related with alterations in sex hormone synthesis and metabolism that can occur during aging, and can affect the hormone environment in postmenopausal women. also age - related changes in vascular estrogen receptors (ers) subtype, structure, expression, distribution, and the signaling pathway in the endothelium and vascular smooth muscle, preexisting cvd conditions, and structural changes in blood vessels architecture have been suggested as possible causes for the failure of hrt in cvd. it also should be noticed that hrt is not only composed by estrogens, but also by a combination of estrogen and progesterone. a recent study demonstrated that a combination of 17--estradiol and medroxyprogesterone acetate aggravates chronic heart failure after experimental myocardial infarction, which can also explain the results from previous studies including whi and hers. the heart is one of the organs with the highest energy demand in the body, which is hardly surprising due to high energetic input required by the contractile apparatus. although the heart is considered an omnivorous organ due to the fact that it can use several substrates for energy generation, including glucose, amino acids, lactate, and ketone bodies, fatty acids are the favored fuel for the cardiac muscle [72, 73 ]. in fact, the adult heart generates between 5070% of its atp from fatty acid beta - oxidation, which occurs mainly in mitochondria, possesses an elaborate system to import and process fatty acids of different lengths [72, 74 ]. in fact, in itself, mitochondrial function is one among different factors that impact the flux of fatty acid beta - oxidation. others include the fatty acid supply itself, which is modulated among other factors by diet, competing substrates for the cardiac tissue, the energy demand and oxygen availability, and the regulation at a nuclear or allosteric level of enzymes which are involved in all steps of fatty acid uptake, esterification, and metabolism. fatty acids can be transported in the plasma as free fatty acids (ffas) conjugated with albumin or as part of triacylglycerol (tag) contained in chylomicrons or very - low density lipoproteins (vldls) [75, 76 ]. ffa concentration in the plasma is highly variable, depending not only on the diet, but also on the developmental state of the organism and if any pathology is present. for example, the amount of ffa in the plasma is known to greatly increase during myocardial infarction and diabetes, which leads to an augmented cardiomyocyte ffa uptake and accumulation, since the concentration of ffa in the plasma is a major determinant for these two events. regardless of the mechanism underlying an acute or chronic accumulation of ffa in the plasma (reviewed in), the end result of cardiomyocyte cytosolic accumulation of fatty acids can differ, depending on a wide range of factors. the first step after entering the cardiomyocyte is conversion to coa esters, through the action of fatty acyl coa synthase (facs). fatty acid uptake by cells is made by membrane proteins with high affinity for fatty acids [79, 80 ], namely, the fatty acid translocase (fat / cd36), the fatty acid binding protein (fabppm) and a variety of fatty acid transport proteins (fatps), as well as by simple diffusion of fatty acids through either the phospholipid bilayer or a pore or channel formed by one or more of the referred fatty acid transporter proteins. upon entering the cell, the rate of utilization is governed by a variety of factors, including malonyl - coa, the ratio acetyl - coa / coa and the availability of other substrates, namely, glucose, lactate, and ketone bodies that can compete with free fatty acids as a source of acetyl - coa. long - term regulation of uptake and utilization requires alterations in expression rates of genes encoding for fatty acid handling proteins. free fatty acids can also by themselves modulate the expression of such genes via nuclear transcription factors such as peroxisome proliferator - activated receptors (ppars). mitochondrial beta - oxidation of long - chain fatty acids starts with its association with coa, forming acyl - coa esters that are transported into mitochondria by carnitine palmitoyl transferase i (cpt - i). beta - oxidation produces in each round one nadh, one fadh2 (as part of an enzymatic complex), and one acetyl - coa, which is further oxidized in the krebs cycle to co2, with the concomitant further generation of three nadh, reduced fad co - factor in succinate dehydrogenase complex, and one gtp. nadh, via nadh dehydrogenase, and succinate dehydrogenase deliver electrons to the remaining electron transport chain complexes which contribute to the generation of a proton gradient used to synthesize atp (figure 2). throughout this whole process, several regulation mechanisms can operate, starting with the transport of the acyl chain to the mitochondrial matrix and ending at the accumulation of end products of the oxidation process, namely, reducing equivalents and ultimately atp levels. the transport process is considered a major player in the control of the flux through beta - oxidation, mostly in intact muscle, since levels of malonyl - coa are kept considerably high. with this type of control, it is possible for the tissues to rapidly adapt to different metabolic demands, such as in muscles. an inhibition of fatty acid beta - oxidation, which as mentioned can occur at several stages, will ultimately result in free fatty acid intracellular accumulation which subsequently will be responsible for poor removal of fatty acids from plasma in any of their forms of transportation. in fact, a possible role has been attributed to female sex hormones in the development of fatty liver pregnancy on the basis of their effect in the reduction of mitochondrial fatty acid oxidation and in regulating cellular energy balance in vivo by regulating the expression of the medium chain acyl coenzyme a dehydrogenase (mcad) gene. besides mitochondrial oxidation, long - chain fatty acyl coa can also be used for the synthesis of intermediates, including tag, diacylglycerol (dag), and ceramide [72, 87 ]. under normal intracellular concentrations, if alterations in normal fatty acid homeostasis occur, which can originate from excessive plasma ffa content or from enhanced facs expression and/or activity, long - chain fatty acyl coa derivatives can accumulate in cells. depending on the tissue, accumulation of some of these intermediates for example, it is known that excessive accumulation of tag in nonadipocyte tissues can result in different negative outcomes including impaired insulin signaling in the liver and skeletal muscle and apoptosis and other metabolic disturbances in the heart [87, 89, 90 ]. dag has also been determined to cause similar effects in the same tissues, including increased insulin resistance observed in a model of rodent high - fat diet. it is interesting to note that both increases in tag and ceramide intracardiac content did not correlate with the increased insulin resistance. ceramide, by its turn, has been demonstrated in different biological models to increase apoptotic signaling in several tissues [9294 ], although evidence is scarcer for the heart. it is interesting to note that ceramide derivatives have been involved in the triggering of the mitochondrial permeability transition pore (mpt pore) and outer - membrane permeabilization [96, 97 ], conditions closely linked with mitochondrial dysfunction and cell death. in opposition, long - chain ceramide species have been shown to inhibit the mpt pore. the discrepancy of results regarding ceramide implicates this lipid species in the control of mitochondrial cell death pathways. from the short description above, it is clear that a balance between ffa cell uptake and metabolism must be reached in order to avoid the accumulation of undesired fatty acid metabolites. also, increased reliance of fatty acids as fuel for cardiac cells has undesired effects, one of them being decreased atp synthesis, resulting from increased atp hydrolysis for noncontractile purposes, increased mitochondrial uncoupling due to increased activity / expression of uncoupling proteins and greater proton futile cycling, creating the so - called oxygen wasting and resulting in several physiological complications [100102 ]. interestingly, inhibition of fatty acid metabolism is proposed to be beneficial for some forms of heart failure. as described above, menopause is a normal consequence of the aging process in women and is accompanied of important physiological and biochemical alterations. there are several evidences in the literature that the content in ffa in the plasma tends to increase during menopause. one particular study performed with 4-vinylcyclohexene - diepoxide- (vcd-) treated rats indicated that progressive loss of ovarian function induced by vcd results in an increase of plasma ffa, which initiated several alterations leading to the development of the metabolic syndrome. this important piece of evidence mimics what is observed in the menopausal women, where an increase in circulating ffa was measured. it is also known that women experience a characteristic increase in circulating lipids at the time of the final menstruation period, although it is difficult to evaluate the component resulting from hormonal alterations and what is the result of the normal aging process [34, 106 ]. the increased ffa was partly reverted by hormone - replacement therapy, showing that, at least in part, it is a hormone - dependent effect. the role of estrogens in fatty acid metabolism is well described and involves different mechanisms [107109 ]. one important effect is that estradiol promotes the channeling of ffa toward oxidation and away from triglyceride storage (figure 3) by upregulating the expression of peroxisome proliferation activator receptor delta and its targets and also by directly and rapidly activating amp - activated protein kinase (ampk). ampk acts as a fuel sensor that increased fatty acid beta - oxidation during higher metabolic demands. the data, although still scarce and largely spread out, indicates that during menopause, fatty acid metabolism is altered. the decrease in estradiol levels may result in decreased fatty acid oxidation and increased accumulation in the adipose tissue, with hormone replacement therapies recovering the pre - menopausal fatty acid status quo. maybe not, one important player in fatty acid metabolism is, as described, the mitochondrion. a proper channeling of fatty acyl - coa and subsequent beta - oxidation is necessary for the energy - generating process. it is clear that a failure of mitochondrial bioenergetics causes an unbalance in fatty acid metabolism, which may result in the accumulation of fatty acyl - coa esters in the cytosol of cardiomyocytes. this phenomenon could result in a larger channeling of fatty acyl - coa esters to the synthesis of the intermediates described above, including tag, dag, and ceramide. it is interesting to recapitulate here that ceramide has been involved in the induction of apoptosis in a variety of biological models [9294 ]. although the relationship between increased ceramide intracellular levels in the menopausal heart and increased apoptotic signaling is still to be determined, several endpoints for increased cardiac fas - dependent and mitochondrial - dependent apoptosis were identified in the hearts of bilateral ovariectomized wistar rats [111, 112 ]. a logical question would be if there is a possible relationship between intracellular lipid metabolism alterations resulting from ovariectomy and enhanced apoptotic signaling in the heart. decreased fatty acid oxidation by mitochondria occurs in a variety of situations, ranging from xenobiotic - induced toxicity to several pathologies. there are many fatty acid oxidation disorders identified in humans, and which affect organs as different as muscle and brain, which result in altered fat deposition and mitochondrial beta - oxidation. defects are commonly present in the mitochondrial machinery that shuttles long - chain fatty acid metabolites to mitochondria, resulting in decreased beta - oxidation. several xenobiotics also alter fatty acid metabolism in different organs, examples are fluorochemicals and the antibiotic tetracycline in the liver. as for the heart, it is now becoming increasingly recognized that alterations in fatty acid uptake and/or beta - oxidation can result in the so - called fatty heart, a largely unrecognized entity for a long time, and which, as described has important cardiovascular complications [89, 118 ]. it has been proposed that mitochondrial function in the heart decreases with the progression of aging. alterations include loss or oxidation of cardiolipin, a tetra - acyl phospholipid involved in the activity of many oxidative phosphorylation enzymes including complex i [119121 ]. this presents a clear determinant of loss of mitochondrial function and also represents a phenotype of mitochondrial membrane aging which impacts both the bioenergetics and several signaling pathways to and from mitochondria. it is also known that aging - dependent cardiac mitochondrial effects are more specific to interfibrillar mitochondria, which is the subpopulation responsible for the majority of energy supply to the myocardium [122, 123 ]. such alterations include decrease respiratory complex activity and increased oxidative stress, while a decreased capacity for beta - oxidation has also been demonstrated in an animal model for aging due to alterations in carnitine palmitoyltransferase i which were suspected to originate from a decrease in cardiolipin content. power in the heart is thus affected with aging, which is further illustrated by a decrease in the nuclear control of mitochondrial biogenesis and function and by increased mtdna deletions frequency found in the aged heart. adding to mitochondrial aging, per se, one has to have in mind that other factors may be operating in the menopausal woman that can contribute to altered mitochondrial function and result in disrupted fatty acid metabolism. for example, the incidence of diabetes, and obesity increases during menopause, which also contributes to accelerate mitochondrial dysfunction [128130 ]. by its turn, the menopausal woman may be under treatment with different medications which may also affect the bioenergetic efficacy of cardiac mitochondria [131, 132 ], especially if other conditions occur at the same time. to summarize, ageing results into a progressive degradation of mitochondrial capacity in the heart, which, in combination with hormonal alterations resulting from menopause and its associated alterations in lipid profile, may result into a progressive decrease in lipid oxidation in mitochondria and increased lipid storage in adipocytes and formation of fatty acyl intermediates in the cytosol of cardiomyocytes (figure 3). the development of insulin resistance, diabetes and obesity can be several faces of the same coin, the increased lipotoxicity in the cardiomyocyte of the menopausal woman. this is a clear avenue for research that still is largely unexplored and deserves attention since menopause is a condition that affects an increasingly number of women, as the general population is progressively aging. if the hypothesis put together in this paper is correct, then prophylactic measures that improve mitochondrial capacity in menopausal women would contribute to decrease cardiovascular risk. in fact, besides hormone replacement therapy, which replenishes estrogens and reequilibrates lipid homeostasis, other cotherapies may help improve the lipid profile in the menopausal woman through different mechanisms. for example, endurance exercise has been demonstrated to increase mitochondrial capacity in the heart [133, 134 ]. in a menopausal setting, twelve weeks of endurance exercise have been demonstrated to provide some benefits in increasing lipid oxidation, besides improving other cardiorespiratory parameters [135, 136 ]. carnitine, which is essential to long - chain fatty acid beta - oxidation, has been shown to recover some of skeletal muscle function and inhibit alterations in ovariectomized rats. nevertheless, to the best of our knowledge, no work on the impact of carnitine on lipid profile and oxidation in the menopausal heart has been provided. cardiac oxidative stress after ovariectomy has also been observed in animal models although evidence for increased oxidative stress in the cardiovascular system is scarce. estrogens per se act as antioxidants, although it is still unclear if estrogen supplementation during menopause is completely without risks for the cardiovascular system [139, 140 ]. also, it is unclear so far if antioxidant supplementations would improve mitochondrial fitness in menopausal women. finally, an interesting alternative was proposed by zern.. the powder was enriched in phytochemicals such as flavans, anthocyanins, quercetin, myricetin, kaempferol, and resveratrol. the results showed alterations in lipoprotein metabolism, oxidative stress, and inflammatory markers, which were all decreased in the treated group. although the heart was not specifically targeted in the study, the results may suggest a positive impact in this organ as well. interestingly, resveratrol is considered an activator of mitochondrial biogenesis in different model systems, acting through sirtuin-1-dependent and independent mechanisms [142144 ]. although there are many loose ends in the story, it appears logical to consider that progressive deterioration of mitochondrial function in the aging woman with menopause contributes to the metabolic alterations observed in the heart, including a decreased capacity for lipid oxidation. a decreased mitochondrial flux of fatty acid beta - oxidation, can result in most cases in the accumulation of toxic intermediates in the cytosol and also of nonmetabolized fatty acids in mitochondria, which leads to further deterioration of mitochondrial function and progressive metabolic changes that can increase cardiovascular risk. not only this line of thought needs to be demonstrated in animal models and humans, but if true, pharmacological, or nonpharmacological strategies must be devised to counteract this metabolic remodeling.	menopause is a consequence of the normal aging process in women. this fact implies that the physiological and biochemical alterations resulting from menopause often blur with those from the aging process. it is thought that menopause in women presents a higher risk for cardiovascular disease although the precise mechanism is still under discussion. the postmenopause lipid profile is clearly altered, which can present a risk factor for cardiovascular disease. due to the role of mitochondria in fatty acid oxidation, alterations of the lipid profile in the menopausal women will also influence mitochondrial fatty acid oxidation fluxes in several organs. in this paper, we propose that alterations of mitochondrial bioenergetics in the heart, consequence from normal aging and/or from the menopausal process, result in decreased fatty acid oxidation and accumulation of fatty acid intermediates in the cardiomyocyte cytosol, resulting in lipotoxicity and increasing the cardiovascular risk in the menopausal women.
there are fewer reports of this in patients with extra hepatic portal vein obstruction (ehpvo), accompanied with cavernous transformation of the portal vein. involvement of the gall bladder by venous collaterals predisposes to inadvertent portal venous haemorrhage during the cholecystectomy. this can be avoided by optimal pre - operative preparation and a cautious surgical strategy. in those with associated obstructive portal biliopathy, a decompressive porto - venous shunt helps to render the peri - hepatic - portal area safer for cholecystectomy and also facilitates the performance of a biliary drainage procedure. we report our experience of laparoscopic cholecystectomy in two cases of symptomatic cholelithiasis associated with portal cavernoma. a 25-year - old young man presented with recurrent right upper abdominal pain for 18 months. abdominal sonography revealed cholelithiasis, a normal biliary tree, cavernous transformation of the portal vein, splenomegaly and significant peri - portal and peri - pancreatic venous collaterals. haematological, coagulation and liver profiles were normal except for raised serum alkaline phosphatase (437 iu / l) and gamma - glutamyl transpeptidase - 1329.3 u / l levels. magnetic resonance cholangio pancreaticograpy (mrcp) revealed mild extrinsic compression of the mid common bile duct (cbd) and no biliary dilatation. a 39-year - old male patient presented with a history of recurrent upper abdominal pain and vomiting for 2 years. on evaluation, he was found then to have cholelithiasis with a portal cavernoma, which were followed - up conservatively. upper gastro - intestinal endoscopy was normal. in view of symptomatic cholelithiasis with non - obstructive portal biliopathy blood was cross - matched and two units of packed cells were arranged on a standby basis. carboperitoneum was created by closed technique at the site of primary laparoscopic access, which was aided by optical guidance. in the first patient, this site was the umbilicus because of splenomegaly and access in the second at the left palmar 's point. subsequent ports were placed under vision to prevent inadvertent injury to collateral venous channels on the abdominal wall for a four - port cholecystectomy. the first patient was found to have a calot 's triangle bordering smooth - surfaced portal cavernoma, minimal gall bladder varices and lateral abdominal wall venous collaterals [figure 1 ]. the second patient had dilated venous collaterals that bordered the calot 's triangle, the cystic duct and both curvatures of the distal stomach [figure 2 ]. laparoscopic view of case 1 showing large smooth - surfaced portal cavernoma abutting the calot 's triangle area and some gall bladder varices too laparoscopic view of case 2 showing clipped varices along the cystic duct the gall bladder was distended and thin walled in both patients, with a normal liver. this was aided by ultrasonic endoshears (harmonic ace, ethicon endo - surgery, usa) and a 5 mm bipolar electro - thermal fusion device (ligasure, covidien ag, usa). critical - view of safety was first created to delineate the cystic artery and the cystic duct discreetly. a 25-year - old young man presented with recurrent right upper abdominal pain for 18 months. abdominal sonography revealed cholelithiasis, a normal biliary tree, cavernous transformation of the portal vein, splenomegaly and significant peri - portal and peri - pancreatic venous collaterals. haematological, coagulation and liver profiles were normal except for raised serum alkaline phosphatase (437 iu / l) and gamma - glutamyl transpeptidase - 1329.3 u / l levels. magnetic resonance cholangio pancreaticograpy (mrcp) revealed mild extrinsic compression of the mid common bile duct (cbd) and no biliary dilatation. a 39-year - old male patient presented with a history of recurrent upper abdominal pain and vomiting for 2 years. on evaluation, he was found then to have cholelithiasis with a portal cavernoma, which were followed - up conservatively. upper gastro - intestinal endoscopy was normal. in view of symptomatic cholelithiasis with non - obstructive portal biliopathy blood was cross - matched and two units of packed cells were arranged on a standby basis. carboperitoneum was created by closed technique at the site of primary laparoscopic access, which was aided by optical guidance. in the first patient, this site was the umbilicus because of splenomegaly and access in the second at the left palmar 's point. subsequent ports were placed under vision to prevent inadvertent injury to collateral venous channels on the abdominal wall for a four - port cholecystectomy. the first patient was found to have a calot 's triangle bordering smooth - surfaced portal cavernoma, minimal gall bladder varices and lateral abdominal wall venous collaterals [figure 1 ]. the second patient had dilated venous collaterals that bordered the calot 's triangle, the cystic duct and both curvatures of the distal stomach [figure 2 ]. laparoscopic view of case 1 showing large smooth - surfaced portal cavernoma abutting the calot 's triangle area and some gall bladder varices too laparoscopic view of case 2 showing clipped varices along the cystic duct the gall bladder was distended and thin walled in both patients, with a normal liver. this was aided by ultrasonic endoshears (harmonic ace, ethicon endo - surgery, usa) and a 5 mm bipolar electro - thermal fusion device (ligasure, covidien ag, usa). critical - view of safety was first created to delineate the cystic artery and the cystic duct discreetly. laparoscopy offers a paradigm advantage for cholecystectomy in the hands of an experienced laparoscopic surgeon. this has been consistently described for laparoscopic cholecystectomy in cirrhotics and in early reports in ehpvo patients with portal cavernomas. in addition, all stratagems in extirpating the gall bladder pathology can be executed by laparoscopy, e.g., varying extents of a cholecystectomy. furthermore, the normal hepatic architecture, coagulation profile, significant varices sparing the gall bladder in our experience, allows us to consider laparoscopic cholecystectomy in these patients. we suggest the use of laparoscopic cholecystectomy by an experienced surgeon in patients with cholelithiasis associated with portal cavernomas related to ehpvo with non - obstructive biliopathy.	a laparoscopic cholecystectomy can be technically challenging with co - existing portal hypertension, as commonly seen with cirrhosis of the liver. extra hepatic portal vein obstruction (ehpvo) although less common, is a significant cause of portal hypertension in india. ehpvo has a unique clinical profile, which differentiates it from portal hypertension associated with cirrhosis of the liver. this impacts therapy in ehpvo algorithmically and operatively. we report two cases of symptomatic gall stones with portal cavernoma. further evaluation revealed non - obstructive portal biliopathy. both underwent a successful laparoscopic cholecystectomy. we highlight the importance of careful operative strategy, diligent haemostasis and the feasibility of performing a laparoscopic cholecystectomy in patients with symptomatic gall stones associated with a portal cavernoma.
since description of neck dissection in late 19th century, modifications have been proposed, practiced, and argued. tracing back the heritage of neck dissection, sequential evolution from a morbid to a cosmetically tailored and oncologically acceptable procedure becomes evident. although several different classifications have been adapted in the past, debate on a balanced and widely acceptable nomenclature continues. lately selective neck dissection (snd) has been the buzz word for regional management in head and neck cancer. shah demonstrated frequency and patterns of regional lymph node metastases from oral squamous cell carcinoma in patients who underwent radical neck dissection. by definition snd although snd is an accepted procedure for pathological staging of clinically node negative (cn0) neck, the house remains divided between elective neck dissection versus a more conservative wait and see policy. the therapeutic role of snd in clinically node positive (cn+) disease is still unclear but is gaining popularity in carefully selected patients [3, 4 ]. the exact protocol for regional management of squamous cell carcinoma of oral cavity is yet to be established. the objective of present study was to report regional control with selective neck dissection in both n0 and n+ previously untreated patients diagnosed with squamous cell carcinoma of the oral cavity. a review of patients who underwent snd between 2003 and 2010 at shaukat khanum memorial cancer hospital and research center was performed. a total of 219 patients who underwent snd for histology proven squamous cell carcinoma (scca) of oral cavity during the study period were included. all patients underwent a comprehensive clinical examination of head and neck followed by mri of the face and neck and chest x - ray. patients were staged according to the ajcc (american joint commission on cancer) guidelines. snd was the mainstay surgical protocol for regional control alongside wide local excision of tumor. for patients diagnosed with scca of upper alveolus, maxillectomy was performed and snd was reserved for patients with clinically or radiologically node positive disease. inclusion criteria for performing snd included tumor > 1.5 cm and tumor thickness of more than 4 mm. adjuvant treatment options such as postoperative radiotherapy (port) or concurrent chemoradiotherapy (crt) were planned for the patients after pathological staging. other indications included patients with tumors crossing midline, involvement of tip of tongue, and extension into the base of tongue or floor of the mouth. the regimen comprised of a combination of 2 drugs : intravenous gemcitabine 1000 mg / m on day 1 and day 8 and cisplatin 75 mg / m on day 1 of each cycle, respectively. after 2 weeks from the second cycle, a response assessment was clinically devised and patients were planned for wide local excision of the tumor along with snd. wide local excision was performed in tongue, lips, retromolar trigone, and buccal mucosa with 1 cm clear margin. patients with squamous cell carcinoma of lower alveolar mucosa underwent marginal or segmental mandibulectomy depending on extent of involvement of lower alveolus. majority of patients with t1 and early t2 tumors (3 cm), t3, and t4 tumors or clinically n+ disease underwent snd i iv. after extraction of neck specimen, sublevels were separated and placed individually in formalin filled containers and sent for histopathological analysis. a template was prepared by pathologist to interpret the report including the number of nodes harvested, size of the fibro fatty tissue, and number of positive nodes. the presence of perineural invasion, lymphovascular invasion, and extra capsular spread was also documented for each level of neck specimen. postoperative radiotherapy (port) was used in patients with pathologically node positive disease, > 1 cm tumor size, > 5 mm tumor thickness, and poorly differentiated tumors. in pathologically node negative patients, 60 gy in 30 fractions was given to the primary site and ipsilateral neck. in pathologically node positive patients, 60 gy in 30 fractions was given to the primary site and bilateral neck. concurrent chemoradiotherapy (crt) was reserved for patients with extra capsular spread, perineural invasion, lymphovascular invasion, and more than 2 positive lymph nodes. expected 5-year regional control was calculated using kaplan - meier curves and significance between variables was determined with log rank test. statistical package for social sciences (spss), version 17, was used for statistical analysis. a total of 219 patients underwent snd of which 158 were clinically node negative and 61 were node positive. median age at presentation for node negative patients was 51 (1376) years and median follow - up was 2.9 (0.079) years. median age at presentation for node positive patients was 50 (2879) years and median follow - up was 1.8 (0.27) years. table 1 summarizes patient characteristics and treatment modalities with respect to clinically node negative and positive patients. there was a significant difference between two groups with respect to site of primary tumor (p = 0.009), treatment received (p = 0.0001), and sublevels dissected (p = 0.002). anterior tongue was the site of primary in 75% patients with n0 neck disease versus 66% with n+ neck disease. surgery alone was the treatment modality in 16% patients with clinically n0 disease and 2% patients with n+ disease. sublevels i iii were dissected in 21% patients with n0 versus 3% patients with n+ disease. out of a total of 52 patients with unknown extracapsular status, 65% patients received postoperative radiation, and 25% received concurrent chemoradiotherapy based on presence of other poor prognostic factors. table 2 represents the clinical and pathological distribution of the study cohort. locally advanced (t3/t4) one hundred and fifty - eight (72%) patients were cn0 at the time of presentation. one hundred and twenty - one (55%) patients had advanced disease (stage iii / iv) on histopathology. mean number of extracted nodes was 50 nodes and a total of 11936 nodes (level 1, 1836 ; level 2a, 2500 ; level 2b, 1600 ; level 3, 3000 ; level 4 ; 3000) were extracted in 219 patients. level ii a was the most commonly involved sublevel in tumors of anterior tongue while level i was most frequently involved in tumors of buccal mucosa and lower alveolus. in table 3, the newly proposed classification of neck dissection was compared with older version. none of the patients who underwent level i thirty - three patients out of 184 who underwent level i iv snd had one or more nonlymphatic structures removed and were clearly demonstrable in new classification. out of these 33 patients, 4 (12%) patients had extra capsular spread on histopathology (ijv = 3, ijv+san = 1). histopathology of resected specimen after induction chemotherapy demonstrated that only 7% patients had t3/t4 tumors. the expected 5-year overall survival for patients who received induction chemotherapy versus those who did not was 69 and 74%, respectively, and was not significantly different (p = 0.4). fourteen (8.8%) patients failed in clinically node negative neck while 8 (13%) failed in clinically node positive patients. out of total 7 patients with extracapsular extension, 3 (42%) patients developed regional failure. median recurrence - free survival in pn+ patients with and without extracapsular spread was 1.6 (0.089) and 1.7 (0.027) years and was not significantly different. a total of 14 patients had ipsilateral failures, including 3 patients that failed both locally and regionally. almost all patients that failed ipsilaterally in the neck were either treated with palliative chemotherapy or symptomatically. there were 38 local, 19 regional, 3 locoregional, and 6 distant failures (not shown). eight patients had occult metastatic disease after snd i iii, that is, 3 patients in level iii, 5 in level ii, and 3 in level i. two patients underwent bilateral snd i iii as they were staged radiologically n2c ; on histopathology they had n1 and n2b disease, respectively. one patient had skip metastasis with pathologically positive node in level iii, escaping levels i and ii. a total of 184 patients underwent snd i iv of which thirteen patients had bilateral neck dissection. ten patients showed skip metastasis of which nine patients had scca oral tongue and one had scca buccal mucosa. level ii b a total of 14 (7.6%) patients had positive lymph nodes in level iv but no isolated level iv involvement was seen. also, 12/14 patients with level iv involvement had more than two positive nodes. five of the fourteen patients had cn0 disease at presentation and on staging mri ten patients had radiologically significant nodal disease. grade, lymphovascular invasion, and pathological n stage were statistically significant for 5-year regional control (table 6). a highly significant difference in regional control was present between n0 and n+ patients with expected 5-year control of 95% and 81%, respectively (p = 0.005). the 5-year overall survival and disease - free survival for the whole group were 73% and 61%, respectively (not shown). table 7 represents the rates of pathological nodal positivity after snd in patients with well differentiated tumors and clinically node negative neck. regional spread of oral cancer continues to be the most significant prognostic factor and decreases survival by 50% [5, 6 ]. studies have shown that lymphatic spread with respect to anatomical subsite can be predicted [8, 9 ]. shah mapped the lymphatic spread for squamous cell carcinoma of upper aerodigestive tract in 501 patients with oral cancer. studies have shown 03% rate of metastatic spread in level v supporting the notion of sparing posterior triangle while performing neck dissection [10, 11 ]. it was also concluded that level v dissection can be avoided even if suspicious lymph nodes are encountered at level iv. in the current study, a marked difference in regional control was observed between node negative and node positive patients who underwent neck dissection. overall, 8% of total patients who underwent level i iv snd had level iv involvement. tongue was the most common site of primary and level ii a or subdigastric level was the most common involved sublevel. overall 22 patients failed regionally with a high preponderance of ipsilateral failures (18 versus 4). regional failure was more common in the first year after surgery and highlights importance of meticulous surveillance in this time period. sublevels of regional failures in neck could not be determined as majority of patients had huge fixed neck recurrences involving more than one sublevels of neck. in addition the prognostic role of extracapsular extension could not be determined due to small number of patients who had extracapsular involvement. there are no set guidelines for management of clinically node negative neck in early oral cancer. studies have been reported both in favor of elective neck dissection and wait and see policy [1214 ]. a high occult metastatic rate of 37%, low socioeconomic status, and distant geographic location of our patients made elective neck dissection a more suitable option in our setting. in critical assessment of supraomohyoid neck dissection, occult metastasis was present in 31% out of a total of one hundred and fifteen patients included in the study. in the current study, iv neck dissection but only 14 patients were found to have pathological evidence of disease in level iv. since the results on the findings of the current study, practice has already been modified and level iv dissection is only performed in patients with clinical nodal disease in level iii / iv in neck in our institute now. radiotherapy either in pre- or postoperative setting has shown its benefit with reduction in the incidence of neck failures by 50% irrespective of the n stage [1719 ]. the choice of pre- or postoperative radiation remains largely institutional with surgeons preferring port as it reduces the operative complications and makes performance of neck dissection relatively easy. adjuvant radiotherapy has also been recommended in clinically node negative contralateral neck to reduce the rate of contralateral neck failure. two trials conducted in europe (european organization research and treatment of cancer (eortc)) and the united states (radiation therapy oncology group (rtog)) have shown better locoregional control in patients with extracapsular spread and/or positive surgical margins who received postoperative chemoradiation [22, 23 ]. chemoradiation has been advocated in presence of poor prognostic factors like stage iii iv disease, perineural infiltration, vascular embolisms, and/or clinically enlarged level iv v lymph nodes. in the current study, tumor grade, extracapsular spread, lymphovascular invasion, and pathological n stage were significant variables for regional control in present study. radical neck dissection (rnd) remained the procedure of choice in node positive patients for greater part of the 20th century. strong in their study showed a regional recurrence rate of 54.3% in node positive patients and 71.3% in patients with positive nodes at multiple levels. this leads to several questions regarding the oncological benefit and morbidity associated rnd. in the past two decades the use of snd has gained popularity in the management of node positive patients partly because of the comparable regional control rate in patients with occult disease undergoing elective neck dissection and also due to an increasing use of port for better disease control. andersen. in their study of 106 patients with 129 therapeutic snd had 9 regional failures. the study included all sites of head and neck region and > 50% patients had n1 disease. in another study on effectiveness of snd in clinically node positive neck including all primary sites of head and neck region, 54 patients underwent snd including 33 patients with pn2/3 disease. there were 2 ipsilateral recurrences and snd showed better disease control but did not reach statistical significance. with an increasing trend of performing limited and site specific lymphadenectomy procedures, a comprehensive classification of neck dissection has been proposed that is logical, simple, and easy to remember. in the current study, the authors have compared the old nomenclature with the proposed classification and found the new classification better at defining precisely the lymphatic and non - lymphatic structures excised. the role of induction chemotherapy in head and neck squamous cell carcinoma before on histopathology, only 1 patient had t4 tumor out of 6 patients with ct4 tumors who received induction chemotherapy. out of 23 patients with ct3 disease who received induction chemotherapy, this favors the role of induction chemotherapy in downstaging of head and neck tumors before surgical resection ; however conclusions can not be drawn due to heterogeneous nature of our cohort. we also made an effort to identify a subgroup of patients in which pathological nodal positivity was absent. almost 90% patients with well - differentiated, t1 tumors and cn0 neck did not have pathological nodal disease after snd. this group could potentially represent a subgroup that might benefit from wait and see policy under close surveillance. further studies are needed to address this issue taking into consideration several other prognostic variables. the current study reports regional failures after snd in previously untreated squamous cell carcinoma of oral cavity in both node negative and positive patients. induction chemotherapy may have a beneficial role in locally advanced head and neck squamous cell carcinomas but this needs to be confirmed in future trials. there might be a subgroup of patients with well - differentiated, clinically node negative t1 tumors that can be safely managed with observation alone. as the role of neck dissection becomes more conservative with ever - expanding application of port and crt providing. superselective neck dissection might become the standard for regional management of neck in oral squamous cell carcinoma.	aim. to share experience with regional failures after selective neck dissection in both node negative and positive previously untreated patients diagnosed with squamous cell carcinoma of the oral cavity. patients and methods. data of 219 patients who underwent snd at shaukat khanum cancer hospital from 2003 to 2010 were retrospectively reviewed. patient characteristics, treatment modalities, and regional failures were assessed. expected 5-year regional control was calculated and prognostic factors were determined. results. median follow - up was 29 (9109) months. common sites were anterior tongue in 159 and buccal mucosa in 22 patients. pathological nodal stage was n0 in 114, n1 in 32, n2b in 67, and n2c in 5 patients. fourteen (6%) patients failed in clinically node negative neck while 8 (4%) failed in clinically node positive patients. out of 22 total regional failures, primary tumor origin was from tongue in 16 (73%) patients. expected 5-year regional control was 95% and 81% for n0 and n+ disease, respectively (p < 0.0001). only 13% patients with well differentiated, t1 tumors in cn0 neck were pathologically node positive. conclusions. selective neck dissection yields acceptable results for regional management of oral squamous cell carcinoma. wait and see policy may be effective in a selected subgroup of patients.
migraine clinically manifests as hemi - cranial throbbing type of pain associated with nausea, vomiting, heightened sensitivity to light (photophobia) and sound (phonophobia) with or without transient neurological symptoms. migraine is one of the common primary headache disorders affecting 13% of the population world - wide. prodromal, headache episode and postdromal phases of migraine are known to impact productivity at work and quality of life, apart from causing cognitive impairment. episodic migraine may lead to chronic migraine, if it is not treated properly it may lead to medication overuse headache and increased risk of suicidal attempt. autonomic nervous system (ans) imbalance explains many of the clinical manifestations of the migraine disorder. autonomic symptoms (such as nausea, vomiting, or diarrhoea, cutaneous vasoconstriction [pallor ], vasodilatation [flushing ], piloerection and diaphoresis) are common during acute migraine headaches. migraine is a chronic disorder with episodic disabilities and it requires long - term management as well as preventive strategies. the treatment of migraine involves both acute and preventive drugs along with non - pharmacological strategies. even though in the last few decades much advancement has occurred in the research field of migraine biofeedback and relaxation have been demonstrated to be useful alternatives to standard medical therapy for both migraine and tension - type headaches. the present study was aimed to evaluate the effect of yoga as adjuvant therapy in patients with migraine. diagnosis of migraine, with or without aura was done according to the guidelines of international headache society, international classification of headache disorders 2 edition (ihs, ichd - ii) after thorough clinical interview, physical and neurological examination by the neurologist. both male and female migraine patients between the age group of 15 - 60 years satisfying the ihs, ichd - ii criteria for migraine without (1.1) or with aura (1.2.1) with a history of at least 2 years of migraine and headache frequency of 5 - 15/month were recruited for the study. recruited patients were explained about the nature of study and written informed consent was obtained. patients were randomized mainly to conventional care only (cc) and conventional care and yoga therapy (y) using a concealed allocation protocol. [study design - figure 1 ] study design. cc - conventional care group, y - yoga+conventional care group migraine patients received conventional care for 6 weeks. patients were advised to maintain headache diary for this period. 1 week after baseline assessment, confirmation of maintaining headache diary was done by telephonic conversation or personal contact. at the end of 6 week post - intervention, assessment was done and headache diary was collected. migraine patients receiving conventional care and yoga therapy had received yoga therapy along with conventional care. patients were scheduled to visit for 30 sessions (5 days a week for 6 weeks) of yoga. they were asked to maintain headache diary for the full duration of the study and was verified during yoga session. at the end of 6 weeks, post - intervention assessment was done and headache diary was collected. the yoga intervention consisted of a daily practice of loosening exercises, breathing exercises, asanas or postures done with awareness and shavasana [table 1 ]. yoga therapy was imparted by a trained yoga therapist at the yoga centre in the institution 's premises. yoga module for migraine patients the subjective variable, headache - specific quality of life was measured as the change in headache - related disability, which was assessed by the headache impact test (hit-6). the total number of headache episodes, intensity of headaches (0 - 10 on visual analog scale) and medication used were noted in headache diary by patients. self - perceived benefit of the intervention was measured at the end of the 6 week of the treatment period in both groups. patients rated the perceived benefit of the therapy on a five point scale ranging from greatly worsened my clinical condition to greatly improved my clinical condition. in addition, they assessed the therapy as more harmful than helpful, neither harmful nor helpful, or more helpful than harmful. autonomic function test was done when the subject was headache - free at least 3 days before and after the test. lead ii electrocardiogram (ecg) and breathing signals were conveyed through analog digital converter (power lab, 16 channels data acquisition system, ad instruments, australia) with a sampling rate of 1024 hz. the data were stored and were analyzed offline using an automatic programme that allowed visual checking of the raw ecg and breathing signals. 15 min basal recordings were stored and later analyzed to obtain both time and frequency domain parameters of heart rate variability (hrv) using hrv analysis software v1.1 (power lab 16 channels data acquisition system, ad instrument, australia). an artefact free 5 min segment was analyzed to obtain time domain (heart rate, standard deviation (sd) of nn interval, square root of the mean of the sum of squares of differences between adjacent r - r intervals) and frequency domain (total power, low frequency power [lf power ], low frequency normalized units, high frequency power [hf power ], high frequency normalized units [hfnu ], sympathovagal balance [svb ] - lf / hf ratio) parameters of hrv. statistical analyses were carried out on statistical package for the social sciences 15.0 software supplied by ibm. results on continuous measurements were presented on mean sd unpaired t - test was used to compare the variables at the baseline characteristics of the two groups of patients. paired t - test was used to compare the clinical, hrv, following 6 week intervention. patients were advised to maintain headache diary for this period. 1 week after baseline assessment, confirmation of maintaining headache diary was done by telephonic conversation or personal contact. at the end of 6 week post - intervention, assessment was done and headache diary was collected. migraine patients receiving conventional care and yoga therapy had received yoga therapy along with conventional care. patients were scheduled to visit for 30 sessions (5 days a week for 6 weeks) of yoga. they were asked to maintain headache diary for the full duration of the study and was verified during yoga session. at the end of 6 weeks, post - intervention assessment was done and headache diary was collected. the yoga intervention consisted of a daily practice of loosening exercises, breathing exercises, asanas or postures done with awareness and shavasana [table 1 ]. yoga therapy was imparted by a trained yoga therapist at the yoga centre in the institution 's premises. yoga module for migraine patients the subjective variable, headache - specific quality of life was measured as the change in headache - related disability, which was assessed by the headache impact test (hit-6). the total number of headache episodes, intensity of headaches (0 - 10 on visual analog scale) and medication used were noted in headache diary by patients. self - perceived benefit of the intervention was measured at the end of the 6 week of the treatment period in both groups. patients rated the perceived benefit of the therapy on a five point scale ranging from greatly worsened my clinical condition to greatly improved my clinical condition. in addition, they assessed the therapy as more harmful than helpful, neither harmful nor helpful, or more helpful than harmful. autonomic function test was done when the subject was headache - free at least 3 days before and after the test. autonomic function tests were carried out in autonomic laboratory, under all standardized conditions. lead ii electrocardiogram (ecg) and breathing signals were conveyed through analog digital converter (power lab, 16 channels data acquisition system, ad instruments, australia) with a sampling rate of 1024 hz. the data were stored and were analyzed offline using an automatic programme that allowed visual checking of the raw ecg and breathing signals. 15 min basal recordings were stored and later analyzed to obtain both time and frequency domain parameters of heart rate variability (hrv) using hrv analysis software v1.1 (power lab 16 channels data acquisition system, ad instrument, australia). an artefact free 5 min segment was analyzed to obtain time domain (heart rate, standard deviation (sd) of nn interval, square root of the mean of the sum of squares of differences between adjacent r - r intervals) and frequency domain (total power, low frequency power [lf power ], low frequency normalized units, high frequency power [hf power ], high frequency normalized units [hfnu ], sympathovagal balance [svb ] - lf / hf ratio) parameters of hrv. statistical analyses were carried out on statistical package for the social sciences 15.0 software supplied by ibm. results on continuous measurements were presented on mean sd unpaired t - test was used to compare the variables at the baseline characteristics of the two groups of patients. paired t - test was used to compare the clinical, hrv, following 6 week intervention. a total of 30 migraine patients were studied under conventional care (group cc) and 30 patients were studied under yoga with conventional care (group y) group. both the study groups were identical in demographic variables such as age, gender distribution, height and weight. no significant differences in mean duration of the illness, frequency and intensity were observed among the two groups [table 2 ]. comparison of demographic and clinical details of patients hit score was found significantly enhanced in group cc (75.43 0.88) compared to group y 66.60 3.21). headache frequency, intensity and hit scores were reduced in both groups of migraine patients [table 3 ]. effect of treatment on clinical variables significant reductions in monthly headache frequency, average pain intensity, and headache - related disability were seen in both the yoga and cc care groups. however, the group y showed improvements in all clinical outcome measures to a greater extent [tables 4 and 5 ]. significant reduction in the frequency of headache observed in the group y compared to group cc even the intensity of headache also reduced significantly in both groups, which was assessed by visual analogue scale (0 being no pain and 10 being the worst pain). at baseline headache intensity was 9.30 1.15 and 8.70 1.26 in group cc and group y respectively. after the end of 6 weeks intervention migraine patients reported headache intensity as 7.73 1.23 in group cc and 2.03 1.29 in group y. the group y has shown significant reduction in heart rate (p < 0.05) compared to group cc. group y has shown more reduction in lf nu and svb than group cc after the intervention but both group have shown statistically significant changes. group y has shown statistically significant increased hf power and hfnu [table 6 ]. comparison of self - perceived benefits of the intervention in two groups of migraine patients comparison of the assessment of therapy by two groups of migraine patients effect of treatment on hrv parameters in two groups of patients in the present study, migraine patients had very severe impact on their life (hit score more than 60) at baseline in both the groups. after 6 weeks of intervention, both group showed statistically significant reduction of hit score. but only yoga with conventional care group has shown headache has little to no impact on patient 's life. the self - perceived benefit of the intervention was measured at the end of the 6-week treatment period. the patients rated the perceived benefit of the therapy on a five point scale ranging from greatly worsened my clinical condition to greatly improve my clinical condition. migraine patients reported that intervention improved their clinical condition except 13.3% patients with conventional care only reported as neutral. in addition, patients assessed the therapy as more harmful than helpful, neither harmful nor helpful, or more helpful than harmful. all patients in yoga with conventional care and 73.3% of conventional care patients told that therapy was more helpful than harmful. this infers that yoga intervention with conventional care has better patient 's improvement and in turn acceptances, than the conventional care only, without any adverse effects. the time domain hrv parameters in yoga with conventional care has shown significant increase in mean r - r interval and reduced heart rate compared to conventional care group at the end of 6 weeks intervention. this denotes an improved vagal tone after yoga intervention. in frequency domain parameters hf power was increased and lf power was reduced with reduced svb (lh / hf ratio) reflecting increased parasympathetic and reduced sympathetic tone in patient with yoga intervention. low frequency and svb (lf / hf ratio) were reduced in conventional care group. considering all hrv parameters, yoga with conventional care however in case of conventional care only reduction in sympathetic tone was noted, without much alteration in the vagal tone. therefore, increasing vagal tone by yoga intervention may improve cardiovascular health and outcomes. along with this the study has demonstrated that migraine patients improved clinically in both groups following 6 weeks of intervention. reduction in the frequency and intensity was more in the yoga with conventional care group. clinical benefits, self - perceived health benefits and improvement in vagal tone with reduced sympathetic tone were found in yoga with conventional care group. for migraine treatment, regular exercise is frequently recommended. many of the studies have reported beneficial effects of aerobic exercise on both frequency and intensity of migraine as well as on the duration of the attacks and on patient 's well - being. reduction in pain, stress and anxiety perception in exercising persons may be due to modification in beta endorphin and hormonal secretion levels. however, around 22% of migraine patients complain as exercise was a trigger factor and hence some patients avoid exercise and were physically less active. in yoga, slower movements or even static muscular exercises are done with mindfulness and during the activities ; person has to think what they are doing during the act. they also have to feel the movements and develop awareness of body and body motion. hence yoga, a slow non - exertional aerobic exercise is more beneficial than pure aerobic exercise. studies have shown that yoga - type of mindful slow exercise is better than aerobic exercise or non - aerobic mindful exercise in enhancing mood and alleviating stress and depression. yoga has been effective in numerous chronic diseases such as asthma, diabetes, arthritis, fibromyalgia, depression, ischemic heart disease etc. yoga is shown improvement quality of life, reduce the episodes of headache attack and medication score in chronic tension type headache and migraine. ans is involved in control of homeostasis as the body 's response to this stress and same stress can be a trigger for migraine. one of the main pain relieving factors in migraine patients is sleep and the sympathetic system drive decreases during sleep. on the other hand, relaxation techniques decrease sympathetic system drive, causing a partial relief in migraine headaches. yoga has shown to reduce stress arousal patterns, reduce stress hormones such as cortisol and bring stable autonomic balance in health and diseases. the mechanism by which yoga practices bring the changes in ans can be explained by the following two hypotheses - in one hypothesis, exercise training improves vagal modulation through angiotensin ii and nitric oxide (no). the mechanism of increased vagal tone by exercise may be due to reduction of angiotensin ii. studies have shown plasma renin activity levels were lower in athletes than in untrained individuals or non - athletes and sedentary individuals. these findings were suggest that athletes with lower plasma renin activity would have lower angiotensin ii and higher associated levels of cardiac vagal activity. no may also play a role in increasing cardiac vagal control and may indirectly inhibit sympathetic influences. exercise training has been found to improve endothelial function and no bioavailability, hence indirectly reduces sympathetic activity. in another hypothesis, voluntary slow deep breathing and exercises reset the ans through stretch induced inhibitory signals and hyperpolarization currents propagated through both neural and non - neural tissue which synchronizes neural elements in the heart, lungs, limbic system and cortex. during inspiration, stretching of lung tissue produces inhibitory signals by action of slowly adapting stretch receptors and hyperpolarization current by action of fibroblasts. both inhibitory impulses and hyperpolarization current synchronize neural elements leading to the modulation of the nervous system and decreased metabolic activity i.e., parasympathetic state, human information processing during yogic exercises altered at cortical level in the hypothalamus and limbic system which in turn modulates the output of ans, hypothalamo - pituitary axis and immune responses. hence yoga along with convention care has definite effect on migraine frequency and intensity, which were assessed by clinical scales and hrv analysis. therefore it can reduce the dosage of drug in the conventional care and reduce the pharmacological adverse effects. significant clinical improvement was observed in both yoga and conventional care group with 6 weeks of intervention. yoga therapy showed an additional beneficiary effect on patients with migraine by reducing frequency and intensity. however, evaluation of autonomic parameters in patient with migraine before and after interventions was the novel one. yoga therapy showed added benefit on autonomic modulation by improving the vagal tone. to conclude	context and aims : migraine is an episodic disabling headache requiring long - term management. migraine management through yoga therapy would reduce the medication cost with positive health benefits. yoga has shown to improve the quality of life, reduce the episode of headache and medication. the aim of the present study was to evaluate the efficacy of yoga as an adjuvant therapy in migraine patients by assessing clinical outcome and autonomic functions tests.subjects and methods : migraine patients were randomly given either conventional care (n = 30) or yoga with conventional care (n = 30). yoga group received yoga practice session for 5 days a week for 6 weeks along with conventional care. clinical assessment (frequency, intensity of headache and headache impact) and autonomic function test were done at baseline and at the end of the intervention.results:yoga with conventional care and convention care groups showed significant improvement in clinical variables, but it was better with yoga therapy. improvement in the vagal tone along with reduced sympathetic activity was observed in patients with migraine receiving yoga as adjuvant therapy.conclusions:intervention showed significant clinical improvement in both groups. headache frequency and intensity were reduced more in yoga with conventional care than the conventional care group alone. furthermore, yoga therapy enhanced the vagal tone and decreased the sympathetic drive, hence improving the cardiac autonomic balance. thus, yoga therapy can be effectively incorporated as an adjuvant therapy in migraine patients.
mammalian development requires both maternal and paternally inherited genomes due to the presence of imprinted genes that are expressed from only one parental allele. imprinted expression is regulated by genomic imprinting, a classic example of epigenetics defined as a heritable change in gene expression caused by mechanisms other than changes in the underlying dna sequence and widely considered to be based on chemical modifications of dna or associated molecules. epigenetic regulation is likely to be a stepwise process where an initial epigenetic signal is recognized by an initiator that then targets changes in gene expression to specific loci. this gene expression state could then be maintained by additional factors, enabling modulation of the epigenetic signal at different steps in the process. in genomic imprinting the initial epigenetic signal is differential dna methylation of the imprint control element (ice) established during gametogenesis by the dnmt3a/3l de novo dna methyltransferase complex [79 ], and maintained on the same parental allele in all subsequent cell divisions by the dnmt1 maintenance methyltransferase. targeting of de novo dna methylation to the ice has been attributed to overlapping transcription at the gnas ice in the oocyte and to discrete sequence elements within the igf2r ice. an ice is associated with at least one, but usually a cluster of imprinted genes (figure 1a). dna methylation is regarded as a repressive epigenetic mark, but dna methylation of the ice is found on the same parental chromosome as the expressed imprinted protein - coding allele. this is because imprinted silencing is triggered by recognition of the unmethylated ice by an epigenetic initiator, whereas recognition is blocked on the other allele by ice methylation. the initiator can be a macro long non - coding (lnc) rna whose expression is controlled by the unmethylated ice (reviewed in), or a ctcf (ccctc - binding factor) insulator complex bound to the unmethylated ice (reviewed in). once imprinted gene silencing is established somatic dna methylation can be recruited and may serve to lock in the silent state ; however, this is a relatively rare event. in contrast to allele - specific somatic epigenetic modifications that are a consequence of imprinted gene silencing, allele - specific ice methylation arises during gametogenesis and is found in all somatic cells irrespective of imprinted expression. a characteristic of imprinted genes is that they show tissue - specific and developmental stage - specific regulation of imprinted expression. thus although genomic imprinting is thought to affect a small subset of mammalian genes, the true number has not yet been identified as only a limited range of tissues and developmental stages have been examined. in some cases tissue - specific imprinted expression results from a locus - specific change affecting only a single gene. however, in other cases a tissue - specific response is likely to be involved as genes from multiple loci are affected. tissue variation in imprinted expression is also likely to arise from modulation of epigenetic steps subsequent to the ice methylation, as this is a constitutive mark present in all somatic cells, at all developmental stages. first, imprinted expression can be lost by gaining dna methylation on the unmethylated allele of the ice in specific tissue types, as occurs for dlk1 and cdh15. widespread de novo methylation during gametogenesis leads to a large number of differentially methylated regions (dmrs) in the preimplantation embryo, most of which are lost by the postimplantation stage due to a gain of methylation on the other allele. however, a distinguishing ice feature is their protection from gain of methylation on the unmethylated allele, indicating that tissue - specific loss of imprinted expression by this mechanism is rare. second, genes can escape imprinted expression because the initiator is not present in a certain tissue. for example, neuron - specific loss of airn lncrna leads to biallelic expression of igf2r in neurons, and a similar mechanism may account for the loss of ube3a imprinted expression in glial cells [2123 ]. for the well - studied igf2r, kcnq1 and gnas clusters the lncrna identified as the initiator is expressed in almost all cell types, indicating that loss of imprinted expression due to absence of the initiator is also rare. third, a change in sensitivity to the initiator can cause a loss or gain of imprinted expression in certain tissues. for example, human igf2 loses imprinted expression in adult liver due to the use of an alternative promoter. this promoter presumably also uses alternative enhancers that are not blocked by insulator activity on the maternal allele, as h19 retains imprinted expression demonstrating that the initiator (insulator complex) is present. increased sensitivity to the initiator has been suggested to explain gain of imprinted expression of flanking genes in extra - embryonic cell lineages (figure 1b). this represents a tissue - specific rather than a locus - specific mechanism and therefore is likely to affect the largest number of genes. indeed genes showing extra - embryonic lineage (exel) specific imprinted expression form the majority of genes showing tissue - specific imprinted expression. extra - embryonic tissues are the placenta, umbilical cord and the yolk sacs that surround the mouse embryo and together provide a maternal interface to supply nutrients, remove waste and send signals that pattern the early postimplantation embryo (figure 1c). cell lineages that contribute to extra - embryonic tissues are derived at different development stages. trophectoderm and primitive endoderm differentiate in the preimplantation embryo at 3.5 and 4.5 days post coitum (dpc) respectively and are collectively known as the extra - embryonic lineages. the trophectoderm contributes to cell lineages in the placenta and parietal yolk sac, while the primitive endoderm contributes to the endoderm layers of the parietal and visceral yolk sacs (figure 1c). other extra - embryonic cell types are derived from the epiblast lineage after gastrulation at 7.5 dpc in the postimplantation embryo. exel specific imprinted expression has only been reported in the placental labyrinth and spongiotrophoblast and visceral yolk sac endoderm. extra - embryonic lineages appear to retain features of the early embryo that are lost in the epiblast lineage. for example, postfertilization the genome undergoes global dna demethylation, although in imprinted clusters methylation of the ice is protected, before global dna methylation increases again in the postimplantation embryo. the extra - embryonic lineages are derived before implantation and maintain a low level of dna methylation. in addition, the early cleavage - stage embryo undergoes imprinted x - inactivation before the paternal x becomes reactivated in the epiblast lineage and then undergoes random x - inactivation in the postimplantation embryo. in contrast, the extra - embryonic lineages retain imprinted x - inactivation throughout embryonic development. these examples show that extra - embryonic lineages retain some of the epigenetic features of the early embryo, which may relate to regulation of exel imprinted expression. this is supported by the example of sfmbt2, which shows imprinted expression in the blastocyst and throughout the early postimplantation embryo that later becomes restricted to extra - embryonic lineages. the repressive polycomb and ehmt2 histone methyltransferase complexes have been shown to be required to maintain silencing of some exel imprinted genes in placenta [3739 ], but why the initiator causes silencing of these genes only in extra - embryonic lineages remains unclear. a comprehensive genome - wide examination of active and repressive histone modifications of an extra - embryonic lineage has not yet been done, but low dna methylation is associated with open chromatin which may allow the initiator to silence more genes in extra - embryonic lineages by extending its influence over a larger chromosomal domain. genes reported to show exel specific imprinted expression have been associated with five imprinted clusters (figure 2), and also include a number of solo imprinted genes that are not associated with a known ice. in these five clusters, genes showing exel imprinted expression are non - randomly distributed lying further away from the ice than other imprinted genes (figure 2). for example, in the igf2r cluster the airn lncrna expressed from the paternal allele acts as the initiator, overlapping and silencing the igf2r gene in all cell types where it is expressed, but also silencing only in extra - embryonic lineages the slc22a2 and slc22a3 genes whose promoters lie between 159 and 237 kb upstream. similarly in the kcnq1 cluster the kcnq1ot1 lncrna acts as the initiator silencing kcqn1, cdkn1c, slc22a18, and phlda2 in multiple cell lineages, while tssc4, cd81, tspan32, aslc2, th, nap1l4 and osbpl5 that lie further away are only silenced in extra - embryonic lineages (figure 2). the igf2 cluster, where the initiator is a ctcf insulator complex, also shows this pattern with h19 and igf2 showing imprinted expression in most cell types, while ins2, which is more distant from the ice, shows imprinted expression only in exel tissues. the grb10 and peg10 clusters follow the same pattern with exel genes lying further away from the ice than other imprinted genes, although there are some exceptions. for example, asb4 shows imprinted expression in multiple embryonic and extra - embryonic tissues, but on the linear chromosome lies further from the putative ice for the peg10 cluster than the exel genes ppp1r9a, pon2 and pon3 (figure 2). however, although linear position on the chromosome may indicate proximity it is unclear how the genes are positioned relative to the ice in three - dimensional space in the nucleus. collectively the data from known exel genes in imprinted clusters indicate that imprinted silencing is extended over broader chromosome domains in extra - embryonic lineages than the embryo. the distribution of known exel imprinted genes in the genome indicates that imprinted silencing is extended in extra - embryonic lineages compared to the embryo. however, only five clusters have been shown to have exel genes whereas over 20 gametic dmrs that are proven or potential ices have been identified, indicating that more exel imprinted genes could be discovered. to determine the extent of imprinted silencing in extra - embryonic lineages a genome - wide unbiased approach is required. imprinted expression can be directly detected by rna sequencing (rna - seq) of f1 crosses between genetically distant mouse strains and identifying single nucleotide polymorphisms (snps) that show the same parental - allele expression bias in reciprocal crosses (figure 3a). this approach was used to investigate exel imprinted expression in placenta, and also to investigate tissue - specific imprinted expression in embryo and at different stages of brain development. the placental studies and the majority of the other tissue - specific studies reported the discovery of a small number of new imprinted genes with the exception of one that reported over 1300 new imprinted transcripts in embryonic and adult brain. however, two subsequent studies that repeated this work failed to confirm most of these novel imprinted transcripts, indicating that the majority were false positives, and providing lessons on the technical limitations of rna - seq and appropriate analysis that also apply to studies to uncover the extent of exel imprinted expression. false positives in detecting imprinted expression by rna - seq can arise from technical issues such as sequencing errors or misalignment of reads, or due to biological variation. therefore, a robust statistical approach is required to identify high - confidence imprinted gene candidates. statistical methods assuming random independent sampling were shown to not accurately model imprinted expression detected by rna - seq, indicating that the false positive rate must be determined empirically to account for systematic biases. to achieve this first an imprinting score was devised that combines deviation from biallelic expression with sequencing coverage to give a statistical confidence value for imprinted expression of each snp. second, a false positive rate was set by determining an imprinting score at which the snps detected in a mock comparison between f1 crosses of the same genotype, where no imprinted expression should be detected, was 5% of the number detected in the reciprocal cross. however, despite implementing this analysis pipeline only 6 of the 11 top candidates identified by rna - seq were validated, emphasizing the importance of biological replicates and validation by independent techniques to confirm imprinted expression. supporting evidence for imprinted expression can be gained from genome - wide parental allele - specific maps of dna methylation and histone modifications of f1 crosses. the repressed allele of some imprinted genes acquire somatic dna methylation, but as the majority do not, it is a poor predictor of imprinted expression. however, differential h3k4me3 and h3k27ac that mark active promoters and h3k36me3 that covers the gene body of expressed genes have been shown to predict imprinted expression. current methods for direct validation of imprinted expression are all based on quantification of a reverse transcription polymerase chain reaction (rt - pcr) product containing a snp in f1 animals, which is a laborious process if a large number of snps need to be validated. restriction fragment length polymorphisms and sanger sequencing can validate strong biases in parental allele expression, but the more sensitive quantification provided by pyrosequencing and sequenom massarray sequencing may be required to confirm subtle biases. determining the extent of imprinted silencing in exel tissues also requires an appropriate study system. placenta contains several lineages that can show exel imprinted expression but may also contain lineages that do not. thus exel imprinted expression may be masked or obscured by biallelic expression in other cell types within an organ (figure 3b). placenta also contains maternal cells from infiltrating maternal blood vessels and blood as well as the tightly associated decidua that can not be completely removed (figure 1c). this maternal contribution can create the false impression of maternal imprinted expression, requiring extra validation steps. these problems could be overcome by isolating a pure exel cell population, which has only been described for the visceral yolk sac endoderm. the distribution of known exel imprinted genes indicates that imprinted silencing by epigenetic initiators is extended in extra - embryonic lineages compared to the embryo. the most parsimonious explanation for increased sensitivity to the initiator in extra - embryonic lineages is that the initiator acts by the same mechanism as in the embryo, but is able to silence a larger chromosomal domain, perhaps due to a different chromatin environment or downstream process. for example, in the igf2r cluster the airn lncrna is the initiator that silences the igf2r promoter by transcriptional interference independent of the lncrna product. in contrast, in placenta the airn rna product recruits and targets the repressive ehmt2 h3k9me2 methyltransferase to the slc22a3 promoter, which is necessary for its silencing. these results could be reconciled by a model whereby airn transcription initiates silencing of exel genes by interfering with the formation of enhancer - promoter interactions, then as a secondary step ehmt2 could maintain imprinted silencing. understanding how more genes become subject to genomic imprinting in exel tissues will not only offer insights into the biology of extra - embryonic lineages, but could identify epigenetic mechanisms that operate to lesser degrees in all somatic tissues. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest of special interest of outstanding interest	gene silencing in imprinted gene clusters is established by an epigenetic initiator that is often a long non - coding (lnc) rna. the clustered organization of known imprinted genes indicates that the initiator extends imprinted silencing over broader chromosomal domains in extra - embryonic lineages compared to the embryo. we propose that extension of imprinted gene clusters may result from known epigenetic differences between extra - embryonic and embryonic lineages that alter the behavior of epigenetic initiators. new rna sequencing technology will enable the full extent of imprinted silencing in embryonic and extra - embryonic lineages to be defined, but appropriate analysis and cell systems are required, which we define here based on a review of recent studies.
sirolimus (rapamycin) is a macrolide lactone that was approved for use as an immunosuppressant in 1999, but not for use in liver transplantation. it suppresses the t - cell response to interleukin-2 by binding to and inhibiting the mammalian target of rapamycin (m - tor). there are reports of benefits and risks for the use of sirolimus (srl), in liver transplantation [217 ]. investigators have reported an increase in renal failure, hepatic artery thrombosis, and overall post - transplant mortality compared to the use of calcineurin inhibitors (cnis) [6, 11, 18 ]. in contrast, other studies report good outcomes and that srl has a renal - sparing effect. our center has routinely used srl for immunosuppression following liver transplantation [1, 1826 ]. we have used srl as primary therapy along with a cni in the early post - operative period starting in january 2000 until it received a black box after that, we converted patients to srl therapy at various time points after discharge from the hospital. internal review of our database showed no increase in morbidity and/or mortality in our srl patients compared to a standard therapy of cni+ enteric - coated mycophenolate sodium (mps). our preliminary data suggested that srl can reduce donor graft rejection and could ameliorate renal injury secondary to increased use of cni. this study seeks to determine if there is an increase risk of complications associated with the use of srl in the first year after liver transplantation. we collected data from all patients who had received srl in the first year after liver transplant. because of previously reported markers of poor outcome associated with the use of srl, this study included overall patient mortality, donor graft loss, and renal function. further, because our previous data suggested that srl may decrease the incidence of rejection, we measured the rates of acute cellular rejection (acr) and steroid resistant rejection (srr) in our patient population. we retrospectively reviewed the university of colorado denver transplant database and collected data on all patients who received a liver transplant between january 2000 and november 2009. this included 688 patients. an independent investigator extracted data from both electronic and paper files. the pattern of immunosuppressant use allowed us to construct five study categories from the 688 patients in the database. patients were assigned to one of the five categories according the immunosuppressive therapy given during the first year following transplantation. patients received a cni + mps at time of discharge (primary therapy) and through the first year of therapy. patients received a cni + mps at time of discharge ; srl was added within the first 6 months and continued through the first year. patients received a cni + mps at time of discharge ; srl was added within the first 6 months and discontinued before the first year. srl was started as primary immunosuppression before discharge from hospital after transplantation combined with other varying therapies including a cni and continued for the first year. srl was started as primary immunosuppression before discharge from hospital after transplantation combined with other varying therapies including a cni and discontinued before the first year. primary endpoints graft failure rate (time to graft failure / death). mortality rate (survival time). graft failure rate (time to graft failure / death). mortality rate (survival time). graft failure rate (time to graft failure / death). mortality rate (survival time). secondary endpoints acute cellular rejection rate (proven by biopsy or clinical parameters).steroid resistant rejection (biopsy proven and treated with thymoglobulin or okt3). graft rejection was diagnosed by liver biopsy or by elevation of liver function test in the absence of other causes of graft dysfunction. if the liver function tests were still elevated after treatment with pulse dose steroids and the patient had not received a biopsy, one was performed. acr that was not responsive to pulse dose steroids and had biopsy proven rejection was then deemed srr and treated with thymoglobulin or okt3.(3)renal function as defined by gfr was recorded at discharge, 1 month, 6 months, and 1 year. acute cellular rejection rate (proven by biopsy or clinical parameters).steroid resistant rejection (biopsy proven and treated with thymoglobulin or okt3). graft rejection was diagnosed by liver biopsy or by elevation of liver function test in the absence of other causes of graft dysfunction. if the liver function tests were still elevated after treatment with pulse dose steroids and the patient had not received a biopsy, one was performed. acr that was not responsive to pulse dose steroids and had biopsy proven rejection was then deemed srr and treated with thymoglobulin or okt3.(3)renal function as defined by gfr was recorded at discharge, 1 month, 6 months, and 1 year renal function as defined by gfr was recorded at discharge, 1 month, 6 months, and 1 year. univariate : we computed the p values for graft failure (failure - free survival) and mortality (patient survival) rate comparisons using the log rank test. the event - free (survival) curves were computed using the kaplan - meier method. for graft failure, mortality, and rejection events, the event rate in any group was computed as (1)event rate = 1000(total number of eventstotal personmonths followup). this rate is in units of events per 1000 person - months (p - mos) of followup and is a summary statistic for the corresponding time to event curve. rate ratios are also reported where the denominator is the rate in the reference control group and the numerator is the rate in the group being compared to the control group. the p values for comparing failure rates and mortality rates are computed using the log rank test. for rejection rate comparisons, this assumption was made (in part) since the time to each acute rejection episode was not available. analysis of deviance results showed that the poisson model is a good fit to the data. a one - way anova was used to compare means for continuous variables at baseline including gfr across the five groups after confirming that a parametric model was appropriate for each on the original scale. the chi - square test / fisher test was used to compute p values for comparing binary variables such as gender, hypertension, diabetes, and any particular diagnosis such as hepatocellular carcinoma (hcc) or hepatitis c virus (hcv). the p value for comparing race / ethnicity was also computed using the chi - square test. the five groups of patients are listed with the patient demographics listed in table 1. the demographic features of patients in groups 2 to 5 were comparable to the control group 1. however, patients differed in their meld score and warm ischemic time (wit). patients in groups 4 and 5 (primary treatment group) had lower meld score (18 versus 22). wit was longer in group 4 patients compared to controls ; however, this was not statistically significant. the conversion groups (2 and 3) had a lower pre - transplant gfr compared to the control group 1. there were fewer transplants done for hcv in group 4 and less for etoh in group 3 patients. graft survival figure 1 depicts plots and tables of graft survival across the five groups represented by failure rates per 1000 person - months followup. there was no statistical difference in graft survival among the five groups of patients. figure 1 depicts plots and tables of graft survival across the five groups represented by failure rates per 1000 person - months followup. there was no statistical difference in graft survival among the five groups of patients. patient survival figure 2 depicts plots and tables of patient survival across the five patient groups represented by failure rates per 1000 person - months follow up. figure 2 depicts plots and tables of patient survival across the five patient groups represented by failure rates per 1000 person - months follow up. acute cellular rejection table 2 depicts acr rates per 1000 person - months of followup. acr rates in the groups 4 and 5 (primary treatment group) where srl was started before discharge (p value.0001 and.0007) are about 50% less than the control group 1 and patients in the conversion group. acr rates in the groups 4 and 5 (primary treatment group) where srl was started before discharge (p value.0001 and.0007) are about 50% less than the control group 1 and patients in the conversion group. steroid resistant rejection table 3 depicts ssr rates per 1000 person - months of followup and reflects the results from acr. the incidence of acr in the primary treatment groups (4 and 5) (p value =.0004 and.038) was about 50% less than the control group 1.since acr and srr rates show clinically and statistically significant differences, the adjusted rates were also computed using poisson regression as above with all 20 covariates included in the model. the 20 covariates were : recipient age, meld score, pre - transplant gfr, bmi, donor age, cit, wit, male gender, ethnicity, cadaveric donor, pre - transplant diabetes, pre - transplant hypertension, hcc, hcv, psc, laennec 's cirrhosis, hbv, aih, nash, and pbc. the adjusted rate ratios are similar to the unadjusted rate ratios showing that the primary treatment group had about half the rejection rate compared to the control group. table 3 depicts ssr rates per 1000 person - months of followup and reflects the results from acr. the incidence of acr in the primary treatment groups (4 and 5) (p value =.0004 and.038) was about 50% less than the control group 1. since acr and srr rates show clinically and statistically significant differences, the adjusted rates were also computed using poisson regression as above with all 20 covariates included in the model. the 20 covariates were : recipient age, meld score, pre - transplant gfr, bmi, donor age, cit, wit, male gender, ethnicity, cadaveric donor, pre - transplant diabetes, pre - transplant hypertension, hcc, hcv, psc, laennec 's cirrhosis, hbv, aih, nash, and pbc. the adjusted rate ratios are similar to the unadjusted rate ratios showing that the primary treatment group had about half the rejection rate compared to the control group. glomerular filtration rate figure 3 depicts the glomerular filtration rate (gfr) (ml / min) for the five groups of patients at the times of hospital discharge, 6 months, and 1 year. as seen in table 2, the median pre - transplant gfr of the conversion groups (2 and 3) (p value =.0136 and.074) was significantly less compared to the control group 1 at time of transplantation. in figure 3, the percent change in gfr was calculated from pre - transplant value to the value for gfr that was obtained at 1 year after transplantation. at 6 months, patients in categories 2 and 4 had a drop in mean gfr (pvalue =.0793 and.0465) that was significantly more than the corresponding drop in controls. at one year, only group 4 had a significantly worse grf than the control group with a change in grf of 17.3% (p value =.0320). while the behavior in group 5 is similar to group 4, the sample size is lower, making the p value larger.at one year, 46 of the original 328 persons in the control group were missing, including 17 who died or were re - transplanted. we assumed that the missing patients had outcomes that were worse than those who remained in the databank. thus, the apparent mean of 6.54 ml / min decrease in gfr in the control group could be an underestimate. the decrease in gfr is likely more than the observed 6.54 ml / min if we included those patients lost to followup. so, the one year mean gfr difference between the control group and the 14 ml / min drop in category 4 patients (srl kept) is likely smaller than the results shown. figure 3 depicts the glomerular filtration rate (gfr) (ml / min) for the five groups of patients at the times of hospital discharge, 6 months, and 1 year. as seen in table 2, the median pre - transplant gfr of the conversion groups (2 and 3) (p value =.0136 and.074) was significantly less compared to the control group 1 at time of transplantation. in figure 3, the percent change in gfr was calculated from pre - transplant value to the value for gfr that was obtained at 1 year after transplantation. at 6 months, patients in categories 2 and 4 had a drop in mean gfr (pvalue =.0793 and.0465) that was significantly more than the corresponding drop in controls. at one year, only group 4 had a significantly worse grf than the control group with a change in grf of 17.3% (p value =.0320). while the behavior in group 5 is similar to group 4, the sample size is lower, making the p value larger. at one year, 46 of the original 328 persons in the control group were missing, including 17 who died or were re - transplanted. we assumed that the missing patients had outcomes that were worse than those who remained in the databank. thus, the apparent mean of 6.54 ml / min decrease in gfr in the control group could be an underestimate. the decrease in gfr is likely more than the observed 6.54 ml / min if we included those patients lost to followup. so, the one year mean gfr difference between the control group and the 14 ml / min drop in category 4 patients (srl kept) is likely smaller than the results shown. only group 2 patients were significantly less than the control group 1 (p value =.0011). all groups at 1 year had a mean level less than 6.0 (ng / ml). our data shows that there is no increase in patient death or donor graft loss when srl is used as primary therapy or when patients are converted to srl following therapy with other immunosuppressants. further, we have no evidence that suggests srl has deleterious effects upon renal function. in contrast, our data suggests that primary therapy with srl, but not conversion to srl, reduced the incidence of acute and steroid resistant rejection in transplant recipients. sirolimus has been widely used in renal transplantation but is not currently approved for use in liver transplantation. there are reports that srl has negative effects on outcome including hat, delayed wound healing, and increased mortality compared to standard immunotherapy [14, 15 ]. our center has used srl therapy after liver transplantation at a number of different time points. few other centers have this large experience using srl in liver transplantation coupled with a nearly complete data set. data from our previous publications did not support claims that the use of srl is associated with negative outcomes [1, 18, 19, 2226 ]. rather, our previous data suggested that mtor inhibitors are safe when used as single therapy or in addition to cni after liver transplant. we observed this when srl was used as a primary immunotherapy or if srl was added later. we administered srl to patients in two regiments : we gave srl immediately after liver transplant or added / substituted srl within the first year following transplantation. there was a range of reasons for different combinations of therapies over the first year mostly from evolving protocols. the conversion and primary treatment groups represents a more heterogeneous collection of immunotherapies and must be interpreted with caution. however, comparison between the categories of patients within both groups did not show significant differences. we did not find differences between patient and/or donor graft survival between our patients who received srl compared to those who received cni + mps. similarly, there were no differences in patient and graft outcome that were related to whether srl was primary or conversion therapy. patients that received srl as primary immunosuppression, defined as srl administered before discharge from the hospital after liver transplant, had less acr and srr rejection. this was true for patients that stayed on srl for one year and for patients that stopped using srl before one year. this finding was statistically significant, and the rates of rejection were 50% less than the control of cni + mps. finally, we reviewed our data to see if there is a difference in renal function when srl is used. srl has been used to reduce the level of cni, thus, protecting the kidney. first, since some patients die or are re - transplanted before 6 months or 1 year, their corresponding missing gfr values create a survival biases. thus, the estimates of mean gfr at one year in particular may be biased because sicker patients that have a high mortality and also a greater reduction in gfr. second, as seen in figure 3, our center keeps the tacrolimus levels in all groups at one year less than six. thus, the lower levels of cni may independently ameliorate the negative effects upon renal function. first, it is a retrospective study and the conversion and primary treatment groups contain a heterogeneous group of practices. however, this data is almost 100% complete and accurately describes our practice patterns and subsequent outcome when using srl in a variety of combinations during the first year. secondly, because it is retrospective and we only reviewed our practice patterns for the first year, our groups are not set up to adequately assess long - term renal outcome. rather, we can only predict kidney function within the first year of transplantation. finally, we do not routinely perform protocol biopsies to diagnose acr, so we could have overestimated the actual incidence of acr in our patient population. however, the same diagnostic criteria were applied to all categories of patients that we studied. therefore, it is unlikely that our diagnostic approach favored the diagnosis of acr in one category of patient compared to another. in conclusion, our goals for this study were to determine if our use of srl during the first year after liver transplant increased mortality or morbidity. surprisingly, srl use as a primary therapy decreased our rates of both acr and srr. based on this review, we will continue to use srl in liver transplant recipients. with the introduction of new mtor inhibitors, such as everolimus,	some studies suggest that sirolimus (srl) is associated with an increased risk of death in liver transplant recipients compared to treatment with calcineurin inhibitors (cnis). we compared patients who received srl or cni in the first year after liver transplant. our database included 688 patients who received a liver transplant. the patients were divided into groups. (1) cni + mps (mycophenolate sodium) at time of discharge. (2) cni + mps at time of discharge ; srl was added within the first 6 months and continued through the first year. (3) cni + mps at time of discharge ; srl was added within the first 6 months and discontinued before the first year. (4) srl as primary immunosuppression. (5) srl as primary immunosuppression and discontinued before the first year. we used mortality and graft loss as the primary measures of outcome. we also quantified renal function using the change in glomerular filtration rate (gfr), the presence of biopsy proven acute cellular reject (acr), and steroid - resistant rejection (srr). there were no significant differences in mortality or graft loss. there was no difference in patient or graft survival. patients that received srl as primary immunosuppression had 50% less rejection compared to controls.
	chromium is found in the environment in two major forms : reduced criii and crvi, or chromate. chromate, the most biologically active species, is readily taken up by living cells and reduced intracellularly, via reactive intermediates, to stable criii species. criii, the most abundant form of chromium in the environment, does not readily cross cell membranes and is relatively inactive in vivo. however, intracellular criii can react slowly with both nucleic acids and proteins and can be genotoxic. we have investigated the genotoxicity of criii in vitro using a dna replication assay and in vivo by cacl2-mediated transfection of chromium - treated dna into escherichia coli. when dna replication was measured on a criii - treated template using purified dna polymerases (either bacterial or mammalian), both the rate of dna replication and the amount of incorporation per polymerase binding event (processivity) were greatly increased relative to controls. when transfected into e. coli, criii - treated m13mp2 bacteriophage dna showed a dose - dependent increase in mutation frequency. these results suggest that criii alters the interaction between the dna template and the polymerase such that the binding strength of the dna polymerase is increased and the fidelity of dna replication is decreased. these interactions may contribute to the mutagenicity of chromium ions in vivo and suggest that criii can contribute to chromium - mediated carcinogenesis.imagesfigure 3.
in species where males hold the resources required for breeding, reproductive behavior is often associated with bourgeois territorial tactics that involve defense of resources in an attempt to monopolize mating opportunities [14 ]. bourgeois tactics are usually adopted by males who achieve competitive superiority (and also attractiveness) thorough behavioral (e.g., antagonistic behaviors) and morphological investments (e.g., large body size). these exclude less competitive males from reproduction. such intrasexual competition for mating may lead to the evolution of alternative reproductive tactics (arts) [5, 6 ]. competitively inferior, less attractive, and smaller subordinate males are unlikely to monopolize opportunities for mating, but they may evade monopolization by bourgeois males by using reproductively parasitic tactics that often involve sneaky behavior and the theft of reproductive efforts by bourgeois males [1, 3, 4, 6 ]. arts inherently give rise to sperm competition, that is, a competition among sperm from different males for the fertilization of ova. subordinate males exhibiting sneaky mating behavior (i.e., sneakers) face a high probability that their sperm will encounter sperm competition (sperm competition risk), as they obtain opportunities for fertilization by taking part in the mating of others. however, this is not necessarily true for bourgeois males, which engage in mating without any rivals (low sperm competition risk) unless sneakers frequently intrude in their mating. the theoretical game model of sperm competition predicts that sneakers are forced to make a greater investment in ejaculate than are bourgeois males due to the increased risk of sperm competition. differential testes investment, as well as behavioral and morphological investments, are found in many species with arts (e.g., [915 ] ; see also [1, 1618 ], for review). arts have been reported in a variety of taxa [4, 6 ] and particularly in fish [1, 3, 16 ]. arts are prevalent and diversified in the lake tanganyika cichlid tribe lamprologini, although such arts can also be found in other lake tanganyika tribes (and references therein). in the lamprologini, arts are typically dichotomous, that is, bourgeois territorial and parasitic sneaky tactics (e.g., cooperative brooders, julidochromis ornatus, julidochromis transcriptus [22, 23 ], and neolamprologus pulcher ; a shell brooder, lamprologus callipterus [3, 2527 ] ; a rock - hole brooder, telmatochromis temporalis). in t. temporalis, piracy tactic (i.e., the takeover of spawning events from territorial males by the largest males) also appears but seasonally. in the shell - brooder telmatochromis vittatus, four reproductive tactics have been reported (sneaker, satellite, territorial, and piracy tactics,), with piracy tactics being dependent on conditions. furthermore, mixed paternity is found in biparental breeders (neolamprologus meeli and variabilichromis moorii), suggesting the presence of parasitic tactics. a lamprologini cichlid, neolamprologus brevis (a synonym of neolamprologus calliurus), is characterized as an obligate shell brooder that spawns and cares for broods inside empty gastropod shells. the wonzye population, which is located in a southern region of the lake, occurs in a wide range of habitats in the littoral zone, from shallow rocky habitats to relatively deep offshore sandy - bottom habitats. however, ecological information for this fish is only available from a particular habitat, shell patches consisting of a number of gastropod shells (mean = 96 shells,). it is not known how this fish uses other habitats. a female l. callipterus occupies a shell for breeding, which lasts for 1014 days, during which period she spawns and subsequently provides brood care. l. callipterus females that are ready to spawn or care for broods are not found in the patches, suggesting that they leave the patches immediately after the completion of a breeding event. n. brevis also breeds in the patches, and a number of females and several males are found at the same time, suggesting that this fish has a multimale polygynous mating system. two other species, neolamprologus fasciatus and t. vittatus, also use shell patches for breeding. l. callipterus is not aggressive toward n. brevis and tolerates their using the unoccupied shells for breeding. mature males of different size classes were found in the shell patches, with larger males floating several dozen centimeters and sometimes more than 1 m above the patches and smaller males residing in the shells. this resembles the bourgeois nesting males and parasitic dwarf males of l. callipterus [18, 26, 36, 37 ]. if parasitic males are present in this fish population, it is expected that there exist discontinuous differences in aggressiveness and relative testes investment (testes mass relative to body mass) between males of the different size classes [8, 15, 18, 38 ]. in this study, we examined habitat use and the presence of arts in n. brevis in the wonzye population. field studies using scuba diving techniques were conducted at wonzye point (843s, 3108e) near mpulungu, zambia, from november to december 2002, and october to november 2007. where this population is found, the ground is covered with rocks from the shoreline to about 9 m depth (rocky habitat) or with sand to about 711 m depth (sandy bottom habitat). in this population, n. brevis occurred in both habitats, each of which was divided into two subhabitats : (1) sandy bottoms that were almost covered with shells at 911 m depth (662.5 shells / m,) (henceforth shell bed or sb) ; (2) sandy bottoms on which shells were widely distributed at 711 m depth (0.12 shells / m,) (henceforth separated shells on sandy bottom or ss) ; (3) midwater aggregations in rocky habitats that often consisted of > 100 individuals, at 49 m depth (henceforth midwater aggregation or ma) ; (4) shell patches of l. callipterus in rocky habitats, which often consisted of > 100 shells, at 49-m depth (shell density = 496.8 shells / m,) (henceforth shell patch or sp). habitat use was determined by differences in size structure, frequency of reproductively active individuals, and behavior among the habitats. to examine the size structure, we captured randomly selected n. brevis using gill nets in three habitats from november to december 2002 (nsb = 60, nss = 63, nma = 34) and in one habitat from october to november 2007 (nsp = 156, see below for details). we brought the collected fish back to the laboratory and measured their standard length (sl) to nearest 0.12 mm and body mass (bm) to nearest 0.002 g. all fish sampled from sandy bottom habitats and midwater aggregations and some of the fish sampled from the shell patches (nmale = 48, nfemale = 31) were dissected immediately after sacrificing among crushed ice or by anesthetizing with eugenol. we then weighed their gonad mass (gm) to nearest 0.002 g and sexed individuals. male maturity was determined from developmental stages of gonads, because testes were either white and enlarged or transparent and threadlike. brood - caring females and females whose ovaries were filled with large, orange - colored (i.e., ripe) eggs were labeled as mature. consequently, as the minimum size of mature females was 31.6 mm sl, we considered females 31.6 mm sl to be sexually mature, even if their ovaries were filled with unripe eggs. for the dissected fish (nsb = 60, nss = 63, nma = 34, nsp = 79), we calculated the gonad - free condition factor (i.e., bm - gm 10/sl) and compared it among habitats and by sex. we also calculated the gonadosomatic index (gsi) of the dissected males as an estimate of testes investment and compared it among habitats (see below for details). to examine the size structure and describe the reproductive ecology of n. brevis we determined the number of males and females in each shell patch, the maturity of males, and the breeding status of females from october to november 2007. we also examined whether there was a spawning cycle in n. brevis, which would be an important aspect of the reproductive ecology of this fish because n. brevis may have a periodic spawning cycle given that the reproductive cycle of the patch - owner species l. callipterus displays weak lunar - related periodicity. additionally, it was important to describe the mating system, as the different sampling days among habitats may have influenced reproductive parameters. in 17 randomly selected shell patches, we counted the numbers of n. brevis individuals that remained in shells in each patch once a week from 19 october to 12 november 2007, during which period a new and full moon occurred twice and once, respectively. consequently, we obtained the counts for a continuous 6-week period in each shell patch. because n. brevis resides in a shell in a head first position, the presence of an individual can be identified by observation of the caudal fin. we therefore visually checked whether n. brevis were present in shells by observing the entrance of shells. however, this visual counting could lead to an underestimation due to missing individuals that had retreated into the inner part of the shells or an overestimation due to the inclusion of males hiding in shells. to confirm the exact number of n. brevis and clarify the presence and absence of fish in a shell, all shells in the shell patches would need to be crushed. because the application of this method is destructive, it was performed in only a portion of the shell patches (n = nine patches), minimizing the impact on the habitats. at the end of the observation (12 november 2007), we counted the number of shells in the nine shell patches by visual inspection and captured all shells and n. brevis individuals that were floating above the shell patches or that remained near the shells (n = 145). in the remaining eight nests, all fish and shells that were sampled were brought back to the laboratory. to determine the exact number of n. brevis the numbers revealed by visual counting and by actual counting (i.e., counting after crushing of shells) were strongly positively correlated (poisson regression model, = 52.42, df = 1, p 0.05), indicating that there was no spawning cycle corresponding to the lunar cycle. actual counting (i.e., counting after the crushing of shells) determined that a mean number of 16.1 n. brevis (sd = 11.3) occurred in a shell patch, with both males and females present (females : mean sd = 12.0 9.0, males : 4.1 3.2, n = 9 patches). of the females, half were brood - caring females (mean number sd = 5.9 6.5, n = 9 patches) and the other half were gravid females that had not yet spawned (6.1 4.4, n = 9 patches). of the males, 40% were floating above the patches (mean number sd = 1.6 0.7, n = 9 patches), whereas 60% remained close to or in the shells (2.6 2.7, n = 9 patches). floating males (mean sd = 60.4 8.3 mm sl, n = 25) were much larger than males residing in shells (35.2 4.6 mm sl, n = 23, glm, f1,46 = 164.63, p malema > malesb = maless > males in a shell within the shell patches, after a bonferroni correction, figure 1). females in the shell beds were in better condition than those in other habitats (glm, f1,88 = 8.89, p < 0.001, after a bonferroni correction, figure 2). likewise, there was a variation in the condition factor for males among habitats : all males in the shell patches were in poorer condition compared with males in other habitats (glm, f4,138 = 75.06, p < 0.001, after a bonferroni correction, figure 2). there was also a great difference in testes investment (estimated by gsi) among habitats : males occupying shells within the shell patches had the greatest testes investment among males, and males in sb had the smallest testes investment (glm, f4,139 = 49.49, p < 0.001, figure 3). females in the shell patches spent more time in shells than did those in sandy - bottom habitats (kruskal - wallis, test, = 15.16, df = 2, p = 0.001, table 1). larger males in the shell patches seldom entered the shells, but this was not the case for the small males (kruskal - wallis, test, = 82.58, df = 4, p < 0.001, table 1). n. brevis individuals usually floated a few meters above the lake bottom in midwater aggregations, whereas in sandy - bottom habitats, individuals foraged in relative close proximity to (several dozen centimeters to a few meters above) the bottom where shells used for shelter were distributed. the feeding frequency of females was lower in the shell patches than in sandy - bottom habitats (negative binomial glm, = 9.78, df = 2, p < 0.01, table 1). in accordance with this tendency of females, male feeding frequency was also lower in the shell patches than in the other habitats (negative binomial glm, = 66.51, df = 4, p < 0.001, table 1). aggressive interactions between n. brevis females occurred only in the shell beds (poisson glm, = 19.71, df = 2, p < 0.001, table 1). aggressive behavior toward other species was observed in sandy - bottom habitats but not in the shell patches (poisson glm, = 16.59, df = 2, p < 0.001, table 1). male attacks on conspecifics were much more frequently performed by males floating above the patches and males in sandy - bottom habitats than by the small males in the shell patches and midwater aggregations (negative binomial glm, = 74.22, df = 4, p < 0.001, table 1). a similar tendency was found in attacks on heterospecifics (poisson glm, = 67.36, df = 4, p < 0.001, table 1). n. brevis obligately uses empty gastropod shells for breeding and seldom uses substrates other than shells for shelter. therefore, the distribution of shells is expected to strongly influence the distribution of n. brevis. n. brevis was found in every habitat of the wonzye population where shells were present. however, breeding females occurred only in the shell patches. this indicates that the breeding events of n. brevis exclusively take place in the shell patches for this population. in contrast, mature males and gravid females were also found in sandy - bottom habitats, although much less frequently than in rocky habitats. both sexes, with the exception of the small males in the shell patches, were smaller in sandy - bottom habitats than in rocky habitats. this suggests that n. brevis spend their early life - history stages in sandy - bottom habitats and migrate to shell patches for reproduction, possibly through shells distributed on the sand bottom between the shell bed and rocky habitats (figure 4). such movements among habitats are often found in fish (e.g., [41, 42 ]) and are favored if the benefits exceed the costs [4345 ]. in n. brevis, the shell patches may be a favorable habitat for breeding compared with other habitats. shell patches contained a number of conspecific and heterospecific females in the wonzye population (this study and), and these patches likely enable more effective avoidance of egg predation through dilution effects than would breeding in sandy - bottom habitats. however, the shell patches may be unsuitable for later growth and survival compared with other habitats. the complex structures of rocks, for example, would disturb food delivery (plankton drifting in the water column) to the shell patches. consequently for feeding, n. brevis would be required to float in the midwater column, where the fish would be exposed to predation risk by piscivorous (e.g., lepidiolamprologus spp. and lamprologus spp.) and scale - eating (perissodus spp.) fish. in contrast, sandy - bottom habitats may provide reduced predation risk and high food delivery near shelters. in these habitats, n. brevis can feed in close proximity to shells because there are no rocks disturbing food delivery, and individuals could hide in the shells immediately in response to the perceived risk of predation. indeed, we found that n. brevis in the shell patches infrequently fed on plankton and consequently had poorer somatic condition compared with n. brevis individuals in other habitats. this suggests that sandy bottom habitats and midwater aggregations may be favored for n. brevis to invest in somatic growth. we therefore hypothesize that n. brevis chooses favorable habitats according to the stage of the life history. however, there is another possible explanation for the movements among habitats. in wonzye, n. brevis females who had finished broodcaring would be obstacle, because they would no longer yield any profits for l. callipterus (such as avoidance of egg predation through dilution effect, see above). if so, l. callipterus would expel them so as to secure shells for their own breeding. then, n. brevis individuals would be forced to migrate to near other habitats, that is, separated shells on sandy bottom. these hypotheses are not mutually exclusive, and it is therefore possible that both factors are responsible for movements among habitats. these males were divided into two types based on morphological and behavioral phenotypes that were clearly differentiated. males floating above the shell patches were larger in body size and exhibited relatively frequent aggressive behavior toward conspecifics. males who usually stayed close to or in the shells were smaller and less aggressive. these discontinuous differences in phenotypes are found in the related shell - brooding cichlids with arts, l. callipterus [3, 26, 37 ], and t. vittatus. in l. callipterus, there are two types of reproductively parasitic males : sneaker males dart toward the shell where spawning takes place and ejaculate from the entrance of the shell and dwarf males wriggle past spawning females and take up residence behind them so as to ejaculate in close proximity to females. in t. vittatus, sneaker males get inside the shell for several seconds or longer and ejaculate, and they then leave the shell immediately. in n. brevis, most sexually mature small males were found solely in shells where spawning had not yet taken place. although these males may be simply hiding in the shells, it is possible that they may have a sit - and - wait tactic for sneaking in which they lie in wait for a female to spawn. because the sit - and - wait tactic enables n. brevis males to position themselves behind females, smaller bodies are required to succeed with this approach. indeed, these males were marginally smaller than the males that remained in shells with females. we also found several males entering the shells after females, some of which had not yet mated. additionally, small male n. brevis individuals were found behind females in shells (i.e., in the innermost parts of the shells,). together, these observations indicate that small mature male n. brevis may have a wide range of parasitic sneaky behavior types so as to steal fertilization opportunities. if the small males really do employ parasitic tactics, their ejaculation should usually occur during mating by territorial males. in this situation, theory predicts that their testes investment is greater than territorial conspecifics. this was the case in n. brevis as well as other lake tanganyika cichlids employing arts [18, 21, 22, 26, 28, 30, 37 ], but some controversy about this remains (see). testes investment among the floating males was the same as for males found in the midwater and sandy - bottom habitats and was much smaller than that of males within the shells of the shell patches. these findings are interpreted as demonstrating the presence of arts, although we did not directly observe the spawning behavior of n. brevis. together with the finding of a number of females in the shell patches, this suggests that n. brevis exhibits multimale polygyny, in accordance with a previous study. this mating system differed from that of other shell brooders found in the shell patches : both l. callipterus and t. vittatus are polygynous. the hierarchy among these species and the reason they share a shell patch remain to be investigated. further studies that include paternity analysis are needed to clarify the mating system of n. brevis. in conclusion, the results strongly suggest that the mating system of n. brevis is characterized by movements among habitats and multimale polygyny with arts. arts seem to be a common attribute in the lake tanganyika cichlid tribe lamprologini, particularly in shell brooders. it is possible that the specific shape of the empty gastropod shells is at least partly responsible for this universality. several studies have suggested that the presence of a space in a breeding substrate that large bourgeois males can not enter is an important factor allowing sneakers to gain access to females [23, 28, 37 ]. as the entrance of the shells is very small (3.3 cm on average in wonzye), entering the shells requires specialized morphologies (e.g., small body size [27, 37 ]). however, territorial males are selected for large size because of the intense male - male competition and, particularly in case of l. callipterus, the requirements of shell transportation (e.g., [4951 ]). this difference in optimal body size between tactics leads to biased access to the shells : can penetrate the spawning substrate to a depth that territorial males can not reach. in accordance with this pattern, in n. brevis, smaller males entered shells, whereas large territorial males seldom entered. we therefore assume that the shape of a breeding substrate is one of the key factors leading to the prevalence of arts among the lake tanganyika shell - brooding cichlids.	alternative reproductive tactics (arts) are found in several lake tanganyika shell - brooding cichlids. field studies were conducted in the wonzye population to examine reproductive ecology and arts in the lake tanganyika shell - brooding cichlid neolamprologus brevis. we discovered that this fish occurred in both rocky- and sandy - bottom habitats, but in rocky habitats, brood - caring females exclusively occurred in shell - patches that another cichlid species created. all n. brevis of both sexes in the patches were sexually mature, whereas immature males and females with unripe eggs were found frequently in sandy - bottom habitats. males in sandy - bottom habitats were smaller, but fed more frequently and were in better somatic condition than males in the patches. similar tendency was found in females. this indicates that n. brevis uses different habitats depending on the stage of its life history, with migration from sandy - bottom habitats to the shell - patches for reproduction. males in the patches exhibited different behavior patterns : floating above the patches and lying in the patches. the former was larger, more aggressive, and invested less in gonads (relative to body size) than the latter. these results accord with those of other shell - brooding lake tanganyika cichlids with arts, and they therefore suggest the presence of arts in n. brevis.
febrile seizures affect 3% of infants between the ages of three months and five years of age and are associated with fever in the absence of intracranial infection or other defined cause. febrile seizures are generally thought to be multifactorial with the genetic component polygenic, as suggested by diminishing risks beyond first degree relatives. as with other genetic forms of epilepsy, there are rare families with presumed autosomal dominant inheritance of febrile seizures [1, 2 ]. these may lead to gene identification potentially offering leads to genes and gene families that may harbour susceptibility variants for the vast majority of febrile seizures with complex inheritance. seizure - related (sez) 6 is a protein of 994 amino acids which is thought to play a role in neuronal cell to cell signalling. normal functioning maintains a balance between dendrite growth and branching to optimize dendritic trees for synaptic connectivity. sez6 was originally cloned following upregulation in mouse neurons after seizure induction using pentylenetetrazole (ptz) stimulation. this raises the question : can disturbances in the amount of transcribed sez6 through naturally occurring mutations predispose to seizures ? yu. subsequently reported a strong association between sez6 and human febrile seizures indicating that sez6 is a susceptibility gene for febrile seizures with complex inheritance. ninety - four (47 males, 47 females) unrelated cases of simple febrile seizure were screened for sez6 mutations in genomic dna isolated from venous blood. diagnosis was based on the observation of a seizure which ceased within approximately three minutes and which did not recur within a 24-hour period. for all cases, seizure onset occurred after three months of age and had ceased in all subjects within five years of age. ninety - six anonymous blood donors from the same caucasian population were used as controls. none of the affected children required lumbar puncture, electroencephalograhy, blood studies, or neuroimaging, consistent with the management recommendations of duffner.. the 17 sez6 exons were pcr amplified using the flanking intronic primers listed in table 1. primers were designed based on the sequence of sez6 transcript variant 1 (ncbi accession number nm_178860). samples were screened by single - stranded conformation polymorphism analysis (ssca) using the gelscan 3000 (corbett research) according to the manufacturer 's instructions. the functional effects of nonsynonymous variants were predicted using the polyphen-2 tool (http://genetics.bwh.harvard.edu/pph2/). nine sequence variants were detected within the protein - coding regions of the sez6 gene (table 2). five of the changes were synonymous and did not change the amino acid, but four of the changes were nonsynonymous and altered the amino acid. two of the missense changes, in exon 2 (c.142 c > a ; p.p48 t) and exon 7 (c.1568 g > a ; p.r523h), are both low - frequency changes but are present in cases of febrile seizures and in controls. they each affect amino acids which are highly conserved across vertebrate species (figure 1). both of these changes were predicted by polyphen-2 to be damaging, with scores of 0.955 and 1.000, respectively. neither change has been reported as a known variant in dbsnp (http://www.ncbi.nlm.nih.gov/snp/). the remaining two missense changes, the exon 8 c.1636 a > g (causing p.t546a) and exon 12 c.2417 t > c (causing p.m806 t), were common, occurring with approximately equal frequency in both febrile seizure cases and controls. they are moderately conserved across vertebrates. both of these changes are listed in dbsnp (rs1976165 and rs12941884, resp.) with frequencies in the european population similar to those seen in our patient and control groups. allele frequencies affecting all coding regions did not markedly differ between febrile seizures (n = 94) and controls (n = 96). there was no evidence in our population, from the sample size examined, for an association of the strength previously reported between sez6 genetic variation and febrile seizures. we also detected an insertion in intron 5 of the sez6 gene (ivs5 + 10 - 11insc) with allele frequencies of 51.6% in patients and 57.3% in controls. the most common of the sez6 variants in the population studied is the ivs5 + 10 - 11insc. this is the same variant reported by yu. as occurring in exon 5 at their position 1435 in the cdna (their genbank accession number gi:20143984). the discrepancy between the position they report for this polymorphism and the position we report arises from differences in the cdna sequences used. in both isoforms of sez6 currently listed in genbank (nm_178860 and nm_001098635), it is likely that this variant is a benign polymorphism, as evidenced by its high frequency among both cases and controls in our study (table 2). the variant is also listed in dbsnp (rs58747412), but the entry does not include population frequency data. reveals that in addition to the intronic variant described above, a threonine to alanine missense variant t546a, observed in four of their patients is in fact the snp rs1976165, which we observed at similar frequency in both patients and controls. we detected the same degree of genetic variation in both the febrile seizure cases and our 96 controls. their control numbers were not of adequate size, and in their febrile seizure cases, they misinterpreted the presence of naturally occurring genetic variations to be multiple pathogenic mutations present only among the febrile seizure patients. data from dbsnp shows that sez6 is a highly variable gene, with 17 nonsynonymous coding snps listed. findings of variants in this gene, therefore, need to be interpreted with caution in the absence of additional data (such as protein alignments, in silico pathogenicity predictions, or functional studies) indicating that the variants are indeed deleterious. polyphen-2 predictions indicate that only three or perhaps four of the eight coding variants reported by yu. are likely to be deleterious. these and our data reporting two additional potentially damaging rare variants are suggestive of a contribution of sez6 to a genetic predisposition to fs in a proportion of cases. the extent of the evolutionary conservation for some of the variants detected warrants further investigation using much larger sample sizes. massively parallel sequencing (mps) now delivers the sensitivity to detect all rare variants. the focus needs to shift to rare variants and statistical developments enabling association tests on rare variants. the polygenic heterogeneity model [10, 11 ] is likely applicable to febrile seizures as well as for genetic generalised epilepsy. it predicts that rare variants conferring genetic susceptibility will be present in both cases and controls ; however, their presence throughout the gene will be significantly higher in cases than in controls if large and adequately powered sample sizes are analysed for the true susceptibility genes. taken together, portions of the data from the present study and from yu. there is no reason to restrict sez6 analyses to febrile seizures since this gene represents a plausible candidate for any seizure disorder based on how it was originally cloned. a significant proportion of febrile seizure cases progress to afebrile seizures suggesting shared genetic determinants between febrile and a febrile seizures. sez6 warrants further investigation as a susceptibility gene for both febrile seizures and the epilepsies which show complex inheritance.	sixty cases of febrile seizures from a chinese cohort had previously been reported with a strong association between variants in the seizure - related (sez) 6 gene and febrile seizures. they found a striking lack of genetic variation in their controls. we found genetic variation in sez6 at similar levels at the same dna sequence positions in our 94 febrile seizure cases as in our 96 unaffected controls. two of our febrile seizure cases carried rare variants predicted to have damaging consequences. combined with some of the variants from the chinese cohort, these data are compatible with a role for sez6 as a susceptibility gene for febrile seizures. however, the polygenic determinants underlying most cases of febrile seizures with complex inheritance remain to be determined.
asian indians appear to have a higher propensity toward developing type 2 diabetes than other race / ethnic groups. india is home to the second - largest population of individuals with type 2 diabetes worldwide (1). furthermore, immigration to developed countries is traditionally associated with higher type 2 diabetes risk (24), and asian indian immigrants have a higher prevalence of type 2 diabetes than the general u.s. population (46). however, given that india has recently undergone rapid economic and nutrition transitions (7,8), it is unclear whether diabetes risk among asian indians immigrants in the u.s. such a comparison of two genetically similar populations living in different environmental settings could shed light on the behavioral and environmental factors associated with increased diabetes risk in this ethnic group. we therefore compared the age - specific prevalence of type 2 diabetes and prediabetes in two current population - based studies of urban asian indians aged 40 years : n = 2,305 residents of chennai, india, using data from the centre for cardiometabolic risk reduction in south - asia study (carrs) (20102011) (9), and n = 757 from the u.s.- based mediators of atherosclerosis in south asians living in america (masala) study (20102013) (10). we also analyzed the relative associations of demographic and anthropometric characteristics on prevalent glycemic status in urban asian indians in both india and the u.s. the design, sampling strategy, recruitment, enrollment, and questionnaire and examination components of the masala and carrs studies have previously been described in detail (9,10). in brief, carrs is a multisite cohort study that recruited participant populations from three urban megacities in india and pakistan (delhi, chennai, and karachi). the baseline examination for this cohort included a representative cross - sectional survey conducted in each city between 2010 and 2011. for the purposes of this study, data were analyzed from the chennai study site only, as this site was the only one to perform an oral glucose tolerance test (ogtt) in order to identify diabetes accurately. households were selected for participation using multistage random sampling technique in order to be representative of the city of chennai (9). a total of 6,920 individuals were screened for participation, of whom 6,906 (99%) provided questionnaire data. fasting plasma glucose was obtained from 5,952 participants (86%) and 2-h post glucose challenge on 4,051 participants. for this study, we limited our population to the 4,865 (70%) participants who were previously diagnosed with diabetes as determined by questionnaire data or who provided fasting and 2-h postchallenge glucose measurements. participants with existing cardiovascular disease as ascertained through self - report (n = 283) and those of age < 40 years (n = 2,277) were excluded from the carrs study for valid comparisons with masala. masala is based on a community - based sample of south asians living in the greater chicago and san francisco bay areas. the masala study was modeled to be similar to the multi - ethnic study of atherosclerosis (mesa) cohort study (11), and only individuals without a known history of cardiovascular disease were eligible. recruitment was conducted using telephone - based recruitment methods, similar to the mesa study (11). sampling frames were created by clinical site (either the university of california, san francisco, or northwestern university) and included all nine counties of the san francisco bay area and the seven census tracts closest to the northwestern university medical center, as well as suburban locations around chicago where census data revealed high proportions of asian indian residents. name, address, and telephone number were obtained for 10,000 households in the targeted census tracts from commercial mailing list companies (infousa, omaha, ne, and marketing systems group, horsham, pa). random samples of south asian surnames from the desired geographic locations were created using a specific cultural coding algorithm to identify 162 ethnicities, 16 ethnic groups, 80 language preferences, 21 countries of origin, and 12 religions using a five - step matching process to classify a person 's first and last name, thereby reducing selection bias among participants with uncommon south asian surnames (10). all participants were screened by telephone and were invited to either the university of california, san francisco, or the northwestern university clinical field center for a 6-h baseline clinical examination. in total, 9,097 households were attempted to be reached. within these households, 3,053 individuals were reached and 1,801 (59%) were eligible for participation (10). of all those eligible, a total of 906 individuals participated in the study. however, for the purposes of our analysis, data were analyzed only for individuals who identified as being born in india (n = 757). details regarding the eligibility criteria, questionnaire, and examination components in carrs and masala are shown in table 1. eligibility, questionnaire, and exam components in carrs and masala in both studies, after at least a 9-h overnight fast, a 75-g ogtt was administered to participants without previously diagnosed diabetes who were willing and able to participate in the glucose challenge. blood samples were obtained from a peripheral vein just before glucose ingestion (time 0) and at 30 and 120 min post glucose challenge for plasma glucose measurements. type 2 diabetes was defined similarly as self - reported use of glucose - lowering medication (either an oral agent or insulin), fasting glucose 126 mg / dl, or 2-h postchallenge glucose 200 mg / dl ; prediabetes was defined as fasting glucose 100125 mg / dl and/or a 2-h postchallenge glucose 140199 mg / dl (12). normal weight was classified as bmi 18.524.99 kg / m, overweight was classified as bmi 2529.99 kg / m, and obese was classified as bmi 30 kg / m. asian - specific cut points for bmi classification were also used for sensitivity analyses (14). prevalence values and 95% cis were estimated by study site, sex, age - group, and bmi category. participant characteristics were stratified by sex and were compared by study using test or anova as appropriate. the non normally distributed variables of fasting and 2-h plasma glucose were log transformed. the effect of location of residence (india or the u.s.) on the odds of prediabetes and type 2 diabetes compared with normal glucose tolerance was assessed using standardized polytomous regression. initially, an unadjusted regression model was created to compare the individual association between study location and prevalent glycemic status. subsequent multivariable models were then created to adjust for covariates including age, sex, blood pressure, waist circumference, educational status, and years since migration to the u.s. all analyses were performed using sas, version 9.4 (sas institute, cary, nc). prevalence values and 95% cis were estimated by study site, sex, age - group, and bmi category. participant characteristics were stratified by sex and were compared by study using test or anova as appropriate. the non normally distributed variables of fasting and 2-h plasma glucose were log transformed. the effect of location of residence (india or the u.s.) on the odds of prediabetes and type 2 diabetes compared with normal glucose tolerance was assessed using standardized polytomous regression. initially, an unadjusted regression model was created to compare the individual association between study location and prevalent glycemic status. subsequent multivariable models were then created to adjust for covariates including age, sex, blood pressure, waist circumference, educational status, and years since migration to the u.s. all analyses were performed using sas, version 9.4 (sas institute, cary, nc). table 2 displays participant characteristics by sex and study. of the 2,305 participants from carrs - chennai, the mean duration of residence in the u.s. for masala study participants was 27.8 10.8 years for men and 26.5 10.8 years for women. participants in the masala study were on average older than those in carrs - chennai and had higher educational attainment. on average, for both sexes, participants in masala were taller and had greater weight and waist circumference measurements than those in carrs - chennai. additionally, men in the masala study had a higher mean bmi than men in carrs - chennai ; however, this was reversed among women. in both studies, fasting glucose was obtained from all participants who were willing to provide a sample ; however, a 75-g ogtt was only administered to participants without a prior diagnosis of type 2 diabetes (masala n = 617, carrs n = 1,674). participants in the masala study had lower log fasting glucose values than participants in carrs - chennai but higher log fasting 2-h glucose values. those in masala also had lower systolic and diastolic blood pressure levels and took more blood pressure lowering medications than participants in carrs - chennai. of those with a prior diagnosis of type 2 diabetes, participants in masala had on average a longer duration since diagnosis. baseline participant characteristics by study center data are mean (sd) or % unless otherwise indicated. log fasting glucose : men, carrs - chennai (n = 1,027), masala (n = 402) ; women, carrs - chennai (n = 1,215), masala (n = 345). log 2-h glucose : men, carrs - chennai (n = 780), masala (n = 323) ; women, carrs - chennai (n = 894), masala (n = 294). age - adjusted type 2 diabetes prevalence was higher among indians in carrs - chennai than those in the masala study both overall (38% [95% ci 3640 ] vs. 24% [95% ci 2127 ]) and by sex (men 36% [95% ci 3339 ] vs. 27% [95% ci 2331 ] ; women 42% [95% ci 3945 ] vs. 23% [95% ci 1928 ]). of participants with type 2 diabetes, 65% of asian indians living in the u.s. age - adjusted prediabetes prevalence was lower in asian indians in chennai than in the u.s. (overall 24% [95% ci 2226 ] vs. 33% [95% ci 3036 ], men 21% [95% ci 1924) vs. 35% [95% ci 3140 ], and women 25% [95% ci 2328 ] vs. 29% [95% ci 2434 ]). these patterns were consistent across age- and sex groups, but differences in type 2 diabetes prevalence by age were more significant in women (fig. the prevalence of diabetes was higher in asian indians living in india than in asian indians living in the u.s. differences in diabetes prevalence between the groups were significant in normal and overweight participants but were not significant in participants who were obese. in all categories of bmi, the prevalence of prediabetes was lower in native asian indians than those in the u.s. and was significantly different in participants with normal bmi. the pattern of higher diabetes prevalence and lower prediabetes prevalence in asian indians living in india than asian indians in the u.s. in all bmi categories was consistent using the asian bmi cut points. however, when using the asian specific cut points, the prevalence of diabetes and prediabetes was most significantly different in participants who were overweight. prevalence of diabetes and prediabetes by study and bmi category. p < 0.05. of the 757 participants from masala, 189 (25%) had origins from one of four of the south indian states of tamil nadu, karnataka, andhra pradesh, or kerala. after restriction of participants from masala to only those with origins from south india, age - adjusted type 2 diabetes prevalence was again higher among indians in carrs - chennai than those in the masala study both overall (38% [95% ci 3640 ] vs. 25% [95% ci 2032 ]) and by sex (men 36% [95% ci 3339 ] vs. 27% [95% ci : 1935 ] ; women 42% [95% ci 3945 ] vs. 25% [95% ci 1534 ]). age - adjusted prediabetes prevalence was again lower in asian indians in chennai than in those in the u.s. with origins from south india specifically (overall 24% [95% ci 2226 ] vs. 33% [95% ci 2639 ], men 21% [95% ci 1924) vs. 36% [95% ci 2745 ], and women 25% [95% ci 2328 ] vs. 27% [95% ci 1938 ]). these patterns were again consistent in all age- and sex groups, but differences in diabetes prevalence between asian indians in chennai compared with asian indians in the u.s. with origins in south india were more significant than differences in prediabetes prevalence between these groups. table 3 shows the association of place of residence (either india [chennai ] or the u.s. [greater san francisco and chicago areas ]) with glycemic status. after adjustment for age, sex, waist circumference, and systolic blood pressure, asian indians in the masala study had a 50% (95% ci 0.40.6) decreased odds of type 2 diabetes but a 20% (95% ci 0.91.5) increased odds of prediabetes than those in carrs - chennai. the inclusion of education and years since migration in multivariable models somewhat attenuated the effect of place of residence on the odds of having diabetes compared with normal glucose tolerance. income could not be assessed in the models, as it was found to be collinear with place of residence. the inclusion of height in multivariable models as a proxy for socioeconomic status prior to migration did not alter the effect of place of residence on the odds of having diabetes or prediabetes compared with normal glucose tolerance between the groups. however, the inclusion of height and education together in multivariable models significantly attenuated the effect of place of residence on the odds of having diabetes. risk factors associated with prediabetes and type 2 diabetes ai, asian indian ; sbp, systolic blood pressure. asian indians living in india (carrs - chennai study) were used as the referent group. few studies have compared asian indians in india to those who have immigrated to the u.s. in this study comparing middle- to older - aged urban asian indians, we found that a community - based sample of asian indians in the u.s. had a lower prevalence of type 2 diabetes but a higher prevalence of prediabetes than asian indians living in urban south india. asian indians in the u.s. also had better blood pressure levels than those in india, possibly explained by their higher usage of blood pressure lowering medications. however, the adjustment for age, sex, waist circumference, and systolic blood pressure did not fully explain the increased odds of type 2 diabetes in asian indians in the carrs - chennai study. it is possible that india is in an early stage of the type 2 diabetes epidemic wherein those who are most susceptible to the disease develop it the earliest (15). have adopted more positive dietary and exercise habits, thereby lowering their risk for progression from prediabetes to overt type 2 diabetes (16). have poorer metabolic profiles than their counterparts in india (17,18), our results indicate that while asian indians in india had lower bmi and waist circumference measurements than those living in the u.s., they still had a higher prevalence of type 2 diabetes even at normal levels of bmi and in both sexes, thereby suggesting a shift in the association between migration and type 2 diabetes risk in this population. paradoxically, both the overall and the age - specific prevalence of prediabetes were lower in asian indians living in india than in the u.s., which may be due to a more rapid conversion through the natural history of disease in asian indians living in india. our results also add strength to the notion that factors besides age and central adiposity play a large role in type 2 diabetes development in asian indians (7) in both developed and developing country settings, since the adjustment for age, sex, waist circumference, and systolic blood pressure did not explain differences in the odds of prediabetes or type 2 diabetes between the two groups. furthermore, while the prevalence of type 2 diabetes was lower in asian indians living in the u.s. than in india, it was still considerably higher than the general u.s. risk factors for type 2 diabetes development such as high - carbohydrate and/or -fat diets and sedentary lifestyles were once considered to influence those who had migrated to developed countries leading to an increased prevalence of diabetes in migrants than in those who remained in developing country settings (17,18). the results of our study are among the first to highlight a higher prevalence of diabetes in individuals living in india than their counterparts who have immigrated to the u.s. it is therefore possible that, given the rapid economic and nutritional transitions currently taking place in india (7,8), these factors now exacerbate risks in asian indians both in india and abroad. it is also possible that with more increased knowledge of beneficial diet and lifestyle choices, migrant asian indians may be shifting toward more health - promoting dietary patterns. a more thorough understanding of the dietary transitions taking place in india and in diaspora indians could provide important insights into the development of type 2 diabetes in nonobese phenotypes. it is possible that asian indians in the u.s. may also have increased knowledge regarding diabetes prevention and greater access to health care (16,22) than asian indians in india. such factors may serve to protect immigrant populations against type 2 diabetes risk ; however, further research is needed in this area. our study directly compared the age - specific prevalence of prediabetes, type 2 diabetes, and the associated risk factors between asian indians living in the u.s. and india. while there were differences in the sampling frames and sociodemographic characteristics between the two studies, both are large population - based samples with similar anthropometric and laboratory measures that are representative of asian indians in large urban centers either in india or in the u.s. additionally, while participants from carrs are primarily of south indian origin and participants from masala migrated from all parts of india, it is possible that the differences in type 2 diabetes prevalence between the groups could be attributed to differences in regional origins. however, when we restricted our analyses to participants from masala with origins in south india only, the finding of a high prevalence of diabetes and a relatively lower prevalence of prediabetes in asian indians from carrs compared with asian indians from masala remained virtually unchanged. these results suggest that the differences in type 2 diabetes prevalence between the groups are likely not attributable to region of origin. furthermore, while there were large differences in education status as well as height between asian indians living in india and the u.s., adjustment for education and height in multivariable models as proxy measures for socioeconomic status prior to migration attenuated the effect of migration on the odds of diabetes between the two groups. these results suggest a possible healthy migrant effect, whereby individuals with greater access to education as well as early maternal and childhood nutrition were more likely to have the means for migration. however, while participants from the masala study had high levels of educational attainment, diabetes prevalence in this group was still considerably higher than that in the general u.s. population (20,21), thereby suggesting that factors besides education attainment play a large role in diabetes risk in asian indians. being that our study directly compares two distinct asian indian populations from differing geographic regions (chennai, india, and the greater san francisco and chicago areas of the u.s.) the results can not be generalized to asian indians living in other parts of india or the u.s. however, several studies have noted an increasingly high prevalence of diabetes in urban india (2325) with recent evidence indicating a rise of diabetes in rural areas of india as well (26). therefore, the high prevalence of diabetes in one urban indian city as reported in this study may be indicative of an even larger burden of disease in india yet to come. furthermore, the diabetes prevalence in masala study participants was similar to what was found in a recently published study of asian indians in michigan (27). however, additional national level data are needed to assess the prevalence of diabetes among asian indians living in the u.s. our findings point to a high prevalence of type 2 diabetes in urban india with a paradoxically low prevalence of prediabetes compared with urban asian indians in the u.s. furthermore, the increased type 2 diabetes prevalence in asian indians in india is evident in both sexes, in all age - groups, and at all levels of bmi and therefore can not be explained by differences in anthropometry or age alone. these findings suggest the need for collaborative longitudinal research efforts between india and the u.s. such collaborations could help identify the gene - environment - lifestyle exposures that underlie the elevated risk for type 2 diabetes development in asian indians.	objectiveto assess the prevalence of diabetes and prediabetes and the associated risk factors in two asian indian populations living in different environments.research design and methodswe performed cross - sectional analyses, using representative samples of 2,305 asian indians aged 4084 years living in chennai, india, from the centre for cardiometabolic risk reduction in south - asia study (carrs) (20102011), and 757 asian indians aged 4084 years living in the greater san francisco and chicago areas from the u.s. mediators of atherosclerosis in south asians living in america (masala) study (20102013). diabetes was defined as self - reported use of glucose - lowering medication, fasting glucose 126 mg / dl, or 2-h glucose 200 mg / dl. prediabetes was defined as fasting glucose 100125 mg / dl and/or 2-h glucose 140199 mg / dl.resultsage - adjusted diabetes prevalence was higher in india (38% [95% ci 3640 ]) than in the u.s. (24% [95% ci 2127 ]). age - adjusted prediabetes prevalence was lower in india (24% [95% ci 2226 ]) than in the u.s. (33% [95% ci 3036 ]). after adjustment for age, sex, waist circumference, and systolic blood pressure, living in the u.s. was associated with an increased odds for prediabetes (odds ratio 1.2 [95% ci 0.91.5 ]) and a decreased odds for diabetes (odds ratio 0.5 [95% ci 0.40.6]).conclusionsthese findings indicate possible changes in the relationship between migration and diabetes risk and highlight the growing burden of disease in urban india. additionally, these results call for longitudinal studies to better identify the gene - environment - lifestyle exposures that underlie the elevated risk for type 2 diabetes development in asian indians.
humans can lead efficient daily lives by adjusting their walking patterns in various ways during exercise. also, general walking does not efficiently control calories, the heart rate, the oxygen consumption, and the respiratory exchange ratio during the activity2. however, nordic walking activates the upper limb muscles and lowers the efficiency of recruitment and weight - bearing of the lower limbs, as the duration of the fatigue lengthens3,4,5. the primary purpose of using a pole is to be able to use the upper limb muscles, which are not normally used in walking, and to facilitate high - intensity exercise with the minimum effort by adjusting the energy consumption of the body6. according to the research of schwameder, nordic walking increased the strengths of the upper and lower limb muscles, and the poles dispersed the body weight ; furthermore, it was found to lower the vertical ground reaction force of the knee and significantly change the knee extension angle7. therefore, the present study analyzed the effects of nordic walking and walking on spatiotemporal parameters and ground reaction force, so that it would be possible to suggest an efficient means of walking for people who walk for health. the subjects were divided into a nordic walking group of 15 subjects and a walking group of 15 subjects. all subjects signed an informed consent form approved by the sehan university institutional review board. the general characteristics of the subjects were measured using an inbody j05 system (biospace, usa). the nordic walking group s average age was 23.2 4.6 years, average height was 167.2 4.3 cm, and average weight was 63.4 4.7 kg. the walking group s average age was 23.8 3.9 years, average height was 169.4 5.2 cm, and average weight was 65.3 5.78 kg. the three - dimensional kinematic and kinetic changes in the joints while walking were analyzed using a six - camera vicon mx motion analysis system (oxford metrics group, oxford, uk) for the gait test of the subjects. markers were placed on each of the subjects lower limbs in the standard places (table 1table 1.marker locationmarker numberlocation1 and 2bilateral psis3 and 4bilateral asis5 and 6midway between the hip and knee, lateral surface of the thigh7 and 8lateral femoral epicondyle9 and 10midway between the knee and ankle, lateral surface of the shank11 and 12lateral malleolus13 and 14calcaneal tuberosity15 and 16proximal to the second metatarsal headasis : anterior superior iliac spine ; psis : posterior superior iliac spine). for analysis of gait, values of cadence (step / min), stride time (seconds), step time (seconds), step length (mm), stride length (mm), and ground reaction force were analyzed. a static test was conducted on the force plate in a stationary standing posture. for a dynamic test, the subjects were asked to walk 12 meters using the walking method more comfortable for them, that is, nordic walking or walking ; after they walked 12 meters more than 10 times, their most natural walking patterns were chosen three times and analyzed. to determine the pole length for nordic walking, each subject s height asis : anterior superior iliac spine ; psis : posterior superior iliac spine the statistical analysis was carried out using a commercial statistics program, pasw statistics for windows version 18.0 (spss, chicago, il, usa). all the data on the measured items were expressed as averages and standard deviations ; to verify the differences in spatiotemporal parameters and ground reaction force, a paired t - test was performed. to verify the statistical significance of each result, comparison of the spatiotemporal gait parameters showed that cadence (118.845.95 steps / min vs. 104.106.32 steps / min), stride length (1.420.10 m vs. 1.300.15 m), and step length (0.720.06 m vs. 0.700.08 m) were increased and stride time (1.010.05 sec vs. 1.16 0.07 sec) and step time (0.510.03 sec vs. 0.590.04 sec) were decreased for nordic walking compared with walking, respecively (table 2table 2.comparison of spatiotemporal parameters for walking and nordic walkingcharacteristicswalkingnordic walkingcadence (steps / min)104.106.32118.845.95stride time (s)1.16 0.071.010.05step time (s)0.590.040.510.03stride length (% ll)1.300.151.420.10step length (% ll)0.700.080.720.06values are shown as the meansd. p<0.05 ; p<0.01 ; p<0.001). values are shown as the meansd. p<0.05 ; p<0.01 ; p<0.001 in the initial stance phase (fz1), walking and nordic walking showed ground reaction forces of 105.109.26% body weight (bw) and 117.8512.70% bw, which were significantly different (p < 0.01). in the mid - stance phase (fx0), walking and nordic walking showed ground reaction forces of 74.277.23% bw and 66.1510.10% bw, which were significantly different (p < 0.01). in the terminal stance phase (fx2), walking and nordic walking showed ground reaction forces of 110.205.27% bw and 114.407.06% bw, which were significantly different (p < 0.05) (table 3table 3.comparison of vertical ground reaction force for walking and nordic walkingwalkingnordic walkingfz1 max105.109.26117.8512.70fz0 max74.277.2366.1510.10fz2 max110.205.27114.407.06values are shown as the meansd. this study was conducted to identify the effects of nordic walking and walking on spatiotemporal parameters and ground reaction force, so that it would be possible to suggest an efficient means of walking for people who perform nordic walking for health. significant differences were in the spatiotemporal gait parameters between nordic walking and walking. in nordic walking, cadence, stride length, and step length were increased compared with walking, but stride time and step time were decreased. they reported that the spatiotemporal parameters were remarkably increased during walking, the time of contact with the ground in nordic walking was decreased, and cadence, step length, and stride length were obviously increased8. in the present study, it is thought that weight dispersion decreased energy consumption and increased the walking speed due to use of the upper limbs in nordic walking, though the propulsive force was adjusted in the lower limb muscles. it is also thought that the calf muscles improved the stability of the ankle joints9 and promoted quick propulsion in the terminal stance phase during nordic walking10. this study analyzed the changes in vertical ground reaction force in nordic walking and walking. the results showed significant differences in the maximum force of the load reactor in the initial stance phase (fz1) (p < 0.01) and in the terminal stance phase (fz2) (p < 0.05). this corresponds with the results of previous studies reporting that the vertical ground reaction force (p < 0.01) and weight - bearing of the lower limbs decreased in nordic walking11, 12. using an electromyogram, force plate, and motion analysis system, chiu and wang analyzed the perceived exertion, muscle activity, and joint angle of the lower limbs and ground reaction force in 30 adults depending on changes in the velocity of walking. they found that walking speed had a clear effect on the vertical ground reaction force : the vertical reaction force was reduced due to increased walking speed in the mid - stance phase (fz0), and the maximum force rose in the load reactor in the initial stance phase (fz1) and the terminal stance phase (fz2). using of nordic walking poles requires an increase in the force of the upper limb muscles to boost the weight - bearing of the body during the stance phase, improves balance and stability, and enhances the metabolism for movement13. this demonstrates that nordic walking is more effective in improving functional capabilities by promoting effective energy use and reducing the lower limb load, as the weight of the upper and lower limbs is dispersed during nordic walking. to apply nordic walking as an exercise program, it is deemed more important to adjust the natural walking speed and weight - bearing of the body. in conclusion, further kinematic and muscular physiological research should be conducted after subjecting the nordic walking subjects to a long - term training program, and a study on the effect of long - term nordic walking on the elderly or people with a pathologic gait is needed in the future.	[purpose ] the purpose of this study was to investigate the effects of nordic walking and walking on spatiotemporal gait parameters and ground reaction force. [subjects ] the subjects of this study were 30 young adult males, who were divided into a nordic walking group of 15 subjects and a walking group of 15 subjects. [methods ] to analyze the spatiotemporal parameters and ground reaction force during walking in the two groups, the six - camera vicon mx motion analysis system was used. the subjects were asked to walk 12 meters using the more comfortable walking method for them between nordic walking and walking. after they walked 12 meters more than 10 times, their most natural walking patterns were chosen three times and analyzed. to determine the pole for nordic walking, each subject s height was multiplied by 0.68. we then measured the spatiotemporal gait parameters and ground reaction force. [results ] compared with the walking group, the nordic walking group showed an increase in cadence, stride length, and step length, and a decrease in stride time, step time, and vertical ground reaction force. [conclusion ] the results of this study indicate that nordic walking increases the stride and can be considered as helping patients with diseases affecting their gait. this demonstrates that nordic walking is more effective in improving functional capabilities by promoting effective energy use and reducing the lower limb load, because the weight of the upper and lower limbs is dispersed during nordic walking.

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